Immunopharmacology of Platelets (Handbook of Immunopharmacology)

Immunopharmacologyof Platelets

THE HANDBOOKOF IMMUNOPHARMACOLOGY Series Editor: Clive Page

King's College London, UK

Titles in this series Cells and Mediators

Systems

Drugs

Immunopharmacology of Eosinophils (edited by H. Smith and tL Cook)

Immunopharmacology of the Gastrointestinal System (edited by J.L. Wallace)

Immunotherapy for Immunerelated Diseases (edited by W.J. Metzger, forthcoming)

The Immunopharmacology of Mast Cells and Basophils (edited by J.C. Foreman) Lipid Mediators (edited by F. Cunningham) Immunopharmacology of Neutrophils (edited by P.G. Hellewell and T.J. Williams) Immunopharmacology of Macrophages and other Antigen-Presenting Cells (edited by C.A.F.M. Bruijnzeel-Koomen and E.C.M. Hoefsmit) Adhesion Molecules (edited by C.D. Wegner)

Immunopharmacology of Joints Immunopharmacology of AIDS and Connective Tissue (forthcoming) (edited by M.E. Davies and J. Dingle) Immunopharmacology of the Heart (edited by M.J. Curtis)

Immunosuppressive Drugs (forthcoming)

Immunopharmacology of Epithelial Barriers (edited by 1L Goldie)

Glucocorticosteroids (forthcoming)

Immunopharmacology of the Renal System (edited by C. Tetta) Immunopharmacology of the Microcirculation (edited by S. Brain)

Immunopharmacology of the Immunopharmacology of Lymphocytes Respiratory System (edited by M. Rola-Pleszczynski) (edited by S.T. Holgate, forthcoming) Immunopharmacology of Platelets (edited by M. Joseph) Immunopharmacology of Free Radical Species (edited by D. Blake and P.G. Winyard) Cytokines (edited by A. Mire-Sluis, forthcoming)

The Kinin System (edited S. Farmer, forthcoming)

Angiogenesis (forthcoming) Phosphodiesteras Inhibitors (edited by G. Dent, K. Rabe and C. Schudt, forthcoming)

Immunopharmaco~gyof P~tekts edited by

M. Joseph Institut Pasteur, Lille, France

A C A D E M I C PRESS Harcourt Brace and Company, Publishers London San Diego New York Boston Sydney Tokyo Toronto

ACADEMIC PRESS LIMITED 24/28 Oval Road London NW1 7DX

United States Edition published by ACADEMIC PRESS INC. San Diego, CA 92101

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All rights reserved No part of this book may be reproduced in any form by photostat, microfilm, or by any other means, without written permission from the publishers A catalogue record for this book is available from the British Library ISBN 0-12-390120-0

Typeset by Mathematical Composition Setters Ltd, Salisbury, Wiltshire Printed and bound in Great Britain by The Bath Press, Avon

Contents Contributors xi Series Preface xv Preface xvii B

Do Platelets have a Role as Inflammatory Cells? 1 Caroline M. H e r d and Clive P. Page

1. 2. 3. 4.

Introduction 1 Platelet Physiology 2 Platelet-derived Mediators 3 Platelets in Haemostasis and Thrombosis 4 5. In vivo Platelet Monitoring 5 5.1 Methodology 5 5.2 Investigation of Platelet Function 5 6. Platelets in Non-allergic Host Defence 6 6.1 Platelets and Bacteria 6 6.2 Platelets and Malignancy 6 7. Platelets and Allergic Inflammation 7 7.1 The IgE Receptor 7

7.2 Platelets, Parasites and Cytotox!c Free Radicals 7 7.3 Platelets and Experimental Inflammation 8 7.4 Platelets and Allergic Asthma 8 7.4.1 Animal Evidence 9 7.4.2 Clinical Evidence 10 7.4.3 ASA-inducedAsthma 11 7.5 Platelets and Rheumatoid Arthritis 11 7.6 Platelets and Skin Inflammation 12 8. Conclusions 12 9. References 12

2 Animal Models for Investigating the Allergic and Inflammatory Properties of Platelets 21 Anthony J. Coyle and B. Boris Vargaftig 1. Introduction 21 2. Methods Available to Assess Platelet Involvement in Experimental Animal Models 22 3. The Role of Platelets in Experimental Models of Acute Inflammation 22 3.1 The Arthus Reaction 22 3.2 The Generalized Swartzman Reaction 23 3.3 Acute Serum Sickness in the Rabbit 23 3.4 Carrageenin-induced Inflammation 23 3.5 ExperimentalHaemarthrosis 23 4. The Role of Platelets in Models of Non-allergic Lung Injury 24 4.1 In vitro Studies 24 4.2 In vivo Studies 24 4.2.1 Microembolism and Direct Lung Injury 24 4.2.2 Experimental Pulmonary Hypertension 24

5. The Role of Platelets in Models of Allergic Lung Injury 25 5.1 Platelet Agonist- and Allergen-induced Bronchoconstriction 25 5.2 The Role of Platelets in Experimental Models of Airway Hyperresponsiveness 25 5.3 Interactions of Platelets with Other Blood Elements 26 5.4 The Role of Platelets in Models of Late Asthmatic Response 26 6. Direct Antigen-induced Activation of Platelets 27 7. The Role of Platelets in Models of Parasitic Infection 28 8. Conclusion 28 9. References 28

vi CONTENTS

The Analysis of L~qand-Receptor Interactions in Platelet Activation 31

0

Michael H. Kroll and Andrew I. Schafer 1. Introduction 31 2. Platelet Physiology 32 3. Activating Ligand-Receptor Interactions 33 3.1 Thrombin 33 3.2 TXA2/PGG2/PGH2 36 3.3 PAF 36 3.4 Collagen 36 3.5 VP 37 3.6 Epinephrine 37 3.7 ADP 37 3.8 5-HT 38 3.9 Other Platelet Receptors That Mediate Activation 38 3.9.1 vWF-GPIb Binding 38 3.9.2 GPIIb-IIIa 39 3.9.3 CD9 39 3.9.4 Other Activation-initiating Receptors 40 4. Activation-induced Changes in Platelet Receptors 40 5. G-proteins 40

0

6.

7.

8. 9. 10.

5.1 Gs]Gi 42 5.2 Gp 42 5.3 Low Molecular Weight G-proteins 42 Intracellular Signalling Pathways 43 6.1 PLC 43 6.2 IP3 and Calcium 43 6.3 PKC 45 6.4 PLA2 46 6.5 Na+/H § Exchange 47 6.6 Other Signal Pathways 48 6.6.1 PLD 48 6.6.2 Tyrosine Kinases 49 6.6.3 Histamine 49 Inhibitory Ligand-Receptor Interactions 49 7.1 Introduction 49 7.2 cAMP 49 7.3 cGMP 49 7.4 Mechanisms of Platelet Inhibition 50 Conclusion 51 Acknowledgements 51 References 51

The Role of Human Platelet Membrane Receptors in Inflammation 67 John L. McGregor

1. 2. 3. 4.

Introduction 67 Platelet Glycoproteins 67 Platelets and Inflammation 68 P-selectins 69 4.1 Structure and Homology 69 4.2 Platelet-Leucocyte Interactions 71 4.3 P-selectin Ligands 72 4.4 P-selectin in Circulation 72 4.5 Platelet-T Lymphocyte Interactions 73 4.6 Transcellular Synthesis of Molecules 73 4.7 Platelet P-selectin in Other Species 74 5. Cytokines and Platelets 74 6. Thrombospondin and CD36 75 0

6.1 A Multifunctional Adhesive Protein 75 6.2 Binding of TSP to Resting or Activated Platelets 75 6.3 TSP Receptors on the Platelet Surface 76 6.4 Sites on TSP Interacting with the Platelet Surface 76 6.5 TSP as a Ligand to Platelet-Monocyte Interactions 76 6.6 Binding of Platelets to Bacteria via TSP 77 7. Platelet Factor 4 77 8. Acknowledgements 77 9. References 78

Platelets in Bacterial Infections 83 C.C. Clawson

1. Introduction 83 2. Platelet Interaction with Non-biological Particulates 84 2.1 Clearance of Particulates from the Circulation 84 2.2 Engulfment of Inert Particles: Phagocytosis or Sequestration? 86 2.3 Influence of Particle Size 87 2.4 Soluble Co-factors of Particle Uptake 88 2.5 Metabolism During Ingestion of Inert Particulates 88

2.6 Platelet Secretion and Aggregation in Response to Inert Particles 89 3. Platelet Interaction with Bacteria in vitro 89 3.1 Aggregometry 90 3.2 Morphology 92 3.3 Influence of Plasma Components 94 3.4 Varied Responses to Different Bacteria 98 3.5 Bacterially Induced Platelet Secretion 100 3.6 Mechanisms of Adhesion and Activation 101

CONTENTS vii 3.6.1 Strep. sanguis Adhesion 101 3.6.2 Platelet Aggregation by Strep. sanguis 101 3.6.3 Ecto-ATPase of Strep. sanguis 102 3.7 Engulfment of Bacteria by Platelets 103 3.8 Fate of the Bacteria 104 3.9 Bacterial Products that Promote or Inhibit Platelet Activation 107 4. Platelet Interaction with Bacteria in vivo 107 4.1 Bacterial Clearance from the Circulation 108 5. Influence of Platelets on Phagocytes 109 5.1 Morphological Observations 109 5.2 Phagocytosis and Killing of Bacteria 109 5.3 Chemotaxis 110

5.4 Phagocytosis of Platelets 110 6. Implications of Platelet-Bacterial Interaction to Human Disease 110 6.1 Inflammation and Tissue Injury 112 6.1.1 Bacterial Endocarditis 112 6.1.2 Adult Respiratory Distress Syndrome 114 6.2 Thromboembolic Disorders and Disseminated Intravascular Coagulation 114 6.3 Atherosclerosis 115 6.4 Thrombocytopenia 115 7. Summary 115 8. References 116

6. Platelets in Parasitic Diseases 125 V6ronique Pancr6 and Claude Auriault 1. Introduction 125 2. Methods 125 2.1 Platelet Isolation 125 2.2 Anti-schistosome Cytotoxicity 126 2.3 Chemiluminescence 126 2.4 Flow Cytofluorometry 126 2.5 Binding ofRadiolabelled IgE 126 3. The Receptor for IgE on Platelets 127 4. Effector Properties of Platelets Towards Helminth Parasites 127 4.1 Schistosomiasis 127 4.2 Filariasis 129 5. Effector Properties of Platelets Towards Other Parasites 129 5.1 Toxoplasmosis 129 5.2 Trypanosomiasis 129 5.3 Malaria 129

7.

6. Other Inducers of Platelet Cytotoxicity 130 7. Regulation of Platelet Effector Function by T Lymphocytes 130 7.1 Activation of Platelets by Lymphokines 130 7.1.1 Role of Interferon Gamma 130 7.1.2 Role of Tumour Necrosis Factor 130 7.2 Suppression of Platelet Cytotoxic Function 131 7.2.1 Role of Platelet Activity Suppressive Lymphokine 131 7.2.2 Role of Ubiquitin 131 8. Regulation of Platelet Effector Function by Monocytes 134 9. Concluding Remarks 134 10. References 134

Platelets in Viral Infections 137 Dorothea Zucker-Franklin

1. Introduction 137 2. Clinical Manifestations 137 3. Direct Effect of Viruses on Megakaryocytes and Platelets 138 4. Thrombocytopenia caused by Immune Mechanisms 145

8.

5. Thrombocytopenia due to Diverse Mechanisms 145 6. Conclusion 146 7. References 147

Platelet-Tumour Cell Interactions 151

Andreina Poggi, Cosmo Rossi, Lucia Beviglia, Roberto Calabrese and Maria Benedetta Donati 1. Introduction 151 2. Platelet-Tumour Cell Interactions: in vitro Studies 151 2.1 Tumour Cell-induced Platelet Aggregation 152

2.2 Platelet-releasedProducts 152 2.3 Platelet-mediated Tumour Cell Adhesion 153 2.4 Adhesive Receptors and Platelet-Tumour Cell Interactions 154

viii CONTENTS 2.4.1 Integrins 154 2.4.2 Selectins 155 2.4.3 IgG-like Molecules 155 3. Platelet-Tumour Cell Interactions: in vivo Studies 156 4. Anti-platelet Drugs and Metastases 157 4.1 Monoclonal Antibodies to c~IIb f13 158 4.2 RGD Peptides 158 4.3 Disintegrins 158 5. Methods 159 5.1 In vitro Assays 159 5.1.1 Mouse Platelet Aggregation 159 5.1.2 Tumour Cell-induced Platelet Aggregation 159 5.1.3 Platelet-Tumour Cell Adhesion 159

5.1.4 Tumour Cell Adhesion to Extracellular Matrix Proteins 159 5.1.5 Tumour Cell Adhesion to Endothelial Cells 160 5.2 In vivo Assays 160 5.2.1 Production of Anti-platelet Serum 160 5.2.2 Ex vivo Platelet Counts 160 5.2.3 Tail Transection Bleeding Time 160 5.2.4 Organ Distribution of SlCr-labelled Platelets 160 5.2.5 Immunohistochemical Localization of Platelets 160 6. Conclusions 160 7. Acknowledgements 160 8. References 161

9. Autoimmune Thrombocytopenias 167 C&ile Kaplan and Gil Tchernia 1. Introduction: Idiopathic or Autoimmune Thrombocytopenia? 168 2. Prevalence 168 3. Clinical Syndrome, Classification and Evolution 168 3.1 The Acute Variety 169 3.2 The Intermittent Variety 169 3.3 The Chronic Form 169 4. Laboratory Testing 169 5. Megakaryocytopoiesis in AITP 170 6. Isotopic Studies 170 7. Immune Abnormalities in AITP 171 7.1 Immune Functions 171 7.2 Autoantibodies and Autoantigens in AITP 171 7.2.1 Detection of Immunoglobulins on Platelets in AITP Patients and Significance of the Tests 172 7.2.2 Techniques for Immunochemical Characterization of Autoantigens 173 7.2.2.1 Immunoblotting 173 7.2.2.2 Radioimmunoprecipitation 173 7.2.2.3 Antigenic Capture Assays 174 7.2.2.4 Molecular Biology 176 7.2.3 Identification of Specific Autoantigens on the Platelet Glycoproteins 176 7.2.3.1 Glycoproteins IIb and IIIa and the IIb-IIIa Glycoprotein Complex 176

7.2.3.2 Glycoproteins IB, IX, and the Ib-IX-V Glycoprotein Complex 177 7.2.3.3 Other Proteins 177 7.2.3.4 Autoantibodies and Platelet Functions 177 7.2.3.5 Autoantigens and Clinical Significance 178 8. Treatment 178 8.1 Acute AITP 178 8.1.1 Steroid Therapy 178 8.1.2 High Dose i.v. IgG 178 8.1.3 Intravenous Rhesus Antibodies (Anti-D) 179 8.2 Chronic AITP 179 8.2.1 Corticosteroids 179 8.2.2 High Dose i.v. IgG 179 8.2.3 Splenectomy 179 8.3 Refractory Chronic AITP 180 8.3.1 Immunosuppressive Drugs 180 8.3.2 Vinca Alkaloid Therapy 180 8.3.3 Colchicine Therapy 180 8.3.4 Danazol Therapy 180 8.3.5 Ascorbate Treatment 180 8.3.6 Anti-D Treatment 180 8.3.7 IFNc~ Therapy 180 8.3.8 Other Therapies 181 8.4 Emergency Treatment 181 8.4.1 Platelet Transfusions 181 8.4.2 Intravenous Methyl Prednisolone Therapy 181 9. Autoimmune Thrombocytopenic Purpura and Pregnancy 181 9.1 Mothers 181

CONTENTS ix 9.2 The Infants 181 9.3 Hidden Maternal Autoimmunity 183 9.4 Asymptomatic Maternal Thrombocytopenia 183 10. Secondary Immune Thrombocytopenic Purpura 183 10.1 Virus-induced Autoimmune Thrombocytopenia 183 10.1.1 RNA Virus Infections 183 10.1.2 RNA Viruses with Reverse Transcriptase Activity 184 10.1.3 DNA Virus Infections 185

10.

10.2 Systemic Lupus Erythematosus 185 10.3 Evans Syndrome 186 10.4 AITP and Malignancies 186 10.4.1 Lymphoproliferative Disorders 186 10.4.2 Solid Malignant Tumours 186 10.4.3 Bone Marrow Transplantation and Thrombocytopenias 186 10.5 Thrombocytopenia and Parasitic Infections 187 11. Acknowledgements 187 12. References 187

The Analysis of Eicosanoids Derived From Platelets 195 Jacques Maclouf and Aida Habib

1. Introduction 195 2. General Considerations 196 2.1 Conceptual Considerations 196 2.2 Analytical Considerations 197 3. Eicosanoids Derived from in vitro Studies 198 3.1 Bioassay 198 3.2 Chromatographic Analysis 198 3.2.1 Extraction 198 3.2.2 HPLC Analysis 199 3.2.2.1 Radioactivity Detection: Exogenous vs Endogenous 199

11.

3.2.2.2 UV Detection 200 3.3 Immunoassays 201 4. Assessment of the in vivo Production of Eicosanoids 204 4.1 GeneralConsiderations 204 4.2 Practical Considerations 205 5. Conclusion 206 6. References 207

The Generation of Free Radicals by Blood Platelets 209 Michel Joseph

1. Introduction 209 2. Oxygen Activation and Free Radical Metabolism 210 2.1 Superoxide Anion 210 2.2 Hydrogen Peroxide 211 2.3 Hydroxyl Radical 211 2.4 Singlet Oxygen 211 2.5 Oxygen Reaction with Free Radicals (Peroxy Radicals) 212 2.6 Peroxides and Lipoperoxides 212 2.7 Enzymes Involved and Cell Localization of Free Radical Production 214 3. Free Radical Generation by Blood Platelets 215 3.1 IgE-induced H202 Production 215 3.2 H202 Production by Platelets from Aspirin-sensitive Asthmatics 215 3.3 Mechanisms of Free Radical Generation by Platelets 216 3.4 Free Radical Generation is not a Side Effect of Platelet Aggregation 216 4. Antioxidant Defence Mechanisms 217

5. 6.

7. 8. 9.

4.1 Endogeneous Protection Against Free Radicals 217 4.2 Exogeneous Defence Against Free Radicals 218 4.2.1 Hydrophobic Scavengers 218 4.2.1.1 Vitamin E 218 4.2.1.2 Carotenoids 218 4.2.2 Hydrophilic Scavengers 218 4.2.2.1 Ascorbate and Glutathione 218 4.2.2.2 Other Scavengers 218 4.3 Inhibitors of Platelet Cytotoxicity 218 4.4 Platelet Defence Mechanisms 219 Some Methods for Monitoring Free Radicals and Their By-products 219 Free Radicals, Diseases and Platelets 220 6.1 Free Radicals and Diseases 220 6.2 Diseases and Platelets 220 Conclusion 221 Acknowledgements 221 References 221

x

CONTENTS

Glossary 227 Key to Illustrations 237 Index 243

Conm'butors C. Auriault Immunologie Cellulaire URA CNRS 1854, Institut Pasteur, B.P. 245, 59019 Lille, France L. Beviglia Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Mafia Imbaro, Italy

R. Calabrese Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Mafia Imbaro, Italy C.C. Clawson Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA

A.J. Coyle Department of Pulmonary Pharmacology, CIBA-Geigy A G , 4002 Basel, Switzerland M.B. Donati Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Mafia Imbaro, Italy

A. Habib INSERM U 348, Hopital Lariboisiere, 6, rue Guy-Patin, 75475 Paris 10, France C.M. Herd Department of Pharmacology, King's College, University of London, Manresa Road, London SW3 6LX, UK M. Joseph INSERM U 416, Institut Pasteur, B.P. 245, F-59019 Lille, France C. Kaplan Service d'Immunologie Leuco-Plaquettaire, INTS, 6 rue A Cabanel, 75015 Paris, France M.H. Kroll Hematology-Oncology, Baylor College of Medicine, 6565 Fannin, MS 902, Houston, TX 77030, USA

J. Maclouf INSERM U 348, H6pital Lariboisi~re, 6, rue Guy-Patin, 75475 Paris 10, France

xii CONTRIBUTORS J.L. McGregor INSERM Unit 331, Faculty of Medicine Alexis Carrel, Institut Pasteur de Lyon, France C.P. Page Department of Pharmacology, King's College, University of London, Manresa Road, London SW3 6LX, UK

A.I. Schafer Chief, Medical Service, Houston VA Medical Center, 2002 Holcombe Blvd. Houston, TX 77030, USA

G. Tchernia H6matologie, H6pital de Bic&re, 94275 Le Kremlin-Bic&re, France

V. Pancr6 Immunologie Cellulaire, URA CNRS 1854, Institut Pasteur, B.P. 245, 59019 Lille, France

B.B. Vargaftig Unite de Pharmacologie Cellulaire, INSERM No. 285, Institut Pasteur, 25 rue de Dr Roux, Paris, France

A. Voggi

D. Zucker-Franklin Department of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA

Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Mafia Imbaro, Italy C. Rossi Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy

Ser es Preface The consequences of diseases involving the immune system such as AIDS, and chronic inflammatory diseases such as bronchial asthma, rheumatoid arthritis and atherosclerosis, now account for a considerable economic burden to governments worldwide. In response to this, there has been a massive research effort investigating the basic mechanisms underlying such diseases, and a tremendous drive to identify novel therapeutic applications for the prevention and treatment of such diseases. Despite this effort, however, much of it within the pharmaceutical industries, this area of medical research has not gained the prominence of cardiovascular pharmacology or neuropharmacology. Over the last decade there has been a plethora of research papers and publications on immunology, but comparatively little written about the implications of such research for drug development. There is also no focal information source for pharmacologists with an interest in diseases affecting the immune system or the inflammatory response to consult, whether as a teaching aid or as a research reference. The main impetus behind the creation of this series was to provide such a source by commissioning a comprehensive collection of volumes on all aspects of immunopharmacology. It has been a deliberate policy to seek editors for each volume who are not only active in their respective areas of expertise, but who also have a distinctly pharmacological bias to their research. My hope is that The Handbook of Immunopharmacology will become indispensable to researchers and teachers for many years to come, with volumes being regularly updated. The series follows three main themes, each theme represented by volumes on individual component topics.

The first covers each of the major cell types and classes of inflammatory mediators. The second covers each of the major organ systems and the diseases involving the immune and inflammatory responses that can affect them. The series will thus include clinical aspects along with basic science. The third covers different classes of drugs that are currently being used to treat inflammatory disease or diseases involving the immune system, as well as novel classes of drugs under development for the treatment of such diseases. To enhance the usefulness of the series as a reference and teaching aid, a standardized artwork policy has been adopted. A particular cell type, for instance, is represented identically throughout the series. An appendix of these standard drawings is published in each volume. Likewise, a standardized system of abbreviations of terms has been implemented and will be developed by the editors involved in individual volumes as the series grows. A glossary of abbreviated terms is also published in each volume. This should facilitate cross-referencing between volumes. In time, it is hoped that the glossary will be regarded as a source of standard terms. While the series has been developed to be an integrated whole, each volume is complete in itself and may be used as an authoritative review of its designated topic. I am extremely grateful to the officers of Academic Press, and in particular to Dr Carey Chapman, for their vision in agreeing to collaborate on such a venture, and greatly hope that the series does indeed prove to be invaluable to the medical and scientific community. C.P. Page

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Preface Although anucleated, blood platelets are extraordinarily efficient cells and still the subject of fruitful investigations in haematology, in vascular physiology, in biochemistry, and more recently in immunology and in physiopathology where their metabolism has been implicated as a source of potential effector compounds. It is challenging for cell physiologists that these blood elements reset so strongly to environmental stimuli, expressing a tremendous and rapid modification of their shape and metabolism, with only their intrinsic and relatively locked properties and without the active machinery of the nucleus. Platelet literature already covers yards of shelves in haematology departments, in blood centre libraries and in atherosclerosis research laboratories. Why produce a new book in such a well-explored area? It is to the credit of Dr Clive Page, the series editor of The Handbook of Immunopharma~ology, that he has focused thought over the last decade, by his own investigations and by a careful survey of the literature, on the involvement of blood platelets outside their classical field of application, and more precisely in physiopathological mechanisms of allergy and inflammation. We have logically let him present an introductory chapter, which summarizes, with great precision what will be found in more detail in the other contributions to this work. Dr Boris Vargaftig, at the Institute Pasteur in Paris, has contributed largely to the development of animal models in allergic and inflammatory disorders, and is particularly well qualified to present here, with the collaboration of Dr Anthony Coyle, a review of such models for investigating the potential implications of platelets in these pathologies. The results they, and others, have obtained with animal substitutes have thrown some light on a possible place for platelets in the cell network of acute inflammation or allergic reactions, and more particularly, asthma. As already stressed above, another fascinating aspect of platelets is the absence of a nucleus, which strengthens the importance of the membrane and receptors in the physiological mechanisms of thrombocyte activation, together with the understanding of the intracellular signalling pathways sustaining their haematological functions. The rapidly moving knowledge in this domain has been carefully covered by the specialists in the matter, Dr Michael Kroll and Dr Andrew Schafer in Houston. Although specifically centred on biochemical

mechanisms leading to aggregation, their extensive analysis brings interesting perspectives to the potential participation of platelets in immunological and physiopathological processes. Their contribution is perfectly complemented by that of Dr John McGregor, in Lyon, which has taken into account the active role played by adhesion molecules and membrane-bound ligands in mediating the platelet involvement in inflammation. Ten years ago, When we observed the efficiency of platelets in killing parasites, we found that Professor Clawson had reported more than 10 years previously, the entrapping and clearance of bacteria in vivo by platelet aggregates. It is an honour to have a contribution from this leading researcher, which gives a striking synthesis of his knowledge on the physicochemical and physiological properties of platelets in their interaction with inert and living particles. It is also a pleasure to have a review on effector functions of platelets towards parasites by the pioneer of the topic in our laboratory, Dr Claude Auriault. More than 10 years ago, he understood very cleverly the importance of in vitro observations by Dr Pierre Viens in Montrral on trypanosme-platelet interactions, and he has developed a limited observation into an incredibly fruitful adventure. To summarize our knowledge in this field, he has worked with Dr Vrronique Pancrr, who has actively participated in the story by discovering a lymphokine inhibiting platelet activation, as well as the efficiency of interferon--), and interleukin-6 in stimulating platelet immune functions. Considering the efficient anti-bacterial and antiparasitic properties of blood platelets, we hoped for a while that they would exhibit similar properties against viruses and tumour cells. However, the next two chapters describe the viral-dependent increase of platelet activation, with damaging consequences for infected animals or patients, and tumoural dissemination. Professor Dorothea Zucker-Franklin, from the New York University Medical Center, and Professor Mafia Benedetta Donati and her colleagues from the Istituto Mario Negri in Italy, were particularly suited to the task of writing them. As platelets are so closely involved in the immune system, as effectors and targets, their disappearance from the bloodstream is associated with severe syndromes. Dr C&ile Kaplan, from the National Blood Transfusion

xvi PREFACE Centre in Paris, has very nicely reviewed all aspects of autoimmune thrombocytopenias. The reader will observe that their characteristics show several similarities with other platelet dysfunctions, such as those observed in intrinsic and extrinsic asthma. The autoimmune processes underlying some idiopathic syndromes therefore deserve our careful consideration in the near future. Finally, two kinds of platelet mediators have been selected as potential partners of the inflammatory and allergic disorders reported throughout this book. Dr Jacques Maclouf, together with Dr A/da Habib, specialists of arachidonate metabolites at H6pital Lariboisi~re in Paris, review the available procedures for identifying and measuring eicosanoids, in biological fluids or in culture supernatants, as an indication of a blood platelet reaction in vivo or in vitro. This difficult approach to the follow-up of platelet involvement is well documented in their present contribution, and the authors bring fascinating perspectives on "transcellular biosynthesis", a concept which could be of potential value for other cells and

physiological situations. The second family of mediators reviewed here is free radicals and their by-products, suspected of sustaining cytotoxic properties of platelets. I have attempted to collect converging reports on plateletassociated biochemical events generating free radicals, and to draw an extensive picture of reactive oxygen metabolism and its regulation. A book entirely devoted to one specific cell might leave the impression that the authors are convinced - and trying to convince readers - of the exclusive importance and role of the particular cell they are investigating in all reported biological events. It may be useful to stress here and I am sure, in doing so, I express the feelings of all contributors of this volume - that this accumulation of "pro-platelet evidence" has to be compared with similar evidence for the role of other cellular components of the immune network, as doubtlessly found in other volumes in this series. -

Michel Joseph

9

Do Platelets have a Role as

Inflammatory Cells? Caroline M. Herd and Clive P. Page

1. 2. 3. 4.

Introduction Platelet Physiology Platelet-derived Mediators Platelets in Haemostasis and Thrombosis 5. In vivo Platelet Monitoring 5.1 Methodology 5.2 Investigation of Platelet Function 6. Platelets in Non-allergic Host Defence 6.1 Platelets and Bacteria 6.2 Platelets and Malignancy 7. Platelets and Allergic Inflammation 7.1 The IgE Receptor

1 2 3 4 5 5 5 6 6 6 7 7

1. Introduction The platelet has been traditionally associated with disorders of the cardiovascular system, a well-recognized cell type actively involved in the maintenance of haemostasis and the initiation of repair after tissue injury. It has generally been accepted that the primary function of platelets is their adhesion to the endothelium or to other components at sites of the injured vessel wall in the initiation of haemostasis. However, it has been ~suggested that the fundamental physiological role of the platelet within the mammalian circulation is in the defence of the host against invasion by foreign organisms (Copley, 1979). In certain lower organisms such as caterpillars, the response to a foreign body involves the adhesion of haemocytes (primitive leucocytes) to the surface of the foreign body, with the subsequent aggregation of further haemocytes. The resultant capsule is morphologically similar to a haemostatic platelet plug, an observation that has led to the suggestion that primary haemostasis in higher mammals Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

7.2 Platelets, Parasites and Cytotoxic Free Radicals 7.3 Platelets and Experimental Inflammation 7.4 Platelets and Allergic Asthma 7.4.1 Animal Evidence 7.4.2 Clinical Evidence 7.4.3 ASA-induced Asthma 7.5 Platelets and Rheumatoid Arthritis 7.6 Platelets and Skin Inflammation 8. Conclusions 9. References

7 8 8 9 10 11 11 12 12 12

is a phylogenetic vestige retained from the behaviour of primitive leucocytes (Nachman and Weksler, 1980). Both experimental and clinical evidence exists to implicate the platelet in natural host defence mechanisms and associated pathological conditions. This cell has only comparatively recently been discussed in the context of allergic processes and immunological mechanisms (Capron et al., 1987; Gresele et al., 1987; Page, 1988). Despite being devoid of a nucleus, platelets possess many of the features of classical inflammatory cells such as polymorphonuclear leucocytes. They are capable of undergoing chemotaxis (Lowenhaupt, 1982), have been shown to phagocytose foreign particles (Mustard and Packham, 1979), contain and release various adhesive proteins, activate complement, interact with parasites, viruses and bacteria, alter vascular tone, enhance vascular permeability and take up, store and metabolize various vasoactive substances (Weksler, 1983). Furthermore, experiments have indicated that platelets have the capacity to release mediators with potent inflammatory or Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

2 C . M . HERD AND C.P. PAGE anaphylactic properties such as the ether-linked phospholipid platelet-activating factor (PAr) or the plateletspecific protein platelet factor 4 (PF4), and so far unidentified factors able to induce histamine release from basophils.

2. Platelet Physiology Platelets are small, anucleate blood elements, and under normal conditions constitute a small fraction of the circulating cells, the platelet count in healthy human blood ranging from 1.3 to 4.0 x 10s platelets/gl. Classically they were thought to be derived from megakaryocytes in the bone marrow by the process of fragmentation (Wright, 1910), however, it has more recently been suggested that megakaryocytes travel to the lung vasculature from the bone marrow where they physically become fragmented following impact with the extensive capillary network (Martin and Levine, 1991). The plasma membrane represents the site of platelet interactions with the external environment and is ultimately involved in the control or generation of the many specialized functional properties of the cell. The platelet surface is a typical bilayer membrane composed of protein, lipids (predominantly phospholipids) and carbohydrate. Platelet surface glycoproteins (GPs) play a primary role in the adhesion of platelets to exposed subendothelial matrix proteins, interaction with ligands such as collagen and thrombin, and exposure of fibrinogen receptors to facilitate aggregation (reviewed in Tuffin, 1991). A number of platelet receptors for ligands of biological or pharmacological significance have been identified. In recent years the major excitatory [including c~2-adrenoceptor, adenosine diphosphate (ADP), serotonin (5-HT2), PAF, thromboxane A2 TXA2), vasopressin (VP), thrombin] and inhibitory [including B2-adrenoceptor, adenosine, prostaglandin D2 (PGD2), prostaglandin I2 (PGI2)] surface membrane receptors of the human platelet have been characterized (reviewed in Tufiin, 1991). Following platelet activation there is exposure of the fibrinogen receptor glycoprotein IIb-IIIa (GPIIb-IIIa) which binds circulating fibrinogen allowing platelet-to-platelet interaction. Another surface receptor that is activated under shear forces within the arterial circulation is the GPIb-IX complex which interacts with von Willebrand's factor and facilitates adherence of the platelet to the vessel wall. Loss of cell surface GPs appears to be a primary mechanism of platelet senescence in vivo (Greenberg et al., 1979). Platelets possess a GP receptor for the third component of complement (C3b) which resembles that located on mononuclear cells (Yu et al., 1986), and Fc receptors for both IgG and IgE antibodies (Rosenfeld et al., 1985; Joseph et al., 1986). The platelet IgE receptor appears not to be associated in any way with the formation of aggregates, but with the ability of platelets to mount a

reaginic antibody-dependent cytotoxic response against helminth parasites such as Schistosoma mansoni, through oxidative killing (as demonstrated in vitro by chemiluminescence; Capron et al., 1986). Just beneath the cell membrane a bundle of microtubules travels the entire circumference of the cell. In the resting state this band is a flexible cytoskeleton exerting tension outward, maintaining both the normal morphometry of the unstimulated platelet and geographical integrity of the organelles. The alteration in platelet shape induced by cell activation is comprised predominantly of a circumferential band of microtubules (the major protein being tubulin; White and Sauk, 1984) and abundant cytosolic actin microfilaments (Boyles et al., 1985). In close configuration with the microtubule band is a microfilament matrix which provides contractile force for the secretion of cell constituents during the platelet release reaction. Two membrane systems weave throughout the cell interior, effectively increasing the platelet surface area. The open canalicular system, a random series of invaginations of the plasma membrane, contains channels which are continuous with the extracellular space, which facilitate secretion from the amine and protein-storage granules during the release reaction and hence serve as a conduit through which endogenous substances pass to the cell exterior. The canaliculi also provide ready access to the interior of the platelet for plasma proteins and other substances (White, 1974). The dense tubular system, derived from megakaryocyte endoplasmic reticulum, is associated with the circumferential microtubule band. This system is implicated as a major site of calcium sequestration, used for the initiation of platelet activation processes. The most numerous organelles held within the platelet cytoplasm are the platelet granules. Dense granules contain ADP and ATP, 5-HT and Ca 2+ . The more numerous cx granules store vasoactive components which have either been synthesized by the megakaryocyte or taken up from the circulation. In addition, they contain a variety of proteins, some platelet specific, which include adhesive proteins, the "anti-heparinoid" PF4, plateletderived growth factor (PDGF), B-thromboglobulin (/5TG), transforming growth factor /3 (TG-B), fibrinogen and clotting factors V and VIII (yon Willebrand's factor). Platelets are capable of only limited protein synthesis. Mitochondria are few in number but contribute significantly to energy metabolism of the cell by providing ATP for the cytoplasmic metabolic pool. Lysosomes, glycogen granules and peroxisomes are randomly distributed throughout the cytoplasm. The platelet lifespan has been estimated at 8-12 days by a variety of radioisotopic labelling techniques (Aas and Gardner, 1958; Najean and Ardaillou, 1969). Destruction of effete platelets is accomplished by macrophages of the reticulo-endothelial system in the spleen, liver and bone marrow.

DO PLATELETS HAVE A ROLE AS INFLAMMATORY CELLS? 3 also for eosinophils (Chihara et al., 1988). The ability of PF4 to activate eosinophils is of interest because it has Platelets are a rich source of a wide range of biologically been suggested that they contribute to the tissue damage active materials that are capable of inducing or aug- observed in asthma which may be associated with airway menting certain inflammatory responses. Such materials hyperresponsiveness (Frigas and Gleich, 1986). Furtherhave been shown to be both preformed mediators stored more, it has been demonstrated that PF4 can increase in either the dense or c~granules and newly formed medi- the expression of Fc IgG and Fc IgE receptors (Chihara ators resulting from the perturbation of membrane phos- et al., 1988). pholipids. These substances may be released from the cell The interesting finding that the immune response supfollowing activation. pressed by lymphoma cells in mice could be restored by 5-HT, stored in large amounts in human platelets, may the injection of mouse serum (Katz et al., 1983), sugcontribute to the inflammatory response via its vasocon- gested an active role of platelets in this phenomenon. It strictor properties and capacity to increase vascular was subsequently shown that the substance that reversed permeability (Majno and Palade, 1961). 5-HT has also the immunosuppression was PF4 (Katz et al., 1985, been shown to stimulate fibroblast growth (Boucek and 1986). The reversal of immunosuppression has been Alvarez, 1970). Adenosine, which can be formed from demonstrated in vitro using cultured mouse spleen cells the nucleotides stored and released by platelets, may play (Katz et al., 1989). The ability of PF4 to reverse this a role in bronchoconstriction (Holgate et al., 1991), and immunosuppression does not appear to be related to its receptors for Adenosive have been shown to be up- ability to bind heparin (Zucker et al., 1989), but may be regulated in allergic rabbits compared with normal rabbits a function of its serine protease activity (Katz et al., (Mustafa et al., 1991). 1986). Human platelets contain, and are capable of synPlatelet-derived growth factor (PDGF) is generally thesizing, histamine (H; Saxena et al., 1989; Mannaioni believed to be the principal mitogen that stimulates cell et al., 1992a) and of taking up the preformed amine with division where vessel integrity has been compromised and an energy-dependent process. H release from human and platelet activation has occurred (Deuel and Huang, guinea-pig blood has been recently demonstrated during 1984). PDGF may also act as a mediator of inflammation aggregation in vitro (Mannaioni et al., 1993). Exogenous and repair by affecting vascular tone (vasoconstriction; H has been shown to dose-dependently enhance platelet Berk et al., 1986), exerting chemotactic effects towards aggregation induced by a variety of stimuli through a monocytes and neutrophils (Deuel et al., 1982) and by Ca2+-dependent, H1 receptor-driven process (Man- activating monocytes (Tzeng et al., 1985) and neunaioni et al., 1992b). H, which is released during platelet trophils (Deuel and Huang, !984). Smooth muscle cells aggregation, potentiates the effect induced by pro- and fibroblasts are strongly attracted to low concentraaggregatory stimuli (Mannaioni etal., 1990, 1991) which tions of PDGF (Grotendorst et al., 1981; Seppa et al., may lead to a positive feedback effect on thrombogenesis 1982; Senior et al., 1983), suggesting that these cells may and on vascular inflammation. Human platelets have migrate to injured sites where subsequent mitogenic been shown to stimulate the release of H from mast cells stimulation further repair processes (Deuel and Huang, and basophils through IgE-dependent mechanisms 1984). Similady, TGF-~3 has been shown to be (Knauer et al., 1984). Thrombin, PAF and collagen can chemotactic for neutrophils and fibroblasts (Wahl et al., liberate this H-releasing substance from platelets (Knauer 1987). PDGF released at sites of continuous vessel wall et al., 1984; Orchard et al., 1986). In addition to causing injury has been suggested to contribute to the vascular eosinophil chemotaxis, platelet-derived histamine- smooth muscle thickening which characterizes cardiovasreleasing factor (PDHRF) has been shown to induce both cular diseases such as atherosclerosis (Ross et al., 1986). early- and late-onset airway obstruction as well as airway Similarly, bronchial smooth muscle hypertrophy is a feahyperresponsiveness in experimental animals (Fisher et ture of the asthmatic lung at autopsy (Ebina et al., 1990; al., 1990; Metzger et al., 1990). Carroll et al., 1993) and it is possible that continuous Platelets contain cationic proteins which can increase platelet activation, recruitment and extravascular diapevascular permeability (possibly by their action on mast desis into the airways with consequent release of cells) (Nachman etal., 1972; Sasaki etal., 1991), in addi- mitogens, could contribute to this feature of asthma. The tion to a cationic protein that cleaves the fifth component role of platelet activation in the induction of myofibroof complement to form a factor which is chemotactic for blast proliferation and bronchial smooth muscle thickleucocytes (Weksler and Coupal, 1973). -ening characteristic of asthma remains yet to be fully PF4, a platelet-specific protein released following elucidated, although PDGF has recently been reported to stimulation, possesses many properties that suggest a role act as a mitogen for airway smooth muscle cells in culture in allergy and inflammation. It stimulates basophils to (Hirst et al., 1992). release H (Brindley et al., 1983) and has been shown not Recent findings that the cytokine RANTES (a member only to be chemotactic for polymorphonuclear leuco- of the IL-8 supergene family), released upon appropriate cytes, monocytes and fibroblasts (Deuel et al., 1981), but stimulation from platelets, is a potent chemoattractant

3. Platelet-derived Mediators

4 C . M . HERD AND C.P. PAGE for both monocytes (Schall et al., 1990) and eosinophils (Kameyoshi etal., 1992), serves as additional evidence for the contribution of platelets to the inflammatory response. Upon cell stimulation and activation, products of the metabolism of membrane arachidonic acid (AA) are synthesized and liberated. TXA2 is a potent vasoconstrictor and bronchial smooth muscle spasmogen (Samuelsson et al., 1978). Prostaglandin F2~ (PGF2~) is a vasoconstrictor whereas PGE2 is a vasodilator and inducer/modulator of pain and fever. 12-Hydroxyeicosatetraenoic acid (12HETE), synthesized by the the platelet-specific enzyme 12-1ipoxygenase (12-LO)on release of AA (Marcus et al., 1984), exerts chemotactic activity towards eosinophils (Goetzl et al., 1977). Platelets have been shown to co-operate with leucocytes to produce chemotactic factors which the cells are unable to synthesize in isolation. Platelet 12-HETE can be metabolized by unstimulated neutrophils to yield 12,20-diHETE, a unique product which cannot be synthesized by either cell alone (Marcus et al., 1984, 1987, 1988). Furthermore, in the presence of activated platelets, leucocytes can produce increased amounts of leukotrienes (LTs) because 12-hydroperoxyeicosatetraenoic acid (12-HPETE), produced by platelets, can stimulate the activity of leucocyte 5-LO (Maclouf et al., 1982). Neutrophils can also utilize AA from stimulated platelets for the synthesis of 5-HETE and leukotriene B4 (LTB4); Marcus et al., 1982), a mediator with a wide pro-inflammatory profile (FordHutchinson, 1990). PAF can also stimulate the synthesis of LTB~ from these cells (Lin et al., 1982). Conversely, platelets may produce LTC4 from LTA4 synthesized by leucocytes via glutathione-S-transferase (Maclouf and Murphy, 1988), a powerful bronchial smooth muscle constrictor and proposed mediator of allergic asthma (reviewed in Piacentini and Kaliner, 1991). Both neutrophils and platelets can release PAF in modest amounts in response to appropriate activation stimuli (Chignard et al., 1980; Lynch and Henson, 1986). However, the presence of a small number of platelets in a suspension of neutrophils results in the generation of significantly increased amounts of PAF, far in excess of that predicted from the individual cell types (Coeffier et al., 1984). Platelet aggregation is observed when mixtures of leucocytes and platelets are stimulated with leucocyte-specific agonists, a response inhibited by PAF antagonists (Oda et al., 1986). PAF is an extremely potent inflammatory agent and has been implicated as a mediator of inflammation and asthma (reviewed in Page, 1988). Neutrophils have been shown to release a factor capable of activating platelets (neutrophilin; Chignard et al., 1986). Platelet activation is also potentiated by neutrophils through the production of hydrogen peroxide (H202) and oxygen free radicals (Canoso et al., 1974). Furthermore, nitric oxide (NO) produced from either vascular endothelial cells, circulating neutrophils or

platelets themselves makes a major contribution to the control of platelet and neutrophil aggregation and disaggregation in vivo (May et al., 1991a).

11

Platelets in Haemostasis and Thrombosis

Platelets play a central role in the prevention of excessive blood loss. Intact blood vessels are lined by haemostatically inert endothelial cells and as a consequence, subendothelial structures do not normally come into contact with flowing blood. Vascular injury (either spontaneous or traumatic interruption of vascular continuity) is the stimulus required to initiate a series of complex and interdependent reactions. Platelet surfaces will adhere to the exposed collagen fibres, through the process of activation of several membrane GPs of the integrin super-family of adhesion receptors. These include the collagen receptor, the GPIa-IIa complex (VLA-2) (c~2~), the fibronectin receptor, GPIc-IIa complex (VLA-5) (asB), the laminin receptor, GPIc'-IIa complex (VLA-6) (a6B), von Willebrand's factor receptor GPIb-IX complex and a vitronectin receptor ~v~3 (Parmentier et al., 1990). Following activation, the platelet membrane integrin GPIIb-IIIa is involved in the spreading of platelets by binding to von Willebrand's factor and fibrinogen. Induction of the membrane adhesion protein of the selectin family, GMP-140 (PADGEM), permits the interaction of platelets with leucocytes (Parmentier et al., 1990). The cells change shape from discoid to a more spherical form, a process mediated by the contractile microtubular system, characterized morphologically by the extension of short and long dendritic pseudopodia (White, 1987). A secretory process ensues, whereby substances stored in platelet granules are extruded from the platelet, i.e. the platelet release reaction. ADP discharged from the dense granules and TXA2 generated by the activation of platelet membrane phospholipase A2 (PLA2) influence the recruitment of additional circulating platelets to clump on those already adhered to the injured site. If the flow conditions are sufficiently disturbed, platelet aggregates form on the vessel wall and serve as a focus for the acceleration of coagulation reactions via platelet factor 3. Contact of blood with the subendothelium and release of the tissue factor (thromboplastin) from the damaged vessels initiates a cascade of proteolytic reactions in the intrinsic coagulation pathway, culminating in the formation of thrombin. The newly formed thrombin acts synergistically with ADP and TXA2 to promote further aggregation of platelets to form an enlarging platelet mass as the haemostatic plug (Zucker, 1980). Thrombin converts fibrinogen, present in plasma and released from platelets, into fibrin monomers which polymerize to stabilize and reinforce the platelet plug. The fibrin meshwork contains platelets and some red and white blood cells. Platelet contractile

DO PLATELETS HAVE A ROLE AS INFLAMMATORY CELLS? 5 proteins thrombosthenin and actomysin are stimulated by thrombin and clot retraction is initiated (Zucker, 1980). Subsequently, plasmin is cleaved from its plasminogen precursor and by its lytic action on fibrin causes the slow dissolution of the clot. Atherosclerosis may occur when platelets deposit in the vicinity of damaged endothelium, and smooth muscle cells proliferate and invade the vessel intimal layer. Lipids and cholesterol accumulate and the plaque is subsequently overgrown by the endothelium. The mitogenic stimulus for smooth muscle cell proliferation appears to be PDGF released from the c~ granules of activated platelets as previously discussed.

5. In vivo Platelet Monitoring Numerous in vitro and in v/v0, techniques have been developed not only for the investigation of platelet function per se but also for the detection of novel drugs that can influence platelet behaviour (reviewed in May et al., 1991b).

5.1

METHODOLOGY

A non-invasive technique for the continuous monitoring of platelets in the circulation has been described by Page et al. (1982), whereby platelets are radiolabelled and externally monitored using scintillation detectors. This methodology has been employed to study platelet function in a variety of experimental animals including guinea-pigs, rats, rabbits, dogs, baboons (reviewed in May et al., 1991b) and horses (Fairbairn et al., 1993). It was adapted from the clinical procedure whereby radiolabelled blood elements can be externally imaged with gamma camera devices. Systemic administration of a platelet agonist causes the formation of aggregates which become trapped in the microvasculature of the pulmonary circulation. This is detected as an increase in radioactive counts by a detector placed over the thoracic region of the animal. This technique is reproducible, many observations can be made in an individual animal and the kinetics of each response can be followed closely (each second if required). Several different anatomical regions can be monitored simultaneously and so platelet function in different vascular beds can be assessed. In addition, the response of other blood elements to various stimuli can also be investigated, e.g. erythrocytes, neutrophils, fibrinogen, albumin. Platelets are isolated from blood, resuspended in modified Tyrode's buffer and incubated with 25-50 gCi 111In-oxine for 90 s at 37~ Unbound radioactivity in the supernatant is removed following centrifugation. The isotopically labelled platelets are resuspended in buffer for i.v. administration to the donor animal. Continuous recording of isotopically labelled platelets in vivo is achieved using collimated sodium iodide crystal

detectors with appropriate spectrometers to monitor radioactivity, a special purpose microcomputer coprocessor to log and process experimental data, a commercial computer to define the experimental protocol and a printer to provide permanent records. Radioactive counts are monitored continuously in various anatomical regions by separate detectors, e.g. thorax, abdomen, leg, head. Signals from these two detectors are amplified within the spectrometer and logged by the recording system which is specifically designed for the collection of such experimental data (AIMS 8000, Mumed Ltd, London, UK). Intravenous administration of a diverse range of aggregatory stimuli such as ADP, collagen, PAF, thrombin, 5-HT and antigen, evoke increases in the counts recorded by the thoracic probe. These increases are attributed to retention ofplatelets within the pulmonary vasculature as confirmed by histological evidence of platelet aggregates within all levels of the pulmonary vasculature of lungs from animals sacrificed when thoracic counts are elevated (Butler et al., 1979; Dewar et al., 1984). This is accompanied by a fall in the count rate in the abdominal probe, which, for ADP, parallels the time course of thrombocytopenia (Barrett et al., 1984), a finding similar to that reported with the use of a different in vivo technique by Smith and Freuler (1973). Such responses are not a reflection of changes in blood flow or blood pooling within the thorax as platelet agonists do not induce significant 111In-labelled red cell accumulation. 5.2

INVESTIGATION

OF PLATELET

FUNCTION Experiments with 111In-labelled platelets have confirmed that platelet-dependent bronchoconstriction is associated with platelet accumulation in the pulmonary vasculature, but additionally have demonstrated a clear dissociation between these two response parameters (Page et al., 1984; Arnoux et al., 1988). Platelet-dependent bronchoconstriction does not follow platelet accumulation within the pulmonary vasculature but rather precedes detectable accumulation (Page et al., 1984), which implies that platelet sequestration per se is not the stimulus for bronchospasm, but some other aspect of platelet activation. Furthermore, several classes of drugs, including the anti-asthma drugs ketotifen and theophylline, inhibit the platelet release reaction in vitro and platelet-dependent bronchospasm in v/v0, but do not affect platelet accumulation within the pulmonary vasculature (Page et al., 1985). These observations indicate that platelet-derived mediators contribute to the bronchospasm as well as, or instead of, physical obstruction of pulmonary vessels by platelet aggregates. By dissociating platelet release and aggregation in vivo, the use of this experimental technique led to the development of a hypothesis that platelet activation plays a central role in the pathogenesis of asthma (Morley et al., 1984).

6 C.M. HERD AND C.P. PAGE The intravenous administration of PAF to guinea-pigs induces pulmonary platelet accumulation, acute bronchoconstriction and increased airway responsiveness, a characteristic feature of asthma. PAF-induced airway hyperresponsiveness has been shown to depend upon the presence of circulating platelets in guinea-pigs (Mazzoni et al., 1985) and rabbits (Coyle et al., 1990b). Activation of platelets by PAF differs from activation by other agonists, since ADP, collagen, thrombin or the TXA2 mimetic U46619, in amounts sufficient to cause comparable pulmonary platelet accumulation in v/v0, do not induce airway hyperresponsiveness (Robertson and Page, 1987; Smith et al., 1989). Therefore, as with the bronchoconstrictor response, the actual pulmonary retention of platelets is not responsible for induction of airway hyperresponsiveness, thus implicating some other property of this cell type. A factor released from platelets has been reported to induce airway hyperreactivity [platelet-derived hyperreactivity factor (PDHF); Sanjar et al., 1989]. The intravenous injection of PAF into guinea-pigs rendered thrombocytopenic by administration of a specific antiplatelet anti-serum, does not induce an acute bronchoconstrictor response nor enhanced airway responsiveness. However, in platelet-depleted guinea-pigs, the supernatant obtained from non-platelet-depleted guineapig platelet-rich plasma (PRP) incubated with PAF, induced airway hyperresponsiveness (Sanjar et al., 1989). The generation of PDHF was inhibited by prior incubation of PRP with the stable prostacyclin mimetic iloprost. The secretion or formation of this mediator of hyperresponsiveness appears to be PAF specific as neither platelet disruption nor activation of platelets with ADP induced its production. The chemical nature of this material remains as yet unidentified. Ketotifen and prednisolone have been shown to inhibit the airway hyperresponsiveness induced by PAF-stimulated platelet supernatants, whereas cromoglycate and aminophylline were without effect (Morley et al., 1989). Similarly, when ketotifen or prednisolone were incubated with PRP prior to the addition of PAF, the injection of supernatants into thrombocytopenic guinea-pigs resulted in reduced airway hyperresponsiveness (Morley et al., 1989).

elements and bacteria or other foreign particles has been known since early this century (see Copley and Witte, 1976; see Copley, 1979). Phagocytosis of foreign particles by platelets may represent one of the mechanisms the platelet employs to remove bacterial invasion. Platelets are capable of adsorption and phagocytosis due to characteristics of their membrane system and inner structure. The ability of platelets to undergo phagocytosis has been observed with yeast, colloidal SIO2, barium sulphate, ferritin and latex particles (Copley and Witte, 1976; Copley, 1979). Foreign particles are captured immediately or rapidly after they enter the bloodstream by the clumping together of platelets which engulf these particles and/or phagocytose them. The clumping of platelets can be induced through the mechanism of ADP liberation from the platelets. Subsequently, these mixed thrombi are eliminated by embolization into the microcirculation of different organs and liberated into the tissues at perivascular sites. Alternatively, mixed thrombi may migrate ultimately into lymph channels. Platelet aggregation can be induced following infection with various bacterial pathogens (Copley, 1979) which can become sequestered in clumps of platelets (Clawson, 1971). As a result of the subsequent platelet release reaction (and possibly also as a result of the production by the aggregated platelets of chemotactic metabolites of arachidonate), the platelet-bacterial aggregates become chemotactic for polymorphonuclear leucocytes and for monocytes. Platelets release bacteriocidal products such as fl-lysin (Hirsch, 1960; Donaldson and Tew, 1977), known to have direct bactericidal activity against a range of organisms, including Bacillus, Clostridia, Micrococcus and Lactobacillus (Nachman and Weksler, 1980). Even though it is not known precisely how bacteria activate platelets, certain products of Gram-negative bacteria such as endotoxin (lipopolysaccharide, LPS) can activate platelets directly and this can be manifested in vivo as thrombocytopenia (Brown and Lachman, 1973) and platelet sequestration into various organs such as the lung, liver and spleen (Cicala and Page, 1992; Endo and Nakamura, 1992; Ford and Longridge, 1993a, b). It has been suggested that by aggregating around invading bacteria, platelets may aid the clearance of the pathogens from the circulation (Nachman and Weksler, 1980), thus reducing the risk of septicaemia.

6. Platelets in Non-allergic Host Defence 6.2 6.1

PLATELETS AND BACTERIA

Evidence exists for the involvement of platelets in nonallergic defence mechanisms such as the removal of bacterial infections. It has long been known that platelets play a role in a number of bacterial diseases and the phenomenon of adhesion between blood cellular

PLATELETS AND MALIGNANCY

Platelet activationis a feature of both malignant disease (Slichter and Harker, 1974) and experimental malignancy (the injection of tumour cell suspensions into laboratory animals; Hilgard, 1982). In addition, injection of tumour cell suspensions known to metastize into the lungs of rats and mice rendered thrombocytopenic,

D o PLATELETS HAVE A ROLE AS INFLAMMATORY CELLS? 7 results in a decrease in the number of metastatic lung colonies found in those animals (Poggi and Donati, 1991). This type of observation had led to the suggestion that platelets have a role in the dissemination of malignant tumours (Hilgard, 1982). It remains plausible that just as platelets isolate and clear bacteria from the circulation as a physiological defence mechanism, the facilitation of the removal of tumour cells by platelets may accelerate a pathological process (Hilgard, 1982). A number of experimental and clinical studies have suggested that anti-platelet drugs may influence the metastatic pattern of tumour spread (Poggi and Donati, 1991), suggesting that the platelets may be a legitimate target for future drugs used in the control of tumours. Furthermore, PDGF has a high degree of sequence homology with one of the main oncogenes implicated in the induction of certain types of tumour (Waterfield et al., 1983). Subcutaneous administration of TGF-B induces a granulation process analogous to that observed during wound repair, suggesting the involvement of this factor in this process (Roberts et al., 1986). Similarly, the release of TGF-/~ following platelet activation could be associated with diseases characterized by abnormal cell growth.

7. Platelets and Allergic Inflammation 7.1

THE IgE RECEPTOR

The demonstration that platelet membranes possess IgE receptors (Cines et al., 1986; Joseph et al., 1986) has given credence to the platelet as an inflammatory cell involved in allergic processes. The identification of a specific IgE receptor on platelets came from the demonstration of cytotoxic functions by platelets from patients infected with the helminth Schistosoma mansoni (Joseph et al., 1983). Furthermore, the role of the platelet IgE receptor in the defence of the host organism against invading parasites is reinforced by the observation that the passive transfer of platelets bearing IgE receptors towards schistosomes to naive rats can protect these animals from parasitic challenge (Joseph et al., 1983). These studies indicated that human platelets can bind IgE in vitro and that the cross-linking of surface-bound IgE with anti-IgE or the specific antigens induces platelet activation and secretion. A specific receptor for the Fc fragment of IgE, the Fc epsilon receptor type II (Fc~RII), has been demonstrated on the platelet membrane, and is of low affinity (10- 7M) compared with that found on mast cell or basophil surfaces [Fc epsilon receptor type I (Fc~RI); 10 .9 M; Joseph et al., 1986], but of comparable affinity to the IgE receptor located on other inflammatory cell types such as alveolar macrophages and eosinophils (Capron et al., 1986). The Fc~RII is associated with the GPIIb-IIIa fibrinogen receptor on the platelet membrane (Capron et al., 1986).

Only a small number of platelets from normal individuals (20-30%) bind IgE, but more than 50% of the platelets from patients with aspirin-induced asthma, allergic patients and patients with parasitic diseases, bind IgE (Maccia et al., 1977; Joseph et al., 1983, 1986; Weksler, 1983).

7.2 PLATELETS, PARASITES AND CYTOTOXIC FREE RADICALS As mentioned earlier, the physiological relevance of the platelet IgE receptor may be associated with a mechanism for aiding the removal of parasitic infections. Platelets have been shown to participate as effector cells in defence mechanisms against helminth parasites (Joseph et al., 1983; Bout et al., 1983). Activation of the IgE receptor by exposure of sensitized platelets to an appropriate antigen has been shown to result in the production of cytotoxic free radicals (Haque et al., 1985; Capron et al., 1987) in sufficient concentrations to kill parasites (Ameisen et al., 1985). Platelets from Schistosoma mansoni-infected patients or rats express direct anti-parasitic killing properties in vitro, which has been in part attributed to the IgE-mediated release of cytotoxic free radicals (Capron et al., 1987). The interaction of platelets with parasites may result in cytotoxic effects on schistosomal and filarial parasites through IgEmediated mechanisms (Weksler, 1983; Joseph et al., 1983). The capacity of platelets to induce cytotoxicity is comparable with that observed with natural killer (NK) cells. Both these cytotoxicities can be inhibited by scavengers of activated oxygen species, although the exact biochemical mechanism underlying this phenomenon remains to be determined (Cesbron et al., 1987). It appears that a distinction may exist between the mechanism of platelet activation resulting in the generation of free radicals and that resulting in granule release. The latter represents classical aggregation, an event normally associated with the contribution of platelets to haemostasis and thrombosis (Page, 1988). Platelets that release free radicals do not aggregate and platelet aggregation itself will inhibit any subsequent free radical release (Page, 1989). This type of activation can be elicited by a range of stimuli thought to be involved in the inflammatory response, including C-reactive protein Simpson et al., 1982; Bout et al., 1986), substance P Damonneville et al., 1990), the complement-derived peptides C3b and C5b-C9 (Henson and Ginsberg, 1981), the eosinophil-specific major basic protein (MBP) (Rohrbach et al., 1990), and the cytokines, interferon 3' (IFN3,; Pancre et al., 1988) and tumour necrosis factor B (TNFB; Damonneville et al., 1990). Anti-allergic compounds such as disodium cromoglycate (Tsicopoulos et al., 1988) and nedocromil sodium (Thorel et al., 1988) inhibit IgE-dependent release of free radicals from platelets, yet these drugs are ineffective against classical platelet aggregation (Lewis et al., 1984). Furthermore,

8 C.M. HERD AND C.P. PAGE the therapeutic efficacy of certain anti-parasite drugs such as diethylcarbamazine may to some extent be related to their ability to generate free radicals from platelets (Cesbron et al. , 1987). It has been shown that a suppressive lymphokine released by activated mononuclear cells can inhibit the production of cytotoxic free radicals by IgE-coated platelets (Pancre et al., 1986). This lymphokine has been termed "platelet activity suppressive lymphokine" (PASL), a heat stable molecule of molecular weight (MW) 15 000-20 000 and a product of a T lymphocyte subpopulation beating the CDs § antigen (Pancre et al., 1986). Furthermore, CD4 § lymphocytes have been observed to release factors including IFN3, which can induce cytotoxic activity in normal platelets (Pancre et al., 1987).

7.3

PLATLETS AND EXPERIMENTAL INFLAMMATION

Several studies have reported the occurrence of platelet accumulation at localized sites of inflammatory lesions or in inflammatory exudates (Cotran, 1965; Kravis and Henson, 1977). Local administration of PAF into the subplantar region of the guinea-pig causes a sustained increase in vascular permeability, which is associated with platelet accumulation (Page et al., 1983), as assessed by the previously described in vivo platelet monitoring technique. Subcutaneous injection of a platelet pellet in the rat has been shown to elicit intense oedema, neutrophil infiltration and the accumulation of myofibroblasts, responses that are not observed following the administration of other tissue homogenates (Braunstein et al., 1980). Similarly, a long-lasting inflammatory response (swelling, tenderness and redness) was observed following injection of platelet extracts into the skin of normal humans (Day et al., 1975). Platelets have been shown to partly mediate the development of inflammatory lesions in experimental animal models, where lung injury provoked by neutrophils following complement activation is reduced as a result of platelet depletion (Tvedten et al., 1985; Ward et al., 1986).

7.4

PLATELETS AND ALLERGIC ASTHMA

Clinically, asthma is characterized by hyperresponsiveness of tracheobronchial smooth muscle to various spasmogens, resulting in the widespread narrowing of the airways. In recent years it has been recognized that asthma is a chronic inflammatory disease associated pathologically with eosinophil infiltration and damaged airway epithelium. These underlying inflammatory events are considered important in the development of the enhanced airway responsiveness observed in

asthmatic individuals. Airway inflammation is a complex event triggered by inflammatory stimuli interacting with primary effector cells resident in the airway, of which numerous cell types have been implicated. Release of inflammatory mediators from these cells may recruit and activate other effector cells, thus augmenting the inflammatory process. Evidence now exists in support of a primary role of the platelet in the pathogenesis of bronchial asthma. Platelets can participate in allergic asthma by acting as inflammatory cells, by releasing spasmogens and/or by interacting with other cells. The phospholipid PAF has been suggested as a mediator of asthma as it can reproduce many of the characteristic features of the disease, including bronchospasm, mucus hypersecretion, increased vascular permeability and increased airway responsiveness, both in experimental animals and humans (reviewed in Page, 1988). PAF may provide the link between platelet activation and allergic asthma (Gresele, 1991) as evidence suggests that the ability of PAF to induce airway hyperresponsiveness and eosinophil infiltration may involve the activation of platelets (Lellouch-Tubiana et al., 1988; Coyle et M., 1990b). PAF is released from a number of inflammatory cells in the lung, including alveolar macrophages, eosinophils and neutrophils. Human alveolar macrophages (Arnoux et al., 1983, 1987) and eosinophils (Lee et al., 1984) are rich sources of PAF and are capable of releasing large amounts in response to activation by IgE-dependent mechanisms. These cell types are present in the airways of asthmatics and are activated following antigen provocation (Metzger et al., 1987; Beasley et al., 1989). Eosinophils obtained from hypereosinophilic patients (including asthmatics) have a much enhanced capacity to generate PAF (Lee et al., 1984). A number of other cell types have been shown to release PAF, including neutrophils (Benveniste et al., 1982b; JouvinMarche etal., 1984b), eosinophils (Jouvin-Marche etal., 1984a), platelets (Benveniste et al., 1982a; Benveniste et al., 1982b; Chignard et al., 1980) and vascular endothelial cells (Camussi et al., 1983), all of which may play a role in the pathophysiology of asthma. Furthermore, isolated lungs from sensitized guinea pigs have been shown to release PAF when challenged with antigen (Fitzgerald et al., 1986). Animal studies have shown that several selective, but structurally unrelated, PAF antagonists inhibit various aspects of asthma pathophysiology, including antigeninduced bronchoconstriction, late phase response, airway hyperresponsiveness, oedema formation, mucus hypersecretion and pulmonary eosinophil infiltration (reviewed in Heuer, 1992). As yet there are few reported clinical studies of PAF antagonists in humans. Pretreatment with BN 52063 has been shown to attenuate the response to PAF in the skin of normal subjects (Chung et al., 1987) and to antigen-induced cutaneous responses in atopic subjects (Roberts et al., 1988b). BN 52063 has also been shown to reduce the bronchoconstrictor response to

DO PLATELETS HAVE A ROLE AS INFLAMMATORY CELLS? 9 inhaled PAF in normal volunteers (Roberts et al., 1988a) whereas WEB 2086 (Adamus et al., 1990) and UK74,505 (O'Connor etal., 1991) completely abolished the response. Furthermore, BN 52063 (Guinot et al., 1987) and BN 52021 (Hsieh, 1991) have been shown to inhibit the immediate bronchoconstrictor response to inhaled allergen. Recent findings with UK-74,505, the most potent PAF antagonist yet studied in humans (Kuitert et al., 1993), confirm preliminary reports with WEB 2086 (Freitag et al., 1991) and MK-287 (Bel et al., 1991) which have shown no effect on the early or late response to inhaled allergen in mild atopic asthmatics or on the subsequent airway hyperresponsiveness. The lack of effect of these PAF antagonists against allergen challenge in humans, despite achieving plasma levels capable of inhibiting ex vivo platelet aggregation induced by PAF, may be due to a number of reasons. Firstly, PAF may not be as important a mediator in asthma as previously thought. Secondly, PAF released in vivo is a family of related compounds, whereas PAF antagonists have been developed as antagonists to PAF C16. It is possible, therefore, that other PAF homologues may be of biological significance. Thirdly, current PAF antagonists have not been designed to penetrate cells and thus may not interact with intracellular receptors. As the bulk of PAF appears to be retained intracellularly in a variety of cell types (Bratton and Henson, 1989), PAF antagonists may need to be able to enter cells or PAF synthesis may need to be inhibited, rather than antagonism of its extracellular effects (Stewart and Phillips, 1989). 7.4.1 A n i m a l Evidence Platelets have been observed to undergo diapedesis into the extravascular tissue of the lungs of guinea-pigs following antigen challenge or treatment with PAF (Lellouch-Tubiana et al., 1985). The extravasated platelets have been observed in close proximity to bronchial smooth muscle and to infiltrating eosinophils. However, treatment of experimental animals with other platelet agonists such as ADP, whilst inducing platelet aggregation in the pulmonary vasculature, does not elicit extravascular diapedesis of platelets and eosinophils (Lellouch-Tubiana et al., 1985), suggesting a possible link between extravascular platelets and eosinophils. Platelets have also been reported in bronchoalveolar lavage (BAL) fluid obtained from allergic rabbits undergoing late-onset airways obstruction following antigen challenge (Metzger et al., 1987). Further evidence that platelets are involved in experimental allergic responses is the detection of markers of platelet activation, such as PF4, in the plasma following antigen challenge in sensitized rabbits (McManus et al., 1979). In several animal species, the intravenous injection of selected platelet agonists induces thrombocytopenia associated with bronchospasm (Vaage and Hauge, 1977; Lefort and Vargaftig, 1978; Vargaftig and Lefort, 1979).

This also occurs in sensitized animals challenged with specific antigen, which appears to be a platelet-dependent phenomenon since platelet depletion protects against the lethal consequences of the antigen provocation (Pinckard et al., 1977; Halonen et al., 1981). In isolated human bronchus, platelet depletion prevents smooth muscle contraction induced by PAF (Schellenberg et al., 1983). Similarly, the intravenous administration of PAF into guinea-pigs induces bronchospasm associated with the accumulation of platelets in the lung (Vargaftig et al., 1980; Page et al., 1984), the bronchospasm is platelet dependent since platelet depletion abolishes the response (Vargaftig et al., 1980). Under these circumstances platelet aggregates have been located histologically (Pinckard et al., 1977; Dewar et al., 1984), and by the use of radiolabelled platelets (Page et al., 1984) within the pulmonary vasculature. It has been suggested that this bronchoconstrictor response is reflex in origin. However, peak changes in lung function largely (> 90%) precede detectable accumulation of lllIn-labelled platelets in the pulmonary vasculature (Page et al., 1984). Furthermore, the pharmacological inhibition of the platelet release reaction (Chignard et al., 1982; Vargaftig et al., 1982) or TXA2 production (Chung et al., 1986) can abrogate the bronchospasm, suggesting the response is related to the release of bronchoactive agents from the platelets. Inhalation of allergen by an appropriately sensitized individual may induce a delayed airway obstruction [referred to as a late-onset response (LOR)], which may be associated with increased airway responsiveness (Cockcroft et al., 1977). The LOR to antigen challenge in IgEsensitized rabbits may be abrogated by prior treatment with a selective anti-platelet antiserum (Coyle et al., 1990a). This phenomenon may be attributable to an interaction between platelets and eosinophils as the antigen-induced pulmonary eosinophil infiltration is inhibited in thrombocytopenic animals (Coyle et al., 1990a). Asthmatic subjects will constrict to airway spasmogens at concentrations far lower than those required to induce a similar degree of bronchoconstriction in normal individuals, i.e. airway hyperresponsiveness (AHR) is a characteristic feature of the asthmatic airway. A number of studies have demonstrated the induction of airway hyperresponsiveness to various stimuli in both experimental animals and humans (reviewed in Page, 1988). In the guinea-pig and rabbit, PAF-induced airway hyperresponsiveness is platelet dependent since it can be abrogated by rendering animals selectively thrombocytopenic by the intravenous administration of a specific lytic anti-platelet anti-serum (Mazzoni et al., 1985; Coyle et al., 1990b). Eosinophils and their products such as MBP have been implicated in the pathogenesis of asthma (Frigas and Gleich, 1986). Platelet depletion has been shown to reduce PAF and antigen-induced eosinophil infiltration into the lungs of normal and allergic animals, respectively (Lellouch-Tubiana et al., 1988; Coyle et al.,

10 C.M. HERD AND C.P. PAGE 1990a, b), suggesting a central role for platelets in the induction of eosinophil accumulation which both facilitates the removal of parasitic infection and contributes to the airway hyperresponsiveness observed in asthma. These experimental observations may be of relevance clinically, where thromboembolic diseases are often associated with the hypereosinophilic syndrome and patients with eosinophilia have coagulation abnormalities (Elonaer-Blanc et al., 1985). The mechanism by which platelets attract eosinophils into the lung may be via the release of the platelet-derived protein PF4 which, as discussed earlier in this chapter, is released upon platelet activation and can exert a powerful chemotactic effect on human eosinophils (Chihara et al., 1988). Treatment of allergic rabbits with an anti-rabbit platelet anti-serum inhibits the ability of antigen to induce late-onset airways obstruction, AHR and the associated infiltration of eosinophils recovered in BAL fluid 24 h following antigen challenge (Coyle et al., 1990a). PAF antagonists have been shown to inhibit the LOR and subsequent increase in airway responsiveness in allergic rabbits (Metzger et al., 1988; Coyle et al., 1989) as well as the eosinophil influx and AHR in sensitized guinea-pigs (Coyle etal., 1988; Smith et al., 1988) following antigen exposure. These findings suggest that antigen-induced release of PAF may play a central role in the platelet activation necessary to initiate the eosinophil infiltration into the airways which, in turn, contributes to AHIL Further evidence in favour of the platelet as an important effector cell in asthma has been provided by in vitro studies where platelets potentiate mucous GP release from tracheal submucosal glands (Sasaki et al., 1989). 7.4.2 Clinical Evidence A number of clinical studies have now revealed that platelet activation is a feature of asthma where there is activation of the allergic response, although this disease is not normally associated with thrombosis (Storck et al., 1955; Knauer et al., 1981; Gresele et al., 1982, 1985, 1987; Traietti et al., 1984; Johnson et al., 1986; Szczecklik et al., 1986; Taytard et al., 1986, 1987; Martin et al., 1987). In certain clinical (Rao and Walsh, 1983) and experimental (Henson and Pinckard, 1977) conditions where there is known to be excessive platelet activation in the circulation, platelets become partially refractory to subsequent stimulation in vitro. In particular, the second phase of platelet aggregation in vitro is often unresponsive to physiological stimuli. A number of studies have reported that platelets from asthmatics behave abnormally in vitro, lacking the second wave of aggregation (Fishel and Zwemer, 1970; Solinger et al., 1973; Maccia et al., 1977; Thompson et al., 1984) or defective release of platelet 5-HT, PF4 (Maccia et al., 1977) and platelet nucleotides (D'Souza and Glueck, 1977) following stimulation with platelet agonists. These in vitro abnormalities are suggestive of overstimulation in v/v0 (Harker et al., 1980; Pareti et al., 1980).

In asthmatic patients the uptake of 5-HT by platelets has been shown to be attenuated, possibly due to previous exposure of platelets to an increased concentration of this amine (Malmgren et al., 1982). Increased plasma levels of 5-HT have been reported in asthmatics (Bakulin and Joffe, 1979), as well as elevated resting levels of cytoplasmic Ca 2§ and inositol triphosphate (IP3) production (Block r al., 1990), findings suggestive of in viv0 platelet stimulation. Thrombocytopenia was first reported to accompany asthmatic attacks in 1955 (Storck et al., 1955). This observation of platelet activation in vivo during provoked or spontaneous asthmatic attacks has also been shown by the detection of circulating platelet aggregates (Gresele ct al., 1982, 1987) or the morphological characterization of activated platelets in the circulation (Traietti et al., 1984). Furthermore, a number of studies have demonstrated the release of two platelet-specific proteins, PF4 and B-TG, into the circulation associated with bronchoconstriction induced by antigen or exercise (Knauer et al., 1981; Greseleet al., 1982, 1985, 1987; Toga r al., 1984; Johnson et al., 1986). The release of these markers is indicative of in vivo platelet activation and in the study of Knauer and colleagues (1981), the increased plasma levels of plateletderived markers occurred in parallel with the bronchoconstriction induced by antigen provocation of allergic asthmatics. Release of PF4 and B-TG was not observed following comparable bronchoconstriction induced by methacholine suggesting that the platelet-derived markers were released as a consequence of the allergic reaction rather than of the bronchoconstriction. Evidence of platelet activation has been reported in plasma obtained r vivo during exacerbations of nocturnal asthma (Morrison et al., 1991; Gresele et al., 1993), which has recently been shown to correspond with AHR (Gresele et al., 1993). In another recent study, PF4 and B-TG in BAL fluid from allergic asthmatics have been demonstrated following antigen challenge (Averill et al., 1992). Platelet products were significantly elevated during the late inflammatory response to antigen and were significantly correlated with elevations in markers of airway permeability (albumin), eosinophil granule proteins [eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPO)] and inflammatory prostanoids (PGE2 and PGF2~). Furthermore, TXA2 release has been shown to accompany the exposure of allergic asthmatics to inhaled antigen by measurement of urinary excretion of TXB2 metabolites (Lupinetti et al., 1989). Release of platelet-derived factors such as PF4, B-TG and TXB2 and altered in vitro platelet aggregatory responses have not been consistently observed (Greer et al., 1984, 1985; Durham r al., 1985; Shephard et al., 1985; Hemmendinger et al., 1989). In other studies pulmonary platelet sequestration was not found to follow antigen challenge in asthmatic volunteers (Ind et al., 1985; Hemmendinger et al., 1989). These negative findings have led some investigators to reject the

D o PLATELETS HAVE A ROLE AS INFLAMMATORY CELLS? 11 proposed role of platelets in asthma. However, numerous other clinical observations support the concept that platelets may be involved in this disorder. In lung tissue removed at autopsy from asthmatics dying from status asthmaticus, abnormal megakaryocytes have been reported to be present in abundance (Slater et al., 1985; Martin et al., 1987), suggestive of a potential abnormality in this system. Platelet survival time in atopic asthmatics is severely shortened, a finding suggestive of continuous cell activation (Taytard et al., 1986). Treatment of asthmatic individuals with anti-asthma drugs such as glucocorticoids and ketotifen has been shown to correct this abnormal platelet survival (Taytard et al., 1987). A recent study reports that in asthmatic subjects the anti-allergy drug nedocromil sodium inhibits platelet activation induced by PAF ex vivo (Roth et al., 1993). Therefore, the efficacy of these drugs may reside in their ability to restore normal platelet behaviour. Shortened platelet regeneration time, an index of in vivo platelet activation associated with accelerated platelet consumption (i.e. increased platelet turnover; Harker, 1978), has been reported in asthmatics undergoing acute asthma attacks (Gresele et al., 1987), and increased bleeding time has been observed in a group of atopic asthmatics (Szczecklik et al., 1986). In addition, altered responsiveness of platelets from allergic patients has been observed by numerous investigators (reviewed by Gresele et al., 1987), the incidence of which was greatest in patients presenting with high serum IgE titres (Maccia et al., 1977). Furthermore, platelet size (Audera et al., 1988), platelet count and platelet mass (Szczeklik et al., 1986) have been found to be increased in asthmatics. Platelets have been reported to accumulate in the microvasculature of the lung in patients undergoing bronchial provocation with allergen (Beasley et al., 1989) and have also been detected by electron microscopy in BAL fluid obtained from allergic asthmatics undergoing late-onset airways obstruction following antigen provocation (Metzger et al., 1987). The extravascular platelets in this clinical situation were observed in close association with other inflammatory cells such as the eosinophil (Metzger et al., 1987). In addition, platelets have been observed undergoing diapedesis in sections biopsied from asthmatics (see Page, 1993). Subepithelial extravasation of platelets together with fibrinous material has been observed at sites of denuded epithelium in bronchial biopsies from symptomatic asthmatics (Jeffery et al., 1989). A recent study reports that platelets from asthmatic subjects migrate in vitro in response to antigen, possibly by interaction with platelet-bound antigenspecific IgE (Zhang et al., 1993). The fate of platelets in the circulation of asthmatics is unknown although overt trapping in the pulmonary vasculature is not a feature of either stable asthmatics or those undergoing bronchoconstriction (Gresele et al., 1987).

7.4.3 ASA-induced Asthma Platelets isolated from patients with acetylsalicylic acid (ASA; aspirin)-induced asthma exhibit an abnormal response to ASA in vitro compared with normal individuals or allergic non-ASA-sensitive asthmatics, generating cytotoxic mediators and oxygen-derived free radicals in the presence of ASA or various nonsteriodal anti-inflammatory drugs (NSAIDs), such as indomethacin (Ameisen et al., 1985). Basophils from ASA-sensitive patients do not release H, and monocytes do not express cytotoxic properties nor any burst of chemiluminescence in the presence of ASA or other NSAIDs. Evidence does not support a role of IgE in this response since serum from patients was unable to passively sensitize platelets removed from healthy volunteers to NSAIDs, as well as the absence of an inhibitory effect of polyclonal or monoclonal antibodies against the Fc,RII. It has been suggested that the abnormal response of platelets from ASA-sensitive asthmatics may reside in the involvement of endogenous prostaglandin H2 (PGH2) in the control of synthesis and/or biological effect of platelet lipoxygenase products (Joseph, 1991). It has been previously shown that sodium cromoglycate and nedocromil sodium could modulate in vitro platelet responsiveness to ASA in ASA-sensitive asthmatics (Thorel et al., 1987), with nedocromil sodium being approximately 500 times more potent in inhibiting the response. Similarly, inhalation of nedocromil sodium by ASA-sensitive asthmatics resulted in a dramatic inhibition ofplatelet responsiveness to ASA (platelet cytotoxicity) when examined ex vivo (Marquette et al., 1990). Since the platelet is the only cell so far shown to respond to ASA or other NSAIDs in ASA-sensitive asthmatics, these findings provide further evidence for a major role of the platelet in this form of bronchial asthma.

7.5

PLATELETS AND RHEUMATOID ARTHRITIS

Platelet participation has been implicated in inflammatory conditions other than bronchial asthma, such as rheumatoid arthritis (RA). Early studies demonstrated platelet thrombi in histologic studies of active lesions in synovial tissue obtained from patients with early and chronic RA (Kulka etal., 1955; Schumacher, 1975). Further evidence that the platelet is involved in some way in RA is the detection of this cell type in synovial fluid (Farr et al., 1984), the detection of small platelet thrombi in acutely inflamed synovial villi of individuals with RA, and the fact that radioactive labelled platelets have been shown to accumulate in inflamed joints (Nachman, 1980). The number of platelets in the synovial fluid has been reported to correlate with various markers of synovial inflammation, and platelet-derived substances have been detected in the inflamed synovial joints (Farr et al., 1983; Zeller et al., 1983). A large percentage of RA

12 C . M . HERD AND C.P. PAGE patients have increased numbers of circulating platelets as well as an apparent relationship between thrombocytosis and several biological and clinical markers of the active disease (Bean, 1965; Selroos, 1972; Hernandez et al., 1975; Hryszko et al., 1975; Hutchinson et al., 1976). Platelet populations from some patients with RA have also demonstrated reduced adhesiveness (Pazdur and Kopec, 1970), increased surface activation (cytoplasmic spreading; Riddle et al., 1981) and an enhanced sensitivity to collagen and adrenaline-induced aggregation in vitro (Colli et al., 1982). Biochemical analysis of platelets isolated from patients with RA has shown an increased content of acid mucopolysaccharides coupled with low serotonin (Kerby and Taylor, 1959) as well as a diminished protein content, a possible decrease in acid phosphatase activity and a reduced amount of connective tissue-activating peptide-III (Smith and Castor, 1978). In vitro studies have demonstrated the ability of IgG rheumatoid factor (IgG RF) in sera obtained from some patients with adult RA to elicit platelet aggregation in PRP isolated from normal subjects (Fink et al., 1979). Furthermore, serum factors, possibly IgG RF or IgGcontaining immune complexes, have been shown to mediate platelet activation in rheumatoid vasculitis (Cunningham et al., 1986). The observation of the involvement of immune complexes in RA, together with the postulated role of PAF in enhancing immune complex deposition and the ability of PAF antagonists to inhibit this deposition (Camussi et al., 1987), suggests that PAF may play an important role in the antibody-mediated platelet activation resulting in host tissue damage. A feature of RA is the presence of large numbers of neutrophils in synovial fluid, and as discussed earlier in this chapter, the platelet-derived cytokine TGF-/~ has been shown to be chemotactic for neutrophils (Wahl et al., 1987). It is widely accepted that cytokines can amplify and perpetuate inflammation in the joints (reviewed in Harris, 1990). TGF-~ is present in both active and latent forms in synovial fluid from patients with RA and its concentrations in such patients are much higher than those in fluid from patients with osteoarthritis (Fava et al., 1989). TGF-g~ appears to counteract many of the effects of interleukin-1 (IL-1), tumour necrosis factor (TNF) and IL-6, while acting synergistically to enhance the effects of other cytokines (Harris, 1990). Elevated levels of other known chemoattractants, including PAF, have also been detected in rheumotoid synovial fluid (Harris, 1990).

7.6

PLATELETS AND SKIN INFLAMMATION

The suggestion that platelets are involved in urticaria came from a microscopic study of a case report of a patient with cold urticaria and vasculitis (Eady et al., 1981). More evidence followed with a study reporting

the detection of PF4 in blood after cold challenge in patients with cold urticaria (Wasserman and Ginsberg, 1984). However, this finding was not substantiated in a more recent study (Ormerod et al., 1988). Evidence exists to support a role for platelets in psoriasis. Platelets taken from psoriatic patients have recently been shown to be hyperresponsive in vitro to collagen and thrombin (Ivey et al., 1993). In these studies, collagen hyperresponsiveness correlated with disease severity. Increased spontaneous platelet hyperaggregability, elevated plasma ~-TG levels, shortened platelet regeneration time and platelet cyclo-oxygenase and glutathione peroxidase enzyme abnormalities have also been reported in patients with psoriasis (Berrettini et al., 1985; Schena et al., 1989; Vila et al., 1990, 1991). Furthermore, a correlation between clinical psoriasis severity and in vitro platelet adhesiveness has been reported (Dandurand et al., 1989). Some investigators have reported that, compared to healthy controls, platelet aggregation is enhanced in patients with Raynaud's phenomenon (Zahavi et al., 1980; Hutton et al., 1984; Biondi and Marasini, 1989; Cuenca et al., 1990). Platelet activation has also been described in conditions such as systemic lupus erythematosis (Dorsch and Meyerhoff, 1980) and systemic sclerosis (Friedhoff et al., 1984), which are associated with secondary Raynaud's phenomenon. These findings suggest that platelet activation may also accompany various inflammatory dermatoses.

8. Conclusion There is overwhelming evidence that platelets are involved and play an active role in primary defence mechanisms such as antibody-dependent cytotoxicity. Inappropriate activation of this system in allergic patients may contribute to eosinophil infiltration and subsequent damage to the host tissue, resulting in the heightened airway responsiveness characteristic of bronchial asthma. Evidence also exists for the activation of platelets in other inflammatory states and thus there is clearly a need to further investigate the role of this cell in conditions other than thrombosis and haemostasis.

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thromboxane B2, 6-ketoprostaglandin FI~ and Bthromboglobulin in antigen-induced asthma before and after indomethacin pretreatment. Br. J. Clin. Pharmacol. 19, 459-470. Simpson, R.M., Prancan, A., Izzi, J.M. and Fiedel, B.A. (1982). Generation of thromboxane A2 and aorta-contracting activity from platelets stimulated with modified C-reactive protein. Immunology 47, 193-202. Slater, D., Martin, J. and Trowbridge, A. (1985). The platelet in asthma [letter]. Lancet i, 110. Slichter, S.J. and Harker, L.A. (1974). Hemostasis in malignancy. Proc. NY Acad. Sci. 230, 252-262. Smith, A.F. and Castor, W. (1978). Connective tissue activation. XII. Platelet abnormalities in patients with rheumatoid arthritis. J. Rheumatol. 5, 177-183. Smith, D., Sanjar, S. and Morley, J. (1989). Platelet activation and PAF-induced airway hyperreactivity in the anaesthetised guinea-pig. Br. J. Pharmacol. 96, 74P. Smith, G.M. and Freuler, F. (1973). The measurement of intravascular aggregation by continuous platelet counting. Bibl. Anat. 12, 229-234. Smith, H.R., Henson, P.M., Clay, K.L. and Larsen, G.L. (1988). Effect of the PAF antagonist L-659,989 on the late asthmatic response and increased airway reactivity in the rabbit. Am. Rev. Respir. Dis. 137, A283. Solinger, A., Bernstein, I.L. and Glueck, H.I. (1973). The effect of epinephrine on platelet aggregation in normal and atopic subjects. J. Allergy Clin. Immunol. 51, 29-34. Stewart, A.G. and Phillips, W.A. (1989). IntraceUular plateletactivating factor regulates eicosanoid generation in guinea-pig resident peritoneal macrophages. Br. J. Pharmacol. 98, 141-148. Storck, H., Hoigne, IL and Koller, F. (1955). Thrombocytes in allergic reactions. Int. Arch. Allergy 6, 372-384. Szczeklik, A., Milner, P.C., Birch, J., Watkins, J. and Martin, J.F. (1986). Prolonged bleeding time, reduced platelet aggregation, altered PAF-acether sensitivity and increased platelet mass are a trait of asthma and hay fever. Thromb. Haemost. 56, 283-287. Taytard, A., Guenard, H., Vuillemin, L., Bouvot, J.L., Vergeret, J., Ducassou, D., Piquet, Y. and Freour, P. (1986). Platelet kinetics in stable atopic asthmatic patients. Am. Rev. Respir. Dis. 134, 983-985. Taytard, A., Vuillemin, L., Guenarg, H., Rio, P., Vergeret, J. and Ducassou, D. (1987). Platelet kinetics in stable asthma patients: effect of ketotifen. Am. Rev. Ra:spir. Dis. 135, 388A. Thompson, J.M., Hanson, H., Bilani, M., Turner-Warwick, M. and Morley, J. (1984). Platelets, platelet activating factor and asthma. Am. Rev. Respir. Dis. 129, A3. Thorel, T., Ameisen, J.C., Joseph, M., Vorng, H., Tonnel., A.B., Marquette, C.H. and Capron, A. (1987). Preventing effect of nedocromil sodium on the abnormal response to aspirin of platelets from aspirin-sensitive asthmatics. Am. Rev. Respir. Dis. 135, A398. Thorel, T., Joseph, M., Tsicopoulos, A., Tonnel, A.B. and Capron, A. (1988). Inhibition by nedocromil sodium of IgE mediated activation of human mononuclear phagocytes and platelets in allergy. Int. Arch. Allergy Appl. Immunol. 85, 232-237. Toga, H., Ohya, N. and Kitagawa, S. (1984). Clinical studies on plasma platelet factor 4 in patients with bronchial asthma. Jpn. J. Allergy 33, 474-479.

20

C . M . HERD AND C . P . PAGE

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0

Animal Modelsfor Investigating the Allerg'c and Inflammatory Propertiesof Platelets Anthony J. Coyle and B. Boris Vargaftig

1. Introduction 2. Methods Available to Assess Platelet Involvement in Experimental Animal Models 3. The Role of Platelets in Experimental Models of Acute Inflammation 3.1 The Arthus Reaction 3.2 The Generalized Shwartzman Reaction 3.3 Acute Serum Sickness in the Rabbit 3.4 Carrageenin-induced Inflammation 3.5 Experimental Haemarthrosis 4. The Role of Platelets in Models of Non-allergic Lung Injury 4.1 In vitro Studies 4.2 I n vivo Studies 4.2.1 Microembolism and Direct Lung Injury

21 5. 22 22 22 23 23 23 23 6. 24 24 24 24

1. Introduction Besides their well recognized role in haemostasis and thrombosis, there is now increasing evidence to suggest that platelets can function as inflammatory cells in their own right and as such, may contribute to a variety of inflammatory disorders. In particularly, platelets may be important effector cells in the pathogenesis of both allergic and non-allergic pulmonary diseases including the Adult Respiratory Distress Syndrome (ARDS) and bronchial asthma. While much of the data suggesting a role for platelet Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

4.2.2

7. 8. 9.

Experimental Pulmonary Hypertension The Role of Platelets in Models of Allergic Lung Injury 5.1 Platelet Agonist- and Allergeninduced Bronchoconstriction 5.2 The Role of Platelets in Experimental Models of Airway Hyperresponsiveness 5.3 Interactions of Platelets with Other Blood Elements 5.4 The Role of Platelets in Models of Late Asthmatic Response Direct Antigen-induced Activation of Platelets The Role of Platelets in Models of Parasitic Infection Conclusion References

24 25 25

25 26 26 27 28 28 28

activation in inflammation has arisen from experimental animal studies, there is an acute lack of animal models to evaluate the inflammatory properties of platelets per se. Those models which are available for assessing platelet activity have focused on identifying functions of platelets relevant to their role in thrombosis, based on techniques designed for investigating the classical aggregatory responses of platelets. As will be discussed below, this may not be the most appropriate property of platelets relevant to their contribution in inflammation. Thus the purpose of this review is to discuss the animal systems that are available to study the allergic and inflammatory Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

22 A.J. COYLE AND B.B. VARGAFTIG properties of platelets and illustrate where possible, how these techniques have increased our knowledge of the role of this fascinating blood element in inflammatory diseases.

0

Methods Available to Assess Platelet Involvement in Experimental Animal Models

Assessment of the number of circulating platelets in the peripheral blood allows a ready and easy determination of whether platelets are undergoing sequestration in a number of vascular beds. It is also possible to investigate platelet accumulation in various organs by histological analysis. For example, lungs excised from experimental animals following intravenous administration of a variety of bronchoconstrictor and inflammatory stimuli, have demonstrated the presence of platelet aggregates in the pulmonary vasculature. However, the methods employed for the routine histological assessment of platelet accumulation are often time consuming, laborious and moreover, relatively insensitive. A more effective and efficient approach has been developed using a non-invasive technique which allows the continuous monitoring of the accumulation of ill In-labelled platelets in vivo. This technique has the advantage that the kinetics of platelet accumulation can be readily studied following intravenous administration of platelet agonists. Furthermore, platelet sequestration in the organ of interest can be compared to a "control site", such as the hindlimb (Page et al., 1982). To control for changes in blood flow, it is also possible to compare accumulation of platelets to that of radiolabelled red blood cells. Modification of this technique has allowed the simultaneous measurement of the lung accumulation of 111In-platelets, 99mTc-erythrocytes and 113In-albumin and to show in vivo sequestration of ~l~In-platelets by a ligand such as N-formyl-methionyl-leucyl-phenylalanine (FMLP), with has no in vitro platelet-stimulating effects (Bureau et al., 1992). This technique has been modified to investigate platelet sequestration in the cranial vasculature following intracarotid administration of platelet agonists (May et al., 1990). Injection of thrombin induced an accumulation of lllin_platelets and l~SIn-fibrinogen, which was sustained up to 3 h after injection. Thus this model may be useful to investigate mechanisms of platelet activation in the cerebral vascular. The disadvantage of this technique, however, is that only a gross pattern of platelet accumulation can be measured, and the precise localization of the platelet sequestration is unclear. Thus, for example, following intravenous injection of plateletactivating factor (PAF), platelet accumulation in the thoracic cavity may reflect, in addition to platelet diapedesis into the airway tissue, platelet adhesion to the

pulmonary and/or bronchial endothelium. Despite this caveat, this technique has been very useful in investigating mechanisms of platelet sequestration and activation, as will be discussed in more detail below. While measurement of either peripheral blood platelet number or accumulation demonstrates that platelets are undergoing activation, their precise role in a particular animal system cannot be determined. One approach to further investigate platelet involvement is by their selective depletion by administration of a cytocidal antiplatelet antibody. Another possibility is the use of agents such as sulphinpyrazone (Chignard et al., 1982) and a combination of aspirin and antagonists to serotonin (5HT) and histamine (Vargaftig et al., 1982) that inhibit the release of platelet-derived products. It has also been possible to measure the expression of various markers on the surface of the platelet. Upon activation, the contents of the granules are secreted with the concomitant fusion of the granule membrane into the plasma membrane. One particular c~-granule protein, originally termed platelet activation-dependent granuleexternal membrane protein (PADGEM; Berman et al., 1986) or GMP-40 (Stenberg et al., 1985) and now referred to as P-selectin (Disdier et al., 1992) becomes expressed on the surface of activated platelets. By preparing a radiolabelled antibody to PADGEM, it was possible to assess platelet activation using radioimaging in an in vivo deep venous thrombosis model in non-human primates (Palabrica et al., 1989). Assessment of platelet function can also be determined ex vivo to investigate whether platelet function has been altered during in vivo treatment. This technique has been applied to a number of animal models. In the adjuvantinduced polyarthritis syndrome in rats, ex vivo assessment of platelet aggregation demonstrated an enhanced responsiveness to adenosine diphosphate (ADP) and collagen during the early stages of disease progression (Lassman et al., 1974). These changes reached a maximum by Day 10, which is the same time secondary lesions and non-injected paw involvement begin to occur. As this process progresses, the platelet abnormalities reverse and disappear. Similarly, following carrageenin-induced acute paw oedema, platelets demonstrate an enhanced in vitro responsiveness to ADP and collagen (Srivastava and Srimal, 1990). These changes in platelet function may therefore reflect the importance of platelets in the early phases of these acute models.

3. The Role of Platelets in Experimental Models of Acute Inflammation 3.1

THE

ARTHUS

REACTION

The classical active Arthus reaction is dependent on the interaction between an intravascular antibody and an

ANIMAL MODELS FOR INVESTIGATING PROPERTIES OF PLATELETS 23 extravascular antigen, the ensuing lesion being characterized by a broad zone of haemorrhagic necrosis at the site of antigen injection. Histological examination of this reaction has demonstrated two phases; the first is associated with an intense neutrophilic vasculitis and perivasculitis, which is then followed by an occlusion of the damaged blood vessel by leucocyte-fibrin-platelet thrombi. While an important role of the leucocyte has been suggested in experimental animals, the role of the platelet is less clear. Early studies suggested platelet involvement, as anticoagulation with heparin prevented the lesion. Moreover, studies by Margretten and McKay (1971) demonstrated that immune depletion of platelets with a specific anti-platelet antibody inhibited the development of inflammation, thrombosis and haemorrhagic necrosis, which was independent of complement depletion. In contrast to the active reaction, other workers have failed to demonstrate any effect of platelet depletion in the reverse Arthus reaction system (Humphrey, 1955). This2difference most likely relates to the nature of the allergenic stimuli, in that in the reverse passive Arthus reaction, the low molecular weight antigen is present within intravascular space and the larger molecular weight antibody is present in the extravascular space, and thus the increase in vascular permeability is independent of platelet-derived permeability factors. In contrast, in the active reaction, as a consequence of antibody-antigen reactions, immune complexes are deposited in the extravascular tissue as a result of platelet-derived factors such as ADP, histamine and 5-HT.

platelet dependent and can be prevented by either antagonists of platelet-derived vasoactive amines or selective platelet depletion (Henson and Cochrane, 1972). Further investigations revealed that this mechanism was due to the release of a soluble mediator from antigenstimulated sensitized basophils (Benveniste et al., 1972). This mediator was named platelet-activating factor (PAF). PAF is an ether-linked phospholipid that has numerous properties appropriate to a mediator of inflammation. Over the last 10 years there has been enormous effort in the investigation of its biological activity in various animal systems. Whilst many of the actions of PAF are platelet dependent, discussion of these are beyond the scope of this chapter and the reader is directed to other sources (Braquet et al., 1987).

3.4

CARRAGEENIN-INDUCED INFLAMMATION

The induction of oedema by carrageenin in the rat is associated with a platelet sequestration at the site of injection (Vincent et al., 1975) and an increased ex vivo platelet responsiveness (Srivastava and Srimal, 1990). However, induction o f necrohaemorrhagic lesions induced by subcutaneous implantation of carrageenin is independent of circulating platelets (Ubatuba and Ferreira, 1976). In contrast, intravenous injection of carrageenin induces thrombocytopenia, hypotension and death which is abrogated by immune depletion of platelets or inhibition of the platelet release reaction (Vargaftig and Lefort, 1977).

3.2 THE GENERALIZED SHWARTZMAN REACTION The generalized Shwartzman reaction can be induced by two intravenous administrations of bacterial endotoxin and is associated with renal glomeruli thrombi and cortical thrombosis. Selective platelet depletion (Margretten and McK~ay, 1969) or prostacyclin infusion (Campos et al., 1983) induced a significant inhibition of the effects of endotoxin suggesting that the platelet plays an important role in this phenomenon.

3.3

ACUTE SERUM SICKNESS IN THE RABBIT

Acute experimental serum sickness in rabbits has been used as a model of tissue injury produced by immune complexes. Following injection with a large dose of antigen such as bovine serum albumin (BSA), immune complexes are formed which circulate in the blood. Following immune complex deposition, there is an induction of vascular lesions which result from an increase in vascular permeability. This increase in permeability is

3.5 EXPERIMENTAL HAEMARTHROSIS Haemorrhage in the cavity of the synovium resulting from either trauma or haemophilia induces an acute inflammatory response in the synovial membrane. After single episodes of haemarthrosis, the acute inflammatory response in the synovial membrane lasts 24-48 h and resolves without subsequent sequelae. When associated with repeated provocations, the acute inflammatory response is followed by synovial proliferation, fibrosis and cartilage destruction. The possible contribution of platelets was investigated by injecting autologous platelets into the synovial cavity of rats (Bignold, 1980). Injection of platelets into the synovium induced an increase in permeability, as assessed by leakage of colloidal carbon, which was maximal at 2 h and persisted for 12 h. The observed increase in vascular permeability was associated with the formation of gaps between venular endothelial cells, but without evidence of frank endothelial damage, suggesting the contribution of platelets was related to the release of platelet-derived permeability factors.

24 A.J. COYLE AND B.B. VARGAFTIG

4. The Role of Platelets in Models of Non-allergic Lung Injury The adult respiratory distress syndrome (ARDS) is a form of non-cardiogenic oedematous lung injury. Despite advances in critical care management of patients with ARDS, the mortality rate remains greater than 50%. One major reason for this lack of progress has been that the underlying mechanisms of this disease are still unclear. The use of animal systems to investigate the underlying pathological process has, however, resulted in the realization of some new concepts in this disease process.

4.1

I N VITRO STUDIES

Several group have investigated the inflammatory properties of platelets using an in vitro technique of perfused isolated lungs. This procedure has the advantage that the lungs can be studied in the absence of extrapulmonary influences, and allows platelets to be perfused to airways as the sole blood element. In addition, it is also possible to directly measure changes in vascular resistance, oedema formation (as assessed by wet to dry weight ratios) and the pulmonary generation of mediators. Perfusion of the lungs with platelets and phorbol myristate acetate (PMA) has been reported to induce oedema formation, associated with an increase in pulmonary arterial pressure (Wang et al., 1991). Products of the cyclooxygenase (CO) pathway were implicated as these parameters were inhibited by indomethacin and imidazole. These observations were supported by the demonstration of an increase in the amount of thromboxane A2 (TXA2) in the perfusate after platelet activation. Likewise, Shoemaker and colleagues (1984) demonstrated that co-perfusion of platelets with Staphylococcus aureus induced a retention of platelets, TXA2 release and an increase in pulmonary artery pressure. Perfusion of lungs with either platelets or S. aureus alone had no significant effect (Shoemaker et al., 1984). Similarly, perfusion of isolated rabbit lungs with platelets in the presence of PAF induced a marked and protracted increase in pulmonary artery pressure (Heffner et al., 1983). The demonstration that platelets contribute to the induction of pulmonary hypertension is supported by in vivo observations that platelet depletion inhibits increased pulmonary artery pressure in a variety of experimental animal models as discussed below. In addition to these reports, there is also evidence that under some circumstances platelets may play an antiinflammatory role and help to preserve the integrity of the vascular endothelium. Thus whole organ oedema has been demonstrated to be enhanced in animals made thrombocytopenic (Lo et al., 1988). Platelet depletion has also been demonstrated to enhance oedema formation induced by cz-naphthylthiourea (Fantone et al., 1984). Similarly in vitro perfusion of isolated rabbit lungs

with human platelets inhibits xanthine oxidase (XO) induced lung injury by an effect which is dependent on the anti-oxidant capacity of the platelet mediated by glucose-6-phosphate dehydrogenase (Heffner et al., 1989). Inhibition of this enzyme resulted in an augmentation of lung oedema through hydrostatic mechanisms mediated by CO products (Heffner et al., 1989).

4.2

I N VIVO STUDIES

4 . 2 . 1 M i c r o e m b o l i s m and Direct L u n g I n j u r y Thrombocytopenia has been reported during lung injury induced by oleic acid, the degree of which parallels the degree of progressive hypoxia. Similarly, infusion of oleic acid (Spragg et al., 1982) or blunt injury to the leg muscles of dogs, results in the sequestration of radiolabelled platelets (Almquist et al., 1983). Induction of platelet aggregation within the pulmonary circulation by platelet anti-serum, collagen and ADP (Vaage et al., 1974) and endotoxin (Stein and Thomas, 1967) has been reported to induce lung injury. These effects most likely relate to the release of platelet-derived factors, rather than direct obstruction of the pulmonary vessels themselves. Characteristically, t h e response to microembolism is increased vascular permeability and focal endothelial damage, although the development of pulmonary oedema is transient and less severe than other forms of experimental ARDS. However, activation of platelets by antigen-antibody complexes has been reported to induce vascular injury and inflammation in rabbits (Hughes and Tonks, 1962). Moreover, vascular injury induced by complement activation in mice is attenuated in animals rendered thrombocytopenic (Tvedten et al., 1985). Nevertheless, there are some reports that fail to support an important role of platelets in oedema formation following microvascular emboli. Thus in chronically instrumented sheep, platelet depletion fails to modify the increase in lung lymph flow (Binder et al., 1980). In addition, platelet depletion fails to inhibit thrombin-induced damage, which appears to be dependent on neutrophil activation as suggested by experiments using selective anti-neutrophil serum (Johnson and Malik, 1985). 4.2.2 Experimental Pulmonary Hypertension Increased vascular resistance and the subsequent development of pulmonary hypertension occur in the first few hours of respiratory failure. Several experimental animal models have been used to investigate the contribution of platelets in this process. Infusion of ADP in dogs induces platelet aggregation, hypoxia and pulmonary hypertension, which are platelet dependent (Bredenberg et al., 1980). Similarly endotoxin infusion in dogs induces elevated pulmonary arterial pressure which is attenuated in animals rendered thrombocytopenic (Bredenberg et al., 1980). The actual mechanisms by which platelets contribute to pulmonary hypertension are unclear, but

ANIMAL MODELS FOR INVESTIGATING PROPERTIES OF PLATELETS 25 appear to be secondary to the release of TXA2 and 5-HT (Heffner et al., 1983). However, there are some conflicting results which suggest that platelets are not involved in this process. Endotoxin infusion in sheep fails to induce a peripheral blood thrombocytopenia (Snapper et al., 1984). Moreover, the pressor response is not modified by prior platelet depletion (McDonald et a/., 1983; Snapper et al., 1984). These studies are in conflict with the studies cited above performed in dogs, where platelet depietion inhibited the development of hypertension. These differences may be related to species differences in platelet production and vascular responsiveness to CO metabolites. In this context sheep platelets are a poor source of TXA2, while dog platelets produce considerably greater amounts. Human platelets produce even greater amounts and therefore it is possible that the contribution of platelet-derived mediators is greater in humans than in experimental animal models of lung injury.

5. The Role of Platelets in Models of Allergic Lung Injury 5.1

PLATELET AGONIST- AND ALLERGEN-INDUCED

BRONCHOCONSTRICTION Intravenous injection of ADP, ATP, PAF and collagen induces a platelet-dependent bronchoconstriction in guinea-pigs (Collier, 1971; Vargaftig and Lefort, 1979; Vargaftig et al., 1980, 1982). In the case of ATP and collagen, bronchoconstriction is also inhibited by aspirin, while that of ADP and PAF is not dependent on the generation of CO metabolites (Lefort and Vargaftig, 1978). Platelet-dependent bronchoconstriction is most likely related to the release of smooth muscle contractile agents from the platelet rather than physical occlusion of the pulmonary microvessels. This has been demonstrated by the observation that a combination of aspirin, 5-HT and mepyramine fails to inhibit ADP-induced thrombocytopenia, but inhibits bronchoconstriction, suggesting that platelets undergo sequestration, but fail to release the granule contents (Vargaftig et al., 1982). In addition, simultaneous continuous monitoring of lXaInlabelled platelets and lung function has demonstrated a temporal dissociation between these two parameters (Page et al., 1982). Similarly in rabbits, infusion of PAF induces changes in lung mechanics that are dependent on platelet activation (Halonen et al., 1985). The mechanisms which underlie acute respiratory anaphylaxis have been widely investigated using a variety of protocols for both sensitization and challenge, both of which have a profound influence on the subsequent pulmonary response. In the rabbit, IgE-dependent anaphylaxis is associated with platelet activation

(Pinckard et al., 1977). A mild thrombocytopenia has also been reported following aerosol provocation of passively immunized guinea-pigs, although the antigeninduced bronchoconstriction was platelet independent (Cirino et al., 1986). Intravenous antigen challenge of active immunized guinea-pigs induces a marked thrombocytopenia associated with an intrathoracic accumulation of radiolabelled platelets (Page et al., 1982). Moreover, histological analysis demonstrated that these platelets had undergone diapedesis and were intimately associated with airway smooth muscle (Lellouch-Tubiana et al., 1985, 1987). Inhibition of platelet function failed to inhibit active anaphylaxis in this model, arguing against a significant contribution of platelets to antigeninduced anaphylaxis (Pretolani et al., 1985). A similar lack of platelet involvement was suggested during IgE anaphylaxis in rabbits following pretreatment with prostacyclin (Halonen et al., 1985). Finally, platelet depletion failed to alter the early asthmatic response to aerosol antigen provocation in an allergic rabbit model (Coyle et al., 1990b). It is noteworthy that the intratracheal injection of antigen to actively immunized or sensitized guinea-pigs is also accompanied by a reduction of platelet counts in blood and by accumulation of radiolabelled platelets in the lungs. The mechanism is probably indirect, but has not been unravelled.

5.2

THE ROLE OF PLATELETS IN EXPERIMENTAL MODELS OF AIRWAY HYPERRESPONSIVENESS

Airway hyperresponsiveness is a characteristic feature of bronchial asthma. While the mechanisms underlying this response are at present unclear, there is considerable evidence to suggest that airway inflammation is important. There is also evidence to suggest that platelets may be involved in this phenomenon. Thus, allergen provocation of asthmatic individuals has been reported to induce platelet activation, as demonstrated by the release of platelet-derived proteins such as platelet factor 4 (PF4) and/3-thromboglobulin (/3-TG; Knauer etal., 1981), and is associated with a prolonged bleeding time (Szczeklik et al., 1986) and shortened platelet survival (Taytard et al., 1986). In experimental animals, platelet activation has also been demonstrated to be associated with an increase in airway responsiveness. Infusion of PAF for I h in guineapigs induces a platelet-dependent increase in non-specific hyperresponsiveness, associated with an intrathoracic accumulation of platelets (Mazzoni et al., 1985; Deeming et al., 1986). Platelet activation has also been suggested to be involved in PAF-induced airway hyperresponsiveness in rabbits (Coyle et al., 1990a). The actual mechanisms by which platelets are involved in this increase in responsiveness are unclear, but appear to be unrelated to the intrathoracic accumulation of

26 A.J. COYLE AND B.B. VARGAFTIG platelets per se, as infusion of other platelet agonists including collagen, ADP and the TX mimetic U46619 induced a comparable, or in the case of collagen, a greater platelet accumulation, but failed to increase airway responsiveness (Robertson and Page, 1987). It should be noted that unlike other platelet agonists, PAF-induced platelet accumulation was prolonged and significant accumulation was measured 90 min after PAF infusion. This phenomenon may be related to the observations by Lellouch-Tubiana and co-workers (1985), that PAF can induce extravascular diapedesis of platelets into pulmonary tissue. Thus it appears possible that PAF can induce activation of platelets which is distinct from that induced by other platelet agonists. To further understand this phenomenon, guinea-pig platelets were incubated with either PAF or ADP for 1 min, and the supernatant injected intravenously into a recipient guinea-pig. PAF-induced platelet activation generated the release of a mediator that was able to increase airway responsiveness (Sanjar et al., 1989). In contrast, injection of supernatants from ADPstimulated platelets had no significant effect on airway responsiveness. This factor was termed platelet-derived hyperreactivity factor (PDHRF) and thus this unstable mediator may be the link between platelet activation and airway hyperresponsiveness. Platelets involvement has also been suggested in the heightened airway responsiveness induced by allergen provocation in an allergic rabbit model (Coyle et al., 1990b).

5.3

INTERACTIONS OF PLATELETS WITH OTHER BLOOD ELEMENTS

The combined presence of neutrophils and platelets at sites of inflammation has been observed both in experimental animal models and in various inflammatory diseases. In experimental animals, platelet-neutrophil interactions have been noted in models of immune complex disease in rabbits (Henson and Cochrane, 1972), hypoxia-damaged arterial endothelium (Jellinek, 1977) and atherosclerosis (Joris and Majno, 1979). However, the precise mechanisms whereby neutrophils and platelets influence each other are unclear. Various platelet-derived products have been demonstrated to possess pro-inflammatory effects. Intradermal injection of platelet-derived cationic proteins induce an infiltration of neutrophils 3 h later (Nachman and Weksler, 1972) and supports the observations in vitro, that PF4 and platelet-derived growth factor (PDGF) are chemotactic for neutrophils (Deuel et al., 1981, Tzeng et al., 1985). An important role of the neutrophil has also been suggested in platelet activation. Data obtained in vitro with human cells demonstrated that cathepsin G is the mediator accounting for this effect (Ferrer-Lopes et al., 1990; Renesto et al., 1990; Evangelista et a/., 1991). Recent evidences indicate that another serine proteinase,

elastase, also participates in neutrophil-induced platelet activation (Selak, 1992). By contrast, similar experiments performed with rabbit cells showed a role for PAF (Co~ffier et al., 1987; Oda et al., 1986). In experimental animals, intradermal injection of zymosan activated plasma, FMLP or endotoxin induced an accumulation of platelets which was inhibited by neutrophil depletion (Issekutz et al., 1983). Similarly, in guinea-pigs in vivo, intravenous injection of FMLP induces pulmonary platelet sequestration, which appears to be dependent on neutrophil activation (Bureau et al., 1992). Platelet activation has also been demonstrated to be an important prerequisite in eosinophil infiltration in a number of animal models. In guinea-pigs, intravenous injection of either PAF or ovalbumin in sensitized animals induced a peribronchial eosinophil infiltration 6 h later, which could be inhibited either by immune platelet depletion or by prostacyclin infusion (LellouchTubiana et al., 1988). In rabbits, PAF-induced cell accumulation was also attenuated in animals rendered thrombocytopenic (Coyle et al., 1990a). Likewise, in IgE-immunized rabbits, platelet depletion inhibited allergen-induced eosinophil, but not neutrophil recruitment (Coyle et al., 1990b).

5.4

THE ROLE OF PLATELETS IN MODELS OF LATE ASTHMATIC RESPONSE

Antigen provocation of allergic individuals induces an acute airways obstruction resulting from smooth muscle constriction and/or airway oedema. In approximately 70% of these individuals, this early asthmatic response (EAR) is followed by a late asthmatic response (LAR), and it is believed that this results from an influx of inflammatory cells into the airways, mainly eosinophils. Immunization of neonatal rabbits within 24 h of birth with ragweed, followed by repeated immunizations every month, results in the preferential expression of IgEspecific antibodies (Shampian et al., 1982; Metzger et al., 1987). Following allergen provocation, immunized animals undergo a response characterized by an EAR, LAR and heightened airway responsiveness, associated with an influx of granulocytes into the airways. Moreover it was suggested, based on depletion/repletion experiments, that this granulocyte infiltration was important in the altered airway physiology (Murphy et al., 1986). However, allergen provocation was also associated with a peripheral blood thrombocytopenia and an increase in the number of platelets recovered from the bronchoalveolar lavage (BAL) fluid, raising the possibility that platelet activation occurs during both the early and late responses (Metzger et al., 1987). Selective platelet depletion using a specific anti-platelet antibody inhibited the LARinduced by allergen challenge (Fig. 2.1; Coyle et al., 1990b). In contrast, the EAR was not modified by

ANIMAL MODELS FOR INVESTIGATING PROPERTIES OF PLATELETS 27

(3 <]

-20

-40

84

2

Time ( h r )

4

6

Figure 2.1 Modification of allergen-induced pulmonary response by anti-platelet serum (open circles) in an allergic rabbit model of asthma. Control animals were treated with normal guinea-pig serum (closed circles). Results are expressed as the mean + SEM of the percentage change from baseline in dynamic compliance over a 6 h period and represent the mean of n = 7 animals. [Reproduced with permission from Coyle et ah (1990).]

platelet depletion demonstrating that mechanisms other than platelet activation are involved in this response.

e

Direct Ant qen-induced Activation of Platelets

Following the identification of low affinity receptors for IgE and IgG on the surface of the platelet, the possibility arose that these cells could not only contribute to allergic diseases as "secondary effector cells", but in fact could also undergo direct activation by the antigen. Indeed studies performed in platelets from allergic individuals demonstrated that activation of the IgE receptor resulted in the release of toxic oxygen free radicals and the subsequent cytocidal effect on parasites (Joseph et al., 1986). The interaction between platelets and allergen has recently been studied in experimental animals by Pretolani and colleagues (1990). Peripheral blood platelets from actively immunized guinea-pigs were purified, washed and subsequently transferred to naive control animals. Twenty-four hours later, animals were provoked intravenously with the allergen and changes in airway obstruction, leucocyte and platelet number recorded. Allergen-induced platelet activation failed to induce bronchoconstriction or any change in the number of circulating platelets, indicating that allergen challenge fails to induce platelet aggregation and the release of the

granular contents of the platelets (Pretolani et al., 1990). In contrast however, there was a marked and rapid drop in the number of circulating leucocytes, which reached a maximum fall of approximately 60% of the control levels, 30 min later (Fig. 2.2). These results are of considerable importance, as they illustrate in vivo that platelets can undergo a form of activation that is distinct from classical platelet aggregation and may be of direct importance in the pathogenesis of allergic disorders. The nature of this allergen-induced platelet-dependent leucopenia is unclear. Early studies using the 5-1ipoxygenase (5-LO) inhibitor, BW755C, suggested that following antigen activation, platelets released products of the 5-LO pathway which then modified either leucocyte and/or endothelial function (Pretolani et al., 1990). However, a subsequent study failed to support this hypothesis, as a more selective 5-LO inhibitor, BWA4C, was ineffective (Coyle et al., 1991). It was speculated that the difference between the efficacy of these two compounds was related to their differential ability to scavenge oxygen free radicals, BW755C being considerably more potent in this respect. Thus it is possible that an analogous situation to that which follows antigen activation of human platelets in vitro occurs, where platelets generate oxygen free radicals, which in this in vivo model could then modify endothelial cell function. While there is no direct evidence at present to support this hypothesis, it is interesting to note that nedocromil sodium (Joseph et al., 1986) and cetirizine (De Vos et al., 1989), agents which inhibit the generation of oxygen free radicals from platelets in vitro, were also effective in inhibiting plateletmediated leucopenia in this model (Pretolani et al., 1990; Coyle et al. , 1991). lO 0 ~= - 10 oo -20 o -30 "~-~_-40 "i "O

-5o "t -6o -7o o

;o

6'o

Time (min)

9'o

Figure 2.2 Time course of the change in platelet or leucocyte count following i.v. administration of ovalbumin in animals that received platelets from immunized animals. Results are expressed as the percentage change from baseline and represent the mean + SEM of n = 6 animals. [Reproduced with permission from Pretolani et ah (1990).]

b

28

A.J. COYLE AND B.B. VARGAFTIG

7. The Role of Platelets in Animal Models of Parasitic Infection Following infection of rats with the schistosomiasis parasite, platelet number in the circulating increased 3-fold reaching a maximum 42 days later (Joseph et al., 1983). Removal of the platelets and determination of their in vitro cytotoxicity demonstrated that platelets had acquired the ability to induce killing of schistosomula. Moreover, passive transfer of platelets from infected animals to normal syngeneic recipients on the day of infection resulted in a significant degree of protection, with a 63% reduction in the total number of worms in the liver 3 weeks later. This effect was dependent of the presence of surface receptors for IgE. The mechanisms of platelet-induced parasite toxicity appears to be dependent on the generation of oxygen free radicals, and thus is a very different type of activation to that which occurs following the induction of platelet aggregation.

8. Conclusion Platelets are usually not considered outside the field of haemostasis and thrombosis, but clearly they have a broader potential, as pro- and possibly anti-inflammatory elements. They also participate in cytotoxic mechanisms, have the ability to respond to direct and indirect provocations and share adhesion proteins with other blood cells. Platelets may constitute a very appropriate target for pharmacological modulation of disease, which, as of yet, has not been sufficiently investigated in the clinical setting.

9. References Almquist, P., Kuenzig, M. and Schwartz, S.I. (1983). The effect of naloxone and cyproheptadine on pulmonary platelet trapping, hypotension and platelet aggregability in traumatised dogs. J. Trauma 23, 405-407. Benveniste, J., Henson, P.M. and Cochrane, J. (1972). Leucocyte dependent histamine release from rabbit platelets: the role of IgE, basophils and a platelet activating factor. J. Exp. Med. 136, 1356-1366. Berman, C.L., Yeo, E.L., Wencel-Drake, J.D., Furie, B.C., Ginsberg, M.H. and Furie, B. (1986). A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. J. Clin. Invest. 78, 130-137. Bignold, L.P. (1980). Importance of platelets in increased vascular permeability evoked by experimental haemarthrosis in synovium of the rat. Pathology 12, 169-179. Binder, A.S., Kageler, W., Perel, A., Flick, M.tL and Staub, N.C. (1980). Effect of platelet depletion on lung vascular permeability after microemboli in sheep. J. Appl. Physiol. 48, 414-420. Braquet, P., Touqui, L., Shen, T.Y. and Vargaftig, B.B. (1987).

Perspectives in platelet activating factor research. Pharmacol. Rev., 39, 97-145. Bredenberg, C.E., Taylor, G.A. and Webb, W.R, (1980). The effect of thrombocytopenia on the pulmonary and systemic haemodynamics of canine endotoxin shock. Surgery, 87, 59-68. Bureau, M.F., De Clerck, F., Lefort, J., Arreto, C.D. and Vargaftig, B.B. (1992). Thromboxane A2 accounts for bronchoconstriction, but not for platelet sequestration and microvascular albumin exchanges induced by fMLP in the guinea pig lung. J. Pharmacol. Exp. Ther. 260, 832-8840. Campos, A., Kim, Y., Azar, S.H., Vernier, R.L. and Michael, A.F. (1983). Prevention of the generalised Shwartzman reaction in pregnant rats by Prostacyclin infusion. Lab. Invest. 48, 705-711. Chignard, M., Wal, F., Lefort, J. and Vargaftig, B.B. (1982). Inhibition by sulphinpyrazone of the platelet-dependent bronchoconstriction due to platelet activating factor (PAFacether) in the guinea pig. Eur. J. Pharmacol. 78, 71-79. Cirino, M., Lagente, V., Lefort, J. and Vargaftig, B.B. (1986). A study with BN 52021 demonstrates the involvement of PAF-acether in IgE dependent anaphylactic bronchoconstriction. Prostaglandins 32, 121-126. CoEffier, E., Joseph, D., Pr&ost, M.C. and Vargaftig, B.B. (1987). Platelet-leukocyte interaction: activation of rabbit platelets by FMLP-stimulated neutrophils. Br. J. Pharmacol. 92, 683-691. Collier, H.O.J. (1971). Prostaglandins and aspirine. Nature, 232, 17-19. Coyle, A.J., Spina, D. and Page, C.P. (1990a). PAF induced bronchial hyperresponsiveness in the rabbit: contribution of platelets and airway smooth muscle. Br. J. Pharmacol. 101, 31-38. Coyle, A.J. Brown, L., Flanahan, R., Page, C.P. and Metzger, J.W. (1990b). The role of platelets in antigen-induced late phase asthma, eosinophil accumulation and heightened airway responsiveness in an allergic rabbit model. Am. Rev. Respir. Dis. 142, 587-593. Coyle, A.J., Lefort, J. and Vargaftig, B.B. (1991). The effect of cetirizine on antigen induced leukopenia in the guinea pig. Br. J. Pharmacol. 103, 1520-1524. Deeming, K., Mazzoni, L., Morley, J., Page, C.P. and Sanjar, S. (1986). Prophylactic anti-asthmatic drugs impair platelet accumulation in the lungs. Br. J. Pharmacol. 87, 37P. Deuel, T.F., Senior, I~M., Chang, D., Griffin, G.L., Heinrikson, R.L. and Kaiser, E.T. (1981). Platelet factor 4 is chemotactic for neutrophils and monocytes. Proc. Natl. Acad. Sci. 78, 4584-4587. De Vos, C., Joseph, M., Leprevost, C., Vorng, H., Tomassini, M., Capron, M. and Capron, A. (1989). Inhibition of human eosinophil chemotaxis and of the IgE dependent stimulation of human blood platelets by cetirizine. Int. Arch. Allergy Appl. Immunol. 88, 212-219. Disdier, M., Morrissey, J.H., Fugate, tLD., Bainton, D.F. and McEver, R,P. (1992). Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway. Mol. Biol. Cell 3, 309-321. Evangelista, V., Rajtar, G., de Gaetano, G., White, J.G. and Cerletti, C. (1991). Platelet-activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by antiproteinases. Blood, 11, 2379-2388.

ANIMAL MODELS FOR INVESTIGATING PROPERTIES OF PLATELETS Fantone, J.C., Kunkel, R.G. and Kinnes, D.A. (1984). Potentiation of cx-naphthylthiourea-induced lung injury by prostaglandin E1 and platelet depletion. Lab. Invest. 50, 703-710. Ferrer-Lopes, P., Renesto, P., Schattner, M., Bassot, S., Laurent, P. and Chignard, M. (1990). Activation of human platelets by C5a-stimulated neutrophils: a role for cathepsin G. Am. J. Physiol. 258, Cl100-Cl107. Halonen, M., Lohman, I.C., Dunn, A.M., McManus, L.M. and Palmer, J.D. (1985). Participation of platelets in the physiological alterations of the AGEPC response and of IgE anaphylaxis in the rabbit. Am. Rev. Respir. Dis. 131, 11. Heffner, J.A., Shoemaker, S.A., Canham, E.M., Patel, M., McMurty, I.F., Morris, H.G. and Repine, J.E. (1983). Acetyl glyceryl ether phosphorylcholine stimulated human platelets cause pulmonary hypertension and oedema in isolated perfused rabbit lungs. J. Clin. Invest. 71,351-357. Heffner, J.E., Cook, J.A. and Halushka, P.V. (1989). Human platelets modulate edema formation in isolated rabbit lungs. J. Clin. Invest. 84, 757-764. Henson, P. and Cochrane, C.G. (1972). Acute immune complex disease in rabbits. The role of complement and a leukocyte-dependent release of vasoactive amines from platelets. J. Exp. Med. 133, 554-561. Hughes, A. and Tonks, tLS. (1962). Intravascular platelet clumping in rabbits. J. Pathol. Bacteriol. 84, 379-390. Humphrey, J.H. (1955). Br. J. Exp. Pharmacol. 36, 268-282. Issekutz, A.C., Ripley, M. and Jackson, J.IL (1983). Role of neutrophils in the deposition of platelets during acute inflammation. Lab. Invest. 49, 716-724. Jellinek, H. (1977). Arterial surface changes examined by scanning (SEM) and transmission (TEM) electron microscopy. Adv. Exp. Biol. 82, 324-329. Johnson, A. and Malik, A.B. (1985). Pulmonary transvascular fluid and protein exchange after thrombin induced microembolism: differential effects of cyclooxygenase inhibitors. Am. Rev. Respir. Dis. 132, 70-76. Joris, I. and Majno, G. (1979). Inflammatory components of atherosclerosis. Adv. Inflam. Res. 71, 71-78. Joseph, M., Auriault, C., Capron, A., Vorng, H. and Viens, P. (1983). A new function for platelets: IgE-dependent killing of schistosomes. Nature, 303, 810-812. Joseph, M., Capron, M., Thorel, T. and Tonnel, A.B. (1986). Nedocromil sodium inhibits IgE dependent activation of rat macrophages and platelets as measured by schistosome killing, chemiluminesence and enzyme release. Eur. J. Respir. Dis. 69, 220-222. Knauer, K.A., Lichtenstein, L.M., Adkinson, N.F. and Fish, J.E. (1981). Platelet activation during antigen-induced airway reactions in asthmatic subjects. N. Engl. J. Med. 304, 1404-1407. Lassman, H.B., Kirby, tLE. and Novick, W.J. (1974). Alterations in platelet aggregation associated with adjuvant arthritis in rats. Pharmacol. Res. Commun. 6, 493-507. Lefort, J. and Vargaftig, B.B. (1978). Role ofplatelets in aspirinsensitive bronchoconstriction in the guinea pig: interactions with salicyclic acid. Br. J. Pharrnacol. 63, 35-42. LeUouch-Tubiana, A., Lefort, J., Pirotsky, E., Vargaftig, B.B., Pfister, A. (1985). Ultrastructural evidence for extravascular platelet recruitment in the lung upon intravenous injection platelet activating factor (PAF-acether) in guinea pigs. Br. J. Exp. Pathol., 66, 345-355.

29

Lellouch-Tubiana, A., Lefort, J., Pfister, A. and Vargaftig, B.B. (1987). Interactions between granulocytes and platelets with the guinea pig lung in passive anaphylactic shock. Correlations with PAF-acether induced lesions. Int. Arch. Allergy Appl. Immunol. 83, 198-205. Lellouch-Tubiana, A., Lefort, J., Simon, M-T., Pfister, A. and Vargaftig, B.B. (1988). Eosinophil recruitment into guinea pig lungs after PAF-acether and allergen administration. Modulation by prostacyclin, platelet depletion and selective antagonists. Am. Rev. Respir. Dis. 137, 948-954. Lo, S.K, Burshop, K.E., Kaplan, J.E. and Malik, A.B. (1988). Role of the platelet in the maintenance of pulmonary vascular permeability to protein. Am. J. Physiol. 254, H763-771. Margretten, W. and McKay, D.G. (1969). The role of the platelet in the generalised Schwartzman reaction. J. Exp. Med. 129, 585-593. Margretten, W. and McKay, D.G. (1971). The requirements for platelets in the active Arthus reaction. Am. J. Pathol. 64, 257-270. May, G.IL, Herd, C.M., Butler, K.D. and Page, C.P. (1990). Radioisotopic model for investigating thromboembolism in the rabbit. J. Pharmacol. Meth. 24, 19-35. Mazzoni, L., Morley, J., Page, C.P. and Sanjar, S. (1985). Induction of airway hyperreactivity by platelet activating factor in the guinea pig. J. Physiol. 365, 107P. McDonald, J., Ali, M., Morgn, E., Townsend, E. and Cooper, J. (1983). Thromboxane synthesis by sources other than platelets in association with complement induced pulmonary leukostasis Am. Rev. Respir. Dis. 134, 62-68. Metzger, W.J., Sjoerdsma, K., Richerson, H.B., Moseley, P. Zavala, D., Monick, M., Hunninghake, G.W. (1987). Platelets in bronchoalveolar lavage from asthmatic patients and allergic rabbits with allergen induced late phase responses. Agents Actions, 21, 151-159. Murphy, K.R., Wilson, M.C., Irvin, C.G., Glezen, L.S., Marsh, W.tL, Haslett, C., Henson, P.M., Larsen, G.L. (1986). The requirement for polymorphonuclear leucocytes in the asthmatic response and heightened airway reactivity in an animal model. Am. Rev. Respir. Dis., 134, 62-68. Nachman, R.L. and Weksler, B. (1972). The platelet as an inflammatory cell. Ann. NY Acad. Sci., 201, 131-137. Oda, M., Satouchi, K., Yasunaga, K. and Saito, K. (1986). Polymorphonuclear leukocyte-platelet interactions: acetylglyceryl ether phosphocholine-induced platelet activation under stimulation with chemotactic peptide. J. Biochem. 100, 1117-1123. Page, C.P., Paul, W. and Morley, J. (1982). An in vivo model for studying platelet aggregation and disaggregation. Thromb. Haemostasis, 47, 210-214. Palabrica, T.M., Furie, B.C., Konstam, M.A., Aronovitz, M.J., Connolly, R., Brockway, B.A., Ramberg, K.L. and Furie, B. (1989). Thrombus imaging in a primate model with antibodies specific for an external membrane protein of activated platelets. Proc. Nail. Acad. Sci. 86, 1036-1040. Pinckard, ILN., Palmer, J.D., McManus, L.M., Shaw, J.O. and Henson, P.M. (1977). Intravascular aggregation and pulmonary sequestration of platelets during IgE-induced systemic anaphylactic shock. Abrogation by platelet depletion. J. Immunol. 119, 2185-2193. Pretolani, M., Page, C.P., Lefort, J., Lagente, V. and Vargaftig, B.B. (1985). Pharmacological modulation of the respiratory

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A.J. COYLE AND B.B. VARGAFTIG

and haematological changes accompanying active anaphylaxis in the guinea pig. Eur. J. Pharmacol. 125, 403-409. Pretolani, M., Randon, J. and Vargaftig, B.B. (1990). Antigen induces leucopenia in non immunised guinea pigs injected with platelets from actively sensitised animals. Br. J. Pharmacol. 100, 185-189. Renesto, P., Ferrer-Lopez, P. and Chignard, M. (1990). Interference of recombinant eglin C, a proteinase inhibitor extracted from leeches, with neutrophil mediated platelet activation. Lab. Invest. 62, 409-416. Robertson, D.N. and Page, C.P. (1987). Effect of platelet agonists on airway reactivity and intrathoracic platelet accumulation. Br. J. Pharmacol. 92, 105-111. Sanjar, S., Smith, D. and Kristersson, A. (1989). Incubation of platelets with PAF produces a factor which causes hyperreactivity in guinea pigs. Br. J. Pharmacol. 96, 75P. Selak, M.A. (1992). Neutrophil elastase potentiates cathepsin G-induced platelet activation. Thromb. Haemost. 68, 570-576. Shampian, M.P., Behrens, B.L., Larsen, G.1. and Larsen, P.M. (1982). An animal model of late pulmonary responses to Alternaria challenge. Am. Rev. Respir. Dis. 126, 493-498. Shoemaker, S.A., Heffner, J.E., Canham, E.M., Tate, R.M., Morris, H.G., McMurty, F. and Repine, J.E. (1984). Staphylococcus aureus and human platelets cause pulmonary hypertension and thromboxane generation in isolated saline perfused rabbit lungs. Am. Rev. Respir. Dis. 129, 92-95. Snapper, J.IL, Hinson, J.M., Hutchinson, A.A., Lefferts, P.L., Ogletree, M.L. and Brigham, K.L. (1984). Effects of platelet depletion on the unanaesthetised sheep's response to endotoxemia. J. Clin. Invest. 74, 1782-1791. Spragg, ILG., Abraham, J.L. and Loomis, W.H. (1982). Pulmonary platelet deposition accompanying acute oleic acidinduced pulmonary injury. Am. Rev. Respir. Dis. 126, 553-557. Srivastava, IL and Srimal, R.C. (1990). Amplification of the platelet response during acute inflammation in the rat. Biochem. Pharmacol., 40, 357-363. Stein, M. and Thomas, D.P. (1967). Role of platelets in the acute pulmonary responses to endotoxin. J. Appl. Physiol. 23, 47-52. Stenberg, P., McEver, ILP., Shuman, M.A., Jacques, Y.V. and Bainton, D.F. (1985). A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J. Cell Biol. 101, 880-886. Szczeklik, A., Milner, P.C., Birch, J., Watkins, J. and Martin, J.F. (1986). Prolonged bleeding time, reduced platelet aggregation, altered PAF-acether sensitivity and increased platelet mass are a trait of asthma and hayfever. Thromb. Haemost. 56, 283-297.

Taytard, A., Guenard, H., Vuillemin, H., Bourot, J.L., Vergeret, J., Ducassou, D., Piquuet, Y. and Freour, P. (1986). Platelet kinetics in stable asthmatic subjects. Am. Rev. Respir. Dis. 134, 983-985. Tvedten, H.W., Till, T.O. and Ward, P.A. (1985). Mediators of lung injury in mice following systemic depletion of complement. Am. J. Pathol. 119, 92-100. Tzeng, D.Y., Deuel, T.F., Huang, J.S. and Baehner, ILL. (1985). Platelet-derived growth factor promotes polymorphonuclear leukocyte activation. Blood, 64, 1123-1128. Ubatuba, F.B. and Ferreira, D.H. (1976). Platelets, Arthus-type reactions and inflammatory mediators. Agents Actions, 6, 483-489. Vaage, J., Aarseth, P., Enge, I. and Hognestad, J. (1974). Changes in pulmonary vascular volumes after induced intravascular aggregation of blood platelets. Acta Physiol. Scand. 92, 84-94. Vargaftig, B.B. (1977). Involvement of the mediators in the interaction of platelets and carrageenin. In "Recent Developments in the Pharmacology of Inflammatory Mediators", Agents Actions, Suppl. 2, pp. 9-39. Birkhauser Verlag, Basle. Vargaftig, B.B. and Lefort, J. (1977). Acute hypotension due to carrageenin, arachidonic acid and slow reacting substance C in the rabbit: role of platelets and nature of pharmacological antagonism. Eur. J. Pharmacol. 43, 125-141. Vargaftig, B.B. and Lefort, J. (1979). Differential effects of prostacyclin and prostaglandin E1 on bronchoconstriction and thrombocytopenia during collagen and arachidonate infusions and anaphylactic shock in the guinea pig. Prostaglandins, 18, 519-528. Vargaftig, B.B., Lefort, J., Chignard, M. and Benveniste, J. (1980). Platelet activating factor induces a platelet dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives. Eur. J. Pharmacol. 65, 185-192. Vargaftig, B.B., Wal, F., Chignard, M. and Medeiros, M. (1982). Non-steroidal anti-inflammatory drugs if combined with an anti-histamine and anti-serotonin agent, interfere with the bronchial and platelet effects of platelet activating factor (PAF-acether). Eur. J. Pharmacol. 82, 121-131. Vincent, J.E., Bonta, I. and Zijistra, F.S. (1975). Accumulation of blood platelets in carrageenin rat paw oedema. Possible role in inflammatory processes. Agents Actions 8, 291-295. Wang, D., Chou, C.L., Hsy, K. and Chen, H.I. (1991). Cyclooxygenase pathway mediates lung injury induced by phorbol and platelets. J. Appl. Physiol. 70, 2417-2421.

3. The Analysis of Ligand-Receptor Interact'ons m Platelet Activation e

9

9

Michael H. Kroll and Andrew I. Schafer

1. Introduction 2. Platelet Physiology 3. Activating Ligand-Receptor Interactions 3.1 Thrombin 3.2 TXA2/PGG2/PGH2 3.3 PAF 3.4 Collagen 3.5 VP 3.6 Epinephrine 3.7 ADP 3.8 5-HT 3.9 Other Platelet Receptors That Mediate Activation 3.9.1 vWF-GPIb Binding 3.9.2 GPIIb-IIIa 3.9.3 CD9 3.9.4 Other Activation-initiating Receptors 4. Activation-induced Changes in Platelet Receptors 5. G-proteins

31 32 33 33 36 36 36 37 37 37 38

6.

38 38 39 39

7.

40 40 40

1. Introduction The analysis of ligand-receptor interactions in platelet activation encompasses principles that are essential for understanding the importance of platelets in hemostasis and thrombosis and are fundamental to many biological systems. The platelet surface has a remarkably dense topography of complex molecules that mediate the cell's recognition of a variety of physical and chemical stimuli. Many of these surface molecules have been characterized as specific protein receptors which, when occupied by Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

8. 9. 10.

5.1 Gs/Gi 5.2 Gp 5.3 Low Molecular Weight G-proteins Intracellular Signalling Pathways 6.1 PLC 6.2 IPs and Calcium 6.3 PKC 6.4 PLA2 6.5 Na +/H + Exchange 6.6 Other Signal Pathways 6.6.1 PLD 6.6.2 Tyrosine Kinases 6.6.3 Histamine Inhibitory Ligand-Receptor Interactions 7.1 Introduction 7.2 cAMP 7.3 cGMP 7.4 Mechanisms of Platelet Inhibition Conclusion Acknowledgements References

42 42 42 43 43 43 45 46 47 48 48 49 49 49 49 49 49 50 51 51 51

ligand, initiate a cascade of transmembranous chemical reactions that effect platelet functional responses. The purpose of this chapter is to review platelet surface receptors for many stimulatory and inhibitory extracellular factors, and to describe the structural requirements of the transmembranous signalling apparatus that mediate the state of platelet activation. This chapter develops several ideas first presented in a related chapter (Kroll, 1993). Many techniques have been applied to the study of platelet receptors. Of fundamental importance are Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

32 M . H . KROLL AND A.I. SCHAFER binding methods employing ligands composed of reporting molecules, such as radioisotopes or fluorescent conjugates (O'Malley and Hardman, 1975). Studies of ligand binding sites often utilize methods for crosslinking ligands to the platelet surface, with subsequent isolation by chromatographic and electrophoresis methods of the plalelet surface receptor for the crosslinked ligand; such studies may also involve immunochemical and lipid analyses. More recently, platelet surface receptors have been investigated using a broad array of molecular cloning and flow cytometry techniques. These techniques have revolutionized the study of platelet receptors and have provided a rapid thrust forward for detailed investigations of structure-function relationships of important platelet surface molecules. In particular, studies of signalgenerating transmembranous platelet receptors have begun to define the biochemistry of guanine nucleotide binding regulatory proteins (G-proteins; Ga) coupled to receptor molecules. Further, because physiologically relevant ligand binding ultimately leads to an intraceUular response, the study of platelet ligand-receptor interactions encompasses analyses of the second messengers that function downstream from receptor activation.

Ec < : : : x : : : : : x ~ - - - ~ v : : : : x : ~

Adhesion

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

SE

Serotonin

TXA 2 k , ADP

Release

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII *ZQ.

Aggregation

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Figure 3.1 Platelet function comprises a triad of responses: adhesion, release (or secretion) and aggregation. These responses arise from the activation of carefully regulated transmembranous signal pathways that result in the generation of a platelet thrombus. EC, endothelial cell; SE, subendothellum; TXA=, thromboxane A=, ADP, adenosine diphosphate.

Nomura et al., 1986; Berridge, 1987; Siess, 1989; Bansal and Majerus, 1990). This transformation occurs to a large extent through classical mechanisms of transmembranous stimulus-response coupling that result in the generation of specific intracellular second messengers (Fig. 3.2). The primary activation pathway of platelets employs two

2. Platelet Physiology Platelets are necessary for normal hemostasis and are participants in the development of pathological thromboses, particularly in the arterial circulation where flow generates elevated wall shear stresses. "Primary hemostasis" is defined as platelet/blood vessel interactions that initiate physiologic thrombus formation. When primary hemostasis is triggered by a pathological stimulus (such as a ruptured atherosclerotic plaque), platelet activation leads to the development of a selfamplifying series of cellular responses that results in the formation of a platelet plug (the so-called "white thrombus"), vaso-occlusion, and ischemia or infarction (Sherman et al., 1986; Kroll and Schafer, 1989; Dutch TIA Trail Study Group, 1991). Platelets circulate in an unactivated state through a complex vascular system lined by a monolayer o f endothelial cells. In response to vessel wall injury, alterations in blood flow or chemical stimuli, platelets manifest a triad of functional responses: adhesion, secretion, and aggregation (Fig. 3.1). These functional responses occur via a series of carefully co-ordinated signals that convert an extracellular stimulus into intracellular chemical messengers that direct specific enzymatic reactions. The state of platelet activation is regulated dynamically by the actions of a diverse array of excitatory and inhibitory extracellular stimuli. Platelets are equipped with specific plasma membrane receptors that organize these various stimuli and transform them into biological responses (Phillips, 1985; Phillips and Shuman, 1986;

Extracellular signal .

.

.

.

.

Receptor , Transducer protein

,

Signal- generating enzyme ,

Precursor

Second messenger Internal effector Platelet response

Figure 3.2 An overview of extracellular signal transduction. A ligand-receptor interaction on the outer leaflet of the plasma membrane is coupled to a signal-generating enzyme complex on the inner leaflet of the plasma membrane by an intramembranous signal-transducing protein. The activity of the signalgenerating enzyme causes the production of intracellular second messengers that direct specific proteins to effect specific cellular responses. Reprinted by permission of the publisher (Grune and Stratton) from Kroll and Schafer AI (1989).

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 33 second messengers derived from enzymatic hydrolysis of inositol phospholipids: inositol 1,4,5-trisphosphate (IP3) and sn-l,2-diacylglycerol (DAG). A major inhibitory signalling pathway in platelets employs cyclic adenosine 3'5'-monophosphate (cAMP) as the second messenger. Both of these pathways are triggered by specific extracellular molecules occupying cell surface receptors, and the second messengers of both pathways are generated intracellularly by the activation of membrane-associated enzymes through receptor-linked changes of heterotrimeric Ga. In each case, the activated signal-generating enzyme converts highly phosphorylated precursors into intracellular second messengers: phospholipase C (PLC) cleaves membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3 and DAG, and adenylyl cyclase converts ATP into cAMP. The second messengers formed by these reactions cause important intracellular responses by inducing conformational changes in target proteins either directly, or indirectly by the activation of protein kinases: IP3 releases calcium (Ca2+), which causes activation of various Ca2+-dependent enzymes (Majerus et al., 1986); DAG causes activation of protein kinase C (PRC, Majerus et al., 1986; Nomura et al., 1986; Berridge, 1987; Siess, 1989); and cAMP causes activation of cAMP-dependent protein kinases (Feinstein et al., 1985). In addition to these second messenger-mediated signal pathways, tertiary biochemical responses branching from the major activation pathways are initiated intracellularly, and these responses fine tune, dampen, or amplify the signals caused by classical second messengers. Thus, a network of carefully regulated reactions governs physiologic and pathophysiologic platelet function. Elucidation of these complex reactions is far from complete, yet knowledge about fundamental mechanisms of platelet activation has contributed substantially to our understanding of human biology. Platelets have often served as the cell type from which basic and general principles of cell signalling have been developed. Furthermore, advances in understanding mechanisms of platelet activation have led to the rational design of pharmacological agents for the prevention and treatment of atherothrombotic diseases.

3. Activating L qand-Receptor Interactions Physiologic platelet activation or inhibition is initiated when an extracellular stimulus interacts with the platelet surface. This interaction often involves a ligand-receptor coupling wherein extracellular molecules bind to one or more specific membrane receptors (Phillips, 1985). There is great diversity in the structure and sites of production of the different extracellular stimuli that regulate platelet function (see Table 3.1). Included are plasma constituents such as proteases (thrombin, plasmin) and

catecholamines; vascular wall products such as prostacyclin (PGI2), nitric oxide (NO) and collagen; platelet products, including adenosine diphosphate (ADP) and serotonin (5-hydroxytryptamine; 5-HT); and molecules potentially derived from multiple blood cell and vascular sources such as ADP, platelet activating factor (PAF), von Willebrand's factor (vWF), prostaglandin endoperoxides (PGG2/PGH2) and thromboxane A2 (TXA2). Soluble extracellular signals that elicit platelet responses through receptor-mediated events can be grouped as: (1) "strong agonists" [including thrombin, prostaglandin endoperoxides, TXA2, collagen, PAF and vasopressin (VP)] that cause platelet aggregation that is not dependent on secretion and that is not inhibited by blocking platelet PGG2/PGH2 synthase ("cyclooxygenase"); (2) "weak agonists" (including ADP, epinephrine and 5-HT) that depend on secretion to effect a full aggregation response; "weak agonists" cause aggregation that demonstrates an irreversible "second wave" blocked by inhibitors of cyclooxygenase (CO; e.g. aspirin); and (3) antagonists (PGI2 and PGD2) that inhibit platelet responses to many different stimuli by causing elevations of platelet cytosolic cAMP. The structures of the platelet receptors for many of these ligands have been identified, but the molecular mechanisms by which ligand binding initiates the receptor signalling function across the plasma membrane remains largely unknown. Analyses of receptor structure demonstrate that a ligand may have more than one receptor and that receptor heterogeneity may be an important mechanism by which an extracellular stimulus is directed to the appropriate physiological response. For example, one c~ thrombin receptor may be linked to activation of PLC, while a separate ~ thrombin receptor may be linked to the inhibition of adenylyl cyclase (McGowen and Detwiler, 1986). Receptor heterogeneity may also occur between cell types, thus providing additional regulation of the organization of an extracellular stimulus into an appropriate multicellular response.

3.1

THROMBIN

The "strong agonist" c~ thrombin is an important extracellular stimulus for platelet activation in vivo (Eidt et al., 1989). Thrombin binding to platelets was first demonstrated by Tollefsen et al. (1974). Subsequent work has established that there are three classes of binding sites having affinities designated as low (590 000 sites/platelet; Ka ~ 2900 nM), moderate (1700 sites/platelet; Ka ~ 11 nM) and high (50 sites/platelet; Ka ~- 0.3 nM). Until recently, the relationship between thrombin's binding and proteolytic activities was uncertain (Davey and Luscher, 1967; Tollefsen et al., 1974; Ganguly and Sonnichsen, 1976). This debate appears to be mitigated by the recent cloning of a functional thrombin receptor. Through these studies, the structure of the receptor for c~ thrombin has been elucidated and

m

_>

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o

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Binding

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3. @

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m Q.

Struchmm

O

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O

Receptor

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I--

Table 3.1 Summary of platelet receptors that mediate activation. See text for further details Effector

Other

=_ ~

I-

Thrombin High affinity

o

(1) o (D

tl)

r-

o~

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8

o

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|

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13

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Primary functional receptor

Gp: pertussis toxin sensitive

PLC

Wide range of published K, and ,8

Uncertain: with 2 10 pg/ml type I collagen PLC is directly activated; at lower concentrations, PLA, is first activated

Mgz+-dependentbinding that is inhibited by Ca2'

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s...

>,

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. . . . . . .

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8

data

c

.,.,

r

E

0 "o

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~m

~r,,

Gq: pertussis toxin insensitive

O

Collagen GPla-lla (integrin a2B,)

Probably not a functional receptor

May "prime' platelets

~

=~o

(r

E

o "(3

et~ .Q

m

342 aa M, 39 000 7 transmembrane domains

Unique proteolytii mechanism creates "tethered peptide ligand"

direct T [Ca2'l,

~.

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9

343 aa 7 transmembrane domains

Nr,..

"(3

~

PAF

0..

e-

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I-

Moderate affinity

-J

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o "o

tD

:=_-

=~

?

PLC Downregulate AC

n

(90

|

Gp, Gi: pertussis toxin sensitive

~

TXA$PGG?/PGH2 High affinity

t~

?

.~3

Low affinity

.c:

425 aa 7 transmembrane domains

~|

Moderate affinity

o

o

.=_

May co-operate with moderate affinity receptor

0

{-

.s

.c:

o

B.

~

i-

.s

o

t~

._o o1 o .o

>.. .c: n

"o e-

r-

Ii)

1-

o x

+

Z

~

+

._~

o

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*-,

8~

ID

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c-

May be coupled to PLA, through a Na+/H exchange and GPllb-llla dependent mechanism 13.

Downregulate AC

O O

Gi: pertussis toxin sensitive .~

B,=300

o

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I=

s

r-

o~|

8

PLC

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K, = 2.5 n~

Physiological relevance is questionable

Gp: pertussis toxin sensitive r-

if)

= 95

Based on clinical evidence

E

o "o

r-

s_

450 AA M, 64 000 7 transmembrane domains

K, = 1 nM

,31

,.I=

r~

.c:

e-.

"(o-

o.

11.

Platelet thrombospondin receptor

'<

ADP Aggregin

E

11) e" L-

I1) 1--

Epinephrine (a2)

o "o

.(3

M, 125 000 7 transmembrane domains

t]) .-~ . _

O

tO m

Vasopression (V,)

"9, ,t, +

,,

= g g =g

M, 62 000

GPVl

=8

r-

81

|

167 000 Mr 130000 M, 85 000-95 000 (highly glycosylated)

%~

m

(9

GPlV (CD36)

a2M,

I1)

.m o

l-

o

~

=

---

o

..~

8

.--

_1

Function unknown

E~

IO

~m

m

"~8

m

|x

o .m if) r-

O e

I~11)

oE

~

o~

S, receptor; Em, measurements in intact platelets complicated by active uptake of ligand ID

O < I1)

I1)

c

~Eg

K, determined by binding to COS cells expressing cloned human I!)

PLC Downregulate AC _J

x

.c_ ~_ |

I

E

._=

o "(3

rt~ L

E o E

Reprinted by permission of the publisher (Blackwell), from Kroll (1993).

Gp, Gi: Pertussis toxin sensitive

9

475 aa K, = 2 n~ 6-60 fmol per 7 transmembrane domains : ,8 mg membrane protein m ~

.c:

c o

(I)

Serotonin (S3

ID o

o8

?

~-9 m

=g

Low affinity

C

8~

~

May be directly coupled to PLAz

=g

ADP binding induces a Ca2+-dependentcleavage that activates GPllb-llla

|L

M, 1WWO

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 35 appears to correspond with the previously described moderate affinity thrombin binding site (Vu et al., 1991a, b; Jones and Jamieson, 1991; Coughlin et al., 1992; Hung et al., 1992; Brass et al., 1992). This structure reveals a 425 amino acid protein that belongs to the family of receptor proteins that have seven transmembranous domains (Dohlman, 1991). The extracellular N-terminal domain possesses a thrombin cleavage site adjacent to a stretch of negatively charged amino acids that interact with the anion-binding exosite of thrombin (Fig. 3.3). Thrombin binding to and cleavage of this receptor results in cellular activation through a mechanism that begins with the exposure of a new "tethered peptide ligand" that binds to some (as yet unspecified) receptor domain probably nearer to the carboxy terminus of the receptor. The soluble ligand peptide is itself a potent stimulus for platelet activation. The molecular events that follow the ligand peptide-receptor interaction and lead to the activation of PLC are being elucidated. Of note is that the thrombin receptor has

Anion-binding exosite binding domain

~

~1r~75 "

homology with adrenergic receptors, and both thrombin and adrenergic receptors are coupled to effector (or signal-generating) enzymes by heterotrimeric Ga (see below). Thrombin activates PLC through a pertussis toxin-sensitive Ga (Gp) and inhibits adenylyl cyclase through a (probably different) pertussis toxin-sensitive Ga (Gi). It appears that c~ thrombin-induced PLC activation is kinetically, rather that concentration, driven. Each cleaved thrombin receptor generates a quantum of second messenger that mediates the functional response (Ishii et al., 1993). Recent advances in understanding the structural requirements for adrenergic receptor interactions with heterotrimeric Ga perhaps can soon be applied to studies of the thrombin receptor (Brass et al., 1991; Kaziro et al., 1991; Liggett et al., 1991; Cotecchia et al., 1992). For example, co-expression of /3 adrenergic kinase 2 (BARK2) with the cloned thrombin receptor results in the phosphorylation of the cytoplasmic tail of the receptor and downregulation of receptor function (Ishii et al., 1994). In addition to

Activating Hexapeptide

NH3 + 61

52 ,,

47

~hrombin Cleavage Site

~,

240

( ~265

( ~336

IV

IV

IVI

35

( ~347

IVII

218

368

387

388

215 210

,

212

,=7o

(Y~ 383

(R ~ 392

298 299

-OOC

300

375

376

- 378

~395 1396 i399 400

425

420

413 412 411 410 406

Figure 3.3 Schematic of the moderate affinity thrombin receptor. This molecule is a member of the family of receptor proteins having seven transmembranous domains. Thrombin binds to this receptor at its cationic domain, comprising amino acids (aa) 52-61, and cleaves at arginine 41/serine 42. This exposes an activating tethered ligand peptide (aa 42-47) that interacts with some part of the receptor probably nearer the C-terminus. Potential glycosylation sites on the extracellular domain are marked. Potential intracellullar phosphorylation sites are designated by the appropriate aa (S, T, Y). The third loop structure is essential for G-protein activation with other receptors (see text). Sites of possible cyteine-linked palmitoyl membrane anchors near the C-terminus are given (C). Reprinted by permission of the publisher (Blackwell) (1993).

36 M . H . KROLL AND A.I. SCHAFER receptor phosphorylation, internalization of the activated receptor may also effect desensitization that is only overcome with receptor resynthesis (Hoxie et al., 1993). Because only a minority of activated thrombin receptors are recycled (Brass et al., 1994) and resynthesis cannot occur in circulating platelets, the irreversible desensitization of the thrombin receptor could have physiological (or pathophysiological) consequences. Based on the interaction of ~ thrombin with the glycocalicin component of glycoprotein Ib (GPIb; Shuman, 1986) and observations that platelets from individuals with Bernard-Soulier syndrome bind less thrombin and demonstrate decreased thrombin-induced aggregation (Jamieson and Okumura, 1978), it has been proposed that GPIbc~ is a functionally significant highaffinity thrombin receptor that couples thrombin binding to platelet activation (Harmon and Jamieson, 1986; Yamamoto et al., 1991). Recent studies of vWF/GPIbinitiated platelet activation demonstrate that the GPIb/V/IX complex may be a signal-transducing receptor (Kroll et al., 1991, 1993; Chow et al., 1992). Further studies are required to clarify the role of the GPIb/V/IX as a functional high-affinity thrombin receptor (Seiler et al., 1991; Coughlin et al., 1992).

3.2 TXA2/PGG2/PGH2 Arachidonic acid (AA) metabolites that stimulate platelets are the prostaglandin endoperoxides (PGG2/PGH2) and TXA2 (Mais et al., 1985; Dorn, 1989; Hanasaki and Arita, 1991). These are considered "strong agonists" because they directly activate PLC. A moderate affinity (Ka ~ 2 nM) endoperoxide/TXA2 receptor appears to be coupled to PLC by a pertussis toxin-insensitive heterotrimeric GTP-binding protein (Gq) that is distinct from that which couples thrombin to PLC (Shenker et al., 1991). There appears to be a second high affinity endoperoxide/TXA2 receptor [Ka 234 pM (Dorn, 1989)] that directly mediates changes in platelet cytosolic ionized calcium ([Ca2+]i) and shape change independent of PLC activity, and may "prime" the platelet for activation by other agonists (Takahara et al., 1990). Neither platelet endoperoxide/TXA2 receptor is coupled to inhibition of adenylyl cyclase. Recent cloning and expression of a 343 amino acid platelet endoperoxide/TXA2 receptor indicates that it, like thrombin, is a member of the family of receptor proteins that have seven transmembranous domains (Hirata et al., 1991). These studies have suggested that an identical species of endoperoxide/TXA2 receptor is present in platelets and vascular cells (Halushka et al., 1987).

3.3 PAF PAF is a complex lipid molecule (1-O-alkyl-2-O-acetyl-snglycerol-3-phosphorylcholine) derived from various inflammatory cells (neutrophils, macrophages, and

eosinophils) as well as from the vascular endothelium and platelets (Zavoico et al., 1990). This molecule is a particularly potent activator of neutrophils, and is a "strong agonist" for platelet activation in vitro (Lapetina and Siegel, 1983), although its physiologic role in platelet-mediated thrombus formation is poorly defined (Chignard et al., 1979; Sturk et al., 1987). PAF has a specific receptor that has been recently cloned from guinea-pig lung (Honda et al., 1991) and human leukocytes (Kunz et al., 1992) and shown to be another member of the family of receptor proteins that have seven transmembranous domains. As would be predicted by its primary amino acid sequence, the PAF receptor (like the thrombin and TXA2 receptors) is coupled to the activation of PLC by a pertussis toxin-sensitive heterotrimeric G-protein (Hwang et al., 1983); like the TXA2 receptor, the platelet PAF receptor is not coupled to the inhibition of adenylyl cyclase (Brass et al., 1991).

3.4 COLLAGEN Platelets are activated by collagen types I, III, IV, V, and VI (types I, III and VI are found in the vessel wall; Fitzsimmons and Barnes, 1985; Morton et al., 1989; Staatz et al., 1990; Rand et al., 1991). Collagen must be in a native triple helical conformation for binding to occur, and platelets are capable of binding both soluble and insoluble collagen. At higher concentrations ( > ~ 10 #g/ml), collagen functions as a "strong agonist" to activate platelet PLC directly (i.e. independent of functional CO activity; Karniguian et al., 1990). At lower concentrations, collagen-induced aggregation of platelets involves a long lag phase during which the release of AA and ADP contributes to the rate and magnitude of aggregation (i.e. collagen functions as a "weak agonist"). At least three platelet receptors for collagen have been identified: GPIa/IIa (Santoro, 1986; Takada and Hemler, 1989; Coller et al., 1989), GPIV (CD36; Tandon et al., 1989a, b) and GPVI (Moroi et al., 1989). Each of these receptors mediates platelet adhesion and secretion and aggregation, suggesting that collagen induces direct adhesion-activation coupling. However, the relative physiologic importance of the different collagen receptors is presently unknown. The platelet Mr 67 000 C lq complement receptor may also function as a collagen receptor (Chiang and Kang, 1982). GPIa/IIa is a heterodimeric member of the integrin family (c~2/~1). Molecular cloning reveals that the larger Ot 2 subunit (GPIa) contains a single transmembrane domain (Takada and Hemler, 1989). Patients congenitally deficient in this platelet protein have been noted to have a mild bleeding diathesis (Niewenhuis et al., 1985). Collagen binding to GPIa/IIa is dependent on Mg 2+ and inhibited by Ca 2+ . GPIa/IIa may become activated in response to ADP and mediate subsequent platelet adhesion to collagen (Kainoh et al., 1992).

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 37 GPIV is a highly glycosylated protein having Mr 85 000-95 000. It has at least one, but no more than two, transmembrane domains. GPIV also functions as the platelet thrombospondin receptor (Asch et al., 1987; Kieffer and Phillips, 1990). In vitro, a monoclonal antibody (mAb) to GPIV activates platelets (Aiken et al., 1990). This may involve tyrosine protein kinases (TPKs) related to the c-src protooncogene, since many src-related proteins are associated with GPIV in the resting platelet membrane (M-M. Huang et al., 1991). The importance of GPIV in vivo is, however, uncertain: up to 11% of healthy Japanese blood donors lack this platelet protein without any detectable clinical effect (Yamamoto et al., 1990). Similarly, there is no evidence that thrombospondin binding to GPIV is a signal transducing event, although in other cell types this has been associated with TPK signalling activity (Asch and Nachman, 1989). The role of GPVI in physiological platelet function is unknown. Its possible importance is based on two clinical observations: one patient with a mild bleeding problem associated with a deficiency of a 62 000 Da membrane protein (GPVI) and absent platelet adhesion and aggregation in response to collagen (Moroi et al., 1989); and another patient with immune thrombocytopenia associated with defective collagen-induced platelet aggregation and serum antibodies to GPVI (Sugyama et al., 1987). The activation pathways initiated by platelet binding to collagen have not been rigorously defined, but include PLC-mediated production of phosphatidic acid (PA) and elevations of [Ca2+]i (Watson et al., 1985; Smith and Dangelmaier, 1990; Karniguian et al., 1990), and may involve expression of a functional GPIIb-IIIa complex (Coller et al., 1989). The role of Ga in collagen-induced platelet activation has, to our knowledge, not been investigated. Recently, a unique inhibitor of collagen-induced platelet activation has been isolated (from the salivary glands of a leech) that may help elucidate the physiological importance of collagen-induced platelet responses in humans (Connolly et al., 1992; Keller et al., 1992).

3.5 VP VP is an extracellular mediator of platelet activation that functions in vitro like other "strong agonists", although its importance in vivo is uncertain. The platelet has a small number (approx!mately 90 binding sites) of specific V1 receptors with Mr 124 000 (Siess et al., 1986; Vittet et al., 1986; Thibonnier e t a / . , 1987). The occupancy of V~ receptors is associated with pertussis toxin-sensitive Ga-mediated activation of PLC but, like the TXA2 and PAF receptors, the platelet V1 receptor is not coupled to the inhibition of adenylyl cyclase (Brass et al., 1991). Molecular cloning of the rat liver V~a receptor demonstrates that this protein is a member of the family of receptor molecules containing seven transmembranous domains (Morel et al., 1992).

3.6

EPINEPHRINE

Human platelets possess only a2 adrenergic receptors. The structure of the platelet a2 adrenergic receptor has been determined (Alexander et al., 1978; Regan et al., 1986; Kobilka et al., 1987). The membrane topology of the platelet c~2 adrenergic receptor provides the structural paradigm (seven transmembranous domains) for the functions of a number of platelet receptors for important extracellular stimulatory ligands, including thrombin, eicosanoids, and PAF (Lefkowitz and Caron, 1988). Each of these ligands stimulates intracellular activation pathways through a transmembranous signal involving a heterotrimeric Ga that interacts with a specific domain (comprised, in part, of the third intracellular loop) of the receptor protein. Epinephrine binding to platelets is associated with an a~rin-sensitive activation of PLC and, therefore, the c~2receptor is not directly coupled to PLC. There is evidence that the O~2 receptor is coupled by a Ga to phospholipase A2 (PLA2) through a mechanism that depends on Na § + exchange and fibrinogen binding to GPIIb-IIIa (as will be discussed further below). Activation of PLA2 causes the release and metabolism of AA, which then leads to the activation of PLC. The a2 receptor is also coupled by a different heterotrimeric Ga to the inhibition of adenylyl cyclase (like thrombin), and this may account in part for the mechanism by which epinephrine "primes" the platelet for activation by other agonists in vitro (Steen et al., 1988) and in vivo (Hjemdahl et al. , 1994).

3.7 ADP Platelets are unique in that the adenine nucleotide ADP, rather than ATP, is preferred by the platelet purinergic receptor, and ADP functions as an important physiologic "weak agonist" while ATP antagonizes ADP-induced platelet responses. Quantitative analyses of ADP binding to platelets have been, until recently, complicated by the rapid hydrolysis of bound ADP by platelet surface ADPases. Using an affinity reagent, one platelet ADP receptor has been partially characterized as an Mr 100 000 protein called "aggregin" (Bennett et al., 1978; Colman, 1990). More recently, the binding sites for ADP have been studied using formalin-fixed platelets and radiolabelled adenine nucleotides (Greco et al., 1991). These studies indicate that there are two ADP receptors (Kal ~- 30 nM, 25 600 sites/platelets; Kd2 ---- 3/zM, 383 000 sites/platelet), with the high affinity receptor identical to the 125 000 Da e~ subunit of GPIIb. The high affinity platelet ADP receptor has even greater affinity for nucleoside trisphosphates which, as a consequence, are potent competitive inhibitors of ADPinduced platelet activation (Greco et al., 1992). ATP binding is also associated with the activation of adenylyl cyclase (Soslau and Parker, 1989). The mechanism by which the platelet ADP receptor

38 M . H . KROLL AND A.I. SCHAFER transduces activation signals is unknown. There is evidence that ADP binding to aggregin induces a Ca 2§ dependent cleavage of this receptor that results in a conformational change affecting the platelet GPIIb-IIIa complex to permit fibrinogen binding (Colman, 1990). This may be associated with PLA2 activation (as with epinephrine), since ADP-induced platelet PLC activation is blocked by inhibiting CO. The ADP/aggregin interaction is essential for epinephrine-induced platelet activation and may contribute to the amplification of platelet responses to many physiological stimuli. As occurs with thrombin and epinephrine, ADP binding to the platelet surface is coupled by a heterotrimeric Ga to the inhibition of adenylyl cyclase; this occurs through a purinergic receptor that is not aggregin but may be the subunit of GPIIb. Its precise characterization awaits further investigations.

3.8

5-HT

5-HT is a "weak" platelet agonist that has received recent attention concerning its potential role in acute coronary artery thrombosis (Willerson, 1991) and as a biological marker for many psychiatric disorders (Wirz-Justice, 1988). Platelets have specific $2 receptors (Geaney et al., 1984; McBride et al., 1987). The human $2 receptor has been cloned and is another member of the seven transmembrane domain receptor family (Branchek et al., 1990; Saltzman et al., 1991). Occupation of this receptor is associated with PLC activation (Pritchett et al., 1988) that is generally dependent on intact AA metabolism (de Chaffoy de Courcelles et al., 1985). The role of Ga in 5HT-induced platelet activation is poorly understood. Quantitative binding studies in intact platelets are difficult because platelets have an active transporter of extracellular 5-HT (with a Km similar to that of neuronal tissue).

3.9

OTHER THAT

PLATELET MEDIATE

RECEPTORS

ACTIVATION

3 . 9 . 1 v W F - G P I b Binding Plasma vWF binding to platelet GPIb as a result of ristocetin (Kroll et al., 1991) or pathological shear stress (Chow et al., 1992) stimulates platelet signalling pathways. This latter observation raises some interesting issues about plalelet activation occurring in vivo. Blood in the circulation generally behaves as a Newtonian fluid capable of generating wall shear stresses (Leonard, 1987). Shear stress can be described as "the force per unit area between laminae", and blood flow can be described as an "infinite number of infinitesimal laminae sliding across one another, each lamina suffering some frictional interaction with its neighbors" (Bird et al., 1960). In humans, physiological levels of shear stress in the arterial circuit reach 25-30 dyne/cm 2 (shear rate of whole blood equal

to 625-750s), and pathological levels (i.e. as in a stenosed coronary artery) may reach >350 dyne/cm 2 (shear rate of 8750 s). Each of the triad of platelet functional responses has been observed to occur as a function of shear stress. Shear stress affects the rate and magnitude of platelet adhesion to artificial and biological surfaces (Folie et al., 1988; Folie and McIntire, 1989; Owens et al., 1990a, b) and induces platelet secretion and aggregation (Hellums et al., 1987). At shear stresses greater than 10-12 dynes/cm 2, these functional responses depend on plasma vWF and platelet GPIb/V/IX and GPIIb-IIIa, but not upon plasma fibrinogen (Peterson et al., 1987; Moake et al., 1988; O'Brien, 1990; Ikeda et al., 1991). ADP, whether released from platelets or derived from red blood cells, contributes substantially to shear stressinduced platelet responses (Alkhamis et al., 1990). Elevations of platelet cAMP or cGMP inhibit shear stressinduced platelet aggregation (Hardwick et al., 1981; Durante et al., 1993), but inhibition of CO metabolism with aspirin has no effect on the initiation of aggregation in response to shear stress (Moake et al., 1988). The vWF-platelet interaction is particularly important for shear stress-induced aggregation, vWF is a multivalent, multimeric plasma protein that is essential for platelet adhesion to the subendothelium of damaged blood vessels (Miller, 1990). vWF has binding sites for platelet GPIba and GPIIb-IIIa, and for various subendothelial constituents, including collagen, vWF bridging the platelet GPIb/V/IX complex to the subendothelium leads to adhesion in vivo and vWF bridging GPIb/V/IX on adjacent platelets leads to the cohesive interplatelet interactions induced by ristocetin in vitro. Under shear stresses /n vitro and /n v/v0, vWF binding to the GPIb/V/IX complex is critically important for platelet adhesion and aggregation (Strony et al., 1990). In the plasma milieu under static conditions, vWF binding to GPIIb-IIIa is minimal (Schullek et al., 1984), but when shear stress is applied to platelets, vWF binds to GPIIb-IIIa as well as to the GPIb/V/IX complex, and this binding contributes substantially to platelet aggregate formation (Weiss et al., 1990). The mechanism by which shear stress induces platelet aggregation is not known, but platelet activation signals appear to play an important role in this response. [Ca 2§ rises in platelets subjected to pathological shear stress (> 30 dynes/cm2), and this depends on vWF binding to platelet GPIba (and to GPIIb-IIIa to a lesser extent) and extracellular Ca 2+ (Chow et al., 1992). Of particular note is that these changes of [Ca2§ contribute greatly to the initiation and maintenance of shearinduced aggregation. Platelet PKC activation also contributes to shear stress-induced platelet aggregation (Kroll et al., 1993), and platelet tyrosine kinases are activated in response to pathological shear stress, although the functional importance of shear associated protein tyrosine phosphorylation is presently uncertain (Razdan et al.,

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 39 1994). As with elevations of [Ca2+]i, PKC and tyrosine kinase activation in response to shear also depends on vWF binding to platelet GPIbc~ and GPIIb-IIIa. The molecular mechanisms of shear stress-induced transmembranous stimulus-response coupling are presently unknown, but appear to be initiated by platelet GPIb. GPIb is a transmembranous heterodimeric leucine-rich glycoprotein having Mr 165 000 (composed of GPIba Mr 143 000 disulfide-linked to GPIbfl Mr 22 000) that forms a non-covalent complex with GPIX and GPV; there are approximately 25 000 GPIb/IX/V complexes per platelet (Roth, 1991; Modderman et al., 1992). The intracytoplasmic domain of GPIb interacts extensively with the platelet cytoskeleton (Roth, 1991) and this may contribute to the redistribution of GPIb to the surface-connected membrane system following platelet stimulation with thrombin (Hourdille et al., 1990). The molecular mechanisms of shear stressinduced vWF[GPIb binding are not yet known. Previous studies suggest that shear stress does not affect the structure of plasma vWF (Moake et al., 1988). Therefore, shear stress may be the physiological (or pathophysiological) equivalent of ristocetin: shear stress may alter some characteristic of the platelet surface GPIb/V/IX complex and permit ligand binding to occur (Roth, 1991). The GPIbcx chain can be lengthened by a genetic polymorphism that adds 13 amino acid tandem repeats to the mucin-like macroglycopeptide region, and this had led to speculation that this alters the susceptibility of the platelet to shear-induced activation (Lopez et al., 1992). Once bound to vVVF, platelet GPIb appears to function as a signal molecule, although the mechanism of GPIbinitiated signalling is not characterized. As discussed earlier, studies of thrombin-platelet interactions have demonstrated that this platelet agonist binds to platelet GPIbcx, but the consequences of this for platelet signal transduction are uncertain. In addition to studies of ristocetin- or shear-induced vXVF binding to GPIbc~, there is indirect evidence in support of the hypothesis that the GPIb/V]IX complex is a signal transducing protein: cAMP phosphorylates the B chain of GPIb (Fox et al., 1987), decreases thrombin .' binding to platelets (Lerea et al., 1987), and inhibits platelet activation (see below). A tenable model therefore emerges: GPIbcz, following shear-induced binding of vWF, undergoes a conformational change that directly, or indirectly through a coupling protein, triggers signals for platelet activation. 3.9.2 G P I I b - H I a Platelet GPIIb-IIIa (c~iib/B3) is a member of the integrin family of proteins (Ruoslahti and Pierschbacher, 1987; Ruoslahti, 1991). It is the single most abundant species of GP on the platelet surface (50 000 copies) and is recognized as the fibrinogen receptor that mediates cohesive platelet interactions (Kieffer and Phillips, 1990). GPIIb (Mr 136 000) is a heterodimer of an extracellular c~ chain

(Mr 125 000) linked by a single disulfde bridge to a B subunit (Mr 23 000) containing one transmembranous domain. GPIIb forms a Ca2+-dependent complex with GPIIIa (Mr 110 000 unreduced) which has 56 cysteine residues, one transmembranous domain and a large disulfide-bound loop which forms a globular structure with an arginine-glycine-asparogine (RGD) recognition site (characteristic of the integrins) near its carboxy terminus (Phillips et al., 1991). Platelet GPIIb-IIIa must undergo an activation-induced conformation change to permit fibrinogen binding mediated, at least, by a "network of signalling reactions involving G proteins, serine/threonine kinases and tyrosine kinases" (Shattil et al., 1992; see below). Isolated platelet membranes may provide a useful system for investigating the molecular regulation of GPIIb-IIIa function (Smyth and Parise, 1993). The role of GPIIb-IIIa in supporting submembranous platelet responses ("bidirectional control") is poorly understood. GPIIb-IIIa undergoes a conformational change following ligand binding (Du et al., 1991) and there is evidence that GPIIb-IIIa of resting platelets binds immobilized fibrinogen leading to platelet spreading (Savage and Ruggeri, 1991) associated with cellular activation (Savage et al., 1992). Consistent with this, Haimovich et al. (1993) have shown that GPIIb-IIIa-dependent platelet binding to a fibrinogen matrix is associated with tyrosine kinase activity, cytoskeletal reorganization, secretion and additional fibrinogen receptor expression. In addition, Chow et al. (1992) showed that vWF binding to GPIIb-IIIa contributes to the level of [Ca2+]i that develops in platelets activated by fluid shear stress. GPIIb-IIIa may also be involved in regulating the direct activation of PLA2 by epinephrine or ADP, and in the regulation of tyrosine kinase activity following platelet stimulation by soluble agonists. Both will be discussed further below. Another platelet integrin is the C~vBScomplex that binds to vitronectin (fibronectin, vVVF and thrombospondin); there is no evidence that this receptor transduces signals for platelet activation (Thiagarajan and Kelly, 1988; Lam et al., 1989). Platelets also have the fibronectin receptor [GPIc-IIa (c~s/B1)] and the laminin receptor [cx6(VAL/i)/B]; no signalling functions have been ascribed to these receptor molecules. 3.9.3 CD9 The 24 000 Da CD9 protein is a relatively high density platelet surface molecule (approximately 25 000 sites]platelet) that may transduce PLC-mediated activation signals following its binding specific mAbs (Hato eta/., 1988; Carroll eta/., 1990). Molecular cloning has revealed a unique structure characterized by five transmembranous domains (Boucheix eta/., 1991; Lanza et a/., 1991). The physiological ligand of CD9 is presently unknown.

40 M . H . KROLL AND A.I. SCHAFER 3 . 9 . 4 O t h e r A c t i v a t i o n - i n i t i a t i n g Receptors The platelet contains Fc receptors designated as the Mr 40 000 Fc~/RII. These Fc3,RII molecules transduce activation signals following antibody binding and may contribute to increased platelet clearance in immune thrombocytopenias (Worthington et al., 1990; Horsewood et al., 1991). Platelet Fc and Clq receptors may also be involved in immune complex formation and complement activation on the platelet surface. The complement membrane attack complex (C5b-9) can directly activate platelets (Wiedmer and Sims, 1991) and lead to membrane shedding with the formation of platelet microparticles (Sims and Wiedmer, 1991). These microparticles are detectable by flow cytometry and can express prothrombinase activity (Sims et al., 1988), although their clinical significance is uncertain (ZuckerFranklin, 1992). The platelet also binds specifically to IgE, possibly through GPIIb-IIIa (Ameisen et al., 1986), but the significance of this in vivo is unknown. Platelets have receptors for plasminogen (Miles and Plow, 1985) and tissue plasminogen activator (tPA; Vaughan et al., 1989), which permit assembly of the fibrinolytic apparatus on the platelet surface in areas of thrombosis (Pasche and Loscalzo, 1991). This local generation of plasmin may, under some conditions, directly activate platelets (Schafer et al., 1986). Plasmin also cleaves platelet GPIb and GPIIb-IIIa, and may thereby inhibit platelet aggregation in response to shear stress (Kamat et al., 1995). Platelets have a heavily glycosylated surface protein of Mr 67 000 (designated PTA1) which, when bound to mAb, initiates aggregation, possibly through an autophosphorylation event (Scott et al., 1989). Platelets express membrane adhesion receptors of the immunoglobulin gene superfamily (reviewed by Springer, 1990). These proteins are also on other cell types and mediate heterotypic cellular interactions that are critically important in immune responses. P-selectin (PADGEM, GMP 140 or CD62) is a Mr 140 000 member of the "selectin" family that is expressed on activated platelets and mediates their binding to leukocytes (McEver et al., 1989). PECAM-1 (CD31) is an Mr 130 000 protein that is constitutively expressed on platelets and may be involved in platelet-endothelial interactions (Newman et al., 1990).

4. Activation-induced Changes in Plate let Receptors Using immunodetection techniques coupled to flow cytometry, many activation-dependent platelet membrane epitopes can be measured (reviewed by Abrams and Shattil, 1991). Such epitopes indude activation-induced conformational changes of a surface protein (GPIIbIIIa); ligand-induced changes of receptor conformation

(GPIIb-IIIa); receptor-induced changes in ligand conformation (fibrinogen); or membrane expression of secreted constituents [lysosomal membrane proteins, or c~granule membrane (P-selectin) or dense granule membrane proteins]. Some of these, like P-selectin (previously termed PADGEM or GMP 140) which mediates platelet-leukocyte interactions, may have important functions. Leukocyte-platelet interactions in whole blood have been measured in vivo by dual staining flow cytometry employing antibodies to P-selectin and a neutrophil antigen (Rinder et al., 1991a, b). Others can be used as markers of platelet activation in vivo. The detection of activated GPIIb-IIIa or P-selectin by flow cytometry may be particularly important measures of platelet activation in clinical conditions (Abrams and Shattil, 1991). Activated platelets are very important in regulating the generation of insoluble fibrin (Walsh, 1987). Activated platelets express specific surface binding sites for soluble clotting factors and thereby promote critically important coagulation reactions: the catalysis of factor X by the factor IXa/factor VIIla complex and the catalysis of prothrombin by the factor Xa/factor Va complex (Ahmad et al., 1989; Furie and Furie, 1988). The presence of platelet procoagulant activities can'be detected by flow cytometry. Activated platelets also regulate natural anticoagulant mechanisms by promoting the inactivation of factor Va by activated protein C (Solymoss et al., 1988) and by releasing two proteins, a 112 000 Da protein that inhibits factor XIa (Smith et al., 1990) and a low molecular weight protein (8500 Da) that also inhibits factor XIa (Cronlund and Walsh, 1992). In addition to reactions that take place on intact activated platelets or result from platelet secretion, activated platelets shed "microparticles" that may either stimulate (by binding factor V and factor VIII and promoting prothrombin activation) or inhibit (by promoting factor Va inactivation) fibrin generation (Abrams et al., 1990; Gilbert et al., 1991; Tans et al., 1991; Thiagarajan and Tait, 1991).

5. G-proteins There are three functionally distinct Ga families in platelets (Kroll and Schafer, 1989; Nagata and Nozawa, 1990; Brass et al., 1991): (1) Gp couples activating ligand-receptor interactions (involving thrombin, eicosanoids, PAF and VP) to the stimulation of phosphoinositide-specific PLC; (2) Gs couples inhibitory prostaglandin-receptor interactions (involving PGI2 and PGD2) to the stimulation of adenylyl cyclase; and (3) Gi couples some agonist-receptor interactions (involving thrombin, epinephrine and ADP) to the inhibition of adenylyl cyclase. These Ga are heterotrimeric complexes (comprised of c~, 3, and -y subunits), which have GDP tightly bound to the a subunit in the basal state (Fig. 3.4; Casey and Gilman, 1988; Neer and Clapham, 1988).

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 41

Pi

GDP

(9 ~1, -GDP

| ,,,,

|l

|

GTP P

playlet inhibition

GTP, Mg 2+

Figure 3.4 General theoretical model by which G-proteins transduce an extracellular stimulus into an intracellular response: (1) in the basal state, GDP is bound to the heterotrimeric G protein (in this case Gs) through a binding site on the (x subunit; (2) an extracellular ligand binding to the G-protein-linked receptor (e.g. the PGI= receptor linked to Gs(x) causes the hydrolysis of GDP from the (x subunit; and (3) in the presence of Mg 2§ GTP occupies the open guanine nucleotide binding site, resulting in the dissociation of the c~ subunit from the G-protein complex. Gsc~ GTP activates adenylyl cyclase, which converts ATP to cAMP. The intrinsic GTPase activity of the G-protein converts GTP to GDP, resulting in the reassociation of the heterotrimer and the cyclic restoration of the basal state. Reprinted by permission of the publisher (Grune and Stratton) from Kroll and Schafer (1989).

Following receptor-ligand coupling, GDP dissociates from the complex. In the presence of normal levels of cytoplasmic GTP and Mg 2+, GTP binds to the open guanine nucleotide binding site on the c~ subunit. This binding of GTP to the c~ subunit results in the dissociation of the ~ subunit from the c~'r complex. The dissociated Gc~.GTP then interacts with the enzyme "signal amplifier" within the inner leaflet of the plasma membrane to convert phosphorylated precursors into second messenger molecules. Heterotrimeric Ga are distinguished by the structure of their c~ subunits. In human platelets, there are at least two Gp proteins, distinguished by pertussis toxin sensitivity and immunoreactivity: Gp is pertussis toxin sensitive, may be structurally identical to Gi and mediates thrombin-induced PLC activation; Gq is pertussis toxin insensitive and mediates TXAz-induced PLC activation (Shenker et al., 1991). There is one Gs in human platelets: Gsc~ is cholera toxin sensitive and mediates PGinduced activation of adenylyl cyclase. There are three

structurally distinct Gi proteins in human platelets: Gic~l, GicO, and Gic~3. Each is pertussis toxin sensitive and mediates agonist-induced inhibition of adenylyl cyclase. The relationship between the subtypes of Gic~ and specific receptors is unknown and, as stated above, a protein with homology to Gic~ may be the Gp in human platelets. Gic~ is involved with the direct activation of PLA2 in other cell types (Bourne et al., 1990), but in platelets the Ga that couples a "weak agonist" (e.g. epinephrine binding to the c~2 adrenergic receptor) to PLA2 is unknown. Gzc~ is another pertussis toxininsensitive Ga that is abundantly present in human platelets (Brass et al., 1991). This protein is homologous to that found in human brain and retina, but its function is presently unknown (Gagnon et al., 1991; Lounsbury et al., 1993). The heterotrimeric structure of Ga provides them with additional versatility through the hydrophobic B~/component that remains a single functional unit after dissociation from the c~ subunit. The /~3' subunit reassociates with the c~ subunit as the intrinsic GTPase

42 M . H . KROLL AND A.I. SCHAFER activity of the c~ subunit hydrolyzes GTP to GDP, thus terminating the Ga-mediated signal (Fig. 3.4). In addition, the/33" subunit may also be the functionally relevant molecular unit regulating the inhibition of adenylyl cyclase (Neer and Clapham, 1988), the activation of adenylyl cyclase (Federman et al., 1992) and the direct activation of PLA2 and PLC (Jelsema and Axelrod, 1987; Kim et al., 1989; Birnbaumer, 1992). None of these effects has yet been demonstrated in human platelets.

5.1 Gs, Gi Ga proteins in platelets that regulate adenylyl cyclase represent the prototypical system that operates to control cAMP generation in response to different extracellular stimuli in a variety of cells and tissues. Plalelet antagonists which operate through the metabolism of Gs include PGIz and PGDz (Jakobs et al., 1985; Houslay et al., 1986). The activation of adenylyl cyclase is counterbalanced by platelet agonist-induced inhibition of the enzyme. In intact platelets, PG-induced cAMP generation is inhibited by pretreatment with thrombin, epinephrine or ADP (Cooper and Rodbell, 1979; Brass et al., 1987, 1988). This agonist-induced inhibition of adenyl cyclase is mediated by Gi. Thus, platelet antagonists raise cAMP by Gs-directed activation of adenylyl cyclase, while some platelet agonists lower cAMP by Gi-mediated inhibition of adenylyl cyclase. The function of Gs and Gi can be altered by modifying their c~ subunits with microbial toxins that function as ADP ribosyltransferases to transfer a minor ADP ribose modification group (from an NAD donor to c~s or c~i; Ueda and Hayaishi, 1985). Cholera toxin ADP ribosylates c~s, causing its activation by preventing the hydrolysis of bound GTP to GDP. Pertussis toxin ADP ribosylates ~i, resulting in an impairment of its ability to interact with the receptor to which it is coupled, and thereby blocking agonist-mediated inhibition of adenylyl cyclase.

5.2 Gp The activation of phosphoinositide-specific PLC in platelets involves at least two functionally separate Ga, Gp or Gq. Gp mediates platelet activation in response to thrombin and possibly also PAF, VP and collagen (Brass et al., 1986, 1991; Lapetina et al., 1986), and Gq mediates platelet activation in response to TXA2 (Shenker et al., 1991). The important function of these Ga in platelet PLC activation is supported by data demonstrating that aluminum fluoride and non-hydrolyzable analogues of GTP (GTP-3"-S, GppNHp), which bypass receptors and cause direct activation of Ga, stimulate the hydrolysis of PIPe in permeabilized platelets (Brass et al., 1988), platelet membrane preparations (Hrbolich et al., 1987) or intact platelets (Brass et al., 1991). Further-

more, a non-hydrolyzable analogue of GDP (GDP-/3-S), which maintains Gp in its non-dissociated and therefore inactive state, inhibits PIPz hydrolysis in response to agonist stimulation. There is also evidence from platelet reconstitution studies that supports the existence of a functional Gp: PLC activity from platelet membrane preparations can be induced by the addition of exogenous Ga (Banno et al., 1987). The functional significance of the structural heterogeneity of Ga mediating PLC activation is understood to some degree. There is good evidence that a unique Ga can affect PLC in a ligand-dependent manner (i.e. Gq and TXA2), indicating the possibility that the heterotrimeric Ga is the switch for specific signal routing. There is also evidence that supports the hypothesis that platelet Gp is the same as Gi, suggesting that ligand binding results in a signal that bifurcates at a Ga "node" towards both PLC activation and adenylyl cyclase inhibition. Finally, the intact f13"subunit appears to have a direct role in initiating and abrogating activation signals. This versatility in signalling function of the heterotrimeric Ga that regulate PLC will be more clearly elucidated as their structural interactions with receptors and signalgenerating proteins are defined (Conklin and Bourne, 1993).

5.3 LOW

MOLECULAR

WEIGHT

G-PROTEINS In addition to the heterotrimeric Ga found in human platelets, there are several distinct Ga with MW between 21 000 and 31 000 Da (Nagata and Nozawa, 1990; Taylor, 1990). Like heterotrimeric Ga, these low MW Ga are predominantly membrane associated, although some are found in the cytosol (Bhullar and Haslam, 1987; Lapetina and Reep, 1987). The functions of platelet low MW Ga are unknown (Nagata and Nozawa, 1988; Ohmori et al., 1988). Some low MW Ga are homologous with ras p21 or other ms-related proteins, suggesting that they may function as molecular switches (Lapetina et al., 1989; Santos and Nebreda, 1989; Karniguian et al., 1993). One membrane-bound low MW Ga, recently identified as rhoA protein (Nemoto et al., 1992), is ADP ribosylated by a botulinum toxin, and ADP ribosylation is associated with enhanced platelet secretion responses to a variety of agonists, suggesting a specific signalling function (Bhullar and Haslam, 1988; Ohmori et al., 1989). A ras-related platelet membrane-associated low MW Ga is phosphorylated by a cAMP-dependent PK, but the functional consequences of this are uncertain (Lapetina et al., 1989). It has been demonstrated in thrombinstimulated platelets, however, that a complex is formed between a ras GTPase-activating protein (Boguski and McCormick, 1993) two src-related tyrosine kinases (see below), GPIIb-IIIa and the membrane cystoskeleton (Fox et al., 1993). This suggests that ras activity, perhaps by regulating the phosphorylation of tyrosine residues on

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 43 contractile proteins, may be an important signal mediating GPIIb-IIIa oligomerization associated with platelet shape change and secretion (Huang et al., 1993).

6. Intracellular S~qnalling Pathways 6.1 PLC The importance of phosphoinositide metabolism in the regulation of cellular stimulus-response coupling was first suggested by Hokin and Hokin (1953). The turnover of platelet phosphoinositides in association with agonist stimulation was initially observed in 1961 (Firkin and Williams, 1961). These phenomena were subsequently determined to be an important mechanism of platelet activation (Rittenhouse-Simmons, 1979; Mauco et al., 1979; Bell and Majerus, 1980; Agranoff, 1986). It is now established that human platelets contain PLC which hydrolyzes PIP2 to the two stimulatory second messengers, IPs and DAG (Marcus et al., 1969; Kawahara et al., 1980; Billah and Lapetina, 1982; Agranoff et al., 1983; Rittenhouse, 1983). There are two membrane and five cytosolic forms of PLC found in human platelets (Nozawa and Banno, 1991). Each catalyzes the hydrolysis of three common phosphoinositides: phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI-4-P), and PIP2. Platelet PLCs differ in their substrate affinities, rates of catalysis, and requirements for Ca 2+ (Chau and Hsin-Hsiung, 1982; Hakata et al., 1982; Banno et al., 1986; Nozawa and Banno, 1991). Many PLCs have been cloned from other tissues and shown to be in one of four families of isoenzyme: PLC-c~, PLC-~, PLC-% and PLC-6, each of which is produced by a discrete gene (Rhee etal., 1991; Ryu etal., 1987; Rebecchi and Rosen, 1987; Suh et al., 1988; Katan et al., 1988; Bennett et al., 1988). There are many subtypes within each family of PLC isoenzymes defined by electrophoretic and immunologic methods. Platelet cytosol contains PLC-/3, PLC-% and PLC-6, as well as two presently unclassified PLC is9forms (Banno et al., 1992). The molecular basis for intrafamilial PLC subtypes is poorly understood, although in some cases subtypes result from proteolytic digestion of a single gene product (Meldrum et al., 1991). The precise molecular mechanisms that control the activation of platelet PLC and determine its substrate specificity and kinetics are presently undefined. The actin binding protein gelsolin inhibits the activity of all platelet cytosolic PLC isoforms (Banno et al., 1992), and low MW Ga, perhaps complexed to a GTPase-activating protein (GAP), may contribute to the activation of platelet cytosolic PLC-3, (Baldassare et al., 1988; Anderson et al., 1990; Hall, 1992). PIP2 is a quantitatively minor platelet membrane phospholipid that is the primary substrate for stimulusevoked PLC activity (Marcus et al., 1969; Kawahara

et al., 1980; Billah and Lapetina, 1982; Agranoff et al.,

1983; Rittenhouse, 1983). The predominant molecular species of platelet PIP2 hydrolyzed by PLC contains the fatty acyl substituents stearoyl (C18:0) and arachidonoyl (C20:4) at the C-1 and C-22 positions, respectively. The glycerol backbone of this phosphoinositide structure, including its sn-l,2 substituents, undergoes recycling (Broekman et al., 1980, 1981; Neufeld and Majerus, 1983). Mass changes in the substrates and products of PLC have been determined, but their correlation with the magnitude of any particular cellular response is not known (Billah and Lapetina, 1982; Rittenhouse and Sasson, 1985; Preiss et al., 1986). The activation of platelet phosphoinositide-specific PLC results in the generation of IPs and DAG. These molecules effect divergent pathways ofplatelet activation: IPs causes elevations of [Ca2+]i and DAG causes the activation of PKC. Ca 2+ mobilization or PKC activation independently result in platelet activation; together, they synergistically stimulate a diversity of platelet responses, including granule secretion and the release of AA from membrane phospholipids (Yamanishi et al., 1983; Kaibuchi et al., 1983; Halenda et al., 1985).

6.2 IP3 A N D CALCIUM Intracellular IPs regulates agonist-induced platelet cytosolic ionized calcium responses (Agranoff et al., 1983; Berridge and Irvine, 1984; Shears, 1989; Majerus et al., 1991; Daniel, 1990; Berridge, 1993). The resting platelet has a [Ca2+]i of approximately 100 nM. Upon agonist stimulation, [Ca2+]i increases to > 1000 nM. There are two pools of calcium in the resting platelet: (1) a cytosolic pool demonstrating rapid turnover (tl12 17 min) regulated by a plasma membrane Ca2+-ATPase (Brass, 1984a, b; Darnanville et al., 1991); and (2) a slowly exchanging pool (tl12 300 min) which is driven into and sequestered within the platelet's dense tubular system by the activity of a Ca2+/Mg2+-ATPase (Statland et al., 1969; Brass and Shattil, 1982; Brass, 1984a, b). It is from this latter pool that calcium is released into the cytosol following agonist stimulation (White, 1972; Gerrard et al., 1974). Plasma membrane exchange of Ca 2+ is important for loading the Ca 2§ storage organelle and, as will be discussed further below, may play a role in agonist-induced changes of [Ca2+]i (Robblee and Shepro, 1976). Experiments in which inositol phosphates are introduced into permeabilized platelets show that IPs in physiologic concentrations causes calcium to be released from its internal storage organelles (Lapetina et al., 1984; O'Rourke et al., 1985; Authi et al., 1986) and that elevated [Ca 2+]i is associated with platelet shape change, secretion and aggregation (O'Rourke et al., 1987). IP3 binds to specific receptors on the dense tubular system. The IP3 receptor from endoplasmic reticulum has been characterized as a tetramer of 260 000 Da GP subunits

44 M . H . KROLL AND A.I. SCHAFER (Ehrlich and Watras, 1988; Ross et al., 1989; Ferris et al., 1989; Furuichi et al., 1989; Mignery et al., 1989; Tsien, 1990). Quantal Ca 2§ release is an intrinsic property of the receptor (Ferris et al., 1992). Recent studies of IPs binding to human platelet dense tubular membranes reveal that there may be two conformational states of the IPs receptor with a shift from the low affinity state (Ka --- 13.2 nM) to the high affinity state (Ka ~- 0.32 nM) caused by [Ca2+]i (Hwang, 1991). In addition, IPs binding to its platelet receptor is modulated by pH, monovalent cations, other divalent cations and guanosine triphosphate (GTP). The regulatory influence of pH may be particularly important, since small increases of pH, such as those that accompany platelet activation, dramatically increase the binding of IP3 to its platelet receptor (Hwang, 1991). Inositol 1,2-(cyclic)-4,5-trisphosphate may also regulate [Ca2§ This is formed within 10 s of thrombininduced platelet activation (Ishii et al., 1986). Its capacity to provoke *SCa release when injected into permeabilized platelets (as does IP3) suggests that it may be functionally important in intact platelets (Wilson et al., 1985). There remains uncertainly regarding the contribution of Ca 2§ influx from the extracellular space to the rise of [Ca 2§ that develops the following platelet activation. The magnitude and duration of [Ca2§ responses to platelet activation are increased in the presence of extracellular Ca 2§ suggesting that the rise of [Ca2§ involves the influx of Ca 2§ (Kroll and Schafer, 1989). Experiments using "strong agonists" (thrombin, TXA2, PAF, 5-HT) and a "weak agonist" (ADP) show divalent cation movement into platelets regulated by a receptoroperated channel (Hallam and Rink, 1985; Tsien, 1990). This channel has been partially purified from thrombinactivated platelets (Zschauer et al., 1988) and electrophysiological studies reveal the presence of receptoroperated Ca 2§ channels in human platelets (MahautSmith et al., 1990, 1992). The physiologic factor(s) that regulate these channels are unknown, but are of great current interest (Putney and Bird, 1993). Of note is that both inositol 1,4,5-trisphosphate and inositol 1,2-(cyclic)-4,5-trisphosphate are metabolized by a 5'-phosphomonoesterase to inactive compounds (Connolly et al., 1985). A significant quantity of inositol 1,4,5-trisphosphate is also immediately phosphorylated by a 3'-kinase to inositol 1,3,4,5-tetrakisphosphate [I(1,3,4,5)P4; Irvine et al., 1986; Choi et al., 1990]. In other cell types, this latter molecule has been shown to cause Ca 2§ influx across the plasma membrane (Irvine et al., 1986; Irvine and Moor, 1987). I(1,3,4,5)P4 has also been shown to induce Ca 2§ sequestration into storage pools in rat liver cells, a process that reverses the IPs-stimulated elevations of [Ca 2 + ]i (Hill et al., 1988). This suggests that the phosphorylation of IP3 to I(1,3,4,5)P4 may serve to actively induce the resequestration of cytosolic Ca 2+ (Hill et al., 1988). I(1,3,4,5)P4

is also metabolized by the 5'-phosphomonoesterase to inositol 1,3,4-trisphosphate, the levels of which peak 60 s following platelet activation and persist for up to 10 min. Inositol 1,3,4-trisphosphate may function to maintain [Ca z § perhaps by affecting transmembranous Ca z+ movement through a specific channel (Daniel, 1990; Daniel et al., 1987). I(1,3,4,5)-P4 can also be metabolized back to IPs by a 3'-phosphomonoesterase, which may also contribute to the maintenance of the platelet CaZ § response (Oberdisse et al., 1990). ADP has been reported to cause a rapid rise (< 1 s) of [CaZ+]i in CO-inhibited platelets unable to hydrolyze PIPz to IPs (Fisher et al., 1985). The ADP-induced Ca z§ response is biphasic, with this initial rise being independent of both IPs and extracellular Ca z§ suggesting the existence of a third pool of mobilizable Ca 2§ (Jones and Gear, 1988). This might represent plasma membrane-bound Ca z§ that is important in weak agonist-induced activation of PLAz (see below). The major binding site for Ca z§ on unstimulated platelets is the GPIIb-IIIa complex (Brass and Shattil, 1984). This has led to the hypothesis that GPIIb-IIIa is involved in the transmembranous influx of Ca 2§ that occurs during platelet activation (Powling and Hardisty, 1985). Experiments using mAbs to block GPIIb-IIIa of platelets (Powling and Hardisty, 1985), or mAbs (Rybak et al., 1988) or synthetic ligand peptides (Rybak and Renzulli, 1989) to block GPIIb-IIIa in liposomes, demonstrate an associated inhibition of transmembranous Ca z § influx. In human erythroleukemia cells (a cell line with many features of megakaryocytes), GPIIb-IIIa appears not to participate directly in agonist-induced changes of [Ca Z§ (Suldan and Brass, 1991), and it appears likely that GPIIb-IIIa plays an indirect role in agonist-induced changes of [CaZ+]i. Recent electrophysiological data suggest that GPIIb-IIIa regulates a platelet plasma membrane Ca2, channel (Fujimoto et al., 1991). The mechanisms by which Ca z§ regulates cellular responses are understood in some detail (reviewed in Rasmussen, 1986; Salzman and Ware, 1989; Tsien, 1990). [CaZ+]i contributes to platelet activation through several effector pathways, including the activation of Ca 2+ [calmodulin-dependent PKs, Ca 2+ -dependent proteases, PLC and PLAE, and PKC. Platelet [Ca2§ is measured by cytosol-trapped fluorophores or photoproteins which exhibit distinct spectral characteristics when bound to Ca z§ . These reagents not only allow measurement of [CaZ§ but may help detect different pools of [CaZ+]i (Grynkiewicz et al., 1985; Tsien et al., 1985; Johnson et al., 1985). Experiments employing these reagents demonstrate that "strong agonists" cause direct elevation of platelet [Ca Z+]i, that the rise of [CaZ+]i in response to ADP and 5-HT is inhibited by blocking CO metabolism, and that the [Ca z§ ]i response to epinephrine can be measured only with the photoprotein aqueorin (Rink et al., 1982; Ware et al., 1986).

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 45 The platelet contains Ca 2+/calmodulin-dependent PKs that are activated by elevated [Ca2+]i. One of these is myosin light chain kinase. Myosin light chain is a 20 000 Da protein that is the major Ca 2+/calmodulindependent PK substrate of platelets; its phosphorylation following platelet activation is easily observed by gel electrophoresis of radiophosphorus-labelled platelets. The phosphorylation of myosin light chain is directly involved in platelet shape change, contraction, and secretion (LeBreton et al., 1976; Daniel et al., 1984; Fox, 1986). Platelet myosin light chain kinase has been partially purified (Hathaway and Adelstein, 1979; Conti and Adelstein, 1991). Ca2 § induces the binding of calmodulin to the kinase, with consequent enzyme activation resulting in the phosphorylation of myosin light chain. The phosphorylation of myosin light chain permits the myosin hexamer to be activated by actin, and this activation generates the forces required for shape change and secretion (Higashihara et al., 1991; Itoh et al., 1992). Phosphorylase kinase is also a Ca2+/calmodulin-dependent PK that contributes to these events by activating phosphorylase, leading to increased cytosolic ATP used to fuel the activation of the contractile apparatus (Gear and Schneider, 1975). In addition to the Ca 2+/calmodulin-dependent responses of activated platelets, other important Ca 2+dependent biochemical reactions contribute to platelet function. There are two Ca2+-dependent proteases in human platelets (calpain I and II) that may play a role in platelet activation (Yoshida et al., 1983; Fox et al., 1985; Schmaier et al., 1986; Mellgren, 1987). Ca 2+dependent protease cleavage of actin-binding protein and talin contributes to platelet cytoskeletal reorganization that is required for platelet aggregation (Fox et al., 1985; O'Halloran et al., 1985). In addition, calpains contribute to agonist-induced platelet procoagulant activity (Fox et al. , 1990). Both platelet PLC and PLA2, which cleaves the esterlinked C-2 position fatty acyl group from phospholipids, are regulated by Ca 2+ . Platelet PLC activation is Ca 2§ dependent, and the level of [Ca2+]i may affect substrate specificity. Activation of PLA2 is Ca 2§ dependent [but calmodulin independent (Watanabe et al., 1986)], and this results in the release of AA (Billah et al., 1980; Rittenhouse-Simmons, 1981; Rittenhouse-Simmons and Home, 1984). PKC is an important regulator of agonistinduced platelet responses (Nishizuka, 1986). Its activation is dependent on [Ca2+]i (Inoue et al., 1977; Minakuchi et al., 1981), and will be discussed further below. Specific intraplatelet inhibitory signals counterbalance CaZ+-mediated platelet activation. The most important of these is cAMP, a molecule that antagonizes all Ca2§ reactions (see below). In addition, a 5'-phosphomonoesterase, as previously discussed, inactivates the IPs signal for Ca 2§ release from intracellular stores by converting IPs (inositol 1,4,5-

trisphosphate) to IP2 (inositol 1,4-bisphosphate; Connolly et al., 1985). There is evidence that PKC phosphorylates and thereby activates the 5'-phosphomonoesterase (Connolly et al., 1985, 1986; Molina y Vedia and Lapetina, 1986). PKC may also cause feedback inhibition of another important Ca 2+mediated response by phosphorylating myosin light chain and thereby inhibiting the activation of the contractile apparatus (Higashihara et al., 1991).

6.3

PKC

PKC is a closely related family of serine/threonine kinases (of Mr ~- 80 000) that requires DAG [of sn-l,2 configuration, with preferred acyl groups of varying lengths and degrees of unsaturation (Lapetina et al., 1985; Bell and Burns, 1991)], Ca 2§ and phospholipid (particularly phosphatidylserine) for its activation (reviewed in Nishizuka, 1988). PKC has been isolated, purified and cloned from non-hematologic and hematologic tissues, including platelets (Knopf et al., 1986; Coussens et al., 1986; Grabarek et al., 1992; Baldassare et a/., 1992; Chang et al., 1993). These studies demonstrate that there are multiple forms of PKC coded for by distinct genes located on different chromosomes, and that some isozymes result from alternative mRNA splicing (Ono et al., 1987; Hubbard et al., 1991). There are nine members of the family of mammalian PKC (Hubbard et al., 1991), of which six are found in human platelets (Grabarek et al., 1991). In stimulated human platelets, PLC-generated DAG is released into the matrix of the plasma membrane and then rapidly recycled back into the phosphoinositides via its phosphorylation to phosphatidic acid catalyzed by the enzyme diacylglycerol kinase (Bishop and Bell, 1986). Membrane-bound DAG triggers the translocation of inactive PKC from the cytosol to the membrane; PKC translocated to the membrane is then activated in the presence of Ca 2+ and phosphatidylserine. DAG increases the affinity of inactive PKC for Ca 2§ such that only small increases of [Ca2+]i are required to effect PKC activation. In agonist-stimulated platelets, however, both DAG and elevated [Ca2+]i synergistically activate PKC (Nishizuka, 1986). Agonist-stimulated accumulation of platelet DAG may be delayed and multiphasic, possibly as a consequence of the sequential hydrolysis of different membrane phospholipids, and this might contribute to the continued activation of PKC and secondary aggregation and secretion (Werner and Hannun, 1991; Werner et al., 1992). Human platelets contain a large amount of PKC and this is activated as a direct consequence of agonistinduced PIP2 hydrolysis. Platelet PKC is also activated independently of PLC by phorbol esters (Castagna et al., 1982) or synthetic DAG (Lapetina et al., 1985). Complement proteins C5b-9 (Wiedmer et al., 1987) and endotoxic lipopolysaccharides (LPS; Romano and Hawiger, 1990) also directly activate platelet PKC and may

46 M . H . KROLL AND A.I. SCHAFER contribute to the pathogenesis of diseases such as disseminated intravascular coagulation. Studies using direct pharmacologic stimulation of PKC demonstrate that PKC activation is associated with platelet aggregation (without shape change), secretion and the release and metabolism of AA (Nunn and Watson, 1987; Kajikawa et al., 1983). In combination with Ca 2§ ionophores, direct PKC activators cause synergistic platelet responses (Mobley and Tai, 1985). The mechanisms by which PKC causes platelet aggregation and secretion are incompletely understood. PKC leads to Ca2§ modification of the membrane GPIIb-IIIa complex which allows it to bind fibrinogen and support platelet aggregation (Shattil and Brass, 1987; van Willigen and Akkerman, 1991). This fibrinogen/GPIIb-IIIa interaction may, as discussed above, lead to the stimulation of signal pathways resulting in secretion (Banga et al., 1986). Platelet secretion could potentially result from PKCmediated effects on [Ca 2§ but there is ambiguous evidence for phorbol ester-induced elevations of [Ca2+]i (Rink et al., 1983; Ware et al., 1985). PKC may also affect platelet responses by modulating the Ca 2§ induced activation of PLA2. The release of AA is primarily dependent on Ca2+-mediated PLA2 activation, but PKC may co-operate with Ca 2§ in this process (Halenda and Rehm, 1987). This latter effect could involve lipocortins (or annexins), a group of Ca 2+ and phospholipid binding proteins with ant/-PLA2 activity (Crompton et al., 1988). It has been suggested that PKC phosphorylates and thereby inhibits platelet lipocortin, thus enhancing Ca2§ PLA2-mediated AA release (Touqui et al., 1986). Physiologic and pharmacologic activation of PKC is associated with the phosphorylation of a 47 000 Da platelet protein termed "pleckstrin' (platelet/eukocyte ckinase substrate) which has served as a useful marker for platelet PKC activation (Imaoka et al., 1983). The identity of this substrate of PKC is uncertain. Purified platelet pleckstrin has been characterized (Imaoka et al., 1983) and an apparently identical protein has been expressed in Escherichia coli from cDNA derived from an HL-60 cell line (Tyers et al., 1988). Based on these studies, some putative functions previously ascribed to it appear to be excluded, including lipocortin (Touqui et al., 1986), the c~ subunit of pyruvate dehydrogenase (Chiang et al., 1987) and the IP3 phosphomonoesterase (Connolly et al., 1985). Its functional and structural relationship to the c~ subunit of Gi (Katada et al., 1985) remains to be determined; it has been demonstrated, however, that pleckstrin modulates actin polymerization in a cellfree system (Hashimoto et al., 1987). Unlike unphosphorylated pleckstrin, the 4 7 0 0 0 D a phosphoprotein permits the elongation of actin filaments necessary for cytoskeletal reorganization. Recent data bank searches demonstrate that there may be a number of proteins containing pleckstrin homology domains (Ferguson etal., 1994) ; future studies are therefore likely

to elucidate the precise structure and function of platelet pleckstrin. Preincubating platelets with phorbol esters or synthetic DAG results in the partial inhibition of subsequent res[~onses to thrombin. PIP2 turnover, IPs generation, Ca t+ mobilization and secretion are attenuated when PKC is activated prior to thrombin stimulation (Zavoico et al., 1985; MacIntyre et al., 1985; Watson and Lapetina, 1985), and relatively selective inhibitors of PKC eliminate this effect of phorbol esters on thrombininduced PLC activity (Tohmatsu et al., 1986; Watson et al., 1988). These observations indicate that PKC may cause feedback inhibition of PLC-mediated PIP2 hydrolysis. This action of PKC diverges from the previously described activating effects of PKC on GPIIb-IIIa and PLA2 (Crompton et al., 1988). In addition, it appears that PKC activation leads to increased levels of platelet PIP and PIP2, which have been associated with increased PLC activity in other tissues (de Chaffoy de Courcelles et al., 1984; Halenda and Feinstein, 1984; Fleischman et al., 1986). Thus, PKC appears to be an important branchpoint for the flow of positive and negative signals discharged from an initiating stimulus, with the activation of PKC contributing initially to platelet activation, and subsequently leading to a separate activation-dampening response. The activity of PKC is primarily controlled by the metabolism of DAG. PKC activity decreases in parallel with the decline in intracellular DAG following platelet stimulation and this is accompanied by dephosphorylation of pleckstrin. DAG is predominantly converted to PA by the action of DAG kinase, and PA is then recycled into the phosphoinositide pool (Bishop and Bell, 1986). A smaller amount of DAG is converted to monoacylglycerols by the action of specific lipases (Huang and Detwiler, 1986). In addition to DAG-mediated PKC regulation, PKC activity is inhibited by elevated levels of platelet cAMP (Kroll et al., 1988), and exogenously added sphingosine and lysosphingolipids inhibit thrombin-induced PKC activity and may be physiologic regulators of PKC (Hannun et al., 1986; Hannun and Bell, 1987, 1989).

6.4 PLA2 The group of phospholipases collectively termed PLA2 play a pivotal rolein platelet stimulus-response coupling (Chang et al., 1987). There are two families of PLA2, secretory and cytosolic. The family of secretory PLA2 is perhaps the best studied; this is comprised of a group of similar Ca 2+-dependent isozymes of Mr ~ 14 000 (White et al., 1990; Scott et al., 1990; Thunnissen et al., 1990). The presence of this PLA2 family in platelets is uncertain. Cytosolic PLA2 comprises the predominant functional PLA2 of platelets. Platelets contain multiple isoforms of cytosolic PLA2 which vary in their specific activity for cleaving the sn-2-acyl bond of different

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 47 phospholipid substrates. At least one of these has been recently cloned and expressed in heterologous cells (Sharp et al., 1991), and shown to be an Mr -~ 100 000 protein that is activated by levels of [Ca 2§ observed in activated platelets (Loeb and Gross, 1986). The major physiologic effect of platelet PLA2 activity is the release of endogenous AA from membrane phospholipid pools. Almost all of the AA released following platelet activation is derived from PLA2-mediated phospholipid hydrolysis. Phosphatidylcholine (PC) is the preferred substrate, but PLA2 also hydrolyzes AA from phosphatidylethanolamine (PE), phosphatidylserine (PS), and, to a lesser extent, PI (Rittenhouse-Simmons, 1981; Prescott and Majerus, 1981). DAG and PA are very minor substrates for platelet PLA2 (Bell et al., 1979; Billah et al., 1981; Mahadevappa and Holub, 1986). Following deacylation, AA is rapidly metabolized to a diverse group of 20-carbon biologically active products (termed eicosanoids) through the CO and lipoxygenase (LO) pathways (Needleman et al., 1986). In human platelets, CO oxygenates AA to the prostaglandin endoperoxides PGG2 and PGH2, which are then converted to TXA2 by thromboxane synthase. These highly labile AA derivatives are potent platelet agonists that bind to specific platelet receptors and, through Gq, activate PLC. Small amounts of AA are also converted to PGD2, PGF2~ and PGE2, less potent inhibitory eicosanoids that may serve to dampen the platelet response. AA is also oxygenated via platelet 12-LO to quantitatively significant 12-monohydroperoxy and 12monohydroxy fatty acids (12-HPETE and 12-HETE, respectively). In v/tr0, these products inhibit a number of platelet responses to AA and its derivatives, but their physiologic relevance appears to be minor (Aharony et al., 1982). Recent data suggest that platelet LO products control cellular volume by opening a K § channel (Margalit and Livne, 1991). Platelets supply eicosanoid substrates to endothelium (Marcus, 1990) and vascular smooth muscle (Hechtman et al., 1991) which convert them to biologically important PGI2. Conversely, platelets can convert neutrophil-derived leukotrienes into proinflammatory lipoxins (Serhan and Sheppard, 1990). PLA2 has a broad specificity for different phospholipids having diverse acyl chain structures. This is particularly relevant to the biological consequences of dietary supplementation with fish oils enriched in w-3 fatty acids, including eicosapentaenoic acid (EPA). Platelet PLA2 hydrolyzes EPA from the C-2 position of membrane phospholipids, and its subsequent metabolism to inactive triene CO products (including TXA3 rather than TXA2) accounts in part for its antiplatelet effects (Needleman et al., 1986). PLA2 is also important in the synthesis of PAF (1-O-alkyl-acetylsn-glyceryl-3-phosphocholine) because it hydrolyzes 1-alkyl-2-acyl-phosphocholine to lyso-PAF (Lapetina and Siegel, 1983), which is then converted to PAF by an

acetyltransferase (Roth, 1986). Platelet levels of PAF probably contribute very little to the state of platelet activation, but released PAF may be important in the recruitment of circulating leukocytes to sites or vascular injury (Sturk et al., 1987). The regulation of platelet PLA2 is complex. Activation is dependent on elevations of [Ca2+]i and independent of calmodulin (Billah et al., 1980; RittenhouseSimmons, 1981; Rittenhouse and Home, 1984; Watanabe et al., 1986). PLA2 activation may be opposed by the endogenous protein inhibitor(s) termed lipocortin, and this effect of lipocortin may be blocked by PKC-mediated phosphorylation (Touqui et al., 1986). In addition, as discussed in the following section, there is evidence that Na +/H § exchange provides membranelevel regulation of PLA2 independent of [Ca2+]i.

6.5

Na + / H + EXCHANGE

The platelet Na+/H § countertransporter maintains basal cytosolic pH (pHi) and becomes activated following platelet stimulation, pHi can be measured using intracellularly trapped H § sensitive fluorophores such as BCECF. Using BCECF, one observes a resting pHi of approximately 7.2 and a 0.1-0.5 pH unit rise above resting pHi occurring in platelets following agonist stimulation (Zavoico et al., 1986). The platelet membrane Na +/H § countertransporter appears to be an Mr ~ 110000 glycoprotein regulated by pHi, [Ca2+]i~ and PKC (Zavoico et al., 1986; Sardet et al., 1990; Kimura et al., 1990). A number of important platelet responses may be regulated by pHi, including the activation of PLA2, GPIIb-IIIa function, [Ca 2§]i and phosphoinositide metabolism. Na § § exchange may be involved in the mechanism by which "weak agonists" activate platelet PLA2. This concept is based on the observations that AA release does not occur without cytosolic alkalinization (Sweatt et al., 1985), and that inhibition of Na +/H § exchange prevents PLC activation in response to weak agonists, which is overcome only by a synthetic prostaglandin endoperoxide (Sweatt et al., 1986a, b). It is, therefore, plausible that weak agonists initiate Na +/H § exchangedependent activation of PLA2 that results in the release of AA (in minute quantities measurable only by mass spectroscopy). This AA is metabolized to prostaglandin endoperoxides and TXA2 that then feedback to directly activate PLC (Fig. 3.5). This may involve the Na +/H § exchange-mediated release of membrane-bound Ca 2§ (IPs-independent), that directly activates PLA2 (Sweatt et al., 1986a, b). It has also been observed that blocking fibrinogen binding to GPIIb-IIIa inhibits epinephrineinduced cytoplasmic alkalinization, suggesting that a functional GPIIb-IIIa complex regulates Na+/H + exchange and that fibrinogen binding to GPIIb-IIIa may be a prerequisite for Na+/H + exchange-dependent PLA2 activation (Banga et al., 1986).

48 M . H . KROLL AND A.I. SCHAFER

// /

/

/

/

/

~ G G ..... ffPKC ~ DG~p- ~

( P I P 2 ~ - " - 3 ~3~Ca2, Ca~ ~ ~.,,~) Shape ~ \ ~ ( ~ ) change

2'/PGH?~.~~ # ~x' A A ~J.~~/~. Na+ \ ~ ~-I~PLA~3~'~ "Weak ) H , ~ ~ ~~-,_'~L" Agon`st'' / AARelease ~ Secretion I ~ ,DP

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Antagonist

Agonist

Figure 3.5 The different pathways of platelet activation. A "strong agonist" binds to a specific receptor and, through a Gp-mediated process, activates PIP=-specific PLC. This results in second messengers that cause the elevation of [Ca=*]~ and the activation of PKC, which together stimulate the release of membrane bound AA and granule constituents (such as ADP). A "weak agonist" binds to a specific receptor and, through a process that depends on Na +/H + exchange, activates PLA=. This results in the release of AA and its metabolism to prostaglandin endoperoxides (PGG2/PGH=) and TXA= that then feed back to directly activate PLC. The metabolism of AA is inhibited by aspirin, which irreversibly blocks the activity of PGG=/PGH= synthase ("cyclooxygenase"), and this is the reason for the effect of aspirin on weak agonist-induced secretion and secondary aggregation. Certain platelet agonists (e.g. ADP) also inhibit the activation of adenylyl cyclase by activating Gi. PLC, phospholipase C; PIP=, phosphatidylinositol 4,5-bisphosphate; DG, dlacylglycerol; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; PLA= phospholipase A=; AC, adenylyl cyclase; PC, phosphatidylcholine. Reprinted by permission of the publisher (Grune and Stratton) from Kroll and Schafer (1989).

As previously stated, IP3-mediated Ca 2+ release is pH dependent (O'Rourke et al., 1985). Early reports indicated that platelet [Ca2+]i is entirely dependent on Na +/H + exchange (Siffert and Akkerman, 1987). More recent data, however, show variable effects of alkalinization on platelet Ca 2+ responses that often depend on the agonist used. Thrombinoinduced changes of [Ca 2+]i are not dependent on cytosolic alkalinization, although pHi might "fine tune" IP3-mediated Ca 2+ release in thrombin-treated platelets (Simpson and Rink, 1987; Zavoico and Cragoe, 1988). On the other hand, platelet [Ca2+]i responses to ADP or the synthetic prostaglandin endoperoxide U46619 are inhibited by at least 50% when platelet Na§ § exchange and resulting cytosolic alkalinization is blocked, indicating that Na+/H § exchange plays an important role in regulating [Ca*]i under some conditions (Siffert et al., 1990). Further studies are required to define precisely how pHi regulates platelet [Ca2+]i. Na +/H § exchange may also regulate platelet signal transduction pathways by affecting the "phosphoinositide cycle". This cycle is the pathway by which DAG is recycled to PIP2 through the following intermediates: PA, PI and PI-4-P. pHi appears to regulate the enzyme DAG kinase (which converts DAG to PA), thereby per, mitting the platelet phosphoinositide cycle to proceed

and perhaps contributing to maintaining thrombininduced aggregation and secretion (Luzzatto et al., 1991).

6.6 OTHER SIGNAL PATHWAYS 6.6.1 P L D Platelets contain a third PL that contributes to platelet activation. PLD cleaves the terminal phosphodiester bond of membrane phospholipids, with PC as its preferred substrate (Rubin, 1988). Recent data suggest that thrombin activates PLD after PLC is activated and that elevated [Ca2+]i mediates PLD activation (IL Huang et al., 1991). PLD causes the direct release of PA, which may be an important signal controlling the activation of PLA2 (Kroll et al., 1989), perhaps by inducing a conformational change in platelet GPIIb-IIIa (Smyth et al., 1991). PA is also formed following PIP2 breakdown by the activity of the enzyme DAG kinase, converting DAG to PA (see above). Because this pathway generates the majority of PA synthesized following platelet stimulation, PLD-initiated signal generation is apparently minimal and contributes very little to the state of platelet activation (1L Huang et al., 1991).

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS 49 6.6.2 Tyrosine Kinases Platelets contain a large amount of protein tyrosine phosphate, indicating the presence of tyrosine kinases and tyrosine phosphatases that may have signalling functions (Gu et al., 1991). In nucleated cells, tyrosine phosphorylation is an important signal for growth factor-induced mitosis. Growth factors bind to specific receptors that are coupled to a tyrosine kinase, and the activation of the tyrosine kinase results in the phosphorylation of the growth factor receptor and its association with phosphatidylinositol-3-kinase. This latter enzyme synthesizes phosphoinositides that have a phosphate group at the D-3 position of the inositol ring (Skolnik et al., 1991). The synthesis of these compounds is associated with nuclear activity that initiates cell growth, although the biochemical mechanisms by which D-3 phosphoinositides mediate this process are currently unknown (Escobedo et al., 1991). Platelets contain relatively large amounts of the tyrosine protein kinase (TPK) pp60 c-src and other "src-related" tyrosine kinases (Golden and Brugge, 1989; Ferrell and Martin, 1989; Dhar and Shukla, 1993). These become activated and phosphorylate many substrates, including the 125 kDa TPK designated "focal adhesion kinase", following platelet stimulation induced by different agonists such as thrombin, collagen, prostaglandin endoperoxides, PAF, ADP and epinephrine (Nakamura and Yamamura, 1989; Gaudette and Holub, 1990; Salari et aL, 1990; Dhar et al., 1990; Golden et al., 1990). The effects of these tyrosine phosphorylation events on platelet activation are poody understood but, as in dividing cells, one consequence of protein tyrosine phosphorylation in platelets is the generation of D-3 phosphoinositides. These are synthesized in platelets following agonist-induced platelet activation, possibly in association with PKC activity or fibrinogen binding to GPIIb-IIIa (Golden et al., 1990; Kucera and Rittenhouse, 1990; King et al., 1991; Sultan et al., 1991). The function of tyrosine kinase-directed synthesis of D-3 phosphoinositides in platelets is not known, but they associate with the cytoskelton (Zhang et al., 1992) and may be involved in cytoskeletal reorganization (Grondin et al., 1991). As described above in sections on GPIIb-IIIa and low MW Ga, src and related proteins translocate to the cytoskeleton of activated platelets (Clark and Brugge, 1993), and may be involved in GPIIb-IIIa mediated "outside-in" signalling. Recent data using the tyrosine phosphatase inhibitor vanadate suggest that platelet tyrosine phosphorylation regulates PLA2 (McNichol et al., 1993), and experiments using the tyrosine kinase inhibitor tyrphostin suggest that tyrosine phosphorylation in response to thrombin may also regulate PLC (Guinebault et al., 1993). 6.6.3 H i s t a m i n e Histamine (H) is a molecule that has pleiotropic biologic effects. Recent studies suggest that histamine may be an

important intracellular signal molecule in human platelets mediating aggregation, although its mechanism of action is not known (Saxena et al., 1989). Platelets contain the enzyme responsible for the synthesis of H (histidine decarboxylase) and it appears that PKC stimulates (and Ca2 § inhibits) its activity (Saxena et al., 1991). H is also an extracellular signal for platelet activation in v/tw0, although its physiological significance is unknown. H binds to a platelet H1 receptor and activates PLA2 through a heterotrimeric Ga-mediated mechanism (Murayama et al., 1990).

7. Inhibitory L

nd- Receptor

Interactions 7.1

INTRODUCTION

Platelet activation is regulated by intracellular signal pathways that attenuate or prevent agonist-induced responses. The major platelet inhibitory pathways of physiological and pharmacological importance are mediated by elevated cytosolic cAMP or cGMP. In addition to cyclic nucleotide-mediated pathways of platelet inhibition triggered by exogenous factors, there are a number of inhibitory intracellular signals that are produced endogenously following platelet stimulation, and that may function to attenuate or terminate the initiating stimulus. These inhibitory signals include the previously described IP3 5'-phosphomonoesterase, lipocortin, platelet LO metabolites and PKC.

7.2

cAMP

cAMP is synthesized when the eicosanoids PGI2 or PGD2 bind to specific platelet receptors and activate adenylyl cyclase. The most important of these is endothelial cell-derived PGI2. There appears to be separate receptors for PGD2 (Schafer et al., 1979) and PGI2/PGE1 (Siegl et al., 1979). Neither receptor has been cloned, but the latter has been partially purified from platelet membranes (Dutta-Roy and Sinha, 1987). Investigations using partially purified PGI2/PGE1 receptors suggest that a single protein of Mr ~ 190 000 has both high affinity ( I ~ - - 9 . 8 riM) and low affinity (Kd ~ 700 nM) binding sites (Dutta-Roy and Sinha, 1987). cAMP probably does not regulate the state of platelet activation under physiological conditions (Nowak and FitzGerald, 1989), but it becomes important in areas of endothelial cell injury (where it functions as a "natural" anti-thrombotic molecule), and pharmacological agents that elevate intraplatelet cAMP are potent platelet inhibitors (Vane et al., 1990).

7.3

cGMP

cGMP is synthesized when nitrovasodilators, including

50 M . H . KROLL AND A.I. SCHAFER the "endogenous nitrovasodilator" nitric oxide [NO; or endothelium-derived relaxing factor (EDRF)], diffuse through the platelet plasma membrane and activate soluble (cytosolic) guanylate cyclase by a receptorindependent mechanism (Brenner etal., 1989). The basal release of EDRF may regulate the state of platelet activation under physiological conditions (Vallance et al., 1989; Durante et al., 1993); and EDRF production in areas of vascular injury (by both endothelium and vascular smooth muscle) may limit the extent of platelet thrombus formation (Vane et al., 1990; Durante et al., 1991; Yao et al., 1992). Pharmacological doses of nitrovasodilators cause elevations of platelet cGMP that are insufficient to effect platelet inhibition, but the coadministration of a reduced thiol donor, such as Nacetylcysteine, produces levels of platelet cGMP sufficient to inhibit platelet function (Horowitz et al., 1983; Stamler et al., 1989).

7.4

MECHANISMS

OF PLATELET

INHIBITION cAMP and cGMP inhibit each component of the triad of platelet functional responses: adhesion, aggregation, and secretion (Feinstein et al., 1985; Radomski et al., 1987; Mendelsohn et al., 1990; Lieberman et al., 1991; Broekman et al., 1991). The molecular mechanisms of cAMP-mediated inhibition are perhaps best studied. The generation of elevated cytosolic concentrations of cAMP, which inhibit platelet responses primarily through cAMP-dependent PKs, results in pleiotropic platelet inhibitory effects involving both the initiation and maintenance of platelet activation (reviewed in Nairn et al., 1985). cAMP decreases thrombin binding to platelets and thereby inhibits one proximal step in platelet activation (Lerea eta/., 1987; Lerea and Glomset, 1987); it inhibits PLC-mediated DAG and IP3 formation (Rittenhouse-Simmons, 1979; Knight and Scrutton, 1984); it inhibits platelet CO, probably independent of Ca 2§ (Schafer et al., 1980); it inhibits the DAG signal for PKC activation by increasing its metabolism to phosphoinositides (Lapetina, 1986) and directly inhibits the activity of PKC (Kroll et al., 1988); and it inhibits many platelet responses that are distal to PKC, such as agonistinduced polymerization of actin and fibrogen receptor expression [which may actually undergo "closure" when PGI2 is added to stimulated platelets (Siess and Lapetina, 1989; van Willigen and Akkerman, 1992)]. The effect of cAMP on actin polymerization is mediated by cAMP-dependent PK phosphorylation of the # subunit of GPIb (Fox et al., 1987; Fox and Berndt, 1989). The most important mechanism by which cAMP inhihits platelets is its antagonism of Ca2+-mediated responses. PLC is the most proximal point in the common pathway leading to changes of platelet [Ca 2+]i, and platelets treated with activators of adenylyl cyclase demonstrate no Ca 2+ response to agonists primarily

because PLC-mediated generation of IPs is blocked (Zavoico and Feinstein, 1984; Feinstein et al., 1985). In addition, cAMP influences both the release and uptake of Ca 2+ from the dense tubular system (DTS). IP3mediated release of Ca 2+ from the DTS may be inhibited by the direct effect of the catalytic subunit of cAMP-dependent PK on the DTS (Enouf et al., 1987b; O'Rourke et al., 1989). Based on observations that adenylyl cyclase stimulators cause a rapid fall in [Ca 2+]i when they are added after thrombin has initiated a Ca 2§ response, it appears that cAMP also stimulates the reuptake of Ca 2§ into the DTS (Kaser-Glanzmann et al., 1977). The mechanism by which this resequestration occurs may involve a cAMP-dependent phosphorylation of some DTS structure regulating this process (KaserGlanzmann et al., 1977; Enouf et al., 1987b). Through its effect on [Ca2+]i, cAMP inhibits a number of other platelet responses, including PLA2-mediated release of AA (as previously described), cytoskeletal assembly (independent of GPIb) and the activity of myosin light chain kinase (Feinstein et al., 1983; Cox et al., 1984). In this last case, there is also a direct inhibitory effect of cAMP-independent of [Ca2+]i: myosin light chain kinase is inhibited when it is phosphorylated by a cAMPdependent PK (Hathaway et al., 1981). The molecular mechanisms by which cGMP inhibits platelet activation are less well understood. Elevated levels of platelet cytosolic cGMP can be induced by nitrovasodilators, nitrosothiol compounds, EDRF or non-hydrolyzable analogs of cGMP, and these are associated with the inhibition of PIP2 hydrolysis and all consequent distal responses, including changes of [Ca2+]i and the activation of PKs (Mendelsohn et al., 1990; Lieberman et al., 1991; Nguyen et al., 1991; Durante et al., 1992). cGMP has also been shown to inhibit PLA2-mediated AA release in human platelets (Vane et al., 1989). cAMP appears to function synergistically with cGMP to effect the inhibition of agonist-induced platelet aggregation (MacDonald et al., 1988), and this may be due to the effect of cGMP on inhibiting the low Km cAMP phosphodiesterase of platelets (Grant et al., 1990; Maurice and Haslam, 1990). An understanding of the synergistic platelet inhibitory effects of prostaglandins and nitrovasodilators in vitro may be useful in developing therapies to inhibit platelet function in vivo. Individually, these drugs demonstrate serious side effects or relative inefficacy that might be overcome when the two classes of compounds are combined. The advantage of these agents is that they inhibit pathways of platelet activation that bypass the effects of aspirin. Thus, effective antiplatelet therapy may be achieved in patients who do not respond to aspirin. Platelet production of cGMP by an endogenous route may also contribute to the state of platelet activation. Platelets contain the constitutive form of the enzyme NO synthase and this may become activated following agonist

ANALYSIS OF LIGAND-RECEPTOR INTERACTIONS stimulation of platelets, resulting in the synthesis of platelet N O and the activation of platelet guanylate cyclase, which could function to dampen the aggregation response (Bredt etal., 1991; Radomski et al., 1990). This hypothesis has been refuted by others (Mollace et al., 1991).

8. Conclusion Platelets are critically important in the physiology of hemostasis and the pathology of thrombosis. In response to vascular injury, platelets adhere to subendothelial components and undergo a series of biochemical processes that culminate in secretion, aggregation, and thrombus formation. Platelet activation results from a complex series of carefully regulated molecular interactions that, under physiologic conditions, govern the location and extent of platelet thrombus formation. When platelet activation is triggered by pathological stimuli, however, these molecular interactions lead to platelet aggregation that may result in ischemia or infarction of vital tissues. The elucidation of mechanisms by which ligand-receptor interactions direct platelet activation or inhibition is important because it may lead to a better understanding of platelet signal pathways that are primarily hemostatic, and platelet signal pathways that are primarily thrombotic. This could then direct the development of truly "lesion-specific" therapies. In addition, further understanding of mechanisms of platelet activation could provide insight into the pathogenesis of thrombosis and atherosclerosis.

9. Acknowledgements The assistance of Deanna Golden and Rita Laddimore in the preparation of this manuscript is most gratefully acknowledged. Supported in part by the National Heart, Lung and Blood Institute, the Research Service of the Department of Veterans' Affairs and The Methodist Hospital Foundation. M.H.K. is an Established Investigator of the American Heart Association.

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van Willigen, G. and Akkerman, J-W.N. (1991). Protein kinase Multiphasic generation of diacylglycerol in thrombinC and cyclic AMP regulate reversible exposure of binding sites activated human platelets. Biochem. J. 282, 815-820. for fibrinogen on the glycoprotein IIb-IIIa complex of White, J.G. (1972). Interaction of membrane systems in blood human platelets. Biochem. J. 273, 115-120. platelets. Am. J. Pathol. 66, 295-312. van Willigen, G. and Akkerman, J-W.N. (1992). Regulation of White, S.P., Scott, D.L., Otwinowski, Z., Gelb, M.H. and glycoprotein IIb/IIIa exposure on platelets stimulated with c~Sigler, P.B. (1990). Crystal structure of cobra-venom phosthrombin. Blood 72, 82-90. pholipase A2 in a complex with a transition-state analogue. Vane, J.IL, Anggard, E.E. and Botting, P.M. (1990). ReguScience 250, 1560-1562. latory functions of the vascular endothelium. New Engl. J. Wiedmer, T. and Sims, P.J. (1991). Participation of protein Med. 323, 27-34. kinases in complement C5b-9-induced shedding of platelet Vaughan, D.E., Mendelsohn, M.E., Declerck, P.J., Van plasma membrane vesicles. Blood 78, 2880-2886. Houtte, E. and Collen, D. (1989). Characterization of the Wiedmer, T., Ando, B. and Sims, P.J. (19S7). Complement binding of human tissue-type plasminogen activator to C5b-9-stimulated platelet secretion is associated with a Ca2*platelets. J. Biol. Chem. 264, 15869-15874. initiated activation of cellular protein kinases. J. Biol. Chem. Vittet, D., Rondot, A., Cantan, B., Launay J-M. and Chevil262, 13674-13681. lard, C. (1986). Nature and properties of human platelet Willerson, J.T. (1991). Serotonin and thrombotic complicavasopressin receptor. Biochem. J. 233, 631-636. tions. J. Cardiovasc. Pharmacol. 17, $13-$20. Vu, T-K.H., Wheaton, V.I., Hung, D.T., Charo, I. and Wilson, D.B., Connolly, T.M., Bross, T.E., Majerus, P.W., Coughlin, S.IL (1991a). Domains specifying thrombinSherman, W.R., Tyler, A.N., Rubin, L.J. and Brown, J.E. receptor interaction. Nature 353, 674--677. (1985). Isolation and characterization of the inositol cyclic Vu, T-K.H., Hung, D.T., Wheaton, V.I. and Coughlin, S.R. phosphate products of polyphosphoinositide cleavage by (1991b). Molecular cloning of a functional thrombin receptor phospholipase C. J. Biol. Chem. 260, 13496-13501. reveals a novel proteo-lytic mechanism of receptor activation. Wirz-Justice, A. (1988). Platelet research in psychiatry. Cell 64, 1057-1068. Experientia 44, 145-151. Walsh, P.N. (1987). Platelet-mediated trigger mechanisms in Worthington, R.E., Carrol, tLC., Boucheix, C. (1990). Platelet the contact phase of blood coagulation. Semin. Thromb. activation by CD9 monoclonal antibodies is mediated by the Hemost. 13, 86-94. Fc-yII receptor. Br. J. Haematol. 74, 216-222. Ware, J.A., Johnson, P.C., Smith, M. and Salzman, E.W. Yamamoto, N., Ikeda, H., Tandon, N.N., Herman, J., (1985). Aequorin detects increased cytoplasmic calcium in Tomiyana, Y., Mitani, T., Sekiguchi, S., Lipsky, tL, Kralisz, platelets stimulated with phorbol ester or diacylglycerol. U. and Jamieson, G.A. (1990). A platelet membrane Biochem. Biophys. Res. Commun. 133, 98-104. glycoprotein deficiency in healthy blood donors: NaKa-Ware, J.A., Johnson, P.C., Smith, M. and Salzman, E.W. platelets lack detectable GpIV (CD36). Blood 76, (1986). Effect of common agonists on cytoplasmic ionized cal1698-1703. cium concentrations in platelets. J. Clin. Invest. 77, Yamamoto, N., Greco, N.J., Barnard, M.R., Tanoue, K., 878-886. Yamazaki, H., Jamieson, G.A. and Michelson, A.D. (1991). Watanabe, T., Hashimoto, Y., Teramoto, T., Kume, S., Naito, Glycoprotein Ib (GPIb)-dependent and GPIb-independent C. and Oka, H. (1986). Calmodulin-independent inhibition pathways of thrombin-induced platelet activation. Blood 77, of platelet phospholipase A2 by calmodulin antagonists. 1740-1748. Arch. Biochem. Biophys. 246, 699-709. Yamanishi, J., Takai, Y., Kaibuchi, K., Sano, K., Castagna, M. Watson, S.P. and Lapetina, E.G. (1985). 1,2-Diacylglycerol and and Nishizuka, Y. (1983). Synergistic functions of phorbol phorbol ester inhibit agonist-induced formation of inositol ester and calcium in serotonin release from human platelets. phosphates in human platelets: possible implications for negaBiochem. Biophys. Res. Commun. 112, 778-786. tive feedback regulation of inositol phospholipid hydrolysis. Yao, S-K., Ober, J.C., Krishnaswami, A., Ferguson, J.J., Proc. Natl. Acad. Sci. USA 82, 2623-2626. Anderson, H.V., Golino, P., Buja, L.M., and Willerson, J.T. Watson, S.P., Reep, B., McConnell, R.T. and Lapetina, E.G. (1992). Endogenous nitric oxide protects against platelet (1985). Collagen stimulates [3H]inositol trisphospate foraggregation and cyclic flow variations in stenosed mation in indomethacin-treated human platelets. Biochem. J. and endothelium-injured arteries. Circulation 86, 226, 831-837. 1302-1309. Watson, S.P., McNally, J., Shipman, L.J. and Godfrey, P.P. Yoshida, N., Weksler, B. and Nachman, IL (1983). Purification (1988). The action of the protein kinase C inhibitor of human platelet calcium-activated protease. J. Biol. Chem. staurosporine on human platelets. Biochem. J. 249, 258, 7168-7174. 345-350. Zavoico, G.B. and Feinstein, M.B. (1984). Cytoplasmic C a 2+ Weiss, H.J., Hawiger, J., Ruggeri, Z.M., Turitto, V.T., in platelets is controlled by cyclic AMP: antagonism between Thiagarajan, P. and Hoffman, T. (1990). Fibrinogenstimulators and inhibitors of adenylate cyclase. Biochem. independent platelet adhesion and thrombus formation on Biophys. Res. Commun. 120, 579-585. subendothelium mediated by glycoprotein IIb-IIIa complex Zavoico, G.B. and Cragoe, E.J. (1988). Ca2+ mobilization can at high shear rate. J. Clin. Invest. 83, 288-297. occur independent of acceleration of Na § § exchange in Werner, M.H. and Hannun, Y.A. (1991). 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65

Zhang, J., Fry, M.J., Waterfield, M.D., Jaken, S., Liao, L., Fox, J.E.B. and Rittenhouse, S.E. (1992). Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets. J. Biol. Chem. 267, 4686-4692. Zschauer, A., van Breemen, C., Buhler, F.IL and Nelson, M.T. (1988). Calcium channels in thrombin-activated human platelet membrane. Nature 334, 705-707. Zucker-Franklin, D. (1992). Clinical significance of platelet microparticles. J. Lab. Clin. Med. 119, 321-322.

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4. The Role of Human P latelet Membrane Receptorsm"Inflammation John L. McGregor

1. 2. 3. 4.

Introduction Platelet Glycoproteins Platelets and Inflammation P-selectins 4.1 Structure and Homology 4.2 Platelet-Leucocyte Interactions 4.3 P-selectin Ligands 4.4 P-selectin in Circulation 4.5 Platelet-T Lymphocyte Interactions 4.6 Transcellular Synthesis of Molecules 4.7 Platelet P-selectin in Other Species 5. Cytokines and Platelets 6. Thrombospondin and CD36

67 67 68 69 69 71 72 72 73 73 74 74 75

1. Introduction Circulating blood platelets rapidly form (within seconds) a thrombus or haemostatic plug at sites of vascular injury to prevent severe bleeding or haemorrhage. These anucleated cells, derived from the fragmentation of megakaryocytes, are also implicated in the occlusion of blood vessels by the formation ofa thrombus triggered by the diseased state of the vessel wall (Marcus, 1990). In addition, platelets are also involved in tissue injury, inflammation and wound healing by attracting and binding leucocytes (Marcus, 1990; McEver, 1991). Platelet-platelet, platelet-extracellular matrix, as well as other cellular interactions that are involved in tissue growth and the defence of the body against infections are mediated by families of adhesion molecules or membrane receptors that include the integrins, selectins, cadherins, leucine-rich glycoproteins (GPs), and immunoglobulin Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

6.1 A Multifunctional Adhesive Protein 6.2 Binding of TSP to Resting or Activated Platelets 6.3 TSP Receptors on the Platelet Surface 6.4 Sites on TSP Interacting with the Platelet Surface 6.5 TSP as a Ligand to Platelet-Monocyte Interactions 6.6 Binding of Platelets to Bacteria via TSP 7. Platelet Factor 4 8. Acknowledgements 9. References

75 75 76 76 76 77 77 77 78

superfamily (Springer, 1990; McEver, 1991; Butcher, 1991; Shimizu et al., 1992; Pardi et al., Hynes, 1992). The aim of this work is to review our knowledge on adhesion molecules and membrane-bound ligands, released from a granules, in mediating the role of human platelets in inflammation.

2. Platelet Glycoproteins A large number of GPs (over 40) are known to be present on the platelet surface. Some of these glycoproteins, such as GPIb-IX, GPIa-IIa (c~2Bs) and GPIIb-IIIa (OeXib~3), are essential for normal platelet adhesion or aggregation to occur (Clemetson and McGregor, 1987). The GPIb-XI complex (member of the leucine-rich GP receptor family), GPIa-IIa (c~2~31, VLA-2), GPIb-IIa (c~sBl, VLA-5) and GPIc*'IIa (c~6/51, VLA-6) (members Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

68 J.L. MCGREGOR of the integrin receptor family) serve as receptors respectively to von Willebrand's factor, collagen, fibronectin and laminin (Rx)th, 1992). Interaction between platelets will occur via GPIIb-IIIa (c~Iib/3S), a member of the integrin receptor family, and adhesive ligands such as fibrinogen and von Willebrand's factor (Marguerie et al., 1987; McGregor and Clemetson, 1988). Platelets adhering to the subendothelium wall will induce circulating platelets to interact with them by secreting activating substances (Kinlough-Rathbone and Mustard, 1987). The thrombus, or haemostatic plug, needed to arrest bleeding in injured vessels, is poorly formed or is absent in patients with diseased platelets showing a congenital absence, reduction or disfunction of certain platelet GP receptors such as GPIb-IX, GPIIb-IIIa or GPIa-IIa (Nurden, 1987). Knowledge derived from the critical role of GPIIb-IIIa in platelet aggregation has greatly helped in generating antithrombotic drugs, directed against the GPIIb-IIIa complex, to be eventually used in thrombolytic therapy (Gold et al., 1990; Savage et al., 1990; Catimel et al., 1991). GPs packaged in ~xgranules (e.g. TSP, fibronectin, fibrinogen, von Willebrand's factor) that are released and

subsequently bound to the platelet surface or fused to the cytoplasmic membrane (GMP-140/CD62/P-selectin, GPIIb-IIIa) (see Fig. 4.1) have also been shown to play a critical role in the stabilization of platelet aggregates (McGregor and Boukerche, 1992).

3. Platelets and Inflammation Platelets, in addition to their vital role in the prevention of haemorrhage, are involved in attracting leucocytes at sites of vascular injury. Moreover, platelets also appear to be in the front line in interacting with organisms, such as bacteria or parasites, that are foreign to the host (Scheld etal., 1978; Joseph etal., 1984; Herrmann etal., 1991). Platelets, in their interaction with the subendothelium, foreign organisms or metastatic cells, release or express on their surface a variety of molecules, such as plateletderived growth factor (PDGF), 12-hydroxyeicosatetraenoic acid (12-HETE), platelet factor 4 (PF,), and transforming growth factor type beta (TGF-/3), that affect neutrophils, eosinophils and monocytes by their chemotactic activity (Turner et al., 1975; Duel et al.,

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STIMULATED- RELEASING PLATELETS Figure 4.1 c~-Granule constituents released and/or bound on the surface of activated platelets. A large number of proteins, such as fibrinogen (Fg), fibronectin (Fn), thrombospondin (TSP), von Willebrand's factor (vWF), platelet-derived growth factor (PDGF), platelet factor 4 (PF4), platelet basic protein (PBP), and /~-thromboglobulin (/~-TG), are stocked in resting platelet c~ granules. On activation of platelets by agonists such as thrombin, collagen or ionophores, these c~ granule proteins are rapidly released (within seconds) in the surrounding medium and/or bound to the platelet surface in the presence of physiological concentrations of calcium. Intecrine family members (PF4, /~-TG) will either bind to the platelet surface or be degraded in the presence of other cells to be converted to potent leucocyte or fibroblast stimulating agents. In addition, glycoproteins that are constitutively part of the (x granule membrane, such as (x,~/33 (GPIIb-Illa) and P-selectin (GMP-140, PADGEM, CD62), fuse with the cytoplasmic membrane to become exposed on the platelet surface.

ROLE OF HUMAN PLATELET MEMBRANE RECEPTORS IN INFLAMMATION 69

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Figure 4.2 Platelet glycoproteins and adhesive ligands mediating leucocyte-platelet interactions. P-selectin expressed by activated platelets will bind to sialyl.Lewis X (sialyl CD15), sialyl-Lewis', heparan sulphate proteoglycans (HSP), suIphatide and possibly to other receptors present on leucocytes. Thrombospondin (TSP) bound to the platelet surface via a number of receptors (CD36, GPla-Ila, GPIIb-Illa) will interact with leucocytes via CD36 (present on monocytes), HSP, C~v~a(vitronectin receptor present on macrophages) or CD11/CD18 (neutrophils). Fibrinogen (Fg) bound to platelet C~qb~aand to leucocyte CD11/CD18 appears to be implicated in inducing an oxidative burst in neutrophils. Platelet factor 4 (PF4) by binding to the platelet and leucocyte surface may also be implicated as a possible ligand in platelet-leucocyte interactions. Platelet membrane-bound IL-1 may be involved in the activation of leucocytes. Ligands, such as fibronectin (Fn) and von Willebrand's factor (vWF), present on the activated platelet surface.may also be involved, as is fibrinogen, in the adhesion of platelets to leucocytes.

1982; Wahl et a/., 1987). Such released or surface expressed GPs may activate, attract and/or allow leucocytes to bind to activated platelets via specific adhesive receptors (see Fig. 4.2). Certain GPs present on the activated platelet surface will not only favour leucocyte-platelet interactions but also act as catalytic sites for the coagulation cascade (see Fig. 4.3) (Bevers et al., 1987; Zimmerman et al., 1991). Some receptors present on platelets or endothelial cells are implicated in the metastatic process, allowing cancer cells to spread from a tumour site to different parts of the human body (Belloni and Tressler, 1990). Extensive evidence is now available to show that platelets interact with leucocytes via at least two adhesion molecules (CD36, P-selectin). Moreover, platelets are known to bind to malignant cells via GPIIb-IIIa (Boukerche et al., 1989). Interaction of platelets with parasites, such as Schistosoma, is also associated with the GPIIb-IIIa complex (Joseph et al., 1986; Ameisen et al., 1986a, b). Neutrophil oxidative burst appears to depend on activated platelet GPIIb-IIIa binding fibrinogen to allow platelet-neutrophil interactions (Ruf et al., 1992). Platelets also express membranebound lymphokine activity (IL-1) and bind a granule-

released components (e.g. PF4, TSP, fibrinogen) that play an important role in inflammation (see Fig. 4.2). Activated platelets are implicated in the inflammatory process by their dual capacity of releasing chemotactic substances and expressing surface receptors capable of binding leucocytes and/or modulating their activity.

4. P-selectins 4.1

STRUCTURE

AND HOMOLOGY

P-selectin, also known as GMP-140 (granuie membrane protein 140 kD; McEver and Martin, 1984), PADGEM (platelet activation-dependent granule-external membrane; Berman et al., 1986) or CD62, was first identified in human blood platelets by McEver and Martin (1984) and Hsu-Lin et al., (1984) using monoclonal antibodies (mAbs). This 140 kD GP was shown to be an integral c~ granule membrane GP component that fuses with the cytoplasmic membrane when platelets are activated and undergo release (Sternberg et al., 1985; Berman et al., 1986). More recently P-selectin was also shown to be

70 J.L. MCGREGOR vWl TSP

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CHONDROITIN SULPHATE PROTEOGLYCAN SYNTHESIS

Figure 4.3 Platelets bound to the surface of monocytes control tissue factor activity, TSP and chondroitin sulphate proteoglycan synthesis. Activated platelets interacting with leucocytes via P-selectin, thrombospondin (TSP), fibrinogen (Fg), and platelet factor 4 (PF4) may modulate the synthesis in monocytes of TSP, tissue factor expression and chondroitin sulphate proteoglycan. In addition, flbrinogen (Fg) bound to platelet m,b/Ssand to leucocyte CD11/CD18 appears to be Implicated in inducing an oxidative burst in neutrophils.

present in the membrane of platelet dense bodies, or granules, together with granulophysin (Israels et al., 1992). Subsequently, P-selectin was also observed to be present in human umbilical vein endothelial cells (HUVEC), and to be rapidly expressed when these cells were activated by agonists such as thrombin, histamine (H), phorbol esters, oxygen radicals, herpes simplex virus (HSV) infection, and complement protein C5b-9 (Hattori et al., 1989; Bonfanti et al., 1989; McEver et al., 1989; Etingen et al., 1991). Expression of P-selectin by HUVEC is transient with a peak reached after 3-10 min following activation and a gradual drop, due to endocytosis, to basal levels by around 30 min (Hattori et al., 1989; Bonfanti et al., 1989; McEver et al., 1989). In contrast, P-selectin expressed by activated platelets remains at the surface for periods of over 60 min (George et al., 1986). P-selectin is expressed in much higher amounts in endothelial cells present in postcapillary venules as opposed to large arteries, arterioles or veins (McEver et al., 1989). In endothelial cells, P-selectin is co-localized with von Willebrand's factor in Weibel-Palade bodies (Bonfanti et al., 1989; Hattori et al., 1989; McEver et al., 1989). P-selectin amino acid composition shows a high number of cystinyl (6.1%), prolinyl (7.2%) and tryptophanyl (2.1%) residues. In addition, N-acetylneuraminic acid, neutral sugar and N-acetylglucosamine residues make up the P-selectin 28.8% carbohydrate (by weight) content. The apparent molecular weight of P-selectin is reduced to 50kDa following enzymatic treatment to remove

N-linked oligosaccarides (Johnston et al., 1989a). Cloning and sequencing of the P-selectin cDNA predicts a cysteine-rich GP with a number of heterogeneous domains. A cleavable signal peptide is present at the Nterminal site, followed by a lectin-like domain, an epidermal growth factor (EGF)-like domain, nine repeats related to complement-binding proteins, a transmembrane segment and a short cytoplasmic region. A number of cDNAs also predict the presence of a soluble form of P-selectin, with a deleted transmembrane domain, and another variant of P-selectin having eight instead of nine repeats related to complement-binding proteins (Johnston et al., 1989b). The human gene encoding P-selectin contains 17 exons, spans over 50 kilobases (kb) and is located on the long arm of chromosome 1 at bands q21-24 (Watson et al., 1990). Each domain of Pselectin, such as the lectin-like domain, the EGF-like domain, nine consensus repeats related to complementbinding proteins, and the transmembrane segment, is encoded by an exon. The two variant forms of P-selectin, lacking the transmembrane domain or having eight instead of nine repeats, appear to be the result of alternative splicing of mRNA (Johnston et al., 1990). The Pselectin cDNA (Johnston etal., 1990) has strong homologies to two other members of the selectin family, Eselectin (also known as endothelial leukocyte adhesion molecule-1 or ELAM-1; Collins et al., 1991) and Lselectin (also known as the lymphocyte homing receptor or Leu-8/TQ1 in humans and gp90 ~EL in mouse; Ord et al., 1990). Sixty per cent of the amino acid sequence

ROLE OF HUMAN PLATELET MEMBRANE RECEPTORS IN INFLAMMATION 71 of the lectin-like and EGF domain of selectins are identical and the consensus repeat regions have a 40% identity. The genes of these two other members of the selectin family (E- and L-selectin) are also located on the long arm of chromosome 1 at bands q21-24, as is Pselectin, and the three genes are packed in a region of no more than 300 kb (Watson et al., 1990). The calciumdependent carbohydrate recognition domain (CRD) of the selectin family, located on approximately 120 amino acids of the lectin-like region, has a strong homology to C-type animal lectins (Halberg et al., 1988; Drickammer, 1988; Weis et al., 1991). Other proteins having a C-type animal lectin region include macrophage receptors dealing with the phagocytosis of pathogens (Taylor et al., 1988), the mammalian mannose-binding proteins that are involved in reactions against pathogens in an immunoglobulin-independent way (Weis et al., 1991), hepatic lectins involved in removing serum GPs from circulation (Halberg et al., 1987) and extracellular matrix proteins with a proteoglycan (White et al., 1985). A sequence motif of 30 conserved amino acids, spread over the 120 amino acids of the lectin-like region, represent the Ca 2+-dependent CRD of C-type animal lectins. Recently the structural importance of this sequence motif of conserved amino acids in the CRD fold of C-type animal lectins was shown by a crystal structure study performed on the CRD of rat mannose binding protein (Weis et al., 1991).

4.2

PLATELET-LEUCOCYTE INTERACTIONS

Neutrophils, monocytes, and human monocytoid (U937) or promyelocytic (HL60) cell lines bind to activated, but not resting, platelets and to purified P-selectin incorporated in phospholipid vesicles, coated on plastic, or to COS cells (a fibroblast-like kidney cell line established" from simian cells) transfected with the P-selectin cDNA. Metabolic activity of leucocytes, U937 or HL60 cell lines was not necessary since interaction with activated platelets occurred when cells were fixed with paraformaldehyde or at 4~ (Jungi et al., 1986; Silverstein and Nachman, 1987; Larsen et al., 1989; Gamble et al., 1990; Geng et al., 1990; Hamburger and McEver, 1990; Corral et al., 1990). The binding of leucocytes, or monocyte-like cell lines, to activated platelets was inhibited by anti-P-selectin polyclonal and monoclonal antibodies and purified P-selectin (Larsen et al., 1989; Parmentier et al., 1991; Geng et al., 1990; Hamburger and McEver, 1990; Skinner et al., 1991; Rinder et al., 1991a; de Bruijine-Admiraal et al., 1992). Thrombin stimulation of platelets in whole blood, in the presence of Arg-Gly-Asp-Ser (RGDS), showed platelets binding to monocytes in a larger number and at a faster rate than to neutrophils (Rinder et al., 1991b). The advantage that monocytes have over neutrophils in binding platelets was

also observed when platelets were stimulated with a combination of two mild agonists (epinephrine/ADP), compared to a strong agonist such as thrombin, which resulted in a reduced expression of P-selectin. This platelet-monocyte or platelet-neutrophil interaction in whole blood was specifically mediated by P-selectin as shown by the inhibition induced by an anti-P-selectin mAb (G1; Rinder et al., 1991a). Activated platelets not only interact with monocytes and neutrophils, via P-selectin, but also with eosinophils, basophils, natural killer (NK) cells and an additional subpopulation of undefined T lymphocytes present in the CD4 § CD8 § subsets (de Bruijine-Admiraal et al., 1992). These P-selectin-mediated interactions between eosinophils, basophils or subpopulations of T lymphocytes and platelets are divalent cation dependent and can be abolished by isolated P-selectin or mAbs directed against this adhesive receptor (Gamble et al., 1990; Geng et al., 1990: Hamburger and McEver, 1990; de BruijineAdmiraal etal., 1992). Half-maximal rate binding ofneutrophils to isolated P-selectin can be achieved at a concentration of 20 mm Ca 2+ . Lower levels of Ca 2§ (2 mM) can be used to obtain half-maximal binding of neutrophils to P-selectin in the presence of 1 mM Mg 2§ which cannot support such interactions on its own (Geng et al., 1990). Two high affinity Ca2+ binding sites, present on isolated P-selectin, induce a change in conformation of the lectin region of this GP that subsequently allows interaction with neutrophils (Geng et al., 1991). Such conformational change of P-selectin exposes a site (present on residues 19-34) on the lectin domain that is recognized by a mAb (G3) that blocks Pselectin-neutrophil interactions. Mg 2+ will also induce a conformational change of P-selectin, binding probably to sites on the protein that are different from those for Ca 2+, but will not allow neutrophil binding in the absence of Ca 2+ . A peptide derived the P-selectin site (present on residues 19-34) exposed as a result of conformational change was effective in blocking neutrophil adhesion to P-selectin (Geng et al., 1991). Subsequently, Geng et al. (1992), using peptides derived from other sites (23-30, 54-63 and 70-79) on the lectin-like domain, observed that they blocked leucocyte binding to P-selectin. Moreover, these three peptides (23-30, 54-63 and 70-79), when conjugated to albumin and immobilized, supported in the presence of Ca 2+, adhesion of cells expressing fucosylated oligosaccharides or myeloid cells. Peptides from E-selectin and L-selectin, derived from regions corresponding to 23-30 and 54-63, also blocked adhesion of leucocytes to P-selectin. Moreover, through the use of intrinsic fluorescence microscopy, Ca 2§ was observed to interact with peptides 23-30 and 54-63 derived from all three selectins (Geng et al., 1992). Such results strongly suggest that peptides 23-30 and 54-63 represent functional sites on the CRD allowing cellular adherence.

72 J.L. MCGREGOR

4.3

P-SELECTIN LIGANDS

Larsen et al. (1990) in their pursuit of a ligand for Pselectin present on monocytes and neutrophils, used a whole series of mAbs directed against receptors present on leucocytes. Only mAbs directed against CD15 blocked the interaction, albeit not completely, between monocytes, neutrophils, U937 or HL60 cells and activated platelets. Moreover, such anti-CD15 antibodies also blocked the interaction between U937 cells and P-selectin expressed by COS cells or incorporated in liposomes. Further, lacto-N-fucopentaose III (Gal B1 ~ 4[Fuc c~1 -, 3] NAcGIc /31 ~ 3Gal /31 ~ 4Glc or Lewis X (LeX; CD15 antigen), purified from human milk, blocked the interactions between platelets and neutrophils or HL60 cells (Larsen et al., 1990). This work, using either anti-CD15 mAbs or purified carbohydrate lacto-N-fucopentaose III, indicated for the first time that CD 15 is a component of the ligand present on leucocytes that allows leucocyte-platelet interactions. Subsequently it was shown that a different panel of anti-CD15 mAbs could not inhibit purified P-selectin or platelet-leucocyte interactions (Moore et al., 1991; de Bruijine-Admiraal et al., 1992). Moreover, neuraminidase treatment of neutrophils, but not eosinophils, basophils or monocytes, completely or considerably inhibited their interaction with activated platelets (Corral et al., 1990; Moore et al., 1991; de Bruijine-Admiraal et al., 1992). Plateletleucocyte interaction was not inhibited, according to de Bruijine-Admiraal et al. (1992), by oligosaccharides sialylLe ~ (SLeX), Le x or mAbs directed against these oligosaccharides. However, the authors observed that the level of SLe x, but not Le x, present on leucocytes could be correlated to their capacity to bind platelets (de BruijineAdmiraal et al., 1992). SLe X, present on neutrophils, monocytes and certain tumour cells, was shown by Polley et al. (1991) to interact with P-selectin and Eselectin. Moore et al. (1991), looking at the binding of purified 12SI-labelled P-selectin to neutrophils, observed that it was dependent on the presence of physiological levels of calcium, was reduced after treatment of neutrophils with different types of neuraminadase, and was not inhibited by the presence of SLeX-bovine serum albumin (BSA) or LeX-BSA. Moreover, binding of 12sIlabelled P-selectin to neutrophils was lost after treatment of cells with trypsin or elastase (Moore et al., 1991). 12SI-labelled P-selectin bound to a single class of receptor (11 000 to 20 000 sites/cell) on neutrophils that were either resting or phorbol myristate acetate (PMA) activated, or HL60 cells (Skinner et al., 1991; Moore et al., 1991). Removal of phosphatidylinositol (PI)-linked proteins from neutrophils did not affect their interaction with activated platelets (de Bruijine-Admiraal et al., 1992). However, the presence of heparin, fucoidin and dextran sulphate 500 000 strongly affected the interaction of neutrophils or HL60 cells with activated platelets or the binding of 12SI-labelled P-selectin to these cells.

Dextran sulphate 5000, I- and k-carrageenan, but not chondroitin 4- and 6-sulphate, affected to a lesser extent the binding of activated platelets to neutrophils or HL60 cells (Skinner et al., 1991). Previous work by the same team (Skinner et al., 1989) showed that P-selectin binds to heparin, a sulphated glycan, and that the Pselectin-heparin interaction was also inhibited by heparin, fucoidin and dextran sulphate 500 000. The results of Skinnner and coworkers (1991) strongly suggest that the P-selectin receptor on neutrophils is identical or very close to a sulphated glycan binding site. In addition, Handa et al. (1991) showed that binding of activated platelets to SLe x or SLe a is inhibited to a certain extent by sulphated glycans. Handa et al.'s (1991) observations suggest that sulphated glycans affect the conformation of the lectin-like domain of P-selectin and hence its ability to bind to SLe x or SLe a. Aruffo et al. (1991) and Todderud et al. (1992) identify sulphatides (3-sulphated galactosyl ceramides) as another ligand to P-selectin not related to sialyl CD15. Sulphatides are expressed on the membrane of granulocytes and a number of tumour cells (Aruffo et al. , 1991). All these observations, coming after the work of Larsen et al. (1990), suggest the presence of a number of different P-selectin ligands on leucocytes, or tumour cells, possibly interacting not just with the lectin-like domain of P-selectin but also with the EGF- and/or complement-like domains. The P-selectin receptor on neutrophils appears to be a protein and not a glycolipid,~ or a PI-linked protein, that is expressed in the absence of any cellular metabolic activity (Moore et al., 1991; de Bruijine-Admiraal et al., 1992). In addition, the sialylated form of Le x (CD15) appears to have a considerable higher affinity for P-selectin than the unsialylated form (Springer and Lasky, 1991). Handa et al. (1991) observed that P-selectin expressing platelets, under their experimental conditions, has a higher degree of binding to SLe a compared to SLe x and minimal or no binding to LeX or Lea.

4.4

P-SELECTIN

IN CIRCULATION

As previously indicated, the P-selectin cDNA predicts the presence (in platelets and endothelial cells) of two transmembrane forms of this receptor, varying in size due to differences in the number of complement-binding domains, and a soluble form having a deleted transmembrane domain (Johnston et al., 1989a). Transmembrane forms of P-selectin are present on platelet microparticles that also express GPIIb-IIIa, GPIb, GPIIIb, GPIa-IIa, and PTA-1. Binding of these P-selectin-expressing microparticles to neutrophils and to myeloid/ monocytoid cell lines (U937, HL60, or RC2a) was inhibited by anti-P-selectin antigen binding fragment (Fab) polyclonal antibody, ethylene diamine tetraacetic acid (EDTA) or sulphated glycans (Burns et al., 1992). Identical agents were previously used to inhibit fluid

ROLE OF HUMAN PLATELET MEMBRANE RECEPTORS IN INFLAMMATION 73 phase P-selectin binding to the above cells (Skinner et al., 1991). The soluble form of P-selectin was detected in human plasma at a concentration of 0.251 + 0.043 gg/ml and 0.175 + 0.063 gg/ml in male and female controls, respectively. Anal~ical gel filtration showed the soluble P-selectin eluting as a monomer in contrast to the detergent-free transmembrane form eluting as a tetramer (Dunlop et al., 1992). Studies performed on the transmembrane, detergent-free, form of P-selectin showed that it can act as an anti-inflammatory agent that inhibits C18-mediated adhesion of tumour necrosis factor-c~ CI~F-ce)-activated neutrophils to resting endothelial cells and the generation of superoxide anions (Gamble et al., 1990; Wong et al., 1991). Neutrophils play an important role in destroying microorganisms via the generation of superoxide anions. However, neutrophil superoxide anion generation can also induce heavy tissue damage at sites of inflammation. It appears that the soluble or transmembrane form of Pselectin, present on platelet microparticles, may play an important role in preventing drastic damage to the vascular system by unaccountable activation of circulating neutrophils.

4.5

PLATELET-T

LYMPHOCYTE

INTERACTIONS In the work of Damle et al. (1992), CD4 § T lymphocytes that have been antigen primed and chronically stimulated, but not freshly isolated cells, bind to a fusion protein of the extracellular domains of P-selectin. de Bruijine-Admiraal et al. (1992), described the binding of a subclass of T lymphocytes (NK cells and an undefined subpopulation of T lymphocytes present in the CD4 § CD8 § subclass) to activated platelets. Moore and Thompson (1992) specifically implicated memory, cells (CD45RO +), in the CD4 § CD8 § subpopulation, in binding to isolated P-selectin. In addition, these authors observed the binding of 12.2 + 4.1% peripheral blood lymphocytes (CD4 § CD8 § and CD16 § to isolated Pselectin. In contrast, a number of teams working in the field have reported no binding of lymphocytes or human cell lines with T (Jurkatt cells) or B lymphocyte-like characteristics (Daudi cells) to activated platelets, Pselectin incorporated in phospholipid vesicles or coated on plastic. One should note that neutrophils, monocytes and human monocyte-like cell lines (U937, HL60), but not lymphocytes, bound to P-selectin incorporated in phospholipid vesicles or coated on plastic (Larsen et al., 1989; Gamble et al., 1990; Geng et al., 1990; Rinder et al., 1991a, b). Purified 12SI-labelled P-selectin bound to neutrophils, monocytes and at very low levels (possibly to contaminating monocytes) to unfractionated lymphocytes but not to a lymphoblast cell line (MOLT 4; Moore et al., 1991). Differences between these observations may be due to the fact, as pointed by Damle et al. (1992), that CD4 + T lymphocytes have to be antigen primed and

chronically stimulated to bind to P-selectin expressed by platelets. Alternatively, T lymphocyte binding to Pselectin may have remained undetected due to a specific subclass, representing a small percentage of the total T lymphocyte population, being involved in such interactions. Neuraminadase treatment, as previously indicated for neutrophils, or the presence of EDTA, soluble sulphated glycan dextran sulphate, fucoidan or heparin, nearly completely inhibits the interaction of CD4 § lymphocytes with P-selectin (de Bruijine-Admiraal et al., 1992; Damle et al., 1992; Moore and Thompson, 1992). Interestingly, P-selectin in tandem with anti-T cell receptor mAbs upregulated the synthesis of granulocytemacrophage colony stimulating factor (GM-CSF) and affected interleukin-8 (IL-8) production by antigenprimed T lymphocytes without having any effect on TNF-ce production (Damle et al., 1992). The overall data presented by the work of de BruijineAdmiraal et al. (1992), Damle et al. (1992), Moore and Thompson (1992) and other workers in this field strongly suggest that activated P-selectin, expressed by activated platelets (or endothelial cells), may be involved in the recruitment of a specific subpopulation ofT lymphocytes at sites of vascular injury, inflammation and interactions with organisms foreign to the host.

4.6

TRANSCELLULAR

SYNTHESIS OF

MOLECULES Monoclonal antibodies directed against P-selectin expressed by platelets, activated by a Dacron graft implanted in an arteriovenous shunt model in baboons, inhibit leucocyte adhesion and the layering of fibrin over the thrombus (Palabrica et al., 1992). This work strongly suggests that under in vivo conditions P-selectin present on the thrombus upregulates tissue factor expression by adhering monocytes, which in turn will initiate fibrin deposition and actively take part in thrombogenesis. Moreover, neutrophils binding to platelets via P-selectin may be linked with the synthesis and conversion of molecules, such as 12-HETE to 12,20-diHETE, that depend on the presence of both cell types (Marcus et al., 1990). In addition, the production of ether-linked phospholipid platelet activating factor (PAF), released by activated platelets or neutrophils in small amounts, can be greatly increased in a mixture of both cell types (Chignard et al., 1980; Lynch and Henson, 1986). In fact, the amount of PAF-acether, which is an important mediator of thrombosis and inflammation, can be doubled when produced jointly by platelets and neutrophils as opposed to each cell separately (Coi~flier et al., 1990). It thus appears that the platelet-neutrophil partnership, involving P-selectin, may be linked with the transcellular production of molecules involved in haemostasis, inflammation, wound healing and thrombosis. However, great care has to be taken in assuming that one receptor, such

74 J.L. MCGREGOR as P-selectin, is involved on its own in the transcellular production of molecules in such cell-cell interactions. Such cell to cell contact was also shown to take place via fibrinogen binding to GPIIb-IIIa on platelets (see Fig. 4.2) and presumably to the CD11/CD18 integrins on neutrophils (Ruf et al., 1992). In these last observations, ADP-activated platelets binding fibrinogen in heparin-rich plasma, under conditions where no release takes place, have been observed to induce an oxidative burst (see Fig. 4.3) in neutrophils (Raaf et al., 1992).

4.7

PLATELET P-SELECTIN IN OTHER SPECIES

Monoclonal antibody LYP20 directed against human platelet P-selectin is the first antibody to be shown to recognize P-selectin on activated rat platelets (Winocour et al., 1992), using ELISA, immunoprecipitation, Western blot and flow cytometry techniques. The glycoprotein band immunoprecipitated by LYP20, from labelled rat platelet lysate, had the same apparent molecular weight as that observed for human platelets. The LYP20 epitope on rat P-selectin is disulphide bridge dependent, as in human platelets, and is not recognized by $12 (another anti-P-selectin mAb; McEver and Martin, 1984). The number of LYP20 binding sites on activated rat platelets (3875 + 750 molecules/platelet) is much higher than that observed on resting platelets (645 + 240 molecules]platelet; Winocour et al., 1992). It appears that LYP20 and $12 are directed against different determinants on P-selectin and that the LYP20 determinant, but not the $12, is preserved on rat P-selectin. LYP20 or its F(ab')2 fragments not only inhibited rosetting of thrombin-activated platelets to U937 cells but also platelet-platelet interaction or aggregation of platelets induced by agonists such as collagen (by 60%) or thrombin (by 50%; Parmentier et al., 1991). Moreover, LYP20 inhibited the interaction of rat or human neutrophils with thrombin-activated rat platelets (Chignier et al., 1993) and the adhesion of leucocytes to the activated HUVEC (Murphy et al., 1993). This inhibitory effect of LYP20, observed on the aggregation of washed platelets or platelets in PRP, had no effect on the binding of fibrinogen to the GPIIb-IIIa complex. The present data, using LYP20, indicate that P-selectin in addition to its role in mediating platelet-leucocyte and endothelial cell-leucocyte interactions may play a certain role in cementing platelet-platelet aggregates (Parmentier et al., 1991). It remains to be seen at what stage of platelet activation P-selectin and other surface or agranule GPs are involved. In that respect it is important to note the work ofW.M. Isenberg, ILP. McEver, Y.V. Jacques and D.F. Bainton (unpublished observations) which shows that P-selectin is the only adhesion molecule found, 15 min after thrombin stimulation, in areas of contact ofplatelets having i~eversibly aggregated. Another rat anti-P-selectin mAb (PB1.3) wasshown by

Mulligan et al. (1992), to protect rat lungs from acute neutrophil-induced injury following systemic complement activation by infusion of cobra venom factor. Presumably PB1.3, by binding to P-selectin, prevents neutrophil adhesion and subsequent extensive damage to activated rat pulmonary lung endothelial cells (Mulligan et al., 1992). The role of activated platelets expressing Pselectin in such a rat lung injury model, remains to be elucidated. Interestingly, PB1.3 mAb was observed to inhibit the interaction of activated rat, rabbit or human platelets with neutrophils. Sequencing of the isolated rat P-selectin cDNA shows extensive homology with the lectin and EGF-like regions of human P-selectin (Sparagano et al., 1993). Mouse P-selectin shows important homologies, but also differences, to human selectin in the complement binding domain regions (Weller et al., 1992).

5. Cytokines and Platelets Platelets activated by a number of agonists, such as thrombin, adrenaline, collagen or ADP, express IL-1 activity (Hawrylowicz et al., 1989, 1991). This proinflammatory cytokine (IL-1) is not released in the supernatant following activation but remains associated with the platelet surface. It remains to be seen ifIL-1 is an integral constituent of the membrane of a granules or other organelles that fuses with the cytoplasmic platelet membrane following activation and release. Hawrylowicz et al. (1989) washed and resuspended platelets at a low calcium concentration and in doing so may have induced platelet release even by a mild agonist such as ADP. Experiments were performed by Hawrylowicz et al. (1989) to show that IL-1 expressed by activated platelets was potent enough in replacing fluid phase IL-1 needed by a T lymphocyte cell line (D10.G4.1) for growth. Moreover, platelet-bound IL-1 significantly upregulated the expression of ICAM-1 in cultured endothelial cells, derived from HUVEC or saphenous veins (SAVEC), and induced the release of IL-1, IL-6 and GM-CSF. Surprisingly, platelet-bound IL-1 induced the expression of ELAM-1 in SAVEC but not in HUVEC. This heterogeneity in the expression of ELAM-1 might reflect the foetal origins of HUVEC compared to SAVEC. Inhibition of such platelet-bound IL-1 biological activity was clearly demonstrated using mAbs directed against IL-la and IL-1/~ (Hawrylowicz et al., 1989, 1991). Resting platelets were observed to be activated through the action of inflammatory cytokines [IL-1B and interferon-3" (IFN3')] and to bind intensely to a monocytic cell line (U937). This binding of platelets to U937 appears to be mediated by P-selectin since an mAb directed against this antigen or EDTA inhibited this interaction. Inflammatory cytokines such as IL-1B and IFN-3, act in synergy with traditional platelet agonist by potentiating the release of serotonin (5-hydroxytryptamine; 5-HT) in the presence

ROLE OF HUMAN PLATELET MEMBRANE RECEPTORS IN INFLAMMATION 75 of a low concentration of thrombin (Todoroki et al., 1991). In contrast, addition of IL-2 to whole blood inhibits platelet aggregation but induces their degranulation. The effect of IL-2 on platelets appears to be indirectly mediated by the presence of mononuclear cells secreting an enhanced amount of an eicosanoid, thromboxane B2 (TXB2) as a result of IL-2 treatment (Oleksowicz et al., 1991). Thus platelets appear not only to be capable of expressing a cytokine (IL-1) on their activated surface but also to have specific receptors for lymphokines, such as IFN--), (Molinas et al., 1987), and be stimulated by inflammatory molecules (IL-lfl and IFN-3,).

6. Thrombospondinand CD36 6.1

A MULTIFUNCTIONAL ADHESIVE PROTEIN

Thrombospondin (TSP) is a multifunctional, calciumsensitive GP first detected in human blood platelets. This 450 kD high molecular weight (MW) GP is secreted from c~ granule upon platelet stimulation (see Fig. 4.1). TSP is synthesized by a large number of cells and is implicated in a variety of cell-cell and cell-matrix interactions. In addition, TSP interacts with heparin, fibrinogen, fibronectin, collagen, histidine-rich GP, plasminogen and thrombin (Lawler, 1986; Frazier, 1987). Research performed over the past decade indicates that TSP plays an important role in haemostasis and inflammation. Several lines of evidence support the role of plateletbound TSP in inflammation. (1) TSP bound to activated platelets, or in solution, has a lectin-like activity. Such a lectin-like activity is implicated in platelet-platelet and platelet-leucocyte interactions, as shown by P-selectin and possibly other secreted platelet GPs (see Fig. 4.2). (2) A number of platelet membrane GPs [CD36, GPIIb-IIIa (OtXIbfl3), GPIa-IIa (Oe2fll), VnR-IIIa (av/3S)] bind released TSP to the surface of activated platelets. The above platelet receptors (GPIIb-IIIa, GPIa-IIa, CD36) have been shown to play a critical role in platelet functions. (3) Monocytes and neutrophils have specific receptors to bind TSP. (4) Monoclonal or polyclonal antibodies directed against TSP or CD36 (also known as GPIIIb or GPIV), one of the TSP platelet receptors on the platelet or monocyte surface, inhibit TSP binding and platelet-leucocyte interactions. On the activated platelet surface TSP is co-localized, using immunoelectron microscopy techniques, with GPIIb-IIIa, fibrinogen and CD36. (5) Platelets, by interacting with monocytes, appear to control the expression of tissue factor activity and TSP synthesis by monocytes. In addition to monocytes or neutrophils a number of cells, such as smooth muscle cells and epithelial cells, synthesize TSP to raise its levels at sites of injury or inflammation. Levels of TSP at

the site of injury are probably greatly increased by the ability of TSP to directly interact with fibrinogen, plasminogen activator, plasminogen, histidine-rich GP (Lawler, 1986; Frazier, 1987) and platelet or monocyte CD36.

6.2

BINDING

OF

ACTIVATED

TSP

TO RESTING OR

PLATELETS

Resting platelets express a limited number of binding sites for TSP (200 to 3000/platelet; Lawler, 1986; Frazier, 1987; Aiken et al., 1986, 1987a, b; Wolff et al., 1986; Legrand et al., 1988; Boukerche and McGregor, 1988). Endogenous TSP present on resting platelets, originating from a small number of activated platelets having released their c~ granule content, binds via its N-terminal region to proteoglycans or sulphated glycolipids (Roberts et al., 1985). Binding of TSP to resting platelets is cation independent, and is inhibited by an mAb (A2.5) directed against the N-terminal heparin-binding region of TSP and by heparin or fucoidin (Gartner et al., 1978, 1980; Roberts et al., 1985; Aiken et al., 1987b). The N-terminal region is known to be involved in the lectin-like activity of TSP (Gartner et al., 1978, 1980) and may be involved, as observed by Legrand et al. (1991), in mediating the interaction of TSP with CD36 and fibrinogen. However, Catimel et al. (1992) in recent work indicated that CD36, does not interact with the isolated N-terminal heparin-binding region ofTSP. A much larger number of binding sites for endogenous (16 000 to 60 000 molecules/platelet) or exogenous TSP (15 000 to 36 000 molecules/platelet), in the presence of calcium and magnesium, are observed on thrombin-activated platelets compared to resting platelets. TSP binds to thrombin-stimulated platelets in a cation-dependent, temperature-sensitive, rapid and saturable fashion (Aiken et al., 1986, 1987a, b; Wolff et al., 1986; Boukerche and McGregor, 1988). The affinity of exogenous TSP for activated platelets is lower (Ka 250nM) than that observed for resting platelets (Ka 50 nM). The differences observed in the number of TSP binding sites, affinity and the presence of cations, between resting and stimulated secreting platelets, indicate the presence of at least two different TSP receptors. The high affinity receptor present on resting platelets can, unlike its low affinity counterpart, bind TSP in the presence of EDTA (Leung, 1984; Aiken et al., 1986, 1987a, b; Wolff et al., 1986; Boukerche and McGregor, 1988). The very large number of endogenous or exogenous TSP molecules present on the activated platelet surface probably indicates the capacity of TSP to interact with c~granule adhesive proteins expressed on the platelet surface as well as with membrane receptors (Lawler, 1986; Frazier, 1987). The ability of TSP to bind to a large number of adhesive proteins appears to also hold true for its capacity to interact with a wide number of platelet membrane GPs.

76 J.L. MCGREGOR

6.3

TSP

RECEPTORS ON THE

PLATELET SURFACE The integrin ~Hb#S (GPIIb-IIIa) was initially implicated as the platelet TSP receptor by observations indicating that anti-GPIIb/IIIa mAbs (Plow et al., 1985) or fibrinogen (Marguerie et al., 1987) affected TSP binding. In addition, this hypothesis was further confirmed by the anti-TSP polyclonal antibody Fab that altered the fibrinogen affinity to GPIIb-IIIa (Leung, 1984). Karczewski etal. (1989), but not Leung and Nachman (1982) or Pytela et al. (1986), observed direct interaction between GPIIb-IIIa and TSP. Subsequently it was shown that activated platelets from patients with Glanzmann thrombasthenia, showing an absence or a severe reduction of GPIIb-IIIa, bound TSP in a normal way (Aiken et al., 1986; Boukerche and McGregor, 1988). The vitronectin receptor (C~v#S), predominantly present on endothelial cells, was shown to bind TSP (Lawler et al., 1988). CD36, a major GP present on the platelet surface, has emerged as one of the TSP receptors. Several lines of evidence point to CD36 as a TSP receptor. (1) Anti-CD36 monoclonal or polyclonal antibodies inhibit TSP binding and platelet aggregation (McGregor et al., 1989; Kieffer et a/., 1989; Beiso et al., 1990; Legrand et al., 1991; Asch et al., 1992). (2) Purified platelet CD36 interacts with TSP in a calcium-dependent and independent manner (Leung, 1984; Asch etal., 1992). (3) Jurkat cells, transfected with CD36 cDNA, bound TSP in a specific and saturable way (Asch et al., 1992). (4) A peptide (CSVTCG) derived from the type I repeat region of TSP interacted with CD36. However, individuals in the Japanese population who have platelets that are deficient in CD36 (Nak a-) showed normal TSP binding (Kehrel et al., 1991). Moreover, another integrin (GPIa-IIa) has also been implicated as a TSP receptor (Tusynski and Kowalska, 1991). It appears that TSP is capable of interacting with a number of platelet membrane receptors; absence of one of them does not seem to prevent TSP from cross-linking adhesive proteins and membrane receptors involved in platelet functions.

6.4

SITE ON

TSP

INTERACTING WITH

THE PLATELET SURFACE TSP is known to interact with the platelet surface via its COOH-terminal domain and its N-terminal domain binds to heparan sulphate proteoglycans (Lawler, 1986; Frazier, 1987; Aiken et al., 1987b). Cleavage by chymotrypsin of the N-terminal (27 kD) domain of TSP leaves a fragment of 140 kD, still bearing the COOHterminal domain, capable of interacting with purifed CD36 in the presence of calcium (Leung, 1984; Lawler, 1986; Frazier, 1987). The interaction of the TSP fragments with CD36 is considerably reduced, but still present, at low calcium concentrations. Such reduction may be the result of the COOH ball-like terminal of TSP

unfolding in the presence of EDTA and losing its binding properties. Catimel et al. (1992), in an attempt to understand why a certain amount of TSP still bound to CD36 in the presence of EDTA, further treated the 120-180 kD TSP piece to obtain a 68 kD TSP fragment lacking the COOH-terminal domain. This fragment was observed to bind to TSP in the presence of EDTA. Moreover, peptide motifs present on the 68 kD fragment, representing the type I repeats, bound to CD36 and inhibited TSP interaction with CD36. In addition, 12SI-labelled YCSVTCG peptide was observed to bind to Jurkat cells transfected with CD36 cDNA. The hexapeptide CSVTCG inhibited TSP binding to activated platelets and reduced platelet aggregation (Asch et al., 1992; Tusynski et al., 1992; Byk and McGregor, 1993). These results suggest that two binding sites are present on TSP, in addition to the N-terminal domain, one present on the COOH domain that is calcium dependent, and the other on the type I repeat that is calcium independent.

6.5

T S P AS A L I G A N D T O PLATELET-MONOCYTE INTERACTIONS

TSP bound to activated platelets via CD36 appears, according to the work of Silverstein and Nachman (1987), Silverstein etal. (1989) and Asch etal. (1987), to act as a ligand allowing the rosetting of activated platelets to monocytes or to a human monocytoid cell line (U937). Glycoprotein CD36 is expressed by platelets, monocytes, endothelial cells and certain tumour cell lines (Asch et al., 1987). An anti-CD36 mAb when incubated with activated platelets, monocytes or U937 cells prevented the rosetting of platelets. Monocytes in direct contact with whole platelets (see Fig. 4.3), but not in their absence, appear to be capable of TSP synthesis (Schwartz, 1989). Monocytes isolated in the absence of platelets will synthesize TSP at levels that are barely detectable by polyclonal or monoclonal antibodies. In contrast, these monocytes show increased TSP synthesis when subjected to increasing number of washed platelets (Schwartz, 1989). Monocytes exposed to platelet lysates show a decrease in synthesized TSP levels. Interestingly, monocytes in the absence of platelets show a reduced level of tissue factor activity compared to those in the presence of platelets (Niemetz and Marcus, 1974). It would thus appear that platelets gathered at a site of injury might bind monocytes via CD36-TSP and/or Pselectin. In addition, by binding platelets monocytes will be greatly influenced to express a higher level of tissue factor activity and synthesize TSP at sites of injury or inflammation. Platelets, by having very high levels of TSP in their ~ granules, may greatly contribute with leucocytes and other cells, to raising the levels of TSP at such sites.

ROLE OF HUMAN PLATELET MEMBRANE RECEPTORS IN INFLAMMATION 77 Such results strongly suggest that platelets, via CD36 and secreted TSP, may modulate the role of monocytes in inflammation, atherosclerosis, and thrombosis. This linkage between platelets and monocytes, involving CD36 and TSP, appears to serve monocytesmacrophages in recognizing neutrophils undergoing apoptosis. Such recognition helps macrophages, using simultaneously CD36 and C~v~3 integrins present on their surface, to bind TSP, in clearing senescent neutrophils at sites of inflammation (Savill et al., 1992). Moreover, TSP bound to human macrophages has also been shown to mediate the killing of squamous epithelial cells (Riser et al., 1989). The TSP receptor on neutrophils appears to be linked to the CD11/CD18 complex (Nathan et al., 1989). TSP released from activated platelets or endothelial cells may be vital for neutrophils in the process prior to extravasation (Suchard et al., 1991). Neutrophils are also capable of synthesizing TSP (Riser et al., 1989; Kreis et al., 1989). Neutrophils activated by N-formyl-methionylleucyl-phenylalanine (FMLP) may be primed by TSP or laminin to generate an oxidative burst. TSP, in addition to P-selectin, helps to modulate neutrophil oxidative metabolic behaviour by binding to specific receptors on polymorphonuclear neutrophils (PMN; Boxer et al., 1990).

6.6

BINDING

OF PLATELETS TO

BACTERIA VIA TSP Staphylococci and streptococci are known to interact with platelets and induce release and aggregation (Clawson et al., 1980). Moreover, binding of staphylococci and streptococci to fibrin is greatly amplified in the presence of platelets. Recent work by Herrmann et al. (1991) has shown that TSP, present on the activated platelet surface or in solution, mediates staphylococcal adherence by binding Staphylococcus aureus.

7. Platelet Factor 4 PF4 bound to a proteoglycan carrier is released from platelets upon activation (see Fig. 4.1) with agonists such as thrombin, collagen or ionophores (Rybak et al., 1989). The PF4 cDNA was isolated and sequenced by Poncz et al. (1987) using an HEL cell line. It shows a high homology to two of its precursors, platelet basic protein and low affinity platelet factor 4 (LAPF4), as well as to B-thromboglobulin (B-TG; Majumdar et al., 1991; Brandt and Flad, 1992). PF4 has structural and biological homology to a new supergene family of proinflammatory cytokines secreted at very low levels and known as intercrines (Oppenheim et al., 1991). This supergene cytokine family includes

neutrophil-activating peptide 1 (NAP-1/IL-8), interferon-gamma inducible protein (IP-10), monocyte chemotactic and activating factor (MCAF), and melanoma growth stimulatory activity (GRO/MGSA). B-TG, connective tissue-activating peptide III (CTAP-III) or neutrophil-activating peptide 2 (NAP-2) and IL-8 are molecules of different sizes obtained by proteolytic cleavage of a common precursor polypeptide known as platelet basic protein (PBP; Brandt and Flad, 1992). The intercrine family can be divided into two subgroups, differing in the presence of additional amino acids between two cysteines or in the immediate vicinity, known either as CXC and CC subgroups, or intercrine c~ and intecrine B (Sager, 1990; Oppenheimet al., 1991). IL-8, PF4, B-TG, GRO]MGSA and IP-10 belong to the CXC or intercrine a subgroup (Sager, 1990; Oppenheim et al., 1991). One of the very interesting properties of PF4, unique to the intecrine group, is its ability to neutralize heparinlike molecules on vascular endothelial cells (Lane et al., 1986; Rybak et al., 1989; Brandt and Flad, 1992). PF4 appears to be very active at certain stages of wound healing as shown by its strong chemotactic effect on fibroblasts and the release of H by basophils (Senior et al., 1983; Brindley et al., 1983; Brandt and Flad, 1992). PF4 is also involved in immunoregulatory activity by preventing the function or induction of T lymphocyte suppressor cells (Gregg et al., 1990). The specialized role of PF4 in tissue repair is also shown by its capacity to activate leucocyte elastase, inhibit collagenase, neovascularization and the proliferation of endothelial cells (Hiti-Harper et al., 1978; Lonky and Wohl, 1981; Brandt and Flad, 1992). In contrast to other CXC intecrines, such as IL-8 and NAP-2, PF4 has a weak chemotactic activity on monocytes and neutrophils (Brandt and Flad, 1992). However, PF4 has been shown, together with PDGF and PGE2, to be involved in stimulating the synthesis of chondroitin sulphate proteoglycan (see Fig. 4.3) in human monocytes (UhlinHansen et al., 1992). Moreover, PF4 is looked upon as one of the substances secreted by platelets that activate eosinophils and as a result is involved in allergic reactions (Burgers et al., 1993). PF4 is known to bind to the surface of activated platelets (Capitanio et al., 1985). The capacity of activated platelets to release PF4 at sites of injury, and express PF4 at their surface indicates a further link implicating platelets in the inflammation and wound healing process.

8. Acknowledgements Supported by ARC (subvention 6586), Ligue Nationale Franqaise contre le Cancer, MGEN. Mrs Isabelle Vastico (INSERM U331) is warmly thanked for secretarial support, as is Mrs Josiane Laignel (CIMAC) for helping in producing a number of the figures.

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E.F., Conway, T.M. and Schwartz, E. (1987). Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line. Blood. 69, 219-223. Pytela, IL, Pierchbacher, M.D., Ginsberg, M.H., Plow, E.F. and Ruoslahti, E. (1986). Member of a family of arg-gly-asp specific adhesion receptors. Science 231, 1559-1561. Rinder, H.M., Bonan, J.L., Rinder, C.S., Ault, K.A. and Smith, B.R. (1991a). Dynamics of leukocyte-platelet adhesion in whole blood. Blood 78, 1730-1737. Rinder, H.M., Bonan, J.L., Rinder, C.S., Ault, K.A. and Smith, B.tL (1991b). Activated and unactivated platelet adhesion to monocytes and neutrophils. Blood 78, 1760-1769. Riser, B.L., Mitra, IL, Perry, D., Dixit, V. and Varani, J. (1989). Monocyte killing of human squamous epithelial cells: role for thrombospondin. Cancer Res. 49, 6123-6129. Roberts, D.D., Haverstick, D.M., Dixit, V.M., Frazier, W.A., Santoro, S.A. and Ginsburg, V. (1985). The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids J. Biol. Chem. 260, 9405-9411. Roth, G.J. (1992). Platelets and blood vessels: the adhesion event. Immunol. Today 13, 224-230. Ruf, A., Schlenk, R~F., Maras, A., Morgenstern, E. and Patscheke, H. (1992). Contact-induced neutrophil activation by platelets in human cell suspensions and whole blood. Blood 80, 1238-1246. Rybak, M.E., Gimbrone, M.A. Jr., Davies, P.F. and Handin, R~I. (1989). Interaction of platelet factor 4 with cultured vascular endothelial cells. Blood 73, 1534-1539. Sager, R~ (1990). GRO as a cytokine. In: "Progress in leukocyte biology: Molecular and cellular biology of cytokines" (eds. J.J. Oppenheim, M.C. Powanda, M.J. Kluger and C.A. Dinarello). 10A, 327-332, Wiley-Liss, New-York. Savage, B., Marzec, U.M., Chao, B.H., Harker, L.A., Maraganore, J.M. and Ruggeri, Z.M. (1990). Binding of the snake venom-derived proteins applagin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb-IIIa complex inhibits receptor functions. J. Biol. Chem. 265, 11766-11772. Savill, J., Hogg, N., Ren, Y. and Haslett, C. (1992). Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J. Clin. Invest. 90, 1513-1522. Scheld, W.M., Valone, J.A. and Sande, M.A. (1978). Bacterial adherence in the pathogenesis of endocarditis. J. Clin. Invest. 61, 1394-1404. Schwartz, B.S. (1989). Monocytes synthesis of thrombospondin: The role of platelets. J. Biol. Chem. 264, 7512-7517. Senior, R.M., Griffin, G.L., Huang, J.S., Walz, D.A. and Deuel, T. (1983). Chemotactic activity of platelets alpha granule proteins for fibroblasts. J. Cell Biol. 96, 382-385. Shimizu, Y., Newman, W., Tanaka, Y. and Shaw, S. (1992). Lymphocyte interactions with endothelial cells. Immunol. Today, 13 (3), 106-112. Silverstein, ILL. and Nachman, ILL. (1987). Thrombospondin binds to monocytes-macrophages and mediates plateletmonocyte adhesion. J. Clin. Invest. 79, 867-874. Silverstein, ILL., Asch, A.S. and Nachman, ILL. (1989). Glycoprotein IV mediates thrombospondin-dependent platelet-monocyte and platelet-U937 cell adhesion. J. Clin. Invest. 84, 546-552.

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Skinner, M.P., Fournier, D.J., Andrews, ILK., Gorman, J.J., Chesterman, C.N. and Berndt, M.C. (1989). Characterization of human platelet GMP-140 as a heparin-binding protein. Biochem. Biophys. Res. Commun. 164, 1373-1379. Skinner, M.P., Lucas, C.M., Burns, G.F., Chesterman, C.N. and Berndt, M.C. (1991). GMP-140 binding to neutrophils is inhibited by sulphated glycans. J. Biol. Chem. 266, 5371-5374. Sparagano, M-H., Chignier, E., ThiUier, A., Gayet, O. and McGregor, J.L. (1993). Isolation and sequencing of rat Pselectin cDNA. Thromb. Haemost. 69, 568. Springer, T.A. (1990). Adhesion receptors of the immune system. Nature, 346, 425-434. Springer, T.A. and Lasky, L.A. (1991). Sticky sugars for selectins. Nature 349, 196-197. Sternberg, P.E., McEver, ILP., Shuman, M.A., Jacques, Y.V. and Bainton, D.F. (1985). A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J. Cell Biol. 101,880-886. Suchard, S.J., Burton, M.J., Dixit, V.M. and Boxer, L.A. (1991). Human neutrophil adherence to thrombospondin occurs through a CDll/CD18-independent mechanism. J. Immunol. 146, 3945-3952. Taylor, M.E., Conary, J.T., Lennartz, M.IL, Stahl, P. and Drickamer, K. (1988). Primary structure of the mannose receptor contains multiple motifs resembling carbohydraterecognition domains. J. Biol. Chem. 265, 12156-12162. Todderud, G., Alford, J., MiUsap, K.A., Aruffo, A. and Tramposch, K.M. (1992). PMN binding to P-selectin is inhibited by sulfatide. J. Leukoc. Biol. 52, 85-88. Todoroki, N., Watanabe Y., Akaike, T., Katagiri Y., Tanoue K., Yamazaki H., Tsuji T., Toyoshima S. and Osawa T. (1991). Enhancement of IL-lb and IFN-gof platelet activation: adhesion to leukocytes via GMP-140/PADGEM protein (CD62).. Biochem. Biophys. Res. Commun. 179, 756-761. Turner, S.IL, Tainer, J.A. and Lynn, W.S. (1975). Biogenesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature 257, 680-683. Tusynski, G.P. and Kowalska, M.A., (1991). Thrombospondin-induced adhesion of human platelets. J. Clin. Invest. 87, 1387-1394. Tusynski, G.P., Rothman, V.L., Deutch, A.H., Hamilton, B.K. and Eyal, J. (1992). Biological activities of peptides and peptide analogues derived from common sequences present in

thrombospondin, properdin and malarial proteins. J. Cell Biol. 116, 209-217. Uhlin-Hansen, L., Langvoll, D., Wik, T. and Kolset, S.O. (1992). Blood platelets stimulate the expression of chondroitin sulfate proteoglycans in human monocytes. Blood 80, 1058-1065. Wahl, S.M., Hunt, D.A., Wakefield, L.M., McCartneyFrancis, N., Wahl, L.M., Roberts, A.B. and Sporn, M.B. (1987). Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc. Natl. Acad. Sci. 63, 943-945. Watson, M.L., Kingsmore, S.F., Johnston, G.I., Siegelman, M.H., Le Beau, M.M., Lemons, ILS., Bora, N.S., Howard, T.A., Weissman, I.L., McEver, ILP. and Seldin, M.F. (1990). Genomic organization of the selectin family of leukocyte adhesion molecules on human and mouse chromosome 1. J. Exp. Med. 172, 263-272. Weis, W.I., Kahn, IL, Fourme, IL, Drickamer, K. and Hendrickson, W.A. (1991). Structure of the calciumdependent lectin domain from a rat mannose-binding protein determined by MAD phasing. Science 254, 1608-1615. Weller, A., Isenmann, S. and Vestweber, D. (1992). Cloning of the mouse endothelial selectins: expression by both E- and Pselectin is inducible by tumor necrosis factor ~. J. Biol. Chem. 267, 15176-15183. White, tLT, Damm, D., Miller, J., Spratt, K., Schilling, J., Hawgood, S., Benson, B. and Cordell, B. (1985). Isolation and characterization of the human pulmonary surfactant apoprotein gene. Nature 317, 361-363. Winocour, P.D., Chignier, E., Parmentier, S. and McGregor, J.L. (1992). A member of the selectin family (GMP140/PADGEM) is expressed on thrombin-stimulated rat platelets in vitro. Comp. Biochem. Physiol. 102A, 265-271. Wolff, IL, Plow, E.F. and Ginsberg, M. H. (1986). Interaction of thrombospondin with resting and stimulated human platelets. J. Biol. Chem. 261, 6840-6846. Wong, C.S., Gamble, J.IL, Skinner, M.P., Lucas, C.M., Berndt, M.C. and Vadas, M.A. (1991). Adhesion protein GMP-140 inhibits superoxide anion release by human neutrophils. Proc. Natl. Acad. Sci. 88, 2397-2401. Zimmerman, G.A., Prescott, S.M. and Mcintyre, T.M. (1991). Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol. Today 13, 93-100.

0

Platelets In " Bacterial Infecti OIlS C.C. Clawson

1. Introduction 2. Platelet Interaction with Non-biological Particulates 2.1 Clearance of Particulates from the Circulation 2.2 Engulfment of Inert Particles: Phagocytosis or Sequestration? 2.3 Influence of Particle Size 2.4 Soluble Co-factors of Particle Uptake 2.5 Metabolism During Ingestion of Inert Particulates 2.6 Platelet Secretion and Aggregation in Response to Inert Particles 3. Platelet Interaction with Bacteria in vitro 3.1 Aggregometry 3.2 Morphology 3.3 Influence of Plasma Components 3.4 Varied Responses to Different Bacteria 3.5 Bacterially Induced Platelet Secretion 3.6 Mechanisms of Adhesion and Activation 3.6.1 Strep. sanguis Adhesion 3.6.2 Platelet Aggregation by

Strep. sanguis

83 84 84 4. 86 87 5. 88 88 89 89 90 92 94

6.

98 100 101 101

101

7. 8.

3.7 Engulfment of Bacteria by Platelets 3.8 Fate of the Bacteria 3.9 Bacterial Products that Promote or Inhibit Platelet Activation Platelet Interaction with Bacteria in vivo 4.1 Bacterial Clearance from the Circulation Influence of Platelets on Phagocytes 5.1 Morphological Observations 5.2 Phagocytosis and Killing of Bacteria 5.3 Chemotaxis 5.4 Phagocytosis of Platelets Implications of Platelet-Bacterial Interaction to Human Disease 6.1 Inflammation and Tissue Injury 6.1.1 Bacterial Endocarditis 6.1.2 Adult Respiratory Distress Syndrome 6.2 Thromboembolic Disorders and Disseminated Intravascular Coagulation 6.3 Atherosclerosis 6.4 Thrombocytopenia Summary References

103 104 107 107 108 109 109 109 110 110 110 112 112 114

114 115 115 115 116

3.6.3 Ecto-ATPase of Strep.

sanguis

i.

102

Introduction

Platelets have three general characteristics that have led to their identification as a host defense cell and being likened to a special form of leucocyte. These characteristics are their propensity to interact with pathogenic organisms, their content of lysosomal products that are released on Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

stimulation, and their metabolic products that contribute to the pool of inflammatory mediators (Nachman and Weksler, 1980). Since platelets normally reside exclusively in the blood circulatory system, their initial and principal contribution as a host defense cell is likely to be their interactions with particulates in the blood stream, including non-biologic materials, antigen-antibody Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

84

C.C. CLAWSON

complexes, viruses, bacteria, or other micro-organisms. Rogers (1960) noted in his review of the subject that "most organisms are less capable of provoking disease when injected intravenously than when administered by any other route". He concluded that, although specific immunity can have a significant influence on bacteremia, the principal mechanisms for removal of bacteria from the bloodstream probably do not depend on a prior exposure to the micro-organism. The following discussion will focus on the interaction of human platelets with bacterial pathogens and the potential that this interaction has for being a detriment or a benefit to the host.

2. Platelet Interaction with Non-biological Particulates The interaction of platelets with bacteria is a particular facet of the platelet's broader propensity to adhere to most foreign materials, especially when present in blood or plasma as particulates. In the simplest view of platelet-bacterial interaction, bacteria may act merely as a passive particulate to be cleared from the bloodstream by the same mechanisms that remove such inert particulates as carbon or quartz. Therefore, this section will examine the interactions of platelets with non-biological foreign particulates as studied in vivo or in vitro. The role of platelets in clearance of foreign materials from the circulation will be reviewed. This will be followed by a more detailed consideration of the platelet's response to contact with foreign particles, the influence of particle size, the platelet's physical and metabolic responses to particulates, and the role of soluble co-factors in platelet-particle reactions. This presentation will set the stage for the later discussion of platelet interactions with bacteria as potential pathogens. While the existence of the blood platelet as an independent cell type was still being debated in the nineteenth century (reviewed in Tocantins, 1938), two observers, Vulpian (1873) and Osier (1874, 1886), commented on the platelet's avidity for adhering to foreign particles and surfaces. These early observations were seminal to all subsequent studies of the platelet's participation in clearance of many foreign materials from the bloodstream. Throughout the first half of this century numerous studies appeared purporting to show a role for blood platelets in clearance of inorganic and biological particulates from the circulation. Although it is not the purpose of this discussion to provide a detailed history and analysis of these studies, an overview of this work is relevant to our current understanding of platelet-bacterial interaction. An understanding of platelet interaction with inert particles will allow us to compare and contrast with the more complex process of platelet-bacterial interaction. This in turn will provide a keener insight to the special qualities of the latter process.

2.1

CLEARANCE

OF PARTICULATES

FROM THE CIRCULATION Hayem, in 1882 (cited in Maupin, 1969), probably made the earliest observations of platelets coating a foreign surface, a thread passed through a blood vessel. Osier reported similar observations both in vivo and in vitro in 1886. He saw that when a ligature was passed through the femoral vein of a dog, filaments of the thread became coated with platelets, or as he termed them, "plaques", within 10 min. He further observed that although a few leukocytes might be entangled among the platelets, "undoubtedly the plaques are the first elements to aggregate about such a foreign body" (Osier, 1886). In the intervening decades numerous studies have appeared on the behavior of platelets in contact with inorganic particulates. Tait and Elvidge (1926) made early, carefully detailed studies of the effects of particulates on platelet counts in vivo and on coagulation both in vivo and in vitro. They ground cleaned fused quartz to a powder, separated the powder into coarse (6-7 gm), medium (3-3.5 #m), and fine (< 1.5 gm) grades, and suspended these in isotonic glucose. In this form they injected various doses of the quartz powders into rabbits, cats, guinea-pigs, and frogs. In the rabbits they followed circulating platelet counts and detected profound falls within 3 min of injection with significant recovery of platelet numbers within a few hours. On a per-milligram basis, fine quartz gave much greater falls in platelet counts than larger particles. They found that the great preponderance of the quartz came to reside in the liver, spleen and bone marrow. They concluded that platelets were probably consumed at these sites along with the particulates, although they offered no direct evidence for this. They believed recovery of circulating platelets was "due to the appearance of a new crop". However, Dudgeon and Goadby (1931), who studied drops in platelet counts after injection of India ink, colloidal silver, or Staphylococcus, concluded that the rapid recovery in platelet counts was due to "breaking up of clumps" and not from "a fresh output from bone marrow". Tait and Elvidge (1926) also added quartz powder to plasma in vitro noting that it would accelerate coagulation of plasma when platelets were present but not when they were absent. They further observed that within a few minutes after injection the animal's blood was hypercoaguable, a condition that they attributed to action of the particles on platelets rather than a direct thrombinlike effect. Later the coagulation time was prolonged corresponding to a demonstrated fall in circulating fibrinogen levels. However, even when the dose of quartz was su~cient to kill the animal, they found no evidence of intravascular thrombosis. These authors confirmed their key observations by repeating them with India ink, barium sulphate, and carmine powders to demonstrate that the results were due to reaction of

PLATELETS IN BACTERIAL INFECTIONS 85 platelets to particulates in general and not specifically to the silicates. Subsequent to these experiments of Tait and Elvidge, numerous other investigators studied various intravasculady injected particulates (reviewed in Maupin, 1969). Bloom and Swensson (1958; Bloom, 1954) were the first to employ electron microscopy in the study of platelet-particulate interactions. They examined whole mounts of spread platelets exposed to very fine powders of quartz, titanium dioxide, cobalt, or carbon and found that particles accumulated in or on the surface of platelets, especially in the region of the central chromomere. They noted the "greater ability of the thrombocytes to gather particles" than leucocytes and concluded that "the thrombocytes may have an important function in elimination of solid particles from the blood" (Bloom and Swensson, 1958). Stehbens and Florey (1960) made direct observations of carbon particles in the blood vessels of living rabbits by means of transparent ear chambers. They noted that after some minutes circulating carbon was largely contained within aggregates of platelets. There was a simultaneous disappearance of free floating platelets. Occasional leukocytes were seen in the aggregates and carbon-coated platelets adhered to leukocytes, but the bulk of the carbon was associated with platelets. The carbon-platelet thrombi clung to vessel walls and blocked some of the small vessels temporarily. After about 2 h circulation improved, thrombi became less numerous, and "platelets and leukocytes which had adhered to the vessel walls began to circulate again". These observations of Stehbens and Florey were extended by Copley and Staple (1962) through their elegant biomicroscopic studies of circulating graphite in the microvasculature of hamster cheek pouches. They noted that 1-2 gm carbon particles and platelets tended to flow together at the periphery of vessels separate from the more central column of red cells in accord with the flow principles of Poiseuille. They also found that, when plasma skimming occurred in the smallest capillaries, carbon and platelets tended to accompany the plasma into these vessels. These phenomena appeared to provide abundant opportunity for platelets to come into intimate contact with the injected particulates. The fate of platelets during the thrombocytopenia seen after particulate injection of rabbits was examined by following 32p-labelled platelets after intravenous injection of India ink (Salvidio and Crosby, 1959, 1960). Within 3 min the initial platelet count of 350 000 had fallen to 6000, and was followed by recovery to 336 000 in 18 h. After recovery the amount of radiolabelled platelets was the same as in uninjected control animals. These investigators concluded that the thrombocytopenia was due to a temporary sequestration of platelets and not due to any significant destruction. By the mid-1960s it seemed clear that most if not all fine particulates formed intravascular complexes with

circulating platelets. It was not clear how these platelet-particulate aggregates escaped being permanently trapped in the microvasculature of the diverse organs and causing significant ischemic damage. The microscopic observation of resumption of circulation after temporary small vessel occlusion by plateletparticulate aggregates and the restoration of platelets to the circulation indicated that such aggregates were not permanently trapped in the microvascular bed. It was also clear that the great majority of particles were eventually found in fixed macrophages of the liver, spleen and bone marrow. In the late 1960s van Aken and colleagues (1968, 1969, 1970) provided a unifying theory that gave an appealing explanation of the role of platelets in clearance of particulates from the circulation. These authors manipulated platelet numbers and fibrinolysis in vivo and observed the effect of these manipulations on clearance rates and organ distribution of carbon. They showed that clearance of carbon particles could be enhanced by increased platelet concentration and slowed by manipulations in vivo that decreased the number or reactivity of circulating platelets. When rabbits were transfused with platelet concentrate 15 min into a carbon clearance experiment, the rate of clearance increased during platelet transfusion and resumed its slower pace when the transfusion was stopped. If platelet-poor plasma was used for transfusion no change in clearance rate was noted. Decreasing platelet counts by an infusion of adenosine diphosphate (ADP) started 10 min after injection of carbon reduced the rate of clearance. Inhibition of platelet aggregability was accomplished by infusions of the platelet inhibitors, adenosine monophosphate (AMP) or adenosine (Ado). The van Aken group also examined the role offibrinolysis on carbon clearance (van Aken and Vreeken, 1970) and found that modification of the fibrinolytic system altered both the rate of clearance and the organ distribution of the carbon. Activation of fibrinolysis by infusion of the fibrinolysins, urokinase or streptokinase, caused slowing of carbon clearance during infusions with a return to normal clearance rates when the infusions were stopped. Similarly, intravenous infusion of fibrin degradation products, which promoted platelet-carbon disaggregation in vitro, markedly slowed carbon clearance. Infusions of the fibrinolytic inhibitor, E-aminocaproic acid (EACA), both slowed the rate of clearance and altered the organ distribution of carbon. With EACA present there was a reduction in carbon particles deposited at 2 h in the spleen, a modest increase in the liver, and a severalfold increase in the amount of carbon in the lungs. From these findings and data from others outlined above, van Aken and Vreeken proposed a unifying hypothesis to explain the events that lead to an injected particulate being removed by fixed macrophages. In the scheme of van Aken and Vreeken (Fig. 5.1), when a particulate is introduced to the circulation, platelet-particle aggregation and disaggregation occur repeatedly in the

86 C.C. CLAWSON

Portalsof Entry

Bacterial

Lungs

Disaggregation

Ischemicactivationof thefibrinolyticsystem Platelet-Bacterial Contact

~)~

~I~ ~ _ ~ .

Disaggregation

Non-RES Organs

,

~

~

Aggregation

/]Bacterial ~ uptake by "RESOrgans

L3

Figure 5.1 Diagram of the possible role of platelets and the fibrinolytic system in the clearance of particulates from the bloodstream after the proposed model of van Aken and Vreeken (1970). By this scheme particulates (here illustrated as bacteria) that gain access to the circulation through breaches in the tissue barriers usually first encounter free platelets and adhere. This primary contact results in the formation of microaggregates made up of platelets and particles. These microaggregates are initially carried to the microvasculature of the lung where their size causes them to be trapped by the smallest vessels and locally obstruct blood flow. The resultant ischemla Induces the release of endothelial activators of fibrinolysis. This allows the break up of the aggregate and recirculation of both the platelets and the particles. The process of aggregation and disaggregation is repeated in the microvascular bed of other tissues until the aggregates by chance enter the reticuloendothellal system. There the particles and adherent platelets are removed from the bloodstream by the fixed phagocytes.

microvasculature of various organs. They emphasized that in the non-reticuloendothelial organs, plateletparticle aggregates will cause a local ischemia that is mild and reversible and leads to activation of fibrinolysis by release of plasminogen activator from vascular endothelium (Tagnon et al., 1946; Kwaan and McFadzean, 1956; Warren, 1964). This local fibrinolysis results in disaggregation of the platelet-particle aggregate. Eventually the particles reach the spleen and liver where there are lower levels of plasminogen activator (Astrup and Albrechtsen, 1957), and will be cleared by fixed macrophages. This model provides an explanation for the rapid restoration ofplatelet numbers, the lack ofischemic tissue damage, and the dearth of permanent particle deposition in non-phagocytic organs seen by earlier investigators of in vivo platelet-particulate interaction. It might be argued that if platelets were absent the great

bulk of particulates would probably find their way to the phagocytic system anyway. This might be true, but normally platelets are present and show a great propensity for aggregation with particulates. Therefore, this platelet behavior and the significance of consequences of this behavior are of interest and possible pathological relevance.

2.2

ENGULFMENT OF INERT PARTICLES" PHAGOCYTOSIS OR SEQUESTRATION?

While examining platelet-particulate aggregation several early investigators came to the conclusion that platelets were capable of engulfing or phagocytosing particles (Tait, 1918; Tait and Gunn, 1918; Stehbens and Florey,

PLATELETS IN BACTERIAL INFECTIONS 87 1960). However, light microscopic examination always left room for doubt. As Stehbens and Florey noted, "The platelets had much more carbon in them or attached to them than individual leukocytes". Even electron microscopy of whole platelets was unable to clarify whether the particles were within or merely attached to the platelet (Bloom, 1954; Bloom and Swensson, 1958). It remained for the application of ultrathin sections to show that particulates often gained access to the interior of the cell (Schulz, 1961, 1968; David-Ferreira, 1964). In his electron micrographs of platelets exposed to colloidal thorium dioxide, either in vivo or in v/tr0, David-Ferreira clearly demonstrated particles to be contained within membrane-bound compartments of the platelet interior. He unequivocally attributed to platelets an "intense phagocytic activity". The use of the term, "phagocytic" indicated an assumption that the process of particle internalization by platelets was analogous to, if not identical with, phagocytosis as practiced by leukocytes and macrophages. For some time thereafter most authors on the topic spoke of platelet phagocytosis without caveat or qualification (Movat eta/., 1965; Mustard and Packham, 1968). Electron microscopy also revealed similar internalization by platelets of other non-biological particulates, e.g. silica (Schulz, 1968), colloidal carbon, and latex (Movat et al., 1965). In 1967 Behnke defined by electron microscopy and ultrastructural cytochemistry the surface-connected or open canalicular system (OCS) of the platelet. He showed that this membrane-bounded channel, which courses through the platelet cytoplasm, is continuous with the surface membrane and remains open to the surrounding media. Ultrafine particles such as ferritin or saccharated iron oxide readily gained access to the OCS, often without apparent activation of the platelet. Some electron micrographs of others (David-Ferreira, 1964; White, 1968) showed thorium dioxide in extended narrow channels that closely resembled OCS illustrations of Behnke. This resemblance suggested that some or all particulate matter may enter the platelet interior through the OCS, and not by formation de n0v0 of phagocytic vacuoles. Our understanding of classic phagocytosis includes formation at the cell surface and subsequent membrane closure of the phagocytic vacuole, thereby isolating its contents from both the cell cytoplasm and the cell exterior. It was not clear that this was what happened in platelet-particulate interactions. White (1972) provided an experiment designed to resolve the issue. He exposed unstirred human platelets in their native plasma to fine latex beads allowing extensive internalization of the particles, and then fixed the platelets for electron microscopy. The fixed platelets were incubated with the electron-dense tracer, lanthanum nitrate. Electron microscopy revealed that lanthanum readily gained access to the membrane-bound, latexcontaining spaces within the platelets. As lanthanum does not cross cell membranes even after they are fixed,

it could only have gained entrance to the latex spaces by channels open to the cell exterior. Since the platelets were fully fixed before exposure to lanthanum, internalization of the lanthanum could not be attributed to any action of the platelets. Therefore, White concluded that engulfment of particulates by platelets is largely, if not exclusively, a process of sequestration within the OCS rather than true phagocytosis (see also White and Clawson, 1981, 1982). Lewis and colleagues (1976) followed platelet-latex mixtures for up to 1 h (longer than White had done) by structural and cytochemical electron microscopy. Initially the latex accumulated in the OCS, but by 60 min some particle-enclosing membranes appeared to have lost their connections to the cell surface, becoming closed vacuoles. These vacuoles contained electron-dense material around latex that was not seen accompanying latex in the OCS. The membranes of these vacuoles differed from the membranes of the OCS and the platelet exterior since the vacuoles lacked a surface granular material when sections were stained with periodatealkaline-bismuth, a stain for glycoproteins. The electrondense material of the vacuoles stained positively for the lysosomal marker, acid phosphatase. Based on their findings these authors concluded that platelets can act as a true phagocyte. (This issue will be revisited in regard to internalization of bacteria by platelet in Section 3.7.)

2.3

INFLUENCE

OF PARTICLE

SIZE

Several investigators have presented findings that indicate that the mode of interaction between platelets and inert particulates varies with particle size. Differences in degree of thrombocytopenia in the experiments of Tait and Elvidge (1926) described above might have been due solely to particle numbers, but they noted only modest falls in platelet counts with coarser quartz even though they used more than 20 times the dose by weight. Other studies of particle-induced thrombocytopenia and light microscopic studies of platelet-particle interaction employed substances of the order of 1/~m diameter. This order of size is most relevant to platelet-bacterial interactions. With these particles there was clear activation of platelets (van Aken and Vreeken, 1970). Several electron microscopic studies have shown that very fine particles such as thorium dioxide or ferritin readily enter the OCS without apparent activation of the platelet (DavidFerreira, 1961; Behnke, 1967; White, 1968; Mant and Firkin, 1972). Comparisons of metabolic requirements have indicated a clear need for energy consumption for ingestion of moderate sized particles such as latex, but little or no energy consumption for uptake of the finest particulates (see Section 2.5). It has been postulated that finer particles may gain access to the OCS by membrane flow (Behnke, 1967). Zucker-Franklin (1981) made a direct comparison between the ways human platelets deal with moderate

88 C.C. CLAWSON sized (0.3/zm latex) and very small (cationized ferritin) particles. The process was followed by electron microscopy of ultrathin sections and freeze-fracture replicas in the presence and absence of metabolic inhibition. She concluded that the platelet has two different pathways of endocytosis. In these experiments latex uptake occurred "by membrane invagination apparently independent of the location of pits believed to represent entrances to the OCS" (Zucker-Franklin, 1981). In contrast, ferritin appeared to enter the OCS only through its normal surface openings. Even where the OCS was greatly distended with ferritin, its surface openings retained approximately nominal diameters of about 25 nm. White (1968) had earlier reached similar conclusions regarding the uptake of another very fine particle, thorotrast.

2.4

SOLUBLE CO-FACTORS OF PARTICLE UPTAKE

In considering the influence of various soluble plasma factors on platelet-particulate interaction, a distinction must be drawn between the initial steps of adhesion and uptake, and subsequent platelet events that accompany secretion and aggregation. This section will focus on the adhesion and uptake of particulates; aggregation and other outcomes will be covered in Sections 2.5 and 2.6. There have been contradictory findings regarding the requirement for divalent cations in particulate uptake by platelets. Early studies indicated that divalent cations are required for ingestion of latex particles, since chelation with ethylenediamine tetraacetic acid (EDTA) prevented uptake of latex (Movat et al., 1965; Mustard and Packham, 1968; Mant and Firkin, 1972). Movat and coworkers (1965) published an electron micrograph of platelet-rich plasma (PRP) with EDTA apparently showing little or no adherence to, nor uptake of latex by platelets. Subsequently, a paper from the same laboratory indicated that EDTA did not diminish adherence of latex to platelets although uptake was diminished (Glynn et al., 1965). Divalent cations are not essential for platelets to adhere to collagen (Santoro, 1988). Other reports have indicated that platelet uptake of latex was not inhibited by the presence of EDTA (Lewis et al., 1976; Zucker-Franklin, 1981). It has been suggested that, even though the ingestion process may be inhibited by EDTA, the adherence of particulates to platelets can still induce the release response (Mustard and Packham, 1968); although EDTA did block the metabolic burst studied by Kuramoto et al. (1970). There is agreement that EDTA does not prevent the uptake of finer particulates such as thorium dioxide (Mant and Firkin, 1972). Human platelets have known receptors for several plasma proteins that may coat particulates such as latex (reviewed in Coller, 1992). These include fibrinogen, fibronectin, vitronectin, von Willebrand's factor, IgG, and some complement components. However, platelet

ingestion of latex particles does not appear to depend on the presence of plasma proteins in fluid media, since washed platelets in protein-free buffer also take up latex (Movat et al., 1965). Glass or methacrylate beads can also adhere to washed platelets in buffer without added serum proteins (Packham et al., 1967). However, when latex is coated with fibrinogen or IgG, the adhesion step is significantly enhanced, but not when it is coated with albumin (Mustard et al., 1967; Mustard and Packham, 1968), for which platelets probably lack a specific binding site.

2.5

METABOLISM

DURING

INGESTION OF INERT PARTICULATES Both glycogenolysis and oxidative phosphorylation occur continuously in the resting platelet and both increase markedly on stimulation (Holmsen, 1990a). ZuckerFranklin (1981) based her conclusion of two separate pathways for particle ingestion by platelets in part on the differences produced by metabolic inhibitors. By inhibiting both oxidative phosphorylation and anaerobic glycolysis she demonstrated that the uptake of the larger particle, latex, was energy dependent while ferritin was readily taken up in the presence of metabolic inhibition. This confirmation of an energy consumption requirement for latex ingestion was in accord with several earlier observations (Movat et al., 1965; Kuramoto et al., 1970; Cooper et al., 1972; Mant and Firkin, 1972). Movat and colleagues (1965) had found, like Zucker-Franklin, that in order to block latex ingestion by platelets it was necessary to inhibit both oxidative phosphorylation and anaerobic glycolysis. Kuramoto and coworkers (1970) examined the time course of both aerobic and anaerobic metabolism for up to 3 h in human platelets exposed to 0.234/~m latex particles. They found that particle ingestion increased both arms of metabolism relatively slowly over the 3 h. Aerobic increases were principally via the citric acid cycle. The hexose monophosphate shunt, which predominates in the post-phagocytic aerobic metabolic burst of neutrophils, was not increased in ingesting platelets. In addition, they showed that energy consumption by latex-ingesting platelets was dependent on particle size. Stimulation of increased energy consumption was maximal with the smallest latex particles tested, 0.088/~m, and was absent with particles of 12 #m or greater. These authors concluded that the platelet's dependence on the citric acid cycle and its slower pace of response make particle ingestion by platelets distinctly different from that of neutrophils. Kuramoto's group also showed that while aggregation was stimulated maximally with very low concentrations of latex, metabolic activity could be further enhanced by addition of larger amounts of particles. Blocking platelet aggregation with AMP or Ado does not appreciably alter latex uptake, indicating that the two

PLATELETS IN BACTERIAL INFECTIONS 89 processes are separable activities, nor does it inhibit either the aerobic or anaerobic energy burst (Movat et al., 1965; Mustard and Packham, 1968; Kuramoto et al., 1970). Inhibition of the cyclooxygenase (CO) pathway also failed to block latex uptake (Lewis et al., 1976). Mant and Firkin (1972) conducted an extensive survey of the influences of metabolic inhibitors on ingestion of particles by human platelets in citrated PRP. For each of the inhibitors tested they contrasted the effects on uptake of 0.176#m latex and colloidal thorium dioxide (10-15 nm, as estimated from electron micrographs). They concluded from these studies that latex uptake required energy primarily from anaerobic glycolysis. In contrast to Movat et al. (1965), they found no decrease in ingestion with inhibition of respiration, and they concluded that platelet ATPases had little or no participation in the process. Incubation at 4~ reduced uptake of latex but not of thorium dioxide. Uptake of the smaller, thorium dioxide particles did not appear to require demonstrable energy consumption; however, various inhibitors gave somewhat conflicting results with this material. The best explanation appears to be that uptake of thorium dioxide was decreased by some of the inhibitory agents because of morphological rather that metabolic events. When an inhibitor appeared to diminish uptake of thorium dioxide, it had also produced significant alteration of platelet structure, especially loss of the surface openings that would preclude even passive admission to the OCS. Disorganization of platelet microtubules with colchicine did not alter uptake of either latex or thorium dioxide, nor did inhibition of CO with aspirin. Cooper and colleagues (1972) also compared the energy consumption of platelets after uptake of 0.234/~m latex or thorium dioxide. In general, their results agreed with those ofKuramoto et al. (1970), but they extended the findings and conclusions in a somewhat different direction. They noted that interiorization of either particulate could be detected by electron microscopy as early as 1 min, while no change in energy use was evident for about 10 min. Lacking this correlation in time between particle uptake and metabolic activation they questioned whether the two could be directly linked as cause and effect. They offered the explanation that degranulation may be a critical intermediate step in initiating augmented energy metabolism. They further suggested that latex particles may enter the OCS, rather than de n0v0 phagosomes, just as thorium dioxide appears to do, and that in neither case should the process be considered true phagocytosis. Thus, although both sized particulates could come to lie in the OCS, these authors would attribute the difference in stimulation of energy usage between latex and thorium dioxide to the special ability of latex to induce the release reaction. In summary, ingestion by platelets of particles in a range of about one-tenth to several tenths of a #m requires energy consumption and can be distinguished from platelet aggregation. Further, there are slow but

appreciable increases in both aerobic and anaerobic metabolic activity of the platelet after particle ingestion. In contrast, uptake of very fine particles such as colloidal thorium dioxide does not require demonstrable energy consumption.

2.6

PLATELET

SECRETION

AGGREGATION

AND

RESPONSE

TO

INERT PARTICLES In addition to the enhancement of energy metabolism by platelets, platelet-particulate interaction can induce the full range of physical phenomena associated with platelet activation. These may be summarized as the steps of shape change, secretion, and aggregation. Each is accomplished by a series of biochemical events on the platelet's surface membrane and in its cytoplasm. More will be detailed of these in the discussion of platelet-bacterial interactions below. For this consideration of effects of inert particles on platelets a more general view will suffice. Packham et al. (1967) reported that in the absence of replacement plasma proteins, glass or methacrylate beads adhered to washed platelets, but secretion was not stimulated. When the beads were coated with immunoglobulin the release reaction was marked. Fibrinogen coating increased adherence, but release was less. They further noted that fibrinogen in the media would inhibit stimulation by IgG-coated beads. Although platelets may use the OCS as a means of taking up particulates rather than forming true phagocytic vacuoles, data have been presented indicating metabolic and physical activity in the platelet analogous to that present in classic phagocytes. Lewis et al. (1976) noted that platelets which had taken up latex particles secreted acid phosphatase from their c~ granules into latex-containing spaces.

0

Platelet Interaction with Bacteria in vitro

Many, but not all, strains of bacterial pathogens are capable of inducing irreversible platelet aggregation with its attendant secretory or release reaction (Clawson and White, 1971a). As reviewed below (Section 4), the aggregation phenomenon has been observed repeatedly in vivo. Numerous authors of these in vivo studies have described platelet-bacterial clumping by light microscopy. Examination of details of platelet-bacterial interaction has been greatly aided by the application of electron microscopy to the problem, and the introduction of nephelometric platelet aggregometry in the early 1960s. The latter instrument records platelet shape change and aggregation in response to an agonist as changes in light transmission during stirring at controlled temperature, usually 37~ Aggregation is indicated by an increase in light trans-

90 C.C. CLAWSON mission due to the presence of fewer, although larger, suspended particles in the light path (Born, 1962; O'Brien, 1962). Findings from in vitro morphologic studies by light and electron microscopy can be correlated with results of platelet aggregometry and biochemical investigations. These will be summarized and interrelated in the sections that follow.

3.1

AGGREGOMETRY

When platelet-activating bacteria such as Staphylococcus aureus 502A are washed and added to PRP at a nominal ratio of 1:1, four phases may be distinguished in the platelet aggregation response as recorded by nephelometry (Fig. 5.2; Clawson and White, 1971a). To obtain reproducible aggregometry results from one sample of platelets to another it is necessary to adjust the platelets to a standard concentration. (For the studies of Clawson and White reviewed here the platelet concentration in PRP was adjusted to 300000/mm3.) In recording platelet aggregation in response to Staph. aureus there was initially a lag period in which no change in the tracing was observed. This was similar to the lag in recorded platelet response to particulate collagen, and contrasts with the immediate response produced by soluble agonists such as ADP. Microscopic examination reveals that contact and adhesion between bacteria and platelets proceeds during

the lag period; therefore, this was dubbed the contact phase of interaction. The first change in the tracing indicating a response came as platelets underwent shape change, which was signalled by loss of rapid oscillation in the tracing that is characteristic of resting discoid platelets. Soon thereafter early adgregation was marked by the initial rise in the tracing as light transmission increased. As irreversible a2gregation was attained, maximal light transmission occurred and the tracing showed wide oscillations due to the variable size of the platelet aggregates. Heat-killed Staph. aureus or a membrane fraction of this organism produced aggregation responses identical to those induced by live bacteria (Clawson et al., 1975). When ADP is used as the platelet agonist, the dose can be adjusted downward to show a reversal of the initial wave of platelet aggregation before secretion of endogenous ADP carries the reaction forward to full irreversible aggregation. When the stimulus was bacteria, progressive lowering of the ratio of microbes to platelets initially prolonged the lag phase, but when aggregation did occur after several minutes, it was complete and in a single wave. At lower ratios (1:70 for Staph. aureus) no recordable aggregation took place, although the narrowed tracing of shape change could be seen. Finally, at still lower ratios ( - 1 " 100 for Staph. aureus) the great majority of platelets remained unstimulated and discoid;

F

\

c: o

. ...,=, t/3 U)

E

(/3

o t==.

I-4--. C

A

B

C

D

E

. ,,==,

_1

I

I minute

I

Figure 5.2 Aggregometer tracing of platelet-bacterial interaction recorded as a change in optical density of a stirred 1 ml sample of human platelets in native plasma at 37~ activated with Staph. aureus 502A at a ratio of one bacterium per platelet. A, oscillating baseline of discoid platelets before addition of the bacteria; B, bacteria added; C, lag or contact phase with no recordable response; D, earliest onset of shape change indicated by loss of the rapid oscillation; E, beginning of the rise in light transmission signalling aggregation; F, rapid oscillation of light transmission indicating formed platelet-bacterial aggregates with plateauing as reaction is completed. [Clawson and White (1971a) with permission.]

PLATELETS IN BACTERIAL INFECTIONS 91

(a)

(b)

"

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~

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.

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.

.

.

.

.

.

~

.

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,~

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iiiili!!iiiiii~"~ ~.:.~:~:,~:~.~~...~:~::~i~:~:~:~!~,~ .~:.~:i~:~

.....~"~';:'~'~~,'~~'~i.~..'..!!!i!ili~ ..

.

ili l .....

i!w

~ ! ii~!~ 84184184 ~!:~i~? !!ii!i!ii~iii~

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17~/i!~!!'~i~i!!i~iiiiiiii!iiiiiiiiiiiiiiii~i~;84

/i!!~i!ii!iii~!!

~ ii 84184184184184184184184184184184184 !! i~!~i~!!i!i!~'!iii~!~i! i~ i.......

......

Figure 5.3 Scanning electron micrographs of the sequence of the interaction of Staph. aureus 502A with human platelets in native plasma. (A) Early contact phase (x 11 000); (B) mid-contact phase with earliest shape change (x 6200); (C) late contact phase (x 5500); (D) full shape-change phase (x 8300); (E) early aggregation (x 5000); (F) irreversible aggregation (x 4300). [From Clawson (1973) with permission.]

92

C . C . CLAWSON

thus the aggregometry tracing was unchanged for 30 min or more. However, even at such a low bacterial challenge, microscopy revealed that microaggregates of a few platelets and bacteria still formed. The presence of these microaggregates indicated that platelet-bacterial interaction could occur even when no mass effect, such as can be recorded on the aggregometer, was present. The microaggregates were comparable to those seen in vivo when bacteria have been injected intravascularly experimentally. Several factors indicated that direct contact and adhesion of Staph. aureus to platelets was required to promote platelet response. Continuous stirring was required in the early minutes. If stirring was stopped after a few seconds of mixing no reaction was seen. Either broth from fluid cultures or incubation media from washed bacteria, filtered free of organisms, failed to stimulate platelets. When examined by scanning electron microscopy only those platelets with adherent microbes showed true shape change.

3.2

MORPHOLOGY

When a strain of bacteria capable of activating platelets, e.g. Staph. aureus 502A, is washed and added to stirred citrated PRP, the bacteria evoke a platelet aggregation in which the bacteria participate. The process has been followed throughout by light microscopy and scanning electron microscopy (Fig. 5.3; Clawson and White, 1971b; Clawson, 1973). Initially unstimulated platelets were clearly discoid, normally granulated, and unclumped. Rapidly, contact between the cell types led to adhesion of bacteria to platelets. The adhesion of bacteria to platelets appeared to occur randomly and was not confined to identifiably specific regions of the platelet disc. The initial morphological response was the development of fine spike-like pseudopods on an otherwise discoid platelet. As platelets entered the true shape-change response, their discoid shape was obliterated by the development of blunt, irregular pseudopods. These initial alterations in form appeared to occur only in those

f <:.%...7..~ ....... ..~

:...~,

,

"+

).

, ~;.

,

>:~%11i

.

.

W

..

Figure 5.4 Transmission electron micrograph of early aggregation between washed human platelets and Staph. aureus 502A fixed at point E on the aggregometry tracing as shown in Fig. 5.2. There is close contact between the bacteria and platelets that have undergone varying degrees of shape change. Some central migration of the platelet granules has occurred, but the granules remain discrete and intact. An example of engulfment of a microbe by a platelet is seen, although complete engulfment cannot be ascertained in a single microscopic section (x 16 000). [From Clawson (1973) with permission.]

PLATELETS IN BACTERIAL INFECTIONS 93 platelets that had bacteria adherent to their surface. Minimal clumping of platelets was seen at this stage of the response. As shape change proceeded aggregation began. There was a lack of synchronization at this point. Some platelets remained discoid with discrete cytoplasmic granules while others had formed well-developed aggregates. Adherent bacteria became enmeshed in forming aggregates. During early aggregation there was a central consolidation of granular material within the platelet cytoplasm visible with light microscopy. The maturing aggregates enlarged by recruitment of resting discoid platelets that did not appear to have had prior contact with the microbes. This resulted in most of the bacteria coming to lie more centrally in the platelet mass. In addition, the central densities seen within platelets of early aggregates were largely lost. Retracing this process by transmission electron microscopy revealed further detail of the morphological response of platelets to bacterial activation (Figs 5.4. and 5.5; Clawson, 1973). At the site of primary adherence a

few strands of the microfibrilar coat of the bacteria formed bridges to the platelet surface. As the platelet was transformed during shape change, it appeared to mold about the organism extending the region of this bridging until in some instances the microbe appeared to be completely engulfed by a single platelet. This was seen only rarely since usually other platelets attached to the microbe or to its adherent platelet so that the bulk of bacteria came to lie within interstices of aggregates. (The issue of bacterial engulfment and its analogy to phagocytosis will be addressed in Section 3.7.) The internal changes, concomitant with the process of shape change, were a central congregation of discrete granules and an inward migration of the peripheral band of microtubules. Maturation of aggregates was marked by molding of platelets to one another and around the bacteria, thereby minimizing intercellular space. At the surface of aggregates many of the blunt pseudopods showed enlargement and internal clearing suggesting that the bulk of the aggregate may have contracted forcing fluid cytoplasm into these surface projections. Secretion of

'~':;..~ ~ ".S

!ii....../ l 99

.q .'~

Figure 5.5 Late aggregation phase of platelet-bacterial interactions between Strep. pyogenes and human platelets in citrated native plasma. T h e specimen was fixed after plateauing of the aggregometry tracing as shown in Fig. 5.2. The platelets have reached a stage of irreversible aggregation with close apposition to each other and to the enclosed bacteria that reside predominantly in the intercellular spaces of the central portion of the aggregate. Studies of the process indicate that the initial platelet-bacterial aggregates recruit other platelets to bind to their exteriors leaving the bulk of the bacteria in the center of the mature aggregate well removed from the suspension media (x6700). [From Clawson (1973) with permission.]

94

C.C. CLAWSON

granular contents to intercellular spaces could be visualized, and a dense central mass formed in each platelet from condensation of the contractile gel of the platelet cytoplasm. Finally, there was loss of most identifiable internal organelles. These morphological changes are not unique to the response of platelets to bacteria. Rather they are typical, except for inclusion of bacteria in the aggregates, of platelet aggregation induced by most agonists of the aggregation response. For a more detailed discussion of platelet ultrastructure the reader is referred to the several reviews of the topic elsewhere (White and Clawson, 1980a; White et al., 1981; White, 1987; ZuckerFranklin, 1990).

3.3

INFLUENCE OF PLASMA COMPONENTS

Platelet response to Staph. aureus was sensitive to the concentration of divalent cations (Clawson and White, 1971a). When EDTA at 0.6 mglml was added to PRP before the bacteria, the entire reaction was modestly slowed, but complete aggregation was attained. With EDTA at 0.75 mg/ml aggregation was blocked, but there was a recordable shape change. At 1.0 mg/ml EDTA the platelet reaction was blocked completely. The ability to partially inhibit platelet response, allowing shape change but not aggregation, indicated that binding of Staphylococcus could still take place at EDTA concentrations that were inhibitory to the reactivity of the platelet. This distinction between adhesion and platelet response

was later examined by Herzberg and co-workers (1983a) who showed that adhesion to viridans streptococci is independent of divalent cations. Plasma proteins influence the interaction of human platelets with bacteria (Clawson and White, 1971b; Clawson et al., 1980b). Since both Staphylococcus and platelets bind fibrinogen, the role of this plasma protein in platelet-bacterial interaction was studied using washed platelets of both humans and rabbits. In order to exclude plasma proteins from the media and from the platelets as possible, triply washed platelets were prepared by flotation on bovine serum albumin (BSA), a modifcation of a method originally devised for separating and concentrating blood monocytes (Bennett and Cohn, 1966; Clawson et al., 1970; Clawson and White, 1971b). The albumin-washed platelets remained discoid with normal ultrastructure (Clawson, 1973), and aggregated well with particulate collagen, ADP, and epinephrine (Ardlie et al., 1970; Clawson et al., 1970; Clawson and White, 1971b; Ra3ssi, 1972; Walsh, 1972). The response of human albumin-washed platelets to Staph. aureus 502A with and without added serum is shown in Fig. 5.6. When serum proteins were omitted, there was a modest prolongation of the contact or lag phase, but once begun, bacterial activation of washed platelets gave aggregometry tracings identical to those with PRP. In contrast, the recorded responses of normal washed human platelets to either soluble or particulate collagen were not influenced by the absence of exogenous fibrinogen. Replacing proteins in the form of 5% pooled human serum resulted in an aggregation response of bacteria with

C 0

gt)

U)

E gr)

C 0

I-

A

!

I minute

I

Figure 5.6 Aggregometry tracings of washed human platelets in HBSS stimulated by Staph. aureus 502A at 1 "1. (A) With 5% pooled serum; (B) without 5% pooled serum. [From Clawson and White (1971b) with permission.]

PLATELETS IN BACTERIAL INFECTIONS 95 washed platelets identical to that in PRP (Figs 5.2 and 5.6). Pooled serum prepared from clotted plasma was used in these experiments; therefore, it was largely free of fibrinogen. The responses of washed normal human platelets to stimulation by Staph. aureus 502A at one microbe to five platelets were compared with and without replacement of exogenous fibrinogen to physiologic levels (2 mg/ml; Fig. 5.7). Without exogenous fibrinogen the lag phase was significantly prolonged as compared to the same bacterial ratio in PRP, but once shape change and aggregation commenced they proceeded normally. When fibrinogen was restored to normal levels (2 mg/ml), response of washed platelets was identical to platelets in native plasma. In contrast, washed platelets without replacement of fibrinogen responded as rapidly and as completely to soluble collagen (40 ~g/ml) as did unwashed platelets in native plasma. However, normal platelets carry their own fibrinogen in their c~ granules, which is released on activation (Kaplan et al., 1979; Sander et al., 1983).

o

Therefore, the opportunity to study the response to Staph. aureus by platelets from a patient with a congenital

absence of both plasma and platelet fibrinogen gave further insight to the role of fibrinogen in the reaction (Fig. 5.8; Clawson et al., 1980b; Clawson and White, 1980). At the nominal 1:1 ratio of bacteria to platelets afibrinogenemic platelets appeared to respond to the bacterial stimulus with a full aggregation tracing after only a modest delay. However, microscopic examination revealed the aggregates to be much smaller than with normal platelets, and many of the platelets remained unaggregated. At reduced ratios of bacteria to platelets (1:2.5 or 1:5) aggregation was usually abolished with fibrinogen-deficient platelets although the bacteria bound to platelets and activation of shape change did occur (Figs 5.8 and 5.9). Replacement of fibrinogen (2 mg/ml) to deficient platelets resulted in a response indistinguishable from that of normal platelets. From these results it may be concluded that fibrinogen is not responsible for binding of Staph. aureus to human platelets nor is it

Washed PLT without added F BGN

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,

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Washed

PLT ~ I A

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T

S. aureus 1-5 PLT

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Collagen 40 pg/ml Soluble

Figure 5.7 Aggregometry tracings showing the effect of replacing exogenous fibrinogen to physiologic levels (2 mg/ml) on responses of washed normal human platelets to stimulation by Staph. aurerus 502A at one microbe to five platelets. Without the fibrinogen (A) the lag phase was significantly prolonged as compared to the same ratio in PRP (C), but once aggregation commenced it proceeded normally. When fibrinogen was restored to normal level (2 mg/ml) the response of the washed platelets (B) was identical to the controls (C). In contrast, the washed platelets without replacement of fibrinogen responded as rapidly and as completely to soluble collagen (40 #g/ml) (D) as did unwashed platelets in native plasma (E). [From Clawson et al. (1980b) with permission.]

c-

ill

O

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i

9

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9

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/

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.

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Normal PLT

S..aureus, to

1"5 "-- ~ - - - r .....!.- . . . . . . . . .

D

S. aureus to

/

I I minute | 17 min. I1 / /

-r-----~, .......................

Figure 5.8 Aggregometry tracings comparing responses of normal (A, B) and afibrinogenemic platelets (C, D) to simulation by Staph. aureus 502A at ratios of 1 : 1 (A, C) and one microbe to five platelets (B, D). With normal platelets the reduced bacterial numbers resulted in a slight prolongation of the lag phase but did not diminish the rate of aggregation (slope of rise). With afibrinogenemic platelets at 1:1 with the bacteria the lag phase was significantly prolonged and the rate of aggregation was slower although complete. When the ratio of the bacterial stimulus was reduced for the afibrinogenemic platelets, there was an extended lag phase followed by the narrowing of shape change, but no aggregation was recorded in 25 rain. [From Clawson et al. (1980b) with permission.] 9 rain.

15 rain.

.............................

~-without FBGN

t

/l__

l/--

S. aureus at 1" 2.5 PLT

with FBGN

g El ~ Or)

_

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5 min.

r 0

o~

_J

I minute

without FBGN

//

.,,,I Soluble Collagen 50 IJg/ml ~

JwIthFBGN

Figure 5.9 Aggregometry tracings showing the effect of replacing exogenous fibrinogen to physiologic levels (2 mg/ml) on responses of afibrinogenemic platelets in autologous plasma to stimulation by Staph. aureus 502A at a ratio of 1 microbe to 2.5 platelets (A, B) or soluble collagen at 50 mg/ml (C, D). [From Clawson et al. (1980bl with permission.]

PLATELETS IN BACTERIAL INFECTIONS 97 of Ehlers-Danlos syndrome that has an associated fibronectin defect (Arneson et al., 1980) were able to respond to either particulate or soluble collagen, but only at several fold greater concentrations than required by fibronectin-sufficient platelets (Fig. 5.10). However, this patient's platelets responded normally to Staph. aureus whether in native plasma or in buffer after washing. These studies indicated that, unlike fibrinogen, fibronectin was not a co-factor in the response of platelets to this species of bacteria. Recently MacFarlane and colleagues (1993) have examined the role of other plasma components in the response of human platelets to Streptococcus sanguis. These

required for platelet activation; however, its presence augments subsequent platelet aggregation. Fibronectin is a highly adhesive molecule that must also be considered as a possible mediator of platelet-bacterial interaction (Packham and Mustard, 1984). Staphylococcus binds fibronectin (Kuusela, 1978), and stimulated platelets exhibit fibronectin binding sites (Ginsberg et al., 1980). The role of fibronectin in platelet interaction with Staph. aureus was also examined by Clawson and colleagues (1980b). Triply washed normal human platelets in protein-free media responded similarly to varied ratios of microbes to platelets whether or not fibronectin was added to the system. Platelets from a patient with a form

1:10 PLT

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f

,m

t

-

Soluble Collagen 7 0 jug/ml(2.3 x t h r e s h o l d )

om

E c b,,

k-

J= OD ...I

le Collagen 100 jug/ml (3.3 x threshold)

~

D~

Collagen

100 .ul (2.5 X threshold )

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Full Strength Particulate Collagen 2 5 )JI ( 6 x threshold )

Figure 5.10 Aggregometry tracings showing the responses of fibronectin-defective platelets in native plasma from a patient with a variety of Ehlers-Danlos syndrome to stimulation by: (A) Staph. aureus 502A at 1 per 10 platelets: (B) soluble collagen at 2.3 times the threshold concentration for normal platelets; (C) soluble collagen at 3.3 times the normal threshold concentration; (D) particulate collagen at 2.5 times the normal platelet threshold; and (E) particulate collagen at 6 times the normal threshold. [From Clawson et al. (1980b) with permission.]

98 C.C. CLAWSON authors found that, as for Staphylococcus, calcium ions and fibrinogen were required for platelet aggregation. Several other plasma components, including IgG, were identified that were not required for aggregation, but would m0d/~ platelet-bacterial response. When IgG was decreased to about half its normal concentration, Strep. sanguisinduced aggregation was enhanced as indicated by a shortened lag time. In contrast, observations by Sullam et al. (1987, 1988, 1990) suggest that IgG may be required for interaction of other stains of viridans streptococci with platelets. In the absence of calcium and fibrinogen, neither IgG nor epinephrine alone or in combination would support Strep. sanguis-induced aggregation, while these four agents together in physiologic concentrations completely restored the aggregation of washed platelets in HBSS. The support of aggregation by IgG could be abolished by prior adsorption of the IgG with intact Strep. sanguis, or by blocking platelet Fc receptors with monoclonal antibody (mAb). Modification of the levels of ~/2microglobulin below 15 ~g/ml or above 20 #g/ml resulted in subdued platelet responses as indicated by prolonged lag times. Fibronectin, C lq, and Cohn fraction VI (glycoproteins) all act to prolong the lag phase of Strep. sangu/s-induced platelet aggregation. Human platelets express receptors both for the Fc fragment of IgG and for the C3 component of complement. Either of these plasma factors may potentiate platelet-bacterial interaction with some strains of microorganisms (Zimmerman and Spielberger, 1975; Hawiger et al., 1979). Packham and colleagues (1967) studied washed platelets in buffer that were stimulated by hemolytic streptococci with and without added IgG. Without IgG there was no stimulation of ADP release by the bacteria, but with added IgG release of ADP was as great as that produced by thrombin. Antigen-antibody complexes or IgG alone gave only about one-third as much ADP release. The role of epinephrine in modulation of platelet reactivity has been of special interest. It has been estimated that the maximum plasma concentrations of epinephrine attainable in vivo are from 3 to 7 nM (Dimsdale and Moss, 1980; Siess et al., 1982), although under some conditions plasma levels may reach 1 #M (Karliner et al., 1981). In this range epinephrine can enhance synergistically platelet response to other agonists such as ADP, thrombin or collagen (Ardlie et al., 1985; Keraly et al., 1988). The studies of MacFarlane et al. (1993) demonstrated that epinephrine also modulated platelet response to Strep. sanguis. They showed that at epinephrine levels of 3 nM, lag times were decreased for half of the 16 individual normal platelet donors tested. It had been shown earlier (Herzberg et al., 1983a) that platelets in native plasma from various individual normal donors exhibit a wide range (1.3-19 min) in lag times of response to Strep. sanguis in a standardized test system. To evaluate further the role of epinephrine in this system, these investigators

compared epinephrine levels in plasma and in platelets from groups of fast (<5 min) and slow (>5 min) responders (Herzberg et al., 1993). They found that mean epinephrine levels in these separate compartments and the combined totals were higher for the fast than for the slow responders. The greatest difference, about 4.5fold, was in platelet levels, while the total was approximately 2.5-fold higher in fast responders. The molecular mechanism(s) responsible for the differing activity of fast and slow responders is yet to be determined. Differences in lag times between fast and slow responders could not be explained by the availability of either ~2adrenoreceptors or secretory ATP since basal levels of these were the same for platelets of both groups; however, the fast responding platelets gave considerably higher levels of secreted ATP following stimulation with bacteria. Addition of epinephrine at a physiologically attainable, but sub-platelet-activating concentration, to platelets of slow responders brought their Strep. sanguisinduced secretion of ATP to levels comparable with those of fast responders.

3.4

VARIED RESPONSES TO DIFFERENT BACTERIA

A large number of bacterial species have been tested for their ability to stimulate platelets (Table 5.1). Using Staph. aureus 502A as a positive comparison standard, Clawson and White (1971a) studied the response of human platelets in native plasma to several other bacterial pathogens (Fig. 5.11). They found a wide spectrum of ability to evoke platelet activation among the six strains compared: Staph. aureus 502A, Streptococcus pyogenes M57, Enterococcus faecalis (formerly Streptococcus faecalis), Streptococcuspneumoniae types 8 and 24, and Escherichia coli. All of these species except type 24 pneumococcus were capable of stimulating stirred human platelets to full irreversible aggregation when added to PRP at a ratio of 1 : 1. The principal difference was in the duration of contact and shape-change phases of the response. The response to the ~/-hemolytic streptococcus, Strep. pyogenes, except for a small increase in lag period, was nearly as potent as to Staph. aureus. Platelets responded a good deal more slowly to both the E. faecalis and the E. coli. With the latter organism the response was borderline at the nominal 1:1 ratio, sometimes failing to produce complete aggregation. The time required for Strep. pneum0nh~e type 8 to induce a platelet response was extended well beyond that of E. coli. Protracted contact and shapechange phases were eventually followed by full aggregation; however, this took about 28 min in the typical tracing shown in Fig. 5.11. The type 24 pneumococcus failed to provide any recordable platelet response. Another staphylococcus, Staph. epidermidis, also failed to evoke any recordable response in human platelets. In the series of studies by Clawson and colleagues outlined above, the standard test organism was Staph. aureus

PLATELETS IN BACTERIAL INFECTIONS 99

Table 5.1

Examples of bacteria reported capable of inducing platelet aggregation in vitro

Species

References

Enterococcus faecalis Escherichia coil Fusobacterium necrophorum Histoplasma capsulatum Listeria monocytogenes Mycobacterium tuberculosis Pseudomonas aeruginosa Salmonella minnesota Salmonella typhimurium Staphylococcus aureus Staphylococcus epidermidis Strepotococcus group B Streptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes Streptococcus sanguis Yersinia pseudotuberculosis

Clawson and White (1971a, b) Clawson and White (1971a, b), Konig et al. (1990) Forrester et al. (1985) Des Prez et a/. (1980) Czuprynski and Balish (1981) Copley et a/. (1955, 1959) Kessler et a/. (1987) Timmons et al. (1986) Mandell and Hook (1969) Clawson and White (1971a, b), Hawiger et al. (1979) Kessler eta/. (1987) Usui et a/. (1987) Herzberg et al. (1983a) Clawson and White (1971a, b) Clawson and White (1971a, b) Herzberg eta/. (1983a) Simonet et al. (1992)

8

A

....

Ul Ul o...

B rC:n ._J

o...

3min 5 min

9rnin

I0 min I

I I minute

Figure 5.11 Aggregometry tracings of human platelets in native plasma stimulated by bacteria at a ratio of 1 1. 9 (A) Staph. aureus 502A; (B) Strep. pyogenes; (C) Entero faecalis; (D) E. coli; (E) Strep. pneumoniae type 8. [From Clawson and White (1971a) with permission.]

100 C.C. CLAWSON 502A, a protein A-producing strain. In another series of experiments these authors compared the 502A strain with Staph. aureus Woods, a non-protein A-producing strain, for their abilities to activate human platelets in native plasma. In addition they also examined the influence of soluble protein A on activation of human platelets (C.C. Clawson, unpublished observations). Initially, when the two strains were compared directly at the usual test ratio of one microbe per platelet, the Woods strain gave a shape-change response but no recordable aggregation. With the 502A strain such a response was found only at extremely low ratios in the range of about one microbe to 70 platelets. When the number of Woods bacteria was increased to a three per platelet, modest aggregation occurred but after a lag phase of 12 min. Further increases in the ratio of the Woods strain to 10 per platelet only shortened the lag phase to 7 min. In contrast, with 502A organisms any excess of bacteria to platelets resulted in lag phase times of well under 1 min. These results strongly suggested that protein A might have been the controlling factor in the observed differences between the two strains of Staph. aureus. However, a series of studies with purified protein A failed to reveal any direct activation of platelets, and its presence neither enhanced nor inhibited any effect on platelet reactivity to other platelet agonists, including these two strains of Staph. aureus. Both human PRP and washed platelets in Hanks' balanced salt solution (HBSS) were tested for direct response to soluble protein A and to protein A bound to sepharose beads without any shape change or aggregation response. Soluble protein A failed to inhibit or enhance platelet reactivity to collagen, thrombin or ADP. From these experiments it was concluded that observed differences between the Woods and 502A strains of Staph. aureus must be accounted for by other variations between the organisms and not by expression of protein A. Usui et al. (1987) studied platelet aggregation by 46 strains of group B streptococci, both serotypable and non-serotypable. They found that only four strains, all of type III, were reactive with human platelets in native serum. Like other bacterial stimulation of platelets, the responses were inhibitable by EDTA, indomethacin, aspirin, and quinacrine, and were sensitive to the ratio of microbes to platelets. Timmons and co-workers (1986) have reported a rough mutant strain (Re595) of Salmonella minnesota, which expresses structural variations of the usual endotoxic lipopolysaccharide, that induced a full platelet secretion and aggregation response. A strain of S. minnesota that produces intact endotoxin produced no platelet aggregation response. Interaction between human platelets and the viridans group of streptococci have been the focus of extensive research because of their special association with bacterial endocarditis. A member of this group, Strep. sanguis, predominates among oral commensal flora. It has been shown that frequently throughout life these organisms

gain access to the circulation through breaches of the normally protective mucosal barriers of the mouth or respiratory and gastrointestinal tracts (Coulter etal., 1990; Kern et al., 1990; Villablanca et al., 1990; Weisman et al., 1990). Douglas and colleagues (1990) studied the ability of 24 strains of oral streptococci to aggregate platelets, and concluded that strains of Strep. sanguis possessed platelet reactivity while Strep. gordonii, Strep. parasanguis, Strep. mitis, Strep. oralis and related taxa did not. Herzberg and coworkers (1983a) compared seven strains of Strep. sanguis and six strains of Strep. mutans for their ability to bind and stimulate platelets. They noted considerable differences among strains of both Strep. sanguis and Strep. mutans in their ability to induce platelet aggregation as measured by the duration of the lag phase for in vitro aggregation responses. In this study various strains of Strep. sanguis and Strep. mutans were tested for their reactivity with fresh platelets in native plasma from several donors individually. All of the reactive strains had been isolated from cases of human endocarditis. Six of seven strains of Strep. sanguis initiated aggregation in from 1.3 min to 19 min among various donors while only two of six strains of Strep. mutans were capable of inducing platelet aggregation and required lag times of about 12 min. Subsequent to their 1983 report, Herzberg and colleagues have examined 85 cases of clinically significant bacteremia caused by viridans streptococci and found that 86% of the strains interacted with human platelets (M.C. Herzberg, unpublished data). In contrast, Douglas et al. (1990) found that of eight strains of Strep. sanguis recovered from cases of endocarditis, only four reacted with platelets. However, in considering platelet reactivity of various strains of viridans streptococcus, it is important to recognize the variability of phenotypic expression that can occur in a single strain when grown in vivo or in culture (see Section 4 and Sommer eta/., 1992). It is also of note that among the initial eight platelet-aggregating strains of viridans streptococci tested by Herzberg et al. (1983b) none was able to activate platelets from all donors tested. Unresponsive normal donors ranged from one out of 17 for one strain to six of seven for another. This is in contrast to Staph. aureus 502A for which no unresponsive normal donors have been found.

3.5

BACTERIALLY PLATELET

INDUCED

SECRETION

The aggregation induced by some strains of bacteria is similar to that of particulate collagen in that it depends on secretion of endogenous ADP to carry it to full aggregation. This was indicated by the effect of the ADPdegrading enzyme, apyrase. When apyrase (5 mg/ml) was added to PRP several minutes before Staph. aureus, platelet shape change occurred normally but no recordable aggregation took place (Clawson and White, 1971a). In contrast to these observations, MacFarlane et al.

PLATELETS IN BACTERIAL INFECTIONS 101 (1994) found that, while ADP secretion may modulate the aggregation response to Strep. sanguis, the response is not dependent upon endogenous ADP secretion. Not only did removal of secreted ADP have little effect on the aggregation response, but platelets from three of six Hermansky-Pudlak donors, which lack dense granules and therefore ADP secretion, gave normal responses to Strep. sanguis. (See Section 3.6 for further discussion of the mechanism of platelet interaction with this organism.) The ability of Staph. aureus to induce platelet secretion of another marker of granule release, serotonin (5hydroxytryptamine; 5-HT), was also studied (Clawson et al., 1975). Normal human platelets in native plasma or in buffer after washing were incubated with 14C-5-HT which allows uptake of this marker of platelet secretion. After 3 min of mixing with either Staph. aureus or other standard platelet agonists, the amount of labelled 5-HT released to the media was calculated as a percentage of the total in the system. The bacteria proved to be as potent or more potent than other aggregating agents (collagen, epinephrine, or thrombin) in promoting platelet secretion. Release of 5-HT occurred concurrently with shape change as recorded on the aggregometer, and in advance o f demonstrable aggregation. Inhibitors of glycolysis or oxidative phosphorylation did not block the platelet secretion response to bacterial stimulation nor subsequent aggregation. Aspirin reduced secretion of 5-HT by about half, but this was not sufficient to inhibit full aggregation. Agents that further reduced secretion [e.g. prostaglandin E1 (PGE1), N-ethylmaleamide] also blocked aggregation. These studies indicated that bacteria are equally as potent as other standard platelet agonists in producing platelet secretion, and that full aggregation response to bacterial stimulation depends on the secretory response.

3.6

MECHANISMS

OF ADHESION

AND ACTIVATION Some of the observations reviewed above indicate that, for some bacteria, adhesion of microbes to platelets might be dissociated from their activation of platelets. While inferential evidence has come from a variety of sources, the model of human platelet interaction in vitro with Strep. sanguis has provided the most detailed elucidation of the process to date (Herzberg et al., 1985). These authors have demonstrated that in this model the process of interaction at the molecular level can be divided into three distinct steps: adhesion, activation, and aggregation. They note further that participation of Strep. sanguis in the process is controlled by expression of three classes of surface determinants that contribute in quite diverse ways to interaction with human platelets. These surface components were shown to be distinct entities by their differing susceptibilities to trypsin digestion, selective inhibition by univalent monospecific antibodies,

and variable phenotypic expression among strains of

Strep. sanguis. Action of these surface determinants of platelet interaction does not require an intact microbe since fragments of cell walls from Strep. sanguis activated platelets equally as well as intact organisms (Herzberg et al., 1983a). This model, as elucidated in the following paragraphs, is currently applicable only to Strep. sanguis; however, it is likely that the phenomenon demonstrated may be a paradigm for platelet activation by other strains of bacteria. 3 . 6 . 1 Strep. s a n g u i s A d h e s i o n While platelet activation by Strep. sanguis requires prior adhesion of microbes to platelets, adhesion of some Strep. sanguis strains can occur without subsequent platelet aggregation (Herzberg et al., 1985, 1990b). For example, strain L74, isolated from dental plaque, appears to express the surface adhesin molecule but lacks the ability to activate platelets. Other Strep. sanguis strains, such as M5, lack both the adhesion and activation determinants. The rate of adhesion of Strep. sanguis to platelets was the same at 4~ and 37~ and modest changes in pH had little effect on adhesion (Herzberg et al., 1983b). Furthermore, binding to the platelet occurred independently of calcium or plasma proteins, while triggering of platelet aggregation required both calcium and fibrinogen (Herzberg etal., 1983b; MacFarlane etal., 1993). The fact that distinct streptococcal components for adhesion and aggregation can be identified suggests that they operate through separate binding sites on the surface of the platelet. The streptococcal adhesin molecules that bind to platelets have been shown to be associated with surface microfibrils of the bacteria (Herzberg et al., 1983b). This location facilitates platelet-bacterial binding, since it has been postulated that the narrow radius of the tips of such microfibrils allow a closer approach to other cell surfaces by overcoming intercellular repulsive forces that are directly proportional to the local radius at the site of contact (Lesseps, 1963; Clawson and Good, 1971). Thus the action of various intercellular adhesive molecules would be facilitated by this location (Hawiger, 1987). It appears that Strep. sanguis strains express multiple adhesins (Hasty et al., 1992). An 87 kD protein has been isolated from Strep. sanguis I 133-79 that can serve as an adhesin for both platelets and saliva-coated hydroxiapatite [Gong and Herzberg, 1989]. Work in progress by Herzberg and co-workers indicates that streptococcal adhesins for platelets are probably oligospecific with more than one epitope capable of binding to platelet surface components [Herzberg, M.C. personal communication]. The platelet surface receptor(s) for streptococcal adhesins has not been identified. 3 . 6 . 2 P l a t e l e t A g g r e g a t i o n b y Strep. sanguis In addition to adhesin(s) for platelets, those strains of Strep. sanguis capable of inducing platelet aggregation

102 C.C. CLAWSON express a second unique surface protein that has been dubbed the platelet aggregation-associated protein (PAAP; Erickson and Herzberg, 1990). PAAP may be released in soluble form from bacteria by sonication or gentle trypsin digestion (Herzberg etal., 1983b), and has been partially characterized (Erickson and Herzberg, 1990). The form of PAAP released by trypsin digestion had a molecular weight of 65 kD, contained 4% carbohydrate by dry weight, and was estimated to contain 586 amino acid residues rich in lysine, glycine and glutamic acid (Erickson and Herzberg, 1990). PAAP is also synthesized by protoplasts of Strep. sanguis and released into the media from whence it has been harvested and purified (Herzberg et al., 1990a). The latter PAAP product had a molecular weight of 115 kD, was 39% carbohydrate, and contained about 681 amino acid residues. When the 115 kD form of PAAP was subjected to trypsin digestion, the resultant product was predominantly the 65 kD form of PAAP. The sonication treatment leaves the bacteria intact except for the removal of most of their microfibrilar coat. Strep. sanguis thus treated largely lose their capacity to induce platelet aggregation indicating that PAAP resides, similarly to the adhesins, with the surface microfibrils (Herzberg et al., 1983b). Soluble PAAP preparations inhibited Strep. sanguis-induced platelet aggregation, apparently by competitively binding to platelet receptors in a manner that failed to meet stereochemical requirements for platelet stimulation. Platelet-aggregating strains of Strep. sanguis treated with a monospecific antibody to PAAP retained their adhesion to platelets, but were inhibited in their ability to act as agonists of platelet aggregation (Erickson and Herzberg, 1990). The variability among strains of viridans streptococci to interact with human platelets was reviewed above (Section 3.4). This variability, coupled with other data, suggests that phenotypic expression of surface adhesins and PAAP by viridans streptococci may both be regulated by exogenous factors and represent adaptive responses of the microbes to environmental alterations (Hillman et al., 1989; Vickerman et al., 1991; Caparon, 1992; Mekalanos, 1992; Sommer etal., 1992; M.C. Herzberg, unpublished data). It may be reasonably argued that the capacity of collagen to act as an agonist of platelet reactivity has a strong survival advantage, and, thus, has been a strongly conserved trait of higher animals. The expression by a microorganism of surface determinants that can mimic the platelet-activating domains of collagen may contribute to the pathogenicity of that micro-organism. Several lines of experiments from Herzberg's laboratory have demonstrated that the ability ofStrep, sanguis to activate platelets is derived from molecular mimicry in which PAAP shares critical platelet-interactive domains with collagen: platelets deficient in surface GPIa do not respond to collagen (Nieuwenhuis et al., 1985), and GPIa-deficient platelets also failed to respond to Strep. sanguis (Soberay et al., 1987). Further, co-incubation of either collagen or

Strep. sanguis with anti-PAAP antibody blocked platelet aggregation response to either agonist (Erickson and Herzberg, 1987). Antibodies to type I or III collagens or to a collagen-like synthetic octapeptide (see below) all interacted with Strep. sanguis to block its induction of platelet aggregation (Erickson et al., 1992) . These observations strongly indicated a close structural similarity, if not identity, between the platelet-agonistic epitopes of PAAP and collagen. It has been shown that some platelet-interactive domains of type III collagen have the amino acid sequence lysine-proline-glycine-glutamic acid-prolineglycine-proline-lysine (Karniguian et al., 1983). Erickson and Herzberg (1987) have demonstrated primary structural similarities between the PAAP component of Strep. sanguis and this platelet-reactive domain of collagen. These authors showed a cross-inhibition of platelet aggregation between a synthetic replica of the above octapeptide and soluble PAAP. Like soluble PAAP, this octapeptide inhibits the platelet response to collagen, presumably by blocking collagen receptors on the platelet surface in a non-agonistic manner. Since a number of platelet-interactive domains have been identified on collagen, amino acid substitutions to the collagenmimicking octapeptide were employed to determine a common structural motif required for platelet interaction (Erickson et al., 1992). As various single amino acid substitutions were made in the octapeptide, a hierarchy for inhibition of platelet response to collagen was established among 24 resultant peptides. Computer modelling was then used to predict the structural implications of the various amino acid substitutions to the plateletinteractive region of type III collagen. From this exercise the authors were able to predict that for a plateletinteractive domain the basic requirements are a negatively charged amino acid flanked by two regions with the potential for B-turn. The several (seven) known plateletreactive sites of bovine and human collagen were also compared for their fit with the predicted requirements. From these collective data the minimal structural motif for the platelet-interactive domains common to collagen was determined to be the amino acid sequence prolineglycine-glutamic acid-(proline or glutamine)-glycineproline. Erickson and Herzberg (1993) then used the ability of soluble PAAP to inhibit platelet aggregation by Strep. sanguis to identify a minimal platelet-interactive domain of Strep. sanguis PAAP. By sequential digestion and testing of fragments for inhibitory effect this domain was localized to a heptapeptide, proline-glycine-glutamic acid-glutamine-glycine-proline-lysine, that conforms with the predicted platelet-interactive structural motif of collagen noted above. It is likely that the cell-surface PAAP contains a number of these epitopes, i.e. it is multivalent.

3.6.3 Ecto-ATPase o f Strep. sanguis The third surface component of Strep. sanguis that has been implicated in their interaction with platelets is an

PLATELETS IN BACTERIAL INFECTIONS 103 ecto-ATPase that is immunologically distinct from the adhesins and PAAP (Herzberg and Brintzenhofe, 1983; Herzberg et a/., 1985; MacFarlane et al., 1994). This ecto-ATPase is associated with the bacterial cell wall, and has been localized, isolated, and distinguished from the F1F0 ATPase of Strep. sanguis cell membrane and other ATPases of the cytoplasm (MacFarlane et al., 1994). Immunoelectron microscopy showed the ecto-ATPase to be associated with the cell wall, surface microfibrils, and divisional septae ofStrep, sanguis. This enzyme can hydrolyze ATP to ADP (Herzberg and Brintzenhofe, 1983). An early event in the response of platelets to most agonists of aggregation is the release of ADP and ATP from the platelet's dense granules. Since ATP is antagonistic and ADP agonistic of further platelet activation, the presence of the ecto-ATPase may serve to shift the balance of the reaction toward enhanced platelet activation. While the role of this enzyme in the metabolic economy of Strep. sanguis is unknown, expression of this ATPase may be considered an added virulence factor for the micro-organism.

3.7

ENGULFMENT OF BACTERIA BY PLATELETS

Examples of engulfment of bacteria by normal platelets have appeared only rarely in the literature of platelet-bacterial interaction; however, bacterial engulfment has been demonstrated under special circum-

=

stances. As noted above, congenitally afibrinogenemic platelets were far more sensitive than normal platelets to the ratio of bacteria to platelets in their aggregation with Staph. aureus (Clawson et al., 1980b; Clawson and White, 1980). Electron microscopy of the reaction demonstrated that, as the ratio was reduced to about one organism to five platelets, most of the platelets that interacted with bacteria remained unaggregated; although they appeared to undergo full shape change and secretion response. These individual reacted platelets also frequently had engulfed adherent bacteria (Figs 5.12 and 5.13). At these ratios bacteria appeared to enter the preexisting OCS in a manner analogous to that described above for inert particulates. Often there was material adjacent to bacteria in the OCS that was similar in texture and density to that of platelet granules suggesting a form of degranulation comparable to that of leucocytes (Figure 5.13). Although direct connections to the exterior or to other segments of the OCS were not always demonstrable in the plane of section, after post-fixation staining with lanthanum nitrate (which does not cross the cell membrane) the bacteria-containing spaces always showed this tracer (Fig. 5.14). If the ratio of bacteria to platelets was 1:1 aggregation was modestly increased, fewer platelets remained free, and bacterial ingestion was less common. When physiologic levels of fibrinogen were replaced in the reaction media, normal aggregation as defined by nephelometry was restored. Nonetheless, examples of bacterial engulfment by fibrinogen-deficient platelets were readily found, although less common than when fibrinogen was omitted and aggregation was less com-

.

Figure 5 12 Platelet from a congenitally afibrinogenemic patient fixed after completion of the aggregation response as indicated by plateauing of the aggregometry tracing. This platelet is an example of the many platelets in this test system that escaped incorporation into a platelet-bacterial aggregate. A microbe appears to have been completely engulfed into the OCS of this platelet (x 27 000). [From Clawson and White (1980) with permission.]

Figure 5.13 Example of possible degranulation into the bacterium-containing space (OCS) of a congenitally afibrinogenemic platelet. Material similar in texture and electron density to platelet (x granules is seen adjacent to the microbe (x 40 000). [From Clawson and White (1980) with permission.]

104 C.C. CLAWSON

Figure 5.14 Congenitally afibrinogenemic platelet with an engulfed bacterium stained with lanthanum nitrate after the Initial fixation and examined without additional electron dense staining. This demonstrates that the membrane-enclosed space in the platelet that contains the microbe remains open to the exterior media since the lanthanum tracer, which cannot cross the cell membrane, has freely gained access to this space (• 42 000). [From Clawson and White (1980) with permission.]

plete. Attempts to foster bacterial engulfment by normal platelets were made by reduced stirring, prolonged incubations, dilution of the ratios to as little as one microbe per 100 platelets, or by washing the platelets free of exogenous plasma proteins, but all failed to induce visible engulfment beyond a few very rare occurrences (Clawson and White, 1980). Mantur and colleagues (1986; Kemona et al., 1986) have reported briefly on a system for quantitating ingestion of bacteria by human platelets. They used PRP anticoagulated with both heparin and citrate, and Staph. aureus 209P incubated with stirring for 10 min. The "extracellular" bacteria were then lysed with lysostaphin. They reported that by light microscopy 0.7% of platelets had ingested bacteria at 6 min with a slight decline at 8 and 10 min. The phagocytic index among the few ingesting platelets was 1.3 microbes per cell. Electron micrographs of their system at times up to 15 min revealed bacteria in spaces that appeared to be within wellaggregated, degranulated platelets. The illustrations are compatible with other studies that have indicated bacterial residence within the OCS. Until recently no further convincing examples of bacterial engulfment by platelets had appeared. In 1992, Simonet et al. reported studies of human platelet inter-

action with an invasin-expressing strain of Yersiniapseudotuberculosis for which they convincingly illustrated uptake of this organism. As previous workers had done, Simonet and colleagues employed an electron-dense tracer that does not cross cell membranes. In contrast to prior studies outlined above, these authors failed to find this tracer in bacteria-containing compartments of platelets. This indicated that, for this organism at least, the bacteria were sealed off from the extracellular media in a manner analogous to true phagocytosis. The Simonet group determined that expression of invasin on the surface of Yersinia was necessary for platelet stimulation. However, when they investigated a strain of E. coli which was also capable of expressing invasin, the bacteria adhered to platelets but were not taken up by them. Thus, there was a dissociation between platelet activation by invasinexpressing Yersinia and its ingestion. The factor(s) necessary for the ingestion phase were not identified. In the early 1970s Fajardo (1973; Fajardo and Tallent, 1974) reported observations by electron microscopy of Plasmodium vivax in platelets of two men with naturally acquired malaria. Fajardo subsequently reported (1979) finding parasites in platelets from 53% of 31 mice experimentally infected with P. berghei Tedeschi et al. (1975) demonstrated by light and electron microscopy Grampositive cocci in the residual circulating platelets of four patients with chronic autoimmune thrombocytopenia. 3.8

FATE OF THE BACTERIA

As reviewed above, bacteria that serve as agonists of platelet response participate in the aggregation and come to lie centrally in platelet-bacterial aggregates. With a strong aggregation response the bacteria are usually trapped between platelets. In other circumstances a small number of the microbes appear to be ingested in a process akin to true phagocytosis. In either case the bacteria are sequestered from the media and are in an ideal position to be exposed to high concentrations of the platelet's secretory products that are released in abundance by bacterial stimulation (Clawson et al., 1975). This raises a question about the fate of bacteria in platelet-bacterial aggregates. Several authors have reported finding bactericidal products of platelets (Gruber and Futaki, 1907; Barreau, 1909; Jacox, 1950; Amano et al., 1952; Kato et al., 1954; Donaldson et al., 1964; Weksler and Nachman, 1971; Donaldson and Tew, 1977; Miragliotta et al., 1988). Probably the earliest report of such a product came from the work of Fodor (1887) in which he described a heat-stable serum component in animals that was active against the anthrax bacillus. This factor was called B-lysin to distinguish it from the complementassociated c~-lysin. Although Gengou (1901)showed that ~-lysin is of cellular origin, its association with platelets was not demonstrated until 1960 (Hirsch, 1960). This platelet-derived protein is active against several strains of

PLATELETS IN BACTERIAL INFECTIONS 105 bacteria ~ncluding Bacillus anthracis, Arthrobacter, other by leukocytes could be minimized. The albumin-washed Bacillus spp., Clostridium, Lactobacillus, Sh~qella, Vibrio platelet preparations had no more than 0.25% leukocyte cholerae, and the prevalent strains of Alicrococcus. It is a contamination, the great majority of which were lymcationic protein of MW 6000 and is stored in the c~- phocytes. In the initial studies platelet aggregates were granules of rabbit platelets (Weksler and Nachman, 1971; osmotically lysed with distilled water to release the bacDonaldson and Tew, 1977). In contrast to other animals, teria, the standard method used in studies of bacterial human platelets appear to contain little or none of this phagocytosis and killing by neutrophils (e.g. Clawson protein. Miragliotta and co-authors (1988) have reported and Repine, 1976). The aggregates appeared to be well antibacterial activity of human platelets against a smooth lysed and surviving bacteria were counted as colonystrain ofSalm0nella typhi Ty-2, but the antibacterial factor forming units by quantitative plate culturing. By this was not identified. They report finding no effect against method it appeared that nearly 80% of Staph. aureus were two rough strains of Salmonella minnesota (R345-Rb and no longer viable after 60 min residence in human platelet aggregates. However, these results did not agree with R595-Re), nor against Staph. aureus. Early investigators isolated another product of horse, morphologic evidence that indicated the bacteria to be rat and rabbit platelets that was active against B. anthracis still quite structurally intact after 1 h in platelet (Gruber and Futaki, 1907; reviewed in Tocantins, 1938). aggregates (Fig. 5.15; Clawson, 1973). Therefore, it was This substance, which Gruber and Futaki called postulated that what were being inferred as individual "plakin", could be extracted from platelets by lysis with organisms by counts of colony-forming units might have distilled water and precipitation in calcium phosphate gel been, in fact, small clumps of bacteria not separated by (Amano et al., 1952). In contrast to ~-lysin, plakin was osmotic disruption. This possibility was examined by active principally against Bacillus species and was not bac- subjecting samples to controlled ultrasonic disruption tericidal for Staphylococcus, E. coli or Vibrio tyrogenus. This after osmotic lysis. Control studies showed bacteria to be product was not found in the platelets of mice, dogs, quite resistant to the level of sonication employed. When the sonication step was added, nearly 100% of the bacsheep, pigs, cattle, or humans. Among others who attributed platelets with bacteri- teria remained viable, and colony counts were not cidal activity were Taniguchi and co-authors (1930). In statistically different from either zero-time cultures or separate experiments they injected rabbits and guinea- 60 min controls that lacked platelets. Similar results were pigs with either nucleated red blood cells of fowl and obtained for washed human platelets with Strep. pyogenes frogs or with several bacterial pathogens. They drew or E. coli and for washed rabbit platelets with blood from the animals at intervals from 2 to 60 min and Staphylococcus. The supernatant of collagen-activated observed that most of the "corpuscles were surrounded platelets also clumped Staphylococcus so that sonication by a mass of blood platelets". The red cells appeared to was necessary to obtain accurate counts by quantitative by undergoing lysis while the platelets remained culturing. It was concluded that aggregated platelets had unharmed suggesting "a picture of fruit being devoured no detectable bactericidal activity On these strains of by a swarm of insects". This process of action against pathogens during up to i h residence deep in the platelet foreign cells and bacteria they dubbed "pepticytosis"; mass. Further, the possibility that bacteria within platelet however, they state that they were unable to demon- aggregates were somehow protected from secretory strate peptocytosis in vitro. In naturally occurring human products released to the media was excluded by incubamalaria and experimental malaria in mice, Fajardo (1979) tion of bacteria with supernatant of platelet-collagen found by electron microscopy intra-platelet structures aggregation. In the latter experiments the bacteria were that they interpreted as partially digested parasites. These again clumped by a platelet product or products, were seen in 50% of the human subjects and 73% of the although remained fully viable. The observation that a product of platelet secretion mice. In the studies of bacterial ingestion by Mantur and colleagues (1986; Kemona et al., 1986) noted above, the could clump the test organisms necessitating sonic disauthors interpreted some of their micrographs as showing ruption for proper quantitation of viability raises other damaged or killed bacteria, but they did not control for issues. One is the proper interpretation of earlier reports possible access of lysostaphin to bacteria-containing ~ of the platelet's bactericidal properties. Where culturing spaces. Also, the bacteria had been taken from late techniques are employed, bacterial death is inferred from growth cultures and no attempt to control for normal a loss of colony-forming units. This method becomes invalid if the organisms cannot be consistently dispersed. bacterial decay was documented. Clawson and White (1971b) examined the survival of Another issue is the identity of the clumping agent itself. Staph. aureus 502A, E. faecalis, and E. coli at 1 h after Since the Staph. aureus used in the experiments of aggregation with washed human or rabbit platelets in Clawson and White (Clawson et al., 1970; Clawson and vitro. Washed platelets were employed in these studies for White, 1971a, b) is a commonly encountered pathogen, two reasons: first, the protein content of the reaction it may be anticipated that most donors have antibodies to mixture could be standardized, usually by addition of 5% this organism, and that these antibodies could be carried pooled human serum; second, contamination of platelets along with the platelets even through several washes.

106 C.C. CLAWSON Even in the absence of specific antibodies, it has been found that when most strains of coagulase-positive strains of Staphylococcus are mixed with plasma or serum they exhibit clumping. This phenomenon was first described by Much (1908) and later characterized in part by Duthie (1955). It has been shown that the process is due to interaction between a "clumping factor" on the surface of the microbe and fibrin monomer complexes (Allington, 1967; Lipinski et al., 1967; Hawiger et al.,

1978). Other organisms are also clumped in the presence of plasma by a similar if not identical mechanism (Tillet and Gardner, 1934:; Duthie, 1955; Yoshida et M., 1979). Subsequently the region of fibrinogen that binds to staphylococcal clumping factor has been identified (Kloczewiak et M., 1987), but the identity of the clumping factor of Staphylococcus remains in doubt. McDevitt and co-workers (1992) have shown that it is not dependent on coagulase itself. Whether or not the

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; :;'

~r

(c) Figure 5.15 Examples of bacteria that have resided in the interior of mature human platelet aggregates for 1 h before fixation for microscopy. There is no evidence of morphologic damage when compared to control bacteria fixed directly in growth phase cultures. (A) Staph, aureus (x 35 200); (B) Strep, pyogenes (x 32 640); (C) E, coli ( x 2 5 600). [From Clawson (1973) with permission.]

PLATELETS IN BACTERIAL INFECTIONS 107 platelet may store this factor and, therefore, carry it through several washings to be later released by activation, is not known. It is clear that even well-washed platelets will release into the media a number of adhesive glycoproteins from their c~-granules that could assist staphylococcal clumping. Those known to date are fibrinogen, fibronectin, laminin, vitronectin, thrombospondin, and osteonectin (Holmsen, 1990b; Coller, 1992). Vercellotti and co-workers (1984) have shown that laminin or fibronectin binds Strep. pyogenes and Strep. sanguis, as well as Staph. aureus, but not a variety of Gramnegative organisms.

3.9

BACTERIAL PROMOTE

PRODUCTS OR INHIBIT

THAT PLATELET

ACTIVATION Thus far this chapter has focused primarily on direct interactions of bacteria with platelets. We must also briefly consider the influence of released products of bacteria that have potential for activating or inhibiting platelets separate from a direct cell-to-cell interaction. A number of specifc agents of bacterial origin have been identified that directly activate platelets. Prototypical among these is the family of lipopolysaccharide endotoxins of Gram-negative bacteria. The multiple complex effects of endotoxins on blood cells and inflammation, which have been extensively studied since first being described over a century ago (Roemer, 1891), are beyond the scope of this chapter. Other bacterial products with platelet effects are also of interest. For example, platelet-activating factor (PAF), which is produced by a variety of cells including leucocytes, macrophages, endothelium and platelets themselves (O'Flahery and Wykle, 1987), is also produced by some bacteria. Denizot and co-workers have identified PAF production by both E. coli (1989), and Helicobacter pylori (1990). In the latter case, supplementation of the growth medium with precursors of PAF, lyso-PAF and acetylCoA, which are present in abundance in mammalian intestine, was required to induce PAF production by three out of five H. py/0r/ strains isolated from gastric biopsy specimens of ulcer patients. Other products of micro-organisms are capable of inhibiting platelet aggregation. More recent examples of these are outer membrane proteins of Yersinia pestis (Leung et al., 1990), an isochromanequinone antibiotic produced by a Thermomonospora species (Patel et al., 1989), and products of Streptomyces species Q-1043 called "placetins" (Ozasa et al., 1990).

4. Platelet Interaction with Bacteria in vivo Periodically throughout most of this century various authors have made a case for including platelets among

the mechanisms that are defensive against bacterial (e.g. Delrez and Govaerts, 1918; Taniguchi et al., 1930; Tocantins, 1938; Salvidio and Crosby, 1960; Maupin, 1969; Copley and Witte, 1976). In 1969 Maupin concluded that "It is permissible to attribute to platelets a defensive role against bacteria, viruses or other foreign particles". In spite of the evidence accumulated to support this viewpoint, Copley and Witte (1976; Copley, 1979) still found it necessary to chide the scientific community for neglecting or ignoring the evidence. As has been outlined above (Section 2.1), considerable in vivo work had been done to study the impact of platelets on inert particles. However, considerably less in vivo work with bacteria has focused on the interplay between platelets and other participant clearance mechanisms. In recent decades there has been a growing acceptance of platelets as an important factor in a variety of human disorders, which has again brought attention to the possible impact of platelet-bacterial interactions on bacterial clearance from the bloodstream. Several facts suggest that platelets are potentially the cell most likely to first contact and interact with circulating microbes. First, platelets are present in large numbers, normally a 10- to 20-fold larger population than the number of circulating phagocytes. Furthermore, studies of cellular distribution within'flowing blood indicate that platelets and bacteria will tend to flow in the same region of the stream. Before the microscope was sufficiently refined to allow true definition of the platelet, Poiseuille (1835) observed that the axial stream of red cells was separated from the walls of capillaries by an annular region of plasma that was free of erythrocytes or leukocytes. This plasmatic zone, as it came to be known, was later shown to be related to the laminar pattern of blood flow (Whitmore, 1968; Goldsmith, 1972). It was demonstrated as early as 1868 that in laminar flow particles tend to separate on the basis of size with the larger distributed more centrally (Vejlens, 1938). In 1886, Eberth and Schimmelbusch, in their paper on experimental thrombosis, published elegant drawings that showed accumulation of platelets in the peripheral plasmatic zone, especially as the rate of flow decreased, while leukocytes moved centrally. Their findings were later extended by Copley and Staple (1962). These latter authors, like Eberth and Schimmelbusch, made direct microscopic observations of small vessels in the living animal. They employed a pneumatic cuff around a portion of hamster cheek pouch that allowed graded and reversible alterations of flow rate. Platelets were seen travelling at the periphery of the axial stream of cells and in the plasmatic zone. When graphite particles the same order of size as bacteria, 1-2 #m and smaller, were injected into flowing blood they tended to move with platelets. In addition, when true plasma skimming took place at the junction of small capillaries, platelets and graphite entered these small vessels leaving erythrocytes and leukocytes behind in the main channel. These direct

108 C.C. CLAWSON observations support the concept that bacteria and platelets, due to similar size and form, tend to flow together and, in fact, at times may be segregated together away from other defensive blood cells. Finally, as has been reviewed above, the platelet surface is very susceptible to adhesion with certain bacteria in fluid suspension, even in the absence of antibody or other opsonic proteins. Once stimulated by this contact, platelets respond very rapidly and sequester the microorganism. In contrast, even with optimal opsonization of bacteria, leukocytes in suspension complete phagocytic engulfment relatively slowly as compared with platelet-bacterial interactions. In the absence of specific opsonins leukocytes take up bacteria in suspension quite poorly (Clawson, 1974) and appear to prefer a surface against which to perform bacterial ingestion (Wood, 1951; Wood et al., 1951; Lee et al., 1983, 1984).

4.1

BACTERIAL

CLEARANCE

FROM

THE CIRCULATION Intravascular aggregation of platelets may be induced in three general ways: activation of the coagulation cascade as in disseminated intravascular coagulation (DIC); exposure of platelets to collagen in the vessel wall; or interaction with circulating particulates such as immune complexes, non-biological particles, or bacteria. In earlier sections of this chapter in vivo platelet-particulate interactions relevant to platelet-bacterial interactions were reviewed. This section will examine the behavior of platelets when placed in contact with circulating bacteria. Probably the first experimental studies of the interplay between septicemia and platelets were those of Levaditi in 1901. Following production of a bacteremia in rabbits with Vibrio cholerae, he found microbes both within leukocytes and clumped with platelets. During the next three decades several authors laid great emphasis on platelets as a primary participant in the removal of microorganisms from the bloodstream. Among these were Govaerts and co-workers (Delrez and Govaerts, 1918; Govaerts, 1921a, b), who examined clumping of bacteria with platelets both in vivo a n d / n v/tr0, concluding that this process made a significant contribution to the elimination of circulating microbes. Teale and Bach (1920) shared this conviction and stated that "the mechanism underlying the removal of the bacteria in the capillaries of the liver, lungs, etc. is the entanglement of the bacteria into loose clumps by a mass of blood platelets". In 1938 Tocantins published an extensive review of all phases of mammalian platelet physiology in which he examined the possible involvement of these cells in host defense. While he reached no firm conclusion on the matter, the case that he presented seems now to have been sufficient to have stimulated further and more extensive investigation of the problem. However, an exception to this viewpoint is found in a paper by Bull and McKee (1922) who studied the clearance of several

strains of bacteria from the blood of normal, immune, and thrombocytopenic rabbits. These authors laid greatest emphasis on the presence or absence of humoral agglutinins concluding that this factor was of prime importance in their results. They dismissed the clumping of bacteria with platelets as "merely incidental" and concluded that "neither the agglutination of bacteria within the blood stream nor their disappearance from it can be ascribed to a function of the platelets". This appears now to have been too sweeping a generalization. Their own data indicate that Staph. aureus had a much greater propensity for clumping with platelets than virulent pneumococci. Further, they noted that clearance of Staph. aureus was actually more rapid from the bloodstreams of thrombocytopenic non-immune animals than from non-immune rabbits with normal platelet levels. They reported nothing on the location of cleared organisms under the various experimental conditions imposed. Nonetheless, the view expressed by Bull and McKee (1922) apparently held sway for the time, and it was some decades before the host defense role of platelets received much attention. Their 1922 paper may be considered as a point of divergence from which studies of platelet hemostatic physiology and the investigation of adaptive immunity seem to have traversed separate paths that only occasionally crossed. The intervening decades, especially the last two, have seen dramatic extensions of our knowledge in both areas. The importance of specific humoral and cellular immune responses has been established as a major facet of host defense. The dominant efforts in platelet research were expended in defining their hemostatic activity, which has now been amply verified; nonetheless, reports of micro-organisms clumping with platelets in vivo and questions regarding their role in host defense continued to appear (Taniguchi et al., 1930; Dudgeon and Goadby, 1931; Raffel, 1934; Houlihan, 1947a, b; David-Ferreira, 1964). Taniguchi and colleagues (1930) found that injection of Staphylococcus into circulation of a rabbit produced a profound thrombocytopenia within 3 min, but within 30min platelets reappeared in the circulation and regained their normal number within hours. As we have seen, this was similar to the observations made with fine quartz particles a few years earlier by Tait and Elvidge (1926). Taniguchi's group also examined the intravascular fate of several species of pathogens: Streptococcus, typhoid bacillus, E. coli and V. cholerae. Almost immediately after injection the micro-organisms were aggregated with platelets and almost no free bacteria could be found. The number of bacteria associated with platelets reached a peak by 5 min, and by 10 min nearly all microbes had disappeared from the circulation. In contrast the number of organisms taken up by leucocytes rose gradually, reaching a peak by 30 min. At all times studied the number of bacteria associated with platelets was far greater than those with leucocytes. They concluded

PLATELETS IN BACTERIAL INFECTIONS therefore that "the corpuscular element of the blood which stands on the first line is not leucocytes, but blood platelets". As we have noted, they further stated that platelets were capable of bactericidal activity through a process of "pepticytosis" (Section 3.8). Copley and Balea (1960; Copley, 1979) observed intravascular platelet-interaction with Mycobacterium directly by biomicroscopy of hamster cheek pouch vessels. Platelet-bacterial aggregates were seen by 10 sec after injection. They also examined the lung and noted capillaries ruptured by platelet-bacterial thrombi with release of microbes into the alveolar space. Perman and co-workers (1975) used dogs made thrombocytopenic by ~-estradiol into which they injected radiolabelled, heatkilled Staphylococcus. The distribution of the organisms was followed by scintillation camera for up to 30 min. It was found that in animals with normal platelet counts organisms passed quickly through the pulmonary circulation and were rapidly taken up by the liver. In contrast, in the thrombocytopenic dogs hepatic uptake was delayed and bacteria were detected in increased numbers and for several minutes longer in their lungs. Restoration of normal platelet counts to estradiol-treated dogs by platelet transfusion eliminated the prolonged residence in the lungs and restored a normal pattern of hepatic clearance. These observations are in agreement with the model of van Aken and Vreeken (Section 2.1) in which platelets aid in transport of foreign particles to the fixed phagocytic system.

5. Influence of Platelets on Phagocytes An important emerging facet of the function of platelets as an inflammatory cell is their influence on the behavior of phagocytes. Evidence is available indicating that platelets are capable of contributing to the pool of chemoattractants for phagocytes and of influencing ingestion and killing of bacteria by these phagocytes. In addition, there is now a growing body of knowledge regarding the "transcellular" eicosanoid metabolism between platelets and neutrophils (Marcus, 1990). The results of this co-operative intercellular metabolism include both enhanced generation of single-cell products and generation of novel products not synthesized by either cell type independently (Serhan, 1991; Maderna et al., 1993; Serhan, 1993). A detailed discussion of this synergistic symbiosis between the platelet and neutrophil is beyond the scope of this chapter, but this metabolic co-operation no doubt supports the influences of platelets on phagocyte functions outlined below.

5.1

MORPHOLOGICAL OBSERVATIONS

Horn et al. (1969) reported accumulations of platelets around fixed macrophages were increased in animals that

109

had been injected with bacteria, and that both platelets and platelet-bacteria complexes were ingested by macrophages. Smith (1972) added Staph. aureus or E. coli to fresh whole human blood and examined the aggregates after 15 min. He found that neutrophils and monocytes were adherent to platelet-bacterial aggregates and had ingested bacteria from the aggregate surface. However, most of the aggregates were too large to be engulfed by a single phagocyte, and the internal bacteria were inaccessible to the phagocytes. Similar observations by light and electron microscopy have been reported for mixtures Staph. aureus with purified washed blood phagocytes and platelets (Clawson, 1974). In the latter studies formation of platelet-bacteria-neutrophil complexes occurred in both the presence and absence of plasma proteins.

5.2

PHAGOCYTOSIS

AND KILLING

OF

BACTERIA It has been noted above that platelets are not capable of independently killing most strains of bacteria. However, platelet-bacterial interaction has been found to enhance the bactericidal activity of phagocytes in vitro and may do so in vivo. Mandell and Hook (1969) mixed Salmonella with mouse peritoneal macrophages in fresh or freezethawed PRP or in platelet-poor plasma. They found that the presence of intact platelets doubled the number of organisms ingested by macrophages. Wilder and Lubin (1973) exposed glass-adherent mouse macrophages to Listeria monocytogenes for 1 h, washed off the uningested bacteria, and then added rabbit platelets. They found that the presence of intact platelets enhanced the bactericidal activity of the macrophages. In this system there was no obvious interaction between the intracellular bacteria and the platelets, and the mechanism for enhanced bacterial killing was not elucidated. Rabbit platelets are rich in /3-1ysin (Weksler and Nachman, 1971), to which Listeria is highly sensitive, but it is not clear that/3-1ysin becomes available to the macrophage in this system. Clawson (1974) studied the influence of plateletbacterial interaction in vitro on killing of Staph. aureus by washed purified human neutrophils (at 50 bacteria per neutrophil) with and without serum replacement. N o bactericidal activity was found in the serum-free system in the absence of platelets, but with platelets present about half of the bacteria that had been ingested by neutrophils were killed in 1 h. When serum was added in the absence of platelets about 40% of the bacteria were killed; with both serum and platelets present 60% were killed. Electron microscopic examination of this system indicated that bacterial killing occurred in neutrophils and not in platelet-bacterial aggregates. Again the mechanism for these observations has not been fully clarified; however, it is known that eicosanoid products of platelets can enhance both degranulation and the metabolic burst of neutrophils (Henderson, 1989). Two products of platelet secretion that augment phagocytosis have been

110 C.C. CLAWSON partially characterized by Sakamoto and colleagues (Sakamoto and Firkin, 1984; Sakamoto et al., 1987; Sakamoto and Yokoya, 1991) as 140-160kD and 290-320 kD proteins that operate via C3 and Fc receptors of the leucocytes. Activated platelets release fibronectin that also may act to enhance neutrophil and monocyte antibacterial functions (Proctor, 1987). Kaplan et al. (1982) have demonstrated that normal platelets can correct the function of neutrophils from the beige mouse analog of the Chediak-Higashi syndrome of humans, probably through a 5-HT-dependent system.

5.3

CHEMOTAXIS

Platelet-bacterial interaction has a material effect on the chemotactic response of human neutrophils. In one series of studies this was tested using normal platelets and neutrophils from which all extracellular plasma proteins were removed by multiple washings that left the cells intact and unreacted (Clawson and White, 1971b; Clawson, 1974). The presence of platelets activated by washed Staph. aureus in protein-free HBSS produced a marked chemotactic response by neutrophils in a Boyden chamber system (Boyden, 1962; Keller, 1966). The chemotactic index was 4-fold greater than that induced by unreacted platelets alone or bacteria alone. The addition of pooled serum produced no further increase in chemotaxis indicating that serum proteins were neither necessary for the response nor did they enhance the response. The chemotaxin produced by plateletbacterial interaction was released into the media since a cell-free supernate of platelet-bacterial aggregation was also chemotactic for neutrophils. It has been demonstrated that platelets are capable of contributing chemotaxins from at least three sources. First, they contain chemotactic products released during the secretory phase of activation. It has been shown that PDGF is a chemotactic stimulus of monocytes and neutrophils (Deuel et al., 1982), activates the metabolic burst of neutrophils (Tzeng et al., 1984), and is also chemoactic for smooth muscle cells (Grotendorst et al., 1981). Platelet factor 4 (PF4) is also a chemoattractant for neutrophils and monocytes (Deuel et al., 1981) and for fibroblasts (Senior et al., 1983). Second, platelets contain a protease that cleaves C5 to CSa, a promoter of leucocyte chemotaxis and activation (Weksler and Coupal, 1973). Finally, activated platelets generate chemotactic eicosanoids, particularly 12-HETE (Turner et al., 1975; Henderson, 1989).

5.4

PHAGOCYTOSIS OF PLATELETS

The formation in vivo of rosettes of platelets about neutrophils, platelet satellitism, with ingestion of platelets has been noted in a variety of pathological conditions (Packham and Mustard, 1984). The phenomenon may be caused by antibodies in some cases or other serum

factors (Greipp and Gralnick, 1976), or due to platelet abnormalities (McGregor et al., 1980). It is not known whether in some instances bacterial contact in vivo might contribute to this process. Under some experimental conditions platelets can enhance the natural adhesiveness of neutrophils and encourage phagocytosis in the absence of specific antibodies. In experiments outlined above (Section 5.2), Mandell and Hook (1969) examined mixtures of macrophages, platelets and bacteria by light microscopy. They found that platelets were not phagocytosed in the absence of bacteria, but with bacteria both were taken up by the macrophages. In the study of Clawson (1974) discussed above (Sections 5.1 and 5.2) only platelets that had been activated by bacterial contact were ingested by the neutrophils. Some investigators have noted that platelets enhance adherence of neutrophils to inert surfaces (Garvin, 1961; Hopen, 1979; Rasp et al., 1981). Rasp and co-workers demonstrated that the presence of platelets significantly increased adherence of neutrophils to nylon fibers with or without the availability of plasma proteins. Their results also indicated that direct association of platelets and neutrophils was, required for the effect, since supernatants of nylonadherent platelets did not increase neutrophil adherence, and scanning electron microscopy demonstrated close proximity of platelets with adherent neutrophils. If the responsiveness of platelets was suppressed by pretreating the platelet donors with aspirin, the augmentation of neutrophil adherence was eliminated.

0

Implications of Platelet-Bacterial Interaction to Human Disease

As has been noted above, bacteria periodically gain access to the bloodstream through disruptions of defensive barriers of mucosal membranes. There is an extensive literature demonstrating detectable bacteremia following dental manipulation or even a period of vigorous chewing (Murray and Moosnick, 1941; Jones et al., 1970; Lineberger and DeMarco, 1973; Hockett et al., 1977; Coulter et al., 1990; Lofthus et al., 1991). Similarly, manipulation of the gastrointestinal (LeFrock et al., 1973), respiratory (Burman, 1960), or genito-urinary tracts (Sullivan et al., 1971) can also induce bacteremia (Levison, 1976). These observations indicate that everyone has numerous episodes of bacteremia in a life time. To this point our discussion has explored the interaction of platelets with bacteria in a manner that would usually be beneficial to the host. In this section emphasis will be placed on implications of these direct interactions to the pathogenesis of human disease. In the experiment reported above (Section 3.2) in which low concentrations of bacteria were added to PRP, the aggregates formed were very few in number and remained composed of but a few cells. Such aggregates could circulate freely through much of the micro-

PLATELETS IN BACTERIAL INFECTIONS vasculature. Similar platelet microaggregates are often found in sepsis (Bick, 1978; Neame et al., 1980). According to the the thesis of van Aken and Vreeken (1970) these small aggregates would disaggregate and reform until they eventually found their way to the reticuloendothelial system where they could be adequately removed by fixed phagocytes. Under usual conditions the interaction of platelets with bacteria in vivo probably operates to the net benefit of the host. However, in altered circumstances the above process may become detrimental to host well-being. We may postulate a variety of stress conditions that would impose deleterious modifications on the normally protective chain of events. The outcome may result in a variety of clinical conditions such as those presented in Table 5.2. For example, platelet aggregation might proceed to an irreversible state due to prior promotion of platelet adhesiveness and]or the aggregates might become larger because of increased numbers of bacteria and/or platelets in a confined blood volume. The aggregates may not properly disaggregate because of inhibited or inadequate fibrinolysis (Astrup, 1969; Holemans and Silver, 1969). The bacteria-laden aggregates could then lodge in capillaries well removed from adequate numbers of phagocytic cells; or circulating platelet-bacterial aggregates might be trapped by a previously formed thrombus on a vessel or

Table 5.2 Platelet-bacterial interaction and disease. A partial listing of pathologic processes in which interaction of platelets with bacteria or bacterial products may be implicated Adult (acute) respiratory distress syndrome Anaphylactic reaction Arteriosclerosis, pathogenesis or complications Bacterial endocarditis Blood or platelet transfusion transmission of infection Brain abscess Cavernous sinus thrombosis Complication of cardiovascular prostheses Complication of shock Diabetic vascular disease or infection Disseminated intravascular coagulation Focal glomerulonephritis Hemorrhage with sepsis in leukemia Infectious thrombophlebitis Myocardial infarction Microvasculitis syndromes Purpura fulminans Schwartzman reactions Stroke Thrombocytopenia Thromboembolic vascular disease Thrombotic thrombocytopenic purpura Toxic shock syndrome Transplanted organ rejection Waterhouse- Friderichsen syndrome Wound infections

111

endocardial surface. Since many strains of pathogens are unharmed by association with platelet aggregates, such bacteria-laden microthrombi could then become a nidus of infection. This, in turn, might lead to dissemination of infection via a thromboembolic pathway. Further harmful influences of such processes might come as a result of the release reaction of platelets. Platelets have been demonstrated to possess substances that may alter or damage vascular endothelium (Hughes and Tonks, 1962; Mustard et al., 1965; Da Prada et al., 1967; Jorgensen et al., 1970; Mustard and Packham, 1970; Nachman et al., 1970; Niewiarowski and Thomas, 1970). Released in locally high concentrations, these would produce vascular damage and inflammation even in the absence of a significant bacterial multiplication. As noted above (Section 5.2), under proper circumstances platelets may assist in destruction of bacteria by neutrophils or macrophages. But, if platelets and bacteria react in a manner that results in large aggregates, which are not small enough to be engulfed by either neutrophils or mononuclear phagocytes, the process may provide a mechanical impediment to removal of organisms by phagocytic cells (Section 5.1). Under such circumstances, accumulation of neutrophils at the site and unsuccessful phagocytosis might well lead to degranulation of neutrophil lysosomes (Henson, 1972) into the surrounding area and provide a further mode of vascular damage and promotion of inflammation. Once activated by bacterial agonists both the platelet and phagocyte individually and in concert can contribute to the pool of inflammatory mediators. From the considerations of various disease states in which platelet-bacterial interactions may contribute to the pathogenesis of the disorder, it appears clear that the ability of an organism to adhere to platelets is among its important virulence factors. It is of interest in this regard that some antibiotics, e.g. fluoroquinolones, affect the adherence of bacteria. Desnottes et al. (1990) report that perfloxacin both diminishes adherence of Staph. aureus, and other susceptible bacteria, to platelet-fibrin complexes and increases their adherence to phagocytes. This modification of the bacterial surface may decrease its tendency to become incorporated into protective platelet aggregates and enhance its availability for phagocytosis. At present these pathogenic implications of plateletbacterial interaction remain largely speculative. Nonetheless, sufficient experimental and clinical data is at hand to make such speculation attractive, and further, to suggest specific avenues of further investigation. Table 5.2 lists some of the disease states or syndromes in which platelet-bacterial interaction might be implicated as a contributor to the pathogenesis of the entity. An extensive literature has accumulated in support of platelet involvement in some of these areas while others remain more tentative. A detailed discussion of each is not necessary here, but a general consideration of a few of these

112 C.C. CLAWSON disorders will serve to point out the implications of platelet-bacterial interactions to human disease.

6.1

INFLAMMATION

AND TISSUE

INJURY Recognition of the platelet as an inflammatory cell has opened many demonstrated and potential varieties of tissue injury to platelet-induced pathogenesis (Silver et al., 1974; Copley, 1979; Nachman and Weksler, 1980; Benveniste and Vargrafig, 1982; Joseph, 1988; Page, 1989). It is not the purpose of this chapter to reiterate the evidence for the view that platelets are an important inflammatory cell. It will suffice for our review of platelet-bacterial interaction to highlight just some of the disorders in which this interaction may potentiate the inflammatory processes. Numerous observations, including many of those from studies outlined above, support the concept of bacterial interaction with platelets being one aspect of the platelet's participation in inflammatory vasculitis and concomitant tissue injury. As we have seen, many strains of bacteria are potent agonists of platelet secretion. Several products of platelet secretion have, in turn, been shown to be important contributors to inflammation. For example, cationic protein(s) from c~-granules are capable of increasing capillary permeability (Nachman et al., 1972). Bacterially induced secretion of ADP from platelets may be central to the initiation of some forms of vasculitis. Mustard and co-workers (Packham et al., 1967; Mustard and Packham, 1970) induced transient platelet aggregates in otherwise normal animals by injections of ADP via the coronary artery for a period of 4-5 min. The majority of animals developed myocardial infarcts; however, if the animals; were first made thrombocytopenic there was no cardiac dysfunction or tissue injury. Histology of the cardiac lesions revealed platelet aggregates in contact with the vessel wall. At the point of platelet contact the endothelium was absent and there were edema and neutrophil infiltrates. The acute glomerulonephritis seen with some bacterial infections may be due to platelet responses to bacteria or their toxins. Lesions similar to focal glomerulonephritis were produced by infusion of ADP into the renal artery (Packham et al., 1967; Mustard and Packham, 1970). Platelets have been implicated in rejection of renal transplants (Mustard and Packham, 1968). At the onset of a rejection episode there is frequently a fall in circulating platelet counts, and radiolabelled platelets accumulate in the transplanted organ. It is thought that some rejection episodes are induced by antigen-antibody complexes or bacterial infections. Behcet's disease is another disorder characterized in part by a severe regional vasculitis in which platelet-bacterial interactions have been implicated (Isogai et al. , 1991).

6.1.1 Bacterial Endocarditis Bacterial endocarditis is the best studied example of platelet-bacterial interaction as a potentially pathogenic mechanism. The disease may be caused by a variety of organisms, but most common are viridans streptococci and Staphylococcus (Durack and Petersdorf, 1977; Wannamaker and Parker, 1977). Although viridans streptococci are a common cause of bacterial endocarditis, the frequency of recovery has varied widely among epidemiologic studies (Roberts et al., 1979; Roberts, 1980). Evidence for a consequential involvement by platelets in this disease comes from pathological, clinical and experimental observations (Clawson, 1977). It has been known for decades that the vegetative lesion of human or experimental bacterial endocarditis is composed largely of aggregated platelets (Angrist and Oka, 1963; Angrist et al., 1967; Garrison and Freedman, 1970; Durack and Beeson, 1972). Electron microscopy by Angrist and others revealed that vegetations of bacterial endocarditis are constructed of a dense feltwork of platelets and fibrin with embedded bacteria and a dearth of leukocytes or erythrocytes. The clinical features are well documented elsewhere and only a few points relevant to platelets warrant mention here. Between 5% and 15% of patients with the disorder have a significant thrombocytopenia (Rabinovich et al., 1965). There may be multiple mechanisms for the fall in platelet counts, but it seems clear that the volume of those platelets that actually form vegetative lesions would not account for a significant thrombocytopenia. Several other clinical features invoke consideration of platelet alterations; these include petechiae, splinter hemorrhages, and thromboembolic phenomena (Johnson, 1976). Experimental evidence for a primary role of platelets in bacterial endocarditis comes from three quarters. First are animal models of bacterial endocarditis in which endocardial damage is induced. Second are observations of platelet interaction with denuded or damaged vascular endothelium. Third are studies of direct plateletbacterial interactions with common pathogens of bacterial endocarditis. The experimental animal models of bacterial endocarditis derive from the clinical observation that most endocarditis occurs under circumstances where a pre-existing endothelial lesion is known or highly likely. Such predisposing conditions include congenital heart disease, rheumatic heart disease, atherosclerosis, mitral valve prolapse, valvular prostheses, and indwelling catheters (Durack and Petersdorf, 1977). Methods employed to induce experimental endocarditis vary from direct mechanical damage of a heart valve to a number of non-specific modes of causing stress in the animal (Sande, 1976). A commonly used model employs a plastic catheter inserted into the heart via a peripheral vessel and allowed to lie within the heart or across a valve long enough to produce endothelial trauma; subsequently

PLATELETS IN BACTERIAL INFECTIONS bacteria may be introduced by various means (Garrison and Freedman, 1970). This method is similar to a model developed a century ago in Germany in which valvular lesions were produced by a sterile sound introduced via the carotid artery followed by inoculation of bacteria into the circulation (Wyssokowitsch, 1886). In experimental models of the disease sterile vegetations can form in the absence of infection and only subsequently become colonized by bacteria (Durack and Beeson, 1972). Moreillon and colleagues (1988) used this model in rats with periodontal disease and induced bacteremia by tooth extraction with subsequent bacterial endocarditis in 18 of 20 animals. An analogous condition may occur in humans when non-bacterial thrombotic vegetations are secondarily infected (Freedman, 1987; Lopez et al., 1987). While it is not always clear what triggers the onset of human bacterial endocarditis, it appears that the two essential initiating events are damage to endothelium and the co-existence of a bacteremia. Damage to the endothelium would usually expose the subendothelial elements that will be rapidly coated with platelets. However, this may not be essential since Ashford and Freiman (1968) have shown that sublethal damage of endothelium can occur that allows initiation of platelet adhesion directly to endothelium. The consensus conclusion that can be drawn from animal models of bacterial endocarditis may be summarized as follows. At the time of the primary alteration of the endothelial barrier, the constant presence of platelets in large numbers favors coating of a seminal lesion with platelets that will be activated by their initial adhesion to the altered endothelium or the subendothelium. A sterile thrombotic vegetation composed largely of a plateletfibrin matrix results, which is then susceptible to colonization by strains of bacteria that possess the requisite binding sites for resting and]or activated platelets or for the fibrin component of the matrix. In this context it is of interest to note the work of Scheld et al. (1981) in which they showed that several common antibiotics (vancomycin, penicillin, tetracycline, chloramphenicol and streptomycin) in sub-minimum inhibitory concentration concentrations reduced adhesion of Staph. sanguis and S. faecalis to a fibrin-platelet matrix in vitro. They also observed that pretreatment of these bacterial strains with sub-MIC levels of the antibiotics greatly reduced their ability to produce bacterial endocarditis in rabbits with damaged aortic valves. It is attractive to many investigators to postulate that interplay between bacteria and platelets, which had previously adhered to damaged endothelium, is the dominant mode of primary pathogenesis in bacterial endocarditis (Glauser, 1984). However, there is a contravening viewpoint that places the initiating event at the level of direct binding of bacteria to altered endothelium or subendothelium (Sullam et al., 1985), with or without participation of some non-cellular agent(s) such as fibronectin, laminin, or dextrans (Scheld et al., 1978;

113

Vercellotti et al., 1984, 1985; Hamill, 1987). The postulate that bacteria which cause endocarditis share a common affinity for such adhesins was examined by Yarnall et al. (1986). These authors studied 15 strains of Streptococcus and Staphylococcus, isolated from clinical endocarditis, for their affinity to bind the Fc region of human IgG, fibrinogen, fibronectin and human Clq, but found no common determinate for these molecules. It has been shown that fibronectin can bind to both the subendothelium (Mustard and Packham, 1984) and common pathogens of endocarditis such as strains of Streptococcus and Staphylococcus (Kuusela, 1978; Vercellotti et al., 1984; Kuusela et al., 1985). These species of bacteria also bind laminin (Vercellotti et al., 1985). Although the endothelial cell produces and exports fibronectin, it apparently normally lacks fibronectin on its luminal surface (Birdwell et al., 1978). Therefore, it would require some alteration of the endothelium or its removal exposing the subendothelium to allow fibronectin binding to occur. Fibronectin with multiple reactive domains might then cross-link bacteria to the subendothelium. However, it has also been demonstrated that, following exposure of the subendothelium, it is rapidly coated with a spreading monolayer of platelets (Mustard and Packham, 1984). Unstimulated platelets have little affinity for fibronectin, .while platelets activated by collagen or thrombin both secrete fibronectin from s-granules and express surface GPIIb-IIIa that acts as receptor for fibronectin (Ginsberg et al., 1980; Mustard and Packham, 1984). Therefore, initial contact between the subendothelium and platelets would appear to be direct and not dependent upon intermediary fibronectin. Once the initial monolayer of platelets is formed, subsequent events may involve the interplay of several mechanisms for development of the vegetation. No doubt fibronectin participates in some of these. Accumulation of further platelets depends on local conditions of flow. If blood flow is rapid and laminar flow persists at the site of the lesion additional platelets do not accumulate and no thrombus develops (Mustard and Packham, 1984). It now seems most likely that endothelial damage results in direct adhesion of platelets to the site of the lesion and favors subsequent development of a full blown thrombus (Honour et al., 1971; Sheppard and French, 1971; Stemerman et al., 1971). Whenever this event occurs prior to or concurrently with a bacteremia, the microbes may be incorporated into the platelet aggregate in two ways: either by direct adhesion to the aggregate surface, or by first binding to circulating platelets forming microaggregates that may then join the fixed platelet-fibrin vegetation. The propensity for a given strain of micro-organism to colonize a sterile vegetation would depend on its expression of platelet binding sites versus expression of binding sites for other components of formed vegetations such as fibrin, fibronectin or laminin. It must also be kept in mind that adherence to

114 C.C. CLAWSON bacteria may differ between resting platelets with their particular set of receptors and activated platelets that express a new set of binding sites. In the case of prosthetic valves or other intracardiac devices the foreign surfaces will also be coated with platelets (Clawson et al., 1980a). Some artificial surfaces coat first with fibrinogen and then platelets adhere in a monolayer (Salzman and Merrill, 1987). Studies of Turitto and Baumgartner (1987) indicated that less than 30% of the surface was covered by platelets, which were only loosely attached and did not spread in up to 40 min. In contrast, Clawson et al. (1980a) found that platinum intracardiac pacemaker electrodes coated extensively with platelets spread in a thin monolayer to which other activated platelets adhered. While no direct role has been elucidated in the pathogenesis of bacterial endocarditis for von Willebrand's factor, another bonding protein contained in platelet c~granules, it is intriguing to note that pigs with von Willebrand's disease are less susceptible to experimental bacterial endocarditis (Johnson and Bowie, 1992; Sullam and Sande, 1992). Since this disorder includes defective platelet adherence as well as the deficiency of von Willebrand's factor, it is not clear which abnormality provides resistance to endocarditis. The third body of experimental data linking platelet-bacterial interactions to bacterial endocarditis comes from direct studies of this interaction as has been characterized previously in this chapter. Here it will suffice to re-emphasize the close correlation between isolation of specific bacterial strains from clinical bacterial endocarditis and the ability of these strains to activate platelet aggregation (Herzberg et al., 1983b; Kessler et al., 1987; M.C. Herzberg, unpublished observations). There is also an association between the severity of the clinical disease and the ability of infecting bacteria to induce platelet aggregation in vitro (Kessler et al., 1987). Whatever the initiating events, it is clear that the bacteria that become incorporated into endocarditis vegetations are in a unique position relative to host defense systems. The selective antibacterial activity of the platelets themselves have been discussed. Those organisms that account for the bulk of clinical bacterial endocarditis are among strains for which human platelets appear to have little or no direct bactericidal activity. The structure of the platelet-fibrin matrix of the vegetations that enclose the bacteria provides a formidable barrier to attack by phagocytes. This matrix may also impede access to the bacteria by antibodies and antibiotics. Consequently, bacteria may be allowed to replicate as microcolonies within the vegetation, which will in turn periodically release microorganisms to the circulation. This would account for the frequent bacteremia that is a common feature of the disease (Glauser, 1984).

remains a medical enigma. It is currently a clinical syndrome for which a pathophysiologic definition is just emerging (Maunder and Hudson, 1991; Hyers, 1991). As now understood, ARDS is characterized by increased permeability of endothelial and epithelial barriers of the alveolus and elevated pulmonary vascular resistance. ARDS is often seen in association with bacterial sepsis (Johanson, 1991), and often the infection is remote from the lungs themselves. Among the theories put forward to explain the profound physiologic changes that accompany this syndrome, many focus on potential roles of the neutrophil and eicosanoid metabolism (Seeger and Lasch, 1987). However, ARDS can occur in extremely neutropenic patients, and animal models with analogous physiologic lung injury do not appear to require neutrophil participation (Glauser and Fairman, 1985; Maunder et al., 1986). Some workers have suggested that platelets may also play a central part in the pathogenesis of ARDS (Bredenberg et al., 1980; Heffner et al., 1983; Shoemaker et al., 1984). While it is not the intent of this chapter to discuss in detail the potential role of platelets in ARDS, a few examples of available data may serve to indicate this role. When fragments of Pseudmnonas were injected into dogs whose platelets had been labelled with SlCr, there were arallel declines in circulating platelets and increases in Cr activity in the lung in the form of reversible platelet aggregates that became temporarily trapped in the pulmonary microcirculation (Myrvold and Brandberg, 1977; Myrvold and Lewis, 1977). Concomitantly cardiac output was decreased and pulmonary vascular resistance increased. From these observations, which are in close accord with the van Aken and Vreeken model, the authors postulated that such microembolization by platelet-bacterial aggregates might be an important contributor to pulmonary complications of sepsis. Shoemaker and coworkers (1984) injected Staph. aureus 502A into isolated-perfused rabbit lungs and noted rapid declines in circulating platelet counts along with rises in pulmonary arterial pressures and increased levels of thromboxane Bz (TXBz), the stable derivative of TXAE, a potent pulmonary vasoconstrictor. Platelets are the major source of TXAE. When TXAz synthetase was blocked by UK 38 485, pulmonary hemodynamic changes, which usually followed intravenous infusion of E. coli in pigs, were greatly attenuated (Svartholm et al., 1987). The authors of the latter study concluded from this model of septic pulmonary injury that TXA2 is one of the important mediators of hemodynamic responses in the septic shock lung syndrome.

6 . 1 . 2 A d u l t R e s p i r a t o r y Distress S y n d r o m e Adult (or acute) respiratory distress syndrome (ARDS)

Bacteria or bacterial toxins have been linked to a variety of thromboembolic diseases. Some of the more clear-cut

6.2

THROMBOEMBOLIC

DISORDERS

AND DISSEMINATED INTRAVASCULAR

COAGULATION

PLATELETS IN BACTERIAL INFECTIONS 115 examples of these are Waterhouse-Fridlichsen syndrome, purpura fulminans, Henoch-Schonlein purpura, and DIC. Others that are less well understood, e.g. the spontaneous intravascular thrombosis of essential thrombocytosis, may eventually be shown to involve plateletbacterial interactions. In discussing thromboembolic disease several authors have pointed out that platelet thrombi can form in circulating blood, free of vessel or cardiac surfaces (Aschoff, 1913; Marder et al., 1987). Might a transient bacteremia be one of the instigating events that would trigger such spontaneous floating thrombi? Certainly infection must be considered as one of the frequent causes ofDIC (Brinkhous et al., 1966). DIC can be induced by a wide spectrum of both Gram-negative and Gram-positive organisms, although in practice the latter have been encountered less often. The WaterhouseFridrichsen syndrome, most usually associated with fulminant meningococcal sepsis, may also be caused by other bacteria. Purpura fulminans, usually seen with streptococcal infection, may also accompany a variety of infections. Marder and co-authors (1987) point out that these latter two disorders may be considered extreme forms of DIC.

6.3

ATHEROSCLEROSIS

One of several theories proposed for the pathogenesis of atherosclerosis involves the accumulation of platelets at an endothelial lesion (Mustard and Packham, 1975; Ross and Glomset, 1976; Mustard et al., 1978; Ross, 1981; Packham and Mustard, 1986; Coller, 1992). As reviewed by CoUer (1992), several lines of evidence can be cited to support this theory. The hypothesis reasons that the process of re-endothelialization over a platelet thrombus would result in a potential site for subsequent arteriosclerotic plaque formation. No doubt a wide spectrum of endothelial damage is possible, from a minor loss of cell surface coats to the full loss of endothelial cells exposing the subendothelial connective tissue. Not all of these degrees of damage would necessarily lead to platelet accumulation. It may be that in some instances altered platelet adhesiveness would also be a prerequisite. Certainly the observation that von Willebrand pigs are resistant to development of atherosclerosis might be due to the defective adherence of von Willebrand platelets. Could platelet-bacterial interaction be an agent for increasing platelet "stickiness" and, thus, contribute to plaque initiation at a site of minimal endothelial damage? PDGF appears to be required for the smooth muscle proliferation that accompanies atherosclerosis (Freidman et al., 1977; Ross, 1981; Packham and Mustard, 1986), again implicating the platelet in the pathogenesis of this disorder.

6.4 THROMBOCYTOPENIA The association of platelet aberrations with clinically

significant septicemia is more apparent than the above suppositions regarding transient, low-grade bacteremia. Thrombocytopenia is associated with a high percentage of both Gram-positive and Gram-negative septicemia (Riedler et al., 1970; George and Aster, 1990). Twothirds of patients with septicemia have platelet counts less that 150 000 while in one-third of the counts are below 50 000 (George and Aster, 1990). While the thrombocytopenia of bacterial sepsis is commonly considered to be due to activation of coagulation, thrombocytopenia is frequent in septicemia even when coagulopathy is not present (Belier and Douglas, 1973; Oppenheimer et al., 1976). DIC is less common in septicemia than is a low platelet count. In one large study of 222 patients, 56% had thrombocytopenia while only 11% had DIC as evidenced by elevated fibrin degradation products, decreased fibrinogen, or depressed clotting factors V and VIII (Kregert et al., 1980). Bacterial infection can potentially affect circulating blood platelets by a variety of mechanisms. Gramnegative endotoxin is well known as a direct agonist of platelet reactivity and in vivo produces thrombocytopenia. Other bacterial toxins have also been implicated in pathological processes that involve, directly or indirectly, platelet thromboses or platelet destruction. Bacteria may contribute indirectly to platelet consumption through interaction of the latter with antigen-antibody complexes. Thrombocytopenia in bacterial infection may also result from direct interaction of platelets with micro-organisms, interaction of platelets with damaged endothelium by infection, initiation of DIC, immune destruction, or suppression of marrow thrombopoiesis (Kelton and Neame, 1987; Shulman and Jordan, 1987).

7. Summary This chapter has attempted to provide an integrated overview of the phenomenon of platelet-bacterial interaction. It has been pointed out that in its simplest form platelet-bacterial interaction may be viewed as a special form of platelet interaction with non-biological particulates. Evidence was presented supporting the concept of platelet-particulate interaction being a mechanism participating in clearance of the particulate from the circulation. The involvement of platelets in the clearance process appears to alter both the rate and organ distribution of particle deposition. When the particulate is a strain of bacteria other factors such as the type of surface components of the microbe and various plasma factors become important determinants of the platelet-bacterial interaction. Numerous examples of clinical disorders have been presented in which platelet-bacterial interaction may be reasonably implicated in the pathogenesis of the disease. These aid in underscoring the significance of in vitro and in vivo studies of platelet-bacterial interaction

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to h u m a n disease. This discussion of the interactions of platelets with bacteria has emphasized that the p h e n o m enon has potential for both beneficial and harmful outcomes for the host. Extension of these investigations to more incisive in vivo conditions and beyond to the disease states of our patients remains for future investigation. It is imperative that we further expand our knowledge o f clinically significant platelet-bacterial interaction so that we may better understand and manipulate this biological double-edged sword.

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Salzman, E.W. and Merrill, E.W. (1987). Interaction of blood with artificial surfaces. In: "Hemostasis and Thrombosis: Basic Principles and Clinical Practice" (eds R~W. Colman et al.) pp. 1335-1347. J.B. Lippincott, Philadelphia. Sande, M.A. (1976). Experimental endocarditis. In: "Infective Endocarditis" (ed. D. Kaye) pp. 11-28. Univ. Park Press, Baltimore. Sander, H.K., Slot, W.J., Bouma, B.N., Bolhuis, P.A., Pepper, D.S. and Sixma, J.J. (1983). Immunocytochemical localization of fibrinogen, platelet factor 4, and beta thromboglubulin in thin frozen jp7. sections of human blood platelets. J. Clin. Invest. 72, 1277-1287. Santoro, S. (1988). Molecular basis of platelet adhesion to collagen. Prog. Clin. Biol. Res. 283, 291-314. Scheld, W.M., Valone, J.A. and Sande, M.A. (1978). Bacterial adherence in the pathogenesis of endocarditis. Interaction of bacterial dextran, platelets, and fibrin. J. Clin. Invest. 61, 1394-1404. Scheld, W.M., Zak, O., Vosbeck, K. and Sande, M.A. (1981). Bacterial adhesion in the pathogenesis of infective endocarditis. Effect of subinhibitory antibiotic concentrations on streptococcal adhesion in vitro and the development of endocarditis in rabbits. J. Clin. Invest. 68, 1381-1384. Schulz, H. (1961). Uber dei phagozytose von kolliodalem SiO2 in thrombozyten. Zbl. Allg. Pathol. 102, 319 (Abstract). Schulz, H. (1968). In: "Electron Microscopy of Blood Platelets and Thrombosis" pp. 57-69. Springer-Verlag, Berlin. Seeger, W. and Lasch, H.G. (1987). Septic lung. Rev. Infect. Dis. 9, 5570-5579. Senior, R.M., Griffin, G.L. and Huang, J.S. (1983). Chemotactic activity of platelet alpha granule proteins for fibroblasts. J. Cell Biol. 96, 382-385. Serhan, C.N. (1991). Lipoxins: eicosanoids carrying intra and intercellular messages. J. Bioenerg. Biomembr. 23, 105-122. Serhan, C.N. (1993). Cell-cell interactions in the generation of eicosanoids: charting the routes and products of transcellular biosynthesis (Editorial). J. Lab. Clin. Med. 121, 372-374. Sheppard, B.L. and French, J.E. (1971). Platelet adhesion in the rabbit abdominal aorta following the removal of the endothelium: a scanning and transmission electron microscopical study. Proc. R. Soc. (Lond.) B 176, 427-432. Shoemaker, S.A., Heffner, J.E., Canham, E.M., Tate, tLM., Morris, H.G., McMurtry, I.F. and Repine, J.E. (1984). Staphylococcus aureus and human platelets cause pulmonary hypertension and thromboxane generation in isolated salineperfused rabbit lungs. Am. Rev. Respir. Dis. 129, 92-95. Shulman, N.R. and Jordan, J.V. (1987). Platelet immunology. In: "Hemostasis and Thrombosis: Basic Principles and Clinical Practice" (eds ILW. Colman et al.) pp. 452-529. J.B. Lippincott, Philadelphia. Siess, W., Lorenz, R., Roth, P. and Weber, P.C. (1982). Plasma catecholamines, platelet aggregation and associated thromboxane formation after physical exercise, smoking or norepinephrine infusion. Circulation 66, 44-48. Silver, M.J., Smith, J.B. and Ingerman, C.M. (1974). Blood platelets and the inflammatory process. Agents Actions 4, 233-240. Simonet, M., Triadou, P., Frehel, C., Morel-Kopp, M-C., Kaplan, C. and Berche, P. (1992). Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin. Infect. Immun. 60, 366-373.

Smith, S.B. (1972). Platelets in host resistance: in vitro interaction of platelets, bacteria and polymorphonuclear leukocytes. Blut 25, 104-107. Soberay, A.H., Herzberg, M.C., Rudney, J.D., Nieuwenhuis, H.K., Sixma, J.J. and Seligson, U. (1987). Responses of platelets to strains of Streptococcus sanguis: findings in healthy subjects, Bernard-Soulier, Glanzmann's, and collagen' unresponsive patients. Thromb. Haemost. 57, 222-225. Sommer, P., Gleyzal, C., Guerret, S., Etienne, J. and Grimaud, J-A. (1992). Induction of a putative laminin-binding protein of Strept0coccusg0rd0nii in human infective endocarditis. Infect. Immun. 60, 360-365. Stehbens, W.E. and Florey, H.W. (1960). The behavior of intravenously injected particles observed in chambers in rabbit ears. Q. J. Exp. Physiol. 45, 252-264. Stemerman, M.B., Baumgartner, H.R. and Spaet, T.H. (1971). The subendothelial microfibril and platelet adhesion. Lab. Invest. 24, 179-186. Sullam, P.M. and Sande, M.A. (1992). Role of platelets in endocarditis: clues from von Willebrand disease (Editorial). J. Lab. Clin. Med. 120, 507-509. Sullam, P.M., Drake, T.A. and Sande, M.A. (1985). Pathogenesis of endocarditis. Am. J. Med. 78, 110-115. Sullam, P.M., Valone, F.H. and Mills, J. (1987). Mechanisms of platelet aggregation by viridans group streptococci. Infect. Immun. 55, 1743-1750. Sullam, P.M., Jarvis, G.A. and Valone, F.H. (1988). Role of immunoglobulin G in platelet aggregation by viridans group streptococci. Infect. Immun. 56, 2907-2911. Sullam, P.M., Payan, D.G., Dazin, P.F. and Valone, F.H. (1990). Binding of viridans group streptococci to human platelets: a quantitative analysis. Infect. Immun. 58, 3802-3806. Sullivan, N.M., Sutter, V.L., Carter, W.T., Atteberg, H.R. and Feingold, S .M . (1971). Bacteremia after genitourinary tract manipulation: bacteriological aspects and evaluation of various blood culture systems. Appl. Microbiol. 23, 1101-1106. Svartholm, E., Bergqvist, D., Lindblad, B., Ljungberg, J. and Haglund, U . (1987) . Pulmonary vascular response to live Escherichia coli: influences of different antiplatelet substances. Circ. Shock 22, 173-183. Tagnon, H.J., Levenson, S.M., Davidson, C.S. and Taylor, F.H.L. (1946) . The occurrence of fibrinolysis in shock, with observations on the prothrombin time and the plasma fibrinogen during haemorrhagic shock. Am. J. Med. Sci. 211, 88-96. Taniguchi, T., Joogetsu, M. and Kasahara, T. (1930). A role of blood platelets against infection. Jpn. J. Exp. Med. 8, 55-64. Tait, J. (1918). Capillary phenomena observed in blood cells: Thigmocytes, phagocytes, amoeboid movement, differential adhesiveness of corpuscles, emigration of leucocytes. Q. J. Exp. Physiol. 12, 1-34. Tait, J. and Elvidge, A.tL (1926). Effect upon platelets and on blood coagulation of injecting foreign particles into the blood stream. J. Physiol. (Lond.) 42, 129-144. Tait, J. and Gunn, J.D. (1918). The blood of Astacusfluviartilis. Q. J. Exp. Physiol. 12, 35-80. Teale, F.H. and Bach, E. (1920). The factors leading to the removal of bacteria from the peripheral circulation by phagocytosis. Proc. R. Soc. Med. (Sec. 3) 13, 77-104.

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metabolic and freeze-fracture studies. J. Cell Biol. 91, 706-715. Zucker-Franklin, D. (1990). Platelet morphology. In: "Hematology" (eds W.J. Williams etal.), pp. 1172-1181. McGrawHill, New York.

9

0

o

9

O

P te ts m Parasitic D seases Vrronique Pancr6 and Claude Auriault

1. Introduction 2. Methods 2.1 Platelet Isolation 2.2 Anti-schistosome Cytotoxicity 2.3 Chemiluminescence 2.4 Flow Cytofluorometry 2.5 Binding of Radiolabelled IgE 3. The Receptor for IgE on Platelets 4. Effector Properties of Platelets Towards Helminth Parasites 4.1 Schistosomiasis 4.2 Filariasis 5. Effector Properties of Platelets Towards Other Parasites 5.1 Toxoplasmosis 5.2 Trypanosomiasis 5.3. Malaria

125 125 125 126 126 126 126 127 127 127 129 129 129 129 129

1. Introduction The research carried out on the effector mechanisms against helminth parasites, and particularly against schistosomes, has revealed that antibody-dependent cellular mechanisms (ADCC) play a key role in worm destruction. A series of experiments demonstrated the participation of inflammatory cells such as macrophages and eosinophils, with the help of anaphylactic antibodies (Abs; IgE or IgG2a), in the immunity developed towards the young larvae of schistosomes, the schistosomula (Capron and Capron, 1986). In these investigations, which led to the identification of IgE receptors on the bone marrow-derived inflammatory leucocytes (Spiegelberg, 1984; Capron and Jouault, 1986), the implication of blood platelets in the killing of young larvae from helminths could be demonstrated (Joseph et al., 1983). This promotion of platelets to the status of cytotoxic effectors has not only opened new perspectives in the Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

6. Other Inducers of Platelet Cytotoxicity 7. Regulation of Platelet Effector Function by T Lymphocytes 7.1 Activation of Platelets by Lymphokines 7.1.1 Role of Interferon Gamma 7.1.2 Role of Tumour Necrosis Factor 7.2 Suppression of Platelet Cytotoxic Function 7.2.1 Role of Platelet Activity Suppressive Lymphokine 7.2.2 Role of Ubiquitin 8. Regulation of Platelet Effector .Function by Monocytes 9. Concluding Remarks 10. References

130 130 130 130 130 131 131 131 134 134 134

potential capabilities of these blood elements against parasites and possibly against other pathogens, but has also made likely their involvement in various pathologies or immunological disorders in which their role had thus far been neglected or at least considered as secondary or indirect. In particular, the demonstration that their cytotoxic functions were mediated in part by IgE through a specific receptor for this isotype suggested their direct participation in the physiopathology of IgE-dependent allergic diseases or hypersensitive states (Capron et al., 1987; Joseph, 1988).

2. Methods 2.1

PLATELET ISOLATION

The procedure for isolation of platelets was carried out at room temperature. Human venous blood (6 vol.) Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

126 V. PANCRfl AND C. AURIAULT collected on ACD-C (1 vol.) was centrifuged for 15 min at 120 g in 5 ml aliquots; the platelet-rich plasma (PRP) was collected in a single volume and centrifuged at 2000 g for 15 min. Avoiding the lowest part of the pellet which was contaminated with red cells, the platelets were resuspended in three washing steps at 2000 g in saline supplemented with citric acid (36 mM), glucose (5 mM), calcium (2 mM), magnesium (1 mM), bovine serum albumin (BSA) (0.35%), and prostaglandin E1 (PEG1) (100 nM). The last pellet was resuspended, and the platelets were counted in a haemocytometer under phase contrast microscopy after a 1:20 dilution with 1% ammonium oxolate and were adjusted to the appropriate concentration. Platelets isolated from normal rats were prepared from 2 ml heparinized blood, diluted with 3 ml heparinized Eagle's minimum essential medium (EMEM), and centrifuged (400 g) for 15 min at 4 ~ Platelets in the supernatant were washed with heparinized EMEM and centrifuged (5000g) for 20 min at 4~ They were counted in a haemocytometer under phase contrast microscopy after a 1:20 dilution in London stain. The observation of undiluted platelet suspensions with interferential contrast microscopy repeatedly confirmed that the leucocyte contamination never exceeded 10 per 106 platelets.

estimated by chemiluminescence. A 1 h incubation of 3 • 107 platelets in 200/zl of phosphate-buffered saline (PBS) with 50 #1 of lymphocyte supernatant or medium was followed after washing by a 30 min incubation of the same platelets with 20 gl IgE-rich serum from allergic asthmatics. Platelets were centrifuged for 15 min at 200 g in the washing medium and resuspended in PBS, pH 7.7. To 5 x 1 0 s platelets in PBS were added luminol (418 mM final concentration), luciferin (30 #M final concentration), horseradish peroxidase (HRP; 720 mU) and the triggering reagent (allergen or anti-IgE) in a final volume of 200 gl. Results, in mV, were the maximum value of light emission obtained during 5 min measurements in a Nucleotimeter 107 (Interbio, 95500 Le Thillay, France). The observed chemiluminescence was a consequence of the stimulation of the platelets themselves and not of contaminating cells, as verified by the absence of light emission with purified leucocytes in the reagents used. The inhibition of luminescence by lymphocyte supernatants was controlled over a 20 min period.

2.4

FLOW CYTOFLUOROMETRY

Blood (9 vols) was collected on ethylenediamine tetraacetic acid (EDTA) 5% in water (1 vol.), and platelets were isolated by the above procedure, with washing in PBS containing 0.01 M EDTA, a buffer also used for the 2.2 A N T I - S C H I S T O S O M E next washing steps. Two saml~les of 107 platelets in CYTOTOXICITY 100 #1PBS were incubated at 37vC, for 30 min each, first The platelets were isolated by the procedure described with 10 #g human myeloma IgE protein, then, after above. At the end of the washing steps, the pellet was washing, with 10 #g mouse monoclonal IgG anti-human resuspended in EMEM. Human (150x106) or rat IgE, and finally, after washing, with 10 gl rabbit fluores(200x 106), platelets from uninfected individuals were cein isothiocyante (FITC)-IgG anti-mouse IgG. For the incubated in flat-bottom microplates (Nunc, Roskilde, detection of IgE bound in vivo on platelets, the first incuDenmark) in 50 gl serum either from normal individuals bation with myeloma IgE was omitted. Occasionally, or from patients and rats with schistosomiasis, 50 #1 of fluorescent F(ab')2 fragment of sheep IgG anti-human lymphocyte supernatant or EMEM, and 75 Schistosoma IgE was used. Labelling of the membrane glycoprotein mansoni schistosomula in 80 gl EMEM. After 24 h at (GP) IIb-IIIa complex, a platelet-specific marker, was 37~ in 5% CO2 in air, motionless and dark dead larvae achieved with AP2 mAb which served as internal stanwere easily distinguished from mobile and refringent dard to platelet fluorescence. living schistosomula by optical microscopy. For IgE identification on rat platelets, the same proceThe experimental procedure was identical for platelets dure was adopted with the appropriate reagents: rat from allergic patients and for platelets from healthy monoclonal IgE, sheep IgG anti-rat IgE, and rabbit donors incubated with 20 #1 serum from allergic asth- FITC-conjugated IgG anti-sheep IgG. matics with high levels of circulating IgE. The killing In each case, after a final washing step, the platelets process, in this latter case, was induced by the addition were resuspended in 500 #1 PBS containing 0.01 M of either anti-IgE (sheep polyclonal anti-human IgE, EDTA, and the percentage of fluorescent platelets was 10 gg IgG/ml) or the allergen specific to the patient measured in a 50-H cytofluorograf (Ortho Instruments, sensitivity (DermatophagofiCespteronyssinus, Apis mellifera, Westwood MA). The background of non-specific Vespula, 10 ng/ml). Each experiment was carried out in binding of the fluorescent Ab on untreated platelets (less duplicate, and results expressed as the mean + SD of dead than 5%) was subtracted from all the results. larvae. 2.5

2.3

CHEMILUMINESCENCE

The generation of oxygen metabolites by platelets was

BINDING

OF RADIOLABELLED

IgE

Platelets were isolated as described above, except that the pellet of the last washing was resuspended in PBS. Eight

PLATELETS IN PARASITIC DISEASES 127 samples of 107 washed platelets in 200 ml PBS were incubated, without stirring, in plastic tubes for 90 min at room temperature with 12SI-labelled IgE (specific activity 150 mCi/mmol), either alone, or after preincubation for 30 min with one of the following unlabelled reagents: polyclonal anti-Fc~R or dimethyl suberimidateaggregated IgE. This preincubation step was followed by washing in the medium described for anti-parasite activity. At the end of the labelling procedure, the platelets were separated from unbound Ig by centrifugation at 8500g for 2 min through 1 ml 25% sucrose in a Microfuge (Beckman, Palo Alto, CA), and the radioactivity of the pellet was measured with a CG4000 gamma counter (Intertechnique, Plaisir, France).

the other (Capron et al., 1986a). Analysis of IgE binding to platelets defined the presence of 800-1000 receptors per platelet with an affinity of 3x 107 M -1, which is very close to the coefficient established for the other nonmast cell leucocytes. In addition to the specificity for IgE, this last observation has established the antigenic community of the receptor in the various leucocyte populations. Electrophoresis of soluble immunoadsorbed IgE receptors has identified a subfraction of 43-45 kD, common to mononuclear phagocytes, eosinophils and platelets, and a platelet-specific subfraction of 32 kD, of higher molecular weight than the 25 kD of mononuclear phagocytes and eosinophils (Capron et al., 1986b).

e

3. The Receptorfor IgE on Platelets As with mononuclear phagocytes and eosinophils, cytofluorometric analysis led to the identification of 10-20% IgE-positive platelets in normal individuals (human and rat), and a large increase of this percentage, up to 50%, in subjects with pathologies showing increased levels of serum IgE, particularly filariasis (Joseph et al., 1986a). The use of radiolabelled IgE and IgG confirmed that the binding operated through a specific receptor, since the labelling by each immunoglobulin was inhibited only by a pretreatment of platelets with aggregated molecules of the corresponding isotype (Joseph et al., 1986b). As experienced for macrophages and eosinophils, inhibition of the IgE binding to human platelets, but not of IgG, was also observed with anti-IgE receptor antibodies, a polyclonal serum against lymphocyte receptor on the one hand (Spiegelberg, 1984), and an mAb against the eosinophil receptor on

Table 6.1

Effector Properties of Platelets Towards Helminth Parasites

(Table 6.1) 4.1

SCHISTOSOMIASIS

Platelets from Schistosoma mansoni-infected patients or, alternatively, platelets from healthy individuals incubated with the IgE-rich serum from S. mansoni-infected patients, expressed high cytotoxic properties in vitro against the parasite (Joseph et al., 1983). The depletion of IgE from immune serum, or the preincubation of the platelets from healthy individuals with unrelated human myeloma IgE, inhibited the further induction of such platelets into anti-parasite effectors. Conversely, IgG depletion of the same immune serum, or preincubation with unrelated human IgG, was unable to inhibit the stimulation of normal platelets. Studies performed in rats confirmed both the direct involvement of IgE and the in v/v0 relevance of this platelet-mediated cytotoxic process (see Fig. 6.1). The serum from immune animals, or an

Effector properties of platelets toward parasites

Target

Inducer

S. mansoni

Antibodies IgE

+

Cytokines IFN~ TNF-ot and -fl IL-6

+ + +

Others C3B SP CRP

+ +

B. malayi Loa Ioa D. viteae

T. gondii

T. cruzi

128 V. PANCRI~ AND C. AURIAULT IgE mAb to S. mansoni (Verwaerde et al., 1987) induced, in platelets from normal rats, the same cytocidal properties as those expressed by platelets from immune animals. Furthermore, the intravenous transfer of platelets from immune to normal recipients led to a high degree of protection against a challenge infection with the parasite.

As a consequence of their interaction with IgE Abs, platelets produced oxygen metabolites, evidenced by chemiluminescence: platelets from rats infected with schistosomes induced the luminescence of a mixture of luminol, luciferin and horseradish peroxidase when incubated with anti-IgE or parasite antigens. The addition of anti-IgG and of unrelated antigens did not

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Figure 6.1 IgE-dependent platelet cytotoxicity toward the young larvae (schistosomula) of Schistosoma mansoni (Photo G. Torpier). (a) One hour incubation of schistosomula with normal rat platelets and serum from S. mansoni-infected rats (42 days of infection with cercariae). The tegument of the schistosomula is still intact but platelets are present around the parasite. (b) After 6 h of incubation, the tegument was destroyed and the parasite died rapidly. This cytotoxic process was abolished when IgE antibodies were eliminated from the sera by immunoabsorption and also when the serum was replaced by an IgE mAb specific for S. mansoni.

PLATELETS IN PARASITIC DISEASES 129 induce significant chemiluminescence (Joseph et al., 1985). Recent data suggest a role for platelets in the extravasation and excretion of parasite eggs in schistosomiasis (Ngaiza and Doenhoff, 1990). Suppression of platelet activity in S. mansoni-infected mice by administering rabbit anti-mouse platelet serum or a selection of "antiplatelet drugs" resulted in a significant reduction of parasite egg excretion. Isolated eggs were also capable of inducing ex vivo platelet aggregation in mice, which was dependent on presensitization with eggs.

supporting the hypothesis of oxygen metabolitedependent generation of more stable lipid peroxides in the cytotoxic process. Finally, the metabolic events leading to these effector functions concerned a different compartment than that involved in platelet aggregation: the IgE-dependent stimulation, inducing cytotoxicity, was inactive in the aggregation of platelets, either washed or in plasma.

0

4.2

FILARIASIS

Effector Properties of Platelets Towards Other Parasites

Very similar observations were made in filarial infections 5.1 TOXOPLASMOSIS where normal rat platelets could be stimulated to kill D/peta/0nema viteae microfilariae with IgE Ab from the The induction of a protective IgE response during rat serum ofD. viteae-infected rats (Haque etal., 1985). This toxoplasmosis has recently been demonstrated. During antifilarial killing mechanism was neither stage nor species Toxoplasma gondii infection, developed in the nude rat specific among filariae, in that microfilariae (L1) of Brug~ model, a signifcant increase of serum IgE was demonma/ay/or of L0a/0a, and infective larvae (L3) of D. viteae strated to correlate with an increase of IgE-bearing or ofB. ma/ay/, were all killed by platelets incubated with platelets (Ridel et al., 1988). The important role of suranti-D, viteae IgE. Platelets taken from B. ma/ayi-infected face IgE on these cells was clearly shown because only patients were able to kill B. malayi microfilariae in vitro. platelets having surface IgE were able to induce a signifiMoreover, platelets from normal donors acquired toxic cant percentage of cytotoxicity toward tachyzoites in properties toward the same target in the presence of vitro, and a significant increase of the survival period when serum from infected patients. Heating as well as IgE the cells were transferred to immunocompromized rats. depletion led to the abolition of the activating properties Recently, it was shown that platelets, even in the absence of such serum, strongly suggesting the involvement of of serum, can exert as strong a cytotoxic activity on T. IgE in this cytotoxic process (Pancr6 et al., 1986). gondii as cytotoxic T lymphocytes (Yong et al., 1991). The demonstration that platelets could be involved in Several lines of evidence indicate that the release of defence mechanisms against filariae led us to reconsider thromboxane A2 (TXA2) is important in this cytotoxic the mode of action of a 40-year-old antifilarial drug, process. diethylcarbamazine (DEC), in the light of potentially active cells. No mechanism had yet been proposed to explain the very rapid and radical activity of this com- 5.2 TRYPANOSOMIASIS pound on blood microfilariae. We have demonstrated Observations made in Trypanosoma musculi-infected that DEC probably exerted its curative effect through mice revealed the in vitro interaction between blood platelets with the additional triggering of a filarial excreplatelets and trypanosomes, leading to death of the tory antigen (Cesbron et al., 1987). The killing mechparasites within a few minutes, and their lysis (Viens anism was Ab-independent and involved the et al., 1983), but no clear-cut identification of a serum participation of free radicals. It appears therefore that factor supporting this activity could be proposed. The platelets may be stimulated into efficient killers by more role of the C3b receptor present on platelet membranes than one triggering signal. in the lysis of sensitized T. cruzi blood stream The parasite killing effect obtained by the IgE-mediated promastigotes has been illustrated (Umekita and Mota, stimulation of platelets could be obtained through a 1989). semi-permeable filter which prevented a direct contact between effectors and targets. Cytocidal mediators have not been unequivocally identified. However, superoxide 5.3 MALARIA dismutase, catalase, and various free radical scavengers totally or partially inhibited cytotoxicity and chemi- Plasmodium fakiparum maturation and growth might luminescence. The blockade of the cyclooxygenase have been inhibited by human platelets in vitro (CO) pathway by aspirin also inhibited the cytotoxic (Peyron et al., 1989), whereas the merezoites themselves properties induced by IgE in platelets. However the dis- were not killed. Although the platelet activation tance created between effectors and targets in the trans- mechanisms are not clearly understood, metabolites filter cytotoxicity made unlikely the direct participation of parasitic origin seemed to be involved in this of short-lived free radical species unlikely, therefore phenomenon.

130 V. PANCRI~ AND C. AURIAULT

6. Other lnducers of Platelet Cytotoxicity

were able to induce, in a dose-dependent manner, the effector functions of the platelets in the absence of antibodies (Pancr~ et al., 1987).

Several other triggering signals were able to induce antiparasite cytotoxicity in vitro. C-reactive protein (CRP), a main serum component of inflammatory reactions, has been shown to stimulate the secretion of cytotoxic compounds from normal platelets (Bout et al., 1986). Normal rat platelets, incubated in rat serum obtained 24 h after subcutaneous injection of turpertine, which is known to increase the serum concentration of CRP, were able to kill schistosome larvae. Platelets collected from rats that had been injected with such a serum 4 h before expressed high cytotoxic activity against the parasites. The same killing could then be obtained with purified CRP, showing a dose-dependent effect. Increased levels of CRP have been identified in the serum of S. mansoniinfected rats between the fourth and fifth week of infection, at the very period of schistosome self-cure by the rats. However, no chemiluminescence could be observed from platelets incubated with CRP. Substance P and its carboxy-terminal fragment SP induce cytotoxic activity of platelets towards the larvae of Schistosoma mansoni, by 90% and 40%, respectively, whereas the modified C-terminal SP, the SP-free acid, exhibits no effect on platelets (Damonneville et al., 1990b). The neuropeptide effects occur at low doses (10 -8 M), and are specific as shown by inhibition studies with a substance P antagonist, D-SP. Binding data obtained after flow cytofluorometry with FITC-SP lead to the conclusion that SP binds specifically to about 20% of the homogeneous population of platelets. Moreover, IgE could modulate the SP-dependent functions of platelets since the pre-incubation with myeloma human IgE or with AP2 mAb - known to inhibit the IgEdependent killing of these cells - leads to a dramatic decrease of the SP-dependent cytotoxic activity of platelets towards the larvae. These findings identify a potent mechanism for nervous system regulation of host defence responses.

7.1.1 Role of Interferon Gamma One of the lymphokines present in stimulated T cell supernatants was clearly identified as interferon gamma (IFN3,), as shown by the neutralization of the induction of platelet cytotoxicity by a monoclonal anti-IFN-r Ab, the detection of IFNy in CD4+/CD8 - lymphocyte supernatants and, finally, the direct cytotoxic effect of pure recombinant IFNy in a dose-dependent manner (Pancr~ et al., 1987). Moreover, the presence of a highaffinity receptor for IFN~, on the platelet membrane has been demonstrated (Molinas et al., 1987). Scatchard analysis of binding data indicates the presence of homogeneous sites estimated in the order of 150-200 with an apparent Kd of 2 x 10-10 M. High affinity receptors for IFNy have also been characterized on the surface of the human megakaryocytic Dami cell line, confirming the presence of receptors for this lymphokine on the platelet precursor hematopoietic cells, the megakaryocytes (Monte et al., 1991). Scatchard analysis indicated the presence of about 11 000 binding sites per cell, with a Kd of 3 x 10-10 M. In vivo experiments showed that passive transfer of platelets preincubated with IFN~, conferred to animals infected 24 h later a significant level of protection compared to the level obtained after transfer of normal platelets (Pancr~ et al., 1990a). The preincubation of immune platelets recovered from infected rats with IFNy increased the level of protection normally obtained with immune cells alone. Interestingly, during the course of rat infection, platelets expressed in vitro direct antiparasite killing properties due to IgE present in the sera of infected rats as described previously. At the same period of infection, the presence of IFNy detected in the sera of infected rats supports the hypothesis of the concomitant involvement of the IgE and IFNy in the induction of platelet effector functions (Pancr~ et al., 1990a). Moreover, a clear additive cytotoxic effect was observed in vitro when IFNy and IgE were tested together on the cells. As shown by cytofluorometry analysis, it appeared that this effect was due, at least in part, to the increase of the expression of the IgE Fc,RII receptors on the platelets, and more precisely due to the increase of number of binding sites for IgE on the platelets from 800 to 1500 sites per cell, as demonstrated by Scatchard plot analysis (Pancr~ et al., 1988c).

7. Regulation of Platelet Effector Function by T Lymphocytes The previously reported data led to the conclusion that platelets appeared as competent immune cells, capable of killing the larvae of parasites. This new immunological platelet activity could be regulated by T lymphocyte factors, as for other immune cells.

7.1

ACTIVATION

OF PLATELETS

BY

LYMPHOKINES Supernatants recovered from S. mansoni antigen or concanavalin A-stimulated CD4+/CD8 - T lymphocytes

7 . 1 . 2 R o l e o f T u r n o u t Necrosis F a c t o r A second inducing activity was detected after isoelectric focusing of stimulated T lymphocyte supernatants. This activity was not neutralized by anti-IFNy mAb, but it was neutralized by anti-tumour necrosis factor (TNF) mAb (Damonneville et al., 1988). Both pure recombinant human TNF-/3 and TNF-a incubated with the platelets acted at very low concentrations in a dose-

PLATELETS IN PARASITIC DISEASES 131 dependent fashion as inducer of platelet cytotoxicity; concentrations as low as 8x 10-12M for TNF-B and 2• 10-1~ for TNF-c~ were active. A clear additive effect was observed when TNF-/3 was incubated with IFNy. In vivo experiments, developed in the rat model, indicated that TNF-c~-stimulated platelets transferred passively to rats induced a significant level of protection against a challenge infection, not observed with platelets treated with interleukin-2 (IL-2), a lymphokine described as having no inducing effect on the platelets (Damonneville et al., 1990a). Thus, TNF could participate in the induction of the protective immune response by activating platelets into killer cells.

a.

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50

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60

70

SUPPRESSION OF PLATELET CYTOTOXIC FUNCTION b..

7.2.1

Role o f Platelet A c t i v i t y Suppressive Lymphokine

In another series of experiments, IgE-dependent effector activity of platelets was demonstrated to be controlled by T lymphocytes. A suppressive lymphokine, called platelet activity suppressive lymphokine (PASL), produced by mitogen-stimulated CD4-/CD8 § lymphocytes, has been shown to inhibit the IgE-dependent platelet cytotoxicity towards the larvae, as well as the chemiluminescence of these blood elements in an IgEanti IgE reaction (Pancr~ et al., 1986). Furthermore, Agspecific stimulated T lymphocyte supernatants from S. mansoni- or B. ma/ay/-infected patients respectively inhibited the IgE-dependent cytotoxicity developed by platelets of the same individuals toward the schistosomula or toward the microfilariae of B. ma/ay/ (Pancr~ et al., 1988). This suppressive lymphokine of platelet functions has a molecular weight of 15 000-20 000, a p I of 4.6, is heat and acid stable, and is sensitive to trypsin and proteinase K, but neuraminidase has no effect on its activity (Pancr~ et al., 1986). The relevance in vivo of these in vitro observations has been established by the following: the protection normally conferred against a challenge infection by a passive transfer of platelets from immune animals to normal rats was completely abolished after treatment of transferred platelets by PASL (Pancr6 et al., 1989) (Fig. 6.2). This type of platelet activation has also been described in allergic disorders such as hymenoptera venom hypersensitivity (HVH; Joseph et al., 1986b; Joseph, 1988) and in aspirin-sensitive asthma (ASA; Ameisen et M., 1985). The IgE-dependent reactivity of platelets in HVH was abolished after immunotherapy and a factor similar to PASL has been identified in sera and in lymphocyte supernatant, from desensitized patients, suggesting the participation of this lymphokine in the mechanisms of rush desensitization (Tsicopoulos et al., 1990). In ASA, the direct, non-IgE-dependent activation of platelets by

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O 0

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30

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DAYS AFTER I N F E C T I O N

Figure 6.2 (a) Production of PASL during experimental infection of rat by S. mansoni. (b) Killing properties of platelets during experimental infection of rat by S. mansoni.

non-steroidal anti-inflammatory drugs was also inhibited by PASL (V. Pancr6, unpublished results). PASL could therefore play a potential role in the regulation of immunologic disorders, particularly allergic diseases, and so represent a molecule of great immunopharmacological interest. 7 . 2 . 2 Role o f U b i q u i t i n In an attempt to clone the suppressive lymphokine of platelet functions (PASL), we have obtained a cDNA clone (Pancr6 et al., 1991) coding for the previously described human ubiquitin-80 amino acid fusion protein (Lund et al., 1985; Fig. 6.3). Ubiquitin was first isolated from bovine thymus and was reported to stimulate differentiation in B and T lymphocytes. Its 76-amino acid sequence is highly conserved

132 V. PANCRI~ AND C. AURIAULT throughout evolution and is identical in amino acid sequence in organisms as diverse as humans and insects. Such intense selective pressure during evolution to maintain the existence and structure of ubiquitin suggests a key role for this molecule in fundamental cellular processes (Monia et al., 1990). At this stage of our work, the question raised was: is ubiquitin PASL, merely a

Jurkat

factor with PASL-Iike activity, or is PASL related to a fam~y of ubiquitins? No effects of ubiquitin on platelets have been previously described. Like PASL, purified ubiquitin can inhibit the cytotoxic properties of platelets and the production of oxygen metabolites by these cells. This molecule is also able to act as a pro-aggregating factor and seems to be of great interest in pathologies

cells

mRNA

Injection in Xenopus oocytes

Test PASL octivity

cDNA Synthesis

( HMLV RT )

cDNA Library

Groups or clones

Ill v i t r o RNA 5ynthesi s

/

Mammalion cells

~

Injection in Xenopus ___necytes

Test PASL act iv i t y

Expression

E. Coil

Yeast

Figure 6.3 Cloning strategy of PASL. Total cellular RNA was prepared from the Jurkat cell line using the guanidine thiocyanate-CsCI method. The mRNA was isolated by chromatography on oligo(dT)-celluIose and after ethanol precipitation was resuspended in water at a concentration of 1/~g//~l. The mRNA was Injected into 20 Xenopus laevis oocytes (at 50 nl per oocyte) and incubations were performed for 48 h at 20~ in Barth's medium. The medium was recovered at 24 h and at 48 h and assayed for PASL activity, cDNA was synthesized using M-MLV reverse transcriptase and the BRL kit according to manufacturer's instructions (Gibco-BRL, CergyPontoise, France). EcoRI linkers were blunt-end ligated to the cDNA, cut with EcoRI and the linker fragments separated by chromatography on Ultrogel AcA 34 (IBF Industrie, Villeneuve-La-Garenne, France). The resulting cDNA was Iigated into Eco Rl-cut pBS plasmid (Stratagene, La Jolla, USA). From the resulting library 5000 individual clones were tested. Plasmid DNA from groups of 25 clones was prepared, linearized by BamHI digestion and used for in vitro transcription using T7 RNA polymerase (Stratagene). Resulting RNA was extracted once with phenol/chloroform, precipited with ethanol, resuspended in water and tested using the oocyte expression assay. One positive group obtained was subsequently divided until a single positive clone was isolated. Plasmid DNA was isolated from the positive clone and the inserts (about 400 and 200 bp) were subcloned into M13 phage and sequenced using the dideoxynucleotide chain termination method.

PLATELETS IN PARASITIC DISEASES 133 Table 6.2

Comparison between PASL and ubiquitin

PASL

Ubiquitin

MW (kDa) Acid sensitivity Heat sensitivity Exceptional stability Species specificity Dose-dependent effect

10 No No Yes No Yes

8.5 No No Yes No Yes

Effect on platelet functions: Cytotoxicity Production of oxygen metabolites Aggregation

Inhibition Inhibition NT

Inhibition Inhibition Helper

Antigen

involving defects in platelet aggregation. Ubiquitin could also have a potential use in the regulation of immunological disorders in which platelets seem to be implicated, such as H V H and ASA, since in both situations ubiquitin is able, as is PASL, to inhibit the cytotoxic function of platelets. In addition to their similar functional properties, ubiquitin and PASL also have the same physicochemical properties (Table 6.2). Another particular characteristic of these two products is the absence of species specificity observed in their action. Human PASL is able to inhibit the properties of human, but also of rat and mouse platelets, and vice versa. Purified bovine ubiquitin acts similarly on human, rat or mouse platelets. The only

Antigen

Mitogen

Mitogen

Adherence

IFNY

TNF 13 TNFo:

+

)

IL6

Figure 6.4

Regulation of platelet effector function by cytokines.

PASL

134 V. PANCRE AND C. AURIAULT difference observed is that a rabbit polyclonal Ab directed against ubiquitin neutralized the PASL activity of purified ubiquitin but failed to inhibit the PASL activity of the lymphocyte supernatant at all the dilutions used. It is possible that the determinants recognized on ubiquitin were absent or modified on PASL. It was previously shown that PASL can bind to a structure on the platelet membrane (Pancr~ et al., 1986). Similarly, very preliminary results demonstrate the existence of a binding structure for ubiquitin on platelets from some subjects. Taken together, our results were very disturbing. We are confronted with two products having the same functional properties but we cannot affirm that they represent the same factor. It is possible that ubiquitin could be excreted by T cells after activation but it is more probable that PASL belongs to the family of ubiquitins.

cytotoxic properties without aggregation, suggesting that they triggered a platelet compartment that was not involved in classical activation by aggregating agents such as thrombine, adenosine diphosphate, or PAF-acether. The hypothesis remains that the killing capacities of platelets were supported by specific subpopulations, either working on their own, or producing secondary mediators for the general participation of non-responding platelet populations. These results bear clear evidence for the integration of platelets into the complex network of cell interactions of various immunologic and inflammatory disorders. Moreover, the participation of lymphokines in the control of platelet effector functions appears as crucial as their implication in the modulation of other effector cell populations, such as macrophages and eosinophils.

8. Regulation of Platelet Effector Function by Monocytes

10. References

A cooperation between monocytes and platelets for the killing of S. mansoni has also been demonstrated (Pancr~ et al., 1990b). Indeed, supernatants obtained after a 24 h adherence of normal human monocytes were able to induce, in a dose-dependent manner, the cytotoxicity of normal human platelets towards the young larvae of S. mansoni in vitro. The physicochemical analysis of the supernatants showed that a factor exhibiting a p I of 4.8-4.9 was responsible for this effect, suggesting a role of IL-6, detected in the supernatant, in this induction (Pancr~ et al., 1990b). This was confirmed by the neutralization of the cytotoxic effect by a polyclonal serum against IL-6 whereas polyclonal sera against IL-I~ or TNF-c~, the other cytokines present in the supernatants, did not modify the cytotoxicity observed. Finally, human recombinant IL-6 induces the platelet cytotoxic function, demonstrating a direct effect of IL-6 on blood platelets (Pancr~ et al., 1990b).

9. Concluding Remarks Since the first model of anti-parasite activity by platelets was reported, different triggering processes have been described that induce these blood elements into efficient killers of pathogens. Besides endogenous compounds, such as specific IgE antibodies, SP, inflammatory CRP, IFN-/, TNF(s), IL-6 and C3b receptor, exogenous chemicals have also been shown to express unexpected activating properties on platelet populations, such as DEC on platelets from patients with filariasis. These various stimuli probably triggered thrombocytes by different pathways, but achieved a common result: the generation of cytocidal molecules able to kill parasites. A general feature of the stimulating signals was apparently to induce

Ameisen, J.C., Capron, A., Joseph, M., Maclouf, J., Vorng, H., Pancr~, V., Fournier, E., Wallaert, B. and Tonnel, A.B. (1985). Aspirin-sensitive asthma: abnormal platelet response to drugs inducing asthmatic attacks. Int. Arch. Allergy Appl. Immunol. 78, 438-448. Bout, D., Joseph, M., Pontet, M., Vorng, H., Deslee, D. and Capron, A. (1986). Rat resistance to schistosomiasis: plateletmediated cytotoxicity induced by C-reactive protein. Science 231, 153-156. Capron, M. and Capron, A. (1986). Rats, mice and men models for immune effector mechanism against schistosomulas. Parasitol. Today 2, 69-75. Capron, M. and Jouault, T. (1986). IgE receptors on human eosinophils and eosinophil heterogeneity. Ann Inst. Pasteur Immunol. 137, 371-374. Capron, M., Jouault, T., Prin, L., Joseph, M., Ameisen, J.C., Butterworth, A.E., Papin, J.P., Kusnierz, J.P. and Capron, A. (1986a). Functional study of a monoclonal antibody to IgE Fc receptor (FcE Ra) of eosinophils, platelets, and macrophages. J. Exp. Med. 164, 72-89. Capron, A., Dessaint, J.P., Capron, M., Joseph, M., Ameisen, J.C. and Tonnel, A.B. (1986b). From parasites to allergy: a second receptor for IgE. Immunol. Today 7, 15-18. Capron, A., Joseph, M., Ameisen, J.C., Capron, M., Pancr6, V. and Auriault, C. (1987). Platelets as effectors in immune and hypersensitivity reactions. Int. Arch. Allergy Appl. Immunol. 82, 307-312. Cesbron, J.Y., Capron, A., Vargaftig, B.B., Lagarde, M., Pincemail, J., Braquet, P., Taelman, H. and Joseph, M. (1987). Platelets mediated the action of diethylcarbamazine on microfilariae. Nature 325, 533-536. Damonneville, M., Wietzerbin, J., Pancr6, V., Joseph, M., Delanoye, A., Capron, A. and Auriault, C. (1988). Recombinant tumor necrosis factors mediate platelet cytotoxicity to Schistosoma mansoni larvae. J. Immunol. 140, 3962-3965. Damonneville, M., Pancr6, V., Capron, A. and Auriault, C. (1990a). Protection of rats against Schistosoma mansoni infection induced by platelets stimulated with the murine recombinant tumor necrosis factor alpha. Int. Arch. Allergy Appl. Immunol. 92, 361-363.

PLATELETS IN PARASITIC DISEASES Damonneville, M., Montr, D., Auriault, C. and Capron, A. (1990b). The neuropeptide substance P stimulates the effector functions of platelets. Clin. Exp. Immunol. 81, 346-351. Haque, A., Cuna, W., Bonnel, B., Capron, A. and Joseph, M. (1985). Platelet-mediated killing of larvae from different filarial species in the presence of D/peta/0nema v/teae-stimulated IgE antibodies. Parasite Immunol. 7, 517-526. Joseph, M. (1988). Platelets in allergy. Clin. Rev. Allergy 6, 191-210. Joseph, M., Auriault, C., Capron, A., Vorng, H. and Viens, P. (1983). A new function for platelets: IgE-dependent killing of schistosomes. Nature 303, 810-812. Joseph, M., Auriault, C., Capron, M., Ameisen, J.C., Pancrr, V., Torpier, G., Kusnierz, J.P., Ovlaque, G. and Capron, A. (1985). IgE dependent platelet cytotoxicity against helminths. In: "Mechanisms of Cell-mediated Cytotoxicity" (eds P. Henkart and E. Martz ), pp. 23-33. Plenum Publishing Corporation, Oxford. Joseph, M., Capron, A., Ameisen, J.C., Capron, M., Vorng, H., Pancrr, V., Kusnierz, J.P. and Auriault, C. (1986a). The receptor for IgE on blood platelets. Eur. J. Immunol. 16, 306-312. Joseph, M., Capron, A., Ameisen, J.C., Caen, J.P., Tsicopoulos, A. and Tonnel, A.B. (1986b). The IgEdependent participation of platelets to cellular mechanisms in allergy. In: "Proc. XII Int. Congr. Allergol. Clinical Immunol.", pp. 135-139. C.V. Mosby, St Louis. Lund, P.K., Moats-Staats, B.M., Simmons, J.G., Hoyt, E., D'Ercole, A.J., Martin, F. and Van Wyk, J.J. (1985). Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesised as a precursor. J. Biol. Chem. 260, 7609-7613. Molinas, E., Wietzerbin, J. and Falcoff, M. (1987). Human platelets possess receptors for a lymphokine: demonstration of high specific receptors for HuIFN-gamma. J. Immunol. 138, 802-806. Monia B.P., Ecker, D.J. and Crooke S.T. (1990). New perspectives on the structure and function of ubiquitin. BioTechnology. 8, 209-215. Montr, D., Wietzerbin, J., Pancrr, V., Merlin, G., Greenberg, S.M., Kusnierz, J.P., Capron, A. and Auriault, C. (1991). Identification and characterization of a functional receptor for interferon-~/ on a megakaryocytic cell line. Blood 78, 2062-2069. Ngaiza, J.R. and Doenhoff, M.J. (1990). Blood platelets and schistosome egg excretion. Proceedings of the So~:iety for Experimental Biology and Medicine, 193, 73-79. Pancrr, V., Auriault, C., Joseph, M., Cesbron, J.Y., Kusnierz, J.P. and Capron, A. (1986). A suppressive Iymphokine of platelet cytotoxic function. J. Immunol. 137, 585-591. Pancrr, V., Joseph, M., Mazingue, C., Wietzerbin, J., Capron, A. and Auriault, C. (1987). Induction of platelet cytotoxic functions by lymphokines: role of interferon gamma. J. Immunol. 138, 4490-4494. Pancrr, V., Cesbron, J.Y., Auriault, C., Joseph, M., Chandenier, J. and Capron, A. (1988a). IgE dependent killing of Brug~ ma/ay/microfilariae by human platelets and its modulation by T cell products. Int. Arch. Allergy Appl. Immunol. 85, 483-486.

135

Pancrr, V., Cesbron, J.Y., Joseph, M., Barbier, M., Capron, A. and Auriault, C. (1988b). IgE dependent killing of Schistosoma mansoni schistosomula by human platelets: modulation by T cell products. Int. Arch. Allergy Appl. Immunol. 87, 371-379. Pancrr, V., Joseph, M., Capron, A., Wietzerbin, J., Kusnierz, J.P., Vorng, H. and Auriault, C. (1988c). Recombinant human interferon-gamma induces increased IgE receptor expression on human platelets. Eur. J. Immunol. 18, 829-832. Pancrr, V., Joseph, M., Capron, A., Delanoye, A., Vorng, H. and Auriault, C. (1989). Characterization of a suppressive factor of platelet cytotoxic functions in human and rat Schistosomiasis mansoni. Clin. Exp. Immunol. 76, 417-421. Pancrr, V., Schellekens, H., Van der Meide, P., Vorng, H., Delanoye, A., Capron, A. and Auriault, C. (1990a). Biological effect of interferon gamma during the course of experimental infection of rat by Schistosoma mansoni. Cell. Immunol. 125, 58-64. Pancrr, V., Montr, D., Delanoye, A., Capron, A. and Auriault, C.(1990b). Interleukin-6 is the main mediator of the interaction between monocytes and platelets in the killing of Schistosoma mansoni. Eur. Cytokine Net. 1, 15-19. Pancrr, V., Pierce, R,J., Fournier, F., Mehtali, M., Delanoye, A., Capron, A. and Auriault, C. (1991). Effect of ubiquitin on platelet functions: possible identity with platelet activity suppressive lymphokine (PASL). Eur. J. Immunol. 21, 2735-2741. Peyron, F., Polack, B., Lamotte, D., Kolodie, L. and Ambroise-Thomas, P. (1989). PlasmMium falciparum growth inhibition by human platelets in vitro. Parasitology 99, 317-322. Ridel, P.R., Auriault, C., Darcy, F., Pierce, tLJ., Leite, P., Santoro, F., Neyrinck, J.L., Kusnierz, J.P. and Capron, A. (1988). Protective role of IgE in immunocompromised rat toxoplasmosis. J. Immunol. 141,978-983. Spiegelberg, H.L. (1984). Structure and function of the Fc receptors for IgE on lymphocytes, monocytes and macrophages. Adv. Immunol. 35, 61-88. Tsicopoulos, A., Tonnel, A.B., Vorng, H., Joseph, M., Wallaert, B., Kusnierz, J.P., Pestel, J. and Capron, A. (1990). Lymphocyte-mediated inhibition of platelet cytotoxic functions during hymenoptera venom desensitization: characterization of a suppressive lymphokine. Eur. J. Immunol. 20, 1201-1207. Umekita, L.F. and Mota, I. (1989). In vitro lysis of sensitized Trypanosoma cruzi by platelets: role of C3b receptors. Parasite Immunol. 11, 561-563. Verwaerde, C., Joseph, M., Capron, M., Pierce, R.J., Damonneville, M., Velge, F., Auriault, C. and Capron, A. (1987). Functional properties of a rat monoclonal IgE antibody specific for Schistosoma mansoni. J. Immunol. 138, 4441-4446. Viens, P., Dubois, R. and Kongshavn, P.A.L. (1983). Platelet activity in immune lysis of Trypanosoma musculi. Int. J. Parasitol. 13, 527-530. Yong, E.C., Chi, E.Y., Fritsche, T.R. and Henderson W.R. (1991). Human platelet-mediated cytotoxicity against Toxoplasmagondii: role of thromboxane. J. Exp. Med. 173, 65-78.

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7. Platelets m

ra l Infecti ons

Dorothea Zucker-Franklin

1. Introduction 2. Clinical Manifestations 3. Direct Effect of Viruses on Megakaryocytes and Platelets 4. Thrombocytopenia caused by Immune Mechanisms

137 137 138

145 146 147

145

i. Introduction It has been known for a long time that viral infections may cause purpura as well as other hemorrhagic manifestations. However, a role for platelets in these bleeding phenomena became appreciated only about 50 years ago. Recognition that platelets may be affected by viruses was contingent on the development of methods for accurate platelet counts, and on the advent of electron microscopy to identify virus particles in bone marrow and circulating blood cells. Subsequently, refinements in immunologic techniques demonstrated that virus infections may cause thrombocytopenia indirectly, i.e. by immune mechanisms, and during the past decade, advances in molecular biology have made it possible to detect viral "footprints" in the megakaryocyte/platelet lineage even in clinically latent, non-proliferative viral infections. Because these distinct developments in the biomedical sciences correspond to some extent with the different mechanisms whereby viruses affect megakaryocytes and platelets, it seems efficacious to consider this subject roughly under the same headings. Accordingly, some examples of early clinical observations which have correlated bleeding phenomena with viral infections will be given first. This will be followed by a discussion of the ultrastructural evidence showir/g that megakaryocytes may provide a particularly hospitable environment for virus storage and replication. The third section will primarily deal with virus-related immune mechanisms that may indirectly affect thrombocytopoiesis as well as Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

5. Thrombocytopenia due to Diverse Mechanisms 6 Conclusion 7. References

platelet lifespan. Lastly, thrombocytopenias that are attributable to a combination of direct and indirect mechanisms depressing platelet counts, especially as exemplified by patients infected with the human immunodeficiency virus (HIV), will be considered in the context of diagnosis and treatment options. It will become apparent that there is a great deal of overlap, because thrombocytopenia associated with viral infections is often multifactorial.

2. Clinical Manifestations Thrombocytopenia is arbitrarily defined as a platelet count below 150 000/#1, but purpura or other bleeding phenomena rarely occur until the count falls below 50 000//~1 or if there is concomitant impairment of platelet function. Petechiae, which are painless, nonblanching purpuric spots less than 3 mm across, usually appear first on the lower extremities but are soon accompanie d by ecchymoses into soft tissues exposed to the slightest pressure or trauma wherever it occurs. Epistaxis and bleeding gums are other signs characteristic of thrombocytopenia. The prototype of a hemorrhagic diathesis caused by a viral infection is represented by dengue hemorrhagic fever. Almost every mechanism known to cause thrombocytopenia, either by underproduction or excessive destruction of platelets, could be gleaned from infection with the dengue virus(es). The epidemics of hemorrhagic fevers, now known to be Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

138 D. ZUCKER-FRANKLIN transmitted by arthropods, were described in ancient Egypt and tropical areas of Southeast Asia. They have become mainly of historic interest. Over the past two centuries, similar well-documented epidemics have occurred worldwide, some of the largest ones perhaps in the United States, Australia, Greece and Japan. Many of these epidemics may have been due to other viruses, such as the Chikungunya virus (Carey, 1971). The subject has been reviewed repeatedly and the interested reader may refer to an extensive literature (Sabin, 1952; Halstead, 1982). Many of the classical studies have, of course, been rendered obsolete, scientifically. Objective scientific data are of more recent origin. The dengue viruses, in particular, have been well characterized immunologically, biochemically, and morphologically, while studies on their biological behavior is still ongoing (see review by Halstead, 1988). It is now recognized that the hemorrhagic form of dengue fever is dependent on prior sensitization with the virus and that, therefore, it is seen only in secondary infections or in primary infection of young children born to dengue-immune mothers (Vasquez, 1983; Ha/stead, 1988). The thrombocytopenia and disseminated intravascular coagulation, which can be rapidly fatal, are attributed to immune complexes coating virus particles and cells lining the vasculature (Putinseva et al., 1986; Funahara et al., 1987a, b). Curiously, it is still not known whether this virus infects megakaryocytes and/or platelets per se. The diverse mechanisms whereby the virus can be responsible for bleeding are included in the appropriate sections below. Other examples of viral infections which, historically, have been recognized to be associated with hemorrhagic phenomena are smallpox, viral hepatitis, and most of the childhood exanthems. The pathogenicity of some of these viruses, e.g. the hepatitis viruses, is very complex, and reaches far beyond the megakaryocyte/platelet lineage (Nakamura et al., 1975; Casciato et al., 1978; Zeldis et al., 1983). The multiplicity of mechanisms responsible for thrombocytopenia in childhood diseases have been well elucidated. They differ from case to case and will be considered among the subjects discussed in the appropriate sections to follow.

3. Direct Effect of Viruses on Megakaryocytes and Platelets Before considering this subject, it should be recalled that platelets are formed within the cytoplasm of megakaryocytes. The megakaryocyte undergoes a series of differentiation steps from a morphologically unidentifiable diploid precursor to a huge polyploid cell measuring 20-60 gm in diameter. Theoretically, viruses could enter the cell at any stage of its differentiation. It has often been suggested that the process of endomitosis and polyploidization which the nuclei of these cells undergo is particularly conducive for viral replication. During differentiation,

the megakaryocyte cytoplasm develops a system of membranes which form tubular structures that demarcate the territories of future platelets (see Fig. 7.1). Particulates taken up from the extracellular medium as well as substances synthesized by the cell may come to occupy these channels. It is now generally recognized that thrombocytopoiesis occurs by fragmentation of megakaryocytes, and not by surface budding (Zucker-Franklin and Petursson, 1984). The size of the resulting platelets depends, in part, on factors controlling the rate of platelet production and the maturity and stage of differentiation of the precursor cell. The process has been described and depicted in detail elsewhere (ZuckerFranklin, 1988). Of importance for the subject under consideration are the following. (1) Platelets are anuclear pieces of cytoplasm which have no DNA and very little RNA and have, therefore, little if any biosynthetic activity. (2) Platelets contain substances synthesized as well as substances endocytosed by megakaryocytes. These include particles which may occupy vacuoles or which may be located in the demarcation membrane system with which platelets are also endowed. In the platelet this is referred to as the surface-connected canalicular system. (3) There are structural and antigenic differences between megakaryocytes and platelet surface membranes (Stahl et al., 1986; Hyde and Zucker-Franklin, 1987). The lastmentioned observation implies that there could be specific viral receptors on the surface of megakaryocytes that are not found on platelets and vice versa, and that immune mechanisms responsible for thrombocytopenia could affect platelets without damaging megakaryocytes. Both these situations could pertain. Reports describing the morphologic detection of viruses in megakaryocytes and/or platelets should be interpreted in the light of these facts. On the one hand, viruses could merely be taken up by these cells without affecting their numbers or function. On the other hand, the uptake of viruses by megakaryocytes or platelets could cause profound thrombocytopenia induced by various mechanisms. Delineation of viruses in thin sections of tissues became possible in the early 1950s following the introduction of heavy metal "staining" into electron microscopy. This coincided with exciting studies associated with the newly discovered murine leukemia and mammary tumor viruses. The abundance of the Friend leukemia virus found within the demarcation membrane system of megakaryocytes was observed early in these studies (De Harven and Friend, 1958, 1960; Dalton et al., 1961). A detailed review comparing the Friend, Gross, Moloney and Manaker-C60 agents is as valid today as it was 30 years ago (Dalton et al., 1961). On the basis of these studies it was concluded that megakaryocytes of the spleen and bone marrow are the best sources of viral particles. The astonishingly large number of particles within a single megakaryocyte may be surmised from Fig. 7.2. Even though these viruses are the recognized cause of

PLATELETS IN VIRAL INFECTIONS

139

Figure 7.1 Detail of a megakaryocyte from a mouse 2 weeks after inoculation with the virus which causes MAIDS. The cell is shown at low magnification to illustrate the demarcated platelet fields (P). Viruses (arrows) are difficult to resolve at this magnification. N, nucleus. Magnification x 7500. Inset shows two virus particles within the demarcation membrane system of a megakaryocyte at higher resolution. Magnification x 51 000.

140 D. ZUCKER-FRANKLIN

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Figure 7.2 (A) Low power magnification of a megakaryocyte isolated from the bone marrow of a mouse infected with the Friend leukemia virus. The abundance of virus particles in this megakaryocyte almost staggers the imagination. The particles are difficult to discern at this magnification which is x 15 000. Therefore, the area indicated by the arrow is depicted at higher magnification in (B). Magnification of (A) is x 15 000. (B) represents the area indicated by the arrow in (A) at higher resolution. Magnification x 30 000.

PLATELETS IN VIRAL INFECTIONS 141 lymphocytic leukemia/lymphoma in mice, the particles are not found as abundantly in lymphocytes. Viral replication within the megakaryocyte lineage is evidenced by ample budding of particles from the internal membranes of the cells (Fig. 7.3). Curiously, budding from the plasma membrane was notably reported absent, and pathologic changes in cell structure were not considered

striking (Dalton et al., 1961). Up to 20% of circulating platelets obtained from leukemic mice contain virus particles within their canalicular system. This is not surprising given the mechanism of thrombocytopoiesis described above. Illustrations of megakaryocytes obtained from mice within a week after infection with the Friend leukemia virus are shown in Figs. 7.2 and 7.3.

Figure 7.3 Higher resolution of details of megakaryocyte cytoplasm showing that almost all virus particles are located within the demarcation membrane system. The cells were isolated from the bone marrow of a mouse 10 days after having been inoculated with the Friend leukemia virus. Proliferation of the virus is evident by viral budding (arrows). Magnification x 50 000.

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PLATELETS IN VIRAL INFECTIONS The huge number of particles seen in a thin section of a megakaryocyte 10 days after inoculation of the virus into a Balb-c mouse is seen in Fig. 7.2. In agreement with earlier observations, budding from the plasma membrane was found to be relatively rare and megakaryocyte morphology appeared to be well preserved. Nevertheless, it has been shown that the number of megakaryocytes decreases within 3 days after infection with the Friend virus, and that megakaryocytes of high ploidy number appear to be infected first (Brown and Axelrad, 1976). It is conceivable that this is related to an entry process which may not be fully developed in less mature cells. Thrombocytopenia becomes apparent within 7-9 days. Clearly, in this instance, the virus must have a direct effect on the development of this cell lineage and immune mechanisms need not be invoked. What is less clear is the nature of the damage inflicted. This has been questioned in the case of rat leukemia induced with a virus of mouse origin. Within 10 days of inoculation, there is an abundant, proliferative accumulation of particles in the megakaryocytes of these animals. The thrombocytopenia develops slowly and does not correlate with the virus load in the precursor cells (de Saint George et al., 1980, 1982). It was postulated that the megakaryocytes play an amplification role in the infectious process. In humans, a direct effect of viruses on the megakaryocyte/platelet lineage is more difficult to decipher. It may be assumed, however, that thrombocytopenia developing within the first week of an acute primary viral infection cannot be an immunologic event, although such a mechanism may come into play during the chronic phase of the same infection. A cogent example is the thrombocytopenia associated with the administration of live measles vaccine. It has been documented that administration of this vaccine is usually accompanied by a very mild subclinical thrombocytopenia (Fig. 7.4). A careful study analyzing serial bone marrow samples following the administration of the vaccine to infants has revealed that the megakaryocyte count decreases within 3 days of inoculation (Fig. 7.4) when many megakaryocytes are vacuolated and their nuclei appear to undergo degeneration (Oski and Naiman, 1966). That the mild drop in platelet count is due to underproduction rather than destruction of platelets is based on the observation that the platelet life span in the circulation is not decreased. Moreover, the administration of inactivated measles vaccine does not change the platelet count (Oski and Naiman, 1966). Additional observations on the effect of measles infection on the platelet count have been published by Morse et al., 1966), Bachand et al. (1967) and Alter et al. (1968), among others. Scattered case reports of early thrombocytopenia associated with viral infections in which excessive destruction or removal of platelets by immune mechanisms has been ruled out, can be found throughout the medical literature. Such reports include Newcastle disease virus in

143

guinea-pigs (Jerushalmy et al., 1963), varicella (Tobin and ten Bensel, 1972; Espinoza and Ruhn, 1974), cytomegalovirus in mice (Osborn and Shahidi, 1973) and humans (Weller and Hanshaw, 1962; Sahud and Bachelor, 1978; Chanarin and Walford, 1973), mumps (Kolars and Spink, 1958; Fama et al., 1965; Graham et al., 1974), infectious mononucleosis (Angle and Alt, 1950; Carter, 1965; Sharp, 1969; Mazza and Magnin, 1975), hepatitis, and parvoviruses (Lefrere and Got, 1987). In congenital as well as acquired cytomegalovirus infections, eosinophilic inclusion bodies and vacuolization of megakaryocytes have been seen on light microscopy when no platelet antibodies or increased platelet destruction could be demonstrated (Chesney et al., 1978). In most of the exanthems, the thrombocytopenia is subclinical and reversible. The devastating amegakaryocytic thrombocytopenia associated with acute viral infections is an extremely rare event (Brook, 1979). It should also be mentioned that infection with hepatitis or parvoviruses is not limited to the megakaryocyte lineage, but usually affects other hematopoietic cell lineages as well. This may result in hypoplastic or even aplastic anemia rather than isolated thrombocytopenia (Foon et al., 1984; Young and Mortimer, 1984). Recently, it has been shown that megakaryocytic cell lines can be productively infected with HIV in vitro (Sakaguchi et al., 1991; Mont~ et al., 1992). Such cell lines were established from bone marrows of leukemic patients. The caveat is that, although the cultured cells have many properties of megakaryocytes freshly isolated from healthy subjects, they do not produce platelets. The mere observation that viruses are internalized by megakaryocytes or platelets in vitro (Zucker-Franklin et al., 1990) does not necessarily imply that the viruses are injurious at this site. However, electron microscopic analysis of bone marrow obtained from patients with acquired immune deficiency syndrome (AIDS) has revealed structural aberrations of megakaryocytes not seen in patients with idiopathic thrombocytopenic purpura. The number of denuded megakaryocyte nuclei in such specimens is very high suggesting that intramedullary damage is taking place (Zucker-Franklin et al. 1989). In the murine model for AIDS, MAIDS (Mosier et al., 1985), we have observed viral budding within 10 days after inoculation of the virus (Fig. 7.5). At this time, the animals do not appear ill. Whether they have subclinical thrombocytopenia has not yet been determined. The human parvovirus B19, which is primarily tropic for erythroid progenitor cells (Young et al., 1984), may also cause thrombocytopenia. Apparently, this virus does not replicate in megakaryocytes, but its presence in this cell line impairs normal thrombocytopoiesis (Srivastava et al., 1990). Newcastle disease and influenza viruses also affect platelets directly in that they adsorb onto the cells and cleave the cell's surface sialic acid residues (Danon et al., 1959; Terada et al., 1966; Turpie et al., 1973).

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PLATELETS IN VIRAL INFECTIONS 145 Vaccinia virus, which lacks neuraminidase, is also able to adsorb to platelets and thereby impairs their ability to aggregate normally (Biket al., 1982).

neuraminidase (Terada et al., 1966). This event is not believed to be a sufficient cause for accelerated platelet clearance. Platelets are not lysed when treated with bacterial neuraminidase. However, when neuraminidasetreated platelets are exposed to autologous serumcontaining antibodies, lysis ensues. A series of elegant studies demonstrated that the lysis of platelets is predominantly due to activation of the classical comple In most cases, virus infections that are accompanied by ment pathway, while some C3 deposition and lysis may thrombocytopenia can be demonstrated to have an also be attributable to activation of the alternative immunologic component. The sine qua non of this pathway (Kazatchkine et al., 1984). On the basis of these process is a shortened platelet lifespan and the futility of and other studies, it has been postulated that platelet desplatelet transfusions. Platelet survival is usually measured truction could be mediated by both antibody and/or with SlCr- or 111In-labelled autologous platelets and complement as well as by antibody-independent C3 normally ranges from 9 to 12 days. Platelets obtained deposition. C3 deposited on platelets will then react with from healthy donors transfused into patients with C3b receptors on cells belonging to the macrophage/ immune thrombocytopenia will be attacked by the same phagocytic system (Nydegger and Kazatchkine, 1984). mechanisms that damage the patients' own platelets. The presence of circulating anti-hemagglutinins elicited The bone marrow usually shows a compensatory by influenza virus infections is very high among the megakaryocytosis. However, since there is considerable healthy population. Since many cross-reactive strains of antigenic similarity between megakaryocytes and platelets this virus exist, immune thrombocytopenias due to it is likely that fully differentiated megakaryocytes are also repeated infections with influenza viruses seem possible. injured by the immune process. Megakaryocyte progenAs mentioned before, the prototype of an immune itors and immature megakaryocytes may not bear the epi- mediated hemorrhagic response to a viral infection is seen tope against which the platelet antibodies are directed, in dengue hemorrhagic fever. Antibody-dependent and may therefore escape damage (Stahl et al., 1986; enhancement of viral uptake and its replication in monoHyde and Zucker-Franklin, 1987). Characteristically, cytes have been studied in great detail (reviewed by Halimmune thrombocytopenia due to viral infection occurs stead, 1982). The dengue antigen complexed with IgG 7-10 days after the primary symptoms of the disease have can be delineated by direct immunofluorescence of been fully established. Antiviral antibodies either bind to platelets in 48% of patients with dengue hemorrhagic platelet-bound viruses or immune complexes consisting fever (Boonpucknavig et al., 1979). However, the perof antibodies and circulating viral antigens subse- centage of positive platelets is not related to the severity quently bind to platelets (Myllya et al., 1969; Dixon et of the thrombocytopenia because platelets are also desal., 1973; Lurhuma et al., 1977; Bik et al., 1982; troyed by disseminated intravascular coagulation (DIC), Kazatchkine et al., 1984). Even normal platelets carry the extent of which varies from patient to patient. Suffice adsorbed immunoglobulin on their surface but when it to say that the marked reduction in circulating C3, C4, measured by binding assays using 12SI-labelled and C5 proteins as well as fibrinogen and other coagumonoclonal antibodies (mAbs) to IgG or 12sI- lation factors suggests that aggregation and sequestration staphylococcal protein A, surface IgG is estimated to be of platelets in the microvasculature contributes to the only a small fraction of the total IgG carried by the cell. catastrophic development of thrombocytopenia in this Most of the immunoglobulin is contained in the c~ disease. granules as are other plasma proteins (George, 1990). The cyclic thrombocytopcnia observed in horses However, it is probable that it is the immunoglobulin on infected with the equine infectious ancmia virus also the platelet surface that plays the crucial role in immune appears to be immune mcdiated. Immune complex thrombocytopenias (for review see Kelton, 1983). Several dcposition on platelcts has becn demonstrated in this investigators have reported that platelets of patients with condition, whcrcas in situ hybridization failcd to show immune thrombocytopenia carry more IgG than platelets cvidcncc of viral sequenccs in thc megakaryocytes of of healthy individuals. In some instances, this has been infected animals (Clabough et al., 1991). shown to consist of specific antibodies. There is usually an inverse relationship between the platelet count and platelet-associated IgG. In addition, antibodyindependent platelet destruction by virus-induced activation of the complement system can occur (Sissons et al., 1980). As already mentioned, myxoviruses, such as influenza, The multiplicity of mechanisms that can be responsible may bind to platelets via specific receptors, i.e. neura- for thrombocytopenia in a chronic viral infection is best minic acid residues which are then cleaved by viral exemplified by infection with HIV. Thrombocytopenia e

Thrombocytopenia Caused by Immune Mechanisms

e

Thrombocytopeniadue to Diverse Mechanisms

146 D. ZUCKER-FRANKLIN may appear years before the development of full-blown AIDS or as a concomitant of other hematologic or nonhematologic manifestations of the disease. The immune mechanisms responsible for the accelerated clearance of circulating platelets were analyzed early in the epidemic (Walsh et al., 1984; Savona et al., 1985; Stricker et al., 1985), and have been reviewed recently (Karpatkin, 1990). There is little doubt that increased plateletassociated IgG, C3, C4, and adsorption of circulating immune complexes, as well as anti-idiotype antibody directed against the antibody to viral surface antigen can be incriminated to a variable extent in different patients. HIV is internalized by freshly isolated megakaryocytes and platelets in vitro (Fig. 7.6) even though these cells have few, if any, CD4 receptors. It has also been demonstrated definitively that HIV infects the megakaryocyte/platelet lineage in viv0 and that considerable structural damage to these cells occurs (ZuckerFranklin and Cao, 1989; Zucker-Franklin et al., 1989b). Apart from the structural injury, the abundance of denuded megakaryocyte nuclei in the bone marrow of HIV-infected individuals bespeaks ineffective megakaryocytopoiesis in the face of a megakaryocytosis which is often seen early in such patients. We have shown by in situ hybridization that megakaryocytes freshly isolated from patients with AIDS harbor proviral sequences (ZuckerFranklin and Cao, 1989; Fig. 7.7A and B). This observation has been amply confirmed (Louache et al., 1991). Moreover, human megakaryocyte cell lines readily

support HIV replication in vitro (Sakaguchi et al., 1991; Mont~ et al., 1992). Obviously, all these observation must be taken into consideration when therapeutic measures are to be chosen. In most virus infections, especially those associated with the exanthems, thrombocytopenia is transient and self-limited. Allogeneic platelet transfusions may, however, be indicated when bleeding is life-threatening and intravascular coagulation has been ruled out. In chronic infections, such as infection with HIV, the choice of therapy must be tailored for each individual patient. Commensurate with the observation that megakaryocytes harbor the virus, it was observed that the administration of azido3'deoxythymidine (AZT) has a beneficial effect on platelet counts within 1-2 weeks of its administration when the drug could not have affected immunoglobulin levels. However, a high dose of AZT may be required. The majority of HIV-infected patients whose clinical symptoms are limited to thrombocytopenia are best managed like patients with idiopathic thrombocytopenia, i.e. with small doses of steroids, and if unresponsive, splenectomy or the administration of intravenous gamma globulin. Remissions in such patients are not unusual.

6. Conclusion As a rule, the writing of a review article is frustrating to

Figure 7.6 Internalization of retroviruses in vitro. (A). Detail of a mouse megakaryocyte co-cultured with cells infected with an amphotropic retrovirus. Arrows indicate viruses within demarcation membrane system. This virus does not appear to replicate. Magnification x 22 000. (Reproduced with permission from Zucker-Franklin et al. (1990).)

PLATELETS IN VIRAL INFECTIONS

147

D Figure 7.7 (A) In situ hybridization of bone marrow from a patient with AIDS utilizing a aSS-labelled HIV RNA probe. Two heavily labelled megakaryocytes are seen in this illustration. (Reproduced with permission from Zucker-Franklin and Cao (1990).) (B) Platelet showing a vacuole containing HIV. Platelets and viruses had been incubated in vitro. Magnification x 28 000.

its author. Space limitations hardly ever permit the inclusion of enough relevant material to call the work complete. This chapter is no exception, but for different reasons. The list of publications dealing with viral infections and platelets is actually very small. More, and equally worthy papers on the subject could have been cited but they would not have broadened the basic concepts conveyed in these pages, nor filled the gaps in our existing knowledge. The concepts gained, to date, may be summarized as follows. (1) Most viruses can enter platelets and their precursor cells, the megakaryocytes, with or without the presence of specific receptors. Such entry may be accomplished by Fc receptor-mediated endocytosis since both megakaryocytes and platelets possess such membrane epitopes. Internalization of viruses may be inocuous, but more likely, causes some damage, although - as exemplified by the measles virus - the damage may be transient and subclinical in nature. (2) Megakaryocytes appear to provide a particularly hospitable environment for virus replication. Why? This is an interesting area for further research. The observation holds true for D N A as well as RNA viruses and has been commented on by many investigators. (3) Viruses may adsorb onto platelets causing their aggregation and degranulation. This initiates the coagulation cascade leading to disseminated thromboses throughout the microvasculature with concomitant depletion of platelets and coagulation proteins. (4) An immune response to

most primary viral infections takes 8 - 1 0 days to develop. The antibodies form immune complexes with the virus, presumably on the platelet surface. Several components of the complement system have also been shown to play a role in this process. (5) In most instances, the course of events is self-limited when the virus is eliminated, but in chronic viral infections, particularly well exemplified by retroviruses such as HIV, the immune response may become the dominant cause for thrombocytopenia. Treatment options should be chosen with the rationale based on the information outlined above.

7. References Alter, H.J., Scanlon, tLT. and Schechter, G.P. (1968). Thrombocytopenic purpura following vaccination with attenuated measles virus. Am. J. Dis. Child. 115, 111-113. Angle, 1LM. and Alt, H.L. (1950). Thrombocytopenic purpura complicating infectious mononucleosis. Blood 5, 449-457. Bachand, A.J., Rubenstein, J. and Morrison, A.N. (1967). Thrombocytopenic purpura following live measles vaccine. Am. J. Dis. Child. 113, 283-285. Bik, T., Sarov, I. and Livne, A. (1982). Interaction between vaccinia virus and human blood platelets. Blood 59, 482-487. Boonpucknavig, S., Vuttiviroj, O., Bunnag, C., Bhamarapravati, N. and Nimmanitya S. (1979). Demonstration of dengue antibody complexes on the surface of platelets from patients with dengue hemorrhagic fever. Am. J. Trop. Med. Hyg. 28, 881-884.

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D. ZUCKER-FRANKLIN

Brook, I. (1979). Disseminated varicella with pneumonia, meningoencephalitis, thrombocytopenia, and fatal intracranial hemorrhage. Southern Med. J. 72, 756-757. Brown, W.M. and Axelrad A.A. (1976). Effect of Friend leukemia virus on megkaryocytes and platelets in mice. Int. J. Cancer 18, 764-773. Carey, D.E. (1971). Chikungunya and Dengue: a case of mistaken identity. J. Hist. Med. Allied Sci. 26, 243-262. Carter, R.L. (1965). Platelet levels in infectious mononucleosis. Blood 25, 817-821. Casciato, D.A., Klein, C.A., Kaplowitz, N. and Scott, J.L. (1978). Aplastic anemia associated with type B viral hepatitis. Arch. Intern. Med. 138, 1557-1558. Chanarin, I. and Walford, D.M. (1973). Thrombocytopenic purpura in cytomegalovirus mononucleosis. Lancet 2, 238-239. Chesney, P.J., Taher, A., Gilbert, E.M. and Shahidi, N.T. (1978). Intranuclear inclusions in megakaryocytes in congenital cytomegalovirus infection. J. Pediatr. 92, 957-958. Clabough, D.L., Gebhard, D., Flaherty, M.T., Whetter, L.E., Perry, S.T., Coggins, L. and Fuller, F.J. (1991). Immunemediated thrombocytopenia in horses infected with Equine infectious anemia virus. J. Virol. 65, 6242-6251. Dalton, A.J., Law, L.W., Moloney, J.B. and Manaker ILA. (1961). An electron microscopic study of a series of murine lymphoid neoplasms. J. Natl. Cancer Inst. 27, 747-791. Danon, D., Jerushalmy, Z. and de Vries, A. (1959). Incorporation of influenza virus in human blood platelets in vitro. Electron microscopic observation. Virology 9, 719-722. De Harven, E. and Friend, C. (1958). Electron microscope study of a cell-free induced leukemia of the mouse: a preliminary report. J. Biophys. Biochem. Cytol. 4, 151-156. De Harven, E. and Friend, C. (1960). Further electron microscopic studies of a mouse leukemia induced by cell-free filtrates. J. Biophys. Biochem. Cytol. 7, 747-754. de Saint-George, L., Baugnet-Mahieu, L., Janowski, M., Van Gorp, U. and Maisin J.R. (1980). Kinetics of the propagation of a murine virus (C57B1 mice) in the lymphoid system and bone marrow of rats. C. R, Seances Soc. Biol. Filial. 174, 845-850. de Saint-George, L., Baugnet-Mahieu, L., Janowski, M., Van Gorp, U., Regniers, L. and Maisin, J.R. (1982). Aspects of megakaryocytic viral production in relation to lymphocytic leukemia in the rat. C. tL Seances Soc. Biol. Filial. 176, 109-114. Dixon, R., Rosse W. and Ebbert, L. (1973). Quantitative determination of antibody in idiopathic thrombocytopenic purpura. Correlation of serum and platelet-bound antibody with clinical response. New. Engl. J. Med. 292, 230-236. Espinoza, C. and Kuhn, C. (1974). Viral infection of megakaryocytes in varicella with purpura. Am. J. Clin. Pathol. 61,203-208. Fama, P.G., Paton, W.B. and Bostock M.I. (1965). Thrombocytopenic purpura complicating mumps. Br. Med. J. 5419, 1244. Foon, K.A., Mitsuyasu, ILT., Schroff, R.W., McIntryre, tLE., Champlin, R. and Gale, R.P. (1984). Immunologic defect in young male patients with hepatitis-associated aplastic anemia. Ann. Intern. Med. 100, 657-662. Funahara, Y., Ogawa, K., Fujita, N. and Okuno, Y. (1987a). Three possible triggers to induce thrombocytopenia in dengue

virus infection. SE Asian J. Trop. Med. Publ. Health 18, 351-355. Funahara, Y., Sumarmo, Shirahata, A., and Setiabudy-Dharma, tL (1987b). DHF characterized by acute type DIC with increased vascular permeability. SE Asian J. Trop. Med. Publ. Health 18, 346-350. George, J.N. (1990). Platelet immunoglobulin G: its significance for the evaluation of thrombocytopenia and for understanding the origin of a-granule proteins. Blood 76, 859-870. Graham, D.Y., Brown, C.H., 3rd, Benrey, J. and Butel J.S. (1974). Thrombocytopenia: a complication of mumps. JAMA 227, 1162-1164. Halstead, S.B. (1982). Dengue: hematologic aspects. Semin. Hematol. 19, 116-131. Halstead, S.B. (1988). Pathogenesis of dengue: challenges to molecular biology. Science 239, 476-481. Hyde, P. and Zucker-Franklin, D. (1987). Antigenic differences between human platelets and megakaryocytes. Am. J. Pathol. 127, 349-357. Jerushalmy, Z., Adler, A., Rechnic, J., Kohn, A. and Vries de, A. (1962). Effect of myxoviruses on the clotting and clot retracting activities of human blood platelets in vitro. Pathol. Biol. 10, 41-48. Karpatkin, S. (1990). HIV-l-related thrombocytopenia. Hematol. Oncol. Clin. N. Am. 4, 193-218. Kazatchkine, M.D., Lambrie, C.IL, Kieffer, N., Maillet, F. and Nurden, A.T. (1984). Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum. J. Clin. Invest. 74, 976-984. Kelton, J.G. (1983). The measurement of platelet-bound immunoglobulins: an overview of the methods and the biological relevance of platelet-associated IgG. Prog. Hematol. 13, 163-199. Kolars, C.P. and Spink, W.W. (1958). Thrombocytopenic purpura as a complication of mumps. JAMA 168, 2213-2215. Lefrere, J.J. and Got, D. (1987). Peripheral thrombocytopenia in human parvovirus infection. J. Clin. Pathol. 40, 469. Louache, F., Bettaieb, A., Henri, A., Oksenhendler, E., Farcet, JP, Bierling, P., Seligrnann, M. and Vainchenker, W. (1991). Infection of megakaryocytes by human immunodeficiency virus in seropositive patients with immune thrombocytopenic purpura. Blood 78, 1697-1705. Lurhuma, A.Z., Riccomi, H. and Masson, P.L. (1977). The occurrence of circulating immune complexes and viral antigens in idiopathic thrombocytopenic purpura. Clin. Exp. Immunol. 28, 49-55. Mazza, J.J. and Maguin, G.E. (1975). Severe thrombocytopenia in infectious mononucleosis. Report of two cases and review of the literature. Wiscon. Med. J. 74, 124-127. Mont~, D., Groux, H., Raharinivo, B., Plouvier, B., Dewulf, J., Clavel T., Grangette, C., Torpier, G., Auriault, C., Capron, A. and Ameisen, J-C. (1992). Productive human immunodeficiency virus-1 infection of megakaryocytic cells is enhanced by Tumor Necrosis Factor-c~. Biood 79, 2670-2679. Morse, E.E., Zinkham, W.H. and Jackson, D.P. (1966) Thrombocytopenia purpura following rubella infection in children and adults. Arch. Int. Med, 117, 579.

PLATELETS IN VIRAL INFECTIONS Mosier, D.E., Yetler, ILA. and Morse III, H.C. (1985). Retroviral induction of acute lymphoproliferative disease and profound immunosuppression in adult C57B1/6 mice. J. Exp. Med. 161,766-784. Myllya, G., Vaheri, A., Vesikari, T. and Penttinen K. (1969). Interaction between human blood platelets, viruses and antibodies. IV. Post-rubella thrombocytopenic purpura and platelet aggregation by rubella antigen-antibody interaction. Clin. Exp. Immunol. 4, 323-332. Nakamura, S., Sato, T, Maeda, T. and Sata, Y. (1975). Viral hepatitis B and aplastic anemia. Tohoku J. Exp. Med. 116, 101-102. Nydegger, U.E. and Kazatchkine, M.D. (1984). The role of complement in immune clearance of blood cells. Springer Semin. Immunopathol. 6, 373-398. Osborn J.E. and Shahidi N.T. (1973). Thrombocytopenia in murine cytomegalovirus infections. J. Lab. Clin. Med. 81, 53-63. Oski, F.A. and Naiman, J.L. (1966). Effect of live measles vaccine on the platelet count. N. Engl. J. Med. 275, 352-356. Putintseva, E., Vega, G. and Fernandez, L. (1986). Alterations in thrombopoiesis in patients with thrombocytopenia produced by Dengue hemorrhagic fever. Nouv. Rev. Fr. Hematol. 28, 269-273. Sabin, A.B. (1952). Dengue: In "Viral and Rickettsial Infections of Man" (ed. T.M. Rivers), 2nd edn, pp. 556-568. Lippincott, Philadelphia. Sahud, M.A. and Bachelor, M.M. (1978). Ctyomegalovirusinduced thrombocytopenia: an unusual case report. Arch. Intern. Med. 138, 1573-1575. Sakaguchi, M., Sato, T., and Groopman J.E. (1991). Human immunodeficiency virus infection of megakaryocytic cells. Blood 7, 481-485. Savona, S., Nardi, M.A., Lennette, E.T. and Karpatkin, S. (1985). Thrombocytopenic purpura in narcotics addicts. Ann. Intern. Med. 102, 737-741. Sharp, A.A. (1969). In: "Platelets, Bleeding, and Haemostasis in Infectious Mononucleosis" (eds ILL. Carter and H.G. Penman), p. 99. Blackwell, Oxford. Sissons, J.G., Oldstone, M.B. and Schreiber R.D. (1980). Antibody-independent activation of the alternative complement pathway by measles virus-infected cells. Proc. Natl. Acad. Sci. 77, 559-562. Srivastava, A., Bruno, E., Briddell, IL, Cooper, IL, Srivastava, C., van Besien, K. and Hoffman, IL (1990). Parvovirus B19induced perturbation of human megakaryocytopoiesis in vitro. Blood 76, 1997-2004. Stahl, C.P., Zucker-Franklin, D. and McDonald, T.P. (1986). Incomplete antigenic crossreactivity between platelets and megakaryocytes: Relevance to ITP. Blood 67, 421-428.

149

Stricker, ILB., Abrams, D.I., Corash, L. and Shuman, M.A. (1985). Target platelet antigen in homosexual men with immune thrombocytopenia. New. Eng. J. Med. 313, 1375-1380. Terada, H., Baldini, M., Ebbe S. and Madoff M.A. (1966). Interaction of influenza virus with blood platelets. Blood 28, 213-228. Tobin, J.D. and ten Bensel, ILW. (1972). Varicella with thrombocytopenia causing fatal intracerebral hemorrhage. Am. J. Dis Child. 124, 577-578. Turpie, A.G., Chernesky, M.A., Larke, ILP.B., Packham, M.A. and Mustard, J.F. (1973). Effect of Newcastle Disease virus on human and rabbit platelets: aggregation and loss of constituents. Lab. Invest. 28, 575-583. Vasquez, A.D., Gonzalez Calasera, I., Cruz Gomez, Y. and Castaneda Morales, M. (1983). Platelet function in dengue hemorrhagic fever [letter]. Acta Haemat. 70, 276-277. Walsh, C.M., Nardi, M.A. and Karpatkin, S. (1984). On the mechanism of thrombocytopenic purpura in sexually active homosexual men. N. Engl. J. Med. 311,635-639. Weller, T.H. and Hanshaw, J.B. (1962). Virologic and clinical observations on cytomegalic inclusion disease. N. Engl. J. Med. 266, 1233-1244. Young, N. and Mortimer, P. (1984). Viruses and bone marrow failure. Blood 63, 729-737. Young, N., Harrison, M., Moore, J., Mortimer, P. and Humphries, ILK. (1984). Direct demonstration of the human par= vovirus in erythroid progenitor cells infected in vitro. J. Clin. Invest. 74, 2024-2032. Zeldis, J.B., Dienstag, J.L. and Gale, ILB. (1983). Aplastic anemia and Non-A, Non-B hepatitis. Am. J. Med. 74, 64-68. Zucker-Franklin, D. (1988). Megakaryocytes and platelets. In: "Atlas of Blood Cells: Function and Pathology" (eds D. Zucker-Franklin, M.F. Greaves, C.E. Grossi, and A.M. Marmont), pp. 559-602. Lea & Febiger, Philadelphia. Zucker-Franklin, D. and Cao, Y. (1989). Megakaryocytes of human immunodeficiency virus-infected individuals express viral RNA. Proc. Natl. Acad. Sci. USA 86, 5595-5599. Zucker-Franklin, D. and Petursson, S. (1984). Thrombocytopoiesis: analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes. J. Cell Biol. 99, 390-402. Zucker-Franklin, D., Termin, C.S. and Cooper M.C. (1989). Structural changes in the megakaryocytes of patients infected with the human immune deficiency virus (HIV-I). Am. J. Pathol. 134, 1295-1303. Zucker-Franklin, D., Seremetis, S. and Zheng, Z.Y. (1990). Internalization of human immunodeficiency virus type I and other retroviruses by megakaryocytes and platelets. Blood 75, 1920-1923.

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Pk telet-Tumour Cell Interact'ons

Andreina Poggi, Cosmo Rossi, Lucia Beviglia, Roberto Calabrese and Maria Benedetta Donati

1. Introduction 2. Platelet-Tumour Cell Interactions: in v/tr0 Studies 2.1 Tumour Cell-induced Platelet Aggregation 2.2 Platelet-released Products 2.3 Platelet-mediated Tumour Cell Adhesion 2.4 Adhesive Receptors and Platelet-Tumour Cell Interactions 2.4.1 Integrins 2.4.2 Selectins 2.4.3 IgG-like Molecules 3. Platelet-Tumour Cell Interactions: in vivo Studies 4. Anti-platelet Drugs and Metastases 4.1 Monoclonal Antibodies to c~iibBs 4.2 RGD Peptides 4.3 Disintegrins 5. Methods 5.1 In vitro Assays 5.1.1 Mouse Platelet Aggregation

151 151 152 152 153 154 154 155 155 156 157 158 158 158 159 159 159

1. Introduction Metastasis is a multistep process, involving vascularization of primary tumour, invasion of capillary or lymphatic vessels, transport, arrest in capillary beds, adherence to vessel wall, extravasation and growth at distant sites (Poste and Fidler, 1980). Clinical and experimental evidence suggests that blood platelets are involved at several steps of this cascade (Ordinas et al., 1990; Poggi and Donati, 1991; Honn et al., 1992b, for reviews). In particular, the adhesion of tumour cells to the vessel wall seems to be favoured by platelet aggregation, which is mediated by specific receptors, also present on leucocytes, endothelium and tumour cells. Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

5.1.2 Tumour Cell-induced Platelet Aggregation 5.1.3 Platelet-Tumour Cell Adhesion 5.1.4 Tumour Cell Adhesion to Extracellular Matrix Proteins 5.1.5 Tumour Cell Adhesion to Endothelial Cells 5.2 In vivo Assays 5.2.1 Production of Anti-platelet Serum 5.2.2 Ex vivo Platelet Counts 5.2.3 Tail Transection Bleeding Time 5.2.4 Organ Distribution of SlCr-labelled Platelets 5.2.5 Immunohistochemical Localization of Platelets 6. Conclusions 7. Acknowledgements 8. References

159 159

159 160 160 160 160 160 160 160 160 160 161

Platelet activation is followed by release of growth factors and inflammatory cytokines, which promote tumour cell migration and proliferation. Monocytes/macrophages, lymphocytes, endothelial cells and tumour cells release similar products, suggesting that tumour cell invasion and metastasis may be the consequence of multiple cell interactions and involvement of different cell functions.

2. Platelet-TumourCelllnteractions: in vitro Studies The role of platelets in tumour dissemination was first proposed by Wood (1958), Gasic and Gasic (1962), and Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

152 A. POGGI E T AL. Gasic etal. (1968)in experimental animal models. Invitro studies subsequently showed that tumour cells were able to induce platelet aggregation. Moreover, it was observed that tumour cell adhesion to the endothelium or to the extracellular matrix was enhanced by activated platelets. Expression of adhesive receptors and secretion of growth factors and cytokines from platelets, leucocytes, endothelial cells and tumour cells would be interrelated mechanisms underlying tumour dissemination and establishment of metastasis.

2.1

TUMOUR CELL-INDUCED PLATELET AGGREGATION

The ability of tumour cells to induce in vitro platelet aggregation was first described by Gasic et al. (1973). Shedding of microvesicles containing lipoproteins and sialic acid, thrombin generation caused by activation of procoagulant factors, and leakage of adenosine diphosphate (ADP) were suggested as mechanisms underlying this process (Jamieson et al., 1987; Poggi and Donati, 1991; Honn et al., 1992b for reviews). A positive relation between the ability to induce in vitro platelet aggregation and metastatic potential has been proposed by several authors (Pearlstein et al., 1980; Donati et al., 1982; Sugimoto etal., 1987). However, this relationship has been questioned (Estrada and Nicolson, 1984).

Table 8.1

Grignani and Jamieson (1988) observed that tumour cell ability to release ADP was irrelevant for tumour cell dissemination. In contrast, Lampugnani and Crawford (1987) found that highly metastatic tumours preferentially induced platelet aggregation through thrombin generation. Moreover, the presence of membrane glycoproteins immunologically related to the platelet glycoprotein (GP) IIb-IIIa on tumour cells has been associated with the tumour cell ability to induce platelet aggregation (Chopra et al., 1988). More recent data indicate that c~-thrombin activates the GPIIb-IIIa o r 0~IXb~3 receptors on tumour cells and that this process mediates tumour cell-induced platelet aggregation and increases lung colony formation in mice (Nierodzik et al., 1991; Wojtukiewicz et al., 1992).

2.2

PLATELET-RELEASED PRODUCTS

Activated platelets release the components of their granules, such as growth factors, cytokines, chemoattractants, adhesive proteins and proteolytic enzymes (Niewiarowski and Holt, 1987). Some of these factors are involved in the metastatic process (Table 8.1). Two mitogenic factors were originally discovered in platelets: platelet-derived growth factor (PDGF; Ross et al., 1974) and transforming growth factor/3 (TGFB; Assoian et al., 1983). Both factors promote cell migration, stimulate cell

Platelet-released products involved in platelet-tumour cell interactions

Platelet-released product

Reference

Growth factors PDGF TGF/~ EGF ECGF HGF

Ross et al. (1974) Assoian et al. (1983) Oka and Orth (1983) Miyazono et al. (1987) Nakamura et al. (1987)

Cytokines and chemokines PF4 /~-TG PBP IL-1 VPF Cytokine RANTES

Niewiarowski and Holt (1987) Niewiarowski and Holt (1987) Niewiarowski and Holt (1987) Hawrlylowicz et al. (1991) Dvorak et al. (1991) Kameyoshi et al. (1992)

Large adhesive proteins and receptors Fibrinogen von Willebrand's factor Fibronectin TSP 140 kDa c~ granule protein (GMP-140)

Keenan and Solum (1972) Koutts et al. (1978) Zucker et al. (1979) Frazier (1987) Stenberg et al. (1985)

Protease inhibitors PAl-1 e2-Antiplasmin

Loskutoff et a/. (1988) Plow and Collen (1981)

PLATELET-TUMOUR CELL INTERACTIONS 153 proliferation, and induce secretion of collagenase and of extracellular matrix proteins (Ross et al., 1986; Sporn and Roberts, 1990). Epidermal growth factor (EGF; Oka and Orth, 1983), endothelial cell growth factor (ECGF; Miyazono et al., 1987) and hepatocyte growth factor (HGF; Nakamura et al., 1987) were also found in platelets. The latter factors seem to have more selected targets, such as endothelial cells, and more specific functions, such as the ability to promote angiogenesis. A new impulse to studies on growth factors was given by the observation that the B chain of PDGF is encoded by the c-sis gene and that PDGF and TGF/3-1ike proteins are secreted from virally transformed cells and from murine and human tumour cells (Goustin etal., 1986; Radinsky, 1991). These factors may stimulate tumour cell proliferation through an autocrine mechanism (Heldin and Westermark, 1989; Lyons and Moses, 1990). Alternatively, they may stimulate inflammatory reactions (Herlyn and Malkowicz, 1991), selective migration (Nicolson, 1991) and angiogenesis around the tumour, with a paracrine mechanism (Weiss et al., 1989; McCormick and Zetter, 1992). The cause-effect relationship between secretion of PDGF and TGF/5-1ike factors and degree of malignancy is not clear (Keating and Williams, 1988; Silver, 1992). An inverse relation has been found between production of TGFB and metastatic ability in a murine model (Giandomenico et al., 1990). Transfection of human cells with the cDNA of active TGF~I caused altered metabolic properties and increased tumorigenicity in nude mice, suggesting that TGF~ may play a role in tumour proliferation (Arrick et al., 1992). The acquisition of growth factor independence at advanced stages of tumour dissemination has been suggested as a cause of malignant progression (Kerbel, 1992). An altered production of cytokines and an abnormal response of tumour cells to these agents have frequently been observed in neoplasia (Herlyn and Malkowicz, 1991). Several tumour cells, such as human osteosarcomas, melanomas and ovarian carcinomas, secrete cytokines, such as interleukin-1 (IL-1; Bennicelli et al., 1989), IL-6 (Watson et al., 1990), granulocyte colonystimulating factor (G-CSF; Lilly et al., 1987) and inflammatory proteins, like monocyte inflammatory protein-1 a n d - 2 (Graves et al., 1989). Platelets release IL-1 (Hawrylowicz et al., 1991) and the cytokine RANTES from their c~granules (Kameyoshi et al., 1992). IL-1 and other cytokines released from platelets modulate the activation of endothelium and take part to malignant progression (Hawrylowicz, 1993). A vascular permeability growth factor (VPF) was also discovered in platelets (Dvorak et al., 1991). Platelet factor 4 (PF4), ~-thromboglobulin (~-TG) and platelet basic protein (PBP) are specific peptides, present in platelet c~granules, whose biological functions have recently been re-evaluated. PF4 was known as the most potent heparin-neutralizing agent and as a powerful chemoattractant for fibroblasts, neutrophils and

monocytes (Deuel et al., 1982). More recently, a high degree of homology has been found between the structures of PF4 and JE peptide, a member of the family of small inducible genes, expressed by PDGF treatment in fibroblasts and smooth muscle cells (Kawahara and Deuel, 1989). PF4 is also a very active immunostimulatory agent in mice (Zucker et al., 1989). In addition, recombinant PF4, devoid of anti-heparin activity, showed angiostatic effect and inhibitory activity in experimental models of metastasis in mice (Maione et al., 1991). Recent evidence suggests that PF4, ~-TG and PBP are members of the family of cysteine-x-cysteine (c-x-c) chemokines or intercrines, peptides with reparative and chemotactic properties (Brandt and Flad, 1992). C-x-c chemokines include several low molecular weight peptides, such as interleukin-8 (IL-8; Van Damme et al., 1988) and melanoma growth stimulatory activity (GRO/MGSA; Moser et al., 1990), which are chemoattractant for monocytes and neutrophils. Finally, platelets release large adhesive proteins, such as fibrinogen (Keenan and Solum, 1972), von Willebrand's factor (Koutts et al., 1978), fibronectin (Zucker et al., 1979) and thrombospondin (Frazier, 1987), whose functions will be discussed next. Platelets also release inhibitors of the fibrinolytic system, such as plasminogen activator inhibitor-1 (PAI-1; Loskutoff et al., 1988) and c~2-antiplasmin (Plow and Collen, 1981) and other protease inhibitors, possibly involved in tumour invasion and metastasis (Niewiarowski and Holt, 1987; Herlyn and Malkowicz, 1991; Duffy, 1992, for reviews).

2.3

PLATLET-MEDIATED CELL ADHESION

TUMOUR

Tumour cells show a selective organpreference to establish metastases (McCarthy et al., 1991; Nicolson, 1991, for reviews). The adhesion of tumour cells to the vessel wall is stimulated by membrane antigens present on activated platelets (Menter et al., 1987; Abecassis et al., 1987). Bastida et al. (1989) found that tumour cell adhesion induced by platelets was increased by high shear forces and inhibited by peptides active as competitors of cell adhesion to fibronectin. Crissmann et al. (1988) showed in morphological studies that tumour cell adhesion was followed by retraction of endothelial cells, exposure of basement membrane and penetration of tumour cells into extravascular spaces. Platelets and leucocytes can mediate the adhesion of tumour cells to the endothelium and to the extracellular matrix through the activation of specific adhesive receptors and the release of cytokines and chemokines (Roth, 1992). Inflammatory cytokines would activate endothelial cells to a proinflammatory and a procoagulant state, thus facilitating tumour cell adhesion (Murphy etal., 1988, Levine and Saltzman, 1990, Lauri et al., 1991). IL- 1 seems to play a special role in this process, since it stimulates procoagulant activity (Nawroth et al., 1986), synthesis of PAI-1 (Nachman

154 A. POGGI E T AL. et al., 1986), release of prostacyclin (Rossi et al., 1985) and exposure of adhesive receptors on endothelial cells (Bevilacqua et al., 1989). This cytokine can exert a prometastatic effect in human and murine tumour models, as shown by Bani et al. (1991). Tumour necrosis factor (TNF) can induce similar effects (Malik, 1992; Hawrylowicz, 1993). Tumour cells and endothelial cells also release cyclooxygenase (CO) and lipooxygenase (LO) products, such as the linoleic acid metabolite, 13hydroxyoctadecanoic acid (HODE), that inhibits tumour cell adhesion to endothelial cells and the arachidonic acid (AA) derivative 12-hydroxyeicosapentanoic acid (HETE), that stimulates cell adhesion (Liu et al., 1991). The adhesion oftumour cells would be dependent on the ratio between 13-HODE and 12-HETE (Buchanan et al., 1990; Chen et al., 1992b, for reviews). Activated platelets release large adhesive proteins, such as fibrinogen, von Willebrand's factor, thrombospondin and fibronectin and the adhesive receptor named platelet activation-dependent granule-external membrane protein (PADGEM; Stenberg et al., 1985). Fibrinogen and von Willebrand's factor have a physiological role for platelet aggregation, adhesion and activation. Their involvement in the process of tumour dissemination is secondary to the interaction of tumour cells, platelets and endothelium with specific receptors, which will be discussed later on. Thrombospondin (TSP), a protein originally discovered in platelets, is endowed with the ability to stimulate adhesion, migration and proliferation of normal and tumour cells (Frazier, 1987). This large adhesive protein seems to play a role in tumour dissemination, as suggested by the observation that TSP enhances the adhesion of human melanoma cells (Tuszynski et al., 1987b) and increases the number of pulmonary metastases, when injected together with B16 melanoma cells in mice (Tuszynski et al., 1987a). Synthetic peptides mimicking the COOH terminal of TSP inhibit platelet aggregation, compete for tumour cell adhesion to substrates and reduce artificial metastases in experimental tumour models (Tuszynski et al., 1992a). High circulating levels of TSP have also been found in patients with malignant diseases, further supporting the role of this adhesive protein for tumour dissemination (Tuszynski et al., 1992b).

2.4

ADHESIVE

RECEPTORS AND

PLATELET-TUMOUR

CELL

INTERACTIONS The increasing amount of knowledge developed in the last few years about the mechanisms underlying cellcell and cell-matrix interactions has increased understanding of the process of tumour dissemination and the development of new antimetastatic drugs. Several classes of adhesive receptors, such as integrins, selectins, IgGlike molecules, cadherins, addressins, etc., have been

identified in platelets, leucocytes, endothelium and tumour cells. We will briefly review the characteristics of some adhesive receptors involved in tumour dissemination. 2.4.1 Integrins Integrins are a family of adhesive receptors, originally identified by the ability to recognize a specific amino acid sequence, arg-gly-asp (RGD), known to be the common cell binding site of several adhesive molecules, such as fibronectin, fibrinogen, yon Willebrand's factor and TSP (Yamada, 1991). Integrins are present in normal and transformed cells and seem to play a role in cell adhesion, migration and differentiation (Hynes, 1992). Integrins are formed by two non-covalently bound chains, named ct and fl, and are divided into several families, characterized by the presence of a specific fl chain. More recently, several new combinations of tx and fl chains have been discovered (Albelda, 1993). Integrins belonging to the fll family are mostly involved in the interactions between cells and extracellular matrix proteins, such as fibronectin, laminin and collagen. Integrins of the f12 family are present on leucocytes and are involved in cell-cell interactions. Integrins of the f13 family comprise the glycoprotein GPIIb-IIIa or integrin c~IIbfl3, which mediates platelet-fibrinogen interactions and platelet aggregation, and the ~vflS receptor or vitronectin receptor, present on platelets and endothelial cells (Phillips et al., 1988). Platelets show also c~2fll, c~sB1, and O~6~1receptors (Hemler et al., 1988). Several integrins have been found in tumour cells and seem to influence tumour cell mobility, adhesion and invasion (Ruoslahti and Giancotti, 1989; Ruoslahti, 1992). aIIbfl3 receptors have been found in human carcinomas and melanomas (Boukerche et al., 1989b), rat Walker 256 carcinosarcoma (Chopra et al., 1988), murine B16 amelanotic melanoma and Lewis lung carcinoma (Chen et al., 1992a). The presence of ~IlbflS receptor seems to be positively related with the platelet aggregating ability in a series of 1316 melanoma subpopulations with different metastatic activity (Honn et al., 1992a). More recently it has been discovered that c~-thrombin enhances the number of alIbflS receptors on B16 melanoma amelanotic cells and that intravenous injection of these cells together with thrombin increases the number of pulmonary metastases in mice (Nierodzik et al., 1991; Wojtukiewicz et al., 1992). However, the exact role of ~Ibfl3 receptors in tumour dissemination has to be clarified. Tumor cells and platelets show several other adhesive receptors, such as integrins of the fll family, which modulate tumour cell adhesion to collagen, laminin and other components of the basement membrane, which may be more relevant for tumour invasion (Ramos et al., 1990). Finally it may be mentioned that the vitronectin receptor C~vflSseems to be a marker of malignancy, since it was associated with vertically invasive or metastatic human melanoma cells (Felding-Habermann et al.,

PLATELET-TUMOUR CELL INTERACTIONS 155 1992). Moreover a positive relation between presence of vitronectin receptor and invasion was observed in several models of human melanoma (Albelda et al., 1990; Gehlsen et al., 1992). Besides Cqlbfl3, platelets possess several other GPs active as receptors for adhesive proteins and involved in pathophysiological functions (Table 8.2). Platelet adhesion to the subendothelium under conditions of high shear stress is mediated by the platelet glycoprotein GPIb-IX complex, which reacts with von Willebrand's factor, released either from platelets or from endothelial cells (Weiss et al., 1978). The injection of a polyclonal antibody to von Willebrand's factor inhibited pulmonary metastases in a murine tumour model, suggesting that platelet adhesion mediated by this receptor may be relevant for metastasis (Karpatkin et al., 1988). It has recently been observed that thrombin, trypsin and other serine proteases are able to stimulate the release of yon Willebrand's factor and PADGEM from cultured endothelial cells, suggesting a new mechanism of amplification of the pro-thrombotic and pro-metastatic activity of thrombin (Collins et al., 1993). Synthetic peptides inhibiting GPIb-IX function have been developed; however, their role for tumour cell dissemination is presently unknown (Dardik et al., 1993). Platelets possess a membrane glycoprotein, named GPIIIb or GPIV, which has been identified as the receptor for TSP (Asch et al., 1987). This GP, also classified as CD36, is multifunctional; its specific role for tumour progression has still to be elucidated (Wyler et al., 1993). Platelet GPV, active as thrombin receptor, also deserves further studies for its potential role in tumour dissemination (Kieffer and Phillips, 1990).

Table 8.2

Platelet adhesive receptors involved platelet-tumour cell interactions

2 . 4 . 2 Selectins Selectins are cell surface receptors with an N-terminal lectin domain, present on platelets, leucocytes and endothelial cells, and characterized by the ability to mediate cell to cell interactions (McEver, 1991, for a review). Platelets release, after thrombin activation, a specific 140 kD c~ granule protein, named GMP-140, PADGEM or P-selectin (Stenberg et al., 1985). Endothelial cells contain P-selectin in the Weibel-Palade bodies and release it after stimulation with thrombin and other agents. Leucocyte adhesion molecule-1 (LAM-1), expressed on neutrophils, monocytes and lymphocytes, and endothelial-leucocyte adhesion molecule-1 (ELAM1), expressed on endothelial cells, belong to the family of selectins (Table 8.3). Selectins are not expressed constitutively but are activated by stimulatory agents, such as ILl (Bevilacqua et al., 1989). Release of P-selectin occurs 5-15 min after platelet activation with thrombin and lasts for 1-2 h, allowing the recruitment of neutrophils and monocytes. P-selectin is involved in the processes of platelet-neutrophil rosetting and neutrophil rolling, which contribute to the initial phases of tumour cell adhesion to the vessel wall. Preliminary data suggest that platelets can form rosettes also around tumour cells and that this process may be relevant for tumour dissemination (Evangelista et al., unpublished results). 2 . 4 . 3 I g G - l i k e Molecules IgG-like molecules are intercellular receptors involved in homotypic and heterotypic cell interactions. Intercellular adhesion molecules-1 a n d - 2 (ICAM-1 and ICAM-2), vascular cell adhesion molecule (VCAM-1), present on activated endothelial cells (Edelman and Crossin, 1991), and platelet-endothelial cell adhesive molecule

in

Receptor

Reference

Selectins PADGEM or P-selectin

Stenberg et al. (1985)

Glycoproteins GPIb GPIIb-Illa GPIIIb or GPIV GPV

Weiss et a/. (1978) Jenning and Phillips (1982) Asch et a/. (1987) Kieffer and Phillips (1990)

Integrins 0~2/~1 ~5~1 Q~6~1 Otllb/~3 O~v/~3

Honn et a/. (1992) Homier et a/. (1988) Homier et a/. (1988) Jenning and Phillips (1982) Gehlsen et a/. (1992)

IgG-like molecules PECAM

Metzelaar et a/. (1991)

Table8.3 Leucocyte and endothelial cell receptors involved in the interactions with tumour cells Receptor

Reference

Selectins P-selectin LAM-1 ELAM-1

Stenberg et a/. (1985) McEver (1991 ) Bevilacqua et a/. (1989)

Integrins (XL/~2(LFA-1) C~i~2 (MAC-l) (xx/~2 (GP-150,95) Cr

Hynes Hynes Hynes Hynes

IgG-like molecules ICAM-1 ICAM-2 VCAM-1 PECAM

Edelman and Crossin (1991) Edelman and Crossin (1991) Edelman and Crossin (1991) Metzelaar et al. (1991)

(1992) (1992) (1992) (1992)

156 A. POGGI ET AL. (PECAM), expressed on platelets and other hae- such as integrins, IgG-like molecules and platelet GPs, are matopoietic cells (Metzelaar et al., 1991) belong to this subsequently activated (Albelda, 1993). As a confamily (Table 8.3). ICAM-1 a n d - 2 are constitutively sequence, platelet aggregation can occur, followed by expressed on leucocytes and endothelial cells, and their release of growth factors and chemokines, which further expression is increased by treatment with cytokines, such stimulate activation of endothelium, exposure of subenas IL-1 or TNF (Dustin and Springer, 1988). The lym- dothelial spaces and migration of tumour cells. At a later phocyte function-associated antigen-1 (LFA-1), a /32 stage, metalloproteases, endoglycosidases and other integrin present on lymphocytes, is a specific receptor for enzymes lead to dissolution of the basement membrane ICAM-1 and mediates lymphocyte-endothelial cell and tumour extravasation (Herlyn and Malkowicz, 1991; interactions. Circulating levels ofICAM-1, P-selectin and Matrisian, 1992). VCAM-1 have been detected in human malignancies and in human tumours implanted in nude mice (Giavazzi et al., 1992; Banks et al., 1993). Platelet-Tumour Cell Interactions: Specific adhesive receptors are selectively activated by in vivo Studies histologically different tumours. For instance, human melanomas and sarcomas recognize VCAM-1 on Alterations of the haemostatic system have been observed endothelial cells by means of the integrin receptor c~4B1 during tumour growth and dissemination not only in (Rice and Bevilacqua, 1989). In contrast, colon carci- experimental models but also in cancer patients (Poggi nomas and myeloid cells possess specific carbohydrate and Donati 1991; Rickles et al., 1992, for reviews). The structures, the sialyl-Lewis X or A antigens, that recog- intravenous injection of tumour cells into mice is accompanied by a marked thrombocytopenia, as a part of nize ELAM-1 on the endothelial cell surface (Walz et al., 1990; Dejana et al., 1992). Distinct adhesive receptors an intravascular coagulation syndrome, characterized also are sequentially activated at the various steps of tumour by low plasma fibrinogen levels and by high fibrinogen dissemination. Selectins are expressed immediately after degradation products. Thrombocytopenia, which can be platelet or endothelial cell activation by thrombin and detected a few minutes after tumour cell injection (Fig. other agents, and contribute to the activation of poly- 8.1), is presumably due to in vivo platelet aggregation morphonuclear cells and the adhesion of tumour cells at induced by tumour cells (Donati et al., 1982). These specific sites of the endothelium. Other adhesive changes are very short-lived and are rapidly reversible receptors present on platelets, tumour cells or leucocytes, after tumour cell injection. The subsequent development @

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MINUTES AFTER B16BL6 INJECTION Figure 8.1 Blood platelet counts after ifitravenous injection of B16BL6 melanoma cells in mice. B16BL6 melanoma cells ( 1 - 4 • 10 s cells/mouse) were injected intravenously in a lateral tail vein of C57BL6 mice. At different time intervals ( 1 - 6 0 min) blood was obtained from the retro-orbital venous plexus by means of capillary vessels and platelet number counted. Each point shows mean of five mice per group. SE did not exceed 10% of mean value.

PLATELET-TUMOUR CELL INTERACTIONS 157 of lung colonies is not accompanied by haematological changes (Poggi et al. , 1981). In contrast, a mild intravascular coagulation syndrome, characterized by microangiopathic haemolytic anaemia, thrombocytopenia and hyperfibrinogenaemia, occurs during spontaneous dissemination in several experimental tumour models (Poggi et al., 1977; Poggi and Donati, 1991). The relevance of these changes for tumour cell dissemination is not clear. Indeed, thrombocytopenia is not abolished by chronic treatment with anti-platelet drugs or anticoagulant agents (Poggi et al., 1981). Moreover, murine fibrosarcoma variants with different metastatic ability do not show a different degree of thrombocytopenia nor a different ability to induce platelet aggregation in vitro (Delaini et al., 1981; Donati et al., 1982). Thrombocytopenia seems to be related to the presence of the primary tumour, as indicated also by normal platelet counts in mice whose primary tumour was surgically removed (Poggi et al., 1981). Under these conditions, thrombocytopenia could be favoured by the release of toxic factors from the primary tumour, such as VPF (Bottazzi et al., 1990), TNF and other cytokines (Herlyn and Malkowicz, 1991). The release of procoagulant and fibrinolytic activity from tumour cells may contribute to tumour invasion and metastasis (Zacharski et al., 1992). Several cancer cells possess tissue factor procoagulant activity, but only highly invasive tumour cells possess factor X activating activity or cancer procoagulant activity (CPA) (Donati and Semeraro 1984; Donati et al., 1990; Gordon, 1992).

Activation of the haemostatic system is followed by release of thrombin and by fibrin deposition around the tumour. Fibrin may play a protective role for the host or provide a network for tumour cell invasion (Poggi et al., 1981; Gorelik, 1992). Accordingly, anticoagulant drugs inhibit experimental metastasis in mice and in some clinical trials (Zacharski et al., 1993). In addition, large adhesive proteins released from tumour cells or present in the milieu, such as fibronectin and TSP, may have a role in tumour progression (Tuszynski, 1987a; Murthy et al., 1993).

4. Anti-platelet Drugs and Metastases The pioneering studies of Gasic and Gasic (1962), Gasic et al. (1968) showed that induction of thrombocytopenia by anti-platelet serum or by treatment with neuraminidase inhibited metastases in experimental animal models. The effect of platelet antiserum was confirmed by Pearlstein et al. (1984) and also by our group (Fig. 8.2). The intravenous injection of B16BL6 melanoma in mice, pretreated with anti-platelet serum, causes a 70% reduction of lung colony formation after 15 days. It is likely that the anti-metastatic effect exerted by the anti-platelet serum may be the, consequence of concomitant alterations of the endothelium, due to the removal of the sialic acid coating, which seems to be important for tumour dissemination, at least in conditions of high shear stress (Kojima et al., 1992). Various anti-platelet drugs

80- ! w u) :::) 0 =E

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Figure 8.2 Effect of mouse anti-platelet serum on B16BL6 lung metastases. Mouse antiplatelet serum (50/d/mouse) was injected intravenously into a lateral tail vein of C57BL6 mice. After 24 h, B16BL6 tumour cells (1 x 10 s cells/mouse) were injected in control and antiserum-treated mice. The latter showed a reduction of platelet counts from 1.8 x 10 e to 2 x 10 s p late lets/ /d of blood. The number of lung metastases was evaluated after 15 days. Each histogram shows mean _+SE of 10 mice per group.

158 A. POGGI ET AL. have been used in the attempt to inhibit metastases (Ordinas et al., 1990; Poggi and Donati, 1991; Honn et a/., 1992b, for reviews). The anti-metastatic effect of aspirin was observed by Gasic et al. (1972), but not confirmed by other investigators (Wood and Hilgard, 1972). Ticlopidine, another potent platelet aggregation inhibitor, was unable to reduce metastasis in a model of spontaneous dissemination (Karpatkin et al., 1988). Rather controversial were the effects of prostacyclin (Honn et al., 1981, Karpatkin et al., 1984), prostacyclin analogues (Costantini et al., 1988) and thromboxane synthetase inhibitors (Honn, 1983; Vicenzi et al., 1987). Calcium channel blockers have successfully been used as antimetastatic agents (Honn et al., 1985). Dipyridamole and the pyrimido-pyrimidine derivative RA233 were active in experimental models (Lichtner et al., 1985). More recently dipyridamole has been shown to be active, alone or in combination with interferon (IFN) and 5fluorouracil, in human patients with non-small cell lung cancer and advanced colorectal cancer (Zacharski et al., 1993). The effect of anti-platelet agents on metastases was often controversial (Honn et al., 1992b). This may be the consequence of the heterogeneity of tumours and administration schedules or may reflect the fact that antiplatelet drugs have multiple effects on different cell targets. More selective agents, whose effects are directed towards specific cell functions, involved in tumour dissemination, have recently been developed.

4.1

MONOCLONAL ANTIBODIES TO O~IIb~3

The monoclonal antibody (mAb) 7E3, a specific inhibitor of the platelet O~IXb~3receptor, was rather active as an inhibitor of human platelet aggregation (Phillips et al., 1988). This antibody reduced lung colony formation after intravenous injection of tumour cells in mice (Karpatkin et al., 1988). Monoclonal antibodies to ~iib~3 also inhibited the adhesion of human tumour cells to fibronectin (Knudsen et al., 1988a) and tumour cellinduced platelet aggregation (Chopra et al., 1988). In addition, Boukerche et al. (1989a) observed a reduction of the growth of human melanoma cells in nude mice, after treatment with LYP18, another mAb to c~Hb~S.

4.2

RGD PEPTIDES

A new impulse to selective inhibition of platelet-tumour cell interaction was given by synthetic peptides with the RGD sequence (Ruoslahti and Giancotti, 1989). The intravenous injection of B16 melanoma cells, together with the peptide gly-arg-gly-asp-ser (GRGDS), significantly inhibited lung colony formation in mice (Humphreys et al., 1986). This effect was attributed to an accelerated disappearance of tumour cells from the lungs of treated mice. Modified peptides, lacking the RGD sequence, such as gly-arg-gly-glu-ser (GRGES), were not

active (Humphreys et al., 1988). Several RGD-containing peptides have successfully been used in experimental models of tumour dissemination (Ugen e t a / . , 1988; Bretti etal., 1989; Saiki etal., 1989). However, the short half-life and the reversibility of the effect of these agents on platelets limit their use as anti-metastatic agents. The effect of RGD-containing peptides on metastasis may be due either to the blockade of platelet-tumour cell interactions or to the inhibition of tumour cell adhesion to fibronectin. To clarify this problem, we studied arg-glyasp-trp (RGDW), a tetrapeptide with a potent inhibitory effect on platelet aggregation and a more limited effect on cell adhesion, in the B16BL6 lung colony assay. RGDW was about 10 times more active in inhibiting ADPinduced platelet aggregation and 12sI-fibrinogen binding to platelets, as compared to arg-gly-asp-ser (RGDS; Charon et al., 1990). RGDW was also more potent than RGDS in inhibiting B16BL6 lung colony formation (Poggi et al., 1988). Arg-gly-gly-trp (RGGW), a tetrapeptide devoid of anti-platelet activity, was also inactive on metastases, suggesting that inhibition of platelet functions was crucial, at least in our model.

4.3

DISINTEGRINS

Disintegrins are a class of snake venom-derived peptides, characterized by the presence of the RGD sequence and a cysteine-rich region. Disintegrins are several orders of magnitude more potent than small RGD peptides as inhibitors of platelet aggregation and cell adhesion (Dennis et al., 1989; Gould et al., 1990, for reviews). Trigramin, a peptide purified from the venom of Trimeresurus gramineus, showed a potent inhibitory effect on the adhesion of human melanoma cells to fibronectin and fibrinogen (Knudsen et al., 1988b). Batroxostatin also caused a potent inhibition of the adhesion of human and murine melanoma cells to fibronectin (Rucinski et al., 1990) and triflavin inhibited the adhesion of B16 melanoma cells to various substrates (Sheu et al., 1992). We found that albolabrin, a disintegfin derived from the venom of Trimeresurus albolabris, was about 2000-fold more active than RGDS at inhibiting mouse platelet aggregation (ICs0:166 nM and 50 #M, respectively) and was also more active than RGDS at protecting mice from pulmonary thromboembolism (Beviglia et al., 1993). Similarly, albolabrin was more active than RGDS at inhibiting B16BL6 melanoma lung colony formation in mice (Soszka et al., 1991). Since albolabrin prevented the accumulation of SlCr-labelled platelets in the lungs of treated mice, it is possible that its anti-metastatic effect may be due to the diversion of platelet-tumour cell emboli from the lungs of treated mice. Eristostatin, a disintegrin with a more selective inhibitory effect on platelet aggregation, was also found to have a more potent inhibitory effect on lung colony formation than .albolabrin (IC50:0.05/zM and 1.0/zM, respectively; Beviglia et al., 1992). Analysis of disintegrin structure revealed that

PLATELET-TUMOUR CELL INTERACTIONS 159 disintegrins with the RGDW sequence, such as eristostatin, have a higher affinity for OqIb~3 receptors than other RGDX sequence, thus explaining their increased activity on platelet functions (Scarborough et al., 1993). The design of even more selective inhibitors of cell functions, such as platelet aggregation or cell adhesion, may improve the effectiveness of these agents on metastases.

5. Methods Several methods have been developed to study platelet-tumour cell interactions in experimental tumour models. Murine or rat tumours of spontaneous origin, such as Lewis lung carcinoma, or chemically induced tumours, such as mFS6 fibrosarcoma, are used. Tumour cells with different metastatic ability have been developed (Giavazzi et al., 1980; Poste and Fidler, 1980). Tumour cells can be injected either intravenously or intramuscularly. Intravenous injection of tumour cells into a lateral tail vein of mice gives origin to metastatic lung nodules, visible after 15-18 days. This model is rather artificial since a huge amount of tumour cells is injected, bypassing the phases of intravasation. In contrast, when tumour cells are injected intramuscularly in mice, a primary tumour grows locally, giving origin to spontaneous metastases in distal organs such as the lungs. Human tumours can be injected either orthotopically (i.e. spleen, intestine) or etherotopically (i.e. subcutaneously or intramuscularly) in athymic nude mice, where they can grow and disseminate. The choice of tumour type and route of injection varies according to the aim of the study. A brief description of methods used for studying platelet-tumour cell interaction is given in the following sections.

5.1 I N VITRO ASSAYS 5 . 1 . 1 M o u s e P l a t e l e t Aggregation Groups of eight to ten mice are deeply anaesthesized with ether and the chest opened. Blood is withdrawn from the heart with a 1 ml syringe prefilled with 0.126 M sodium citrate (1:10 v:v). Blood is centrifuged at 160g for 20 min, to obtain platelet-rich plasma (PRP). About 0.2 ml of PRP (0.8-1.2x 1 0 6 platelets]#l) are obtained from a single mouse. Platelet aggregation is performed by means of an Elvi 840 aggregometer (Elvi-Logos, Milano, Italy), with 400 gl PRP (approximately 4 x 10 s platelets/#l), 5-10 gl ADP (5-15 gM final concentration) or collagen (5-20 #g/ml final concentration) and 5-10 gl phosphate-buffered saline (PBS), albolabrin (10-300 nM final concentration), eristostatin (4-20 nM, final concentration) or RGDS (20-120/~M final concentration), dissolved in PBS. Results are expressed as ICs0, i.e. the molar concentration of peptide causing 50% inhibition of platelet aggregation (Beviglia et al., 1993).

5.1.2

Tumour CeU-induced Platelet Aggregation

Subconfluent cultures of tumour cells are detached by 1-3 min incubation with 0.25% trypsin/0.02% EDTA, washed twice with Hank's balanced salt solution (HBSS) without Ca2+/Mg 2+ at 4~ and suspended at a final concentration of 2 x 1 0 6 cells/ml in the same buffer. Tumour cell suspension is kept on ice and used no later than 1 h after collection. Blood is collected via cardiac puncture from ether anaesthesized mice, using 10 #1 of heparin (5-10 U in saline) per 1 ml blood. Blood is centrifuged to obtain PRP. Aliquots of PRP are preincubated with buffer or peptides at 37~ in the aggregometer for 3 rain and then tumour cell suspension (1 x 106 cells/100/zl) is added and aggregation recorded under stirring (Beviglia et al., 1993). 5 . 1 . 3 P l a t e l e t - T u m o u r Cell Adhesion Mouse platelets ( 5 x 1 0 s platelets/ml) are prepared as described above and washed with a solution of HEPESTyrode buffer plus 2 gM PGE1. Platelets are mixed with a suspension of 1 0 6 t u m o u r cells in HEPES-Tyrode buffer and incubated on a rocking platform for 15 min at room temperature. In some experiments, platelets can be previously activated with 0.5 #/ml of thrombin. The mixture is washed twice with HEPES-tyrode buffer and centrifuged at 900 rpm for 15 min. The final suspension is examined by phase-contrast microscope to count the number of platelet-tumour cell clumps. 5.1.4

T u m o u r Cell A d h e s i o n t o E x t r a c e U u l a r Matrix Proteins Tumour cells are detached b y brief incubation with 0.25% trypsin/0.02% EDTA. Trypsin activity is neutralized by addition of soybean trypsin inhibitor (1 mg/ml) or 10% foetal calf serum in Dulbecco's modified Eagle medium (DMEM). Tumour cells are centrifuged at 300 g and suspended in serum-free DMEM at a density of 5 x 10S/ml. Aliquots of 100 gl/well (5 x 104 cells/well) are plated in 96-well plates, previously coated with human fibronectin (5-7/zg/ml), laminin (5-10 #g/ml), type I or type IV collagen (70 gg/ml), RGD peptides (50-70 gg/ml) or albolabrin (1-25 gg/ml). Coating is obtained by incubation of cell plates with adhesive proteins for 2 h at 37~ or overnight at 4~ followed by incubation with 1% bovine serum albumin (BSA) for 30 min at 37~ and repeated washings with phosphatebuffered saline (PBS) without Ca 2+ /Mg 2+ . Tumour cells are mixed with the disintegrins or RGD peptides, at appropriate concentrations, added to the plates, and incubated for 1-2 h at 37~ Non-adherent cells are removed by washing three times with PBS with Ca2+/Mg 2+ and adherent cells are detected by the addition of 0.5% cristal violet for 5 min at room temperature. The developing blue colour is recorded at the optical density of 550 nm with a Titertek ELISA reader. The

160 A. P O G G I E T A L . intensity of the colour reaction is proportional to the number of adherent cells. Alternatively, adherent cells are fixed with 10% formaldehide for 30 min, stained with methylene blue (dissolved in 0.01 M sodium borate) for 30 min and washed four times with 0.01 M sodium borate. The staining is extracted with a 1:1 solution of 0.1 M HCI and ethanol in a volume of 200 #l/well. After at least 1 h, the absorbance is read with an ELISA reader plate at optical density of 630 nm (Soszka et al., 1991). T u m o u r Cell A d h e s i o n t o Endothelial Cells Subconfluent cultures of tumour cells are incubated with 0.3/zCi/ml 5-[12sI]iodo-2'-deoxyuridine (2 mCi/mmol, Amersham International). After 24 h, cells are rinsed thoroughly, harvested with 0.02% EDTA/0.25% trypsin, washed with HBSS without Ca2+/Mg 2§ and incubated with the endothelial cells. The amount of precipitated radioactivity is measured with a gammacounter (Laud et al., 1991). 5.1.5

5.2

IN

VIVO ASSAYS

5.2.1 Production of Anti-platelet S e r u m PRP, obtained from 15-20 mice (about 109 platelets), is washed as described previously and suspended in 2 ml of Tyrode solution without Ca 2 + /Mg z + . PRP is then injected in a marginal ear vein of a New Zealand rabbit, weighing 2-2.5 kg. The injection is repeated after 2 weeks. After a further 2 weeks, blood is taken from the ear of the ether-anaesthesized rabbit; serum is obtained by centrifugation at 1200 g for 10 min. The anti-platelet activity of the immune serum is challenged by i.v. injection in a lateral tail vein in mice, followed by platelet counts. The injection of 50 ~l of immune serum in mice causes a platelet drop from 1.2-1.4 x 106 platelets/~l to about 2 x 10 s platelets/#l after a few hours. The effect lasts for 24 h. 5 . 2 . 2 E x vivo P l a t e l e t C o u n t s Groups of mice are injected i.v. in a lateral tail vein with tumour cells, suspended in PBS (1-4 x 10 s cells/mouse), with or without albolabrin or RGD peptides, at a final volume of 0.2 ml/mouse. At different time intervals (1-60 min), blood is taken from the retro-orbital venous plexus by means of a capillary vessel or by puncture of a lateral tail vein. Usually 5-20 #1 of blood are collected and diluted with the Unopette System (Becton Dickinson, Novate Milanese, Milan, Italy). Platelets are counted with a haemocytometer by phase contrast microscopy.

5.2.3 Tail T r a n s e c t i o n B l e e d i n g T i m e Groups of mice are injected with 0.1 ml of PBS or albolabrin (10/~g/mouse) or RGDS (1.5 mg/mouse) in PBS in a lateral tail vein. After 1 min, a cut of 2 mm of

the tail keeping time is stopped

tip is performed and blood leakage observed, the tail in physiological saline at 37vC. Bleeding recorded as the time when blood leakage has for more than 30 s (Dejana et al., 1982).

5.2.4

O r g a n D i s t r i b u t i o n o f 51Cr-labelled Platelets Approximately I x 109 mouse platelets are incubated with Slchromium as sodium chromate (100/~Ci, Amity PG, Milan) in i ml Tyrode's buffer, pH 7.4, without calcium and magnesium, with 2 nM prostacyclin and 0.35% BSA for 90 min at 37~ SlCr-labelled platelets are washed twice and injected i.v. in mice, at a concentration of 30-80 x 106 at 0.1 ml/mouse; after 5 min tumour cells are injected as previously described. The animals are sacrificed by ether anaesthesia after a further 3 min; blood and organs are obtained, washed in 70% ethanol for 72 h and radioactivity measured (Soszka et al., 1991). 5.2.5

Immunohistochemical Localization of Platelets Mice are ether-euthanized. The lungs are excised, washed and fixed with Bouin's fluid for 6 h and then kept in 70% ethanol until paraffin embedding. Sections (5/~m thick) are stained with haematoxylin-eosin. Immunohistochemical staining is performed with the avidinbiotin complex (ABC) procedure, using a commercial immunoperoxidase kit (Vectastain elite, Vector). A rabbit anti-factor VIII related antigen (von Willebrand's factor) antiserum is used as the primary antibody to stain platelets and vascular endothelium (Beviglia et al., 1993).

6. Conclusions Platelet interactions with tumour cells have been described both in vitro and in vivo. Platelet activation, with subthreshold doses of thrombin or other agents released by cancer cells, can stimulate tumour cell adhesion to the vessel wall, with a mechanism involving activation of selectins, integrins, IgG-like molecules and other adhesive receptors. Similar receptors present on neutrophils, monocytes and endothelial cells take part to this process, with a concerted action. Growth factors, cytokines, chemokines and other products released after platelet aggregation and activation may further stimulate the adhesion of tumour cells to activated endothelium. Although more experimental work is needed, new approaches aimed at inhibiting receptor-mediated platelet adhesion and activation are promising for the prevention and cure of metastasis.

7. Acknowledgements The authors' studies mentioned in this paper have been performed with contributions from the Italian Associ-

PLATELET-TUMOUR CELL INTERACTIONS ation for Research on Cancer (A.P. 1992) and the Italian National Research Council, Convenzione CNRConsorzio Mario Negri Sud. L.B. is the recipient of a fellowship from the Regional Council of Abruzzo and the Commission of European Communities (CEE/Regione Abruzzo). We thank Prof. Stefan Niewiarowski, Temple University, Philadelphia, PA, USA, for invaluable help in discussion and the gift of disintegrins. Miss Teresa Di Camp'li and the staff of the Gustavus and Louise Pfeiffer Library helped prepare this manuscript.

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Introduction: Idiopathic or Autoimmune Thrombocytopenia? Prevalance Clinical Syndrome, Classification and Evolution 3.1 The Acute Variety 3.2 The Intermittent Variety 3.3 The Chronic Form Laboratory Testing Megakaryocytopoiesis in AITP Isotopic Studies Immune Abnormalities in AITP 7.1 Immune Functions 7.2 Autoantibodies and Autoantigens in AITP 7.2.1 Detection of Immunoglobulins on Platelets in AITP Patients and Significance of the Tests 7.2.2 Techniques for Immunochemical Characterization of Autoantigens 7.2.2.1 Immunoblotting 7.2.2.2 Radioimmunoprecipitation 7.2.2.3 Antigenic Capture Assays 7.2.2.4 Molecular Biology 7.2.3 Identification of Specific Autoantigens on the Platelet Glycoproteins 7.2.3.1 Glycoproteins IIb and IIIa and the IIb-IIIa Glycoprotein Complex

Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

7.2.3.2 Glycoproteins Ib, IX, and the Ib-IX-V Glycoprotein Complex 7.2.3.3 Other Proteins 7.2.3.4 Autoantibodies and Platelet Functions 7.2.3.5 Autoantigens and Clinical Significance

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Treatment 8.1 Acute 8.1.1 8.1.2 8.1.3

AITP Steroid Therapy High Dose i.v. IgG Intravenous Rhesus Antibodies (Anti-D) 8.2 Chronic AITP 8.2.1 Corticosteroids 8.2.2 High Dose i.v. IgG 8.2.3 Splenectomy 8.3 Refractory Chronic AITP 8.3.1 Immunosuppressive Drugs 8.3.2 Vinca Alkaloid Therapy 8.3.3 Colchicine Therapy 8.3.4 Danazol Therapy 8.3.5 Ascorbate Treatment 8.3.6 Anti-D Treatment 8.3.7 IFNc~ Therapy 8.3.8 Other Therapies 8.4 Emergency Treatment 8.4.1 Platelet Transfusions 8.4.2 Intravenous Methyl Prednisolone Therapy Autoimmune Thrombocytopenic Purpura and Pregnancy 9.1 Mothers 9.2 The Infants

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C. KAPLANANDG. TCHERNIA 9.3 Hidden Maternal Autoimmunity 9.4 Asymptomatic Maternal Thrombocytopenia 10. Secondary Immune Thrombocytopenic Purpura 10.1 Virus-induced Autoimmune Thrombocytopenia 10.1.1 RNA Virus Infections 10.1.2 RNA Viruses with Reverse Transcriptase Activity 10.1.3 DNA Virus Infections 10.2 Systemic Lupus Erythematosus

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1. Introduction: Idiopathic or Autoimmune Thrombocytopenic Purpura? Idiopathic thrombocytopenic purpura (ITP), the most common haematologic disorder, is due to the destruction of circulating platelets coated by antibodies, and affects children as well as adults (Harrington et al., 1951, 1953). It was first described by Werlhof in 1775 under the name of Aforbus maculosus hemorrhagicus, when it occurred suddenly in an adult female with severe visceral haemorrhage co-existing with cutaneous haemorrhage (Werlhof, 1775). Advances in the understanding of this autoimmune disease have been particularly evident during recent years with the development of fundamental immunology, as well as advances in molecular biology for identification and characterization of autoantibodies and autoantigens. Despite this progress, much remains to be learned about the breakdown of immune regulation, and the best ways of management of this common syndrome, which carries the risk of life-threatening haemorrhages. Until the 1950s, this syndrome was described as idiopathic. The autoimmune basis of ITP emerged when Harrington and coworkers showed that the platelet destruction was provoked by a circulating factor. Fresh whole blood or plasma collected from thrombocytopenic patients and infused into non-thrombocytopenic recipients caused a prompt and sometimes dramatic decrease of circulating platelets. This effect, maximal within the first hours, disappeared after 4 - 7 days and was associated with the gammaglobulin fraction of plasma (Harrington et al., 1951). From this description, assays were developed to characterize this anti-platelet factor. It was shown that 66% of serum obtained from patients gave a positive reaction in the in vitro platelet agglutination tests. In the presence of complement, lysis of platelets could also be observed (Tullis, 1956). The humoral factor involved in ITP as well as in thrombocytopenic infants delivered by mothers with ITP was shown to be potentially an anti-platelet antibody

10.3 Evans Syndrome 10.4 AITP and Malignancies 10.4.1 Lymphoproliferative Disorders 10.4.2 Solid Malignant Tumours 10.4.3 Bone Marrow Transplantation and Thrombocytopenias 10.5 Thrombocytopenia and Parasitic Infections 11. Acknowledgements 12. References

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(Shulman et al., 1964). Similarities between ITP and thrombocytopenia caused by drug antibodies or isoantibodies suggested its immune nature (Shulman et al., 1964a, b, 1965a). This factor was found in the 7S gammaglobulin fractions of the plasma, had the capacity to destroy autologous as well as homologous platelets, and appeared to be species specific. It could be absorbed on platelets and its effect was reproducible and dose dependent. The role of the spleen was evidenced by the observation that splenectomized individuals were resistant to the thrombocytopenic effect of ITP plasma, and using SlCr-labelled platelets (Shulman et al. 1965b). The sensitized platelets were sequestrated in the spleen, but with higher sensitization a liver sequestration was observed. In this respect, the term autoimmune thrombocytopenic purpura (AITP) better reflects the pathogenesis of this disorder.

2. Prevalence The incidence of AITP is unknown: the disease is often transient and can remit spontaneously. Moreover moderate thrombocytopenia can exist for a long time without any clinical expression, especially if patients do not experience surgery or severe trauma. A number of cases are found through fortuitous or systematic sampling. During the last decades the emergence of systematic automated platelet counting in most countries where blood tests are performed has considerably increased platelet count screening and thus diagnosis for mild AITP. It has been estimated that AITP develops in 1/10 000 people per year (Bussel, 1990), with a higher incidence in females.

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Clinical Syndrome, Classification and Evolution

AITP typically presents as the onset of haemorrhagic symptoms in otherwise healthy persons. Numerous

AUTOIMMUNE THROMBOCYTOPENIAS petechiae can be present, associated with ecchymosis. Epistaxis is common. Haemorrhagic blisters of the lips or the buccal mucosa as well as retinal haemorrhage are indicative of severe thrombocytopenia and must lead to therapeutic decisions. In children posterior nasal bleeding, especially while sleeping, can be swallowed and lead to severe anaemia. Bleeding of the gastrointestinal or of the urinary tract are infrequent. The most serious problem is intracranial haemorrhage which occurs in 0.5-1% of children after diagnosis (Woerner et al. 1981; Robb and Tiedeman, 1990). Its incidence in adults is not known. When present it is associated with a high mortality (Bussel, 1990). AITP is usually classified into three categories: acute, chronic and intermittent.

3.1

THE ACUTE VARIETY

This is most often seen in children, with a peak incidence between 2 and 6 years of age. It is generally a benign, selflimited condition with, in 80-90% of cases, a spontaneous recovery within a matter of days or weeks. It often follows a seasonal viral illness, most often upper respiratory tract infection occurring within the 4 weeks before the child presents with thrombocytopenia. Vaccination, infectious mononucleosis, varicella, or various non-diagnosed erythemas can also be incriminated. The male:female ratio is 1. Approximately 10-20% of children with acute AITP will develop the chronic variety. There are no well-defined criteria for predicting such an evolution. It has however been observed that cases with a history of symptoms lasting for more than 2 weeks at presentation and a decreased platelet count 4 weeks after diagnosis could more frequently have a chronic evolution (Robb and Tiedeman, 1990).

3.2 THE INTERMITTENT VARIETY This may occur in children as well as adults and may be spaced by intervals of several years free from disease during which both the platelet count and survival are normal.

3.3

THE CHRONIC FORM

This occurs most often in adults, and is persistent, lasting years or a lifetime. It has a female : male ratio of 2.1-4.1 (Karpatkin, 1985). The evolution of chronic thrombocytopenia is unpredictable. Even after a favourable outcome it can relapse spontaneously or after any antigenic stimulation such as viral infection or vaccination (Kelton, 1981). Moreover, similar antigenic triggering can result in a transient increase in platelet counts as well as relapse or aggravation. Transiently normal platelet counts have been observed in chronic thrombocytopenia after viral

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hepatitis or other infectious diseases (Kurata et al., 1982; Schmidt et al., 1983; Tertian et al., 1986). It is now widely accepted that even after medical treatment or splenectomy some patients who have recovered normal platelet count are in a chronic state of compensated thrombocytolysis with a decreased yet persistent rate of destruction and an increased production. Apparent relapses are only a disruption in such an equilibrium.

4. Laboratory Testing The characteristic features are isolated thrombocytopenia and normal to increased numbers of megakaryocytes in the bone marrow. The platelet population is heterogeneous, with increased platelet volume corresponding to large platelets which are probably "stress" platelets (Karpatkin, 1985). Anaemia is present only when bleeding is important or when autoimmune haemolysis is associated. Platelet counting by blood cell automatic counters is mostly reliable. However one must remember that most of these counters are accurate only within a given range around normal values. Each laboratory must be aware of its limits in accuracy and perform the appropriate controls if necessary. In some situations platelet counting can be erroneous; for example, circulating particles of volume within the platelet volume range can spuriously increase the platelet count and mask a thrombocytopenia; this can occur with extreme microcytosis or red cell fragmentation. On the contrary, in vitro platelet consumption in inefficient anticoagulation of the sample or aggregation by EDTA (Berkman et al., 1991) can lead to spurious thrombocytopenia. In any case an initial diagnosis of thrombocytopenia must be verified by phase control microscopy after microsampling, and by examination of the platelet size, morphology and density on blood smears. Decreased platelct count should be accompanied by a cautious evaluation of the coagulation status. Disseminated intravascular coagulation has to be ruled out: in that case, thrombocytopenia is associated with a decrease in coagulation factors. The association with any coagulation abnormality can heighten the risk for bleeding and call for adapted management. An autoimmune biological assessment should be performed before treatments such as corticosteroids or gammaglobulins. Storing frozen serum samples can be of a great help for further immunological or viral investigations which did not appear to be mandatory at presentation. The need for bone marrow examination has been the subject of controversy, especially in children. Most groups do not feel it is mandatory in acute AITP when thrombocytopenia is isolated without abnormalities of red cell indices or white cell differential, and when a rise in platelet count is observed either spontaneously or after i.v. IgG therapy. However if clinical presentation is atypical or if corticosteroids are

170 C. KAPLAN AND G. TCHERNIA prescribed, bone marrow examination before treatment is usual, although debatable (Corrigan, 1988; Halperin and Doyle, 1988). In adults with chronic AITP, bone marrow aspiration and/or histology has to be performed to search for associated disorders such as lymphoid diseases. There is no close correlation between clinical presentation and platelet count. Most patients will have minimal bleeding symptoms even with platelet counts below 50 000/mmS. Other can exhibit bruising and bleeding in spite of moderate thrombocytopenia because of associated platelet functional abnormalities. Most authors consider that in chronic AITP platelet counts above 30 000/mm3 without anaemia or overt cutaneous or mucosal haemorrhage do not require any treatment in the absence of trauma or need for surgery. Bleeding time is often less useful than a good clinical examination.

5. Megakaryocytopoiesisin AITP Platelet production has been reported to be elevated in most patients with AITP, consistent with the abundant number of megakaryocytes visualized on bone marrow smears. Moreover immunomorphometric studies on trephine biopsies have shown this increase to concern predominantly immature forms, leading to the hypothesis of a reduced time for megakaryocyte production and platelet release (Thiele et al., 1991). Increase in platelet production as discerned by SlCr platelet turnover was estimated to be 2-5 times normal (Braneh6g et al., 1974, 1975). However it was subsequently shown that in patients with moderate thrombocytopenia platelet production rate could be in the normal range (Stoll et al., 1985). Moreover, studies using 111Inlabelled platelets have identified a subset of patients with severe disease as reflected by low platelet count and diffuse platelet sequestration who exhibit a decrease in platelet production (Heyns et al., 1986). In vitro studies have produced conflicting data which probably reflect the heterogeneity of the disease and/or the differences in the techniques used in different laboratories: platelets and megakaryocytes share identical antigens and megakaryocytopoiesis may be impaired in AITP by autoantibodies directed against these antigens. A complementdependent IgG mediating in vitro inhibition of megakaryocytopoiesis has been demonstrated in the serum of a patient with chronic refractory AITP. In that case increase in bone marrow megakaryocytes found at the onset of the disease, subsequently evolved towards an absence of megakaryocytes on bone marrow biopsy. Megakaryocytes reappeared after plasmapheresis and vinblastin treatment (Hoffman et al., 1985). In acute AITP several groups have reported an increase in megakaryocyte progenitors (CFU-Mk) and in the number of cells per colony (de Alarcon et al., 1987; Abgrall et al., 1992). In contrast, chronic AITP can

exhibit a decrease in CFU-Mk (Abgrall et al., 1992). However in chronic adult AITP an increase of in vitro megakaryocyte formation has also been demonstrated. Moreover the serum of the patients could enhance megakaryocyte formation of control marrow cells, although no decrease in inhibitors such as ~thromboglobulin (~-TG) or increase in megakaryocytes growth factor such as interleukin-6 (IL-6) could be demonstrated (Bellucci et al., 1991). Other groups have found that although the CFU-Mk pool was quantitatively unchanged the number of CFU-Mk in S phase was increased, correlating with the increase in mature megakaryocytes (Dan et al., 1990). Finally one must bear in mind that in AITP the platelet count is the result of a balance between destruction of circulating platelets and their bone marrow production. Destruction of circulating platelets depends mainly on the nature and the amount of the autoantibody, but is also influenced by the reticulo-endothelial cell function in the ability of tissue macrophages to clear antibodycoated platelet antigens (Kelton et al., 1984). Production of platelets depends on stimulation of the megakaryocytopoiesis related to the feedback mechanism initiated by thrombocytopenia, on the number of megakaryocyte progenitors in cycle, and on the inhibition of their differentiation by immunologically mediated mechanisms. In that regard, acting on platelet production by the bias of growth factor could, in the following decade, become an adjuvant therapy (Gordon and Hoffman, 1992).

6. Isotopic Studies Isotopic evaluation of mean platelet survival and turnover is a valuable approach for chronic AITP whenever an accurate evaluation is needed in order to confirm the diagnosis or to weigh therapeutic decisions. Most groups favour the use of 111In as a radionucleide for platelet labelling: its affinity for platelets versus that for plasma proteins is satisfactory; the irradiation delivered to the patients, due to a shorter physical period, is of a lesser magnitude than that delivered by S1Cr; and scintillation cameras can be used. The test is always performed with autologous platelets. Isolation of circulating platelets is the first step of the procedure as 111In has no specificity for platelets. Thus a sufficient number of circulating platelets is needed, especially in children where the amount of blood sampling is limited. A thrombocytopenia under 2 0 0 0 0 / m m s makes the labelling hazardous. The dose used for such an exploration averages 20-100 #Ci according to the age of the patient. The whole body irradiation (70 mrads for 100 #Ci) is below the doses delivered for most X-ray explorations. The parameters studied are the following. (1) The radioactivity recovery 30 and 180 min after

AUTOIMMUNE THROMBOCYTOPENIAS injection. Normal recovery ranges between 40% and 80%; this can be decreased in case of hypersplenism or severe thrombocytopenia. (2) The mean survival obtained by an extrapolation of the radioactivity disappearance curve as assessed on daily sampling. The magnitude of the decrease in survival and the intensity of thrombocytopenia can lead to an appreciation of thrombopoiesis in bone marrow.

(3) External counting establishes the ratio of splenic radioactivity versus that of other areas such as heart or liver and thus indicates the site ofplatelet destruction. (4) Platelet production rate is calculated according to platelet count, % recovery, platelet mean cell life and estimated blood volume, and expressed as platelets/ kg/day (Ozsoylu et al., 1976; Stoll et al., 1985). Isotopic study is the only mean to establish a diagnosis of compensated thrombocytolysis state where an increased destruction of circulating platelets is counterbalanced by an increased megakaryocytopoiesis leading to a persistently normal platelet count (Karpatkin et al., 1971; Ozsoylu et al., 1976). In some cases it can correct an erroneous diagnosis when the mean survival time is normal, leading to the search for a pure production defect such as familial thrombocytopenia (Buchanan et al., 1977). Further, it can give an indication of the adverse effect of the autoantibodies on megakaryocytes. In that case a defect in platelet production rate can be found as well as a reduced survival time. Finally, it can be predictive for the result of splenectomy: if platelet destruction is only splenic, splenectomy outcome is considered to be favourable in 90% of cases, while a more diffuse destruction will lead to only 50% of normal platelet count recovery after surgery (Gugliotta et al., 1981; Najean et al., 1991).

7. Immune Abnormalities in AITP Normal individuals possess lymphocytes secreting antibodies reacting with a wide variety of self antigens with no identifiable deleterious consequence. At the opposite extreme, autoimmune diseases, characterized by a breakdown of physiological self-tolerance, lead to pathological manifestations. This condition could result from a variety of mechanisms such as genetic susceptibility, dysregulation in the T cell compartment, polyclonal activation of B and T cells for example after viral infection, dysregulation in the idiotype-anti-idiotype network (Shoenfeld, 1990), or molecular mimicry [cross-reaction between a specific determinant of an infecting agent and a similar host sequence (Oldstone, 1987)].

suspected since impaired blastogenesis was observed with mitogens. Several authors have reported abnormalities in the distribution of peripheral blood lymphocytes. These include decrease in CD4 (T helper-inducer) :CD8 (suppressor cytotoxic) ratios, with a more severe perturbation in women (Mylvaganam et al., 1985), an increase in the number of CD4 4, CD8 § (Mizutani et al., 1985), in activated T cells (Mizutani et al., 1987) and in CD5 § B cells in peripheral blood and spleen lymphocytes (Mizutani et al., 1991). No correlation was found between the platelet count and the number of CD5 § circulating B cells. In vitro experiments showed the capacity of isolated splenic CD5 § B cells from two of five patients to produce high levels of IgM platelet bindable antibodies after stimulation with Staphylococcus aureus Cowan 1; this was not the case for C D 5 - B cells isolated from the same spleen or for CD5 § B cells from other non-immune disorders. It was speculated that this could partly correspond to the in vivo situation where platelet-associated IgM are detected on the platelet surface. These data are in favour of an involvement of CD5 § B cells in the pathophysiology of AITP. When the T cell function was assessed in vitro, abnormal autologous mixed lymphocyte reaction was documented (Zinberg et al., 1982) A defect in suppressor T lymphocyte function associated with a complement fixing antibody has been identified in some chronic AITP patients seropositive for the Epstein-Barr virus (Hymes and Karpatkin, 1990). This dysregulation leads to abnormal production of anti-platelet autoantibodies. The abnormal T helper lymphocyte reactivity observed after in vitro stimulation of AITP lymphocytes by normal platelets devoid of HLA antigens by acid treatment, i.e. the capacity of CD4 § helper cells to secrete IL-2 (Semple and Freedman, 1991), was associated with important changes in the peripheral lymphocyte distribution: marked reduction of suppressor-inducer T cells and increase in CD4 + leu 8- T helper cells (of B cell differentiation), and activated T and B cells, compared to controls. The abnormal response of CD4 + T cells could be explained by escape from the thymic selection, antigen mimicry stimulating a normal T cell repertoire, or abnormalities in the regulation network, consistent with a decreased T suppressor cell function in such patients (Hymes and Karpatkin, 1990). The enhanced antiplatelet antibody production could also result from an abnormal proliferation of B cells due to the defective natural killer (NK) activity in these patients. In conclusion, an abnormal regulation in the immune network leads to the production ofanti-platelet autoantibodies which could recognize their putative epitopes on the major platelet membrane glycoproteins (GPs). 7.2

7.1

IMMUNE FUNCTIONS

In AITP abnormal cell-mediated immunity has been

171

AUTOANTIBODIES AUTOANTIGENS

AND IN AITP

Platelet sensitization by antibodies leading to platelet

172 C. KAPLAN AND G. TCHERNIA destruction by the reticulo-endothelial system is thought to be due to specific platelet antibodies. The anti-HLA antibodies, although HLA class I antigens are present on the platelet surface, do not play a role in AITP. Many difficulties have been encountered in attempting to detect anti-platelet antibodies, and different assays for the detection and evaluation of immunoglobulins on platelets were developed. The detection of antibodies in the patient's serum is of great value for the identification of the autoantigen. The first indication of a potential specific target in AITP was the demonstration, employing the platelet suspension immunofluorescence test, that antibodies from AITP patients reacted with normal platelets but failed to react with platelets from type I Glanzmann thrombasthenia (GT) patients (van Leeuwen etal., 1982), as these platelets are devoid of the major GP complex IIb-IIIa. Since then, attempts have been made to identify and characterize specific antigens. As the autoantigens seem to be public antigens, it is possible to use random as well as autologous platelets for the different tests, particularly when patients are severely thrombocytopenic. Different methods, such as immunoprecipitation (Devine and Rosse, 1984), immunoblotting (Beardsley et al., 1984a), and immunocapture by monoclonal antibodies (mAbs) have been applied (Woods et al., 1984a, b; Kiefel et al., 1987) It is essential to emphasize that every method has its own advantages and disadvantages, and that the study of autoimmunity probably needs a combination of different approaches in order to understand the interactions of the autoantibodies and their putative epitopes. The most recent technologies, synthetic peptides (Fujisawa et al., 1991) and transfection experiments (Fujisawa et al., 1992), have led to the dissection of epitopes on GPs. The aim of this characterization is not only the molecular approach to autoimmunity, but also the hope that a correlation could be established between the autoantigens and the clinical condition, improving management of patients. 7.2.1

Detection o f Immunoglobulins on Platelets in AITP Patients and Significance o f t h e Tests

Since it was suggested that a circulating anti-platelet factor present in the purified IgG fraction of the plasma was responsible for accelerated platelet destruction, by analogy with haemolytic anaemia, numerous techniques equivalent to the "Coombs test" (Coombs et al., 1945) have been developed for the measurement of plateletassociated IgG (Dixon et al. 1975; Cines and Schreiber, 1979; LoBuglio et al., 1983; Kelton et al., 1989). However, many issues remain unresolved concerning the significance of these tests and their results (less than 1% of total platelet IgG is detected on the cell surface). In general these studies have shown that plateletassociated IgG are increased in almost all AITP patients

as compared with normal individuals (McMillan, 1981; Karpatkin, 1985). The severity of the thrombocytopenia has been found to be correlated or not with the increase in platelet-associated IgG (Cines and Schreiber, 1979; LoBuglio et al., 1983; Kelton et al., 1989). Finally, elevated platelet-associated IgG have been reported in patients with non-immune thrombocytopenias (MuellerEckhardt et al., 1980; Kelton et al., 1982b; MuellerEckhardt, 1988; Kelton et al., 1989; George, 1989). It has been speculated that the low specificity of these tests was due to different factors, such as anticoagulants (von dem Borne et al., 1986) or platelet fragments coprecipitating with washed platelet samples, but conflicting results have been obtained (Kelton et al., 1985; Holme et al., 1988). Other causes of elevated platelet-associated IgG in nonimmune thrombocytopenias could be related to the binding of immune complexes to the platelet Fc receptor, or alterations of the platelet membrane exposing cryptic antigens reacting with subsequently occurring antibodies. Recent data showing the presence of specific antiplatelet antibodies in AITP patients, in their sera, and/or by elution from their platelets, could be an argument in favour of platelet surface IgG representing anti-platelet antibody. Thus the measurement of platelet-associated IgG could be used as a screening test for immune thrombocytopenias (Sinha and Kelton, 1990). Other studies have focused on the potential value of the total content of IgG in the platelets of thrombocytopenic patients (George, 1990). An increase of total platelet IgG concentration in AITP patients has been reported to correlate with the clinical evolution and inversely with the platelet counts. As it has been demonstrated that the majority of the platelet IgG was located in the c~ granules, the reason for such an elevation in AITP patients, as compared to controls, was questionable. It was suggested that in AITP patients, as in other causes of peripheral platelet destruction, the circulating platelets are large "stress" platelets with a higher content of c~ granules. This was in fact observed in AITP and in a few cases of thrombotic thrombocytopenic purpura. Moreover, in non-immune thrombocytopenias due to decreased platelet production, no elevation of the mean total amount of platelet IgG was reported. Therefore, an increased total platelet IgG content may be comparable to the presence of reticulocytes in regenerative anaemia (George, 1990). Further studies are needed to confirm these hypotheses. In conclusion, despite all these reservations, we think that the evaluation of total platelet IgG reflecting the presence of young platelets, and moreover the evaluation of platelet-associated IgG representing probably in part anti-platelet antibody are clinically useful assays in patients with suspected AITP. Of course, these results must be interpreted in conjunction with clinical data and other biological results such as identification of the implicated autoantigens.

AUTOIMMUNE THROMBOCYTOPENIAS

syndrome (BSS) could be very informative for the characterization of autoantigens. In BSS the platelets are devoid of the GPIb-IX-V glycoproteins. Immunoblotting tests with either the whole platelets or the membranes have helped to establish the presence of anti-cytoplasmic or anti-membrane autoantibodies which can be of importance for understanding the pathophysiology. With this technique a variety of autoantibodies against GPIIb, IIIa or cytoplasmic components have been evaluated, but not all autoantibodies can be visualized. Immunoblotting which involves the use of denatured antigens treated with SDS and boiled, is likely to destroy conformationdependent epitopes and will only allow detection of linear epitopes (Fig. 9.1). In some instances, many bands are revealed and true positive reactions are difficult to assess. Because of these disadvantages we think that immunoblotting is more suitable for confirmatory purposes and for alloantibodies than for autoantibodies.

7.2.2 Techniques for Immunochemical Characterization of Autoantigens 7.2.2.1 Immunoblotting This technique has been used to identify the antigens present on platelet GPs (Beardsley et al., 1984a) In this approach the whole platelets, or only their membranes, are solubilized in sodium dodecyl sulphate (SDS), then the GPs are separated according to their molecular weight by SDS-polyacrylamide gel electrophoresis (PAGE) (Fig. 9.1). After vertical separation, the GPs are transferred to a nitrocellulose sheet. The GPs, immobilized on the nitrocellulose, can react with antibodies present in the serum or the plasma. After incubation, the Ag-Ab complex is revealed by an anti-human globulin reagent labelled with radioactive probe or coupled to an enzyme which will catalyse a colour reaction. Molecular weight standards are run at the same time to determine the molecular weight of the identified positive protein. With this technique, autologous platelets used as targets and incubated with the F(ab')2 fragments of the circulating immunoglobulins allowed the demonstration of true autoantibodies in patients. Negative results using platelets from patients with inherited defects such as Glanzmann's thrombasthenia or Bernard-Soulier

SDS-PAGE electrophoresis

7. 2.2.2 Radioimmunoprecipitation In this technique, antibodies react with radioactive labelled platelet suspensions (Fig. 9.2). The antibodysensitized platelets are solubilized after washing. After precipitation by protein A-sepharose and washing, SDS is added to each pellet and the solution is electrophoresed

Immobilized Proteins Incubation with Test Plasma/Serum on Nitrocellulose

Electrophoretic Transfert pad gel nitrocellulose

High

3

MW

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/

Results

Labelled anti-human globulin reagent /I

J

-Low MW

Human autoantibody (Aab)

7

L

+

i ~ "~ ~

-~ ------=.--

Native proteins

+

SDS

Platelet proteins (Antigen = Ag)

Denatured proteins

v

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Conformation dependent epitope

&&

_

-

- - -

,/

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Destruction of the conformation dependent epitope Figure 9.1

173

Schematic representation of immunoblotting.

,~ s s ~

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Linear epitope

Ag-Aab complex

174 C. KAPLAN AND G. TCHERNIA 1st STEP

l=s I surface labelled platelets

+

7,

Test serum/plasma (autoantibodies)

2nd STEP

l=Sl Antigen-lgG Autoantibody complex Jbilization. if= v-

d=d.

3rd STEP

ProteinA-sepharose + l=51-antigen-lgG autoantibody complex

4th STEP SDS and SDS-PAGE electrophoresis + autoradiograph

Results

Figure 9.2 Schematic representation of radioimmunoprecipitation. by SDS-PAGE. After drying, the gels are autoradiographed and the molecular weight of the detected bands assessed by comparison with standards (Fig. 9.2). This technique was essentially used for testing circulating antibodies, but recently platelet antigens binding plateletassociated autoantibodies have been revealed using a direct immunoprecipitation procedure (Tomiyama et al., 1989). One limitation of this test is the necessity to radiolabel the antigen with 12sI or SH, which is not possible for all the platelet membrane proteins. This assay is more suitable for confirmation or identification of an unknown antigen than for routine purpose. Both immunoblotting and radioimmunoprecipitation assays are technically demanding and difficult to use in a routine laboratory.

7.2.2.3 Antigenic Capture Assays For clinical applications, capture assays using murine mAbs specific for defined platelet GPs have the advantage

of sensitivity, and the more recent of them allow the measurement of both platelet-associated and circulating autoantibodies. They are adaptable for large-scale screening. They allow identification of a mixture of antibodies. Their first application was the microtitre well assay (Woods et al., 1984a, b). The microtitre wells are coated with mAbs which fix platelet GPs from a platelet triton lysate. The human plasma under investigation is incubated in these wells, and after washing positive binding is revealed with a radiolabelled monoclonal antihuman IgG. The principal disadvantage of this technique is that binding of the autoantibodies occurs after platelet lysis, and during this procedure, some target antigens lose their conformational epitope inducing false negative results. To overcome this problem, the immunobead assay (McMillan et al., 1987) and the MAIPA test (Kiefel et al., 1987) were developed (Fig. 9.3). In these techniques, binding of the autoantibodies occurs before platelet solubilization, preventing the loss of epitopes

AUTOIMMUNE THROMBOCYTOPENIAS

175

I M M U N O B E A D ASSAY

MAIPA ASSAY

1st STEP: Platelet sensitization

Anti GP lib-Ilia Anti GP Ib-IX Anti GP lb-IX Human Aab Moab Human Aab

Anti GPllb-llla Human Aab

Anti GP Ib-IX Human Aab

2nd STEP: Lysis eQm

|ml

~mmm|

3rd STEP: Capture of the immune complex

IY

II

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I

4th STEP: Revelation Labelled anti-human Anti-mouse

globulin reagent

Immunoglobulin

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It Results

Figure 9.3

-

t +

t i t -

+

Schematic representation of antigenic capture assays: comparison between the MAIPA and the immunobead assays.

during this procedure. In the immunobead assay the sensitized platelets, after being lysed, react with a defined mAb which immobilizes the immune complex; while in the MAIPA assay, the mAb is added in the same step as the human antibody to be tested. In this last case, the immune complex is attached to the microtitre well with an antibody antimouse IgG. In both assays the prerequisite is the availability of mAbs specific for the considered GP. It must be mentioned that some immunologically important autoantibodies can be missed in routine testing if the GPs are altered during the preparation, are sensitive to proteolytic degradation, or if the anticoagulant induces a conformational change in the

epitope by chelating the calcium (Kuijpers et al., 1992). Some other disadvantages must also be mentioned: false negative results can be obtained if there is competition for the same epitope between the autoantibody and the mAb, or if there is steric hindrance between these antibodies, or if the epitope recognized by the mAb is modified by lysis. To avoid this, different mAbs must be used. Another problem with the MAIPA is the presence of human antibodies reacting with mouse immunoglobulins, inducing false positive results. To avoid this artefact, sequential incubation with the human serum, then after washing, with the mAbs, has been proposed, but low al~ity antibodies can be lost during the washing.

176 C. KAPLAN AND G. TCHERNIA Whatever the assay, the background signal must be low and the specific signal strong to easily determine the clear cut-off point of positivity. In conclusion, for the diagnosis of autoimmunity it is necessary to use simple, rapid and reproducible tests. The assays must be applicable for large-scale screening but each one has its limitations and the appropriate one should be chosen after comparison and evaluation. At present it appears that a combination of methods must be used for detecting all types and classes of specific antibodies. 7.2.2. 4 M o l e c u l a r Biology The recent advances in molecular biology and technological applications have made a major contribution to improved understanding of the structure of autoepitopes. The molecular structure of autoantigens could be determined by doning and sequencing. Despite some limitations (recombinant proteins may be different from the native forms because of the absence of posttranslational processes, destruction of some epitopes due to the procedure itself), these new tools are beginning to be applied in the study of anti-platelet autoimmunity. Using synthetic peptides and/or transfection experiments, localization of autoepitopes on the GPIIIa glycoprotein has been published (Fujisawa et al., 1991, 1992; Kekomaki et al. 1991a). 7.2.3

I d e n t i f i c a t i o n o f Specific A u t o a n t i g e n s on the Platelet Glycoproteins

Glycoproteins H b a n d H I a a n d the l I b - H I a Glycoprotein ~ p l e x In 1982 platelet eluates or sera from 42 patients with AITP were tested in an immunofluorescence test (van Leeuwen et al., 1982). Thirty-five eluates (83%) reacted with all the platelets from the panel but not with the platelets from GT patients. Fourteen sera (33%) showed the same results. The immunoglobulins were IgG, IgM or a combination of both. To avoid the possibility that a positive reaction with normal but not with GT platelets was due to alloantibodies directed against the GPIIIa alloantigenic system Zw (PI A, HPA-1) the eluates were tested against a panel of Zw a or Zw b subjects. No differences were observed in the strength of the reaction, confirming that the target in AITP was not located on the same epitopes. Since this report, different methods have been used to detect specific antibodies reacting with IIb-IIIa: microtitre well assay (Woods et al., 1984b), radioimmunoprecipitation (Woods et al., 1984b; Devine and Rosse, 1984) and immunoblotting (Beardsley et al., 1984a). With these techniques, autoantibodies reacting specifically with GPIIIa or GPIIb could be demonstrated (Beardsley et al., 1984a; Tomiyama et al., 1989). In the case of GPIIb, the localization of the autoantigen did not differ from the epitope of the Bak a (HPA-3) alloantigen: 7.2.3.1

the 65 kD COOH-terminal fragment of the GPIIbc~ was equally recognized. With immunobeads and the MAIPA assays allowing the measurement of either platelet-associated autoantibody or plasma autoantibody, the implication of GPIIb-IIIa in chronic AITP has been confirmed (McMillan et al., 1987). GPIIb-IIIa could be implicated either alone or associated with GPIb-IX when a large series of sera (81) was investigated in the MAIPA assay (Kiefel et al., 1991). More recent works have been reported concerning the localization of the autoantigens on the GPIIIa, as probably as many as one-half of patients with AITP have autoantibodies directed against the GPIIb-IIIa complex, and this complex is implicated in platelet physiology mediating platelet aggregation. Epitopes were characterized using either purified GPIIb-IIIa in an immunoblot assay, or synthetic peptides corresponding to different regions of the GPIIIa. In a series of 39 patients with primary AITP, 43.6% had antibodies against GPIIb-IIIa or the 50kD chrymotrypsic fragment of GPIIIa (Kekom/iki et al., 1991a). This fragment is recognized by a strong autoantibody considered as a prototype. Among these patients, the majority (31/39) were considered as chronic AITP sufferers. A total of 48.4% of them had antibodies against GPIIb-IIIa and/or 50 kD. In acute AITP, two of eight patients demonstrated antibody activity, and this was directed only against the 50 kD fragment. The same reactions were also observed in secondary AITP as in non-immune thrombocytopenias. From this report it can be concluded that the 50 kD cysteine region of GPIIIa is frequently involved but not restricted to immune thrombocytopenia, and that some autoantigens only found associated with this fragment are probably cryptic or neoantigens. The role and significance of the antibodies, and the implication of these antigens in the pathology, remains to be determined. In another study of 13 patients with chronic and severe AITP, the putative epitopes have been explored with synthetic peptides (Fujisawa et al., 1991). An antibody binding to the peptides forming most of the carboxyterminal region of the GPIIIa, thus presumably the cytoplasmic domain, was observed in the positive specific reaction. The anti-peptide antibody could represent either the most or only a major part of anti-GPII-IIIa in plasma autoantibody. After transfection experiments with GPIIb and whole or modified GPIIIa, it was shown that the carboxyterminal region was important for maximal binding of the autoantibodies and not only when these antibodies reacted with the peptides of this region. In another report (Fujisawa et al., 1992), concerning the epitopes recognized by the platelet-associated autoantibodies, it was found that they were not dependent on the carboxy-terminus of GPIIIa, accessibility of which on intact platelets seemed to be very low. In a same patient, plasma or platelet-associated autoantibodies

AUTOIMMUNE THROMBOCYTOPENIAS against GPIIIa can recognize different epitopes on this molecule. The circulating antibodies could be considered not as primary but as secondary antibodies, emerging when inaccessible structures are exposed after cell destruction, probably mediated by primary antibodies. The presence of these circulating autoantibodies could indicate a particular stage of the disease. If it is confirmed that severe disease is associated with a particular epitope, the presence of autoantibodies against this epitope could represent an important marker in the survey of patients. 7.2.3.2

Glycoproteins Ib, I X a n d the I b - I X - V Glycoprotein ~ p l e x Using similar methods to those used for the characterization of autoantibodies against GPIIb-IIIa (Woods et al., 1984b; Szatkowski et al., 1986; McMillan eta/., 1987; McMillan, 1990; Kiefel et al., 1991) the presence of autoantibodies against the GPIb or the GPIb-IX complex was evaluated. The GPIb-IX complex plays an essential role in the first step of haemostasis, platelet adhesion to the vascular subendothelium (Roth, 1991). In a series of 106 chronic AITP patients, only three gave positive reactions in the microtitre well assays when their plasma was incubated with an mAb specific for GPIb (Woods et al., 1984a), and this was confirmed by the immunoprecipitating procedure, where a band correlated with the position of GPIb was revealed on the autoradiograms. With the immunobead assays (McMillan et al., 1987), eight of 59 patients had plateletassociated GPIb autoantibody and positive anti-GPIb plasma autoantibodies was found in 11 of the 59 patients. Thus, considering these studies, the appearance of anti-GPIb antibodies during the course of AITP was considered to be less frequent than anti-GPIIb-IIIa. But in a recent work it has been reported that anti-GPIb-IX antibodies were as common as those against antiGPIIb-IIIa (Kiefel et al., 1991). The association of both is not a rare event; this was the case for 22 patients out of 81. The same observation was made when the plateletassociated antibodies were considered. Blocking experiments with different mAbs against various epitopes on the GPIb-IX complex suggest that the autoantigens are in close proximity to the insertion of this complex in the platelet membrane, as described earlier (Szatkowski et al., 1986)

7.2.3. 3 O t h e r Proteins Apart from the so-called major membrane GPs, other proteins have been implicated as target for autoantibodies in AITP. Using immunoblot procedure, immunoprecipitation or ELISA assays (MAIPA or derived techniques), analysis of patients' sera or plasma revealed the presence of autoantibodies directed against various proteins, including the 62 kD collagen receptor (Sugiyama et al., 1987), GPIV (Pfueller et al., 1990), VLA-2 (Gerber et al., 1989), and other proteins with apparent molecular weights ranging from 50 to 240 kD (Lynch and Howe,

177

1986), or in a more narrow range 50-60 kD and 90 kD (Rock et al., 1990), especially in acute, steroid-resistant AITP. 7.2.3. 4 A u t o a n t i b o d i e s a n d Platelet Functions The major platelet membrane glycoproteins, GPIb-IX and GPIIb-IIIa play a crucial role in haemostasis, adhesion and aggregation by acting as receptors or adhesive proteins. Congenital abnormalities of the platelet membrane GPs have contributed to a better understanding of the role of these proteins. Although classic BSS or GT are inherited disorders, acquired defects can cause similar conditions. Not all autoantibodies induce a functional defect, but a subset have been shown to interfere with normal platelet function. An acquired BSS caused by autoantibody against GPIb has been documented (Devine et al., 1987). Platelet immunoprecipitation experiments demonstrate the existence of a plasma antibody precipitating a protein band corresponding to GPIb. The patient's plasma inhibited the ristocetin agglutination test. Patients with acquired disorder of platelet function associated with the presence ofautoantibodies against the GPIlb-IIIa complex have been reported (Balduini et al., 1987, 1992; Meyer et al., 1991). These patients had a severe bleeding tendency. Functional studies showed that the platelet aggregation in response to ADP and collagen was impaired, but in contrast to the congenital defect, the GPIIb-IIIa was present as normal on the patient's platelet membrane. By radioimmunoprecipitation, crossed immunoelectrophoresis or immunoblotting, an autoantibody reacting with the GPIIb-IIIa complex was documented. By competition experiments, it was shown that patient's plasma inhibits the binding of mAb to GPIIb-IIIa. No interaction was seen with the binding of anti-GPIa-IIa, GPIb-IX or GPV mAbs, but in this patient, a defect in platelet adhesion was also described (Balduini et al., 1992). Two AITP patients had auto-GPIIIa antibodies, interfeting with the binding of fibrinogen to stimulated platelets (Beardsley et al., 1984b). One patient with AITP and defective collagen-induced platelet aggregation had a plasma antibody which could immunoprecipitate a platelet membrane protein of 62 kD (Sugiyama et al., 1987). On the other hand, association of AITP and induced platelet aggregation or thrombosis has also been documented: a patient with AITP and arterial thrombosis has been described (Jackson et al., 1989). The platelet aggregation activity, mediated by direct interaction of the F(ab')2 portion of the antibody, was considered to be located on GPIIb by immunoprecipitation experiments. In another case, an autoantibody activating normal platelets and reacting with a protein of 36 kD has been reported (Yanabu et al., 1991). This antibody induced platelet aggregation and ATP secretion in normal PRP.

178

C. KAPLAN AND G. TCHERNIA

This factor was not present during the recovery period. No aggregation was observed with GT platelets or platelets with cyclooxygenase (CO) deficiency. The antibody bound to a protein of 36 kD in Western blotting. The platelet aggregation was mediated through GPIIb-IIIa and depended on ADP secretion of the dense granules.

7 . 2 . 3 . 5 A u t o a n t i g e n s a n d Clinical Significance Due to progress in technology, target antigens have been now identified in a large series of patients. If it was supposed that the GPIIb-IIIa complex was more often implicated, it has been shown now with different assays that other proteins are also common in this pathology. The finding of specific antigens argues in favour of autoimmunity, but there are still difficulties in choosing the best assay for diagnosis. Is the presence of a specific antibody a marker of evolution or of severity of the disease? With the development of ELISA assays, it seems that the same targets are found in acute as in chronic AITP. But the interpretation of the results must be made with caution. After demonstration of diversity ofautoantibodies in the same patient, and the discrepancy between platelet-associated and circulating autoantibodies (Fujisawa et al., 1992), questions arise concerning the value and significance of the presence of these antibodies in the pathogenesis and in the course of AITP: do particular antibodies/antigens correlate with a severe haemorrhagic tendency or thrombosis? In fact a subset of antibodies interfere with normal platelet functions. If it was assumed that GPIb-IX was implicated in severe disease, no correlation was found recently between autoantigens and severity (Kiefel et al., 1991). Therefore it is important to develop systematic large-scale assays and new technologies for the biological diagnosis of AITP to solve several issues: (1) pathogenesis of autoantibodies; (2) clinical significance of such antibodies; (3) definition of optimal management for the patients in regard to evolution and haemostatic complications. This could lead to analysis of whether or not a relationship does exist between the antigen and the haemostatic disorder, and the severity of the disease. Further, this could reveal new ways to develop the biological diagnosis of autoimmunity, and the understanding of the molecular defect associated with autoimmunity could offer new therapeutic alternatives.

8. 8.1

Treatment ACUTE AITP

Mostly seen in children with a peak incidence between 2 and 6 years of age, this is generally a benign, self-limiting condition with spontaneous recovery within a matter of days to a week. In that case the aim of the treatment is

only to shorten the risk period by increasing platelet count before spontaneous increase takes place. 8.1.1 Steroid Therapy For many years most paediatricians have used this therapy at a dose o f l - 2 mg/kg as initial treatment for 10-28 days (Buchanan et al., 1985). In most cases this leads to a stable increase in platelet counts within a few days and to a more rapid disappearance of petechiae and bleeding symptoms. Steroids should only be used as an initial therapy. In some patients when the dose is tapered the platelet count can drop and clinical expression may reappear: in that case the evolution towards chronic AITP as well as subsequent spontaneous resolution can be observed. In any case, steroids should not be given for a prolonged period and should the evolution be prolonged other therapeutic approaches must be proposed. Initial steroid treatment does not change the total duration of the illness and probably does not reduce the number of patients whose disease becomes chronic (McElfresh, 1975). This treatment has been subject to controversy. For most paediatricians the decision of initial steroid therapy is based individually on a combination of platelet count and clinical factors (Dubansky and Oski, 1986). Others, owing to the rarity of intracranial bleeding, have suggested that initial treatment should not be prescribed provided that patients are under close control until the platelet count begins to rise (Lusher et al., 1984). More recently it has been shown (Bellucci et al., 1988) that low dose steroids (0.25 mg/kg/day) has the same efficacy in terms of long-term results, i.e. percentage of chronic forms after 6 months follow-up, as conventional dosage.

8.1.2 High Dose i.v. IgG Ten years ago this became a major therapeutic alternative to steroids. Intravenous IgG has been shown to increase transiently the platelet count in patients with acute or chronic AITP (Imbach et al., 1981, 1984). The most commonly recommended dosages are 0.4 g/kg/day for 5 days in a 4 h infusion or 1 g/kg/day. The increase can occur in the 12-72 hours following the infusion. In most cases of acute AITP spontaneous favourable outcome is reached during the time of the therapeutic response. The major limitations are the price of the product, the time necessary for its administration in out-patients and the problems of viral safety. The exact mechanism of the beneficial effect of i.v. IgG is unknown. Several modes of action have been proposed (Imbach, 1991), such as an interference with macrophage Fc receptors (Fehr et al., 1982), modifications of the T cell subsets and of the B cell function (Tsubakio et al., 1983; Delfraissy et al., 1985), anti-idiotypic binding to idiotypic determinant on the target cells and its antibody (Rossi and Kazatchkine, 1989; Berchtold et al., 1989a), and feedback inhibition of release or binding of mediators.

AUTOIMMUNE THROMBOCYTOPENIAS A randomized multicentre study on 108 children with untreated acute AITP has compared i.v. IgG and steroids as initial treatments. The effects were identical for rapid responders but patients requiring more than initial treatment responded better if randomized to IgG (Imbach et al., 1985). Intravenous IgG is currently, in some countries, routine treatment for acute AITP on presentation. In a recently published retrospective study, intracranial haemorrhage-related deaths were observed in the period before the availability of i.v. IgG (Robb and Tiedeman, 1990). In other countries, they are only considered for patients in whom a rapid rise in platelet count is deemed essential, that is before surgery, after significant trauma, or because of life-threatening mucosal or internal haemorrhage (Buchanan, 1985, 1987).

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days after onset of therapy and reaches a maximum within 2 - 3 weeks. Normal platelet counts will only be obtained in less than 25% of cases (McMillan, 1981). If so, or if a significant increase even without normalization has been observed, 'the dose should be tapered with extreme caution and patience. In most cases relapse occurs after discontinuation of therapy or at various dose thresholds, according to the patient. In other patients a satisfactory, although below normal, platelet count can be maintained off therapy or with very low doses every other day.

8.2.2 High Dose i.v. IgG

Initial therapy is aimed at attaining a complete lasting response. If this is unsuccessful, subsequent treatment should be directed at maintaining "safe platelet counts", i.e. above 2 0 - 4 0 x 109/1 according to the individual clinical expression (Berchtold and McMillan, 1989). Three main treatments are proposed. Most patients with longlasting severe chronic AITP will experience all three at different times in the course of the disease.

This will lead in very few patients to a long-lasting stabilization of platelet count in chronic AITP by the bias of a hypothetical immunoregulation by the idiotypic network. In most cases however a sharp rise is obtained but the response duration will not exceed 1-6 weeks before relapse occurs. However, i.v. IgG is of major interest in two settings: (1) if a transient increase in platelet count is necessary, e.g. for surgery, delivery, or intercurrent events such as travel obligations; (2) in some patients regular i.v. IgG infusions can maintain a "safe platelet count" and a normal life for several months, with an injection on an out-patient basis every 3 - 6 weeks. Moreover in some of these patients the interval can gradually be increased. This strategy, although costly, can provide the necessary time to learn the degree of tolerance and the natural history of the disease before discussing splenectomy. In some cases the association of steroid therapy with i.v. IgG can be effective after failure of either alone. Intravenous IgG can be started during conventional dose of steroids, which will be tapered if an increase in platelet count is obtained. Such treatments, even if they have previously failed, can be prescribed again after splenectomy if a relapse has occurred: their efficiency can be increased.

8 . 2 . 1 Corticosteroids Corticosteroids are known to reduce the ability of phagocytes to digest intracellular particles and destroy bacteria and to decrease phagocytosis of platelets by macrophages. Therefore treatment with costicosteroids has been proposed as an equivalent to "medical splenectomy" in that it prevents the sequestration by the spleen of damaged or antibody-coated platelets (Karpatkin, 1985). Moreover, steroid therapy could inhibit antibody production to a previously recognized antigen and increase the catabolic rate o f circulating IgG, and even at low dose (e.g. 0.25 mg/kg/day) they have been shown to enhance capillary resistance (Karpatkin, 1985). Prednisone 1 mg/kg/day can be started on diagnosis. Clinical improvement as shown by reduced cutaneous expression and bleeding, can be observed within the first 24-48 h, even if the increase in platelet count is not yet obvious. Improvement in platelet count occurs within the 3 - 7

This results in a much higher cure rate than any medical regimen. The overall response rate averages 70% with 14% relapse, resulting in a stable remission rate of 60%. An additional 12% of these patients achieve a stable partial response. Therefore about 72% of patients have a beneficial response (Den Ottolander et al., 1984; Pizzuto and Ambriz, 1984; Berchtold and McMillan, 1989). According to various reports, several features could help to predict the results, such as previous steroid responsiveness (Weinblatt and Ortega, 1982), short survival and high turnover of labelled platelets (Siegel et al., 1989), or high post-operative rise (> 500x 109/1) in platelet count after surgery. However there is no foolproof way to predict which individual patient will have a response to splenectomy. Some surgical failures or relapses can be related to persistent accessory spleen, although secondary splenunculectomy has not been shown to be regularly

8.1.3

Intravenous Rhesus Antibodies (Anti-D) Intravenous rhesus antibodies have been shown in some preliminary studies to have a beneficial action in rhesuspositive children with acute AITP. The advantages are the lower cost when compared to i.v. IgG and the short duration of the infusion (30 min period on an outpatient basis). However conflicting data have appeared (Bussel et al., 1991a) which probably reflect a different mode of sensitization of the donors for the obtaining of anti-D, and further evaluations have to be conducted.

8.2 CHRONIC AITP

8.2.3 Splenectomy

180 C. KAPLAN AND G. TCHERNIA efficient in such cases (Reid et al., 1986; Akwari et al., 1987). Operative morbidity of splenectomy is uncommon. If steroid therapy or i.v. IgG has been previously shown to be transiently efficient they should be prescribed before surgery in order to allow a safe procedure. Otherwise platelet transfusions should be ready for use and infused immediately after clamping of splenic vascular predicles if surgical haemostasis is difficult to achieve. However the hazard of post-splenectomy severe infections, mostly related to Streptococcus pneumoniae, Haemop h i lus i nfluen zae , Neisseria meningitidis, which can lead to death even several years after surgery, has severely restrained the indications of splenectomy in all conditions including AITP (Krivit, 1977; Brivet et al., 1984; Evans, 1985; Hollis et al., 1987; Holdsworth etal., 1991). It should be emphasized that in a large series of AITP in children the mortality risk of overwhelming post-splenectomy infection can be as important as that of CNS haemorrhage (Walker and Walker, 1984). In any case all patients, whatever their age, should receive appropriate vaccination before surgery and regular oral penicillin prophylaxis should be prescribed for life. If regular compliance to treatment cannot be obtained patients must be advised to take oral penicillin for 7-10 days in case of 'flu or seasonal ear/nose/throat infection, and the necessity of regular revaccinations explained. In cases of high fever they should immediately refer to an appropriate centre. This particular risk is a cause of controversy, and explains why splenectomy, which everyone agrees to be the most frequently effective treatment, may be proposed early in the course of the disease. (Berchtold and McMillan, 1989), after 6 months (McClure, 1975) or restricted to particular cases with refractoriness to all medical treatments, persistent bleeding tendency leading to major discomfort or risk for cerebral haemorrhage, or unacceptable side effects of medical treatments. In any case splenectomy in infancy or early childhood should be considered with major reluctance and only in cases of severe thrombocytopenia with overt clinical expression.

8.3

REFRACTORYCHRONIC AITP

There is at present no definitive treatment for refractory chronic AITP. The plethora of suggested solutions simply reflects the fact that refractory idiopathic thrombocytopenic purpura often remains refractory (Rosse, 1984).

8.3.1 Immunosuppressive Drugs Azathioprine and cydophosphamide have been reported to be effective after failure of steroid therapy and splenectomy (Finch et al., 1974). However, as in other nonneoplastic disorders, their use has been considerably restrained by the risk of acute non-lymphoid leukaemia (Krause, 1982).

8.3.2 Vinca Alkaloid Therapy Vinca alkaloids have been widely used. They bind to platelets and can thus be selectively delivered to macrophages either after in vitro loading of allogenic platelets (Ahn et al., 1978) or after slow infusion and in vivo loading of the patient's own platelets (Ahn et al., 1984). However, whichever way used, vinca alkaloids have not fulfilled their early promise (Rosse, 1984). 8 . 3 . 3 C o l c h i c i n e Therapy This treatment, probably acting through the bias of a decreased clearance of opsonized platelets secondary to inhibition of microtubule-dependent events in macrophages, was advocated in 1984 (Strother et al., 1984). The response rate was 29% in a small group of patients, but no further report has confirmed a regular efficacy of this treatment. 8 . 3 . 4 D a n a z o l Therapy Conflicting results have been published for this therapy, which has been claimed to be a good alternative to splenectomy in elderly patients, especially in women (Ahn et al., 1989). Its effects could be mediated by a modification of cell membrane or by an immune modulation. Some responses occur as late as 10 months after initiation of therapy, therefore requiting 10 months of therapy for full evaluation. Combined therapy with low dose glucocorticoids is well tolerated and in responders, steroids can be reduced in dosage or discontinued (Ahn et al., 1989). However, due to side effects many groups have not tested long duration therapy and the overall impression is rather disappointing (Fenaux et al., 1990). 8.3.5 Ascorbate Treatment Ascorbate has been reported to be beneficial for refractory AITP by Brox (1988). Platelet count increased in seven out of 11 patients on a single daily dose of 2 g. However no convincing data have appeared since this first publication. 8 . 3 . 6 A n t i - D Treatment In contrast, although not yet widely used, anti-D could turn out to be a suitable alternative for rhesus-positive patients with chronic refractory thrombocytopenia, at a cost which approximates 10% of that for i.v. IgG. Anti-D treatment seems to be especially effective in children. It has already been used as maintenance therapy (Bussel et al. 1991a). A response has been observed in 92% of patients with a mean duration of 5 weeks and in most cases there was a second response to a subsequent injection (Andrew et al., 1991). Moreover when a response was obtained, intramuscular anti-D could maintain the increment in platelet count (Gringeri et al., 1992). 8 . 3 . 7 IFNc~ Therapy For short courses at low doses (Proctor et al., 1988;

AUTOIMMUNE THROMBOCYTOPENIAS Hudson et al., 1992) interferon c~ (IFNcz) has been used by several groups but still needs further evaluation.

8.3.8 Other Therapies In severe refractory immune thrombocytopenia attempts have also been made at removing circulating antibodies with techniques such as plasmapheresis (Branda et al., 1978) or more recently extracorporeal protein A immunoadsorption, which appears to be a promising technique (Snyder et al., 1992).

8.4

EMERGENCY TREATMENT

Whatever the clinical course of AITP, acute or chronic, patients with significant mucosal bleeding or extremely low platelet counts (< 5-10 x 109/1) must be considered as at risk for cerebral bleeding, and immediately hospitalized and treated.

8.4.1 Platelet T r a n s f u s i o n s Platelets should be administered only in an emergency: 6 - 8 random units can be transfused as often as necessary (every 6 h) in an attempt to control bleeding. Some groups prefer continuous infusion of platelets (1-2 U/h). In spite of their rapid destruction in the patient, transfused platelets can protect against life-threatening bleeding but should only be used for a short period of time, while waiting for the beneficial effect of other simultaneous treatment such as i.v. IgG (Berchtold and McMillan, 1989).

8.4.2 I n t r a v e n o u s M e t h y l P r e d n i s o l o n e Therapy High dose corticosteroids have been mostly used in childhood chronic AITP (Menichelli et al., 1984) at a dose of 15 mg]kg/day for 3 days, but have also been shown to be effective in adult patients (von dem Borne et al., 1988) at a dose of I g methyl prednisolone i.v. for 30 min daily for 3 days. The response is transient and such treatment, if justified by the severity of bleeding, should not become a long-term steroid treatment with pulses of treatment repeated to maintain an effect (Lilleyman, 1984). This treatment can be used in an emergency, after failure of i.v. IgG or simultaneously with i.v. IgG if haemorrhage is life threatening. In such patients plasmapheresis (Hoots et al., 1986) or extracorporeal immunoadsorption are proposed, although technically difficult in severely thrombocytopenic patients, and in some patients emergency splenectomy has to be performed, such as when neurosurgery for CNS bleeding is needed (Akwari et al., 1987).

9. Autoimmune Thrombocytopenic Purpura and Pregnancy Chronic AITP is frequent in women and thus often

181

associated with pregnancy. Platelet antibodies, being usually of the IgG class, can cross the placenta, recognize foetal platelets as targets, and induce thrombocytopenia which can cause bleeding. During the last three decades, the association of AITP and pregnancy has led to many controversial discussions, most of them still unsettled.

9.1

MOTHERS

The frst major concern is with thrombocytopenic mothers. In the fifties their mortality rate was high and the question was whether these patients should be advised to conceive and whether abortion or splenectomy should be discussed during pregnancy (Tancer, 1960). However, owing to the improvement of obstetric and medical management and to the successive availabilities of steroid therapy (O'Reilly and Taber, 1978) and of i.v. IgG (Tchernia etal., 1984; Lavery etal., 1985), most of the maternal problems have been solved and no maternal death related to thrombocytopenia has been reported since 1960. Provided that follow-up of such pregnancies is carried out by obstetricians who are aware of the management of bleeding disorders, pregnancy should not be discouraged in such patients. If maternal thrombocytopenia is severe before delivery, or if bleeding occurs during pregnancy, steroid therapy or i.v. IgG or the association of both can be prescribed. It must be stressed that foetal thrombocytopenia can be observed whatever the maternal status: a woman who has AITP can be in a state of compensated thrombocytolysis. For that reason past history of platelet disorders or bleeding episodes should be sought by all obstetricians, even though the routine biological status appears to be normal. Girls and women who have experienced AITP should be advised that subsequent pregnancies even after years of normal platelet count, will require an appropriate follow-up.

9.2

THE INFANTS

During the sixties, major concern was focused on the high mortality rate among infants born to mothers with AITP, which was estimated to range between 10% and 20% (Murray and Harris, 1976; O'Reilly and Taber, 1978). Morbidity was also important. Both death and neurologic sequelae were due to intracerebral bleeding initiated during delivery by the trauma of labour. In contrast, antenatal bleeding is seldom reported and although stillbirth is not uncommon in the previous obstetric history of women with AITP, the antenatal risk for foetuses appears to be far less important than it is in neonatal alloimmune thrombocytopenia (Kaplan et al., 1992). Thrombocytopenia is often moderate at birth and worsens during the first week of life with a nadir on day 4-6, preceding a spontaneous resolution which will occur within 10-60 days. This postnatal accentuation of

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C. KAPLAN AND G. TCHERNIA

thrombocytopenia could be related to a transient foetal hyposplenism illustrated by the presence of pocked erythrocytes and Howell-Jolly bodies or to a functional immaturity of the macrophages (Speer et al., 1986). However, unless initiated during delivery, postnatal bleeding is absent or moderate and most of the time easy to manage. Therefore the infants at risk for severe bleeding and especially intracranial haemorrhage are only those who exhibit an antenatal severe thrombocytopenia (< 50 x 109 platelets/l). The vast majority of infants born to mothers with AITP will notexhibit severe thrombocytopenia at birth and will not need any help other than regular platelet counts until normalization and possibly low dose steroid therapy or i.v. IgG if required for any clinical event or for a surgical procedure (Chirico et al., 1983). Parents must be aware of the necessity of an appropriate follow-up for the next pregnancy. It must be stressed that many studies report an overall percentage of severely thrombocytopenic infants, regardless of the age of onset of severe thrombocytopenia, which leads to an overestimation of the risk. In our experience the percentage of severe thrombocytopenia at birth averages 10-15%. However the ratio of thrombocytopenic neonates, when compared to the first studies in this field, appears to have markedly decreased in recent reports. This is probably due to the fact that mothers with low platelet counts deliver thrombocytopenic infants more often than mothers with stabilized AITP, and that only the former appeared in the first publications. Ultimately, concern must be focused only on the severely thrombocytopenic foetuses, at risk for intracerebral bleeding related to a passive and transient disease in an otherwise normal infant. For many years, all groups involved in the field have been searching for a clinical or a biological maternal parameter predictive of the occurrence or the absence of foetal thrombocytopenia. However all clinical or biological indicators which have been claimed as possibly relevant have been subsequently proved not to be. Women with or without previous splenectomy have an equal risk of delivering thrombocytopenic offspring (Scott et al., 1983). Thrombocytopenic women have a slightly higher risk of having affected infants, but the overlap between the groups (Kelton, 1983) is more important than previously assumed. Antibody titres in maternal serum, transiently considered as predictable in some studies (Kelton et al., 1982a; Cines et al., 1982; Logaridis et al., 1983; Kelton, 1983), have been shown to be disappointing when used on a larger scale (Scott et al., 1983; Laurian et al., 1987). However a thrombocytopenia, when first diagnosed during pregnancy, and associated with negative results on circulating antibody testing could indicate a minimal foetal risk (Samuels et al., 1990; Bussel et al., 1991b). If foetal thrombocytopenia cannot be predicted, the next logical step is to try to protect the infants from birth injury. Systematic Cesarean section in any parturient who

has experienced AITP has been advocated (Murray and Harris, 1976). However this led to many unnecessary Caesarean sections, with cord blood samples exhibiting normal or moderately decreased platelet counts (Laros and Kagan, 1984). Recognition of severe foetal thrombocytopenia at the onset of labour has then considered: foetal scalp sampling proposed in 1978 (Ayromlooi, 1978) and widely used during the following years provides platelet count by direct sampling performed at the initiation of labour and gives the opportunity to decide on Caesarean section if thrombocytopenia is severe (Scott et al., 1980). However scalp blood samples are often contaminated by amniotic fluid which leads to microcoagulation in vitro and to spurious low platelet counts (Tchernia, 1988). Once this practice evolved from a clinical investigation to a routine procedure, the incidence of such artefactual thrombocytopenias increased and led to Caesarean section for non-thrombocytopenic infants: there is generally agreement to reject the procedure (Christiaens and Helmerhorst, 1987). Finally, percutaneous umbilical blood sampling (PUBS); (Daffos et al., 1983; Hobbins et al., 1985) became accepted in 1983-1989. This is a reliable procedure which, if performed during the last week of gestation, provides a good correlation between antenatal and cord blood or postnatal counts (Daffos et al., 1988; Moise et al., 1988; Scioscia et al., 1988; Kaplan et al., 1990) However, it is an invasive procedure with risks depending on the dexterity of the obstetrician. It should only be performed in referral centres, where the overall estimated lethal risk of PUBS varies from 0 to 0.2% (Daffos et al., 1988; Pielet et al., 1988). Nevertheless, the low incidence of severe foetal thrombocytopenia (10-15%) and the fact that not all severely affected infants will develop an intracerebral bleeding must be taken into account. For these reasons controversy persists and some authors favour no active procedure (Sacks 1986; Burrows and Kelton, 1989; Cook et al., 1991). Antenatal treatments, if shown to be effective in all cases and with no secondary adverse effects for mothers and foetuses, would be a logical answer to the many dilemmas. Low dose steroid therapy prescribed to the mother was first considered as ineffective (Heys, 1966) and then claimed to be effective in a small group of patients (Karpatkin et al., 1981). Subsequently all groups experienced negative results with this treatment (Yin and Scott, 1985; Christiaens et al., 1990); moreover it has been shown that if such treatment could decrease the amount of IgG bound to maternal platelets, and raise their platelet count, it would consequently increase the free serum antibodies, making them available for transplacental passage (Cines et al., 1982). High dose i.v. IgG has been prescribed to some thrombocytopenic mothers in order to raise their platelet count before delivery (Tchernia et al., 1984). These mothers gave birth to normal as well as to thrombocytopenic infants (Lavery et al., 1985). However it was suggested that such

AUTOIMMUNE THROMBOCYTOPENIAS treatments were possibly not effective on foetal platelets because transplacental passage of IgG is slow and i.v. IgG had been administered shortly before delivery (McNabb et al., 1976; Pitcher-Wilmott et al., 1980; Smith and Hammarstrom, 1985). In fact the low incidence of severe thrombocytopenia at birth impedes any definitive appreciation of such antenatal treatments, unless early foetal samples obtained before the initiation of therapy can be compared to posttreatment platelet counts after late or cord blood sampling. We have performed such controls in some patients and failed to illustrate any regular effect either of low dose steroid therapy or of i.v. IgG (Kaplan et al., 1990). Other therapeutic trials should be conducted in some patients in referral centres, after informed consent. Should they confirm the failure of antenatal treatments, there would be no further justification for early PUBS in AITP.

9.3

HIDDEN

MATERNAL

AUTOIMMUNITY The search for the aetiology of neonatal thrombocytopenia has led to the identification of women with no past history of acute or chronic AITP who however exhibit specific anti-platelet circulating antibodies and compensated thrombocytolysis on isotopic studies. As thrombocytopenia can recur in offspring, such women when identified should enter the same follow-up during pregnancy as overt AITP (Kaplan et al., 1991).

9.4

ASYMPTOMATIC

MATERNAL

THROMBOCYTOPENIA This represents the last controversial issue. It has been shown that some pregnant women, especially during the last trimester of gestation, exhibited moderate thrombocytopenia which resolved after delivery (Burrows and Kelton, 1988). The precise mechanisms and the foetal risk of asymptomatic maternal thrombocytopenia (AST) are yet poorly known. Moreover, sometimes chronic AITP can be first discovered during pregnancy and considered as AST (Burns, 1988; Copplestone, 1988; Kaplan et al., 1990). In most of the recent studies the two entities are analysed together, which probably leads to an undervaluation of the foetal risk in AITP, and can be responsible for the present tendency to avoid invasive procedures and carry out vaginal delivery despite the risk of severe thrombocytopenia. AST is probably a heterogeneous disorder which reflects the extreme variation of a physiological process (Fay et al., 1983), a pathological event (Giles and Inglis, 1981), or a combination of the two, disrupting a long-lasting equilibrium such as compensated thrombocytolysis. Its incidence is estimated to average 6% (Rasmus et al., 1989). The low incidence of obstetric or foetal morbidity leads to the erroneously consideration of AST as an insignificant event and to leaving women without post-partum assessment.

183

Although the foetal risk is definitely less important than in women with a past history of overt AITP (Samuels et al., 1990), it is probably not absent (Kaplan et al., 1990). We believe that such patients should be further explored and be considered for a platelet life span study (Yvart et al., 1988), especially if another pregnancy is planned or if thrombocytopenia, even moderate, does not resolve. Finally we consider that when a diagnosis of AITP has been previously established, on classical criteria in a pregnant woman, antenatal PUBS should be discussed and can avoid neonatal intracerebral haemorrhage during labour. Even if this event has a low incidence, all groups who have experienced it do not wish such a preventable accident to occur again. The occurrence of aggressive thrombocytopenia in a previous child must also be taken into account, as clinical expression is often similar in siblings (Busse! et al., 1991b). In contrast, moderate thrombocytopenia of unknown origin, when occurring during the last trimester of pregnancy, will probably not require special management. However this entity is ill defined and calls for better clarification. No clear-cut recommendations can be given for borderline cases such as moderate thrombocytopenia early in pregnancy, and individual decisions have to be achieved according to biological and obstetric criteria and the availability of PUBS in experienced hands.

10. SecondaryImmune ThrombocytopenicPurpura 10.1

VIRUS-INDUCED AUTOIMMUNE THROMBOCYTOPENIA

Evidence for the immune nature of the thrombocytopenia occurring after viral infection remains difficult. The relevance of autoantibodies to pathogenesis is not yet determined. A further difficulty is in the fact that the appearance of autoantibodies after viral infection is often detected when the virus is no longer present. The mechanisms inducing autoimmunity after viral infection could be molecular mimicry, production of anti-idiotype antibodies, enhanced expression of the major histocompatibility complex (MHC) class I and class II molecules, disturbance in the host immune response and changes in endogenous antigen (Oldstone, 1989; Shnattner and Rager-Zisman, 1990). Thrombocytopenia probably due to autoimmunity has been described alone, or associated with anaemia and neutropenia during the course of infections due to different classes of virus. Only in a few cases have specific known antigens been identified. 10.1.1 RNA Virus Infections Paramyxoviridae (measles, mumps) have been rarely the cause of autoimmune thrombocytopenia. It is suspected

184 C. KAPLAN AND G. TCHERNIA in most cases that thrombocytopenia results either from a direct action of the virus and/or deposition of immune complexes on platelets or their defective production. In cases of congenital or acquired rubella, thrombocytopenia is not a rare event (Bayer et al., 1965). In congenital rubella, it seems that a defective platelet production is the main aetiology. In acquired rubella, platelet antibodies have been detected in the serum or directly associated with the platelets. A mechanism analogous to drug-dependent thrombocytopenia has been suggested (Myllyl~i et al., 1969). An autoimmune mechanism has been suggested to cause thrombocytopenia observed after combined vaccination against measles, mumps and rubella. In reported cases, it has been shown that the platelet destruction was peripheral and in some patients, specific assays demonstrated that the antigens recognized by the anti-platelet antibodies were localized on GPIIb-IIIa (Kekom~iki et al., 1991b). 10.1.2

R N A Viruses w i t h Reverse Transcriptase Activity In this category the most important virus associated with haematological disorders is the human immunodeficiency virus (HIV). Thrombocytopenia is frequent and is considered to be one of the criteria of the AIDS-related complex. The first description of immune thrombocytopenia similar to the classic autoimmune thrombocytopenic purpura was reported in homosexual men in 1982 (Morris et al., 1982). Since then, thrombocytopenia in HIV patients has been observed in children as well as in adults, the incidence varying among asymptomatic patients from 0-10% to up to 40% in symptomatic adults. Among thrombocytopenic patients, a significantly higher prevalence in males and intravenous addicts as well as patients with advanced disease has been detected (Murphy et al., 1987; Rossi et al., 1990). In children, thrombocytopenia could be the critical manifestation of infection; 30% of children with AIDS have been reported to be thrombocytopenic (Shannon and Ammann, 1985). Thrombocytopenic patients are not at greater risk for the development of AIDS than HIV-positive nonthrombocytopenic patients (Holzman et al., 1987; Oksenhendler and Seligmann, 1990). Although the haemorrhagic tendency in HIV patients is usually mild or absent (Walsh et al., 1985; Abrams et al., 1986; Aboulafia and Mitsuyasu, 1991), it could be an important cause of mortality and morbidity in children (Labrune et al., 1989). Some cases of life-threatening haemorrhages have been reported in adults (Oksenhendler and Seligmann, 1990; Ragni et al., 1990; Rossi et al., 1990), especially in HIV thrombocytopenic haemophiliacs (Ragni et al., 1990). The mechanisms leading to thrombocytopenia in HIVinfected patients are probably multiple: it has been demonstrated that HIV induces a B cell polyclonal

activation that can lead to the production of multiple autoantibodies (Lane et al., 1983). Thus it is likely that anti-platelet antibodies are also induced by the virus. Specific platelet autoantibodies and circulating immune complexes have been reported as well as the direct or indirect role of the virus itself. Originally, the mechanisms involved were thought to be different in homosexual, narcotic addict and hemophiliac thrombocytopenic purpura (Ratnoff et al., 1983; Walsh et al., 1984; Savona et al., 1985; Karpatkin, 1988). Analysis of serum immune complexes in homosexual patients and narcotic addicts revealed the presence of high titres of IgG anti-F(ab')2 antibodies (Yu et al., 1986). Further, it was shown that the serum polyethylene glycol-precipitable immune complexes contained anti-HIV-1 antibody as well as anti-idiotypic antibody. The same results were obtained with platelet eluates. Neither HIV-1 antigen nor proviral DNA were detectable. These data were in favour of the deposition of antiHIV antibody and anti-idiotypic antibody on the platelets contributing to the elevation of the plateletassociated IgG (Karpatkin et al., 1988). Recently, it has been documented with a sensitive immunoassay that autoimmune anti-idiotype-like antibody directed against HIV-1 GP-120 was found in the sera ofHIV-1 seropositive homosexual or drug addict patients. The anti-HIV-1 GP antibody and the anti-idiotype-like antibody are present in a polyethylene glycol (PEG) precipitable macromolecular complex containing IgG/IgM and C3. No independent binding of afffinity-purified anti-HIV-1 GP-120 antibody or anti-idiotype-like antibody was observed, but they do bind as a complex in a saturable manner. This complex could probably account for the elevation of platelet-associated Ig and contribute to the thrombocytopenia due to platelet destruction (Karpatkin and Nardi, 1992). However, other investigations have suggested that the thrombocytopenia was due to platelet-reactive autoantibodies. Attempts were made to define target platelet antigen in HIV patients. Sera from homosexual patients, thrombocytopenic or not, were tested by immunoblotting. Almost all the sera reacted with a 25 kD protein on normal platelets (Stricker et al., 1985). However, this finding was retracted later on, as normal serum samples gave the same results and other investigators failed to find a specific antibody against the 25kD protein among their HIV-l-positive patients (Shuman et al., 1991). In other HIV patients the platelet eluates reacted positively with normal platelets but failed to bind to type I Glanzmann's disease platelets (van der Lelie et al., 1987), suggesting that the epitope could be localized on the platelet membrane GPIIb-IIIa (Bettaieb et al., 1989) as in AITP. Finally, a broad spectrum of platelet antigens has now been recognized. Thus, the antigenic patterns differ markedly between patients (Magnac et al., 1990). It has been suggested that molecular mimicry between HIV and platelet antigens could exist. It has been shown that

AUTOIMMUNE THROMBOCYTOPENIAS 185 a platelet-eluted antibody recognized both the GPIIIa and HIV envelope GP-120 (Oksenhendler and Seligmann, 1990). Recently, it was clearly demonstrated that an antiplatelet antibody in an HIV-seropositive drug addict patient, specifically recognized an epitope shared by HIV GP-160/120 and the platelet GPIIb-IIIa. It is not known if cross-reactive antibodies are a frequent phenomenon at the origin of platelet destruction observed in HIV-seropositive thrombocytopenic patients (Bettaieb et al., 1992). Platelet-bound immunoglobulins found not only in asymptomatic and symptomatic HIVinfected patients but also in non-HIV-infected sexually active homosexual men suggest that stimulation by other viruses could also account for platelet autoimmunity (Klaassen et al., 1990). Direct involvement of progenitor cells might be an important factor in the cytopenias. If a decreased number of progenitor cells and/or abnormal regulation of cell growth is observed (Stella et al., 1987), other data support the hypothesis of a direct role of the virus itself in the pathophysiology of HIV-associated thrombocytopenia because megakaryocytes (Mks) have ultrastructural abnormalities and express viral RNA (Zucker-Franklin and Cao, 1989). Investigations have been carried out to detect HIV transcripts or proteins in fresh or cultured Mks from I-IIV-seropositive thrombocytopenic patients (Louache et al., 1991). Positive results were obtained with heterogeneity among patients, and in a given patient only a fraction of the Mks clearly expressed HIV transcripts. On the other hand, no positive results were found in the Mks differentiating in vitro from the CFU Mks, despite some of the native Mks being positive. The main hypothesis could be infection of the Mks only during terminal differentiation in the marrow, or that infection of CFU Mks disables them from differentiating in vitro. Due to Mk infection by HIV virus, viral antigens may be present in the platelets at low density, explaining their non-detection but leading to platelet destruction by anti-HIV antibodies. On the other hand, this infection could facilitate cross reactivity between the virus and the platelet antigenic determinants, as has been already mentioned (Bettaieb et al., 1992). Moreover, malignancies, viral, bacterial and protozoal infections and cytotoxic drugs or antibodies may be implicated as well as specific autoimmunity in the mechanisms of thrombocytopenia in HIV-infected patients. 10.1.3 D N A Virus Infections During the course of these infections, autoimmunity is not a rare finding. The herpes viruses are good candidates for the induction ofautoimmunity not only by molecular mimicry but also by polyclonal activation of human B cells. In thrombocytopenia related to varicella zoster virus, specific circulating anti-platelet autoantibody has been

identified in children with its epitope located on GPV (Beardsley et al., 1985). Thrombocytopenia complicating infectious mononucleosis due to Epstein-Barr virus has been described either on its own or associated with autoimmune anaemia and neutropenia (Smith et al., 1963), which is in favour of autoimmunity. Although thrombocytopenia could result from different mechanisms, attempts have been made to demonstrate the presence of anti-platelet autoantibodies. It has been demonstrated by immunoblotting that in some cases, these antibodies recognize epitopes principally located on GPIIb. In other cases, they are directed against intracellular antigens (Winiarski, 1989). The mechanism for cytomegalovirus-induced thrombocytopenia is not clear and to date there is no report of identification of the specific target on platelets. The human parvovirus B19 causes a transient aplastic crisis due to its cytotoxic effect against erythroid progenitor cells. A moderate thrombocytopenia of central origin seems to be common in parvovirus primary infection. Following B19 infestation, thrombocytopenia may also be due to the non-structural protein (NS-1)-mediated cytotoxicity of Mks (Srivastava et al., 1990), but immune thrombocytopenic purpura in this affection is also documented (Foreman et al., 1988; Hanada et al., 1989; Lefr~re et al., 1989). The presence of true autoantibodies during documented viral infections supports the hypothesis of a viral-induced autoimmunity, although the precise mechanism is still unclear. The identification of the autoimmune process, and the better understanding of this mechanism, may optimize therapeutic approaches and prevent in some cases the deleterious effect of alterations in immunity.

10.2

SYSTEMIC LUPUS ERYTHEMATOSUS

Systemic lupus erythematosus (SLE), which can be considered as the prototype of immune complex disease in humans, is characterized by circulating autoantibodies directed against nuclear cytoplasmic, cell membrane and other antigens. Thrombocytopenia is frequently encountered in SLE. It has been considered that one-third of patients are thrombocytopenic but the platelet count rarely falls below 100 x 109/1 (Rothfield, 1981). Peripheral destruction of thrombocytes is well known in this disease. Since in most cases an increase in plateletassociated IgG was observed (Mueller-Eckhardt et al., 1980), it was suggested that the destruction of platelets might be dependent on the coating of platelets with specific antibodies (Karpatkin et al., 1972) or on the binding of immune complexes (Dixon and Rosse, 1975; McMillan, 1983). It is important but sometimes difficult to establish the precise mechanism responsible, and it is likely that both mechanisms are involved in some patients. A large number of autoantibodies have been demonstrated in SLE, and the structure of the platelet

186 C. KAPLAN AND G. TCHERNIA antigen in SLE-related immune thrombocytopenia remains obscure. Interest has focused on the anticardiolipin antibodies; a strong statistical correlation between thrombocytopenia and increased anticardiolipin levels has been reported (Harris et al., 1985a, b). These authors suggested a direct role for these antibodies in platelet destruction in some patients by interactions with the platelet membrane. In favour of the binding of immune complexes, evidence for in vivo immune complex-platelet interaction involving antiDNA antibodies in SLE with nephritis has been presented (Frampton et al., 1986). Using immunoblotting, identification of autoantigens has been possible. Different platelet proteins have been detected; among them proteins of 66 and 108 kD have been localized in the cytoplasmic fractions of platelets in three out of nine patients studied (Kaplan et al., 1987). The precise role of such antibodies has not yet been determined. They could be a new marker associated with disease activity. In other series, antibodies against 80 and 120 kD were detected in all the 10 patients tested, and were absent from the sera of normal individuals and only infrequently found in patients with AITP (Howe and Lynch, 1987). It has been shown that these antigens could be intact and fragmented vinculin, and may be found in 67% of patients with primary or secondary AITP and in 40% of normal subjects (Brox et al., 1988). Using an antigen-specific assay, autoantibodies to platelet GPs were evaluated in patients with diseaserelated immune thrombocytopenia (Berchtold et al., 1989b). In three cases with SLE, two patients had elevated platelet-associated IgG and circulating antiplatelet antibodies. The target of these antibodies was GPIIb-IIIa. It was shown after absorption of the plasma with platelets than in one case of SLE with immunemediated heating loss, the activity against GPIIb-IIIa completely disappeared without altering the level of anticochlear antibody activity, demonstrating the existence of various antibodies with no cross-reactivity reactions. The anti-platelet antibodies identified in this report did not differ from those in AITP patients, and they probably play a role in the aetiology of the thrombocytopenia. For the therapy of immune thrombocytopenia associated with SLE, corticosteroids, vincristine, danazol or plasmapheresis should be considered before splenectomy, results of which are poor in this condition (Hall et al., 1985). It must be emphasized that transitional forms between AITP and SLE may exist. It is important to look for additional autoantibodies in AITP: they have been described directed against nuclear antigens (Panzer et al., 1989), phospholipids (Harris et al., 1985b) or against various tissue antigens (Conley and Savarese, 1989). AITP and autoimmune haemolytic anaemia (AIHA) could be associated in subclinical or overt SLE (Miescher et al., 1976). If the additional antibodies are of high titres, particularly against nuclear antigens, it has been shown that

the patients are likely to develop SLE (Miescher et al., 1992). Recently, AIHA or thrombocytopenia associated with lupus parameters have been described in patients with no evolution towards SLE; these transitional forms between autoimmune blood disorders and SLE must be recognized (Miescher et al., 1992).

10.3

EVANS SYNDROME

This was described in 1951 as the association of"primary thrombocytopenic purpura" and acquired haemolytic anaemia. The close association of the two processes suggests a common autoimmune mechanism. Neutropenia can occur. Evans syndrome has been reported in association with SLE or various allied systemic diseases (scleroderma, mixed connective disorder, sarcoidosis). Self-limited course is rare: steroid therapy or splenectomy can lead to a transient remission but relapses are usually observed (Evans et al., 1951; Pui et al., 1980).

10.4

AITP AND MALIGNANCIES

1 0 . 4 . 1 L y m p h o p r o l i f e r a t i v e Disorders Thrombocytopenia is a common complication. In most cases it is presumed to be due to marrow infiltration, hypersplenism or chemotherapy. However in some patients it has been shown to be immune mediated and related to the peripheral destruction of platelets: Mks are abundant on bone marrow smears, spleen enlargement is moderate or absent, and in some cases shortened platelet survival and/or elevated platelet-associated IgG have been demonstrated (Kaden et al., 1979). As such thrombocytopenia is only an illustration of the wide variety of immune function disorders observed during the course of lymphoproliferative diseases and, in contrast with bone marrow infiltration-related thrombocytopenia, has no adverse prognostic significance. It is often associated with autoimmune haemolytic anaemia syndrome. It has been mostly reported in chronic lymphocytic leukaemia and in non-Hodgkin lymphoma. In Hodgkin's disease it can occur several years after the initial treatment and does not necessarily imply a recurrence (Cohen, 1978). However a diligent search for active disease is mandatory (Berkman et al., 1983). 1 0 . 4 . 2 Solid M a l i g n a n t T u m o u r s In various tumours such as carcinomas or germ cell cancer, thrombocytopenia can be observed without evidence of bone marrow tumour involvement or hypersplenism, or consumption coagulopathy. However the immune mechanism has not been clearly established in all cases (Garnick and Griffin, 1983; Bellone et al., 1983).

10.4.3 Bone Marrow Transplantation and Thrombocytopenias After allogeneic as well as after autologous bone marrow grafting, humoral autoimmunity against platelets has

AUTOIMMUNE THROMBOCYTOPENIAS been observed (Bierling et al., 1985; Benda et al., 1989; Marmont, 1992). The principal issue is to distinguish thrombocytopenia due to decreased platelet production and that due to immune pathology. The conditioning suppressive therapeutic regimen and viral infections after the graft can impair megakaryocytopoiesis. When thrombocytopenia persists for a long duration after bone marrow graft, the role of graft versus host disease has to be considered, and could be at the origin of developing autoimmunity. Another aetiology for immune thrombocytopenia is the adoptive autoimmunity clinically identical to AITP. Some reports describe this occurrence with the identification of anti-platelet antibodies in the donor as well as in the patient. In one case, although antibody was present in the donor, thrombocytopenia was only observed in the recipient, with good response to i.v. IgG and no subsequent relapse (Minchinton et al., 1982). In another case, splenectomy was the only way to achieve remission (Spruce et al., 1983). In one report, thrombocytopenia was observed after the remission of red cell aplasia following ABO incompatibility between donor and recipient. Platelet antibody was present, co-existing with an increase in platelet production documented by elevated megakaryocytopoiesis (Marmont, 1994). Recently a case of transmission of AITP by solid organ transplantation was documented (Friend et al., 1990). The donor had AITP and died of intracerebral haemorrhage. With the development of specific tests for evaluating the presence of true anti-platelet antibodies, it has been shown that the same targets as in AITP could be implicated: GPIIb-IIIa and GPIb. Thus it could be considered that donor autoreactive B cell clones and immune dysregulation in the recipient could lead to immunologically mediated destruction of platelets.

10.5

THROMBOCYTOPENIA AND PARASITIC INFECTIONS

The majority of patients with malarial infection have thrombocytopenia. It was postulated that DIC was responsible but most patients with malaria do not have DIC. In contrast elevated levels of platelet-associated IgG have been reported which return to normal as the thrombocytopenia resolves and while the patient continues on anti-malarial drugs. The increased platelet-associated IgG could represent immune complexes adsorbed to platelet Fc receptor or immunoglobulin binding to plateletbound malarial antigen (Kelton et al., 1983).

11.

Acknowledgements

The assistance of Professor Reuben Mibashan and of Monique Dehan in the preparation of this manuscript is most gratefully acknowledged. We thank T. Caetano for

187

secretarial assistance. This work was supported in part by the Fondation de France 1992.

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10. The Analysis of Eicosanoids Derived From P latelets Jacques Maclouf and _&ida Habib

1. Introduction 2. General Considerations 2.1 Conceptual Considerations 2.2 Analytical Considerations 3. Eicosanoids Derived from in vitro Studies 3.1 Bioassay 3.2 Chromatographic Analysis 3.2.1 Extraction 3.2.2 HPLC Analysis 3.2.2.1 Radioactivity

195 196 196 197 198 198 198 198 199

1. Introduction Under normal circumstances, platelets circulate passively as they flow along the vascular tree lined by a monolayer of endothelial cells. The major function of these cells is however to seal breaks in blood vessels after recognition of small damages in the endothelial lining from the vasculature. The process by which platelets cease to be tranquil, discoid components of blood and rapidly respond to specific or non-specific stimuli after adhesion, shape change, activation and secretion is the result of loss of balance between excitatory and inhibitory signals. Most of these responses are caused by translation of these extracellular signals into a remarkably narrow number of intracellular messengers. Activation also involves the fusion of membranes of the open canalicular system and of the intracellular granules to the plasma membrane to become confluent with the plasmalemma that will allow the release reaction. In addition, the plasma membrane provides lipid substrates for the production of arachidonic acid (AA), the precursor of eicosanoids as well as other intermediates Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

Detection: Exogenous vs Endogenous UV Detection

199 3.2.2.2 200 3.3 Immunoassays 201 4. Assessment of the in vivo Production of Eicosanoids 204 4.1 General Considerations 204 4.2 Practical Considerations 205 5. Conclusion 206 6. References 207

implicated in platelet activation. Because lipids are involved in platelet reactivity, analysis of these substances either representing a structural component or a metabolic function have to be investigated at the resting state or after appropriate activation. In this chapter only the latter aspect of the formation and analysis of soluble lipid mediators originating from AA will be addressed. Under normal conditions, activation of platelets results in the formation of three major eicosanoids, i.e. thromboxane A2 (TXA2), 12(S)-hydroxy5,8,10-heptadecatrienoic acid (HHTrE; deriving from the transformation of the unstable cyclooxygenase (CO) intermediate prostaglandin endoperoxide PGH2) and 12-hydroxyeicosatetraenoic acid (12-HETE), which is the result of a 12-lipoxygenase (12-LO). The role of TXA2 as an amplifying signal of platelet activation and for the recruitment of vicinal platelets is well established; in contrast, the functions of 12-HETE are still elusive. Although, strictly speaking, eicosanoids derived from platelets correspond to those described above (i.e. constitutive metabolism), data from recent years have pointed out that in a multicellular environment, the Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

196 J. MACLOUF AND A. HABIB metabolic potential of these cells can be dramatically modified. Under such situations, the production of platelet-derived metabolites resulting from cellular coincubates does not correspond to the sum of each individual cell metabolism; this mode of formation has been termed "transcellular metabolism". In this section, we shall briefly describe such situations when these "indirect platelet metabolites" are synthesized and detail some aspects related to their analysis. Historically, the first example of such metabolism was described from the observation that [3H]AA-labeled platelets stimulated in the presence of aspirin-treated endothelial cells (i.e. having an irreversibly inactivated CO) produce [SH]6keto-PGFl~, the stable hydrolysis product of prostacyclin in addition to the normal production of plateletspecific metabolites (Marcus et al., 1980). Such formation originates from the transfer of platelet-derived endoperoxides (donor cells) to prostacyclin synthase of endothelial cells (acceptor cells). Marcus (1990) subsequently coined the term "transcellular biosynthesis" to describe this process. Other examples of this metabolism in platelets incubated with other cells have been described (reviewed in Maclouf et al., 1989). Platelet-derived 12-HETE can be metabolized by the 5-LO of activated neutrophils into the dioxygenation product, 5(S),12(S)-dioHETE, a stereoisomer of leukotriene B+ (LTB4) with little biological activity (Borgeat et al., 1981). Conversely, 5HETE formed by the neutrophils can also be metabolized into 5(S),12(S)-di-HETE by the platelet 12-LO. During similar co-incubations, platelet activation occurring in the presence of resting neutrophils generated 12,20-di-HETE as a result of the metabolism of 12HETE by cytochrome P450 activity of the neutrophils (Fitzpatrick and Murphy, 1989). Interestingly, during these conditions, no 5(S),12(S)-di-HETE could be detected suggesting that the formation of this metabolite depends strictly on the activation of 5-LO (Marcus et al., 1984). Another recent example of transcellular metabolism has been identified for the formation of leukotriene C4 (LTC4), a potent vasoconstrictor of small and large vessels. Co-incubation of platelets in the presence of activated neutrophils results in the formation of this metabolite which neither cell alone can produce (Maclouf and Murphy, 1988). Such a property of platelets is very unusual since the metabolic capacity of these cells to produce LTC+ does not depend on their activation. In addition this reaction is not affected by aspirin, conferring to platelets the potential to generate a potent vasoconstrictor in spite of the CO blockade. This reaction is only limited by the amount of leukotriene A4 (LTA4) generated by the vicinal neutrophil. In vitro, platelets have a huge capacity to transform neutrophil-derived LTA4 into LTC4, probably in excess to that of neutrophils to generate LTA4 (Habib and Maclouf, 1992). The mode of biosynthesis of this compound (transfer of substrate

from neutrophil to platelets or vascular cells) and subsequent activity on its cellular target (e.g. constriction of vascular smooth muscle cell) constitutes an amazing example of paracrine sequences. Because lipid mediators have an important role in the amplification of the signal-transduction signalling (autocrine mechanism) and in the recruitment or stimulation of vicinal cells (paracrine function), analysis of these substances either in vitro on isolated cells or as a reflection of the in vivo activation is an important issue. Analysis of lipid constituents of platelets is a challenging task since both remodelling of the structural species as well as the production of autacoids derived from their metabolism can occur artefactually during the simple process of blood collection. Because of the extreme sensitivity of the cells to activation, great care should be taken to standardize blood collection, use the correct anticoagulant ethylenediamine tetraacetic acid (EDTA), citrate, ACDC or ACD-A (acid citrate dextrose anticoagulant, National Institutes of Health, formula A and C) supplemented with prostaglandin E1 (PGE1) or prostacyclin (PGI2), indomethacin or aspirin), as well as in handling the samples as rapidly as possible in order to avoid undesired production of metabolites in vitro. These steps are additional to the care taken for the analytical procedures. Most of the methods used to investigate lipid mediators from platelets are quantitative and we will deliberately focus on these because they help to establish the relationships between the production of a mediator and its effector function. In addition, the procedures described here can be applied to other cells. Excellent references for analysis of various lipid compounds (as well as other related topics such as protein purification or assays, enzyme activity or receptor functions, etc.) can be found in the series Methods in Enzymology, Vols 86 and 187 (Academic Press).

2. General Considerations 2.1

CONCEPTUAL CONSIDERATIONS

Not strictly inherent to this chapter, but very relevant to autacoids is the question of what to measure and when. This specific consideration represents probably the major difference between the quantitative assessment of hormones vs local mediators. Measurement, for example, of thromboxane B2 (TXB2) as a reflection of TXA2 production is a good index of platelet activation in vitro under controlled circumstances. However, the possibility that this compound can be formed artefactually during platelet preparation - which may occur during blood sampling - is a serious drawback for its use as a reliable index of in vivo production by measuring plasma levels. It has been pointed out by others (Patrono et al., 1986) that the capacity of platelets to generate TXB2 in 1 ml human blood that has been allowed to clot is roughly

ANALYSIS OF EICOSANOIDS 197 300-400 ng/ml, which corresponds to the total amount of TX generated per 24 h in a normal subject. As little as 0.1% of platelet activation during blood collection is enough to account for the hypothetical circulating concentrations of this mediator, ranging from 10 to 300 pglml as compared to the calculated circulating 2 pg in peripheral blood. Additionally, the production of TXB2 generated under controlled studies of in vitro platelet activation can be seriously affected if the subject has ingested aspirin or other non-steroidal antiinflammatry drug (NSAID) 1-7 days prior to blood collection (FitzGerald et al., 1983a). These considerations are totally independent from the quality (i.e. correct use and validation) of the assays. Consequently, for these reasons measurement of plasma values of eicosanoids is very seldom useful, except in very well-defined conditions (brief, controlled stimulation, veno-arterial gradient, etc.). In contrast to these disadvantages, work from recent years has established that the measurement of urinary metabolites represents a reliable index to evaluate the in viv0 production of TX or PGI2 deriving from platelets or vascular cells (Patrono et al., 1989). Very recently the same approach has been found to be valid for the analysis of urinary metabolites of LTC4 excreted from the vascular compartment (Maclouf et al., 1992). This approach has the advantage of being non-invasive and also independent from the previously mentioned artefacts inherent to uncontrolled in vitro biosynthesis during the collection of biological samples. However, because LTC4 is an important mediator of asthma and allergy, the significance of LTF_4 measured in urine as a reliable index of a vascular event can only be considered after having ruled out any susceptibility of the subject to allergy. In all cases measurement of these compounds and interpretation of the values has only a significant meaning in the clinical and/or therapeutic context provided by independent criteria.

2.2

ANALYTICALCONSIDERATIONS

Eicosanoids are polyunsaturated molecules that require some special attention to avoid artefactual degradation that may irreversibly alter their structure. Supernatants from cell incubates or other biological fluids (i.e. urines or plasma or serum) should therefore be kept at - 2 0 ~ for short term storage (i.e. up to 4-6 weeks) and at - 7 0 ~ for longer periods of time. This is also very critical for stock solutions of standards that will be relied upon for accurate quantitation; standards should be dissolved in degased solvents such as methanol:water (50: 50, v/v) and kept at -70~ Intermediate solutions should be kept frozen (-20~ and discarded, at regular intervals (e.g. every 6 months or every year). Special care should be taken for compounds possessing ~-hydroxyketones (e.g. PGE2 or PGD2) which confer a strong instability to these molecules and allow subsequent degradation into

dehydrated molecules such as PGB2 or PGA2 from PGE2 or9-de;;Yd-elolxkye~l:k~to:hydrh;yro~;;~~2-,~,ostprno~teacid acid from PGD2 (Stehle, 1982). In addition, the recent discovery of a series of PGF2~-like compounds that can be formed ex vivo and non-enzymatically from AA in plasma or urine during storage (Morrow et al., 1990) justifies the addition of antioxidants such as butylated hydroxytoluene (0.002%) to the samples. This agent will prevent the in vitro formation of these compounds as well as degradation of peptidoleukotrienes. For most standards the user must depend upon the indications provided on the label of the bottle by the manufacturer. Specific compounds can be purchased with reliable quality from several suppliers (Cayman Chemicals, Ann Arbor, MI, USA; Biomol Research Laboratories, Plymouth Meeting, PA, USA; Cascade Biochem Ltd, Reading, UK). However the purity of other products such as hydroxy-acid derivatives (HETE or HHTrE) or LTC4 (see later) can also be verified by high performance liquid chromatography (HPLC) and quantitated by their UV absorbance using established spectra and coefficients of absorbance (Table 10.1). AA utilized in cellular incubations requires regular purification prior to use in order to avoid the formation - and use - of a peroxidized substrate. A simple purification consists of the use of a small chromatographic open column of 0.5 g silica gel column (Silicar CC4, AR, Mallinckrodt) in 1% diethyl ether in hexane. AA, dissolved in hexane, is deposited on the column and elution is performed using 5 ml 10% ether in hexane (v/v) into a preweighed tube. The amount of AA is determined by gravimetry, weighing the same tube after drying the solvent. It can be stored, dissolved in 100% degased ethanol and the stock solution stored frozen at - 7 0 ~ in aliquots; the solution in use should be kept at - 2 0 ~ and brought at room temperature in the dark (i.e. aluminium foil) prior to utilization. This solution should be discarded every 2 weeks if used frequently. For all these compounds great care should be taken to avoid contamination. All solutions should be pipetted with clean syringes carefully rinsed or using disposable polypropylene tips. The absence of degradation of standards such as the

Table 10.1

Ultraviolet extinction coefficients of some eicosanoids

Compound PGB2 and related 5-, 12-, 15-HETEs HHTrE LTB. 5(S), 12(S)-di-HETE LTC4, LTD4, LTE4

Xmax

(molar extinction coefficient ~) 278 235 232 270 268 280

(19 000) (30 500) (33 400) (50 000) (50 000) (40 000)

198 J. MACLOUF AND A. HABIB various hydroxy-acid derivatives (HHTrE or HETE) or LTs can be verified by HPLC using first a small analytical run to localize the compound. If needed, purification can be done subsequently after collecting all the effluent, in large fractions at first and using smaller volume fractions (e.g. 0.5 ml for a 1 ml/min flow) when getting closer to the retention time of the compound. Quantitation of the purified materials is performed by UV spectroscopy.

0

EicosanoidsDerivedJ om in vitro Studies

These methods focus on monitoring the synthesis of AA metabolites to study the capacity of platelets to produce them in defined conditions of activation. The same approach can be used to measure the generation of these compounds in cellular co-incubations, e.g. under situations when transcellular metabolism occurs.

3.1

BIOASSAY

Often, when dealing with small quantities of biologically active compounds, detection and quantitation of platelet-derived lipid substances were initially performed using biological tests based on their activities. Bioassay has contributed to the discovery that TXA2 was a mediator generated upon platelet activation which possessed a potent rabbit aorta-contracting activity (RACS). It is unquestionable that the importance of these compounds would never have been discovered from the structure elucidation of the chemically stable, biologically inactive TXB2. The discovery of TXA2 and its important role in platelet activation has highlighted a need for quantitative evaluation. TXA: is very unstable (tl/2--30 s in buffer) and although perfectly fitted for bioassay, it is rapidly hydrolysed into the stable but inactive TXB2. With the availability of easy-to-use immunoassays that can monitor the production of TXA2 reflected by the quantitative measurement of TXB:, this technique is no longer employed. Similarly, bioassay has helped to confirm the identification of new biosynthetic properties of platelets in their ability to produce LTC4 upon addition of LTA4 (Maclouf and Murphy, 1988). For those readers interested by the technique, information can be found in Folco and Sala (1987) and Vigano et al. (1990).

3.2

CHROMATOGRAPHIC ANALYSIS

3.2.1 E x t r a c t i o n Before separation by chromatographic procedures, or for most quantitative analyses of complex biological milieux, the sample must be prepared for analysis. Because all the operations related below will involve evaporation of the organic phase, and therefore concentration, it is necessary that all solvents should be of HPLC grade. Most of the

eicosanoids can be extracted by organic solvents by taking advantage of the existence of free carboxyl group of these substances to separate them from fats lacking this function (e.g. glycerides). Most of the time, proteins are removed by adding 4-5 volumes of ethanol during 30 min in order to precipitate insoluble material, which is subsequently removed by centrifugation. All eicosanoids derived from platelets will be found in that phase. The ethanol is evaporated under vacuum and the sample dissolved in water adjusted to pH 8-8.5 with NaOH or NH4OH which transforms eicosanoids into their sodium or ammonium salts. Glycerides or fatty acids can be extracted by petroleum ether. The water is acidified to pH 3-4 with formic acid and it is subsequently extracted twice with diethyl ether or ethyl acetate. The phase should then be washed with water until neutral and evaporated to dryness prior to chromatographic purification. However, substances such as peptido leukotrienes cannot be extracted using this procedure and it becomes very awkward when it comes to the extraction of large volumes of sample. This last decade has seen the widespread use of solid-phase extraction which makes the preparation of the samples faster, easier and more automatable. This technique provides a double goal: partial purification of the sample and concentration of the analytes which, without this enrichment, could not be evaluated no matter how specific and sensitive the means of detection. The most commonly encountered solidphase extraction sorbent is based on porous silica, the surface of which has been modified by chemically bonding a layer of organic molecules such as C18. Because the molecules to be extracted will interact with the phase in several different ways, such as hydrogen bonding, dipolar interactions or electrostatic (ionic) attraction, this technique brings the separating power of liquid chromatography and the speed of ordinary extraction techniques to the sample preparation step. In its most simple use, the cartridge (Bakerbond Octadecyl, J.T. Baker Inc., Phillipsburg, NJ or C18 SepPak Plus, Water/Millipore, Milford, MA, USA) is wetted by 3-4 ml of methanol (for the extraction of the chemically unstable LTs, it can be washed first with 3-4 ml of a methanol:water solution (50: 50, v/v) containing 0.5% EDTA, in order to remove metal traces, followed by 4 ml of pure methanol) and then rinsed in 5 ml of HPLC grade water; it is essential that the cartridge is kept wet during these procedures. In samples containing proteins (serum, plasma, albumin-containing buffers), their precipitation should be made prior to extraction. This is usually done by adding 2-3 volumes of methanol (or acetonitrile) to the sample and allowing the proteins to precipitate for a few hours (or overnight) at -20~ The protein pellet is removed by centrifugation (15-20 min at 1500g). The sample to be extracted (urine, methanolic supernatant of plasma, supernatant of cell incubate) can be run on this cartridge provided the methanol

ANALYSIS OF EICOSANOIDS 199 content (if any) of the sample remains low (10-20% 3.2.2.1 Radioactivity Detection: Exogenous vs Endogenous methanol at most). Because for solid-phase extraction the volume matters little, the supernatant of the protein The simplest approach is to incubate platelets with precipitate can be diluted with distilled water to bring the exogenous isotopic dilutions of [SH]- or [14C]-labelled organic solvent concentration to less than 10-20%. Acid AA. After incubation, the reaction is stopped by addition (usually 0.1% acetic acid or another volatile weak organic of pre-cooled methanol or citric acid to pH 3 or by cenacid such as formic acid) can be added to the samples in trifuging the platelets after addition of ice-cold EDTA order to keep the carboxyl group of eicosanoids totally 0.077 M (1 vol. per 9 vol. platelets). The supernatant non-ionized. After washing the cartridge with 10 ml can be analysed directly by reverse phase HPLC (Russell water, elution can be performed by a small volume of and Deykin, 1979) or after extraction with organic solmethanol (2-3 ml) in polypropylene tubes in which vents or solid phase cartridges followed by analysis by adsorption is reduced. The extract can be dried under TLC (Salmon and Flower, 1982). Profiling analysis of the vacuum for subsequent purifications or analysis (HPLC, constitutive oxidative metabolism of AA by CO/TX synthin-layer chromatography (TLC), immunoassays). thase or by 12-LO can be obtained as confirmation that Powell (1982) has taken advantage of both reverse phase all peaks observed derive from the metabolism of AA. or normal phase properties of these silica cartridges to This technique can be used for screening the effect ofvarobtain a selective elution of the different categories of ious drugs on the different pathways: at the level of oxymetabolites. In the first stage, the stationary phase was genases or on thromboxane synthase. Because it utilizes used for its reverse phase properties (i.e. C18 groups of exogenously supplied, and defined, isotopic dilution, this the phase and aqueous mobile phase of the sample and/or technique can be quantitative by counting the radioacsolvent); in subsequent steps, after removal of the water, tivity found in the individual metabolites (TXB2, PGs or it was used as normal phase chromatography (i.e. taking the hydroxy fatty acids 12-HETE and HHTrE; Sors et advantage of the free remaining silica groups of the solid al., 1978). Figure 10.1 shows the HPLC profile of phase). Under these last conditions, elution is performed human platelets incubated with exogenous [14C]-AA in different conditions. The upper panel (A) shows control by increasing the solvent polarity. incubation; as can be seen, platelets generate TXB2, 3 . 2 . 2 H P L C Analysis HHTrE and 12-HETE as the main metabolites. TreatThis method allows a rapid, reproducible separation and ment of platelets by indomethacin totally abolishes the purification of closely related compounds; it has become CO activity thus resulting in an increased production of widely utilized for the purification of biological samples 12-HETE and a rise in unconverted AA (panel B). from complex matrixes prior to quantitation by However, incubation of platelets with various amounts immunoassays (see later). It is also quantitative although of the TX synthase inhibitor imidazole dose-dependently detection of compounds remains a major limitation inhibits the formation of TX and shows an increase of because of a relative lack of sensitivity at the detector PGE2 and PGF2~ as a consequence of the accumulation level. Due to the great variety of conditions which can be of PGH2 and its transformation by isomerases combined used, this technique is extremely powerful for the separ- to non-enzymatic hydrolysis (panels C and D). Another approach uses the labelling of phospholipids ation of compounds. Column and particle sizes support (normal and reverse phase) and eluting solvents are all by incorporation of labelled AA in the platelet phoscritical variables which can be manipulated for optimal pholipids; this is usually done by incubating washed resolution. The more usual columns are 4.6 x250 mm platelets with either [ 14 C ] - o r [3H]-AA for 1-2 h. with particle sizes of 5/~m for analysis or 10 #m for purifi- Labelled cells are washed with a buffer containing 1-2% cation. Although the use of 3 #m particles and capillary albumin in order to remove the unincorporated substrate columns has brought a tremendous increase in resolution (usually less than 10%). Challenge of cells with the and sensitivity we do not believe that they are as versatile appropriate inducer (e.g. thrombin, ionophore), allows to support both analysis and purification purposes on study of substrate liberation from endogenous sources complex biological media. The normal phase (straight and its subsequent oxidation as evaluated by analysis of phase) support is used for separation or analysis of the various radioactive compounds including nonnon-polar metabolites (HETE, HHTrE, AA) using a metabolized AA (Russell and Deykin, 1979). The study mobile phase mostly of hexane containing a small per- of the formation of the different metabolites is done by centage of 2-propanol (isocratically or in a gradient) TLC or HPLC. This method can also, theoretically, and 0.01-0.1% of acetic acid. This phase also pro- allow study of the distribution of AA in the different vides a good resolution between 12-HETE from its phospholipid pools before and after stimulation of the 12-hydroperoxy precursor (Porter et al., 1979). same samples. However, other studies have shown that, However, the most common techniques use reverse relatively short-term labelling (a few hours) in order to phase C18 silica columns because they also allow separ- preserve the cellular functions of platelets, did not allow ation and analysis of very polar components such as the label to reach equilibrium between the different phospholipid pools (Capriotti et al., 1988), contrasting with peptidoleukotrienes.

200

J. MACLOUF AND A. HABIB

cells in culture that can be incubated over 24 h prior to stimulation. Simple cell labelling cannot provide a definitive answer to the origin of the prostanoid precursor pool because of the complex manner in which arachidonate species are shuttled among various phospholipids when cells are activated. More complex protocols of pulse label (from 5 min to a few hours) and calculations of the specific activities of the different compounds under these conditions should be performed. Careful interpretation of the results should be made, especially in order to draw

A

Control

quantitative information on the metabolism of AA by this technique. Nevertheless, this approach has been used extensively in biochemical and pharmacological investigations.

3.2.2.2 U V Detection The most convenient conditions are related to the analysis of metabolites having intrinsic UV absorption properties (i.e. allowing direct detection) such as the various HETEs or H H T r E which absorb at 235 nm with a

B

+ indomethacin

==

E>.

p

D

+ imidazole. 1 mM

3

4 2

=2

15

15

Retention time (min)

Retention time (min)

Figure 10.1 Reverse phase HPLC chromatogram of the metabolites contained in the supernatant of human washed platelets incubated with [14C]AA. Platelets, 1 ml, 0.3 x 107/ml were Incubated with 0.048 #Ci of [14C]AA. After 5 rain, the reaction was stopped by addition of 2 vols of ice-cold methanol. After overnight at -20~ the incubates were centrifuged for 15 min at 3000 g. The supernatant was collected, diluted in water (10-15% methanol, final concentration) and extracted on a C18 cartridge (Bakerbond); elution was performed with 3 ml of 100% methanol. The samples were dissolved in 1 ml 10% methanol in water and centrifuged in an Eppendorff centrifuge for 20 min at 10 000 g. The supernatant (20%) was injected onto an Ultrapack octadecyl reversed phase column (5 #m, 250 x 4.6 mm) using an LDC gradient HPLC system (GM 4000, LDC, Riviera Beach, FL); a Guard-pack precolumn (Waters Instruments) was also used. A non-linear gradient started with 100% solvent A (acetonitrile: water: acetic acid, 10 : 90 : 0.01, v/v/v) going to 100% of solvent B (100% acetonitrile containing 0.01% acetic acid): 0 - 1 0 rain, linear to 20% B; 10-20 min, linear to 45% B; 20-28 min, linear to 80% B; 30-34 min, linear to 100% B; re-equilibration was started at 35 min. Detection of radioactivity was performed using an on-line HPLC scintillation detector (Ramona 90, Raytest, Germany). (A) Control platelets with [14C]AA. (S) 5/~.M indomethacin. (C) 100 FM imidazole; (D) 1 mM imidazole. Peak 1 corresponds to TXB2, peak 2, PGE2, peak 3, HHTrE, peak 4, 12-HETE and peak 5, AA.

ANALYSIS OF EICOSANOIDS 201 molar extinction coefficient of 30 000 due to their conjugated dienes. LTs have an even better coefficient since the triene structure of LTC4 or other peptidoleukotrienes provides a coefficient of 40 000 at 280 nm and that of LTB4 is 50 000 at 270 nm. As seen above, platelets have the capacity to convert LTA4, derived from the 5-LO of neutrophils, into LTC4 by transcellular metabolism. Production of this compound can easily be assessed by HPLC (Fig. 10.2) or by immunoanalysis (see later). The excellent absorbing properties of LTs in UV renders this method very appropriate for the measurement of these substances. In this case, quantitation is better appreciated by the use of the internal standard method. This technique requires the addition to the biological sample of a defined component with known concentration for comparative determinations. The substance used for this purpose should never be found as a normal component of the solute and should completely resolve from sample component peaks. Since the internal standard is of defined constants, I

Platelets, 30 min

-

2

3

l

0

20

4

Retention time (min)

40

Figure 10.2 Reverse phase HPLC separation of LTA4 metabolites after incubation with human washed platelets. Platelets (5 x 108/mi) were incubated in a physiological buffer at pH 7.4, containing 1 mg/ml albumin. After 30 min, the reaction was stopped by addition of 2 vols of ice-cold methanol containing 200 ng 19-hydroxy-PGB2 as an internal standard. After overnight at - 2 0 ~ the sample was centrifuged and an aliquot was injected to a C18, 5 ~m column (same equipment as Fig. 10.1). A non.linear gradient programme was used: 0 - 6 min, linear to 35% B; 6 - 3 2 min, linear to 65% B; 3 2 - 3 3 min, linear to 100% methanol, with solvent A (methanol: water: acetic acid 40:60 : 0.05, v/v/v, buffered to pH 5.7 with ammonium hydroxide) to 1000/0 solvent B (100% methanol). Peak 1 corresponds to 19-OH-PGB2, peak 2 to LTC4 and peaks 3 and 4 to the non-enzymatic hydrolysis and methanolysis products of LTA4, respectively.

it will compensate for variations in losses during purification or analysis (e.g. for peptidoleukotrienes), as well as sample injection size and any manipulations of the samples. The mechanism for quantitation is based upon comparison of the peak heights (or areas) for both the metabolite(s) and the internal standard. In the analysis, such as is shown in Figure 10.2, 19-OH-PGB2 and LTC4 have obvious differences in their physicochemical natures. It is therefore necessary to better define the response factor by mixing (and injecting) known concentrations of pure internal standard with various concentrations of LTC4. Such measurements allow to define the concentration ratio for which the two compounds (internal standard and LT) have a linear range of response; concentrations of the two should be in the same range. Another problem specific to the analysis of LTs concerns the degradation ofpeptidoleukotrienes on the column by contaminants suspected to be trace metal elements (Wescott et al., 1984). Extensive washing of the column and HPLC components (e.g. tubing, fritts) with a solvent containing 0.5% EDTA (20-40 ml) usually suppresses the problem. EDTA in the solvent is not recommended because of the possibility of salt precipitation in the pumps if left unused and consequent irreversible damage to the seals and other components of the pumps. Another alternative for those metabolites that do not possess a UV absorbing function consists of the derivatization of the compounds (i.e. covalent attachment of chromophores on reactive functions of the eicosanoid to be studied, mostly the carboxylic group) with a sensitive detectable probe such as the para-substituted 'phenacyl esters. This approach has been used successfully for prostanoids (Morozowich and Douglas, 1975; Fitzpatrick, 1976) or fluorobenzyloximes on the carbonyl group (Fitzpatrick et al., 1977). However, none of these latter procedures has enjoyed widespread use owing to the problem of derivatization leading to a range of sensitivity well below that attained by other techniques such as immunoassays or gas chromatography-mass spectroscopy (GC-MS). The use of a radioactive detector online (see above) is also convenient to localize the products, confirm the presence ofAA-related metabolites and give a relative quantitation of the different compounds resulting from the incubation of the cells with radiolabelled AA or the use of prelabelled cells. The use of sensitive diode-array detectors also allows good confirmation of the identity of the compounds by establishing the spectrum specific to the intrinsic W - a b s o r b i n g moieties (diene structures of HETEs or H H T r E ; triene structures of LTs). They are however still rather expensive pieces of equipment and, if one wants to use it for quantitation, less sensitive than conventional fixedwavelength detectors.

3.3

IMMUNOASSAYS

When first introduced for the measurement of eicosa-

202

J. MACLOUF AND A. HABIB

noids, this technique provided an easy and reproducible biochemical monitoring of platelet activation. Measurement of TXB2 allows a good quantitative evaluation of platelet stimulation in vitro under controlled conditions (e.g. different inducers in the absence or presence of molecules interfering at different steps of platelet activation); under most circumstances, it reflects their biochemical capacity to generate lipid signalling molecules. Eicosanoids belong to the class of haptens; the raising of antibodies therefore necessitates covalent coupling to a macromolecular carrier such as serum albumin, keyhole limpet haemocyanin or thyroglobulin in order to perform immunizations. For the interested reader, many excellent reviews have described the production of antibodies and specific protocols for eicosanoids (Ciabattoni, 1987). After raising the antisera, the technique requires the use of a labelled antigen to detect antibodies and evaluate the formation of antibody/antigen complexes (see later). In its early phases, the technique suffered from serious misuse due to little concern on the relative absence of specificity and several factors intrinsic either to the chemical instability of some of the molecules (e.g. PGE2). The relative importance of non-specific interfeting factors such as pH, ionic environment, temperature, anticoagulants, damage to the tracer, etc. is exacerbated by the low concentration of eicosanoids (fM) in complex biological matrices and has been reviewed elsewhere (Granstrrm and Kindahl, 1978). Analysis of these substances in complex biological matrices having a high lipid environment, e.g. plasma, may require an extraction step to assay trace amounts of some metabolites; it can be followed by chromatographic purifications (see later). 500

Since TXB2 is the major platelet metabolite, we shall emphasize analysis of this compound, although in some situations it is of interest to monitor other products such as PGE2 or PGD2. Various radioimmunoassays for TXB2 have been described (Fitzpatrick, 1982). Most rules that apply for one compound are also valid for others. The majority of research or commercially available reagents for TX or related compounds are similar with respect to their sensitivities or specificities. The amount of TX generated by platelets upon in vitro activation is elevated; consequently most investigators use direct immunoassays to measure platelet production in vitro (suspended in plasma or in buffer) or after purification for urine analysis (see later). It is important to have a critical evaluation of the limits of specificity of the assay as cross reactivities defined with structurally known compounds may not be very relevant for the analysis of biological fluids. Few people have access to sophisticated independent methods such as GC-MS (see later) or can afford to make a comparison between results obtained by direct measurements and those where the samples have been extensively purified. A simple test of the validity of an assay can be performed by analysing samples under conditions in which the results can be anticipated, such as the loss of immunoreactivity contained in the supernatant of platelets stimulated in vitro when the cells have been pretreated with an established CO inhibitor such as aspirin or indomethacin (see Figure 10.3). Suppression or modulation of immunoreactivity by drugs that are known to inhibit or alter the biosynthesis of metabolites constitutes a serious indication that detection of immunoreactivity in control cells corresponds to the analyte to be measured.

-

I TXB2 PGE 2

400

300

\\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\

200

I00

0

1

2

3

4

5

Figure 10.3 Analysis of TXB2 and PGE2 by enzyme immunoass.ay of supernatants of washed human platelets. Platelets suspended in plasma were incubated with AA in the absence or presence of indomethacin or of various concentrations of imidazole. 1, Unstimulated control platelets; 2, control platelets incubated with 0.6 mM AA; 3, same as 2 with 5 FM indomethacin; 4 and 5, same as 2 in the presence of 0.1 and 1 mM imidazole, respectively. For details on enzyme immunoassay, see Fig. 10.4.

ANALYSIS OF EICOSANOIDS 203 In addition to specificity, sensitivity can also be a legitimate concern that may hamper the use of the assay. In some cases, the antibody (i.e. low affinity) and/or tracers (i.e. insufficient specific activity) can limit the sensitivity. Another option is the selection of an alternative label. [SH]TXB2 or PGs were the first easy-to-use ligands because of a long shelf-life and availability at high specific radioactivities (e.g. > 120 Ci/mmol, 4.4 TBq/mmol). However labels such as [12SI]histamine or tyrosyl methyl ester coupled to TXB2 have been developed (Maclouf et al., 1976, 1978; Sors et al., 1977). They are now commercially available (Amersham or New England Du Pont) and present several advantages: (1) they have a higher specific activity ( > 1 0 times that of tritium, i.e. 2200 CilmA, 80.5 TBq]mA); (2) they avoid problems of quenching and use of costly liquid scintillation cocktails and require a short time for counting (usually i min); (3) they represent a cheap way of circumventing the problems inherent to radioactive waste because their relatively short half-life (2 months) allows the user to let radioactivity decay in a storage place. Because of the absence of appropriate positions in which the molecules could be labelled directly without drastically changing their structure, eicosanoids have to be conjugated to another molecule (usually using an active ester of the PG

100

-

80

-

60

-

or TX that will be hydrolysed by an amine, e.g. tyrosyl methyl ester or histamine) to give an amide (Maclouf et al., 1978). The purified conjugate can be stored for several months; an aliquot is taken to perform the iodination. Usually, labelling with 12sI is done using the chloramine-T method. Labelled compound is purified from unlabelled and other side-reaction products to keep the high specific radioactivity of the iodine (2000-4000 Ci/mmol). Based on a literature survey and from our own experience, labelling of peptidoleukotrienes such as LTC4 or LTE4 with iodine cannot be achieved very easily. Enzymatic tracer represents probably the only alternative for obtaining a labelled LT other than tritium. Recently, non-isotopic alternatives have been introduced (Hayashi et al., 1981, 1983; Miller et al., 1985; Pradelles et al., 1985; Hiroshima et al., 1986) with obvious advantages in the context of tightened legislation to limit the use of radioactivity in the labs as well as because of increasing costs of radioactive disposal and the need for special facilities. Enzyme labels provide a good alternative for immunoassays of eicosanoids in a lab not approved to handle radioactive elements. Also, their long shelf-life represents an advantage over the use of radioactive isotopes with high specific activities such as 12sI, -

2

-

1 O

v

-

o

1:13 40

0

-

o -

--1

20-

0

I

I

10

100

"8

-2 1000

TXB e (pg/ml) Figure 10.4 Enzyme immunoassay (EIA) standard curve for TXB2 using acetylcholinesterase covalently linked to TXB2 as tracer. (Pradelles et al., 1985). Known amounts of TXB2 ( 0 . 1 2 - 2 5 pg/well) were added to separate wells of the microtitre plate previously coated with mouse monoclonal anti-rabbit IgG antibody (2/~g/well). Enzyme tracer (50 #1) and specific antibody (50/~1) were added. After overnight incubation at 4~ TBX2 bound to the antibody was separated from free TXB2 washing the plate using an automatic washer (Labsystems, Helsinki, Finland). The plates were automatically filled with a medium containing the synthetic substrate, acetyl thiocholine, and EIIman's reagent, 5,5'-dithiobis-2-nitrobenzoic acid, using an automatic dispenser. The production of a yellow coloured product was measured at 414 nm by a plate reader (Titertek MuItiskan, Labsystem). The standard curve is run using duplicates and calculation are made by fitting the standard curve (or of TX in samples) with a non-linear curve fitting programme.

204 J. MACLOUF AND A. HABIB

v

0 0 . r--.i

""9

20

0

0

f,..,i 9 CJ ot-i

9

0

0

1

I

I

I

10

100

1000

TXB

2 (pg/ml)

Figure 10.5 Precision profile of analysis TXB2 by EIA. The precision profile was established on each concentration of TXB2 from six dose-response curves run on different occasions; results are expressed in terms of the coefficient of variation (i.e. interassay variation, CV, %) vs the logarithm of the dose.

which has a relatively short half-life raising the problem of frequent labelling. The use of enzyme immunoassay (EIA) techniques for eicosanoids has expanded: (1) because the sensitivity of the assays is equal to that achieved with corresponding radioimmunoassays; (2) the labels possess a very long shelf-life; (3) they provide the possibility of using microtitre plates allowing full automation. These assays should gain increased popularity in the scientific/medical community as such reagents have become commercially available from several companies (Advanced Magnetics, MA, Amersham, UK or Cayman Chemical, Ann Arbor, MI, USA). Figure 10.4 shows a dose-response curve of TXB2 using an enzymatic tracer. When this curve was run on six different occasions and all values averaged, the coefficient of variation was low at all concentrations except at both extremities of the curve (Fig. 10.5). For the measurement of in vitro production of LTs, measurement of LTC4 may be biased by rapid metabolism. In plasma/blood, peptidase activities have been shown to very rapidly degrade LTC4 into a mixture of LTD4 and LTE4; measurement of the latter may be more appropriate to evaluate the synthesis of peptidoleukotrienes. For that purpose, the conversion of residual LTC4/LTD4 into LTE4 can be done after addition of gamma glutamyl transpeptidase and leucine amino peptidase to the biological sample prior to any extraction or purification (Heavey et al., 1987; Antoine et al., 1991).

4. Assessmentof the in vivo Production of Eicosanoids 4.1 GENERAL CONSIDERATIONS TXA2 plays an important role in the various loops of amplification of platelet activation. In v/v0, its function in pathological situations is demonstrated by the beneficial therapeutic use of aspirin (Willard et al., 1992), an NSAID which exerts its effect by irreversibly acetylating CO (Smith, 1992). It seemed logical to try to use TX measurement as an index of the state of pathology in which aspirin had a beneficial impact, i.e. cerebrovascular accidents, prevention of stroke, unstable angina, etc. Because of the various problems encountered with measuring circulating blood levels, it was rapidly realized that plasma values are useless (see previous discussion). Recent studies, however, combining metabolism and pharmacology, have succeeded in establishing that some urinary metabolites can serve as reliable indexes ofplatelet activation/involvement in vivo. TXB2 undergoes two main metabolic pathways in humans; one is f-oxidation resulting in the formation of 2,3-dinor-TXB2 and the other involves dehydrogenation of the hemiacetal alcohol group at C-11, resulting in the formation of a series of metabolites with a &lactone ring structure such as 11dehydro-TXB2 (reviewed in Patrono et al., 1992). This latter metabolite has a substantially longer plasma half-life and is excreted at a higher rate than 2,3-dinor-TXB2

ANALYSIS OF EICOSANOIDS 205 As mentioned before, these compounds exist in minute amounts in complex biological matrices. Interfering material is mainly due to unrdated substances (e.g. nonspecific materials interfering in the antigen/antibody reaction) that exceed the metabolites to be assayed by several order of magnitude, or because of structural relations (e.g. urine where 15-20 metabolites for a given eicosanoid can be present). Purification prior to analysis is therefore a prerequisite to measure urinary samples, regardless of the specificity of the antisera or the specific activity of the tracer. This purification can be achieved for prostanoids using TLC. Immunoassays of urines are performed after extraction/purification steps in order to eliminate nonspecific interfering background as well as for concentration purposes. Solid phase extraction with a C18 cartridge (see above) is the most popular technique used to concentrate the urine sample (1-20 ml, i.e. enough to get above background; although it depends on the sensitivity of the assay, a volume corresponding to 0.1 mmol creatinine is usually enough). This is usually followed by TLC purification (Patrono et al., 1985; Lellouche et al., 1990). Because urinary extracts can very easily clog HPLC systems, we prefer to use thin layer plates for routine analysis in the purification of metabolites derived from CO. The plates (Silicagel G, 20 x 20 cm) are washed in methanol: chloroform (1:1, v/v) and activated by heating for 1 h at 110~ prior to application of the samples. The biological extracts, dissolved in 100 ~1 of methanol: chloroform (30: 70, v/v), are applied in a

(Catella and FitzGerald, 1987). Similar work has established that 2,3-dinor-6-keto-PGFl~ is the major urinary metabolite of PGI2 (Brash et al., 1983). In contrast, urinary TXB2 and 6-keto-PGFl~ are thought to reflect mainly renal origin. Measurement of urinary metabolites has turned out to be invaluable to support the involvement of platelets in the pathology of cardiovascular diseases or to follow anti-platelet therapy as reviewed in Patrono et al. (1992). As seen earlier, platelets, and vascular cells, have a major capacity to metabolize LTA4 derived from neutrophils into LTC4. This compound, which possesses potent vasoconstrictor properties, may play an important role in vaso-occlusive episodes. Because this pathway is not sensitive to aspirin, the role of platelets in vasospasm can remain potentially important (Carry et al., 1992). Similarly to the metabolic analysis of PGI2 and TX, recent studies have shown that LTE4 represents a good reflection of the production of LTC2 by the vascular route (Maclouf et al., 1992).

4.2

PRACTICAL CONSIDERATIONS

All initial methods that have established metabolic studies have been performed by GC-MS, which remains a physicochemical method of reference. This technique is still in use in labs equipped with these instruments and it has also been used to validate immunoassays (Wescott et al., 1986; Ciabattoni et al., 1987; Lellouche et al., 1990). We shall however deliberately focus on immunoassays because they are more easily accessible.

Standards

Extracts

~Solvent front

/

C

I .

9

9

9

9 D

q~,

dlnor-6-keto-PGF~. 11-dehydro-TXB2

Scraping zon~B I.. .

I

:

.

o O

6-keto-PGF,~ TXB~ dinor-TXB2

~Start

Figure 10.6 Setting of the TLC plate for the separation of urinary metabolites of TXA2 and PGI2. The plate was developed using chloroform : methanol: acetic acid : water (90 : 8 : 1 : 0.8, v/v/v). Visualization was done using a 3.5% phosphomolybdic spray after carefully masking the adjacent lanes containing the biological samples. Three zones were scraped off corresponding to: A, dinor-TXB2, B, TXB2, 6-keto-PGFl,, 11-dehydro-TXB2; C, 2,3-dinor-6-keto-PGF1,.

206

J. MACLOUF AND A. HABIB

linear spot (approximately 2.5 cm, four samples/plate, see Fig. 10.6); corresponding standards (1-2/~g each) are spotted in an isolated lane as dots. The spot size should be kept to a minimum using a capillary and if multiapplications are needed, it is essential that the first application is totally dry before the second is performed. Great care should be taken to avoid contamination by standards of the lanes containing the urinary extracts (as even 0.1% contamination is overwhelmingly high compared to the quantity contained in the biological samples); the chromatographic tank should be rinsed carefully with methanol between the different runs. After migration, the lanes corresponding to the samples are masked with aluminium foil and the compounds located by visualization of standards using a colorimetric reaction (spraying 3.5% phosphomolybdic acid in ethanol and heating the zone with a hot airgun which gives bluish spots on a yellow-green background). As can be seen, the TLC system achieves separation of the metabolites (Fig. 10.6) and completes purification from unrelated substances. At this stage, we usually tape the lane containing the standards in order to avoid contamination. Three zones are scraped corresponding to: dinor-TXB2 for the first one; TXB2, 6-keto-PGF~ and 11-dehydroTXB2 for the second one; and finally dinor-6-ketoPGFI~. Elution of the compounds off the phase can be done by adding directly the assay buffer to the different silica fractions; tubes are centrifuged in an Eppendorf centrifuge (12 000g) and the supernatants collected. After careful vortexing the immunoassays are performed on these eluates. In order to simplify the assay procedure and because TXB2 and 6-keto-PGFl~ are resolved from their closely structurally related parent dinorcompounds, the cross-reactivity of these compounds with the TXBz/6-keto-PGFI~ antisera can be used (Lellouche et al., 1990); i.e. anti-TXB2 antiserum using TXB2 or dinor-TXB2 standards and an anti-6-ketoPGFI~ antiserum with 6-keto-PGFl~ or dinor-6-ketoPGFI~ standards, respectively; the third immunological system is 11-dehydro-TXB2. A radioactive substance (usually [aH]TXB2) is added prior to all steps in order to correct for recovery assessment of the purification; appropriate corrections should be made depending on the recovery of a given metabolite compared to that of TXB2 (Lellouche et al., 1990). Procedures validated by GC-MS have demonstrated the reliability of such methods when handled properly (i.e. with purification prior to the assays; Ciabattoni et al., 1987, Lellouche et al., 1990). For those compounds such as LTs that cannot be purified on TLC because of the nature of the interactions between their peptidic moiety and normal silica, reversephase HPLC provides a good alternative (Heavey et al., 1987; Antoine et al., 1991). Urine should be collected using an antioxidant such as 5-hydroxy-Tempo (1 mm) to prevent degradation of LTs. Extraction is performed as above using cartridges pretreated with EDTA, and great

care should be taken to centrifuge (usually at 12 000 g, in a polypropylene tube, for 10 min) and filter (on a 0.2-0.4 #m filter) the dry extract resulting from the solid phase extraction prior to chromatography. This will avoid damage to the column due to an unwanted accumulation of residues, followed by a rapid increase in pressure as well as to a deterioration of the chromatographic parameters of the column. As mentioned above, it is important to flush the column with EDTA every 6-8 runs in order to chelate those trace elements responsible for the degradation of these substances. This is critical in the context of the low amounts of these substances contained in the urine. Analysis of urinary LTE4 is performed similarly to that of TX and PGI2. Briefly, after addition of [aH]LTE4 for recovery, urine is concentrated using solid-phase extraction (using a cartridge washed with methanol/EDTA, see above) and quantitation of this compound by EIA or radioimmunoassay (RIA) can be performed after HPLC purification (Heavey et al., 1987; Antoine et al., 1991). Measurement of eicosanoids contained in aliquot fractions of a PO01 of urine stored a - 7 0 ~ for each series of samples constitutes a good control to test the reproducibility of the technique over time.

5. Conclusion Under defined conditions (i.e. /n v/tr0 cell incubations), measurement of the production of these biologically active lipids is relatively simple to perform and to interpret in relation to the cell from which they have been generated. Their use as markers of in vivo situations remains more delicate. Due to their relative short half life in urine (a few hours after their formation in v/v0), it is unlikely that they can be used as predictive indexes (Patrono et al., 1984). For diagnosis, it is obvious that they remain subordinate to other clinical and biological information. However, their usefulness becomes apparent when studied in a dynamic state [i.e. multisampling with defined time intervals exemplified for the studies on unstable angina (Fitzgerald et al., 1986)]. When considering the formation of lipid mediators occurring via transcellular biosynthesis, the demonstration for such metabolism will be indirect because of the involvement of several cells and also because only one cell type (i.e. donor) needs to be activated in order to give rise to the compound. Yet, it is very likely that under pathological situations that involve several cells, this process will occur. Pharmacological evidence has already demonstrated the possibility of transfer of unstable substrate (i.e. PGH2) from one cell to the other in vivo (Nowak and FitzGerald, 1989). TX synthase inhibitors provoke an accumulation of PGH2 and a reduction of the production of TX metabolites in urine; this is accompanied by an increase of the metabolite derived from PGI2 as a result of a metabolic shift of platelet-

ANALYSIS OF EICOSANOIDS derived PGH2 towards the PGI2 synthase from the vascular cells. Interest in the analysis of metabolites derived from AA has been renewed by the growing evidence that they possess unique properties. As biochemical markers of cell activation under in vitro conditions they provide insights on the synthesis/function of autacoids, but they can also serve as indexes to comprehend in vivo situations in a multicellular environment occurring in pathophysiological events (cardiovascular or thrombotic episodes), or to evaluate a drug treatment (monitoring of an anti-platelet therapy).

6. References Antoine, C., Lellouche, J.P., Maclouf, J. and Pradelles, P. (1991). Development of enzyme immunoassays for leukotrienes using acetylcholinesterase. Biochim. Biophys. Acta 1075, 162-168. Borgeat, P., Picard, S., Vallerand, P. and Sirois, P. (1981). Transformation of arachidonic acid in leukocytes. Isolation and structural analysis of a novel dihydroxy derivative. Prostaglandins Med. 6, 557-570. Brash, A.IL, Jackson, E.K., Saggese, C.A., Lawson, J.A., Oates, J.A. and FitzGerald, G.A. (1983). The metabolic disposition of prostacyclin in man. J. Pharmacol. Exp. Ther. 226, 78-87. Capriotti, A.M., Furth, E.E., Arrasmith, M.A. and Laposata, M. (1988). Arachidonate released upon agonist stimulation preferentially originates from arachidonate most recently incorporated into nuclear membrane phospholipids. J. Biol. Chem. 263, 10029-10034. Carry, M., Korley, V., Willerson, J.T., Weigelt, L., FordHutchinson, A.W. and Tagari, P. (1992). Increased urinary leukotriene excretion in patients with cardiac ischemia. In vivo evidence for 5-1ipoxygenase activation. Circulation 85, 230-236. Catella, F. and FitzGerald, G.A. (1987). Paired analysis of urinary thromboxane B2 metabolites in humans. Thromb. Res. 47, 647-656. Ciabattoni, G. (1987). Production of antisera by conventional techniques. In: "Radioimmunoassay in Basic and Clinical Pharmacology" (eds C. Patrono and B.A. Peskar), pp. 23-68. Springer-Verlag, Berlin. Ciabattoni, G. Maclouf, J., CateUa, F., FitzGerald, G.A. and Patrono, C. (1987). Radioimmunoassay of ll-dehydrothromboxane B2 in human plasma and urine. Biochim. Biophys. Acta 918, 293-297. Fitzgerald, D.J., Roy, L., Catella, F. and FitzGerald, G.A. (1986). Platelet activation in unstable coronary disease. N. Engl. J. Med. 315, 983-989. FitzGerald, G.A., Oates, J.A., Hawiger, J., Maas, R.L., Roberts, L.J., Lawson, J.A. and Brash, A.IL (1983a). Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man. J. Clin. Invest. 71,676-688. FitzGerald, G.A., Pedersen, A.K. and Patrono, C. (1983b). Analysis of prostacyclin and thromboxane biosynthesis in cardiovascular disease. Circulation. 67, 1174-1177.

207

Fitzpatrick, F.A. (1976). High performance liquid chromatographic determination of prostaglandins F2a, E2 and D2 from in vitro enzyme incubations. Anal. Chem. 48, 499-502. Fitzpatrick, F.A. (1982). A radioimmunoassay for thromboxane B2. Methods Enzymol. 86, 286-297. Fitzpatrick, F.A. and Murphy, ILC.M. (1989). Cytochrome P450 metabolism of archidonic acid, formation and biological actions of "epoxygenases" derived eicosanoids. Pharmacol. Rev. 40, 229-241. Fitzpatrick, F.A., Wynalda, M. and Kaiser D.G. (1977). Oximes for high performance liquid and electron capture gas chromatography of prostaglandins and thromboxanes. Anal. Chem. 49, 1032-1035. Folco, G.C. and Sala, A. (1987). Bioassay of eicosanoids. In: "Biology ofIcosanoids" (ed. M. Lagarde), pp. 217-226. Colloque INSERM 152. Granstr6m E. and Kindahl, H. (1978). Radioimmunoassay of prostaglandins and thromboxane. In: "Advances in Prostaglandins and Thromboxane Research" (ed. J.C. Frolich), pp. 119-210. Raven Press, New York, 5. Habib, A. and Maclouf, J. (1992). Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells. Arch. Biochem. Biophys. 298, 544-552. Hayashi, Y., Yano, T. and Yamamoto, S. (1981). Enzyme immunoassay of prostaglandin F2~. Biochim. Biophys. Acta 663, 661-688. Hayashi, Y., Ueda, N., Kazushige, Y., Kawamura, S., Ogushi, F., Yamamoto, Y., Yamamoto, S., Nakamura, K., Yamashita, K., Miyzaki, H., Kato, K. and Terao, S. (1983). Enzyme immunoassay of thromboxane B2. Biochim. Biophys. Acta 75, 322-329. Heavey, D.J., Soberman, ILJ., Lewis, 1LA., Spur, B. and Austen, K.F. (1987). Critical considerations in the development of an assay for sulfidopeptide leukotrienes in plasma. Prostaglandins 33, 693-708. Hiroshima, O., Hayashi, H., Ito, S. and Hayashi, O. (1986). Basal levels of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay. Prostaglandins 32, 63-80. Lellouche, F., Fradin, A., FitzGerald, G.A. and Maclouf, J. (1990). Enzyme immunoassay measurement of the urinary metabolites of thromboxane A2 and prostacyclin. Prostaglandins 40, 297-310. Maclouf, J. and Murphy, ILC.M. (1988). Transcellular metabolism of neutrophil-derived leukotriene A4 by human platelets. A potential cellular source of leukotriene C4. J. Biol. Chem. 263, 174-181. Maclouf, J., Pradel, M., Pradelles, P. and Dray, F. (1976). 12sI derivatives of prostaglandins: a novel approach in prostaglandin analysis by radioimmunoassay. Biochim. Biophys. Acta 431, 139-146. Maclouf, J., Sors, H., Pradelles, P. and Dray, F. (1978). Prostaglandin methyl esters improve the sensitivity of iodinated histamine-prostaglandin radioimmunoassay standard curves. Anal. Biochem. 87, 169-176. Maclouf, J., Fitzpatrick, F.A. and Murphy. 1LC.M. (1989). Transcellular biosynthesis of reactive intermediates of the arachidonic acid cascade. Pharmacol. Res. 21, 1-7. Maclouf, J., Antoine, C., De Caterina, IL, Sicari, 1L, Murphy, 1LC., Patrignani, P., Loizzo, S. and Patrono, C. (1992) Entry rate and metabolism of leukotriene C4 into the vascular compartment in healthy subjects. Am. J. Physiol. 263, H244-H249.

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Marcus, A.J. (1990). Thrombosis and inflammation as multicellular processes, pathophysiologic significance of transcellular metabolism. Blood 76, 1903-1907. Marcus, A.J., Weksler, B.B., Jaffe, E.A. and Broekman, M.J. (1980). Synthesis of prostacyclin from platelet-derived endoperoxides by cultured human endothelial cells. J. Clin. Invest. 66, 979-986. Marcus, A.J., Sailer, L.B., Ullman, H.L., Broekman, M.J., Islam, N., Oglesby, T.D. and Gorman, ILR. (1984). 123, 20dihydroxy eicosatetraenoic acid produced by thrombin- or collagen-stimulated platelets. Proc. Natl. Acad. Sci. USA 81, 903-907. Miller, D., Sadowski, S., DeSousa, D., Maycock, A.L., Lombardo, D.L., Young, R.N. and Hayes, E.C. (1985). Development of enzyme-linked immunosorbent assays for measurement of leukotrienes and prostaglandins. J. Immunol. Methods 81, 169-185. Morozowich, W. and Douglas, S.L. (1975). Resolution of prostaglandin p-nitrophenacyl esters by liquid chromatography and conditions for rapid, quantitative pnitrophenylacylation. Prostaglandins 10, 19-40. Morrow, J.D., Hill, K.E., Burk, ILF., Nammour, T.M., Badr, K.F. and Roberts, L.J. (1990). A series of prostaglandin F2like compounds are produced in vim in humans by a noncyclooxygenase, free radical catalyzed mechanism. Proc. Natl. Acad. Sci. USA 87, 9383-9387. Nowak, J. and FitzGerald, G.A. (1989). Redirection of prostaglandin endoperoxide metabolism at the platelet-vascular interface in man. J. Clin. Invest. 83, 380-385. Patrono, C., Preston, F.E. and Vermylen, J. (1984). Platelet and vascular arachidonic acid metabolites, can they help detect a tendency towards thrombosis? Br. J. Haematol. 57, 209-212. Patrono, C., Ciabattoni, G., Remuzzi, G., Gotti, E., Bombardieri, S., Di Minno, O., Tartarelli, G.A., Simonetti, B.M. and Pierucci, A. (1985). Functional significance of renal prostacyclin and thromboxane A2 production in patients with systemic lupus erythematosus. J. Clin. Invest. 76, 1011-1018. Patrono, C., Ciabattoni, G., Pugliese, F., Pierucci, A., Blair, I.A. and FitzGerald, G.A. (1986). Estimated rate of thromboxane secretion into the circulation of normal humans. J. Clin. Invest. 77, 590-594. Patrono, C., Ciabattoni, G. and Patrignani, P. (1989). Biochemical indices of arachidonate metabolism by platelets and vascular endothelium in vim. In: "Platelets and Vascular Occlusion" (eds C. Patrono and G.A. FitzGerald), pp. 193-212. Raven Press, New York.

Patrono, C., Dav'l, G. and Ciabattoni G. (1992). Thromboxane biosynthesis and metabolism in relation to cardiovascular risk factors. Trends Cardiovasc. Med. 2, 15-20. Porter, N.A., Logan, J. and Kontoyiannidou, V. (1979). Preparation and purification of arachidonic acid hydroperoxides of biological importance. J. Org. Chem. 44, 3177-3181. Powell, W.S. (1982). Rapid extraction of arachidonic acid metabolites from biological samples using octadecylsilyl silica. Methods Enzymol. 86, 467-477. Pradelles, P., Grassi, J. and Maclouf, J. (1985). Enzyme immunoassays of eicosanoids using acetylcholine esterase as label, an alternative to radioimmunoassay. Anal. Chem. 57, 1170-1173. Russell, F.A. and Deykin, D. (1979). The use of high pressure liquid chromatography (HPLC). for the separation of radiolabeled arachidonic acid and its metabolites produced by thrombin-treated human platelets. I. The validation of the technique. Prostaglandins 18, 11-18. Salmon, J.A. and Flower, R.J. (1982). Extraction and thin layer chromatography of arachidonic acid metabolites. Methods Enzymol. 86, 477-493. Smith, W. L. (1992). Prostanoid biosynthesis and mechanisms of action. Am. J. Physiol. 263, F181-F191. Sors, H., Maclouf, J., Pradelles, P. and Dray, F. (1977). The use ofiodinated tracers for a sensitive radioimmunoassay of 13,14dihydro-15-keto prostaglandin F2,. Biochim. Biophys. Acta 486, 553-565. Sors, H., Pradelles, P., Dray, F., Rigaud, M., Maclouf, J. and Bernard, P. (1978). Analytical methods for thromboxane B2 measurement, Validation of radioimmunoassay by gas-liquid chromatography-mass spectrometry. Prostaglandins, 16, 277-290. Stehle, ILG. (1982). Physical chemistry, stability and handling of prostaglandins E2, F2,,, D2 and I2: a critical summary. Methods Enzymol. 86, 436-458. Vigano, T., Crivellari, M.T., Mezzetti, M. and Folco, G.C. (1990). Preparation of human and animal lung tissue for eicosanoid research. Methods Enzymol. 187, 621-628. Wescott, J.Y., Clay, K.L. and Murphy, ILC. (1984). Decomposition of leukotriene C4. J. Allergy Clin. Immunol. 74, 363-368. Westcott, J.Y., Chang, S., Balazy, M., Stene, D.O., Pradelles, P., Maclouf, J., Voelkel, N.F. and Murphy, ILC. (1986). Analysis of 6-keto-PGF~,, 5-HETE and LTC4 in rat lung, comparison of GC/MS, RIA and EIA. Prostaglandins 32, 857-873, 1986. Willard, J.E., Lange, tLA. and Hillis, L.D. (1992). The use of aspirin in ischemic heart disease. N. Engl. J. Med. 327, 175-181.

11. The Generate'onof Free Radicals

by Blood P latelets Michel Joseph

1. Introduction 2. Oxygen Activation and Free Radical Metabolism 2.1 Superoxide Anion 2.2 Hydrogen Peroxide 2.3 Hydroxyl Radical 2.4 Singlet Oxygen 2.5 Oxygen Reaction with Free Radicals (Peroxy Radicals) 2.6 Peroxides and Lipoperoxides 2.7 Enzymes Involved and Cell Localization of Free Radical Production 3. Free Radical Generation by Blood Platelets 3.1 IgE-induced H202 Production 3.2 H202 Production by Platelets from Aspirin-sensitive Asthmatics 3.3 Mechanisms of Free Radical Generation by Platelets 3.4 Free Radical Generation is not a Side Effect of Platelet Aggregation

209 210 210 211 211 211 212 212

214 215 215 215 216 216

1. Introduction In the past decade, the attention of cell physiologists, biochemists, and pathologists has been focused on the involvement of free radicals in various cell models (Emerit and Chaudi~re, 1989), and in pathological (Halliwell et al., 1992) or toxicological processes (Aust et al., 1993). In the same period, new journals have been published to cover the field of reactive oxygen species, lipid peroxidation, or oxidative stress and damage in physiological and pathological situations. In parallel, new methods have Immunopharmacology of Platelets ISBN 0 - 1 2 - 3 9 0 1 2 0 - 0

4. Antioxidant Defence Mechanisms 4.1 Endogeneous Protection Against Free Radicals 4.2 Exogeneous Defence Against Free Radicals 4.2.1 Hydrophobic Scavengers 4.2.1.1 Vitamin E 4.2.1.2 Carotenoids 4.2.2 Hydrophilic Scavengers 4.2.2.1 Ascorbate and Glutathione 4.2.2.2 Other Scavengers 4.3 Inhibitors of Platelet Cytotoxicity 4.4 Platelet Defence Mechanisms 5. Some Methods for Monitoring Free Radicals and Their By-products 6. Free Radicals, Diseases and Platelets 6.1 Free Radicals and Diseases 6.2 Diseases and platelets 7. Conclusion 8. Acknowledgements 9. References

217 217 218 218 218 218 218 214 218 218 219 219 220 220 220 221 221 221

been developed to analyse free radical generation in cells, tissues, and in biological fluids. Finally, a series of research has widened our knowledge on natural and synthetic antioxidant compounds, with the purpose of finding original and more efficient means of regulating the detrimental effects of oxygen activation in cellular redox systems. Based on chemical reactions known for years, if not decades, the pathways leading to free radical production in biological models have gradually found applications in cell systems: the capacity of mononuclear phagocytes, Copyright 9 Academic Press Limited All rights of reproduction in any form reserved.

210

M. JOSEPH

polymorphonuclear neutrophils and eosinophils t o generate such metabolites has been explored and described, essentially as a tool for anti-microbial and cytotoxic mechanisms (Babior, 1984). The enzymatic machinery supporting this metabolism has been extensively investigated and is now largely understood: it appears as a general feature of all cells, and is basically designed for physiological processes. The pathological effects induced by oxidative metabolites are mainly the consequence of disregulated pathways in the normal involvement of free radicals in cell physiology, and of unadapted levels of protective scavengers or natural antioxidant compounds at the site of free radical generation. In this perspective, like other cells, blood platelets contain all the necessary molecular and structural complexes to produce such oxidizing and reactive compounds. This chapter will therefore consider: (1) the metabolic pathways leading to free radical generation in general; (2) platelets as a source of reactive oxygen metabolites; (3) antioxidant protection systems and platelets as scavengers or modulators of free radicals; (4) some methods of identifying oxidative metabolites; and finally (5) the involvement of free radicals in pathological processes.

0

2.1

Oxygen Activation and Free Radical Metabolism

Free radicals are molecules or atoms with one or more unpaired electron on their external orbitals, instead of the paired electrons with antiparallel spins which establish or allow the stable covalent linkage in most biological molecules. As they tend to restore electron parity, these compounds are very reactive and short-lived. However, a free radical can give its unpaired electron (reducing

Singlet oxygen lX;O2

l~

Hydroxyl ion OH-

;q

~" pKa=4.8

e-

02 Ground-state oxygen

When oxygen accepts one electron, generally by the activity of enzymes such as xanthine oxidase (XO; Kuppusamy and Zweier, 1989) or NAD(P)H oxidase, it is converted to the perhydroxyl radical (HO~) or its ionized form, the superoxide anion (O~; Fridovich, 1986). Both have a remaining unpaired electron. The pKa of the ionization is 4.8. Therefore, at neutral and alkaline pH, superoxide anion predominates. When the pH is lowered the protonated form increases in

O2"-

t

~II

SUPEROXIDEANION

Superoxide anion

1AO2

,'0:0"

radical) to a non-radical structure, or it can receive an electron (oxidizing radical), or it can combine with a non-radical molecule. In each of these situations, a new free radical is generated, initiating a chain reaction which only ends when the transferred unpaired electron is captured by a scavenger. The pivotal compound in the initiation and development of free radical reactions is molecular oxygen. Oz, in its ground state, is a very stable, neutrally charged diradical with two unpaired electrons of parallel spins. Its beneficial effect in biochemical processes resides in its property of being the final electron acceptor of the respiratory chain, by successive steps of one-electron transfer through cytochrome oxidases in mitochondria, leading from 02 to H20. In this process, free radical intermediates are usually not released from the active site of the enzyme. The four-electron reduction of oxygen is shown schematically in Fig. 11.1. However, in many instances the straightforward fourelectron transfer is not achieved and only partial reduction occurs, giving rise to activated forms of oxygen and free radicals. The chemical and biochemical constraints of oxygen activation have been recently reviewed (Chaudi~re, 1994).

'~ @-

e-

HO2 9

Perhydroxyl xadical

:O:H ee

-O:H ee

ee

e-

H202

HO"

Hydrogen

Hydroxyl radical

peroxide

H:0:H

9r

H20 Water

Figure 11.1 Schematic picture of the four-electron reduction of oxygen. The intermediate compounds between 02 and H20. normally not released by the active site-of cytochrome oxidase in milochondria, are under the narrow control of antioxidant mechanisms outside mitochondria. These metabolites of oxygen reduction are produced and used physiologically as antimicrobial weapons but induce cell and tissue injury when produced in excess or under dysregulated control (oxidative stress).

GENERATION OF FREE RADICALS BY PLATELETS 211 concentration. 02 + NADPH --, O~ + NADP § + H §

catalysed dismutation of superoxide anion. However, H202 can be produced by the divalent reduction of oxygen, accepting two electrons, without a superoxide intermediate. Such a direct double reduction is observed with glucose oxidase, several dehydrogenases, uricase, monoamine oxidase, flavin- or cytochrome P450dependent monooxygenase. Even XO, which forms superoxide, reduces directly a portion of molecular oxygen to H202 without detectable superoxide formation.

(1)

Superoxide is also generated by the autoxidation of mitochondrial electron carriers, or in the presence of exogenous compounds such as quinones or paraquat, or under the action of radiation. The superoxide anion is both a reductant and an oxidant. As a reductant, O~ gives up an electron and is reoxidized to oxygen. This happens in the popular superoxide assay, based on the ferricytochrome c reduction, or in the formation of blue, insoluble tetrazolium salts. As an oxidant, O~ gains an electron and is reduced to H202, for example in the oxidation of epinephrine. Two superoxide molecules may interact, one being oxidized and the other reduced, in a dismutation reaction which produces 02 and H202. This dismutation is spontaneous at acidic pH, such as in lysosomal environment of mononuclear phagocytes, and takes place between the protonated form (HO~) and the non-protonated form (O~) when these molecules coexist in approximately equal amount (Fig. 11.2a). At neutral and alkaline pH, when the spontaneous dismutation declines, the reaction may be catalysed by superoxide dismutases (SODs); (McCord and Fridovich, 1969; Fridovich, 1986). SODs are metalloenzymes, with copper and zinc in cytosol and mitochondria, and manganese in mitochondria. An iron SOD is found in bacteria. The enzyme-mediated dismutation implies the alternate reduction and oxidation of the metal component of the enzyme (Fig. 11.2b).

2.3

The transfer of one electron to H202 induces the generation of the highly reactive hydroxyl radical (HO'). Since its production is inhibited by catalase, SOD and HO" scavengers and stimulated by H202, it was postulated that HO" generation implied the interaction of O~ and H202 (Ferradini et al., 1978; Liochev and Fridovich, 1994). However the direct interaction of these two compounds is kinetically blocked under biological conditions, unless trace metal serves as an oxidationreduction catalyst (Fenton reaction; Fig. 11.3), similarly to the copper-dependent dismutation of superoxide in Fig. 11.2. Traces of unprotected iron complexes, present in most, if not all biological systems, allow therefore the production of HO" by the Haber-Weiss cycle (Haber and Weiss, 1934) based on the Fenton reaction (Halliwell, 1978).

2.4 2.2

HYDROGEN PEROXIDE

O2""

+

H+

~''~

O2""

+

HO2.

"#

HO2"

+

H+

4,,,e

2 0 2 o- + 2H +

b)

HO2. O2

202"'+

HO2"

(dismutation)

H202

-I~

02"'+

(2) +

02 +

S O D - C u ++ + O2"" + H + SODH-Cu + +

SINGLET OXYGEN

When one of the unpaired electrons of the ground-state configuration of molecular oxygen shifts to an orbital of higher energy, with an inversion of spin, singlet oxygen

As described in Fig. 11.2, H202, which is not a free radical, is mainly generated by the spontaneous or enzyme-

a)

HYDROXYL RADICAL

(3) (4)

H202

(5)

S O D H - C u + + 02

(6)

H+

-e S O D - C u + +

2H +

,,e

202

+ H202

+ H202

(7) (8)

Figure 11.2 Spontaneous (a) and enzyme-mediated (b) dismutation of superoxide anion radical. Superoxide anion is both reductant and oxidant. As a reductant (with cytochrome c or NBT salt) the unpaired electron is returned and superoxide reoxidized to molecular oxygen. Oxygen produced during the spontaneous dismutation [equation (3)] is transiently in the form of singlet oxygen [equation (12) in Fig. 11.4]. As an oxidant (on epinephrine for example) superoxide anion gets one electron and is reduced to hydrogen peroxide. Spontaneous dismutation occurs at acidic pH [equation (2)] when an equal amount of O[ and -HO2 co-exist; under neutral or basic conditions the dismutation is induced by superoxide dismutase.

212

M. JOSEPH O 2""

+

Fe ++ O2""

+ +

Fe + + +

"~

O2

+

H202

-k

Fe+++

H202

-~

02

Fe + + +

+

OH"

(9) +

OH-

+

HO.

(10)

HO.

(1 i )

Figure 11.3 Hydroxyl radical formation by the Haber-Weiss cycle basedon the Fenton reaction [equation (10)]. The oxygen molecule generated in equation (9) is transiently in the singlet oxygen state [see equation (14) in Fig 11.4].

is formed (Fig. 11.1). In the sigma form (1~O2) the electrons of opposite spin occupy different orbitals, whereas in the delta form ('AO2) the paired electrons occupy the same orbital. Their respective lifetimes in solution are 10-11 S (~) and 2/~s (A). The shift of the unpaired electrons to their stable orbitals generates thermal decay, light emission or chemical reaction, sustaining the potential damaging properties of excited oxygen. The chemical reactivity of singlet oxygen is mostly due to the delta form. Its generation implies one or the other oxygen metabolite described above (Fig. 11.4). Superoxide anion is likely to produce singlet oxygen during spontaneous dismutation (Fig. 11.2a) or during its interaction with H202 in the Haber-Weiss reaction (Fig. 11.3), where resulting 02 molecules might be transitorily singlet oxygen; it can react with HO" to produce singlet oxygen and O H - ; and it can react with diacyl peroxides. Finally, H202 probably generates singlet oxygen during its interaction with hypochlorite at alkaline pH (Fig. 11.4).

2.5

OXYGEN REACTION WITH FREE RADICALS (PEROXY RADICALS)

Oxygen reacts very strongly with organic free radicals by capturing the unpaired electron to mate with one of its own, generating a peroxy radical (Fig. 11.5a; Ingold, 1969). Peroxy radicals may recombine (Fig. 11.5b), with the formation of a very unstable tetroxide bridge R O O ' O O R which fragments and produces singlet oxygen. Singlet oxygen reacts spontaneously with stable organic compounds, as energetically as hydroxyl radical. Peroxy radicals can also strip hydrogen from organic compounds, initiating the free radical cascade (Figs 11.5c and 11.6). Therefore oxygen exerts its toxic effects by interferring with the stabilizing paired recombination of neoformed free radicals.

2.6

PEROXIDES AND LIPOPEROXIDES

Polyunsaturated fatty acids represent crucial components

Spontaneous dismutation of superoxide : 02"

+

HO2. +

H § "~

102 +

H202

(12)

OH"

(13)

Superoxide + Hydroxyl radical : 02"

+

102

OH."#

§

Superoxide + Hydrogen peroxide : 02""

+

102

"~

H202

+ OH" +

HO.

(14)

Superoxide + Diacyl peroxides : 202""

+

O 0 R~O:O~R

-t

2102 +

0

2R~O-

(15)

Hydrogen peroxide + Hypochlorite : H202

+

OOl-

.a

102

+

r

+ H20

(16)

Figure 11.4 Singlet oxygen formation. The excitation of oxygen happens when one of the unpaired electrons shifts from an orbital of lower energy to one of higher energy and undergoes an inversion of spin. The return of the shifted electron to its stable orbital in ground state triplet oxygen molecule is accompanied by emission of light. Equation (16) is mediated by myeloperoxidase (see Fig 11.7).

GENERATION OF FREE RADICALS BY PLATELETS

213

a) Peroxyl radical formation:

R.

+

.OO.

"~

R : O O . or

ROO.

(1 7 )

b) Tetroxide bridge: ROO.

+

ROO:OOR

ROO.

( 18 )

or c) Free radical cascade" ROO- + RI:H ~

(19)

ROOH + R l o

R lo + R2:H

~

RI:H

R2o + R3:H

~

(20)

+ R2. R2:H

+

R3 .... e t c

Figure 11.5 Peroxyl radical formation in the presence of molecular oxygen. 02 is the agent of the propagation reaction, part of the free radical cascade developed in Fig. 11.6 for polyunsaturated fatty acids, and summarized in equations (19) and (20) of the present figure for any carbon-centred free radical.

Initiation

Q H eo

H ee

,%

,.

A

Q

~ H ol

/,;',,

Q H oo

/

Q H oo

.% : A

H eo

~-.

|

~,

,=,.o,:rot, J.delocal , sat , o n <EP .".

~- d~)~...j;o o....//

oo eo

eo

H H

o.~,

"~

Q

Figure 11.6 Membrane peroxidation and autoxidation of polyunsaturated fatty acids (PUFA). The unpaired electron of peroxy radicals ROO" is scavenged by vitamin E and lipid hydroperoxides ROOH destroyed by GSH peroxidases. The Iipoperoxide R" which results from the electron delocalization is a conjugated diene, a compound specific for free radical injury, which allows the identification of Iipoperoxidation in living organisms and biological fluids.

214

M. JOSEPH

of the cell membrane architecture and function. Through the presence of several double bonds in their carbon chain, which permit an easy electron delocalization, they allow the generation of stable free radicals (Girotti, 1985). As shown in Figure 11.6, an unstable free radical breaks one double bond of a fatty acid chain (KI) by abstracting a hydrogen with its electron. A conjunction process - with electronic resonance between four carbons - and a transfer of the unpaired electron give rise to a stable lipid free radical. In the presence of oxygen a peroxy radical will appear and attack another neighbouring fatty acid (R2), by stripping again one hydrogen with its electron. While the peroxy radical becomes a lipidic hydroperoxide, stable in the absence of iron, the new free radical will in turn keep up the free radical cascade. In the presence of trace metal, a Fenton reaction takes place which produces alkoxy radicals, R O ' , free radical species as aggressive as the hydroxyl radical.

2.7

radical cascade is linked to its ability to capture one electron, via oxidases (XO, NAD(P)H oxidase). About 5% of oxygen reduced into water at the mitochondrial level is reduced by a monoelectronic step, inducing free radicals. Superoxide anion is produced at a high rate by the autoxidation of mitochondrial electron carriers: flavoproteins, iron-sulphur proteins and quinols. Autoxidation of ubiquinol, such as mitochondria and membrane ubiquinone, proceeds via a semiquinone intermediate generating superoxide anion. In cytoplasmic membranes, NAD(P)H oxidase is the main generator of O~. NAD(P)H oxidase is present in platelets at high specific activity (Leoncini et al., 1991) (see Fig. 11.7). In unstimulated cells, NAD(P)H oxidase is composed of membrane and cytosolic factors which need to be assembled into the plasma membrane for the full expression of the enzymatic activity. Among the membrane proteins which allow the anchoring of cytosolic proteins, an iron-containing subunit of cytochrome b24s (also referred to as cytochrome bssg) is necessary, as well as a flavoprotein for the electron transfer from NAD(P)H to oxygen (Saran and Bors, 1989). In some abnormal situations, such as post-ischaemic reperfusion and, far more, exposition to radiation, a very high yield of O~ is

ENZYMES INVOLVED AND CELL LOCALIZATION OF FREE RADICAL PRODUCTION

The central role of oxygen in the initiation of the free

HMPS NADPH+H +

G6P

10

NADP +

102A HO* I

FAD

FADH 2

Fe + + C y t b _ 2 4 5 ~~_

Fe Cytb-245 j _+++

,,

c~o-

s,"

02

NADPH-oxldase

v

,~

#

~

,

~..

"~er

S6

%

/ _<~ '

,

.x~

I

~

'

'

% l

" -.

f

HIdPS

66P f

NADP +

C1-

~~q~RSP._

NADPH+H

+

SH~~auct.se~~6S

H2.02Glutath/oneperox/dase.H20

D(P)H+H § R5P

"

~0CI

I I

%

v2 ~o GS'n~ ' 7~ " ~o+ G ~ '. ~ o

h.~\r gOl-'~ . ~

%

#

~r- " . . . . . . I I

e ~

H 20 + 0 2

Figure 11.7 Metabolic pathways and enzymes regulating the four-step reduction of oxygen. Two crucial metabol|tes NADPH2 and GSH support a steady flow from 02 to H20. The well-balanced availability and function of three enzymes (SOD, GSH peroxidase and catalase) ensure optimal antioxidant effects in and outside cells (as summarized in Fig. 11.8).

GENERATION OF FREE RADICALS BY PLATELETS 215 generated. Another superoxide anion-generating system is the NO synthase complex, which normally produces NO and L-citrulline from L-arginine, but generates O~ and H202 in the absence of L-arginine (Mayer et al., 1991; Heinzel et al., 1992; Pou eta/., 1992). Constitutive NO synthase is found in platelets (Radomski et al., 1991). H202 is mainly produced by SOD, moderately by cytochrome P450-dependent monooxygenase and at a low level by monoamine oxidases. HO" generation requires the presence of free transition metals (iron or copper) through the Fenton reaction. Nearly 70% of iron in adult mammals is complexed with haemoglobin, 10% with myoglobin, a few per cent is found in ironcontaining enzymes and transport proteins such as transferrin or lactoferrin; and the residual iron is linked to intracellular storage proteins such as ferritin or haemosiderin (Ryan and Aust, 1992). Acidic pH conditions (phagolysosomes in phagocytes), superoxide anion, ascorbic acid or semi-quinone radicals, dissociate ferritin or inhibit its biosynthesis giving rise to free reduced Fe 2§ available for the Fenton reaction [reaction (10) in Fig. 11.3]. For their part, peroxides dissociate haemoglobin (Gutteridge, 1986.) Because of its extreme reactivity, HO" will react close to its site of production. The cell or tissue damage will therefore depend upon the availability and concentration of metal ions mediating HO" formation (Halliwell and Gutteridge, 1986). Similarly, traces of iron or copper will decompose lipid peroxides into highly cytotoxic aldehydes (Esterbauer et al., 1982). The above mechanisms require specific metabolic pathways and their corresponding enzymes. Is there indication that blood platelets are able to produce such metabolites and, if so, do platelets use one or the other of these procedures?

11

Free Radical Generation by Blood Platelets

As described in Chapter 8 of this book, blood platelets from humans or rat express cytotoxic functions against parasites in vivo and in vitro. Various stimulating processes can lead to these properties. The first triggering agent described was associated to anti-parasite IgE antibody, bound to Fc~ specific receptors on the platelet membrane, and inducing a transduction signal when crosslinked by parasite antigens (Joseph et al., 1983). In an effort to identify potential cytotoxic components generated by these blood elements, the possibility that platelets could produce free radicals was investigated. Lucigenin for superoxide anion and luminol for hydrogen peroxide were used a s probes for chemiluminescence experiments: we failed to measure any activated oxygen metabolites by this method. At that time an enhancing procedure for chemiluminescent

ELISA and peroxidase-labelled antibody was published, using a mixture of luminol luciferin and hydrogen peroxide, a technique which gave a 600-1000-fold increase of the chemiluminescence induced by peroxidase when compared to that produced with luminol or luciferin alone (Whitehead et al., 1983). We assumed that with a constant amount of peroxidase, luminol, and luciferin, the chemiluminescence would be proportional to the hydrogen peroxide produced by platelets. However a crucial parameter of the chemiluminescence procedure was the platelet concentration in the test tube, which had to be a compromise between a sufficient number of platelets to give a detectable signal, and a moderate number to avoid the quenching of emitted light by excess turbidity. A final concentration of 3 x 106 platelets/ml appeared optimal. 3.1 IgE-INDUCED H202 PRODUCTION Platelets from rats or from human patients infected with the helminth parasite Schistosoma mansoni generated H202-dependent chemiluminescence when triggered with antigenic extracts of schistosomes or with anti-IgE, antibody, whereas irrelevant antigens or anti-IgG antibody induced very limited luminescence (Joseph et al., 1985). Similarly, platelets from allergic patients produced H202-mediated luminescence in the presence of specific allergens or anti-IgE antibody, but not with unrelated allergens or anti-IgG antibody (Joseph et al., 1986). The role of IgE in the triggering process was confirmed by the chemiluminescence obtained after the passive sensitization of normal platelets with the serum of allergic patients and the cross-linking of IgE receptors by specific allergens or anti-IgE, a stimulation which disappeared after IgE depletion of the serum. Furthermore a strong correlation was observed between chemiluminescence and cytotoxic functions expressed by platelets under the same experimental conditions. Catalase, which destroys H202 molecules, thus avoiding production of HO', inhibited both the chemiluminescence and the killing properties of platelets. Electron spin resonance of human platelet preparations with dimethyl pyrroline oxide (DMPO) spin trapping gave a positive signal for HO" production when platelets were incubated in IgE and anti-IgE (Kitagawa et al., 1992; Deby et al. personal communication).

3.2

H 2 0 2 P R O D U C T I O N BY PLATELETS FROM ASPIRINSENSITIVE ASTHMATICS Platelets isolated from patients with aspirin-induced asthma (AIA) expressed, in vitro, cytotoxic properties against parasite larvae in the presence of aspirin (acetylsalicylic acid; ASA) or non-steroidal anti-inflammatory drugs (NSAIDs; Ameisen et al., 1985). In the same conditions, these platelets generated H202-mediated chemiluminescence, with a high correlation with cytotoxic properties

216 M. JOSEPH and in all AIA patients tested. The production of H202 was also observed by Pearson and Suarez-Mendez (1990) but in a smaller percentage of AIA patients (32%). In contrast, a reduced generation of reactive oxygen was reported elsewhere in the same ASA intolerance expressed by asthmatics (Schmitz-Schumann et al., 1989).

3.3

MECHANISMS OF FREE RADICAL GENERATION BY PLATELETS

The link between platelet chemiluminescence (---reactive oxygen metabolites) and killing properties (---cytotoxic mediators) is difficult to understand, observing that platelets may kill parasites across polycarbonate filters at a distance from their targets (Joseph et al., 1985), or that supernatants of platelets stimulated with IgE (allergy) or incubated with ASA (ASA intolerance) keep their cytocidal properties towards parasites, even after storage, if frozen (Ameisen et al., 1985). Oxygen reactive metabolites are too short-living to explain such properties. The generation of lipid peroxides and of toxic aldehydes could be an alternative solution to the relatively long-lasting effects of platelet stimulation. In fact, recent experiments show that such metabolites seem to be present in platelet supernatants (in preparation). Using the reduction of cytochrome c, it has been demonstrated that platelets, in the presence of various agonists of classical platelet activation (i.e. leading to aggregation), also generate superoxide anion, a mechanism partially inhibited by SOD (Leoncini et al., 1991). These compounds - AA, calcium ionophore, phorbol myristate acetate (PMA) or thrombin - induced the oxidation of dichlorofluorescin (DCFH), trapped inside the cells, to fluorescent DCF, by intraplatelet-generated H202. N-Ethylmaleimide, an inhibitor of glutathione (GSH) peroxidase, but not 3-amino-l,2,4-triazole, an inhibitor of catalase, enhanced the oxidation of DCFH (Maresca et al., 1992). The increased superoxide anionmediated chemiluminescence of platelets after the stimulation of AA metabolism could be inhibited by blockade of the LO pathway, but not the CO pathway (Jahn and Hansch, 1990). In the same investigation, the depletion of platelet GSH peroxidase resulted in reduced synthesis of the LO product 12-hydroxyeicosatetraenoic acid (12HETE) as well as in reduced chemiluminescence. The authors suggest a link between O~ generation, the LO pathway and the GSH cycle. The prostaglandin metabolism in platelets was described as a source of hydroxyl radical (Sagone et al., 1980; Singh et al., 1981). 3.4

FREE RADICAL NOT

GENERATION

IS

A SIDE EFFECT OF

PLATELET

AGGREGATION

However the association of aggregation with the generation of reactive oxygen metabolites must be viewed with

great care: as far as the free radicals and cytotoxicity can be considered as two faces of the same phenomenon, it has been shown that, in v/tr0, thrombin and plateletactivating factor (PAF) mediated either cytotoxicity at low concentrations or aggregation at higher concentrations, with no overlapping of the respective ranges (Tran et al., 1992). In fact, these two effects could never be produced simultaneously. Killing functions and aggregating properties seem therefore to be two separate mechanisms of the platelet biology. It was also shown that triggering platelets with low concentrations of one aggregating agent - or with cytotoxicity inducers (IgE) impaired very significantly the aggregation mediated by the other agent, ruling out some kind of tachyphylaxis. The concept of a dual antinomic function of platelets is strengthened by the possibility of inducing cytotoxic expression against schistosome larvae with purified platelet membrane stimulated with IgE/anti-IgE or IgE/aUergen. Aggregation inhibited almost completely the IgE-dependent cytoloxic process- even with platelet membranes isolated from disrupted aggregates - whereas membranes from disrupted unaggregated platelets expressed significant cytotoxicity. Therefore, as these effects could be produced not more successively than simultaneously, it implies, in some step, a diversion in the metabolic processes leading to one or the other function. The above considerations may be summarized by the experimental observation, made over years of platelet isolation and cytotoxicity, that when platelets aggregated during the isolation procedure they could no longer be induced into cytotoxic effectors, and conversely the cytotoxic process led platelets to an inhibitory stage for aggregation with usual aggregating agents. We have proposed the term of platelet "stimulation" for the induction of cytotoxicity to avoid confusion with platelet "activation" which commonly points to aggregation. It has been reported by others (Hecker et al., 1991) that, whereas low concentrations of H202 (10#M) synergized arachidonate-induced platelet aggregation, a 10-fold increase of the peroxide led to reversible aggregation and even to the inhibition of aggregation if platelets were preincubated for 2 min with H202. It must also be kept in mind that among normal products of AA metabolism in platelets can be found prostaglandin G2 (PGG2) and 12-hydroperoxyeicosatetraenoic acid (12-HPETE), two hydroperoxy compounds, which could have some effects similar to H202. Preincubation of platelets with 12HPETE inhibited AA induced aggregation, possibly through the activation of guanylate cyclase (Halliwell, 1987). The generation of free radicals by agents triggering c);totoxic properties is a surface mechanism expressed in the extracellular compartment. This concept is confirmed by a comparison between the DCF-dependent intraplatelet fluorescence and the luminol[luciferin H202-mediated extracellular chemiluminescence investigated on the same platelet populations: calcium

GENERATION OF FREE RADICALS BY PLATELETS 217 ionophore triggered only intraplatelet fluorescence and no extracellular chemiluminescence, whereas IgE/ allergen or ASA, on platelets corresponding to these respective sensitivities, stimulated only extraplatelet chemiluminescence and no intracellular fluorescence (Dahinden et al., unpublished observations).

4. Antioxidant Defence Mechanisms To minimize free radical damage, two lines of protection are available to cells: endogeneous mechanisms (mainly enzymes) and exogeneous compounds (free radical scavengers).

4.1

ENDOGENEOUS PROTECTION AGAINT FREE RADICALS

Insofar as reactive oxygen species represent the starting trigger of free radical cascade in cells and tissues, three enzymatic systems can potentially limit the damaging consequences of an exaggerated production of these compounds (Fig. 11.8). SODs play a central role in catalysing the spontaneous one-electron dismutation of superoxide: they produce 02 and H202. Protection by SOD is incompletely achieved if HzOz is not subsequently degraded. This is the function of catalase, which generates oxygen and water by a two-electron dismutation of HzO2. However in many cells (hepatocytes, endothelial cells, glial cells or myocytes), catalase concentration is very low, localized in peroxisomes, and frequently unavailable for H202 dismutation. Thus, in most tissues, the degradation of hydroperoxides is obtained by GSH peroxidase. This enzyme, which reduces H202 to water, and organic hydroperoxides to alcohols, is a tetrameric protein containing an essential atom of selenium at the active site. In patients with AIA, a lowered selenium serum concentration has been reported together with a reduced platelet GSH peroxidase activity (Pearson et al., 1991), a link

i

Oz

t

Superoxide dismutase

[I

e- -

0~."

2H +

e-

Ill

"

which was not observed by others (Schmitz-Schumann et al., 1989).

Cu-Zn SOD activities in platelets from asthmatic patients were significantly higher than those from normal healthy subjects and significantly higher in atopic than in non-atopic asthmatics (Kurosawa et al., 1993). These authors observed no significant differences in platelet enzyme between ASA-sensitive and ASA-tolerant patients. They had also shown previously (Kurosawa et al., 1991) that platelets from normal healthy donors were able to inhibit in vitro the superoxide generation by neutrophils stimulated with N-formyl-methionyl-leucylphenylalanine (FMLP) or PMA, whereas platelets from asthmatic patients failed to inhibit the free radical production by neutrophils. Platelet GSH peroxidase activity is the most sensitive index of selenium status in patients with chronic gastrointestinal disease receiving long-term total parenteral nutrition with or without selenium supplementation (Lane et al., 1987; Sando et al., 1992). With 100 #g selenium intake per day, GSH peroxidase activity is saturated in red cells and plasma and almost saturated in platelets (Alfthan et al., 1991). GSH peroxidase exerted a protective role on adriamycin-perfused hearts in rats (Nakano et al., 1989). The recycling of reduced GSH from glutathione disulphide (GSSG) generated in the antioxidant process is achieved through NADPH-dependent glutathione reductase (Koufos and Sagone, 1980; Fig. 11.7). Glutathione reductase is a flavin-containing enzyme which is inactivated by NADPH and activated by GSSG: during oxidative stress increased amounts of GSSG activates the enzyme, while the NADPH-mediated reduction of glucose-6-phosphate dehydrogenase (G6PD) induces the production of NADPH through the hexose monophosphate shunt (HMPS). Finally, among enzymes playing a role in detoxication, glutathione-S-transferases may be in charge of the catabolism of organic hydroperoxides, especially in the case of selenium deficiency (Lawrence et al., 1978;

Glutathione peroxidase

H202

Catalase

H+

e-

-

HO"

H+

e-

=HzO

I

Figure 11.8 Antioxidant enzymes. All three enzymes are not localized in the same compartments of cells: SODs are either in the cytosol (Cu-Zn) or in mitochondria (Mn); GSH peroxidase is cytosolic; catalase is mainly found in peroxisomes.

218

M. JOSEPH

Mannervik and Danielson, 1988; Ketterer and Meyer, 1989; Pickett, 1989; Rushmore and Pickett, 1993).

4.2

EXOGENEOUS DEFENCE AGAINST FREE RADICALS

Any compound with the potential capacity of scavenging free radicals will be a valuable tool in the protection of cells and tissues against deleterious effects of these toxic species, at least if its reaction does not generate new free radicals and if the rate of scavenging is higher than the free radical cascade (Deby et al., 1984). According to the biochemical compartment, scavengers are either hydrophobic or hydrophilic. 4.2.1

H y d r o p h o b i c Scavengers

4.2.2.1 V i t a m i n E Vitamin E (or c,-tocopherol, c~-TH) yields a long-lived free radical upon hydrogen abstraction (~-T') stopping the free radical cascade (Witting, 1980; Liebler et al., 1986), especially with peroxy radicals (ROO'). However, the reaction produces one molecule of hydroperoxide (ROOH) per each termination reaction. An optimum efficiency of vitamin E requires therefore the simultaneous degradation of such hydroperoxides, a reaction carried out by a membrane-bound GSH peroxidase pathway (Maiorino et al., 1989). Vitamin C (ascorbate) can regenerate a-TH from e~-T', a process which occurs at the lipid/water interface (McCay, 1985). The resulting semi-hydroascorbate free radical is stable enough to slowly disappear through dismutation into ascorbate and dehydroascorbate (Bendich et al., 1986). 4.2.1.2 Caro~s Carotenoids, especially B-carotene, show free radical scavenging properties synergistically with vitamin E (Burton and Ingold, 1984; Krinsky, 1989). These compounds are extremely efficient quenchers of singlet oxygen and are therefore crucial against this compound in oxidative stress, or in inflammation where singlet oxygen is a by-product of the reduction of PGG2 to PGH2.

4.2.2 Hydrophilic Scavengers Besides enzymes, which were considered above (Section 4.1), some water-soluble molecules are active scavengers of free radicals. 4.2.2.1 Ascorbate a n d Glutathione Ascorbate and glutathione, as reducing agents, are both able to cope with an acute peroxide overload, show excellent free radical scavenging properties (Bodannes and Chan, 1979; Rose and Bode, 1993), and prevent the oxidation of vitamin E in platelets (Vatassery et al., 1989). At physiological pH, ascorbate acts as an electron donor, whereas glutathione may act either as a hydrogen donor

or as an electron donor via GS-. Most hydrophilic free radicals can react with A H - , producing ascorbate free radical A" with a long life time (Bendich et al., 1986). As shown in Fig. 11.7, in the presence of GSH, superoxide anion plays the role of a free radical sink (Winterbourn, 1993). This GSH-mediated protection depends however on the presence of SOD, and vice vcrsa. Glutathione also regulates the oxidation of cysteine residues in proteins: the GSH/GSSG ratio affects the ratio of thiols and disulphides in proteins and thus their optimal structure and functions. The intraplatelet concentration of GSH is normally from 3 to 5 mM (5% of which is in the form of GSSG). Aggregation induces a 20% fall of GSH, which is normalized through the hexose monophosphate shunt and the cocomitant production of NADPH. 4. 2 . 2 . 2 Other Scavengers Other scavengers are also active in hydrophilic compartments: uric acid (Becker, 1993), mannitol, bioflavonoids (such as quercetin or rutin), bilirubin, allopurinol and oxypurinol (Moorhouse et al., 1987), and even glucose (Sagone et al., 1983) are efficient hydroxyl radical scavengers. Ceruloplasmin, a plasmatic copper protein which oxides Fe 2+ to Fe 3+ , reduces the Fenton reaction and the subsequent generation of hydroxyl radical.

4.3

INHIBITORS OF PLATELET CYTOTOXICITY

As reported above, the implication of oxygen metabolites in biological systems can be deduced from the inhibition of their effects by oxidoreductases or by free radical scavengers. In platelet models, superoxide dismutase and catalase inhibited IgE- and aspirin-dependent cytotoxicity and chemiluminescence, but not diethylcarbamazine-mediated killing activity on microfilariae (Cesbron et al., 1987), whereas peroxidase inhibited the effects of all 3 agonists on platelets. In the same models of platelet-dependent cytotoxicity, a-tocopherol showed inhibitory effects down to 10 -1~ M, with an IC50 at 0.5 x 10 -9 M. The activity of such low concentrations of the scavenger could be linked to its hydrophobic properties which might have concentrated molecules within cell membranes where free radicals themselves were produced. Intraplalelet a-tocopherol was consumed within the 30 min post-stimulation. Hydroxyl radical scavengers, such as mannitol, benzoate or uric acid, significantly inhibited the IgE-mediated killing of parasites as well as the ESR spectrum of HO'-mediated spin trapping. Finally, it was observed that Fe § down to 10 -11 M, increased the IgE- and diethylcarbamazinedependent platelet cytotoxicity, a free radical amplification process which was abolished by the iron chelators ophenanthroline (10 -6 M) or deferoxamine.

GENERATION OF FREE RADICALS BY PLATELETS 219

4.4

PLATELET DEFENCE MECHANISMS Besides their potential capacity to generate free radicals, platelets also have a role in modulating free radical effects through scavenging properties (Salvemini and Botting, 1993). GSH peroxidase and glutathione regulate the ASA metabolism of human platelets by reducing HPETE in HETE (Hill et al., 1989). Glutathione level in platelets correlates with their vitamin E content, and a decrease of GSH induces a decrease of e~-tocopherol (Calzada et al., 1991). In the elderly (> 68 years), platelet vitamin E and GSH peroxidase activity were significantly depressed together with increased lipid peroxidation, especially lipid hydroperoxides, resulting from 12-LO activity (Vericel et al., 1992). In rat, an acute iron load resulted in platelet hyperactivity, in part associated to free radical generation [67% increase in plasma malondialdehyde (MDA) and 60% decrease in plasma vitamin E]. After vitamin E supplementation, the effects of acute iron load on platelets were significantly decreased (Polette and Blache, 1992). In humans, the platelet level of c~tocopherol followed the dietary intakes of vitamin E, although to a lesser extent than in red cells (Saito et al., 1992). Human platelets attenuate oxidant oedema in isolated perfused rabbit lungs through mechanisms dependent on platelet glutathione (Zamora et al., 1991) but not by a direct scavenging of H202 (Godwin and Heffner, 1992). Selenium-dependent GSH-peroxidase has implications in platelet functions by regulating the biosynthesis of prostanoids (Perona et al., 1990).

5. SomeMethodsfor Monitoring Free Radicals and Their By-products A number of methods have been developed to identify and quantify, in vitro and in vivo (or more precisely ex v/v0), the generation of free radicals or their by-products (excellent review in Hageman et al., 1992). The presence of products of free radical damage in biological fluids is representative of the in vivo generation of reactive oxygen species. However limited information can be drawn about the cellular origin of free radicals involved. Very similar methods can be used for isolated cells, by either monitoring the direct generation of free radicals or analysing free radical by-products in cell supernatants. The best method is based on electron spin resonance spectroscopy after adduction of free radicals with a suitable spin trapping agent, such as 5,5'-dimethyl-1pyrroline-N-oxide (DMPO), or c~-phenyl-N-tert-butyl nitrone (PBN). Each free radical gives a distinctive spectrum (Rosen and Finkelstein, 1985; Buettner, 1987).

Spectrocolorimetric and spectrofluorimetric methods, with lower sensitivity than electron spin resonance however, offer alternative techniques for direct oxygen metabolite generation, together with high sensitivity chemiluminescence techniques. A survey update of luminometers is regularly published (Stanley, 1992, 1993). In the presence of superoxide anion ferricytochrome c is reduced to ferrocytochrome (Butler et al., 1975; Simic etal., 1975; Koppenol etal., 1976), and nitroblue tetrazolium salt (NBT) is reduced to a blue formazan precipitate (Baehner and Nathan, 1968), whereas lucigenin produces chemiluminescence (Williams and Cole, 1981). With H202, DCFH is oxidized to DCF (Burow and Valet, 1987; Robinson et al., 1988), whereas the sensitive assay performed for platelets with luminol/luciferin-dependent chemiluminescence in the presence of horseradish peroxidase (HRP) has been useful (Joseph et al., 1986), and the luminol-dependent chemiluminescence has been widely used for various leucocyte preparations (Allen and Loose, 1976; Briheim et al., 1984). These techniques, based on the direct measurement of free radicals, cannot be applied to the in vivo evaluation of free radicals. In that particular case, the detection, in biological fluids, of by-products of reactive oxygen-induced damage to cells and tissues gives the opportunity to identify: (1) conjugated dienes, which are primary products of free radical-induced lipoperoxides, detected by high performance liquid chromatography (HPLC) (Recknagel and Glende, 1984); (2) malondialdehyde, a by-product of lipoperoxides, measured by spectrophotometry or by spectrofluorimetry after adduction with thiobarbituric acid (TBA) (Wong, 1987; Kosugi et al., 1989) or by reversed-phase HPLC and UV detection after adduction with dinitrophenylhydrazine (DNPH) (Ekstr6m et al., 1988); (3)aldehydic products of lipid peroxidation, identified after derivatization with DNPH (Esterbauer and Zollner, 1989; Esterbauer et al., 1991); and finally (4) alkanes (pentane or ethane) identified and quantified in expired air by gas chromatography (Schaeffer, 1989). The determination, in cells and biological fluids, of enzymatic (SOD, catalase and GSH peroxidase; Beauchamp and Fridovich, 1971; Aebi, 1984; Maiorino et al., 1990) and non-enzymatic antioxidants (vitamins C and E, glutathione, urate and carotenoids; Reed et al., 1980; Buttriss and Diplock, 1984; Desai, 1984; Hochstein et al., 1984) defines the oxidant status of the subject. A comparison of the actual level with normal levels of antioxidants gives a good correlate of their free radical-dependent consumption in vivo. After appropriate stimulation, isolated platelets and their supernatants can be submitted to these various techniques. Investigations in this field are presently in progress in different research areas and clinical trials. New insights should emerge on the ability of these blood elements to generate free radical compounds.

220 M. JOSEPH

6. Free Radicals, Diseases and Platelets 6.1

FREE RADICALS A N D DISEASES

Reactive oxygen species appear more and more implicated in a number of clinical situations (Halliwell, 1991; Gutteridge, 1993) linked to inflammation (Halliwell, 1982; Biemond et al., 1984; Ward et al., 1988; Barnes, 1990; Henrotin et al., 1992), to immune disorders (Monti et al., 1992; Murrell, 1993), to ischaemia reperfusion (McCord, 1985; Engler, 1989; Ambrosio and Chiariello, 1991; Ferrari et al., 1992), carcinogenesis (Salonen et al., 1985; Menkes etal., 1986; Orr etal., 1988; Goldstein et al., 1989; Sahu, 1991), ageing (Pryor, 1982; Harman, 1988; Suzuki, 1993), pregnancy (Wickens et al., 1981) and to various diseases concerning nearly all organs: lungs (Joseph et al., 1980; Johnson et al., 1981; Cluzel et al., 1986), heart (Hess etal., 1982; Dousset etal., 1983; Ellis et al., 1984), cardiovascular system (Das, 1992; RiceEvans and Bruckdorfer, 1992; Halliwell, 1993a, b; RiceEvans and Burton, 1993), kidneys (Baud and Ardaillou, 1986; Greene and Paller, 1991; Ito et al., 1992), liver (Lewis and Paton, 1982; Comporti, 1985), gastrointestinal tract (Babbs, 1992; Harris et al., 1992; Vandervliet and Bast, 1992), brain (Gutteridge, 1992), nervous and neuromuscular system (Zemlan et al., 1989; Floyd and Carney, 1992; Halliwell, 1992a, b; Olanow, 1992; Bondy and Lebel, 1993), or skin (Braverman and Fonferko, 1982a, b). In v/v0, the rate of production of superoxide anion, H201 or lipid peroxides is generally not by itself sufficient to induce significant damage to cells, tissues and organs, but the onset of cellular injury increases the availability of metal ions, and thus amplifies the generation of aggressive free radical species (Halliwell and Gutteridge, 1985, 1986).

6.2

DISEASES AND PLATELETS

The participation of blood platelets in the occurrence of such clinical disorders is often considered as very limited if not impossible due to the vascular localization of thrombocytes. On the one hand, it can be underlined that diseases in narrow association with the cardiovascular system - from the heart to universally distributed capillaries in nearly all tissues and organs - represent a large proportion of human pathologies, therefore leaving to platelets a wide area for exerting potential injury through reactive oxygen species - for example, suggestion has been made that platelets could have part in one or the other form of migraine (Cotrim et al., 1993; D'Andrea et al., 1994). On the other hand, blood platelets have been found occasionally outside the bloodstream in various inflamed tissues submitted to oedema and blood cell influx, where they could also produce deleterious effects (Kravis and Henson, 1977; Pinckard et al., 1977; Page et al., 1984; Metzger et al., 1987; Beasley et al., 1989). It must also be kept in mind that platelets are the second

largest blood cell population after red cells, representing approximately one-third of the leucocyte volume. However, demonstration of the direct involvement of platelets in such processes needs further investigations. Only indirect evidence has been reported in some cases. We have described above that platelets, and no other leucocyte, isolated from patients with ASA-sensitive asthma generated in vitro free radicals (at least H202) in the presence of ASA, whereas platelets from normal individuals or platelets from allergic patients not only were unable to produce such metabolites but were inhibited "normally" by ASA in their cytotoxic functions (Ameisen et al., 1985). Furthermore, when ASAsensitive asthmatics were desensitized to ASA during the refractory period which follows a salicylate diet, their platelets became insensitive to ASA in vitro. The focus on a platelet abnormality in this intrinsic form of asthma opens interesting perspectives on a possible involvement of platelets in this pathology (Ameisen, 1986). In another systemic manifestation of allergy, i.e. Hymen0ptera venom hypersensitivity, in which lethal shocks are unfortunately frequent, isolated platelets generated free radicals and cytotoxicity in the presence of specific venoms (bee or wasp, according to the patient sensitivity; Capron et al., 1987). As for ASA-sensitive platelets, the in vitro reactivity of platelets to venom disappeared as soon as specific immunotherapy was undertaken in hypersensitive patients, i.e. in the very first days of the desensitizing process (Tsicopoulos et al., 1988). Platelets, and their ability to generate free radicals, have also been implicated in the activity of the microfilaricidal drug diethylcarbamazine, which induces in vivo the lysis of circulating microfilariae within a few minutes after its ingestion, but is totally inactive on these parasite larvae in vitro, unless blood platelets are added to the mixture (Cesbron et al., 1987). The generation of hydroxyl radical by platelets in the presence of diethylcarbamazine was identified by electron spin resonance with DMPO spin trapping. In 41 patients with coronary heart disease the concentration of total platelet malondialdehyde, a by-product of lipid peroxidation, was increased compared to values in healthy subjects, and all three antioxidant enzyme activities of patient platelets (SOD, GSH peroxidase and catalase) were significantly decreased (Buczynski et al., 1993). Similarly, platelet GSH peroxidase was significantly reduced in 45 patients with chronic renal failure, in correlation with a lower serum selenium concentration. The decrease of both parameters was still greater if patients presented simultaneously cardiovascular complications. These observations are relevant to the accelerated atherosclerosis reported for patients with chronic renal failure (Girelli et al., 1993). However platelet participation in the physiopathology of the diseases considered here seems to be linked more to a reduced capacity of thrombocytes in scavenging free radicals than to a direct production of reactive oxygen metabolites.

GENERATION OF FREE RADICALS BY PLATELETS

7. Conclusion The suggestion is made that an active role for blood platelets in the generation of deleterious molecules and subsequent cell and tissue damage in mammals may have been underestimated, in part as a consequence of the univocal concept which has been prevalent until recently that platelet functions were restricted to haemostasis and its disorders, in other words to physiological and pathological aggregation. The purpose of the present chapter, and others in this book, was to collect information on more widespread capacities for these blood elements with their possible implication in pathological disorders. The direct demonstration of reactive oxygen production by platelets in vivo is still lacking, as well as its involvement in the various pathologies in which free radicals could be playing a role. But one may wonder, stressing this particular parameter of the cell biology and biochemistry, if such a criticism would not be relevant for any other cell. As always in the history of science, the future will provide the answers.

8. Acknowledgements I am very much indebted to Professor Carol Deby and Doctor JoEl Pincemail (University of Liege, Belgium) for their help in electron spin resonance spectroscopy and useful discussions on methods to identify the generation of free radicals by blood platelets in our immunological models. Thank you also to Doctor Jean-Yves Cesbron, in Lille, whose dynamism has largely allowed the fruitful collaboration with the Liege group.

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Glossary Note: This glossary is up to date for the current volume only and will be supplemented with each subsequent volume. ~1, az receptors Adrenoceptor subtypes eq-ACT C~l-Antichymotrypsin eq-AP cq-antiproteinase also known as C~l-antitrypsin and c~-proteinase inhibitor al-AT al-Antitrypsin inhibitor also known as c~-antiproteinase and c~proteinase inhibitor ~I-PI c~-Proteinase inhibitor also known as C~l-antitrypsin and c~l-antiproteinase az-M c~2-macroglobulin A Absorbance AI, AII Angiotensin I, II A Angstrom AA Arachidonic acid aa Amino acids AAb Autoantibody ABAP 2',2'-azobis-2-amidino propane Ab Antibody Abl Idiotype antibody Ab2 Anti-idiotype antibody Ab2a Anti=idiotype antibody which binds outside the antigen binding region Ab2~ Anti-idiotype antibody which binds to the antigen binding region Ab3 Anti-anti-idiotype antibody Abcc Antibody dependent cellular cytotoxicity A B A - L - G A T Arsanilic acid conjugated with the synthetic polypeptide L-GAT AC Adenylate cyclase ACAT Acyl-co-enzyme-A acyltransferase ACAID Anterior chamber-associated immune deviation ACE Angiotensin-converting enzyme ACh Acetylcholine ACTH Adrenocorticotrophin hormone ADH Alcohol dehydrogenase Ado Adenosine ADP Adenosine diphosphate ADPRT Adenosine diphosphate ribosyl transferase AES Anti-eosinophil serum

Ag Antigen AGE Advanced glycosylation end-product AGEPC 1-O-alkyl-2-acetyl-snglyceryl-3-phosphocholine; also known as PAF and APRL AH Acetylhydrolase AID Autoimmune disease AIDS Acquired immune deficiency syndrome A/J A Jackson inbred mouse strain ALP Anti-leukoprotease ALS Amyotrophic lateral sclerosis cAMP Cyclic adenosine monophosphate also known as adenosine 3', 5'-phosphate AM Alveolar macrophage AML Acute myelogenous leukaemia AMP Adenosine monophosphate AMVN 2,2'-azobis (2,4dimethylvaleronitrile) ANAb Anti-nuclear antibodies ANCA Anti-neutrophil cytoplasmic auto antibodies cANCA Cytoplasmic ANCA pANCA Perinuclear ANCA AND Anaphylactic degranulation ANF Atrial natriuretic factor ANP Atrial natriuretic peptide Anti-I-A, Anti-I-E Antibody against class II MHC molecule encoded by I-A locus, I-E locus anti-Ig Antibody against an immunoglobulin anti-RTE Anti-tubular epithelium AP-1 Activator protein-1 APA B-azaprostanoic acid APAS Antiplatelet antiserum APC Antigen-presenting cell APD Action potential duration apo-B Apolipoprotein B APRL Anti-hypertensive polar renal lipid also known as PAF APUD Amine precursor uptake and decarboxylation AR Aldose reductase AR-CGD Autosomal recessive form of chronic granulomatous disease ARDS Adult respiratory distress syndrome

AS Ankylosing spondylitis ASA Acetylsalicylic acid also known as aspirin 4-ASA, 5-ASA 4-, 5-aminosalicylic acid ATHERO-ELAM A monocyte adhesion molecule ATL Adult T cell leukaemia ATP Adenosine triphosphate ATPase Adenosine triphosphatase ATP~s Adenosine 3' thiotriphosphate AITP Autoimmune thrombocytopenic purpura AUC Area under curve AVP Arginine vasopressin /~1, B2 receptors Adrenoceptor subtypes /$2 (CD18) A leucocyte integrin B2M B2-Microglobulin B-TG/3-Thromboglobulin BT/BB1 Known to be expressed on B cell blasts and immunostimulatory dendritic cells BAF Basophil-activating factor BAL Bronchoalveolar lavage BALF Bronchoalveolar lavage fluid BALT Bronchus-associated lymphoid tissue B cell Bone marrow-derived lymphocyte BCF Basophil chemotactic factor B-CFC Basophil colony-forming cell BCG Bacillus Calmette-Gu6rin BCNU 1,3-bis(2chloroethyl)- 1-nitrosourea bFGF Basic fibroblast growth factor Bg Birbeck granules BHR Bronchial hyperresponsiveness BHT Butylated hydroxytoluene b.i.d. Bis in die (twice a day) Bk Bradykinin Bkl, Bk2 receptors Bradykinin receptor subtypes also known as B1 and B2 receptors Bk2 receptor Bradykinin receptor subtype BI-CFC Blast colony-forming cells

228

GLOSSARY

B-lymphocyte Bursa-derived lymphocyte BM Bone marrow B M C M C Bone marrow cultured mast cell B M M C Bone marrow mast cell B O C - F M L P Butoxycarbonyl-FMLP bp Base pair BPB Para-bromophenacyl bromide BPI Bacterial permeability-increasing protein BSA Bovine serum albumin BSS Bernard-Soulier Syndrome SlCr Chromium s~ C1, C2...C9 The 9 main components of complement C1 inhibitor A serine protease inhibitor which inactivates C l r / C l s C l q Complement fragment lq C l q R Receptor for Clw; facilitates attachment of immune complexes to mononuclear leucocytes and endothelium C3a Complement fragment 3a (anaphylatoxin) C3a72-77 A synthetic carboxyterminal peptide C3a analogue C3aR Receptor for anaphylatoxins, C3a, C4a, C5a C3b Complement fragment 3b (anaphylatoxin) C3bi Inactivated form of C3b fragment of complement C4b Complement fragment 4b (anaphylatoxin) CABP CA binding protein; plasma protein which acts as co-factor to factor I inactivate C3 convertase CSa Complement fragment 5a (anaphylatoxin) CSaR Receptor for anaphylatoxins C3a, C4a and C5a CSb Complement fragment 5b (anaphylatoxin) C,2, C,3, C,4 Heavy chain of immunoglobulin E: domains 2, 3 and 4 Ca The chemical symbol for calcium [Ca z§ i Intracellular free calcium concentration C A H Chronic active hepatitis C A L L A Common lymphoblastic leukaemia antigen C A L T Conjunctival associated lymphoid tissue CaM Calmodulin cAMP Cyclic adenosine monophosphate also known as adenosine 3', 5'-phosphate C A M Cell adhesion molecule CAP57 Cationic protein from neutrophils CAT Catalase CatG Cathepsin G

CB Cytochalasin B CBH Cutaneous basophil hypersensitivity CBP Cromolyn-binding protein CCK Cholecystokinin C C R Creatinine clearance rate CD Cluster of differentiation (a system of nomenclature for surface molecules on cells of the immune system); cluster determinant CD1 Cluster of differentiation 1 also known as M H C class I-like surface glycoprotein C D l a Isoform a also known as nonclassical M H C class I-like surface antigen; present on thymocytes and dendritic cells C D l b Known to be present on thymocytes and dendritic cells C D l c Isoform c also known as nonclassical M H C class I-like surface antigen; present on thymocytes CD2 Defines T cells involved in antigen non-specific cell activation CD3 Also known as T cell receptorassociated surface glycoprotein on T cells CD4 Defines MHC class II-restricted T cell subsets CD5 Known to be present on T cells and a subset of B cells; also known as Lyt 1 in mouse CD7 Cluster of differentiation 7; present on most T cells and NK cells CD8 Defines MHC class I-restricted T cell subset; present on NK cells CD10 Known to be common acute leukaemia antigen C D l l a Known to be an c~ chain of LFA-1 (leucocyte function antigen-I) present on several types of leucocyte and which mediates adhesion CD11c Known to be a complement receptor 4 c~ chain. CD13 Aminopeptidase N; present on myeloid cells CD14 Known to be a lipid-anchored glycoprotein; present on monocytes CD15 Knawn to be Lewis X, fucosylN-acetyllactosamine CD16 Known to be Fc~ receptor III CD16-1, CD16-2 Isoforms of CD16 CD19 Recognizes B cells and follicular dendritic cells CD20 Known to be a pan B cell CD21 C3d receptor CD23 Low affinity FcER CD25 Low affinity receptor for interleukin-2 CD27 Present on T cells and plasma cells CD28 Present on resting and activated T cells and plasma cells

CD30 Present on activated B and T cells CD31 Known to be on platelets, monocytes, macrophages, granulocytes, B-cells and endothelial cells; also known as PECAM CD32 Fc-y receptor II CD33 § Knawn to be a monocyte and stem cell marker CD34- Known to be a stem cell marker CD35 C3b receptor CD36 Known to be a macrophage thrombospondin receptor CD40 Present on B cells and follicular dendritic cells CD41 Knawn to be a platelet glycoprotein CD44 Known to be a leucocyte adhesion molecule; also known as hyaluronic acid cell adhesion molecule (H-CAM), Hermes antigen, extracellular matrix receptor III (ECMIII); present on polymorphonuclear leucocytes CD45 Known to be a pan leucocyte marker CD45RO Known to be the isoform of leukosialin present on memory T cells C D 4 6 Known to be a membrane cofactor protein CD49 Cluster of differentiation 49 CD51 Known to be vitronectin receptor alpha chain CD54 Known to be Intercellular adhesion molecule-1 also known as ICAM-1 CD57 Present on T cells and NK subsets CD58 A leucocyte functionassociated antigen-3, also known to be as a member of the B-2 integrin family of cell adhesion molecules CD59 Known to be a low molecular weight HRf present to many haematopoetic and nonhaematopoetic cells CD62 Known to be present on activated platelets and endothelial cells; also known as P-selectin CD64 Knawn to be Fc3, receptor I CD65 Knawn to be fucoganglioside CD68 Present on macrophages CD69 Knawn to be an activation inducer molecule; present on activated lymphocytes CD72 Present on B-lineage cells CD74 An invariant chain of class II B cells CDC Complement-dependent cytotoxicity cDNA Complementary DNA CDP Choline diphosphate C D R Complementary-determining region

GLOSSARY 229 CD~ Common determinant xx CEA Carcinoembryonic antigen CETAF Corneal epithelial T cell activating factor CF Cystic fibrosis Cf Cationized ferritin CFA Complete Freund's adjuvant CFC Colony-forming cell CFU Colony-forming unit CFU-Mk Megakaryocyte progenitors CFU-S Colony-forming unit, spleen CGD Chronic granulomatous disease cGMP Cyclic guanosine monophosphate also known as guanosine 3', 5'-phosphate CGRP Calcitonin gene-related peptide CH2 Hinge region of human immunoglobulin CHO Chinese hamster ovary CI Chemical ionization CIBD Chronic inflammatory bowel disease CK Creatine phosphokinase CKMB The myocardial-specific isoenzyme of creatine phosphokinase CI The chemical symbol for chloride CL Chemiluminescent CLA Cutaneous lymphocyte antigen CL1816 Anti-ICAM-1 monoclonal antibody CLC Charcot-Leyden crystal CMC Critical micellar concentration CMI Cell mediated immunity CML Chronic myeloid leukaemia CMV Cytomegalovirus CNS Central nervous system CO Cyclooxygenase CoA Coenzyme A CoA-1T Coenzyme A - independent transacylase Con A Concanavalin A COPD Chronic obstructive pulmonary disease COS Fibroblast-like kidney cell line established from simian cells CoVF Cobra venom CP Creatine phosphate Cp Caeruloplasmin c.p.m. Counts per minute CPJ Cartilage/pannus junction Cr The chemical syn~ol for chromium CR Complement receptor CR1, CR2 & CR4 Complement receptor types 1, 2 and 4 CR3-a Complement receptor type 3-or CRF Corticotrophin-releasing factor CR/-I Corticotrophin-releasing hormone CRI Cross-reactive idiotype CRP C-reactive protein CSA Cyclosporin A CSF Colon)~-stimulating factor

CSS Churg-Strauss syndrome CT Computed tomography CTAP-III Connective tissueactivating peptide CTD Connective tissue diseases C terminus Carboxy terminus of peptide CThp Cytotoxic T lymphocyte precursors CTL Cytotoxic T lymphocyte CTLA-4 Known to be co-expressed with CD20 on activated T cells CTMC Connective tissue mast cell CVF Cobra venom factor 2D Second derivative Da Dalton (the unit of relative molecular mass) DAF Decay-accelerating factor DAG Diacylglycerol DAO Diamine oxidase D-Arg D-Arginine DArg- [HypS,DPhe 7] -BK A bradykinin Bz receptor antagonist. Peptide derivative of bradykinin DArg- [HypS,ThiS,DTicr,Tic s] -BK A bradykinin B2 receptor antagonist. Peptide derivative of bradykinin DBNBS 3,5-dibromo-4-nitrosobenzenesulphonate DC Dendritic cell DCF Oxidized DCFH DCFH 2',7'-dichlorofluorescin DEC Diethylcarbamazine DEM Diethylmaleate desArg9-BK Carboxypeptidase N product of bradykinin desArgl~ Carboxypeptidase N product of kallidin DETAPAC Diethylenetriaminepentaacetic acid DFMO c~-Difluoromethyl ornithine DFP Diisopropyl fluorophosphate 9 DFX Desferrioxamine DGLA Dihomo-~/-linolenic acid DH Delayed hypersensitivity DHA Docosahexaenoic acid DHBA Dihydroxybenzoic acid D H R Delayed hypersensitivity reaction DIC Disseminated intravascular coagulation DL-CFU Dendritic cell/Langerhans cell colony forming DLE Discoid lupus erythematosus DMARD Disease-modifying antirheumatic drug DMF N,N-dimethylformamide DMPO 5,5-dimethyl-l-pyrroline N-oxide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid D-NAME D-Nitroarginine methyl ester DNase Deoxyribonuclease

DNCB Dinitrochlorobenzene DNP Dinitrophenol Dpt4 Dermatophagoides pteronyssinus allergen 4 DGW2, DR3, DR7 HLA phenotypes DREG-56 (Antigen) L-selectin DREG-200 A monoclonal antibody against L-selectin ds Double-stranded DSCG Disodium cromoglycate DST Donor-specific transfusion DTH Delayed-type hypersensitivity DTPA Diethylenetriamine pentaacetate DTT Dithiothreitol d v l d t Rate of change of voltage within time e Molar absorption coefficient EA Egg albumin EACA Epsilon-amino-caproic acid EAE Experimental autoimmune encephalomyelitis EAF Eosinophil-activating factor EAR Early phase asthmatic reaction EAT Experimental autoimmune thyroiditis EBV Epstein-Barr virus EC Endothelial cell ECD Electron capture detector ECE Endothelin-converting enzyme E-CEF Eosinophil cytotoxicity enhancing factor ECF-A Eosinophil chemotactic factor of anaphylaxis ECG Electrocardiogram ECGF Endothelial cell growth factor ECGS Endothelial cell growth supplement E. coli Escherichia coli

ECP Eosinophil cationic protein EC-SOD Extracellular superoxide dismutase EC-SOD C ExtraceUular superoxide dismutase C EDss Effective dose producing 35% maximum response EDs0 Effective dose producing 50% maximum response EDF Eosinophil differentiation factor EDL Extensor digitorum longus EDN Eosinophil-derived neurotoxin EDRF Endothelium-derived relaxing factor EDTA Ethylenediamine tetraacetic acid also known as etidronic acid EE Eosinophilic eosinophils EEG Electroencephalogram EET Epoxyeicosatrienoic acid EFA Essential fatty acid EFS Electrical field stimulation EG1 Monoclonal antibody specific for the cleaved form of eosinophil cationic peptide

230

GLOSSARY

EGF Epidermal growth factor EGTA Ethylene glycol-bis(/3aminoethyl ether) N , N , N ' , N ' tetraacetic acid EHNA Erythro-9-(2hydroxy-3-nonyl)-adenine EI Electron impact EIB Exercise-induced bronchoconstriction eIF-2 Subunit of protein synthesis initiation factor ELAM-1 Endothelial leucocyte adhesion molecule-1 ELF Respiratory epithelium lung fluid ELISA Enzyme-linked immunosorbent assay EMS Eosinophilia-myalgia syndrome ENS Enteric nervous system EO Eosinophil EO-CFC Eosinophil colony-forming cell EOR Early onset reaction also known as EAR EPA Eicosapentaenoic acid EpDIF Epithelial-derived inhibitory factor also known as epitheliumderived relaxant factor EPO Eosinophil peroxidase EPOR Erythropoietin receptor EPR Effector cell protease EPX Eosinophil protein X ER Endoplasmic reticulum ERCP Endoscopic retrograde cholangiopancreatography E-selectin Endothelial selectin formerly known as endothelial leucocyte adhesion molecule-1 (ELAM-1) ESP Eosinophil stimulation promoter ESR Erythrocyte sedimentation rate e.s.r. Electron spin resonance ET, ET-1 Endothelin, -1 ETYA Eicosatetraynoic acid FA Fatty acid FAB Fast-electron bombardment Fab Antigen binding fragment F(ab')2 Fragment of an immunoglobulin produced pepsin treatment FACS Flow activated cell sorter factor B Serine protease in the C3 converting enzyme of the alternative pathway factor D Serine protease which cleaves factor B factor H Plasma protein which acts as a co-factor to factor I factor I Hydrolyses C3 converting enzymes with the help of factor H FAD Flavine adenine dinucleotide FapyAde 5-formamido-4,6diamino-pyrimidine

FapyGua 2,6-diamino-4-hydroxy-5formamidopyrimidine FBR Fluorescence photobleaching recovery

Fc Crystallizable fraction of immunoglobulin molecule Fc-y Receptor for Fc portion of IgG Fc~RI Ig Fc receptor I also known as CD64 Fc-yRII Ig Fc receptor II also known as CD32 FcrRIII Ig Fc receptor III also known as CD16 Fc,RI High affinity receptor for IgE Fc,RII Low affinity receptor for IgE FcR Receptor for Fc region of antibody FCS Foetal calf (bovine) serum FEV1 Forced expiratory volume in 1 second Fe-TPAA Fe(III)-tris [N-(2pyridylmethyl)-2-aminoethyl] amine Fe-TPEN Fe(II)-tetrakis-N,N,N',N'(2-pyridyl methyl-2-aminoethyl)amine FFA Free fatty acids FGF Fibroblast growth factor FID Flame ionization detector FITC Fluorescein isothiocyanate FK.BP FK506-binding protein FLAP 5-1ipoxygenase-activating protein FMLP N-Formyl-methionylleucyl-phenylalanine FNLP Formyl-norleucylleucyl-phenylalanine FOC Follicular dendritic cell FPLC Fast protein liquid chromatography FPR Forrnyl peptide receptor FS cell Folliculo-stellate cell FSG Focal sequential glomerulosclerosis FSH Follicle stimulating hormone FX Ferrioxamine 5-FU 5-fluorouracil Ga G-protein G6PD Glucose 6-phosphate dehydrogenase GABA -r-Aminobutyric acid GAG Glycosaminoglycan GALT Gut-associated lymphoid tissue GAP GTPase-activating protein GBM Glomerular basement membrane GC Guanylate cyclase GC-MS Gas chromatography mass spectroscopy G-CSF Granulocyte colonystimulating factor GDP Guanosine 5'-diphosphate GEC Glomerular epithelial cell GF-1 An insulin-like growth factor GFR Glomerular filtration rate

GH Growth hormone GH-RF Growth hormone-releasing factor Gi Family of pertussis toxin sensitive G-proteins GI Gastrointestinal GIP Granulocyte inhibitory protein GlyCam-1 Glycosylation-dependent cell adhesion molecule-1 GMC Gastric mast cell GM-CFC Granulocyte-macrophage colony-forming cell GM-CSF Granulocyte-macrophage colony-stimulating factor GMP Guanosine monophosphate (guanosine 5'-phosphate) Go Family of pertussis toxin sensitive G-proteins GP Glycoprotein gp45-70 Membrane co-factor protein gpg0 MF'L90 kD glycoprotein recognized by monoclonal antibody MEL-14; also known as L-selectin GPIIb-IIIa Glycoprotein IIb-IIIa known to be a platelet membrane antigen GppCH2P Guanyl-methylene diphosphanate also known as a stable GTP analogue GppNHp Guanylylimidiodiphosphate also known as a stable GTP analogue GRGDSP Glycine-arginine-glycine-aspartic acid serine-proline Gro Growth-related oncogene GRP Gastrin-related peptide Gs Stimulatory G protein GSH Glutathione (reduced) GSHPx Glutathione peroxidase GSSG Glutathione (oxidized) GT Glanzmann Thrombasthenia GTP Guanosine triphosphate GTP-~-S Guanarine 5'O-(3thiotriphosphate) GTPase Guanidine triphosphatase GVHD Graft-versus-host-disease GVHR Graft-versus-host-reaction H Histamine H1, H2, Ha Histamine receptor types 1, 2 and 3 H202 The chemical symbol for hydrogen peroxide Hag Haemagglutinin Hag-l, Hag-2 Cleaved haemagglutinin subunits-1, -2 H & E Haematoxylin and eosin hIL Human interleukin Hb Haemoglobin HBBS Hank's balanced salt solution HCA Hypertonic citrate H-CAM Hyaluronic acid cell adhesion molecule HDC Histidine decarboxylase

GLOSSARY 231 HDL High-density lipoprotein HEL Hen egg white lysozyme HEPE Hydroxyeicosapentanoic acid HEPES N-2-

[Hyp3]-BK Hydroxyproline derivative of bradykinin [Hyp4]-KD Hydroxyproline derivative of kallidin

Hydroxylethylpiperazine-N'-2-ethane sulphonic acid HES Hypereosinophilic syndrome HETE 5,8,9,11,12 and 15 Hydroxyeicosatetraenoic acid 5(S)HETE A stereo isomer of 5-HETE HETrE Hydroxyeicosatrienoic acid HEV High endothelial venule HFN Human fibronectin HGF Hepatocyte growth factor HHTrE 12(S)-Hydroxy-5,8,10heptadecatrienoic acid HIV Human immunodeficiency virus HL60 Human promyelocytic leukaemia cell line HLA Human leucocyte antigen HLA-DR2 Human histocompatability antigen class II HMG CoA Hydroxylmethylglutaryl coenzyme A H M W High molecular weight HMT Histidine methyltransferase HMVEC Human microvascular endothelial cell HNC Human neutrophil collagenase (MMP-8) HNE Human neutrophil elastase HNG Human neutrophil gelatinase (MMP-9) HODE Hydroxyoctadecanoic acid HO- Hydroxyl radical HO2" Perhydroxyl radical HPETE, 5-HPETE & 15-HPETE 5 and 15 Hydroperoxyeicosatetraenoic acid HPETrE Hydroperoxytrienoic acid HPODE Hydroperoxyoctadecanoic acid HPLC High-performance liquid chromatography HRA Histamine-releasing activity HRAN Neutrophil-derived histamine-releasing activity H R f Homologous-restriction factor HRF Histamine-releasing factor HRP Horseradish peroxidase HSA Human serum albumin HSP Heat-shock protein HS-PG Heparan sulphate proteoglycan HSV, HSV-1 Herpes simplex virus, -1 3HTdR Tritiated thymidine 5-HT 5-Hydroxytryptamine also known as Serotonin HTLV-1 Human T-cell leukaemia virus-1 HUVEC Human umbilical vein endothelial cell

rain Indium ll~ Ia Immune reaction-associated antigen Ia+ Murine class II major histocompatibility complex antigen IB4 Anti-CD18 monoclonal antibody IBD Inflammatory bowel disease IBMX 3-isobutyl-l-methylxanthine IBS Inflammatory bowel syndrome iC3 Inactivated C3 iC4 Inactivated C4 ICs0 Concentration producing 50% inhibition ICAM Intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 Intercellular adhesion molecules-i, -2, -3 cICAM-1 Circulating form of ICAM-1 ICE IL-115-converting enzyme i.d. Intradermal IDC Interdigitating cell IDD Insulin-dependent (type 1) diabetes IEL Intraepithelial leucocyte IELym Intraepithelial lymphocytes IFA Incomplete Freund's adjuvant IFN Interferon IFNcx, IFNB, IFN? Interferons c~, /3, -y Ig Immunoglobulin IgA, IgE, IgG, IgM Immunoglobulins A, E, G, M IgG1 Immunoglobulin G class 1 IgG2a Immunoglobulin G class 2a IGF-1 Insulin-like growth factor Ig-SF Immunoglobulin supergene family IGSS Immuno-gold silver stain IHC Immunohistochemistry H-IES Idiopathic hypereosinophilic syndrome IxB NFxB inhibitor protein IL Interleukin IL-1, II-2...IL-8 Intedeukins-1, 2...-8 IL-lt~, IL-1B Intedeukin-lce,-1~ ILR Intedeukin receptor IL-1R, IL-2R; IL-3R-IL-6R Interleukin-l-6 receptors IL-1Ra Interleukin-1 receptor antagonist IL-2RB Interleukin-2 receptor/3 IMF Integrin modulating factor IMMC Intestinal mucosal mast cell i.p. Intraperitoneally IP~ Inositol monophosphate IP2 Inositol biphosphate IPs Inositol 1,4,5-trisphosphate

IP4 Inositol tetrakisphosphate IPF Idiopathic pulmonary fibrosis IPO Intestinal peroxidase IpOCOCq Isopropylidene OCOCq I/R Ischaemia-reperfusion IRAP IL-1 receptor antagonist protein IRF-1 Interferon regulatory factor 1 Is, Short-circuit current ISCOM Immune-stimulating complexes ISGF3 Interferon-stimulated gene Factor 3 ISGF3a, ISGF~ c~, ~/subunits of ISGF3 IT Immunotherapy ITP Idiopathic thrombocytopenic purpura i.v. Intravenous K The chemical symbol for potassium

Ka Association constant kb Kilobase 20KDHRF A homologous restriction factor; binds to C8 65KDHRF A homologous restriction factor, also known as C8 binding protein; interferes with cell membrane pore-formation by C5b-C8 complex Kcat Catalytic constant; a measure of the catalytic potential of an enzyme Ka Equilibrium dissociation constant kD Kilodalton KD Dissociation constant KD Kallidin Ki Antagonist binding affinity K/gi7 Nuclear membrane antigen KLH Keyhole limpet haemocyanin Km Michaelis constant KOS KOS strain of herpes simplex virus

Xma~Wavelength of maximum absorbance LAD Leucocyte adhesion deficiency LAK Lymphocyte-activated killer (cell) LAM, LAM-1 Leucocyte adhesion molecule, -1 LAR Late-phase asthmatic reaction L-Arg L-Arginine LBP LPS binding protein LC Langerhans cell LCF Lymphocyte chemoattractant factor LCR Locus control region LDH Lactate dehydrogenase LDL Low-density lipoprotein LDV Laser Doppler velocimetry LeX(Lewis X) Leucocyte ligand for selectin LFA Leucocyte function-associated antigen LFA-1 Leucocyte function-associated antigen-I; also known to be a member of the/3-2 integrin family of cell adhesion molecules

232

GLOSSARY

LG/3-Lactoglobulin LGL Large granular lymphocyte L H Luteinizing hormone L H R H Luteinizing hormonereleasing hormone LI Labelling index LIS Lateral intercellular spaces LMP Low molecular mass polypeptide L M W Low molecular weight L-NOARG L-Nitroarginine LO Lipoxygenase 5-LO, 12-LO, 15-LO 5-, 12-, 15-Lipoxygenases LP(a) Lipoprotein(a) LPS Lipopolysaccharide L-selectin Leucocte selectin, formerly known as monoclonal antibody that recognizes murine L-selectin (MEL-14 antigen), leucocyte cell adhesion molecule-1 (LeuCAM-1), lectin cell adhesion molecule-1 (LeCAM-1 or LecCAM- 1), leucocyte adhesion molecule-1 (LAM-1) LT Leukotriene LTA4, LTB4, LTC4, LTD4, LTE4 Leukotrienes A4, B4, C4, D4 and E4 Ly-1 § (Cell line) LX Lipoxin LXA4, LXB4, LXC4, LXD4, LXE4 Lipoxins A4, B4, C4, D4 and E4 M Monocyte M3 Receptor Muscarinic receptor subtype 3 M-540 Merocyanine-540 mAb Monoclonal antibody mAb IB4, mAb PB1.3, mAb R 3.1, mAb R 3.3, mAb 6.5, mAb 60.3 Monoclonal antibodies IB4, PB1.3, R 3.1, R 3.3, 6.5, 60.3 MABP Mean arterial blood pressure MAC Membrane attack molecule Mac Macrophage (also abbreviated to M~) Mac-1 Macrophage-1 antigen; a member of the/5-2 integrin family of cell adhesion molecules (also abbreviated to McI,1), also known as monocyte antigen-1 (M-l), complement receptor-3 (CR3), CD11b/CD18 MAF Macrophage-activating factor MAO Monoamine oxidase MAP Monophasic action potential MAPTAM An intracellular Ca 2§ chelator MARCKS Myristolated, alanine-rich C kinase substrate; specific protein kinase C substrate MBP Major basic protein MBSA Methylated bovine serum albumin MC Mesangial cells

MCAO Middle cerebral artery occlusion M cell Microfold or membranous cell of Peyer's patch epithelium MCP Membrane co-factor protein MCP-1 Monocyte chemotactic protein-1 M-CSF Monocyte/macrophage colony-stimulating factor MCT Tryptase-containing mast cell MCTc Tryptase- and chymasecontaining mast cell MDA Malondialdehyde MDGF Macrophage-derived growth factor MDP Muramyl dipeptide MEA Mast cell growth-enhancing activity MEL Metabolic equivalent level M E M Minimal essential medium MG Myasthenia gravis MGSA Melanoma-growthstimulatory activity M H C Major histocompatibility complex MI Myocardial ischaemia MIF Migration inhibition factor mIL Mouse interleukin M I / R Myocardial ischaemia] reperfusion MIRL Membrane inhibitor of reactive lysis mix-CFC Colony-forming cell mix Mk Megakaryocyte MLC Mixed lymphocyte culture MLymR Mixed lymphocyte reaction M L R Mixed leucocyte reaction mmLDL Minimally modified lowdensity lipoprotein MMC Mucosal mast cell MMCP Mouse mast cell protease MMP, MMP1 Matrix metalloproteinase, -1 MNA 6-Methoxy-2-napthylacetic acid MNC Mononuclear cells McI, Macrophage (also abbreviated to Mac) MPG N-(2mercaptopropionyl)-glycine MPO Myeloperoxidase MPSS Methyl prednisolone MPTP N-methyl-4-phenyl- 1,2,3,6tetrahydropyridine MRI Magnetic resonance imaging mRNA Messenger ribonucleic acid MS Mass spectrometry MSS Methylprednisolone sodium succinate MT Malignant tumour M W Molecular weight N a The chemical symbol for sodium NA Noradrenaline also known as norepinephrine

NAAb Natural autoantibody NAb Natural antibody NAC N-acetylcysteine N A D H Reduced nicotinamide adenine dinucleotide NADP Nicotinamide adenine diphosphate N A D P H Reduced nicotinamide adenine dinucleotide phosphate NAF Neutrophil activating factor L-NAME L-Nitroarginine methyl ester NANC Non-adrenergic, non-cholinergic NAP Neutrophil-activating peptide NAPQI N-acetyl-p-benzoquinone imine NAP-I, NAP-2 Neutrophilactivating peptides -1 and -2 NBT Nitro-blue tetrazolium NC1 Non-collagen 1 N-CAM Neural cell adhesion molecule N C E H Neutral cholesteryl ester hydrolase NCF Neutrophil chemotactic factor NDGA Nordihydroguaretic acid NDP Nucleoside diphosphate Neca 5'-(N-ethyl carboxamido)-adenosine NED Nedocromil sodium NEP Neutral endopeptidase (EC 3.4.24.11) NF-AT Nuclear factor of activated T lymphocytes NF-xB Nuclear factor-xB NgCAM Neural-glial cell adhesion molecule NGF Nerve growth factor NGPS Normal guinea-pig serum N I H 3T3 (fibroblasts) National Institute of Health 3T3-Swiss albino mouse fibroblast NIMA Non-inherited maternal antigens NIRS Near infrared spectroscopy Nk Neurokinin NK Natural killer Nk-1, Nk-2, NK-3 Neurokinin receptor subtypes 1,2 and 3 NkA Neurokinin A NkB Neurokinin B NLS Nuclear location sequence NMDA N-methyl-D-aspartic acid L-NMMA L-Nitromonomethyl arginine N M R Nuclear magnetic resonance NO The chemical symbol for nitric oxide NOD Non-obese diabetic NOS Nitric oxide synthase c-NOS Ca2*-dependent constitutive form of NOS i-NOS Inducible form of NOS NPK Neuropeptide K

GLOSSARY 233 NPY Neuropeptide Y NRS Normal rabbit serum NSAID Non-steroidal antiinflammatory drug NSE Nerve-specific enolase NT Neurotensin N terminus Amino terminus of peptide 1AO2 Singlet Oxygen (Delta form) 11202 Singlet Oxygen (Sigma form) 0 ~ - The chemical symbol for the superoxide anion radical OA Osteoarthritis OAG Oleoyl acetyl glycerol OD Optical density ODC Ornithine decarboxylase ODFR Oxygen-derived free radical ODS Octadecylsilyl OH- The chemical symbol for hydroxyl ion 9OH The chemical symbol for hydroxyl radical 8-OH-Ade 8-Hydroxyadenine 6-OHDA 6-Hydroxyguanine 8-OH-dG 8-Hydroxydeoxyguanosine also known as 7,8-dihydro-8oxo-2'-deoxyguanosine 8-OH-Gua 8-Hydroxyguanine OHNE Hydroxynonenal 4-OHNE 4-Hydroxynonenal OT Oxytocin OVA Ovalbumin ox-LDL Oxidized low-density lipoprotein ~I,a Apical membrane potential P Probability P Phosphate PaO2 Arterial oxygen pressure Pi Inorganic phosphate p150,95 A member of the ~-2integrin family of cell adhesion molecules; also known as CD11c PA Phosphatidic acid pA2 Negative logarithm of the antagonist dissociation constant PAF Platelet-activating factor also known as APRL and AGEPC PAGE Polyacrylamide gel electrophoresis PAI Plasminogen activator inhibitor PA-IgG Platelet associated immunoglobulin G PAM Pulmonary alveolar macrophages PAS Periodic acid-Schiff reagent PBA Polyclonal B cell activators PBC Primary biliary cirrhosis PBL Peripheral blood lymphocytes PBMC Peripheral blood mononuclear cells PBN N-tert-butyl-~-phenylnitrone PBS Phosphate-buffered saline PC Phosphatidylcholine

PCA Passive cutaneous anaphylaxis pCDM8 Eukaryotic expression vector PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction PCT Porphyria cutanea tarda p.d. Potential difference PDBu 4c~-Phorbol 12,13-dibutyrate PDE Phosphodiesterase PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor PE Phosphatidylethanolamine PECAM-1 Platelet endothelial cell adhesion molecule-I; also known as CD31 PEG Polyethylene glycol PET Positron emission tomography PEt Phosphatidylethanolamine PF4 Platelet factor 4 PG Prostaglandin PGAS Polyglandular autoimmune syndrome PGD2 Prostaglandin D2 PGE1, PGE2, PGF2, PGF2~, PGG2, PGH2 Prostaglandins El, E2, F2, F2~, F2, H2 PGF, PGH Prostaglandins F and H PGI2 Prostaglandin I2 also known as prostacyclin PaO2 Arterial oxygen pressure PGP Protein gene-related peptide Ph i Philadelphia (chromosome) PHA Phytohaemagglutinin PHD PHD [8(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-5,10 heptadecadienic acid] PHI Peptide histidine isoleucine PHM Peptide histidine methionine Pi Inorganic phosphate PI Phosphatidylinositol PI-3,4-P2 Phosphatidylinositol 3, 4-biphosphate PI-3,4,5-P3 Phosphatidylinositol 3, 4, 5-trisphosphate PI-3-kinase Phosphatidylinositol-3-kinase PI-4-kinase Phosphatidylinositol-4-kinase PI-3-P Phosphatidylinositol-3-phosphate PI-4-P Phosphatidylinositol-4-phosphate PI-4,5-P2 Phosphatidylinositol 4,5-biphosphate PIP Phosphatidylinositol monophosphate PIP2 Phosphatidylinositol biphosphate PK Protein kinase PKA, PKC Protein kinases A and C PKG cGMP-dependent protein kinase, protein kinase G PL Phospholipase

PLA, PLA2, PLC, PLD Phospholipases A, A2, C and D PLN Peripheral lymph node PLNHEV Peripheral lymph node HEV PLP Proteolipid protein PLT Primed lymphocyte typing PMA Phorbol myristate acetate PMC Peritoneal mast cell PMN Polymorphonuclear neutrophil PMSF Phenylmethylsulphonyl fluoride PNAd Peripheral lymph node vascular addressin PNH Paroxysmal nocturnal hemoglobinuria PNU Protein nitrogen unit p.o. Per os (by mouth) POBN c~-4-Pyridyl-oxide-N-t-butyl nitrone PPD Purified protein derivative PPME Polymeric polysaccharide rich in mannose-6-phosphate moieties PRA Percentage reactive activity PRD, PRDII Positive regulatory domain, -II PR3 Proteinase-3 PRBC Parasitized red blood cell proET-1 Proendothelin-1 PRL Prolactin PRP Platelet-rich plasma PS Phosphatidylserine P-selectin Platelet selectin formerly known as platelet activationdependent granule external membrane protein (PADGEM), granule membrane protein of MW 140 kD (GMP-140) PT Pertussis toxin PTCA Percutaneous transluminal coronary angioplasty PTCR Percutaneous transluminal coronary recanalization Pte-I-I4 Tetrahydropteridine PUFA Polyunsaturated fatty acid PUMP-1 Punctuated metalloproteinase also known as matrilysin PWM Pokeweed mitogen Pyran Divinylether maleic acid q.i.d. Quater in die (four times a day) QRS Segment of electrocardiogram 9R Free radical R15.7 Anti-CD18 monoclonal antibody RA Rheumatoid arthritis RANTES A member of the IL8 supergene family (Regulated on activation, normal T expressed and secreted) RAST Radioallergosorbent test RBC Red blood cell

234

GLOSSARY

RBF Renal blood flow RBL Rat basophilic leukaemia RC Respiratory chain RE RE strain of herpes simplex virus type 1 REA Reactive arthritis REM Relative electrophoretic mobility RER Rough endoplasmic reticulum RF Rheumatoid factor RFL-6 Rat foetal lung-6 RFLP Restriction fragment length polymorphism RGD Arginine-glycine-asparagine rh- Recombinant human - (prefix usually referring to peptides) RIA Radioimmunoassay RMCP, RMCPII Rat mast cell protease, -II RNA Ribonucleic acid RNase Ribonuclease RNHCI N-Chloramine RNL Regional lymph nodes ROM Reactive oxygen metabolite RO" The chemical symbol for alkoxyl radical ROO- The chemical symbol for peroxy radical

ROP Retinopathy of prematurity ROS Reactive oxygen species R-PIA R-(1-methyl-1phenyltheyl)-adenosine RPMI 1640 Roswell Park Memorial Institute 1640 medium RS Reiter's syndrome RSV Rous sarcoma virus RTE Rabbit tubular epithelium RTE-a-5 Rat tubular epithelium antigen a-5 r-tPA Recombinant tissue-type plasminogen activator RW Ragweed S Svedberg (unit of sedimentation density) SALT Skin-associated lymphoid tissue SAZ Sulphasalazine SC Secretory component SCF Stem cell factor SCFA Short-chain fatty acid SCG Sodium cromoglycate also known as DSCG SCID Severe combined immunodeficiency syndrome sCR1 Soluble type-1 complement receptors SCW Streptococcal cell wall SD Standard deviation SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM Standard error of the mean SGAW Specific airway conductance

SHR Spontaneously hypertensive rat SIM Selected ion monitoring SIRS Soluble immune response suppressor SIV Simian immunodeficiency virus SK Streptokinase SLE Systemic lupus erythematosus SLe ~ Sialyl Lewis X antigen SLO Streptolysin-O SLPI Secretory leucocyte protease inhibitor SM Sphingomyelin SNAP S-Nitroso-Nacetylpenicillamine SNP Sodium nitroprusside SOD Superoxide dismutase SOM Somatostatin also known as somatotrophin release-inhibiting factor SOZ Serum-opsonized zymosan SP Sulphapyridine SR Systemic reaction sr Sarcoplasmic reticulum SRBC Sheep red blood cells SRS Slow-reacting substance SRS-A Slow-reacting substance of anaphylaxis STZ Streptozotocin Sub P Substance P T Thymus-derived a-TOC c~-Tocopherol t,lu Half-life T84 Human intestinal epithelial cell line TauNHCI Taurine monochloramine TBA Thiobarbituric acid TBAR Thiobarbituric acid-reactive product TBM Tubular basement membrane TBN di-tert-Butyl nitroxide tBOOH tert-Butylhydroperoxide TCA Trichloroacetic acid T cell Thymus-derived lymphocyte TCR T cell receptor off3 or ~,[6 heterodimeric forms TDI Toluene diisocyanate TEC Tubular epithelial cell TF Tissue factor Tg Thyroglobulin TGF Transforming growth factor TGFa, TGFfl, TGFfll Transforming growth factors cx,/3, and fll Tn T helper cell Ti-io T Helper o Tilp T helper precursor TH0, Till, Til2 Subsets of helper T cells

THP-1 Human monocytic leukaemia Thy 1 + Mufine T cell antigen t.i.d. Ter in die (three times a day) TIL Tumour-infiltrating lymphocytes TIMP Tissue inhibitors of metalloproteinase

TIMP-1, TIMP-2 Tissue inhibitors of metalloproteinases 1 and 2 Tla Thymus leukaemia antigen TLC Thin-layer chromatography TLCK Tosyl-lysyl-CH2Cl TLP Tumour-like proliferation Tm T memory TNF, TNF-cx Tumour necrosis factor, -c~ tPA Tissue-type plasminogen activator TPA 12-0tetradeconylphorbol-13-acetate TPCK Tosyl-phenyl-CH2Cl TPK Tyrosine protein kinases TPP Transpulmonary pressure TRAP Thrombospondin related anomalous protein Tris Tris(hydroxymethyl)aminomethane TSH Thyroid-stimulating hormone TSP Thrombospondin TI'X Tetrodotoxin TX Thromboxane TXAu, TXB2 Thromboxane A2, B2 Tyk2 Tyrosine kinase U937 (cells) Histiocytic lymphoma, human UC Ulcerative colitis UDP Uridine diphosphate UPA Urokinase-type plasminogen activator UTP Uridine triphosphate UV Ultraviolet UVA Ultraviolet A UVB Ultraviolet B UVR Ultraviolet irradiation UW University of Wisconsin (preserving solution) VAP Viral attachment protein VC Veiled cells VCAM, VCAM-1 Vascular cell adhesion molecule, -1, also known as inducible cell adhesion molecule MW 110 kD (INCAM-110) V'F Ventricular fibrillation VIP Vasoactive intestinal peptide VLA Very late activation antigen beta chain; also known as CD29 V'LA a2 Very late activation antigen alpha 2 chain; also known as CD49b VLA a4 Very late activation antigen alpha 4 chain; also bumm as CD49d VLA a6 Very late activation antigen alpha 6 chain; also kno,m as CD49f VLDL Very low-density lipoprotein V max Maximal velocity V rain Minimal velocity VN Vitronectin VO~ The chemical symbol for vanadate vp Viral protein VP Vasopressin VPB Ventricular premature beat

GLOSSARY VT Ventricular tachycardia vWF von Willebrand factor

WI Warm ischaemia XD Xanthine dehydrogenase XO Xanthine oxidase

W Murine dominant white spotting mutation WBC White blood cell W G A Wheat germ agglutinin

Y1182A A monoclonal antibody detecting a cytoplasmic antigen in human macrophages

ZA Zonulae adherens ZAS Zymosan-activated serum zLYCK Carboxybenzyl-Leu-Tyr-CH2Cl ZO Zonulae occludentes

235

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to Illustratz'ons

Suppressor lymphocyte

Helper lymphocyte

Killer lymphocyte

Bacterial or Tumour cell

Plasma cell

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Neutrophil passing through vessel wall

238

KEY TO ILLUSTRATIONS

Resting neutrophil

Resting eosinophil

Smooth muscle

Smooth muscle contraction

Endothelial cell permeability

Activated macrophage

Activated neutrophil

Activated eosinophil

Smooth muscle thickening

Normal blood vessel

Resting macrophage

Nerve

KEY TO ILLUSTRATIONS 239

Damaged epithelium

Intact epithelium

Normal submucosal gland

Intact epithelium with submucosal gland

Hypersecreting submucosal gland

I

Normal airway

Bronchospasm

Oedema

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Airway hypersecreting mucus

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Activated platelet

240

KEY TO ILLUSTRATIONS Activated basophil Resting basophil

r--

Activated mast cell Resting mast cell

Activated chondrocyte

Resting chondrocyte

Fibroblast

~~#,p,," Dendritic cell/ Langerhans cell

Cartilage

Arteriole Venule

KEY TO ILLUSTRATIONS 241 Inflamed venule

Microcirculatory system

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Index c~-thrombin, activation of GPIIb-IIIa receptor 152 c~-tocopherol 218 WlIb/33 receptor see glycoproteins, GPIIb-IIIa receptor N-acetylneuraminic acid 70 acute serum sickness, rabbit 23 adenosine 3 pl. aggregation 88-9 adhesion of bacteria, pathologic processes involving pl.-bacterial interactions 111 adhesive receptors, pl.Dtumour cell interactions 154-6 ADP ADP/aggregin interaction 38 aggregation 9 binding sites 37 ecto-ATPase 102-3 secretion, bacterially induced 112 ADP receptors 34 high]low affinity 37-8 signal transduction 38 as weak agonists to pl. 37 ADP ribosyltransferases 42 /3 adrenergic kinase-2 (BARK) 35 aggregin receptor 34, 37 aggregometry reproducible 90-2 see also platelet(s), aggregation AIDS and autoimmune thrombocytopenias 184 murine 139 thrombocytopenia in 146 airway hyperresponsiveness animal models 6, 25-6 late-onset response (LOR) 9 albolabrin 158 allergens, anti-schistosome cytotoxicity 126 allergic asthma see asthma allergic disorders allergic inflammation 7-12 animal models 21-30 aspirin-sensitive asthma 11, 131 hymenoptera venom hypersensitivity 131, 220 allopurinol, hydrophilic scavenger 218 3-amino- 1,2,4-triazole 216

AMP, pl. aggregation 88-9 anaphylaxis, acute respiratory 25 animal models acute respiratory anaphylaxis 25 allergic and inflammatory lesions 21-30 acute inflammation 22-3 acute serum sickness, rabbit 23 Arthus reaction 22-3 available methods 22 carrageenin-induced inflammation 23 haemarthrosis 23 polyarthritis syndrome in rats 22 Schwartzman reaction 23 ARDS, role of Pseudomonas 114 bacterial endocarditis 112-13 induction of thrombocytopenia 108-9 melanoma 156, 157 parasitic infection 28 phagocytosis 109 pl.-tumour interactions 159-60 tumour cell aggregation, methods 159-60 von Willebrand's disease 114 antibodies, see also monoclonal antibodies antibody-dependent cellular cytotoxicity (ADCC), parasitic infection 125 antigen-induced activation of pl. 27 antigenic capture assay 174-6 antioxidants defence mechanisms 217-19 enzymes 217 antiplatelet antibodies 168, 171-2 antiplatelet serum, in vivo production method 160 antiplatelet therapy alternative to aspirin 50 and metastases 157-9 Ap2 mAb, labelling GPIIb-IIIa receptor (pl. antigen) for cytofluorometry 126, 130 apyrase, ATP and aggregation 100 arachidonic acids 216 metabolism, phospholipase A2 46-7 radiolabelling 199-200

ARDS animal models 21, 24 role of pl. 114 arthritis experimental haemarthrosis 23 polyarthritis syndrome in rats 22 rheumatoid arthritis 11-12 Arthrobacter, /3-1ysin 104-5 Arthus reaction 22-3 ascorbate hydrophilic scavenger 218 refractory chronic AITP 180 aspirin antimetastatic effect 158 aspirin-induced asthma see asthma, ASA (aspirin)-induced and IgE 129 asthma airway hyperresponsiveness 9 animal models 25-6 allergic provocation 25 animal models bronchoconstriction, pl. agonist and allergen-induced 25 early asthmatic reponse (EAR) 26 late asthmatic reponse (LAR) 26-7 PAF as mediator 9-10 ASA (aspirin)-induced 11,215-16, 220 cytotoxic free radicals 11, 215-16 BAL fluid, pl. in 11 late response 26-7 nocturnal, platelet activation 10 PAF as mediator 8-11 animal evidence 9-10 clinical evidence 10-11 thrombocytopenia, first reports 10 atherosclerosis endothelial damage 111, 115 renal failure and 220 see also cardiovascular disease ATP apyrase, and aggregation 100 binding, adenylyl cyclase 37 autacoids 195-208 autoantigens clinical significance 172, 178 immunoblotting 173 recombinant techniques 176

244

INDEX

autoimmune thrombocytopenias 167-94 AIDS and 184 Evans syndrome 186 parasitic infections 187 secondary immune thrombocytopenic purpura 183-5 systemic lupus erythematosus 185-6 virus-induced 183-5 autoimmune thrombocytopenic purpura (AITP) acute, and chronic forms 169 autoantigens 172 CD5+ B cells 171 clinical syndrome 168-9 emergency treatment 181 GPIIb-IIIa receptor complex 176-7 IgG detection 172 immune abnormalities 171-8 isotopic studies 170-1 laboratory testing 169-70 and malignancies 186-7 megakarocytopoiesis 170 pl. counts 170 in pregnancy asymptomatic maternal 183 hidden maternal autoimmunity 183 infants 181-3 mothers 181 prevalence 168 secondary 183-5 treatment acute AITP 178-9 Rhesus antibodies (anti-D) 179 steroids or IgG 178 chronic AITP 179-80 corticosteroids 179, 181 high-dose i.v. IgG 179 splenectomy 179-80 emergency treatment 181 refractory chronic AITP 180-1 azathioprine 180 azidothymidine (AZT), and pl. counts 146 B lymphocytes, CD5+ 171 Bacillus anthracis, B-lysin 104-5

bacteria adherence, pathologic processes involving pl.-bacterial interactions 111 in vivo, historical background 107-8 bacterial endocarditis 112-14 and von Willebrand's factor 114 bacterial infections 83-124 non-allergic host defence 6-7 bacterial interactions with platelets, in v/tr0 89-107 adhesion and activation 101-3 aggregometry 90-2 engulfment of bacteria 103-4

fate of bacteria 104-7 bacterial products affecting pl. 107 influence of plasma components 94-8 morphological response 92-4 platelet secretion 100-1 J variations in response 98-100 bacterial interactions with platelets, in v/v0 107-9 bacterial clearance from circulation 108 human disease 110-16 phagocytes 109 bacteriocides fl-lysin 6 released by pl. 6 BAL fluid, pl. in 11 batroxostatin 158 beige mouse analog, ChediakHigachi syndrome 110 Bernard-Soulier syndrome 177 bilirubin, hydrophilic scavenger 218 bioassay, eicosanoids 198 blood flow bacterial endocarditis 113-14 shear stress 38-9 BN-52063 8-9 bone marrow, in situ hybridization, HIV infection 147 bone marrow transplantation 186-7 bronchoconstriction, pl. agonist and allergen-induced, animal models of lung injury 25 Brug/a ma/ay/127, 129 BW755C, inhibition of lipoxygenase 5-LO 27 BWA4C, inhibition of lipoxygenase 5-LO 27 Clq receptors 40 C3b 7 GP receptor 2 C5, chemotactic factor 3 C5b-C9 7 C18, adhesion of TNFc~-activated neutrophils 73 C-reactive protein, induction 130 calcium GPIIb-IIIa complex 44 and IP-3 43-5 regulation of cellular responses 44-5 calcium ionophore 216 cAMP inhibition of pl. activation 50 as second messenger, IP-3 33 synthesis 49 canalicular system (OCS) 87, 141 carbohydrate recognition domain (CR-) 71 carbon see non-biological particulates cardiovascular disease atherosclerosis, endothelial damage 115

CAD malondialdehyde 220 shear stress of blood 38 see also atherosclerosis carotenoids 218 carrageenin-induced inflammation 23 catalase 215 cathepsin G, neutrophils 26 CD4+ antigen priming 73 and IL-2 171 CD4+/CD8+ 73, 171 CD4+]CD8-, IFN release 8 CD4-]CD8+, PASL production 131 CD5+, B lymphocytes 171 CD8+ antigen, PASL 8 CD9 protein 39 cloning 39 CD11]CD18 complex integrins 74 thrombospondin receptor 77 CD15 72 CD16+ 73 CD31 (PECAM-1) 40, 155 CD36 (GPIV) receptor 34 and thrombospondin 75-7 CD62 see GMP- 140 ceruloplasmin, reduction, Fenton reaction 218 cetirizine, action on pl. s 27 cGMP inhibition of pl. activation 50 pl. production 50-1 synthesis 49-50 Chediak-Higachi syndrome, beige mouse analog 110 chemokines, pl.-released products, list 152 chemotactic factors complement C5 3 see also 5-HETE, 12-HETE, 12,20-diHETE chemotaxis, neutrophils 110 Chikungunya viral infection 138 chondroitin sulphate proteoglycan 70 synthesis 77 chromatographic analysis of eicosanoids 198-9 radioactivity detection 199 circulation, bacterial clearance 108-9 Clostridium, fl-lysin 104-5 coagulation reactions, regulation by activated pl. 40 colchicine, refractory chronic AITP 180 collagen, hyperresponsivenss 12 collagen receptor 34, 36-7 GPIa-IIa complex 4 complement see C1; C3, etc. coronary artery disease see cardiovascular disease corticosteroids, treatment, refractory chronic AITP 179, 181 cranial vasculature, pl. sequestration 22

INDEX cyclooxygenase 154 cyclophosphamide 180 cytokines activation of pl. 74-5 pl.-released products, list 152 proinflammatory see intercrines RANTES 153 regulation of pl. effector function 133-4 cytotoxic free radicals see free radicals danazol, refractory chronic AITP 180 defence mechanisms see host defence dengue viral infections 138, 145 dense tubular system (-TS) 50 dextran sulphate 72 diapedesis 25 dichlorofluorescein 216 diethylcarbamazine in filariasis 129 hydroxyl radicals 220 dimethyl pyrroline oxide 215 dinitrophenylhydrazine 219 D/petakmema viteae 127, 129 dipyridamole, antimetastatic action 158 disease, human and free radicals 220 pathologic processes involving pl.-bacterial interactions, list 111 pl.-bacterial interactions 110-16 see also specific conditions

disintegrins 158-9 disseminated intravascular coagulation 114-15 animal models, tumour models 156-7 DNA virus infections, virus-induced autoimmune thrombocytopenias 185 E-aminocaproic acid (EACA) 85 EDTA 126 chelation, and uptake of latex 88 Ehlers-Danlos syndrome, fibronectindefective pl. 97 eicosanoids 195-208 analysis 197-8 characteristics 197-8 concepts 196-7 derived from in vitro studies 198-204 in vivo production, assessment 204-6 UV extinction coefficients 197 ELAM-1, tumour cell interactions 155 EMEM, pl. isolation procedure 126 endocarditis, infective 112-14 endothelial cell growth factor (ECGF), properties 153 endothelial cells damage atherosclerosis 115

by pl. 111 HUVEC, SAVEC, upregulation of ICAM-1 74 endothelium-derived relaxing factor (nitric oxide) 49-50 Enterococcus faecalis, pl. responses 98-100 eosinophil, role in pl. activation 26 eosinophil granule proteins (E-N) 10 epidermal growth factor, properties 153 epinephrine oxidation 211 in pl. reactivity 98 epinephrine receptor 34 characteristics 37 eristostatin 158 erythrocytes, [99m] Tc-labelled, lung accumulation, animal models 22 erythroleukemia cells, human, GPIIb-IIIa receptor 44 Escherichia coli

pl. responses 98-100 survival 105, 106 N-ethylmaleimide 216 Evans syndrome 186 fatty acids, PUFA, membrane peroxidation 213 Fc epsilon receptor see Ig Fenton reaction Haber-Weiss cycle 218 reduction of ceruloplasmin 218 ferritin, uptake 88 fetal thrombocytopenia 181-3 fibrin, generation, regulation 40 fibrinogen, and pl. aggregation response 89 fibrinogen receptor 7 fibrinolytic system, interactions with pl., clearance of particulates 86 fibronectin, GPIIb-IIIa as receptor 113 filariasis 127, 129 FITC 126 flow cytofluorometry 126 fluoroquinolone, adhesion of bacteria 111 fluorouracil, antimetastatic action 158 FMLP 217 pl. accumulation 26, 77 pl. sequestration 26 foreign particles see non-biological particulates free radicals ASA-induced asthma 11 defence mechanisms endogenous protection 217-18 exogenous defence 218 superoxide dismutases 217 defined 210 generation by pl. 209-25 enzymes involved 214-15 mechanisms 216

245

and human disease, list 220 monitoring 219-20 electron spin resonance spectroscopy 219 oxygen activation and 210-15 oxygen reaction with 212-14 and parasites 7-8 partial reduction 210 pl., scavenging properties 219 Friend leukemia virus 138-41 fucoidin 72 Fusobacterium necrophorum, pl. induction 99 G-CSF, tumour cells 153 G-proteins 40-3 Gp, Gs and Gi 40-2 LMW Ga 42-3 transduction of intracellular stimulus, schema 41 GAP see GTPase-activating protein Glanzman thrombasthenia 172 glossary 227-35 glucose, hydrophilic scavenger 218 glutathione, hydrophilic scavenger 218 glutathione (reduced) recycling 217 renal failure and 220 glutathione-S-transferases 217-18 glycoproteins c~/33 receptor see glycoproteins, GPIIb-IIIa receptor collagen receptor 4, 36-7 GP receptors 2 GPIa-IIa receptor 4, 34, 36, 68 deficiency, clinical features 36 GPIa-IIb 67, 68 GPIb 4, 38-9, 155, 177 GPIb-IIa 67, 68 GPIb-IX 67, 68, 176, 177 GPIb-IX-V complex 177 GPIb-V-IX complex 39 GPIc~ 39 GPIc-IIa 67, 68 GPIIa-IIIa 155 GPIIb 176 GPIIb-IIIa receptor (pl. antigen) 39, 68 e~-thrombin activation 152 and AITP 176-8 complex with calcium 44 human erythroleukemia cells 44 labelling for cytofluorometry 126 oligomerization 43 receptor for fibronectin 113 see also integrins GPIIbc~ 34 GPIIIa 176 GPIIIb 155 GPIIIb see CD36 GPIV 34, 36, 37, 155 characteristics 37 GPIX 177 GPVI 34, 36, 37 possible role 37

246

INDEX

glycoproteins (continued) inflammation 67-8 packaged in granules 68 of pl. surface 67-8, 75 GPIIb-IIIa receptor see glycoproteins, GPIIb-IIIa receptor granule membrane proteins (GMP) cGMP, synthesis 49-50 GMP-140 (PADGEM) antibody to 22 selectins 4, 40 co-granules, activated pl. surface 2, 68 GRGDs 158 growth factors, pl.-released products, list 152 GTPase-activating protein complexed to Ga 43 ras 42 guanylate cyclase, aggregation response, damping 51 Haber-Weiss cycle, formation of hydroxyl radicals 212 haemarthrosis, animal models 23 haemorrhagic phenomena, viral infections 137-8 haemostasis 4-5 haptens, eicosanoids 202 HBSS 110 S. aureus aggregation 94, 98 tumour cell aggregation 159 Helicobacter pyh)r/107 helminths see parasitic infection; schistosomiasis heparin 72 hepatocyte growth factor (HGF) 153 5-HETE 4, 68, 73, 154 UV detection 200-1 12-HETE 4, 73, 216 function 195 metabolism 196 12,20-diHETE 4, 73 HHT 195 histamine intracellular signalling 49 synthesis 3 histidine decarboxylase 49 Histoplasma capsulatum, pl. induction 99 HIV and autoimmune thrombocytopenias 184-5 in situ hybridization of bone marrow 147 thrombocytopenia in 146 host defence allergic inflammation 7-12 historical background 108 mechanisms antioxidants 217-19 endogenous/exogenous protection 217-18 non-allergic pl. and bacteria 6 pl. and malignancy 6-7

5-HT see serotonin human disease see disease;, see also specific conditions

human parvovirus B19 185 hydrogen peroxide generation 211 IgE-induced 215 pl. from asthmatics with aspirin sensitivity 215-16 hydrophilic scavenger molecules 218 hydrophobic scavenger molecules 218 5-hydroxyeicosapentaenoic acid see HETE hydroxyl radicals 211 formation, Haber-Weiss cycle 212 13-hydroxyoctadecanoic acid (HODE) 154 hymenoptera venom hypersensitivity 131,220 ICAMs ICAM-1, endothelial cells 74 IgG-like molecules 155 idiopathic thrombocytopenic purpura see autoimmune thrombocytopenias Ig Fc fragments Fc epsilon receptor 7, 40, 98 FcPRII 40

IsE and aspirin 129 identification on pl. 126 IgE-induced hydrogen peroxide generation 215 radiolabelled, binding 126-7 IgE receptor 2, 7, 27, 127 cytotoxic free radical production 7 historical background 125 IgG detection, autoimmune thrombocytopenic purpura (AITP) 172 IgG-like molecules 155 pl., in patients with immune thrombocytopenia 145 rheumatoid factor 12 treatment, acute AITP 178 treatment, chronic AITP 179 IgG receptor 27 illustrations, key to all illustrations 237-41 immune mechanisms, causing thrombocytopenia 145 immune response, restoration 3 immune thrombocytopenic purpura 183-5 see also autoimmune thrombocytopenic purpura immunoassays 201 urines 205-6 immunoblotting, autoantigens 173 [111] In-labelled platelets animal models 22 investigations of function 5-6

inflammation acute, experimental models 8, 22-3 glycoproteins 67-8 human pl. membrane receptors 67-82 see also named receptors and tissue injury 112-15 inositol phosphates inositol 1,2-(cyclic)-4,5trisphosphate, regulation of calcium 44 inositol 1,4,5-trisphosphate (IP-3) 33 receptor, characterization 43-4 intracellular signalling pathways 43-5 see also phosphoinositides integrins cDll/CD18 complex 74 list 155 membrane GPs 4 pl.-tumour cell interactions 154-5 see also glycoproteins (esp.) GPIIb-IIIa intercrines, CXC 77 interferons antimetastatic action 158 IFN~, refractory chronic AITP 180 IFNP serotonin release 74 T lymphocyte regulation of pl. effector function 130-1 interleukins activation of pl. 74-5 IL-1 expression by pl. 74 release 153 upregulation of ICAM-1 74 IL-lc~ 174 IL-1B 74 IL-2, and CD4+ 171 IL-6, monocytes 134 IL-8 77 IL-8 supergenes, RANTES 3-4 NAP-2 77 intracellular signalling pathways IP-3 and calcium 43-5 phospholipase C 43-9 protein kinase C 45-6 iron load, pl. hyperactivity 219 JE peptide, and platelet factor-4 (PF4) 153 ketotifen, inhibition of pl. release reaction 5 fl-lysin 104-5 lanthanum, access to pl. interios 87 late-onset response (LOR), airway hyperresponsiveness 9 latex uptake, chelation with EDTA 88 leukocyte adhesion molecules, LAM-1, tumour cell interactions 155 Lactobacillus,

INDEX leukocytes bacterial uptake 108 leukocyte-platelet interactions 40, 71 primitive 1 leukotrienes LTA4, neutrophil 196 LTC4, metabolism of eicosanoids 196 LTB4, LTC4 4, 204 Lewis lung carcinoma 154 ligand-receptor interactions in pl. activation 31-66 activation-induced changes 33-40 G-proteins 40-3 inhibitory 49-51 intracellular signalling pathways 43-9 strong/weak agonists 33 lipid mediators, eicosanoids 196 lipoxygenase 5-LO inhibition with BWA4C 27 pathway 216 Listeria monocytogenes

phagocytosis 109 p l . induction 99 L0a/0a 127, 129 lucigen chemiluminescence 219 luminol/luciferin chemiluminescence 219 lung injury animal models allergic lung injury 25-6 bronchoconstriction, pl. agonist and allergen-induced 25 microembolism 23 non-allergic lung injury 24-5 pulmonary hypertension 24-5 in vitro studies 24 oedema PASL 131 pl. attenuation 219 lymphokines, PASL 131 lymphoproliferative disorders 186 LYP-20 74 /~-lysin 6 origin 104-5 lysostaphin 105 MAIDS (murine AIDS) 139, 143-4 MAIPA, antigenic capture assay 174-6 malaria 129 malignancies autoimmune thrombocytopenias 186-7 non-allergic~host defence 6-7 see also platelet-tumour cell interactions; tumour cells malondialdehyde 219, 220 in CAD 220 Manaker-C60 agents 138 mannitol, hydrophilic scavenger 218 measles virus, vaccination 142, 143

megakarocytopoiesis, autoimmune thrombocytopenias 170 megakaryocytes differentiation 138 and platelets, viruses, effects 138-45 melanoma B16BL6 line (mice) 156, 157 pl. counts 156 mepyramine 25 metastases see malignancies; platelet-tumour cell interactions; tumour cells microembolism, and lung injury, animal models 23 microtubules 2 platelet shape 2 migraine 220 mitogenesis, platelet-derived growth factor 3 Moloney virus 138 monoclonal antibodies Ap2, labelling GPIIb-IIIa receptor (pl. antigen) for cytofluorometry 126, 130 LYP20 74 MAIPA assay 174-6 radiolabelled, binding assays 145 monocyte inflammatory protein-1 153 monocytes binding pl. 71 chemotactic stimulation by PDGF 110 regulation of pl. effector function 134 thrombospondin as ligand to pl.-monocyte interactions 76-7 murine AIDS 139, 143-4 Mycobacterium t~erculosis, pl. induction 99 Na+-H § exchange, countertransporter 47-8 nedocromil sodium, action on pl. 11, 27 neutrophil-activating peptide (NAP-2) 77 neutrophilin 4 neutrophils chemotactic response 110 chemotaxis 110 phagocytosis of pl. in presence of bacteria 110 platelet activating factor (neutrophilin) 4 in rheumatoid arthritis 12 role in pl. activation 26 nitric oxide (endothelium-derived relaxing factor) 49-50 nitrovasodilatols 49-50 non-allergic lung injury role of pl. in animal models 23-4 in vivo, in vitro studies 23-4

247

non-biological particulates aggregation response 89 clearance from circulation 84-6 engulfment, phagocytosis or sequestration 86-7 historical background 84-6 influence of particle size 87-8 metabolism during ingestion of inert particulates 88-9 pl. adherence 84 pl. secretion 89 soluble co-factors of particle uptake 88 oleic acid, lung injury 24 open canalicular system (OCS) 87, 141 oxygen reduction, enzymes involved 214-15 singlet 211-12 oxypurinol, hydrophilic scavenger 218 PADGEM see (P)-selectins parasitic infection 125-36 animal models 28 and autoimmune thrombocytopenias 187 and cytotoxic free radicals 7-8 particulates see non-biological particulates PECAM-1 (CD31), IgG-like molecules 40, 155 pepticytosis 105 perfloxacin, adhesion of bacteria 111 peroxides, generation 211,212-14 phagocytes, bacterial interactions, effects of platelets 109-10 phagocytosis by pl. 6, 110 chemotaxis 110 and killing of bacteria 109-10 or sequestration, engulfment Of non-biological particulates 86-7 phorbol myristate acetate 216 lung injury 24 phosphatidic acid, formation from PIP2 48 phosphoinositides DAG generation 43 IP-3 and calcium 43-5 phosphatidylinositol 1,4,5trisphosphate (PIP3) 33, 43 regulation of calcium 44 phosphatidylinositol-3-kinase 49 phosphatidylinositol-4, 5bisphosphate (PIP2) 33, 43 regulation of cellular stimulus-response coupling 43 phospholipase A2 arachidonic acid metabolism 46-7 generation of TXA2 4 secretory and cytosolic 46 specificity for phospholipids 47

248

INDEX

phospholipase C activation 37 forms 43 intracellular signalling pathways 43 phospholipase D, initiation of signal generation 48 phospholipids, radiolabelling 199-200 physiology of platelets 2, 32-3 placetins 107 plakin 105 plasmatic zone 107 plasminogen activator inhibitor-1 153 plasminogen receptor 40 Plasmodium falciparum 129 Plasmodium vivax, engulfment of parasites 104 platelet(s) see also other entries, below, commencing with platelet

activation, antigen-induced 27 activation in allergic disorders 131 activation pathways schema 48 strong and weak agonists 48 aged, destruction 2 aggregation ADP 9 animal models 159 and free radical generation 216-17 induction by pathogens 6 platelet-tumour cell interactions 152 ticlopidine inhibition 158 antigen-induced activation 27 bacterial interactions in vitro 89-107 cationic proteins 3 characteristics 1-2 count 2 cytotoxicity anti-schistosome 126 inhibitors 218 other inducers 130 defence role see host defence destruction 2 effector function 130 engulfment of bacteria 103-4 fate of bacteria 104-7 generation of free radicals 209-25 granules 2, 68 hyperactivity, iron load 219 hyperlipidaemia 2 immunohistochemical localization 160 in inflammation see inflammation inhibition, cAMP and cGMP 50-1 interactions with: eosinophils 26 neutrophils 26 isolation procedure 125-6 lifespan 2 microtubules 2

monitoring in vivo investigations of function 5-6 methods 5 open canalicular system (OCS) 87, 141 origin 2 physiology 2, 32-3 primary activation pathway 32 release reaction 111 secretion, bacterial interactions 100-1 senescence 2 suspension, preparation 125-6 transfusion 181 ultrastructure 2 platelet activating factor 34, 36 antagonists BN-52063 and BN-52021 8-9 UK-74505 8-9 characteristics 36 in vivo administration, animal models 6 LTB4 4 LTC4 4 mediation of airway hyperresponsiveness 25-6 PAF-acether 73 platelet activation-dependent granule external membrane protein see(P)-selectins (PADGEM) platelet activity suppressive lymphokine (PASL) 8, 131 cloning strategy 132 ubiquitin and PASL 132-3 platelet adhesive receptor, pl.-tumour cell interactions, list 155 platelet aggregation-associated protein (PAAP) 101-2 platelet-bacterial interactions, disease, human 110-16 platelet basic protein (PBP) 153 platelet-derived growth factor (P-GF) chemotactic stimulation, monocytes 110 mitogenesis 3 properties 152-3 platelet-derived histamine releasing factor (PDHRF) 3 platelet-derived hyperreactivity factor (PDHRF) 6, 26 platelet-derived mediators 3-4 see also arachidonic acid; chemotactic factors; histamine; neutrophilin; PF-4; plateletderived growth factor; RANTES; serotonin platelet-endothelial interactions 40 platelet factor-4 (PF4) affinity to fl-thromboglobulin 77 airway hyperresponsiveness 25 in asthma, following antigen challenge 10 characteristics 77 in inflammation 68

intercrine family 77 and JE peptide 153 low affinity (LAPF4) 77 platelet-specific protein 3 properties 153 platelet-leukocyte interactions 40, 71 P-selectins 71 platelet receptors activation-induced changes 40 human 67-82 surface membrane receptors 2 see also specific receptors platelet-T lymphocyte interactions 73 platelet-tumour cell interactions 151-65 animal models 159-60 in vitro and in vivo assays 159-60 antiplatelet drugs, and metastases 157-9 in vitro studies 151-6 in vivo studies 156-7 pl.-released products, list 152 pregnancy autoimmune thrombocytopenic purpura (AITP) 181-3 asymptomatic type 183 prostacyclin, inhibition of pl. aggregation 158 prostaglandin endoperoxides 33, 36 prostaglandin IG2 49 protein kinase C intracellular signalling pathways 45-6 pharmacologic action 46 stimulation of histidine decarboxylase activity 49 Pseudomonas

pl. induction 99 role in ARDS, animal models 114 psoriasis, and pl. 12 PTA1 40 pulmonary hypertension, animal models 24-5 purpura viral infections 137 see also autoimmune thrombocytopenic purpura (AITP) pyrimido-pyrimidine RA233, antimetastatic action 158 quercetin, hydrophilic scavenger 218 rabbit, acute serum sickness 23 radioimmunoprecipitation 173-4 radiolabelling, for monitoring pl. function in vivo 5, 160 ragweed, and IgE 26 RANTES 153 IL-8 supergenes 3-4 ras GTPase-activating protein 42 Raynaud's disease, pl. aggregation 12 receptors see ligand-receptor interactions in pl. activation red cell aplasia 187

INDEX renal transplantation, rejection, role of pl. 112 retroviruses, internalization 146 RG- peptides 158 RG- sequence 154 Rhesus antibodies (anti-D) refractory chronic AITP 180 treatment, acute AITP 179 rheumatoid arthritis 11-12 ristocetin, von Willebrand factor-GPIb binding 38 RNA virus infections with reverse transcriptase activity 184 virus-induced autoimmune thrombocytopenias 183-5 rulin, hydrophilic scavenger 218 Salmonella spp. fla-lysin 105 pl. induction 99 scavenger molecules 218 Schartzman reaction, animal models 23 schistosomiasis 125, 127-9 anti-schistosome cytotoxicity 126 effector properties of pl. 127 and IgE receptor 7, 125 and IgE-induced peroxide production 215 schistosomula, role of pl. in animal models 28 substance P and 130 SDS-Page electrophoresis, radioimmunoprecipitation 173-4 E-selectin 71 L-selectin 71 P-selectins amino acid composition 70 in animal models 74 in circulation 72-3 gene, human 70-1 GMP-140 (PADGEM) 4, 22, 40, 69, 154 HUVEC 70 ligand 72 in man 67-74 monoclonal antibodies against 73-4 platelet-leukocyte interactions 71 radiolabelling 72 structure and homology 69-71 tumour cell interactions 155 variant forms 70-1 septic shock lung syndrome, TXA-2 in 114 sequestration, or phagocytosis, engulfment of non-biological particulates 86-7 serotonin release, IFN-b-induced 74 S. aureus induction of secretion 101 serotonin receptor 34, 38 shear stress, pl. aggregation 38-9

shear stress-induced transmembrane stimulus-response coupling 39 shear stressed platelet aggregation 38-9 Sh~qella, fl-lysin 104-5 signal transduction extraceUular, overview 32 intracellular signalling pathways 43-9 see also ligand-receptor interactions in pl. activation skin inflammation, and pl. 12 sodium cromoglycate, in vitro pl. responsiveness to ASA asthma 11 splenectomy, treatment, chronic AITP 179-80 src-related tyrosine kinases 42 Staphylococcus aureus 209P, engulfment of bacteria 104 502A pl. aggregation 92-7 pl. responses 98-100 survival 105, 106 aggregometry 90-2 bacterial endocarditis 112-14 IgM pl. bindable antibodies 171 interaction with pl. 77 cation concentration 94 phagocytosis 109 pl. induction of secretion 101 Staphylococcus epidermidis pl. induction 99 pl. responses 98 steroids, treatment, chronic AITP 179 Streptococcus bacterial endocarditis 112-14 interaction with pl. 77 Streptococcusfaecalis, survival 105 Streptococcus mutans, pl. induction 99 Streptococcus pneumoniae pl. induction 99 types 8 and 24 98-100 Streptococcus pyogenes pl. induction 99 pl. interactions 93 pl. responses 98-100 survival 105, 106 Streptococcus sanguis adhesion 101 aggregation 98, 101-2 ecto-ATPase 102-3 pl. induction 99 pl. reponse 97-8 substance P 130 sulphinpyrazone 22 superoxide anion 210-11 superoxide dismutases 211 defence mechanisms, free radicals 217 systemic lupus erythematosus 185-6 pl. aggregation 12 systemic sclerosis , pl. aggregation 12

249

T lymphocytes platelet-T lymphocyte interactions 73 regulation of pl. effector function 130-4 INFb130 TNFc~, fl 130-1 suppression of pl. cytotoxicity function 131-4 PASL 131 ubiquitin 131-4 see also CD tail transection bleeding time 160 [99m]Tc-labelled erythrocytes, lung accumulation, animal models 22 theophylline, inhibition of pl. release reaction 5 Thermonwno~ra, inhibition of pl. aggregation 107 thiobarbituric acid 219 thorotrast, uptake 88 thrombin 33-6 a-thrombin, activation of GPIIb-IIIa receptor 152 thrombin receptor 34 cloning 35-6 schema 35 thrombocytopenia amegakaryocytic, acute infections 143 animal models 108-9, 156 causes asthma 10 autoimmune 167-94 bacteremia 115 diverse causes 145-6 immune mechanisms 145 induced by Gram-negative bacteria 6 induced by pl. agonists 9 induction in animal models 108-9 measles virus vaccination 143 clinical features 137-8 defined 137 fetal 181-3 see also autoimmune thrombocytopenic purpura (AITP) thromboembolic disease 114-15 fl-thromboglobulin (fl-TG) 153 airway hyperresponsiveness 25 in asthma, following antigen challenge 10, 25 thrombosis 4-5 thrombospondin bacteria-pl, binding via 78 binding to resting or activated pl. 75 and C-36 75-7 characteristics 75 as ligand to pl.-monocyte interactions 76-7 properties 154

250

INDEX

thrombospondin (continued) receptors on pl. surface 76 site interacting with pl. surface 76 synthesis 70, 75 thromboxane A2 (TXA-2) 34, 36 bioassay 198 dogs 25 function 4 generation of phospholipase A2 4 receptor, high]medium affinity 34 release, Staph. aureus 24 in septic shock lung syndrome 114 sheep 25 see also prostaglandin endoperoxides thromboxane B2 (TXB-2) analysis 202-4 index of pl. activation 196-7 metabolic pathways 204-5 thromboxane synthetase inhibitors 158 ticlopidine, inhibition of aggregation 158 tissue injury, and inflammation 112-15 tissue plasminogen activator 40 tnf~, and monocytes 134 tnfc~, 8, T lymphocyte regulation of pl. effector function 130-1 TNF~-activated neutrophils, adhesion to C18 73 c~-tocopherol 218 Toxoplasma gondii 127, 129

transforming growth factor-~ (TGF-3), metastasis 152-3 triflavin 158 trigramin 158 Trypanosoma cruzi 127, 129 tumour cells adhesion to ECM 159 adhesion to endothelial cells 160 interactions with pl., see also platelet-tumour cell interactions metastases, and antiplatelet drugs 157-9 pl. adhesion 153-4 pl. aggregation, models 159-60 see also malignancies; metastases tyrosine kinases 49 tyrosine phosphorylation 49 ubiquitin isolation and cloning 131-2 and PASL, compared 132-4 UK-74505 9 uric acid, hydrophilic scavenger 218 urines, immunoassays 205-6 urticaria, and pl. 12 valves, prosthetic, endocarditis 114 vascular permeability haemarthrosis 23 increase, pl.-induced 3 vasculitis bacterially induced secretion of A-P 112

pl.-bacterial interactions 112 vasopressin receptor 34 characteristics 37 VCAM, IgG-like molecules 155 V/br/0, #-lysin 104-5 vinca alkaloids, refractory chronic AITP 180 viral infections 137-49 effects on megakaryocytes and pl. 138-45 thrombocytopenias associated, list 143 virus-induced autoimmune thrombocytopenias 183-5 vitamin E 218 VLA-2 see GPIa-IIb VLA-5 see GPIb-IIa VLA-6 see GPIc-IIa von Willebrand factor binding to GPIb 38 platelet interactions 38 von Willebrand's disease, animal models 114 Weibel-Palade bodies, vWf 70 xanthine oxidase 210 Yersinia pestis, inhibition of pl. aggregation 107 Yersinia pseudotuberculosis engulfment of bacteria 104 pl. induction 99