The Aetiology of Deep Venous Thrombosis: A Critical, Historical and Epistemological Survey

The Aetiology of Deep Venous Thrombosis

P. Colm Malone • Paul S. Agutter

The Aetiology of Deep Venous Thrombosis A Critical, Historical and Epistemological Survey

P. Colm Malone Formerly of the University of Birmingham UK

ISBN 978-1-4020-6649-8

Paul S. Agutter Theoretical and Cell Biology Consultancy Glossop, Derbyshire UK

e-ISBN 978-1-4020-6650-4

Library of Congress Control Number: 2007938450 © 2008 Springer Science + Business Media B.V. No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Cover illustration: Micrograph of a thrombus anchored to the base of a venous value cusp, reproduced with permission from Sevitt, S. (1974) Br. J. Surg. 61, 641–649 Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Preface

What we now call ‘deep venous thrombosis’ (DVT) has been studied in diverse ways during the last 200–300 years. Each of these approaches contributes to a full modern understanding of aetiology. Therefore, much of this book is a historical survey of the field. However, our remit is broader than the title might suggest: the evolution of ideas about DVT is typical in many ways of medical biology as a whole. Thus, although the aetiology of DVT may seem a narrow topic for a monograph – it implicitly excludes arterial thrombosis and marginalises prophylaxis, therapy, and even such clinically significant sequelae as pulmonary embolism – we hope to engage the reader in a much more general inquiry. Our historical investigation reveals a 160-year-old schism between two contrasting philosophies of medical and biological research, a schism that is particularly – but by no means uniquely – relevant to the study of DVT. In principle, these philosophies should be complementary rather than competing. So while we wish to elucidate the aetiology of DVT per se, we are also concerned with a more abstract and wide-ranging issue: the future accommodation or rapprochement between two conceptual and methodological traditions. To be more specific: present-day ideas about the occurrence, cause and treatment of DVT are dominated by a ‘consensus model’ that was authoritatively articulated during the early 1960s. This model attributes venous thrombosis to a combination of ‘hypercoagulability’, ‘stasis’ and ‘intimal injury’, and presumes on this basis to sharev common ground with the framework dubbed ‘Virchow’s triad’. In fact, as historical exegesis reveals, the consensus model originated and developed as a by-product of the century-long haematological/biochemical investigation of bleeding diatheses and in vitro studies of blood coagulation that began with the studies of Buchanan in the 1830s and was carried forward in the work of Schmidt and his successors. Scientifically, it owes little to Virchow; philosophically, it owes even less. An up-todate re-evaluation of Virchow’s actual contribution to the study of thrombosis and embolism is clearly indicated. The two contrasting approaches to research are clearly identifiable in this aspect of the history of medicine: the ‘mechanistic’ viewpoint, pioneered by Cartesians such as Hoffmann, La Mettrie and Boerhaave, re-articulated by du Bois Reymond and his associates in the mid-19th century, and reflected today in (e.g.) the consensus

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model of DVT; and the ‘pathophysiological’ viewpoint, rooted in Harvey’s work, articulated by (e.g.) Hunter, Virchow and Lister, and nowadays marginalised. We suggest that in view of the significance of the work of Virchow and his successors, the ‘pathophysiological’ or ‘vital-materialist’ approach to DVT – and to other areas of medicine and biomedical1 research – merits reflection, debate and reconsideration. Fundamentally, whereas pathologists sought for a century to find and explain the cause of DVT – and manifestly failed to do so – proponents of the consensus model after the Second World War effectively marginalised ‘cause’ and concentrated on therapy and pharmaceutical prophylaxis. The clinical value of this approach is not in question, but it should not be allowed to conceal the gap that it leaves in our understanding of aetiology. The mechanistic approach has enabled the coagulation (fibrinogenesis) process to be elucidated, and leukocyte and platelet congregation and the responses of cells to hypoxia to be characterised in cell-biological and molecular terms. All these are necessary for understanding the aetiology of DVT, but collectively they are not sufficient. The Virchowian, ‘pathophysiological’ literature shows that the circulatory impairment crucially associated with DVT is not reduced linear blood flow rate but non-pulsatile movement, and that ‘endothelial injury’ as a cause of DVT is essentially confined to the parietalis endothelium of the valve cusp leaflets. The ‘mechanistic’ literature articulating the consensus model ignores these crucial points. As it stands, the consensus model tends to confound ‘cause’ with ‘predisposing factors’, obfuscates the circulatory factors tending towards thrombosis under the misleading term ‘stasis’, and loses sight of the fundamental role of venous valves in the formation of thrombi. More generally: the consensus model does not wholly satisfy the epistemological criteria for an account of DVT aetiology. We argue a case for reconsidering the ‘valve cusp hypoxia hypothesis’ (VCHH), which appears to meet these criteria more completely. The VCHH is in the pathophysiological or ‘vital-materialist’ mould of Harvey, Hunter, Virchow and Lister; i.e. its focus is on malfunction at the physiological level. However, as we endeavour to show, it can be reconciled productively with mechanistic accounts of the blood coagulation mechanism and the molecular biology of the venous endothelium. We believe that this reconciliation leads to a novel, intellectually productive and clinically useful account of DVT: an illustration of the unification of traditions that we consider desirable in biomedical research as a whole. We review the epistemological/metaphysical overtones of the history and their significance for biomedical research in general in an appendix. 1 This adjective is of quite recent coinage (it is not recorded in the 1968 Shorter Oxford Dictionary but is to be found, along with the noun ‘biomedicine’, in the 1998 edition of Chambers Dictionary), but its use is now widespread.

Acknowledgments

We are indebted to colleagues past and present who directly or indirectly influenced the work described in this book or commented on draft chapters. Their contributions have been invaluable, but we accept full responsibility for the opinions expressed in the following pages. In particular, we wish to express our gratitude to Dr. Jürgen Lawrenz (University of Sydney) for his guidance on the history and philosophy of science; Professor Charles Warlow (Western General Hospital, Edinburgh) for aspects of the history of thrombosis research; Professor Vladimir Matveev (Russian Academy of Sciences) for information about the history of cell biology; and Professor Fedor Lurie (University of Hawaii) for discussions of the dynamics of venous valves. Professor Emeritus K.W. Walton provided research facilities and ongoing advice for two decades at the Department of Experimental Pathology at Birmingham University, and gave helpful criticisms of the manuscript. Professor Emeritus Ian Silver (University of Bristol) made indispensable contributions to the key experiments and discussed the valve cusp hypoxia hypothesis over 30 years. Dr. Chris Morris performed the transmission electron microscopic studies. Mr. Liam Murphy has been a faithful discussant for over 40 years, and latterly suggested that we include a synopsis of the book as a means of introducing newcomers to ideas quite different from those they have learned. We also thank Dr. Peter Walton (Dendrite Clinical Systems Ltd., Henley on Thames) for permission to reproduce Fig. 11.2; Mr. John Scurr and Dr. Carolyn Fisher for their encouraging comments on the draft outline of this book; Diane Blackmore of the Birmingham University Medical School Photographic Unit for her reproductions of micrographs and line drawings; Bethan Carter, Beata Kloska and Emma Shaw of the Royal Society of Medicine Library for their expertise in tracing obscure references; and the staff of the Barnes Library at Birmingham Medical School for almost four decades of service. The late Dr. Gordon Cumming (Queen Elizabeth Hospital and Midhurst Research Centre) suggested the crucial carbon monoxide model experiments. The late Mr. John Hamer (Queen Elizabeth Hospital) was a major participant in the experimental studies. The late Dr. Simon Sevitt demonstrated venous valve pocket thrombosis clinically in 1959, collaborated in the initial work on the hypothesis, carried out and discussed the histology of the animal experiments, and, before he

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died, bequeathed a number of his published and unpublished micrographs of venous thrombi in valve pockets. The late Dr. Michael Hume gave unstinting and much-appreciated support at all times. We first met during discussions of biological transport mechanisms with our friend and colleague Professor Denys Wheatley (Director, BioMedES, Inverurie) in the late 1980s. Had Denys not introduced us, this book would not have been written. Nor would it have been completed without the patience and forbearance of our families, for which we remain unreservedly grateful. July 2007

P. Colm Malone Paul S. Agutter

Synopsis

During the course of this book, two quite different approaches to the study of deep venous thrombosis (DVT) are evaluated and ultimately reconciled. At the outset, some of the material may be unfamiliar; for instance, the distinction between clot and thrombus is often not recognised, though it is fundamental to an understanding of DVT aetiology. We therefore begin by outlining the plan of the book and indicating the ‘end-point’ reached in Chapters 11–13. Chapter 1 is a general introduction: ●

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The frequency and incidence of deep venous thrombosis (DVT) and the major known predisposing factors are summarised, and ‘traveller’s thrombosis’ is mentioned. The main pathological consequences of the condition are noted: pulmonary embolism, the main cause of mortality; and post-thrombotic syndrome (a general label for the morbid sequelae, involving valve incompetence and leukocyte infiltration). What we term the ‘consensus model’ of DVT, which attributes the condition to some combination of hypercoagulability, stasis and endothelial injury, is then introduced; some variants of this model are outlined. A short critique of the consensus model is given, followed by a conjecture about its lasting prominence in the field, and this is followed by a critical discussion of the phrase ‘Virchow’s triad’. An alternative model of DVT is likewise briefly sketched. Because both the consensus model and the alternative viewpoint have deep historical roots, we argue that historical exegesis is necessary to explain their respective origins and development and to establish a rational understanding of the aetiology of DVT.

Most of Chapter 2 is devoted to a review of the blood coagulation mechanism as it is understood today. No matter how the aetiology of DVT is considered, blood coagulation is clearly involved at some point in the process: ●

The development of the blood coagulation ‘cascade’ concept after the Second World War is reviewed in conjunction with the then-increasing clinical use of

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anticoagulant and (subsequently) thrombolytic treatments. Those developments accompanied the emergence of the consensus model. We believe that the consensus model arose from a research tradition essentially unrelated to thrombosis, and that its articulation (a) effectively ousted the thencurrent pathophysiological viewpoint dating back to Virchow and (b) marginalised studies of the venous endothelium in relation to DVT. During this process, Virchow’s explicit distinction between ‘clot’ and ‘thrombus’ fell out of use. Virchow had likened thrombi to clots, but he realised the limitations of his analogy and insisted that the histology of thrombi proves that they form in flowing blood, not in static blood (as clots do). Some additional semantic points are raised for later discussion and development.

The theme of Chapter 3 is ‘hypercoagulability’: ●

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The common assumption that Virchow envisaged ‘hypercoagulability’ as a cause of venous thrombosis is refuted. Two uses of the word ‘hypercoagulability’ are distinguished: a general sense, which entails circular reasoning, and a specific sense, for which we substitute the synonym ‘thrombophilia’. The consensus hypothesis that ‘hypercoagulability causes DVT’ yields three predictions. One of these is shown to be weakly corroborated by early studies on ‘experimental thrombi’. The second and third predictions are evaluated through a review of the literature on hereditary and acquired thrombophilias and are shown not to be supported by the available evidence. It is concluded that thrombophilias increase the likelihood of DVT but are not to be considered ‘causal’. This is followed by a critical discussion of the clinical value of laboratory tests for thrombotic tendencies. The need for an alternative to the consensus model of DVT aetiology is re-emphasised.

We begin the historical exegesis in Chapter 4: ●

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Two approaches to biomedical investigation are distinguished: the ‘(patho)physiological’, associated with Harvey, Hunter and Virchow, and the ‘mechanistic’, associated with Boerhaave, du Bois Reymond and the consensus model of DVT. The origins of both traditions are traced to the 17th century, the Scientific Revolution and the emergence of natural philosophy. Their developments into the 18th and early 19th centuries are outlined. We identify the lines of investigation that led on the one hand to the elucidation of the blood coagulation mechanism, and on the other to the seminal contributions of Virchow. We take particular care to distinguish both mainstream traditions from the oncemisleading notion of ‘vital force’, and we consider other possible sources of semantic confusion.

Synopsis

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Chapter 5 pursues the first of these two lines of investigation, which led to the elucidation of blood coagulation: ●

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Studies of blood coagulation had originated among the ‘animal chemists’ of the 18th and early 19th centuries, but were placed on a firmer footing in the 1830s by Andral’s pioneering compilation of medical knowledge and by Buchanan’s experimental studies. From that time, the history of blood coagulation research can be divided into four arbitrary phases. This chapter concerns the first two phases; the third and fourth phases, covering the period between the Second World War and the present, were explored in Chapter 2. Phase 1 extended from Buchanan’s publications to the maturation of Schmidt’s ‘classical hypothesis’, when the action of thrombin was identified and the existence of prothrombin and antithrombins was inferred. These advances were underpinned by a philosophical movement known as ‘mechanistic materialism’, which (inter alia) marginalised the role of cells in blood function by focusing attention on the soluble components of the plasma. Phase 2, covering roughly the first half of the 20th century, saw the identification of prothrombin and of an ever-increasing number of coagulation factors, and the discoveries of heparin, vitamin K and dicoumarol. By the end of this period, the mechanistic-materialist character of coagulation research was becoming entrenched. Finally, we discuss the origin of this philosophical movement in a schism that took place around 1847. This schism primarily involved Emil du Bois Reymond, the leading protagonist of mechanistic materialism, and Rudolf Virchow, the pioneer of cellular pathology and an exponent of the ‘pathophysiological’ approach to biomedicine.

Chapter 6 pursues the second of the two lines of investigation mentioned in Chapter 4. The focus is on Virchow’s studies of thrombosis and embolism: ●

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The ‘doctrine of Cruveilhier’ is discussed in order to evaluate the then-customary use of the word ‘phlebitis’ in relation to what we now call ‘thrombosis’. Virchow’s life, times and philosophy are reviewed, and we emphasise his complete familiarity with the work of Boerhaave and Hunter as well as that of Cruveilhier and the early 19th-century microscopists who followed him. Virchow synthesised those contributions to knowledge by demonstrating that pulmonary emboli originate by metastasis (the word he used in this context) of peripheral venous thrombi to the pulmonary artery. He proved that thrombus ‘embolia’ form in moving, not static, blood; illustrated thrombi apparently associated with the cusps of venous valves (both parietal and ostial); distinguished clearly between thrombi and clots; and surmised that oxygen is required for thrombosis. Many authors have queried the concept termed ‘Virchow’s triad’. We concur that it perhaps originated in a misapplication of his work. We discuss Virchow’s opposition to Cruveilhier and his ‘pre-microscopy’ philosophy of biology and medicine.

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Synopsis

Later developments in the Virchowian tradition of investigation are reviewed in Chapter 7: ●

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The words ‘inflammation’, ‘phlebitis’ and ‘pus’ are immensely problematical. They have denoted different entities, and carried markedly different connotations, in different historical eras. These shifts of meaning make 21st-century interpretation of 18th- or 19th-century publications very difficult. Failure to take account of this difficulty has led, and will continue to lead, to persistent misunderstandings. The prime casualty of these semantic problems is the once-prominent association of ‘phlebitis’ with venous thrombosis. Indeed, resolution of that misleading association is a key outcome of our historical exegesis. The second interpretation problem to be resurrected is the involvement of leukocytes (alongside platelets) in the aetiology of DVT, re-focusing attention on the origin of venous thrombi on the central surfaces of valve cusps. The approach to DVT aetiology implicit in this discussion will be developed progressively in the following chapters.

The themes of Chapter 8 are the meaning of ‘stasis’, the variables of venous blood flow, and the significance and functioning of venous valves in DVT: ●

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The notion that ‘blood stasis’ is a causal or contributory factor in DVT has Galenic connotations and connections, which are intellectually problematical. In the literal English sense (i.e. absolute cessation of movement), ‘stasis’ entails local or organismic death. It has been shown experimentally to be ‘anti-coagulatory’ and must therefore presumably be ‘anti-thrombogenic’. The use of ‘stasis’ to denote ‘retarded flow’ or ‘interrupted flow’ is confusing not only for semantic and historical reasons, but also because it focuses attention on mean blood velocity in veins rather than on the pulsatility of blood movement. We contend that the (temporary) cessation of pulsatility in venous blood movement is instrumental in DVT. The history of the discovery of venous valves, and Harvey’s work in establishing their function, is the background to our reassessment of the evolution and proper interpretation of the ‘stasis’ concept. Our contention is that sustained non-pulsatile (streamline) blood flow in veins results in local hypoxaemia in affected venous valve pockets (VVP), the refilling of which furthers the initiation of a potentially thrombogenic sequence of events.

Chapter 9 focuses on the valves and valve pockets: ●

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Our key presumption is that the first stage in the aetiology of DVT is sustained underperfusion of the VVP during extended periods of non-pulsatile flow. Therefore, the structure, function and pathology of venous valves are discussed. The questions to be answered are: why and how does underperfusion of a VVP potentially lead to thrombosis; and where in the valve – and how – is thrombus formation initiated? Valve morphology and function, and certain findings at post-mortem examination, provide the key evidence that VVP are indeed the sites of venous thrombogenesis.

Synopsis

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The pathological circumstance arises when long-underperfused VVP result in endothelial hypoxia, specifically of the inner (parietalis) surface of the valve cusp(s), and that repetition and persistence of such special conditions may initiate DVT. On the presumption that this hypothesis is broadly valid, we calculate the approximate time needed for non-pulsatile venous flow to become pathogenic.

The idea that valve cusp endothelial hypoxia is instrumental in the aetiology of DVT is extended in Chapter 10: ●

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Hypoxic death of the valve cusp parietalis endothelium during phases of streamline local venous blood flow is treated as instrumental in thrombogenesis. This hypothesis accords with some mid-20th-century literature on the pathogenic effects of endothelial hypoxia. Published micrographs of venous thrombi are reviewed to elaborate this hypothesis and to establish the stage-by-stage process of thrombogenesis. Our account also relates to the induction of DVT-like lesions by non-fatal carbon monoxide poisoning, to the margination of leukocytes on hypoxic endothelia, to Aschoff’s autopsies of First World War victims, and to recent conflicting evidence about ‘traveller’s thrombosis’.

The valve cusp hypoxia hypothesis is stated in full in Chapter 11: ●

The VCHH describes the aetiology of DVT in terms of: ● ● ●

Venous valve cusp form and function. Hypoxic necrosis of the VVP parietalis endothelium. The active response of viable leukocytes and platelets to the hypoxic or dead endothelium.

It emphasises the gradual, sequential nature of venous thrombogenesis and focuses particularly on why and how blood that entered a VVP ‘alive’ may become ‘dead’ if it is not expelled until some hours later. ● ●

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Experimental confirmation of the premises and predictions of the VCHH is reviewed. We discuss the sharp distinctions between the VCHH and the consensus (haematological) model in scientific character, medical implications and philosophical connotations. DVT ‘risk factors’ are explained in the light of the VCHH. The prophylactic applications of the hypothesis are discussed.

In Chapter 12, the VCHH is supplemented and enriched by recent discoveries in the molecular biology of endothelial cells and their responses to hypoxia: ●

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Gene expression patterns in endothelial cells (EC), presumably including those of the valve cusp parietalis, change in response to hypoxia and other challenges. These changes, the signalling networks involved and the consequences for cell phenotype have been elucidated in considerable detail, providing a mechanistic underpinning for the VCHH. In particular, they define mechanisms for: ● The increased congregation and anchoring of leukocytes and platelets on the hypoxic area;

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The effects of activated neutrophils on the injured vascular endothelium; Enhanced blood coagulation in the immediate neighbourhood A significant part of the molecular-biological literature in this field concerns the effects of hypoxia on vasodynamics. We presume that this has limited relevance to the aetiology of DVT, though it must be pertinent to perfusion of venous vessel walls via the vasa venarum. We hope that this assimilation of ‘mechanistic’ findings into the ‘pathophysiological’ VCHH exemplifies how biomedicine might adopt a re-unified approach to problems, as outlined in the preface and discussed further in Chapter 4. ● ●

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Chapter 13 illustrates the value of an integrated, a priori account of the value of DVT by explaining the variable condition of post-mortem blood: ●

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Post-mortem blood may either consist of semi-solidified reddish masses (resembling either clots, thrombi or their combinations), or be entirely ‘white’ thrombi; or, in occasional cases, be wholly liquid and incoagulable. Such variations have confused pathologist observers, and we suggest an alternative explanation in terms of the VCHH (not an original explanation, as will be seen). The central question is whether it can ever be rightly imagined that intravascular blood coagulates after death. This question was intensely debated in the early 20th century but was then resolved ad hoc. To attempt a resolution, we re-explore the First World War controversy, reconsider the forensic and judicial interpretations/ implications of the states of post-mortem blood, and re-draw some clinical inferences. This leads to the conclusion that ‘post-mortem’ thrombi or clots – when they are not wholly absent – are invariably agonal thrombi.

The Appendix reflects on the philosophical underpinnings of the study of DVT throughout history and in the present day.

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Synopsis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 1

Chapter 2

Introduction to the Study of Deep Venous Thrombosis ........

1

1.1 1.2 1.3 1.4 1.5 1.6

Incidence............................................................................. Pathology ............................................................................ Aetiology: The Consensus Model ...................................... The Dominance of the Consensus Model .......................... ‘Virchow’s Triad’................................................................ An Alternative Viewpoint...................................................

1 3 4 7 8 9

The Coagulation Cascade and the Consensus Model of DVT ............................................................................

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2.1 2.2 2.3

11 13 16

2.4

The Cascade Model of Blood Coagulation ........................ The Origin of the Consensus Model of DVT..................... The Coagulation Cascade Today ........................................ 2.3.1 Platelet Activation and Congregation: Local Vasoconstriction ..................................................... 2.3.2 The Contact (‘Intrinsic’) System............................ 2.3.3 The Tissue Factor (‘Extrinsic’) System ................. 2.3.4 Fibrinogenesis ........................................................ 2.3.5 Fibrinolysis ............................................................. 2.3.6 Controlling Coagulation ......................................... Disorders of Coagulation ...................................................

17 19 20 20 23 25 27

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Chapter 3

Chapter 4

Contents

Hypercoagulability ....................................................................

31

3.1 Introduction ...................................................................... 3.2 The ‘General’ Use of the Term ‘Hypercoagulability’...... 3.3 Early Evidence for ‘Hypercoagulability’ Conditions ...... 3.4 Testing the ‘Hypercoagulability’ Hypothesis ................... 3.5 Inherited Thrombophilias ................................................. 3.6 Acquired Thrombophilias................................................. 3.7 Thrombophilia and DVT .................................................. 3.8 Testing for Thrombophilias .............................................. 3.9 Implications for Prophylaxis and Therapy ....................... 3.10 Reflection..........................................................................

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Historical Roots .........................................................................

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4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9

Chapter 5

Two Approaches to Biomedical Research ....................... Semantic Issues ................................................................ The Ancient World ........................................................... ‘Binary Oppositions’ in 17th–18th-Century Medicine .... Harvey and the ‘Physiological’ Approach ....................... The 18th Century: Solidism, Humoralism and the Work of Boerhaave .............................................. Hunter and Hewson .......................................................... Late 18th- and Early 19th-Century Studies of Blood Chemistry .......................................................... The 18th-Century Pioneers of Haemostatics and Haemodynamics ........................................................

Coagulation and its Disorders: A History of Haematological Research ..................................................... 5.1 5.2

5.3

5.4 5.5

Introduction ...................................................................... Phase 1: 1835–1893 ......................................................... 5.2.1 Haemophilia and the Study of Coagulation ......... 5.2.2 Buchanan .............................................................. 5.2.3 Buchanan’s Influence: The Impact of Mechanistic Materialism .............................................. 5.2.4 The ‘Classical Hypothesis’ of Blood Coagulation ........................................... Phase 2: 1893–1947 ......................................................... 5.3.1 Prothrombin and its Conversion to Thrombin .......................................................... 5.3.2 Heparin, Vitamin K and the Dominance of In Vitro Studies................................................. Philosophical and Semantic Considerations .................... Reflective Anamnesis .......................................................

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57 57 58 59 59 60 63 64 64 65 66 67

Contents

Chapter 6

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Virchow and the Pathophysiological Tradition in the 19th Century ................................................................... 6.1 6.2

Introduction ........................................................................ Cruveilhier .......................................................................... 6.2.1 Previous Insights into Thromboembolism ............. 6.2.2 Cruveilhier’s Contribution ...................................... Other Formative Influences on Virchow ............................ Resolving the Conflict: Virchow’s Synthesis ..................... Virchow on the Structure of a Thrombus ........................... Virchow on Oxygen and Thromboembolism ..................... Virchow versus Cruveilhier ................................................ The Possible Source of ‘Virchow’s Triad’ ......................... Reflective Anamnesis .........................................................

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The Pathophysiological Tradition after Virchow ...................

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6.3 6.4 6.5 6.6 6.7 6.8 6.9 Chapter 7

7.1

7.2 7.3 7.4 7.5 7.6 Chapter 8

71

Problems of Nomenclature: ‘Phlebitis’ and ‘Inflammation’ .................................................................... 7.1.1 ‘Pus’........................................................................ Leukocytes, Phagocytosis and Thrombosis........................ Platelets............................................................................... The Persistence of the ‘Phlebitis’ Concept ........................ Continuation of the Pathophysiological Perspective: Welch and Aschoff ......................................... The Role of Leukocytes Reconsidered ..............................

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Interrupted Circulation: The ‘Stasis’ Hypothesis and the Significance of Venous Valves ..................................... 103 8.1 8.2 8.3 8.4 8.5 8.6

Introduction ........................................................................ The Maturation of the Circulation Hypothesis .................. Connections with the Revolution in Mechanics................. The Discovery of Venous Valves ....................................... The Significance of Venous Valves .................................... 8.5.1 The Venous Valves and DVT ................................. The Persistence and Misleading Character of the ‘Stasis’ Concept ....................................................... 8.6.1 Inherent Difficulties in the ‘Stasis’ Dogma ........... 8.6.2 The Survival of the ‘Stasis’ Dogma in the 19th Century ................................................. 8.6.3 ‘Stasis’ and the Consensus Model of DVT Aetiology ....................................... 8.6.4 Sevitt on the Aetiology of DVT ............................. 8.6.5 The Consensus Model and the VVP as Sites of Thrombogenesis ..............

103 104 106 107 109 110 111 111 112 113 114 115

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8.7

8.8 Chapter 9

Is the Current (Mis)Use of ‘Blood Stasis’ Equivalent to Virchow’s ‘Interrupted Circulation’? ......... 8.7.1 Pulsatile Blood Movement in Veins..................... 8.7.2 Compression of Veins in the Soles of the Feet ........................................ 8.7.3 Effects of Standing, Sitting and Lying on the Dynamics of Lower Limb Veins ............... Towards the Valve Cusp Hypoxia Hypothesis: I – Altered Blood Movement ...........................................

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Underperfusion of Valve Pockets and the Initiation of DVT ....................................................................................... 121 9.1 9.2 9.3

9.4 9.5

9.6 9.7

9.8

Introduction ...................................................................... Thrombi Originate in the Venous Valve Pockets ............. The Morphology and Pathology of Venous Valves .......... 9.3.1 Venous Valve Morphology ................................... 9.3.2 Valve Pathology: The Formation of Nascent Thrombi within VVP ......................... 9.3.3 Can the Venous Return Circulation and Valve Function be Correlated? ...................... The Valve Cycle and the Effects of Non-Pulsatile Flow .................................................................................. Relevance of Venous Blood Rheology ............................. 9.5.1 Relevance of the Vasa Venarum ........................... 9.5.2 Flow Patterns within VVP ................................... 9.5.3 Implications for the Formation of Pro-Thrombotic Nidi ....................................... Implications for Compression Prophylaxis ...................... Towards the Valve Cusp Hypoxia Hypothesis: II-VVP Hypoxaemia......................................................... 9.7.1 Hypoxic Injury to the VVP Cusp Endothelium Is Potentially Thrombogenic: a Proposal ............................................................. 9.7.2 VVP Hypoxaemia and Hypoxic Injury to the Parietalis Endothelium .................... 9.7.3 Testing the Predictions ......................................... The Lesson of History ......................................................

121 122 125 127 129 132 133 134 135 137 137 138 139

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Chapter 10 The Role of Endothelial Hypoxia in DVT ............................. 147 10.1 10.2

Oxygen, the Venous Endothelium and Thrombosis....... Hypoxaemia, the Vascular Endothelium and Thrombosis .............................................................. 10.2.1 Association of DVT with Endothelial Hypoxia......................................... 10.2.2 Endothelial Hypoxia and Thrombus Formation in VVP ............................................

147 148 149 151

Contents

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10.2.3 10.2.4

10.3 10.4 10.5 10.6 10.7

Chapter 11

The Significance of Ostial Valves .................... Interpreting Micrographs of Venous Thrombi: The Tendency of Thrombi to Embolise ................................... Carbon Monoxide Poisoning and Anaemia ................... Endothelial Hypoxia and Leukocyte Margination ......... Aschoff on the Coagulation of Cadaver Blood .............. Hypoxaemia and ‘Traveller’s Thrombosis’ .................... Overview: Articulating the Valve Cusp Hypoxia Hypothesis .......................................................

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The Valve Cusp Hypoxia Hypothesis .................................... 169 11.1 11.2

11.3

11.4

11.5 11.6

11.7

Introduction .................................................................... 11.1.1 Criteria for an Aetiological Hypothesis ........... The General Aetiological Sequence: The ‘Trinity’........ 11.2.1 ‘Interrupted Flow’ and Underperfusion of VVP.............................................................. 11.2.2 The Specific Involvement of the Valve Cusp Parietalis Endothelium ....... 11.2.3 Blood Cell Congregation and Blood Coagulation ..................................... 11.2.4 Pathological Consequences .............................. Experimental Support for the VCHH............................. 11.3.1 Polarographic Demonstration of VVP Hypoxaemia during Non-Pulsatile Flow .......... 11.3.2 Experimental Venous Thrombi Induced by a Non-Invasive Technique ............. Clinical Implications ...................................................... 11.4.1 The Risks of Sleeping for Long Periods in the Sitting Position ....................................... 11.4.2 Simpson’s Cases ............................................... 11.4.3 Anaesthesia....................................................... 11.4.4 Crucifixion........................................................ 11.4.5 Varicose Veins .................................................. ‘Risk Factors’ for DVT Reconsidered in the Light of the VCHH .................................................................. Prophylaxis ..................................................................... 11.6.1 Our Theory-Based Estimate of 1.5–3 h ............ 11.6.2 Lister’s Experience ........................................... 11.6.3 Normal Tourniquet Practice ............................. 11.6.4 Published Traveller’s Thrombosis Data ........... 11.6.5 Intermittent Positive Pressure Compression (IPPC) of Feet or Legs ............... 11.6.6 The Animal Experiments of Hamer and Malone (1984) ........................................... Reflection .......................................................................

169 170 171 172 173 174 177 178 178 178 183 183 185 187 188 188 189 190 191 191 191 192 192 194 194

xx

Chapter 12

Contents

Molecular Changes in the Hypoxic Endothelium ................ 195 12.1 12.2 12.3

12.4 12.5 12.6

12.7

12.8 12.9 12.10 12.11 Chapter 13

Endothelial Cell Physiology ........................................ The VCHH and the Molecular Responses of EC to Hypoxia .................................................................... Phenotypic Changes in EC under Hypoxic Conditions .................................................................... 12.3.1 Phenotypic Changes Consequent on Egr-1 Induction ........................................................ 12.3.2 Elk-1 and SRF ................................................ 12.3.3 Other Regulators of Egr-1 Expression ........... Erg-1, Hypoxia and DVT ............................................. Thrombin and the PARs ............................................... Interactions Between Platelets and the Hypoxic Endothelium ................................................................. 12.6.1 Platelet Congregation and Implications for DVT .............................. 12.6.2 Leukocyte–Platelet Complexes in the Circulation and their Association with the Hypoxic Endothelium ...................... Endothelial Hypoxia and the Congregation of Leukocytes ................................................................... 12.7.1 Monocytes and Macrophages......................... 12.7.2 Neutrophils ..................................................... 12.7.3 Effects of Leukocytes on Injured Endothelium ................................. The Endothelium and Coagulation .............................. The Endothelium and Vasomotor Tone ....................... A Further Comment on ‘Risk Factors’ ........................ The Unification of Approaches ....................................

195 197 197 199 200 202 204 204 206 206

207 208 209 210 211 212 216 218 218

Cadaver Clots or Agonal Thrombi? ...................................... 221 13.1 13.2 13.3 13.4

13.5

Can Blood Coagulate in a Cadaver? ............................ An Early 20th-Century Debate .................................... Aschoff on ‘Post-Mortem Clots’ ................................. The Debate Reconsidered in the Light of the VCHH . 13.4.1 Death from Acute Respiratory Failure........... 13.4.2 Death from Circulatory Failure...................... 13.4.3 Summary: The Condition of the Blood Post-Mortem Depends on the Mode of Death .................................... Consequences of Positing that All Thrombi Are Agonal .......................................................................... 13.5.1 Possible Post-Mortem Changes .....................

221 222 224 226 226 227

228 228 229

Contents

xxi

13.5.2 13.6

Summing up the Argument: Judicial Implications ........................................ 229 Therapeutic and Prophylactic Implications.................... 230

Appendix: Science, Medicine and Philosophy.............................................. 233 A.1 A.2

A.3

A.4

A.5

The Two Approaches to Medical Biology........................... The Philosophical Background to the Schism of the 1840s ......................................................................... A.2.1 The Aftermath of the Scientific Revolution ........... A.2.2 The Empiricist Tradition......................................... A.2.3 Hume: The Achilles’ Heel of Empiricism.............. A.2.4 Kant ......................................................................... A.2.5 Naturphilosophie and its Influence on Philosophy and Science ............................................................. A.2.6 Schopenhauer .......................................................... A.2.7 Significance for 19th-Century Physiology and Pathology ......................................................... A.2.8 The Opening of the Schism .................................... A.2.9 The Origin and Development of Mechanistic Materialism ............................................................. Mechanism Versus Vitalism: The Distinctiveness of Vital-Materialism............................................................. A.3.1 The Mechanism-Vitalism Debate and its Implications ................................................. A.3.2 Alternatives to Mechanism are Often Misrepresented........................................ A.3.3 ‘Extreme’ Mechanism: The 19th-Century German Materialists.............................................................. The Modern Dominance of the Mechanistic Approach ...... A.4.1 The Metaphysical Dichotomy in Early 20th-Century Biology and Medicine .......................................................... A.4.2 Some Contributing Factors to the Hegemony of Mechanism............................. A.4.3 Molecular Motion Versus Bulk Transport: The ‘Newtonianism’ of Biochemistry... Rapprochement between the Mechanistic and Vital-Materialist Approaches ...............................................

233 234 235 235 236 237 238 239 239 240 241 243 243 245 245 247

247 248 249 251

References ........................................................................................................ 255 Author Index ................................................................................................... 295 Subject Index .................................................................................................. 305

Chapter 1

Introduction to the Study of Deep Venous Thrombosis

Abstract The frequency and incidence of deep venous thrombosis (DVT) and the major known predisposing factors are summarised, and ‘traveller’s thrombosis’ is briefly discussed. The main pathological consequences of the condition are outlined: pulmonary embolism, the main cause of mortality; and post-thrombotic syndrome (a general label for the morbid sequelae, involving valve incompetence and leukocyte infiltration). What we term the ‘consensus model’ of DVT, which attributes the condition to some combination of hypercoagulability, stasis and endothelial injury, is introduced; some variants of this model are outlined. A brief critique of the consensus model is given, followed by a conjecture about its sustained prominence in the field, and this is followed by a critical discussion of the phrase ‘Virchow’s triad’. An alternative model of DVT is briefly outlined. Because both the consensus model and the alternative viewpoint have deep historical roots, we suggest that historical exegesis is necessary to explain their origins and development and to establish a rational understanding of the aetiology of DVT.

Keywords Consensus model of DVT, endothelial injury, hypercoagulability, venous stasis, Virchow’s triad

1.1

Incidence

Deep venous thrombosis (DVT)1 is a significant and costly health care and social problem, a common post-operative complication and a serious threat to the patient’s general recovery (Peterson 1986). The average population incidence is about 0.5 per 1,000 person-years (Fowkes et al. 2004) but increases markedly with age

1 Many excellent reviews of the subject are available. We would refer the reader particularly to Browse et al. (1988b), which is now somewhat out of date, but gives a comprehensive list of references.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

1

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1 Introduction to the Study of Deep Venous Thrombosis

(Kniffin et al. 1994). Men are at slightly greater risk than women (Cushman et al. 2004) and Asian populations have a slightly lower incidence than other ethnic groups (Klatsky et al. 2000). DVT is statistically more common in obese and pregnant women, in hypertensives, in smokers and in those with varicose veins. Specifically, it complicates injuries, major surgical procedures, cardiac disease, various infections, neoplasias, autoimmune diseases (notably systemic lupus erythematosus, SLE), genetic defects affecting haemostatic factors, inflammatory bowel disease, endovascular irritation/injury from intravenous (IV) lines, ‘acquired hypercoagulability’ syndromes, and – most notoriously, and perhaps linking many or all the above – prolonged inactivity during decubitus, paresis, or paralysis of extremities (Geerts et al. 2004; den Heijer et al. 1998; Martinelli et al. 1998; Samama 2000; Heit et al. 2001; Galli et al. 2003; White et al. 2003; Alikhan et al. 2004). Jick et al. (1969) reported a statistical association with blood group A. The incidence is higher when more of the aforementioned factors are present concurrently, so it is widely held that DVT is a ‘multifactorial’ condition (e.g. Rosendaal 2005). However, it may be misleading to equate combinations of predisposing factors with ‘cause’. Many cases are considered idiopathic. The condition became headline news recently. Initially dubbed ‘economy class syndrome’, first recognised half a century ago (Emonson 1997), it was subsequently called ‘traveller’s thrombosis’ when that alternative was suggested by the UK House of Lords. Large-scale studies (e.g. Scurr et al. 2001; Jacobson et al. 2003; Schwarz et al. 2003; Cannegieter et al. 2006; see also Scurr 2002) have shown that long flights slightly increase the likelihood of DVT; there is a small exponential increase of incidence with travelling time (Lapostolle et al. 2004). However, nearly all the ‘traveller’s thrombosis’ cases reviewed by these authors occurred in passengers who had some combination of predisposing factors. These factors (see above) contribute jointly and severally to the incidence of DVT in the general population much more than ‘traveller’s thrombosis’ per se. Moreover, there is good evidence that prolonged sitting increases the likelihood of thrombosis in the deep leg veins (Homans 1954), irrespective of travel. An interesting and entertaining article by Dexter (1973) suggested a relationship between the rate of venous thromboembolism and the use of chairs throughout history.2 DVT remains most common in patients who require protracted bed-rest, especially after surgery involving a prolonged period of general anaesthesia. An aetiological model must explain both the incidence pattern and the ‘multifactorial nature’ of the disease; i.e. it must account for the ‘risk factors’.

2 This may suggest an explanation for the racial differences in the incidence of DVT. In many Asian societies, people sit on the floor rather than on chairs. For both postural and gravitational reasons, the venodynamic effects of this may be less favourable for thrombus formation; cf. Chapters 8 and 11.

1.2 Pathology

3

Fig. 1.1 Different seating conditions with decreasing risk of venous thrombosis. An elderly person dozing while sitting upright (top) is at greater risk than a younger person semi-reclining (middle), and the risk may be considerably lower for someone adopting the lotus position (bottom). The reasons are discussed in chapters 8 and 11

1.2

Pathology

Potentially thromboembolic DVT usually arises in one of the large deep veins of the lower limb and can be associated with significant morbidity and mortality. Around two thirds of hospitalisations are first-time episodes and the remainder are recurrent (Anderson et al. 1991). Unlike superficial venous thrombosis, which can present painfully, DVT tends to be asymptomatic unless either (a) fractured thrombi metastasise as emboli or (b) morbid sequelae develop. (a) Embolism is the commonest cause of DVT-related mortality. Cina et al. (1996) reported that there are at least 100,000 deaths per annum from pulmonary

4

1 Introduction to the Study of Deep Venous Thrombosis

embolism in the United States (around 0.1% of all patient mortalities). Emboli are observed in over 10% of unselected autopsies, and recurrent venous thrombotic and thromboembolic episodes are quite common (Freedman 1998). Mortality from untreated pulmonary embolism has been estimated at 30% (Carson et al. 1992). It is worth recalling that Virchow’s pioneering scientific study of DVT related entirely to the concept and category of ‘embolia’ and the novel related phenomenon of metastatic pulmonary embolisation. We shall discuss his work in Chapter 6. (b) Local DVT-associated morbidity is much less discussed than embolism but it may impair a patient’s quality of life by causing permanent disability. Morbid sequelae, collectively labelled ‘post-thrombotic syndrome’, usually begin with limb oedema caused or aggravated by raised venous pressure distal to a thrombus, which increases the rate of capillary filtration. Although this oedema may subside within a few months in about half of all cases – through thrombolysis and recanalisation3 (Killewich et al. 1989) – untreated DVT can result in chronic residual venous obstruction, incompetence or obliteration of affected venous valves, or both. This was recognised a century and a half ago (Gay 1866) and described in detail by Edwards and Edwards (1936). Chronic venous disease is a common sequel of valvular incompetence: leukocyte infiltration of injured valves may irreversibly alter their morphology and physical characteristics (Nicolaides 2005). The possible aetiological significance of this venous valve cusp infiltration has been widely overlooked or misunderstood.

1.3

Aetiology: The Consensus Model

Since the early 1960s, most explanations of DVT aetiology have revolved around ‘hypercoagulability’ and ‘stasis’. For example, Sherry (1962) wrote: ‘For over a century, leading pathologists, investigators and clinicians have continued to emphasize the importance of alterations in blood flow, damage to the vessel wall, and changes to the coagulability of blood as the major factors responsible for thrombus formation in vivo’. Comerota et al. (1985) commented: ‘The cause of postoperative DVT is considered to be changes in blood coagulation, stasis of blood within the veins, and injury to the vein wall’. Peterson (1986) declared: ‘Virchow’s triad of stasis, vessel injury, and hypercoagulability remains a valid explanation of the pathogenesis of thrombus formation’. Burroughs (1999) wrote: ‘The cause of thrombosis is often unknown but is universally ascribed to part of Virchow’s triad: stasis, hypercoagulability, and intimal injury’. Rosendaal (2005) stated: ‘The modern era of

3 Recanalisation results from fibrinolysis (Chapter 2) within the thrombus. A product of fibrin degradation, D-dimer, is considered to have negative diagnostic value (e.g. Lee and Hirsch 2002); that is, normal D-dimer levels can be taken to exclude a diagnosis of DVT, but elevated levels, though possibly suggestive, do not confirm the diagnosis.

1.3 Aetiology: The Consensus Model

5

understanding the etiology of thrombosis began with the pathologist Virchow, who in the mid-1800s postulated three major causes of thrombosis: changes in the vessel wall, changes in the blood flow, and changes in the blood composition’. Davies and McNicol (1983), Brott and Stump (1991), Salzman and Hirsh (1994), Hirsh and Crowther (2000) and numerous other authors have used similar terminology. Collectively, they articulate the modern consensus view that DVT is caused by some combination of (1) ‘hypercoagulability’, either systemic or local (Mammen 1992), (2) ‘stasis’ of the venous blood, and (3) injury to the vein wall intima, specifically the endothelium. Medical graduates will recognise this scheme as ‘Virchow’s triad’. Several variants of this consensus model have appeared during the last 4–5 decades. They all presume that venous thrombi result from disturbance of the ‘delicate balance of haemostatic machinery’ (Thiagarajan 2002) and differ only in their relative emphases on the three individually conceived ‘Virchow’s triad’ elements. Comerota et al. (1985) noted that the blood-composition element had been explicated in terms of platelet activation, the coagulation4 cascade and the suppression of endogenous fibrinolysis. The haemodynamic entity they called ‘stasis’, had, they implied, been demonstrated by (a) measurements of diminished venous blood velocity in anaesthetised or otherwise supine patients and (b) retention of contrast material in soleal sinuses and valve pockets (cf. McLachlin et al. 1960). Comerota et al. said that the vein-wall-injury component of the ‘triad’ was ‘more controversial’ and associated it (perhaps questionably) with vasodilatation accompanying the coagulation cascade in the aftermath of surgical trauma. More recent variants of the consensus model have envisaged a different balance among the putative aetiological elements. For instance, Bulger et al. (2004) still accepted that ‘activated coagulation’ is the primary contributor to venous thrombosis, but they considered ‘endothelial injury’ to have recovered some of its once-central significance. The pertinence of such injury is apparent from the increased incidence of DVT after tibial fracture (Nylander and Semb 1972) and after disturbance of the femoral vein during hip replacement (Stamatakis et al. 1977). The cellular and

4

The commonplace word ‘coagulation’, as used in this context, has a long history. In its familiar English sense it pictures an analogy between the semi-solidification of blood and the hardening of boiled or chemically treated egg internum. The idea that blood coagulation is in any way affected by or related to heat was disproved by Hewson and Hunter in the late 18th century. Hunter speculated that it might be more analogous to other functions of living tissues (see Chapter 4). Further to his speculation (re. muscle contraction), we may reflect that eggs are also semi-solidified by prolonged warmth, not just by heat or chemical action; kept warm and cosy they hatch into different kinds of solid objects, chickens. It is reasonable to consider blood semi-solidification as analogous to hatching rather than the hardening of cooked eggs, since the former results from a living process rather than the physico-chemical effects of heat alone. Be that as it may, it would be difficult to review and discuss the literature in this field without using ‘coagulation’ in its accepted sense. Moreover, the literal etymological meaning of coagulate (‘to drive together’) is a not inappropriate description of either platelet association or fibrin polymerisation. In much of this book, however, we shall use ‘semi-solidification’ instead, since this term carries no adventitious connotations.

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1 Introduction to the Study of Deep Venous Thrombosis

molecular changes consequent on endothelial injury have been elucidated by recent research (see Chapter 12). Bulger et al. relegated ‘stasis’ to ‘a largely permissive factor’. They also drew attention to the evolution of cellular and molecular changes in growing thrombi. They surmised that acute DVT reflects the balance between recurrent thrombotic events and processes restoring the vein wall (specifically the intima), both of which affect the subsequent lesion. This view was popular in the 19th century: DVT is a sustained, putatively continuous, multi-step process rather than a circumscribed event. We agree that thrombosis involves a sequence of steps, each of which is necessary for the pathological outcome. The proposed dynamic interrelationships among coagulation, altered circulation and the vein wall have received increasing attention since 1990. Mammen (1992) noted that endothelial damage stimulates the coagulation cascade; conversely, he posited that the factors involved in both blood coagulation and fibrinolysis might induce endothelial injury. (Mammen reiterated the questionable view that local vasodilatation resulting from the ‘pooling of slowly-moving blood’ could injure the intima. This is logically and physiologically incongruous but it should not detract from his paper as a whole.) Blann (2003) surmised that endothelial injury leading to local fluctuations in vasoconstriction and vasodilatation could also be thrombogenic. However, as recently as 2005, we find the following remark: ‘The major predisposing factors to venous thrombosis are activation of blood coagulation and venous stasis. … Nevertheless, wall damage may predispose to venous thrombosis in special circumstances’ [our emphasis] (Badimon and Badimon 2005). Since the historical record shows that venous thrombosis has defied human understanding through two centuries of thought and experimentation, we must eschew oversimplified aetiological models. Nevertheless, it is widely agreed that venous thrombi typically originate in the venous sinuses and valve pockets of the lower limb when blood movement is interrupted/retarded because the ‘muscle pump’ of immobile patients’ legs is contracting less frequently (McLachlin et al. 1960). Alternatively, some writers assert that an injured intima may itself impair circulation and cause interruption or retardation of flow. Hamby (2005), for instance, wrote: ‘Sometimes … blood clots [sic] will form when blood flow merely slows down and becomes sluggish, or when the interior walls of blood vessels become damaged or roughened, though no wound has occurred. … Clots usually form when blood flow becomes sluggish, as when there is roughness or scar tissue along the interior walls of a blood vessel that slows down the blood flow’. Each of these (and other) variants of the consensus model has been associated with advances in knowledge, but not necessarily with better understanding.5 Briefly, our criticisms are as follows.

5

Interestingly, some writers now quote the elements of ‘Virchow’s triad’ as causes of both arterial and venous thrombosis (e.g. Makin et al. 2002). They imply that an understanding of arterial thrombosis might elucidate the aetiology of DVT. However, the two pathologies are distinct, since (e.g.) blood ‘stasis’ cannot a priori be a causal factor in arterial thrombogenesis – whatever is claimed about relative vertical/reflux slowing of circulation. Poole and French (1961) indicated

1.4 The Dominance of the Consensus Model

7

First, it is an implicit postulate of the post-1960 consensus model that DVT is primarily a disorder of haemostasis6, and is by that token a haematological condition. We will dispute this. Certainly the formation and growth of a thrombus must involve blood semi-solidification at some stage, but it does not follow that the coagulation mechanism is perturbed. It could be, and often is, acting or responding normally (see chapter 3). It has not been convincingly demonstrated that the primary pathogenic factor in DVT is blood semi-solidification. It may be an intermediate step in a succession of events that ultimately causes thrombosis. The genesis of silent autochthonous thrombi cannot be seen unless and until we devise an experimental test-bed that produces such thrombi. Second, in most accounts, the model remains imprecise. What evidence is there that blood must be ‘more-than-normally-coagulable’ for DVT to arise? In what sense is ‘stasis’ to be conceived? And what sort of ‘vessel injury’ or ‘intimal injury’ is invoked and/or involved? Some recent literature throws light on these questions, but they still require careful scrutiny. We shall address them during the course of this book. Third, even if these three components of the consensus model are considered relevant – and interpretable – how are they supposed to be linked or to ‘cooperate’ in the production of a venous thrombus? Few writers have attempted to address this problem systematically, and no integrated account so far proposed has been experimentally corroborated or attained general acceptance in the field. Fourth, in glaring contradiction to standard teaching and presumptions, the consensus model is not attributable to Virchow.

1.4

The Dominance of the Consensus Model

In view of these possible weaknesses, why has the consensus model achieved and retained such prominence? One plausible answer, which we shall consider further in Chapter 2, is that some workers in the third quarter of the 20th century opted to abandon the seemingly fruitless search for an aetiological – a priori – solution to the problem of thrombosis and opted to focus on empirical prophylactic and therapeutic (ad hoc) advances in anticoagulation and related techniques. Such a decision perhaps subordinated aetiological (‘why’) accounts to descriptions of effective therapeutic (‘how’) methods. Prophylaxis against, and treatment of, DVT are health care issues of the utmost importance. Clinicians rely primarily on anticoagulants (especially low-molecular clear differences between arterial and venous thrombi. Since our aim in this book is to explain deep venous thrombosis, we can provisionally sideline arterial thrombosis. 6 The word haemostasis is unproblematic – it denotes the curtailment and then stoppage of bleeding from a site of injury – but it is important to make the relationship with coagulation (see previous footnote) explicit. Coagulation or blood semi-solidification, while undoubtedly part of the mechanism by which haemostasis is achieved, is not ‘haemostasis’. Haemostasis is the end, coagulation is one of the means. The words are not interchangeable, but they are used loosely in some publications.

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1 Introduction to the Study of Deep Venous Thrombosis

weight heparin) and thrombolytic agents; compression stockings and thrombectomy are important approaches but have mostly been considered supplementary ‘afterthe-event’ measures (Wilkins and Stanton 1953; Burroughs 1999; Thiagarajan 2002; Ramzi and Leeper 2004; Krishnan et al. 2004; Kyrle and Eichinger 2005). It is beyond doubt that anticoagulant and thrombolytic treatments have saved many lives and will continue to do so; their clinical value is not in question. But we suggest that it is precisely because the literature in the field has been dominated by discussions of prophylaxis and treatments, with no more than fig-leaf speculations about aetiology, that the consensus model has acquired its general character. Since our intention in this book is to halt and to reverse the perceived trend, we shall focus our attention on aetiology and treat prophylaxis and therapy only as corollaries.

1.5

‘Virchow’s Triad’

It is trivially true, and has long been recognised, that DVT must entail some combination of malfunctions in ‘vessel wall, blood movement and blood coagulation’. However, it is curious that this truism has come to be labelled ‘Virchow’s triad’. The eponym has become prominent in clinical, pathological and thrombosis research circles, but there are several reasons to consider it problematical. First, its origin is obscure, because it was unquoted (and presumably unknown) in textbooks or research papers before the 1950s, about 100 years after Virchow’s seminal publications. Secondly, Virchow’s personal concern was with the life-threatening effects of venous thrombi – i.e. with their metastasis as emboli – not with their causes. These points have been recognised by several authors (Anning 1957; Brinkhous 1969; Owen 2001; Brotman et al. 2004; Dickson 2004). Thirdly, Virchow was not the first to investigate what is now called ‘DVT’. It is true that he introduced the words ‘thrombus’ and ‘embolus’, but that was to evaluate – and to reconcile as far as possible – the divergent accounts offered by his predecessors, notably Boerhaave (Burton 1746), Hunter (1793) and Cruveilhier (Long 1929).7 In particular, he was concerned to refute the notion of ‘phlebitis’ (the Greek translation of Hunter’s ‘inflammation of the internal coats of veins’ promulgated by Cruveilhier). Fourthly, Virchow never so much as implied, still less invoked, the concept of ‘hypercoagulability’. Moreover, he explicitly rejected the ‘doctrine’ that stasis has a causal role in thrombosis and embolism (Virchow 1846–1856; Virchow 1856). In other words, Virchow’s writings provide no justification whatever for associating his name with the current consensus model of DVT; rather the contrary. Since some readers will question these remarks, we shall explore them in greater detail in Chapter 6.

7

Virchow did not invent the word ‘thrombus’ but he gave it a new and precise meaning.

1.6 An Alternative Viewpoint

1.6

9

An Alternative Viewpoint

Since we question the consensus model because it treats DVT as a primarily haematological condition, it is to be expected that the alternative model we propose (cf. Malone 1977) does not treat DVT as a primarily haematological condition. We shall direct Chapters 8–10 to the development of this model and evaluate it in Chapter 11. According to this viewpoint, the initiating pathological process is necrosis of the valve cusp endothelium, a likely consequence of hypoxaemia in the valve pocket/sinus. This can lead to local accumulation of phagocytic leukocytes and platelets in and on the parietalis endothelium of the flimsy valve cusps, with progressively disruptive effects on their function. Under certain (definable) circumstances, this may result – ultimately – in local initiation of the coagulation cascade, which in turn may, but may not, lead to thrombosis. Once a bona fide thrombus begins to form in a vein, its growth can be accelerated by positive feedback effects. This hypothesis, which has been experimentally corroborated (e.g. Hamer et al. 1984), explains the frequency of pulmonary embolism and chronic venous disease. Two of its important premises are: (1) that venous thrombi can (and usually do) form in blood that is normally coagulable, both systemically and locally; (2) that the blood must be flowing, not static, for thrombi to form (Malone and Agutter 2006). In the years following its publication and experimental corroboration, interest in the phenomenon of ischaemia-reperfusion injury (IRI) flourished, not least because IRI is of great significance in transplant surgery. The underlying mechanism of IRI as it is understood today somewhat resembles that described by our alternative model, though the former relates primarily to the heart and arteries, the latter exclusively to the veins. Both the consensus model and our alternative proposal have evolved from a history of thought and experimental pathophysiological and haematological research dating back to the 17th century. The two approaches may appear divergent, maybe even conflicting, but we believe that that they must be amalgamated and unified if there is to be further progress towards understanding DVT. In practice, ideas cannot be spliced without a full historical exegesis to elucidate their origins, development and philosophical character. For this reason, much of the book (Chapters 4–9) will emphasise the history of ideas in biomedicine. However, we shall begin with a more detailed examination of the consensus model and in particular the blood coagulation mechanism as it is understood today.

Chapter 2

The Coagulation Cascade and the Consensus Model of DVT

Abstract The development of the blood coagulation ‘cascade’ concept after the Second World War, together with the increasing clinical use of anticoagulant and (later) thrombolytic treatment, is briefly reviewed and related to the origin of the consensus model. It is suggested that the consensus model arose from a research tradition that was essentially unrelated to thrombosis, and that its articulation entailed an effective, if unintended, suppression of Virchow’s pathophysiological viewpoint (as defined in the preface to this book) and also marginalised studies of the venous endothelium in relation to DVT. It is notable that Virchow’s explicit distinction between ‘clot’ and ‘thrombus’ was not preserved during this process; Virchow had hypothesised that thrombi are analogous to clots, but he knew the limitations of the analogy. The remainder of the chapter is devoted to a more or less detailed overview of the coagulation cascade as it is understood today. These details are included because no matter how the aetiology of DVT is considered, it is clear that the blood coagulation mechanism is involved at some point in the process. Some brief semantic points are raised for later discussion and development.

Keywords Anticoagulants, coagulation, control of coagulation, fibrinogenesis, platelets

2.1

The Cascade Model of Blood Coagulation

The idea of a ‘coagulation cascade’ was adumbrated in the late 1930s (Chapter 5),1 but did not achieve definitive form until after the Second World War. For a long time, the most challenging problem persisted: the circulating blood contains all the

1 In principle, any causal explanation in science is a ‘cascade’, i.e. a sequence of events of which the consequent of one is the antecedent of the next. In biochemistry, however, ‘cascade’ has taken on a more limited range of denotation, and the blood coagulation cascade remains one of the preeminent exemplars as well as the earliest to be elucidated.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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2 The Coagulation Cascade and the Consensus Model of DVT

biochemical ingredients needed to enable it to coagulate, but it remains fluid (Eagle 1938). It coagulates very rapidly when a vessel wall is injured, but only at the immediate site of injury. The third edition (1962) of Biggs and MacFarlane’s Human Blood Coagulation and its Disorders reveals the contemporaneous focus of interest among workers in the blood coagulation field. For the first 40 years of the 20th century, said Biggs and MacFarlane, the mechanism of blood coagulation had seemed ‘largely comprehensible’, and the ‘theory’ (hypothesis) of Schmidt and Morawitz2 ‘has been confirmed many times … finally by the Quick one-stage prothrombin time in the study of haemorrhagic states. … The only major disadvantage was its failure to account for the coagulation defect in haemophilia – after all, a rare disease’. The authors gave a detailed account of the cascade model as then conceived, discussed diatheses haemorrhagica at length, but devoted only eight of the book’s 360 text pages to thrombosis. The accelerated elucidation of haemostatic lesions during the 15 years prior to 1962 was presaged in the work of Owren (1947). Owren began the practice of distinguishing the coagulation factors by Roman numerals, and he identified a type of bleeding diathesis in which tissue extracts could not activate3 prothrombin. This proved that there was more than one kind of ‘haemophilia’ and led to the discovery of factor V (Owren 1947, 1950; Fantl and Nance 1948; cf. also Quick 1943; Ware et al. 1947). The ‘failure to account for the coagulation defect in haemophilia’ was about to end, though with unanticipated complications. Addition of ‘antihaemophilic globulin’ (factor VIII) to haemophiliac blood in vitro restored the normal clotting time, but this did not happen in the type of ‘haemophilia’ discovered by Owren (factor V deficiency). Pavlovsky (1947) and others also observed that blood samples from some haemophiliac patients normalised the clotting rates from others, indicating deficiencies of different factors. By 1954, the literature was distinguishing between ‘haemophilias’ and ‘haemophilioid states’, factor IX had been identified, and Christmas disease had been recognised and named (Biggs et al. 1952; Aggeler et al. 1952; Graham and Brinkhous 1953; Koller 1954). The cascade model was acquiring its now-familiar form. Owen and Bollman (1948) found the first evidence that dicoumarin did not cause prothrombin deficiency, as previously suspected: the one-stage prothrombin time of blood from dicoumarin-treated patients was shortened by normal serum. This finding led to the identification of factor VII 4 years later (Mann 1949; Owen et al. 1951; Koller et al. 1952), and a functional relationship was indicated between

2

See Chapter 5 for this aspect of history. The terms ‘activate’ and ‘activation’ are unavoidable, but they are potentially equivocal and misleading. A protein molecule, e.g. an enzyme, is said to be ‘activated’ when endogenous chemical modification confers biologically relevant properties on it. For the last 50 years, coagulation has been regarded as a paradigm example of an ‘activation cascade’, characterised by a sequence of protein activation events. In contrast, a cell is said to be ‘activated’ when it ceases to be quiescent and moves, or secretes, or associates with other cells, in a new way. In a poetic sense, these two uses of ‘activate’ are analogous; but mechanistically, activation of a molecule and activation of a cell are obviously quite different. The distinction should be kept in mind. 3

2.2 The Origin of the Consensus Model of DVT

13

factor VII and Owren’s factor V. Another distinctive haemorrhagic state investigated during the 1950s led to the identification of factor X (Denson 1958). The remaining coagulation factors were discovered during the following decade. Factors XI, XII and XIII had not been unequivocally identified by the time of Biggs and MacFarlane’s 1962 edition, the factor activation processes had not been biochemically characterised, and the mechanisms controlling fibrinogenesis and fibrinolysis were not yet understood. However, the presently accepted model of coagulation and the associated explanations of bleeding diatheses (not thrombosis) were broadly established. We have focused on the third edition of Biggs and MacFarlane because it was published at almost exactly the time when the consensus model of DVT was first fully articulated. This may not have been a coincidence; it seems to us to reveal the true character of the consensus model. Noticeably, when De Nicola (1979) reflected on ‘Thirty years of studies of blood coagulation (1935–1965) – The renaissance period of blood coagulation’,4 he did not stipulate ‘30 years of thrombosis studies’. Still later, in an authoritative work, Browse et al. (1988b) would reminisce: ‘Not until the discovery of the coagulation cascade did the modern era of thrombosis research begin’. These quotations are highly suggestive.

2.2

The Origin of the Consensus Model of DVT

The notion that venous thrombosis might result from a combination of increased coagulability and ‘stasis’ was suggested in a survey by Ochsner et al. (1950), but it was to be a further 10 years before the consensus model was stated explicitly (Sise et al. 1962; Mustard et al. 1962, 1963; Wessler 1962, 1963). How should we interpret the pronouncement that ‘Not until the discovery of the coagulation cascade did the modern era of thrombosis research begin’? It is obvious that clots, haemostatic plugs and thrombi have similar compositions (though not structures), and coagulation must be involved in thrombus formation. But while the cascade model may explain coagulation, it is no more likely per se to explain DVT than is Heisenberg’s Uncertainty Principle. A haemostatic plug forms when a vessel is injured; it rapidly and efficiently seals the leak, leaving the vessel patent. But the formation of a thrombus is entirely intravascular and partially or wholly occludes the vessel. The phenomena are quite different. A special scenario has to be imagined: if ‘hypercoagulability’ were postulated as the pathological antithesis of haemophilia, and if drastically slowed

4 His ‘renaissance period’ was evidently initiated by Quick’s work on prothrombin in test-tube blood (Quick 1945). It might have been fairer to trace it to the experiments of Bordet and Delange and of Nolf in the period 1912–1928 (see Chapter 5), which showed that prothrombin could not be converted to thrombin as straightforwardly as the ‘classical hypothesis’ of Schmidt and Morawitz had predicted.

14

2 The Coagulation Cascade and the Consensus Model of DVT

blood circulation were to allow such ‘hypercoagulability’ to act extremely locally in a delimited part of the vascular tree, then knowledge of the coagulation cascade could become focally relevant to the aetiology of DVT. This, in effect, is what the first proponents of the consensus model presumed. Why did this model arise when it did, rooted in a research tradition that had everything to do with the study of bleeding diatheses and little or nothing to do with thrombosis? Although we can only speculate retrospectively, we believe that the creation of the ‘hypercoagulability-and-stasis’ model was closely linked to the success of anticoagulants in preventing thrombotic lesions in patients undergoing prolonged bed rest, which had been established on independent, empirical grounds soon after Owren’s 1947 paper was published (van der Veer et al. 1950; van der Veer 1951; Crane 1951; Wessler 1952). There were other factors, of course: for example, it was known that platelets became more numerous and more ‘adhesive’ after surgery, when the risk of DVT is increased (Payling Wright 1942). However, major breakthroughs in the understanding of haemostasis, precise biochemical pinpointing of haemophilia and related conditions, and the opening of prospects for specific and effective (albeit ad hoc) treatments for thrombosis, must have created a heady intellectual climate. In such a climate, it was not surprising that the clinical success of anticoagulation was interpreted by clinicians and researchers as ‘normalisation of an excessive tendency of the blood to coagulate’ (cf. Owen 1958). In other words, we suspect that what became the consensus model of DVT was primarily an attempt to explain, in haematological language, why anticoagulant therapy works. It was to be another 3 years before an actual ‘hypercoagulability’ condition was characterised in a few isolated cases of familial venous thrombosis (Egeberg 1965). In retrospect, to have inferred aetiology from therapeutic success might seem akin to attributing a bacterial infection to endogenous antibiotic deficiency, but in the haematological research climate of the early 1960s it was perhaps understandable. However, no such reasoning was ever explicitly stated. Instead, the ‘triad’ framework misleadingly attributed to Virchow was subtly amended and used as a stick to beat the proponents of the older tradition.5 Both Wessler and Mustard et al. wrote scholarly reviews of Virchow’s work and its aftermath, but they viewed it in a historically distorting mirror. The primary focus of the consensus model was no longer the cause or aetiology of DVT, which became a secondary, and eventually a suppressed, consideration.6 The biochemical account of coagulation attained lasting hegemony in the thrombosis field and, to accommodate it, the centuries-long

5

PCM recalls its use in the context of DVT pathology in the late 1950s: but biochemically orientated haematologists (‘coagulationists’) invoked and popularised the phrase ‘Virchow’s triad’ in their pioneering papers of 1962–1963. Yet Virchow’s great intellectual legacy had contributed nothing to the evolution of knowledge of haemostasis (see Chapters 5 and 6). 6 PCM also recalls a phone conversation with Duncan Thomas during the 1990s in which DPT said: ‘It’s good to hear that some are still interested in aetiology’. By implication, the majority were not interested in aetiology because such interest had been suppressed by the consensus and was presumed lost.

2.2 The Origin of the Consensus Model of DVT

15

endeavour to find a physiological explanation was sidelined. (It was never expressly repudiated. It simply fell from favour and became yesterday’s concern.) The reaction of contemporary clinical pathologists to this ‘paradigm shift’ seems to have been baffled and discontented. Witness the address to the Royal College of Surgeons by Pulvertaft (1947): ‘…students of physiology are often a little puzzled to find so many of their masters weaving a tortuous and highly individual path through the polysyllabic maze of blood coagulation. … The penultimate word on the question [i.e. the aetiology of thrombosis] was written by Welch in 1897 [sic] in Allbutt’s System of Medicine, and much that has been written since then has been a bungaloid growth rather than an architectural synthesis: on reading his excellent study, one feels that students would be well advised to remember that there were great men before the Agamemnons of modern medicine’.7 In a similar spirit, de Nicola (1979) complained that nomenclature in the field was confusing and suggested a lack of conceptual clarity. The maturing consensus model replaced the classical picture of the aetiology of DVT. The new generation was taught a novel version of Virchow’s approach to the subject, which had guided clinical thrombosis research for a century. Apperly et al. (1951), Hallwright (1951), Henderson (1951), Hill (1951) and Stanton (1955) wrote accounts of the management of venous thrombosis and pulmonary embolism that balanced the new approaches with traditional ones; but the hegemony of the biochemical-haematological standpoint had become apparent in Barker (1959) and Kinley and Colan (1965). The new picture did not become dominant immediately. Ogston (1987) admitted that ‘[H]istological changes in the vein wall at the site of a venous thrombus have been difficult to confirm. … It is probable that the majority of venous thrombi in the legs are not the result of local trauma’. However, on the next page, he commented critically on the views of the consensus proponents, referring particularly to the experimental thrombi of Wessler (1955) and others (which we shall discuss further in chapter 3). He wrote: ‘The conclusion reached was that a combination of venous stasis and coagulation activation, local or systemic, is responsible for the initiation of venous thrombosis. The thrombi produced in these experiments histologically resemble blood clots rather than thrombi, but following exposure to flowing blood there is an accumulation of cellular elements and fibrin on its surface’ [our emphasis]. Many proponents of the consensus model sought to eliminate the venous endothelium from consideration in respect of DVT because it was judged peripheral to the haematological viewpoint, though there were significant dissenters (e.g. Ashford et al. 1967). Thomas (1987) stated that to prune ‘Virchow’s triad’ to a ‘duet’ would eliminate the need to ‘presume’ that a vein wall lesion contributes to thrombosis. He then admitted that ‘a school of thought’ still believed that endothelial

7 ‘Vixere fortes ante Agamemnona’: Horace, Ode IV, ix = ‘Ours is not the only era to have had great men’. Pulvertaft famously saved Winston Churchill’s life in Tunis when he became gravely ill in 1943 (Jenkins 2001).

16

2 The Coagulation Cascade and the Consensus Model of DVT

lesions play a part in the process, but dissociated himself from so heterodox a creed.8 At the time, Thomas appeared to have a plausible case. To their puzzlement, and despite intensive studies of serial histological sections, 20th-century clinical researchers of the older tradition had failed to corroborate their belief that the vein wall must be relevant to thrombosis. Unlike Thomas and other proponents of the consensus model, they regarded ‘venous lesions’ as the missing part of the jigsaw. We shall discuss this puzzle in detail in Chapters 9 and 10, but the conclusion we shall reach is essentially simple: the lesions are not lesions of the vein wall but – as Virchow’s studies had suggested in the 1850s – of the valve cusps, which are so fragile that they are degraded or only partially recognizable in histological preparations.

2.3

The Coagulation Cascade Today

Studies of coagulation have burgeoned since the 1960s and the consensus model has evolved concomitantly. Progress has been especially notable on three fronts: elucidation of the biochemical mechanisms of platelet activation, understanding of the control of fibrinogenesis and fibrinolysis, and renewed interest in the role of the vascular endothelium in coagulation and its regulation. Haemostasis is now considered to proceed in three main stages. First, platelets accumulate within and around the site of injury to initiate a platelet plug9; the leaking vessel is constricted during this process (primary haemostasis), curtailing local circulation. The vasoconstriction response seems to be evolutionarily older than platelet plug formation or its sequelae (cf. Clarke 1968). Second, the activated platelets initiate the biochemical cascades (MacFarlane 1941, 1964) that generate a web of cross-linked fibrin; this stabilises the platelet plug (secondary haemostasis). Finally, much more slowly and concomitantly with wound healing, the fibrin web is attacked, the plug is dispersed and its fragments are removed by phagocytosis. During the last 10–20 years it has become clear that coagulation is a ‘solidphase’ process. In their inactive forms, the coagulation factors are soluble plasma proteins; they become active only when they are immobilised by attachment to the platelet plug bound to the vessel wall. Also, coagulation is considered to be regulated by enzymes bound to membrane surfaces or secreted by

8

Barrett (1924) showed that even when pathogenic bacteria were injected directly into veins, they did not produce thrombi unless they were attached to the endothelium by ‘threads’, and this might have suggested that Virchow’s descriptions were correct; but Thomas and other contemporary authors seldom cite older literature. 9 Welch seems to have been the first author to use the term ‘plug’ in relation to the (haemostatic) sealing of a blood vessel. However, Virchow described an incipient venous thrombus as a ‘plug’ – in a footnote, relating to his Fig. 69, on p. 232 of Thrombose und Embolie. This might have been the first usage.

2.3 The Coagulation Cascade Today

17

endothelial cells. The in vitro ‘clotting’ model of coagulation, in which all events occur in the liquid phase, seems to have become increasingly less pertinent to the in vivo process; a fortiori, the old puzzle about the precise localisation of blood semi-solidification appears to have been solved.

2.3.1

Platelet Activation and Congregation: Local Vasoconstriction

Although the following events are better established in arteries than in veins, exposure of subendothelial collagens I and III at sites of intimal injury seems to be the general primary trigger for the congregation and attachment of platelets10 (Triplett 2000). Attachment appears to be mediated by tissue fibronectin and by von Willebrand Factor (vWF) (Meyer and Larrieu 1970; Caen and Levy-Toledano 1973), which links the collagen fibrils to platelet receptors (Sixma and Schiphorst 1980). vWF is synthesised by endothelial cells as well as megakaryocytes, and is found in subendothelial connective tissue (Jaffe et al. 1974; Nachman and Jaffe 1975). It also binds and stabilises circulating or endothelially manufactured factor VIII (Nachman and Jaffe 1975). Platelet congregation is controlled by circulating adrenalin, endothelial cell ADPases, locally produced nitric oxide and eicosanoids (Marcus and Safier 1993). Figure 2.1 summarises the network of intracellular biochemical events now considered responsible for platelet congregation and activation. Thrombin binds to receptors on the platelet membrane (Colman 1990), initiating a classical G-protein signalling pathway (Hung et al. 1992; Brass et al. 1994) leading to thromboxane A2 production (Carey et al. 1982)11 and intracellular calcium release. Calcium stimulates myosin light chain kinase, changing the platelet’s morphology, destabilising its granules (Opstvedt et al. 1986) and increasing its motility. These changes, described the early 1960s, were originally ascribed to phagocytosis by the platelets (David-Ferreira 1961, 1964; Movat et al. 1965). The liberated granule contents include vWF, ADP and the vasoconstrictor serotonin (Marcus 1965; White 1968; Gerrard et al. 1977; White and Clawson 1980). Thromboxane A2 and ADP are platelet activators; thromboxane A2 is also a vasoconstrictor (Michal and Motamed 1976; Davies et al. 1978). Thus, activation

10

The verbs ‘attach’ and (particularly) ‘aggregate’, which are normally used in this context, connote passivity on the part of the platelets. It behoves us to remember that platelets are living cells. The processes described here are active responses to injury, involving chemotaxis and ‘swarming behaviour’ of a kind that is more popularly recognised among social insects. Platelets are not passive. ‘Congregate’ seems a more apt verb than ‘aggregate’. 11 The cycloxygenase required for the production of thromboxane and other eicosanoids from arachidonic acid is irreversibly inhibited by aspirin. This is believed to be the main reason for the anticoagulant effect of aspirin.

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2 The Coagulation Cascade and the Consensus Model of DVT

Fig. 2.1 Outline of pathways involved in platelet activation, congregation and secretion

of a single platelet is sufficient in principle to initiate a chain reaction, attracting and activating more platelets and inducing more local vasoconstriction; they are particularly responsive to ADP (Gaarder et al. 1961). Phosphatidylserine and phosphatidylinositol are exposed on the platelet surface (Naka et al. 1983) and are crucial in forming the tenase complex, originally named ‘prothrombinase’ (Owren 1954; Lamphear and Fay 1992).

2.3 The Coagulation Cascade Today

19

To form a platelet plug, fibrinogen must adhere to platelet surface glycoproteins,12 notably GPIIb and GPIIIa (Lee et al. 1981; Herrmann et al. 1983; Ware 2004), promoting further secretion of granule contents. This does not occur while the platelets are circulating; the two glycoprotein receptors must be ‘primed’ by ADP in order to bind fibrinogen, which then interlinks numerous activated platelets (Peerschke 1985). Another granule component, the glycoprotein thrombospondin (Lawler et al. 1978), is a cofactor in this congregation process (Leung and Nachman 1982) and also helps to anchor the platelet plug to the subendothelial connective tissue (Lahav et al. 1982). It is interesting to compare this summary of platelet function (Fig. 2.1) with earlier accounts (e.g. Poole 1964; French 1967; French and Barcat 1968; White 1971; Lowenhaupt et al. 1973; Born 1987). The platelet is no longer regarded as a hypothetical source of coagulation factors, as it was a century ago (Chapter 5); the platelet plug is the active locus of fibrinogenesis.

2.3.2

The Contact (‘Intrinsic’) System

Two complementary, interacting, synergistic cascade systems are implicated in coagulation, dubbed contact (intrinsic) and tissue-factor (extrinsic). The contact system is usually initiated when the plasma touches a negatively charged ‘foreign’ surface. In vivo, this is usually exposed collagen or membrane glycosaminoglycans. Ex vivo, it can be almost any surface, such as glass, which is believed to be why blood clots rapidly when it is shed. Four plasma proteins have been specifically implicated in the initiation process or contact phase: high-molecular weight kininogen (Waldmann et al. 1975; Webster et al. 1976), prekallikrein (Fletcher et al. 1959; Sherry and Colman 1968), factor XI (Frick 1954; Gailani 1994) and factor XII (Chevallier et al. 1956; Ratnoff and Davie 1962). No pathology is known to result from deficiencies of any of these factors except XI. Contact induces the conversion of prekallikrein to kallikrein, which hydrolyses factor XII to XIIa.13 Factor XIIa has three known proteolytic effects (Silverberg and Kaplan 1982): (i) it catalyses the conversion of prekallikrein to kallikrein, an example of positive feedback amplification; (ii) it hydrolyses high molecular weight kininogen, causing local release of bradykinin, leading to vasodilatation and

12

Glycoproteins are usually classified as I, II, III, etc. on the basis of relative molecular mass (‘I’ denotes the largest molecules). Within each size class, molecular species are distinguished as a, b, c, etc. 13 The activated form of a haemostatic factor is represented by a lower-case letter a after the identifying Roman numeral. This has been the practice since Owren’s seminal publications. All the proteinases involved in haemostasis are highly selective: they cleave only a very few, very specific protein substrates at just one or two peptide bonds. Less-selective proteinases at large in the blood stream, even locally, would wreak havoc.

20

2 The Coagulation Cascade and the Consensus Model of DVT

therefore an increased supply of platelets and coagulation factors to the site of injury; and (iii) it activates factor XI. In the presence of calcium ions released from activated platelets, factor XIa catalyses the activation of factor IX (Fujikawa et al. 1974), one of several plasma proenzymes that contain a γ-carboxyglutamyl linkage14 (McGraw et al. 1985). Concurrently, factor VIII is activated by minute amounts of thrombin. The tenase complex (see above) is then assembled on the activated platelet surfaces: factor VIIIa binds to the exposed phosphatidylserine and phosphatidylinositol molecules and forms the binding site for calcium ions and factors IXa and X. Factor IXa rapidly activates factor X (Solum 1999), and fibrinogenesis is initiated (Broze and Majerus 1980; Rosing et al. 1985; Mann 2003). Current understanding of the contact system, along with fibrogenesis per se (see below), is summarised in Fig. 2.2.

2.3.3

The Tissue Factor (‘Extrinsic’) System

Tissue factor (TF) is a subendothelial cell-surface glycoprotein with a high affinity for phospholipids (Nemerson and Pitlick 1972). Factor Xa and thrombin activate factor VII, and factor VIIa in conjunction with TF catalyses the activation of factor X (Mariani and Mandelli 1983). This is another positive feedback loop, amplifying the fibrinogenic response. Factor VIIa and TF also activate factor IX: another amplification device, and another link between the ‘intrinsic’ and ‘extrinsic’ systems (Bajaj et al. 1983). The TF/VIIa complex can apparently activate enough factor X for fibrinogenesis to proceed at a normal rate without input from the contact system (Broze and Majerus 1980; Zur et al. 1982) (Fig. 2.3).

2.3.4

Fibrinogenesis

Factor Xa bound to the platelet plug is the key to thrombin production (Jesty and Nemerson 1976; van Dieijen et al. 1981; Rosing et al. 1985). Prothrombin is attached to platelet surfaces by activated factor Va15 (Esmon et al. 1974). Factor Xa cleaves the immobilised single-chain 72 kDa prothrombin molecule in two places, liberating a small peptide fragment and generating two polypeptide chains joined

14 Formation of γ-carboxyglutamyl linkages requires vitamin K and is inhibited by Warfarin and analogues. Factors VII and X, as well as prothrombin, also contain vitamin K-dependent γ-carboxyglutamyl linkages (Suttie 1993). 15 The numbering appears non-systematic because the chronological order of discovery and naming of the factors did not parallel their functional sequence. To make matters worse, factor Va was not originally recognised as a product of factor V; it used to be called ‘factor VI’. Also, factor IV turned out to be calcium ions. So the labels ‘factor IV’ and ‘factor VI’ are no longer used.

2.3 The Coagulation Cascade Today

21

Fig. 2.2 The contact system and fibrinogenesis

by a disulphide bridge (Rybak et al. 1981). This cross-linked, two-chain product is active thrombin (factor IIa). It catalyses the formation of fibrin (factor Ia) from the glycoprotein fibrinogen (factor I) and is a significant activator of factors XI, VIII,

22

2 The Coagulation Cascade and the Consensus Model of DVT

Fig. 2.3 The tissue factor system and fibrinogenesis

V and XIII (Brummel et al. 2002) and the primary activator of platelets. It has several other pertinent effects, which will be discussed later. Fibrinogen comprises six polypeptide chains (Soderqvist and Blomback 1971). The intact protein is highly soluble because four of the six chains contain large

2.3 The Coagulation Cascade Today

23

numbers of acidic residues, concentrated at the ends of the chains but conferring a substantial net negative charge on the whole molecule. Thrombin removes sequences rich in these acidic residues (the fibrinopeptides; Blomback 1966) from platelet-bound fibrinogen. The resultant molecule, fibrin, carries a much smaller net charge and is therefore much less soluble. It aggregates spontaneously to form a regular ‘molecular spider-web’ entrapping the platelet plug (Wiltzius et al. 1982) and other blood cells. This array is structurally weak. Factor XIIIa cross-links the individual fibrin molecules covalently, making the ‘spider-web’ much more robust (Kanaide and Shainoff 1975). Factor XIII activation appears to be a multistep process, significantly slower than fibrinogenesis. The principal components of a ‘mature’ haemostatic plug are therefore considered to be activated platelets and cross-linked fibrin. Early studies on the structure of the fibrin web (Lendrum et al. 1962) revealed most of the morphological features acknowledged today.

2.3.5

Fibrinolysis

Fibrinolysis is usually discussed as an adjunct to coagulation (Flute 1965) for the following reasons (Fig. 2.4). First, since coagulation is a response to injury, discussion can be extended to the initiation of tissue repair, of which fibrinolysis is considered a concomitant. Secondly, the factors involved in fibrinolysis are activated, indirectly, by the events leading to fibrinogenesis, so mechanistically it is part of the same process. Thirdly, there is a venerable (though not necessarily tenable) school of thought that low-level fibrinogenesis is omnipresent in the circulation and must be accompanied by a compensating level of fibrinolysis if the blood is to remain fluid (MacFarlane and Biggs 1948; Åstrup 1958). Wright (1952) repeated the 19th-century speculation that thrombi are being formed and dissolved continuously throughout life. The healthy endothelium is supposed to counteract spontaneous blood coagulation by expressing membrane-bound thrombomodulin and by secreting factors such as tissue plasminogen activator (stimulating fibrinolysis) and ADPase (inhibiting platelet activation); see, e.g. Parise and Phillips (1986), Woldhuis et al. (1992). The key component in fibrinolysis is plasmin, another serine proteinase. It is formed from an inactive soluble precursor, plasminogen (Fearnley 1953; Ouchi and Warren 1962; Robbins et al. 1967), and catalyses the degradation of cross-linked fibrin. The degradation products are soluble and are digested by non-specific proteinases. Plasmin action has to be regulated so that its onset is slow (otherwise the haemostatic plug may be degraded too quickly), and it is efficiently inhibited once fibrinolysis is complete. Plasminogen binds strongly to both fibrinogen (platelet-bound and in the plasma) and fibrin (in the haemostatic plug), so when it is activated it is already

24

2 The Coagulation Cascade and the Consensus Model of DVT

Fig. 2.4 Plasmin activation and fibrinolysis

closely juxtaposed to its substrate. The activation depends mainly16 on tissue plasminogen activator (tPA), which is released from proliferating vascular endothelial cells during wound repair (Åstrup and Permin 1947; Todd 1959). Inactive tPA is activated when it binds to fibrin, and the process is controlled by plaminogen activator inhibitors (PAIs: Chapman et al. 1982; Rijken et al. 1984). The fibrin/tPA/ plaminogen complex is therefore the site of plasmin production (Prentice et al. 1969). Plasmin cannot attack the fibrin web while the thrombin-activated

16 Many other tissues may also produce tPA. The plasminogen activator secreted by, e.g. kidney cells and first identified in urine during the early 1950s might be less physiologically important; this activator, ‘urokinase’, does not bind tightly to fibrin.

2.3 The Coagulation Cascade Today

25

fibrinolysis inhibitor (TAFI) is active (see below), but as thrombin levels decline, TAFI becomes inactive and plasmin-catalysed fibrin degradation begins. This combination of checks and balances is believed to account for the requisite slow onset of fibrinolysis. In due course, fresh living phagocytes remove the fragmenting mass of fibrin and dead cells. Unmodified plasminogen recombines preferentially with fibrinogen (Berg and Korsan-Bengtsen 1963). Plasmin is quickly inactivated by another plasma protein, α2-antiplasmin (Mannucci 1979). tPA is inactivated by at least four PAIs (Thorsen and Phillips 1984; Hekman and Loskutoff 1985); PAI-1 is considered especially relevant to vascular disease (Paramo et al. 1985; Agirbasli 2005). Other inhibitors include interleukin 1, thrombospondin (a histidine-rich plasma glycoprotein) and platelet products, but the physiological importance of these is not certain. It was long suspected that fibrinolysis products inhibit thrombin (e.g. Niewiarowski et al. 1959). Fibrin can also be degraded, independently of plasminogen, by elastase released from leukocytes (Plow 1980).

2.3.6

Controlling Coagulation

Thrombin activity is critical not only for several stages in coagulation, but also for initiating protection and repair processes (Fig. 2.5). In addition, it activates the above-mentioned inhibitor of fibrinolysis, TAFI (Hryszko et al. 2001), which makes the cross-linked fibrin resistant to plasminogen. Through G-protein signalling pathways, thrombin promotes the release of interleukins IL-1 and IL-6 and various cellular adhesion molecules, notably ICAM-1 and VCAM-1 (Sugama et al. 1992). The interleukins are integral to the immune response; the adhesion molecules are crucial for initiating tissue repair and also participate in endothelial interactions with leukocytes. In short, thrombin not only fulfils many different roles in haemostatic plug formation, it also participates in mounting an immune response and in initiating tissue repair. Therefore, zero thrombin activity in the plasma would result in zero coagulation and inadequate protection and repair following injury; but high or even moderate circulating thrombin levels would cause excessive, almost certainly fatal, disseminated fibrinogenesis. Precise local control of thrombin activity is obviously a prerequisite for mammalian life. Low levels activate platelets and factors V and VIII; moderate levels inactivate them. When the coagulation process is well established and substantial amounts of prothrombin have been locally converted to thrombin, factors Va and VIIIa are locally rapidly inactivated. This seems to be one reason why haemostatic plug formation is self-limiting: the terminal product of amplification, thrombin, switches off the activation cascades. It does so by occupying and cleaving the endothelial cell receptor thrombomodulin (e.g. Esmon et al. 1982). This activates protein C, which degrades the platelet-bound factors Va and VIIIa (Esmon and Owen 1981; Salem et al. 1983; Fulcher et al. 1984; Guinto and Esmon 1984). Protein C, another vitamin Kdependent molecule (Esmon et al. 1976), is a major regulator of coagulation. Its

26

2 The Coagulation Cascade and the Consensus Model of DVT

binding to factor Va is enhanced by another plasma constituent, protein S (Walker 1980), which appears to act in concert with one or more regulators of the complement pathway. Protein S can also be inactivated by thrombin, suggesting a negative feedback loop in the control of coagulation. There is another possible link with the complement pathway: the major complement inhibitor C1 also inactivates kallikrein and factors XIa and XIIa (McConnell 1972). Several plasma proteins attenuate thrombin activity non-specifically (Ganrot 1969): α2-macroglobulin, α1-antitrypsin and heparin cofactor II. However, the specific thrombin inhibitors, antithrombins I-IV, anticipated by Schmidt (see chapter 5), are considered more important (Klein and Seegers 1950; Hensen and Löliger 1963). Of these, antithrombin III (ATIII) is especially significant because it also inhibits factors XIIa, XIa, IXa and Xa, effectively closing down the contact system. ATIII is inhibited by fibrin and activated by heparan sulphate on the blood vessel endothelium. Heparin is an effective substitute for heparan sulphate, which explains its anticoagulant effect (Blackburn et al. 1984).

Fig. 2.5 The control of thrombin activity and of coagulation

2.4 Disorders of Coagulation

27

It is widely believed that the main regulator of the tissue factor system, and hence of coagulation, is the tissue factor pathway inhibitor (TFPI), also known as lipoprotein-associated coagulation factor (LACI), extrinsic pathway inhibitor (EPI), tissue factor pathway inhibitor (TFPI) or anticonvertin (Wagner et al. 1955). TFPI is a multimeric protein that binds to the TF/factor VIIa/factor Xa complex (Broze et al. 1988), inhibiting factors VIIa and Xa. This blocks the positive feedback amplification wrought by VIIa and concomitantly down-regulates prothrombin activation.

2.4

Disorders of Coagulation

This overview of haemostasis indicates the continuing growth of knowledge since the 1960s. The coagulation process is now understood in great detail, though the continuing expansion of knowledge will inevitably add further features. Many hereditary disorders of blood coagulation have been characterised: one or other of the coagulation factors is either absent, or present at low (or occasionally high) level, or defective. Some prominent examples are summarised in Table 2.1, which also gives alternative, often archaic, names for the factors. Table 2.1 reflects the historical emphasis on bleeding diatheses (‘hypocoagulability’ conditions), which are at best only indirectly relevant to DVT. Elucidation of these conditions was the primary driving force behind the study of blood coagulation (Chapter 5), and blood coagulation itself is broadly pertinent to the subject of this book. ‘Hypercoagulability’ conditions, of which relatively few are specified in Table 1, have been identified more recently. According to the consensus model, ‘hypercoagulability’ is integral to the causation of DVT. This will be the topic of Chapter 3.

–

Fletcher factor Contact activation factor; Fitzgerald factor; Flaujeac Williams factor Hageman factor

Plasma thromboplastin antecedent Christmas factor; antihaemophilic factor B; plasma thromboplastin component Antihaemophilic factor A; antihaemophilic globulin

Von Willebrand factor

Prekallikrein High MW kininogen

Factor XI

Tissue factor (TF)

Stuart-Prower factor

Factor II

Factor I

Factor III

Factor X

Prothrombin

Fibrinogen

Factor VIII

Factor IX

Factor XII

Alternative name(s)

Fibrin

Thrombin

Factor Xa

Protein cofactor

Proteinase

Proteinase

Multimeric protein Forms insoluble mesh, the core of the haemostatic plug

Many activities in coagulation and related functions

Afibrinogenaemia, hypofibrinogenaemia, dysfibrinogenaemia

–

–

–

Haemophilia A (X-linked disorder)

Haemophilia C (autosomal recessive) Haemophilia B (Christmas disease)

–

Proteinase Proteinase

Von Willebrand disease (commonest bleeding disorder in humans) – –

Deficiency disease(s)

Subendothelial glycoprotein, also in platelets Proteinase –

Comment

Subendothelial glycoprotein Activated by tenase complex on Proteinase platelet surface; activates prothrombin

Along with calcium ions, anchors tenase complex on platelets Cofactor for VII

Factor VIIIa

–

Activates factor X in tenase complex

Binds exposed collagen; activates Factor XI Activates factor IX

Activates XII and kininogen Local vasodilatation

Binds platelets to collagen

Function

Factor IXa

Factor XIa

Factor XIIa

Kallikrein Bradykinin

–

Active form

Table 2.1 Coagulation factors and some associated hereditary pathologies

Factor

28 2 The Coagulation Cascade and the Consensus Model of DVT

–

–

–

Extrinsic pathway inhibitor; tissue factor pathway inhibitor; anticonvertin

Protein C

Antithrombin III

Protein S

LACI

–

–

–

–

Plasmin

–

Plasminogen

Factor XIIIa

–

Protransglutaminase; fibrin stabilising factor; fibrinoligase

Thrombomodulin –

Factor XIII

Inactivates Va and VIIa

Activates protein C

Inhibits thrombin, XIIa, XIa, IXa and Xa

Inactivates Va and VIIIa

Degrades fibrin

Cleaved by thrombin; activates protein C

Cross-links and stabilizes fibrin mesh

Autosomal recessive condition leading to delayed bleeding

Proteinase inhibitor

Protein cofactor

Proteinase inhibitor

Proteinase

Proteinase

–

Protein S deficiency, autosomal (dominant?)

Autosomal dominant deficiency associated with higher risk of thromboembolism

(1) Autosomal (recessive?) deficiency; (2) failure to bind to defective factor Va. Both associated with increased incidence of DVT

–

Endothelial cell surface – protein

Protein ligase

2.4 Disorders of Coagulation 29

Chapter 3

Hypercoagulability

Abstract The claim that Virchow attributed venous thrombosis to ‘hypercoagulability’ is refuted. Two uses of the word ‘hypercoagulability’ are distinguished: a general sense, which entails circular reasoning, and a specific sense, for which the synonym ‘thrombophilia’ is substituted. Three predictions of the hypothesis that ‘hypercoagulability causes DVT’ are identified. One of these is shown to be weakly corroborated by early studies on ‘experimental thrombi’. The others are evaluated through a review of the literature on hereditary and acquired thrombophilias and are shown not to be supported by the available evidence. The conclusion – that thrombophilias increase the likelihood of DVT but cannot be considered ‘causal’ – is followed by a critical discussion of the clinical value of laboratory tests for thrombotic tendencies, and the need for an alternative to the consensus model of DVT aetiology is re-emphasised.

Keywords Acquired thrombophilias, anticoagulants, experimental thrombi, inherited thrombophilias, laboratory testing

3.1

Introduction

Twenty years after the consensus model was first articulated, Davies and McNicol (1983) contended that ‘hypercoagulability is unproved … undemonstrated, in man’. Such sceptical caveats have been few. Proponents of the consensus model habitually attribute the belief that ‘hypercoagulability causes DVT’ to Virchow (Browse et al. 1988b made the claim explicitly1). However, Virchow never used the term ‘hypercoagulability’, nor did his successors (see Chapter 6). Some writers claim that

1 ‘Category 3 [of ‘Virchow’s triad’] – “changes in blood constituents” – is hypercoagulability.’ The claim seems both historically and scientifically bizarre; it has no foundation in Virchow’s writings.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

31

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3 Hypercoagulability

he implied the concept in Thrombose und Embolie when he surmised (not implausibly) that the concentration of fibrin was a potential determinant of thromboembolism and that blood could be made ‘more adhesive’ by adding oil, paste or other substances. But the effect of ‘increased adhesiveness’ is more readily interpreted in terms of interactions with the endothelium than of enhanced blood coagulation. As for the fibrin(ogen) concentration, Virchow was clearly speculating because he was in no position to present evidence for either a systemic or a local increase.

3.2

The ‘General’ Use of the Term ‘Hypercoagulability’

Some uses of ‘hypercoagulability’ in the earlier literature appear to imply circular reasoning, which may be represented as follows. ‘Let us presume that thrombosis results from inappropriate (aberrant) blood coagulation; therefore, if thrombosis occurs, the patient’s blood must have an inappropriate tendency to coagulate, either locally or systemically; so by definition, the patient has a “hypercoagulability” (local or systemic)’. Such reasoning was perhaps seductive when detailed characterisation of the blood coagulation process was novel and anticoagulant therapy/prophylaxis was newly established and successful, but it is obviously unsound. Loose interpretation of Virchow (above) does not rescue the flawed logic. Neither does a posteriori reasoning along the lines: ‘since anticoagulants protect against thrombosis and act by down-regulating the coagulation mechanism, it follows that thrombosis must result from a pathological up-regulation of the coagulation mechanism’. When ‘hypercoagulability’ is mentioned in discussions of DVT, it is important to ascertain whether it is intended in this misleading, circular sense, or whether it denotes one or more of the inherited or acquired thrombophilias that have been well described and characterised. To avoid ambiguity, we shall use ‘hypercoagulability’ to denote the general concept, with its attendant risk of circularity, and ‘thrombophilia’ to denote any one of the specific clinical conditions.

3.3

Early Evidence for ‘Hypercoagulability’ Conditions

Familial ‘thrombophilia essentialis’, predicted by Andral in the 1830s, was recognised later in the 19th century by Armand Trousseau and was independently described by George Elgie Brown and Kaare Kristian Nygaard. However, it remained a medical curiosity until the 1950s. The main symptom is intermittent claudication (i.e. arterial rather than venous occlusion) with reduced bleeding times and in vitro clotting times. The syndrome can progress to thrombosis of the abdominal and pelvic vessels, and to haematuria and collapse. It is sometimes called the ‘Nygaard-Brown syndrome’ (e.g. Monto et al. 1952); the synonym ‘Trousseau’s syndrome’ is archaic. It is difficult to relate thrombophilia essentialis to any

3.4 Testing the ‘Hypercoagulability’ Hypothesis

33

aetiology relevant to DVT; other and better-characterised conditions are more obviously relevant. The earliest papers on DVT-related ‘hypercoagulability’ (e.g. Ochsner et al. 1950; Soulier and le Bolloch 1950; Waldron and Duncan 1950; Eisenmenger et al. 1952; Monto et al. 1952; Warren 1953) were written while the cascade model of blood coagulation was maturing. These early papers mention ‘apparent hereditary defects’, high ACTH levels, hepatotoxicity, ‘certain cancers’, silicosis, diabetes, and ‘rebound’ from anticoagulant therapy among the causes of DVT. However, they generally seem to imply the circular reasoning identified above: the supposition that ‘positive’ as well as ‘negative’ disorders of haemostasis might complete a spectrum of coagulabilities from hypo to hyper, with physiological normality in the centre of the imagined continuum. As we observed in Chapter 2, many workers in the field appear to have accepted this imagery as ‘established fact’ by the 1960s. Significant references from the period include Mustard et al. (1962), Sise et al. (1962), Wessler (1963) and Sise (1964).2

3.4

Testing the ‘Hypercoagulability’ Hypothesis

Consider the proposition ‘the likelihood (frequency) of thrombosis is higher in X than in the bulk population if, and only if, the blood in X is hypercoagulable’, where X is a patient or group of patients. Three predictions follow: (1) If normally coagulable blood is rendered hypercoagulable, then the likelihood of thrombosis is increased; (2) If the blood is not hypercoagulable, then the frequency or likelihood of thrombosis should be that of the ‘normally coagulable’ general population; (3) If thrombosis occurs (perhaps frequently or recurrently) in an individual patient or group of patients, then the blood of that individual or group will prove to be, or to have been, hypercoagulable. Prediction (1) is the weakest: the hypothesis would be dented if it were falsified, but corroboration would provide only circumstantial support. Predictions (2) and (3) are stronger: corroboration would establish the hypothesis beyond reasonable doubt, but the hypothesis would have to be abandoned if either or both were disproved. Testing these predictions requires appropriate statistical evaluation of extensive bodies of data, but publications in the area are numerous, which reduces

2 We should emphasise that while we have major reservations about indiscriminate use of the word (and the idea of) ‘hypercoagulability’, these authors have made extremely valuable empirical contributions to this field. Mustard pioneered anticoagulant therapy, with emphasis on atherosclerosis, and has added significantly to knowledge of platelet function; see e.g. Packham and Mustard (2005). Sise, among other achievements, characterised hyperfibrinogenaemias (e.g. Sise 1973), and Wessler identified many factors that increase the likelihood of thromboembolism (e.g. Wessler 1992).

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3 Hypercoagulability

the difficulty. We shall evaluate the second and third predictions before the end of this chapter. The first prediction was tested directly on ‘experimental thrombi’ soon after the hypothesis was embraced. These ‘experimental thrombi’ were induced by injuring the venous intima, often grossly, occasionally more subtly, e.g. by passing an electric current through the vessel wall (Day et al. 1977). Morphologically, they seldom resembled autochthonous thrombi, unlike the experimental thrombi discussed by Welch (1899). Some were propagating ‘red thrombi’; others were platelet accumulations; in still others, the first event appeared to be fibrin deposition (for reviews see Poole and French 1961; Sevitt 1973; Ogston 1987; Browse et al. 1988b). Chandler (1958) produced artefacts more closely resembling thrombi by ‘passively’ circulating blood in a closed loop of plastic tubing on a rotating turntable, and this technique became widely used for creating experimental thrombi. Later techniques involved injecting coagulation-inducing substances into ‘low blood velocity’ veins; semi-solid masses broadly resembling thrombi resulted. Various substances were used, including celite (Thomas et al. 1963), bacterial endotoxin (Thomas and Wessler 1964), ellagic acid (Botti and Ratnoff 1964) and, more interestingly, serum that contained activated coagulation factors – or purified factor Xa (Wessler and Yin 1969). Subsequently, Aronson and Thomas (1985) showed that injected pro- and anticoagulants had the expected effects in such experiments. The approach was based on studies of static models (ligated blood vessels) and the presumed creation of artificial ‘hypercoagulability’ (e.g. Mitchell 1964; Hunt et al. 1966; Pitney 1972; Strachan et al. 1974). Hume et al. (1970) discussed the earlier experiments in detail. Overall, the results were consistent with the first prediction, so the general ‘hypercoagulability’ hypothesis was weakly corroborated. However, endothelial injury in these studies was general, not focused in specific regions such as the venous valve cusps. We shall discuss ‘experimental thrombus’ models further in Chapter 7.

3.5

Inherited Thrombophilias

Logically, any thrombophilia must involve either a chronic positive stimulus to thrombus formation, or a defective inhibitory one, or defective fibrinolysis. There are several recent reviews of this field (e.g. Keeling 2001; Thorneycroft and Goldzieher 2003; Mazza 2004; Rosendaal and Reitsmer 2004; Rosendaal 2005). They report that the major inherited thrombophilias are statistically associated with: (a) Family histories of thrombosis; (b) First thrombosis occurring during the first 40 years of life; (c) Frequent recurrences. The most reliable information has come from cross-sectional studies of families with one or more of the inherited disorders. Their significance in arterial thrombosis as opposed to DVT is debatable (Grant 2004; Reitsma 2004).

3.5 Inherited Thrombophilias

35

Historically, the characterisation of hereditary thrombophilias was interwoven with the sequencing of the coagulation cascade and the biochemical elucidation of platelet function (e.g. Arkin and Hartman 1979; Goodenough et al. 1983; Owen et al. 1983; Schafer 1985). The significance of abnormal factor VII levels was recognised in the early 1960s (Godal et al. 1962), and hyperfibrinogenaemia, defective fibrinolysis and factor XI abnormalities in the late 1960s/early 1970s (Egeberg 1967; Isaacson and Nilsson 1972). Since then, several inherited thrombophilias have been characterised. Most of them arise from defects in regulators of coagulation. The earliest thrombophilia to be defined clearly was antithrombin III (ATIII) deficiency (Egeberg 1965). ATIII is low or deficient in approximately 0.01–0.1% of the general population but in 1–4% of DVT patients (Grünberg et al. 1975), indicating that it increases the likelihood of DVT (Nagy and Losonczy 1984). Several ATIII mutants have been identified, some of them clinically severe (Thaler and Lechner 1981). Most affected individuals are heterozygotes, with ATIII levels around 40–70% normal; homozygous deficiency is rare (Demers et al. 1992). The most common inherited thrombophilias result from autosomal dominant defects in the protein C pathway. Protein C deficiencies of at least two kinds (Lane et al. 1996) perturb the normal control of fibrinogenesis; they occur in some 0.1– 0.2% of the general population but in 3–5% of DVT patients, so they increase the population frequency of venous thrombosis (Griffin et al. 1981; Esmon 1984; Broekmans 1985). Protein S, the main cofactor of activated protein C (APC), has abnormally low levels in only 3–5% of DVT patients, again as the result of more than one mutation (Borgel et al. 1997). However, the general population prevalence is unknown, so it is difficult to assess its effect on the likelihood of thrombosis (Comp et al. 1984; Bertina 1985). Homozygous protein C and protein S deficiencies are statistically associated with neonatal purpura fulminans. In general, protein C and S deficiencies entail less risk of DVT to a patient than does ATIII deficiency (Heijboer et al. 1990). However, around 33–50% of hereditary thrombophilia patients have normal circulating levels of APC, protein S and ATIII; they have a point mutation in the factor V gene (arginine-506 is replaced by glutamine in the mature protein; Dahlbäck et al. 1993; Dahlbäck 1995). This point mutation, termed factor V Leiden or ‘APC resistance’ (Alhenc-Gelos et al. 1994; De Stefano and Leone 1995), markedly attenuates the susceptibility of factor Va to inhibition by APC. The likelihood of DVT3 is high in homozygotes (Rosendaal et al. 1995). The mutation is found in 3–5% of the general population among Caucasians but has not been observed among the native populations of the Far East or Sub-Saharan Africa (Rees et al. 1995). Elevated levels of coagulation factors also increase the statistical frequency of venous thrombosis, though the population incidences seem fairly low. Excessive factor VIII has been implicated in some cases (Koster et al. 1995), particularly

3

APC resistance does not, however, seem to constitute a risk for arterial thrombosis.

36

3 Hypercoagulability

when the plasma level of TAFI or of plasminogen activator inhibitor-3 is also raised (Meijers et al. 2002). Because factor VIII levels are linked to blood group (Jeremic et al. 1976), this might explain the alleged relationship between blood group A and the incidence of DVT (Jick et al. 1969). Both increased prothrombin levels and the G20210A mutant form of the prothrombin gene increase the likelihood of DVT (Poort et al. 1996). Similar effects have been reported for increased factor IX and factor XI levels in the plasma (van Hylckama Vlieg et al. 2000; Meijers et al. 2000), and there are suggestions that the same might apply to factor XIII (Swiatkiewicz et al. 2002). More than one of these abnormalities may be found in a single affected family. Perhaps a transcription factor regulating the expression of several coagulation factor genes may be abnormal, or there may be a defect in the system for posttranslational modification of the coagulation factors (Kaufman 1998). Plasma homocysteine levels are elevated in certain hereditary as well as acquired thrombophilias. The most common hereditary causes are defects in the methylene tetrahydrofolate reductase or cystathionine-β-synthetase genes, which encode enzymes essential for homocysteine metabolism (Skovby 1989). Hyperhomocysteinaemia has been observed in some 10% of patients suffering a first episode of venous thrombosis (Falcon et al. 1994). The aetiology is not understood; homocysteine may induce endothelial cell injury or prevent protein C activation (Sainani and Sainani 2002) and appears to mediate the expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes (Zeng et al. 2003), which may be relevant to the aetiology of thrombosis proposed in this book; see Chapter 12. Other known hereditary thrombophilias are relatively rare. Various forms of hyperfibrinogenaemia (Briët et al. 1985) and defects in fibrinolysis such as plasminogen deficiency (Girolami et al. 1986) have been implicated in DVT. Heparin cofactor II deficiency is another minor risk (Tollefsen 2002).

3.6

Acquired Thrombophilias

Acquired deficiencies of antithrombin III, proteins C and S and other anticoagulant factors have been described in cases of (e.g.) advanced hepatopathology, nephritic syndrome and prolonged anticoagulant therapy (Green 1988; Corda et al. 1991; Kemkes-Matthes 1992; Bertina 1999). Acquired hyperhomocysteinaemia has been reported; it is usually caused by deficiencies of the vitamins (B6 and B12) that generate cofactors in homocysteine metabolism, or by drugs such as methotrexate that interfere with folate metabolism (Skovby 1989). However, the most common acquired thrombophilia is antiphospholipid syndrome (APS) (Asherton and Hughes 1989). Circulating antibodies against prothrombin and β2-glycoprotein-1 (or perhaps other plasma proteins and lipoproteins) are found in patients with histories of venous or arterial thrombosis, thrombocytopaenia and spontaneous abortion (Forastiero et al. 1997). DVT is more commonly associated

3.7 Thrombophilia and DVT

37

with circulating anti-lupus antibodies and phospholipid-dependent coagulation, while arterial thrombosis is predominantly associated with anti-cardiolipin antibodies. Most individuals with APS are otherwise healthy, but the condition occurs in patients with SLE and other autoimmune disorders, HIV infection or lymphomas. The link between antiphospholipid antibodies and thrombosis is not clear, but individuals with APS seem to have lower than normal protein S levels (e.g. Morange et al. 1997). Circulating lupus antibodies are held to suppress prostacyclin synthesis, but the evidence is not compelling. One possible mechanism entails binding of the antibodies to platelet membranes, increasing the likelihood of forming a tenase complex without the normal physiological ‘priming’ of the platelets. Alternatively, the antibodies might interfere with APC action at the platelet surface, or cause direct injury to the endothelial cells. Such speculations have been discussed in the literature (Field et al. 1999; Rand 2002). Other alleged causes of acquired thrombophilia include paroxysmal nocturnal haemoglobinuria, nephrotic syndrome, various myeloproliferative disorders, and malignancies and cancer chemotherapy (not necessarily involving inhibitors of folate metabolism). The mechanisms are generally obscure.

3.7

Thrombophilia and DVT

Most studies suggest that antithrombin III, protein C and protein S deficiencies increase the incidence of DVT about 25-fold compared to the general population (Ely and Gill 2005). Factor V Leiden (APC resistance) elevates the incidence less than 10-fold, but it frequently occurs in combination with other defects and aggravates them (Gomez and Laffan 2004). However, there is no simple, regular correspondence between any thrombophilia (inherited or acquired) and DVT or other pathological sequel. This contrasts with the simple, regular and specific correspondences between defective haemostatic factors and bleeding diatheses. About 30% of patients with deep venous thrombosis or pulmonary embolism have a thrombophilia (Deitelzweig and Jaff 2004), which means that – in effect – about 70% do not. This 70% may include some yet-unidentified thrombophilias, but in most cases we must infer that the blood is normally coagulable. Taking the best available evidence, therefore, the third prediction of the general hypercoagulability hypothesis is not supported: a preponderance of thromboembolism sufferers have no identifiable thrombophilia. The second prediction is not supported either: familial studies show that not all carriers of inherited thrombophilias develop DVT (see the reviews cited above). The conclusion is inevitable: although thrombophilias increase the incidence of DVT, ‘hypercoagulability’ is not a prerequisite for thrombosis. These conclusions tend to invalidate the consensus model in its most familiar forms. Whereas a ‘hypocoagulable state’ (bleeding diathesis) constitutes a systemic functional abnormality and is revealed by haemorrhages occurring throughout the vascular bed, no ‘hypercoagulable state’ exists that causes all a mammal’s blood to coagulate

38

3 Hypercoagulability

(as some snake venoms do). Thrombosis is invariably localised (or ‘locally studded about’ in disseminated vascular thrombosis). This simple observation challenges the presumption that any ‘systemic hypercoagulable state’ causes thrombosis. DVT seems likely to result from ‘pathophysiological’ local triggering of the normal, physiological coagulation process, though thrombophilias potentiate such triggering. Of course, thrombophilias and other predisposing factors (such as those listed at the beginning of Chapter 1) interact synergistically; the likelihood of DVT in an individual patient increases with the number of ‘risk factors’ (O’Shaughnessy et al. 2005, 2007). Thus, a factor V Leiden sufferer who uses oral contraceptives has a statistically greater chance of thromboembolism (Vandenbroucke et al. 1994). However, we contend as a matter of principle that no amalgamation of predisposing factors can constitute a ‘cause’ of DVT, as Rosendaal (2005) implies; it can only increase its likelihood. Increased likelihood is clinically very important, and statistical studies that evaluate the increase in likelihood are potentially valuable. But to regard combinations of thrombophilias and other predisposing factors as ‘causal’ is scientifically and philosophically wrong. Combinations of predisposing factors do not constitute aetiology; rather, an aetiological hypothesis must explain why they are predisposing factors.

3.8

Testing for Thrombophilias

These arguments are widely ignored; testing for hereditary thrombophilias has become almost routine in hospitals. Some 25,000 tests for factor V Leiden alone are conducted in the UK every year, and wider population screening has been mooted (Keeling 1998). On the face of it, the mortality risk associated with DVT (perhaps 2% in younger patients and 10% or more in older ones) might seem to justify the expense of testing and perhaps prophylaxis. However, there is little evidence that such indiscriminate testing benefits patients (cf. Deykin et al. 1977). The statistical connections between thrombophilias and the incidence of DVT tell us little about the likelihood that an individual patient will become a victim of thrombosis (Levi 2004). DVT is likely to ensue in any patient under the right combination of circumstances, and some authorities doubt whether tests for thrombophilias have any clinical value (compare e.g. Baglin et al. 2003; Ehrenforth et al. 2004; Kamphuisen and Rosendaal 2004). These are not academic quibbles. They have serious practical, clinical and economic implications. A diagnosis of hereditary thrombophilia alone provides no compelling justification for increasing the intensity or duration of anticoagulant therapy. There is no evidence that discontinuing anticoagulant therapy puts hereditary thrombophilia patients at greater risk of recurrence of thrombosis than anyone else. There is no clear evidence that screening of women for hereditary thrombophilias can establish the desirability or otherwise of oral contraceptive use, pregnancy or HRT (Inman and Vessey 1968; Morssink et al. 2004; Fabregues et al. 2004). Even if an inherited predisposition to thromboembolism is established, interactions with environmental factors make it impossible to predict the likelihood that an individual will suffer an episode of DVT (Gallerani et al. 2004; Rosendaal

3.9 Implications for Prophylaxis and Therapy

39

2005). Therefore, testing for hereditary thrombophilia in an individual DVT patient seems to afford no clear benefit for clinical management (Baglin 2001a; Caprini et al. 2004). Personal history seems to be more useful than family history. Current evidence does not appear to justify the expense of testing first degree relatives for hereditary thrombophilias. Given a pathophysiological trigger, the probability of DVT in an individual with an identifiable thrombophilia may be greater than that in a normal individual. But identification of the pathophysiological trigger – locally altered blood flow (Chapters 8 and 9) – is more relevant than the diagnosis of thrombophilia for establishing future risk and for making clinical management decisions. It is certainly more relevant to understanding the aetiology of venous thrombosis.

3.9

Implications for Prophylaxis and Therapy

Although these issues belong to the domain of thrombosis management rather than aetiology, we address them briefly here because of their importance in clinical practice. Speaking for most clinicians, Alving et al. (2003) remarked: ‘The key to the prevention and treatment of venous and arterial thrombosis is anticoagulant and antiplatelet therapy’. They echoed Sevitt (1967): ‘The sheet-anchor of therapy is a course of an oral anticoagulant drug’. Those developments have saved many lives and have improved the quality of life for thrombosis sufferers. Nevertheless, the side effects associated with long-term use raise questions about the desirability of withdrawing treatment in some cases, notwithstanding the associated risk of recurrence of thrombosis. Anticoagulant prophylaxis and therapy became popular in the era that saw the rise of the ‘hypercoagulability’ concept (e.g. Murray 1947; Johnson and McCarty 1959; Sise et al. 1961; Mayer et al. 1963). The benefits are unquestionable and unquestioned.4 Anticoagulants have a long history: Hewson described in the 1770s how ‘butchers shed blood into salt to keep it liquid and fresh’ (because clotted/semisolidified blood putrefied rapidly when it lost its access to atmospheric oxygen and became necrotic); and the medicinal leech Hirudo medicinalis was used in the middle ages and before. But modern anticoagulant treatment is a 20th-century development. Heparin was discovered in 1916–1918 and dicoumarol in the 1930s. Ridker et al. (2003) gave a particularly interesting account and critical assessment of the history of Warfarin treatment, and Markwardt (2002) wrote a fascinating review of hirudin (the active extract of H. medicinalis).

4

Nevertheless, we now have a situation in which patients who have suffered minor thromboses are required take anticoagulants for the rest of their lives, though the risk of thrombosis recurrence is significant for only a few of them. Fear of litigation dissuades many doctors from stopping anticoagulant treatment, even when they may suspect that it is increasing rather than decreasing the risk to the patient’s general health and well-being.

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3 Hypercoagulability

Fibrinolytic therapy was first explored in the 1950s (see references above and Chapter 2) and for some years has been used as routine prophylaxis against DVT in broadly selected patients. Prophylactic anticoagulation in cases entailing prolonged patient immobility, such as fracture of the neck of the femur, began around 1960. It was tested and justified in a seminal paper by Sevitt and Gallagher (1961); most practitioners accepted it on empirical grounds and no formal biochemical justification was suggested or sought.5 Notwithstanding the explosion of thrombophilia testing in recent years, most uses of anticoagulant and fibrinolytic therapy remain prophylactic, as they were forty or more years ago. Patients likely to endure decubitus bed rest are given regular thrombolytic agents or anticoagulants without laboratory testing for a ‘thrombotic tendency’. Most physicians regard such prophylactic anticoagulation as fully justified, and surveys of DVT incidence in long-term hospital patients support their view, though there have been questions about its clinical value and costeffectiveness (Salzman and Davies 1980; Wells and Forster 2001; Baglin 2001b; Torn et al. 2004). Physicians, surgeons and haematologists apparently deem it unnecessary (or too expensive) to establish that a patient’s blood is ‘hypercoagulable’ before they decide clinically to exhibit Warfarin or heparin. This strongly implies that they do not give anticoagulants with the aim of reducing the ‘coagulability’ of ‘hypercoagulable blood’. Rather, they give anticoagulants to reduce the normal coagulability of normal blood to ‘below normal’. This, in turn, supports the obvious and reasonable inference that normal coagulability of normal blood is sufficient for DVT to occur.

3.10

Reflection

Although majority opinion in the field seems to doubt the clinical value of diagnosing particular thrombophilias, the characterisation of these conditions has helped to elucidate several aspects of the coagulation mechanism and its regulation, and has led to the identification of individuals at statistically higher-than-average risk of venous thrombosis. A sound aetiological account of DVT must (inter alia) be able to explain the empirical evidence that has accrued about each type of thrombophilia and the associated risk. However, we have disputed the opinion that hypercoagulability is causal in DVT. Since this opinion is implicit in the haematological ancestry of the consensus model, we need an alternative aetiological account that is not fundamentally haematological but can accommodate relevant haematological data. The search for such a model, and for a better understanding of the character of the consensus model, requires substantial historical exegesis. We shall begin this exploration in Chapter 4.

5

Interestingly, Ahlberg et al. (1968), Jansen (1972) and others showed that 500 ml intravenous dextran during operation prevented postoperative thromboembolism, recalling Aschoff’s opinion about the thrombogenic effects of hypovolaemia (see later chapters). Dextran is believed to have no anticoagulant side effects (Bergentz 1978; Negus and Ruckley 1980), and we concur with others that it acts to maintain circulating blood volume during surgical procedures (see Chapters 8 and 11).

Chapter 4

Historical Roots

Abstract Two historically disjoint approaches to biomedical investigation are identified: the ‘(patho)physiological’, associated with Harvey and Virchow, and the ‘mechanistic’, associated with Boerhaave, du Bois Reymond and the consensus model of DVT. The origins of both traditions are traced to the 17th century, the Scientific Revolution and the dawn of natural philosophy. Their developments into the 18th and early 19th centuries are outlined. The lines of investigation that led on the one hand to the elucidation of the blood coagulation mechanism, and on the other to the seminal contributions of Virchow, are identified. Care is taken to distinguish ideas in both mainstream traditions from belief in a ‘vital force’, and other possible sources of semantic confusion are discussed.

Keywords Animal chemistry, haemodynamics, mechanistic philosophy, microscopy, pathophysiology

4.1

Two Approaches to Biomedical Research

During the first three chapters we have referred to work of earlier times (notably Virchow’s), made semantic and philosophical observations and mentioned an alternative approach to the aetiology of DVT. We shall now start to develop these passing references into a historical review, explaining our doubts about the consensus model and laying the foundations for the proposed alternative. In the first two sections of this chapter we reflect on the broader issues involved. Feinstein (1999), concerned that laboratory research and clinical practice were drifting apart, appealed for the rebuilding of the ‘pathophysiological bridge between bench and bedside’. Malone and Agutter (2003) argued that the Feinstein’s concern applies with particular relevance to DVT, contending that it too suffers from dichotomous thinking in severed historical traditions. In brief: the ‘mechanistic’ tradition in DVT research came to fruition in the biochemical and haematological studies of the 19th and 20th centuries that culminated in our current approach to blood coagulation and the consensus model (Chapters 2 and 3). Its roots are in the P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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‘medical physics’ of du Bois-Reymond and his peers and, beyond them, in the work of Boerhaave and his followers; it can legitimately be traced to Descartes. The other, ‘pathophysiological’, tradition1 is rooted in the conceptual contributions and investigative work of Hunter, Virchow, Welch and 20th-century pathologists such as Aschoff. This tradition focuses on function and malfunction at the macroscopic level. It can be traced to Harvey’s seminal work on the circulation of the blood. There has been, and still is, little productive dialogue between these two conceptual and methodological traditions. Proponents of the consensus model often seem to be unaware of the alternative viewpoint, or perhaps consider it passé, or offer distorted interpretations that imply that their work and approach has satisfactorily encompassed its achievements. We dissent from this view (which will be discussed further in the appendix). Our alternative model derives mainly from the ‘pathophysiological’ tradition, but our aim is to help forge a rapprochement between the two approaches to research and practice, not only in the limited field of venous thrombosis but in biology and medicine as a whole.

4.2

Semantic Issues

Rigorous use of words is a sine qua non of scientific discourse. The need for rigor becomes yet more acute when contrasting perspectives2 are explored and publications from different periods of history are juxtaposed and compared. What a word denotes depends on theoretical presumptions underlying its use, so the same word might have different meanings – connotations as well as denotation – in different contexts. Moreover, denotations of words (scientific terms in particular) may change remarkably over time. Since the language of theory is inherently metaphorical, and injudicious usage can insidiously substitute ‘is’ for the implicitly metaphorical ‘as if’, all scientists must be keenly aware of the philosophical faux pas that Whitehead (1925) dubbed

1 We intend ‘pathophysiological’ to be understood in a general sense, i.e. indicating physiological malfunction, as in a blood flow rate that is significantly above or below the normal physiological range. 2 Seen from one point of view, what we term ‘approaches’ or ‘perspectives’ or ‘frames of reference’ might have been called ‘paradigms’ by Kuhn (1970). Like Kuhn’s ‘paradigms’, they have their origins in seminal discoveries and encompass particular theoretical notions and methodologies, and most of the activities associated with any single ‘approach’ seem to correspond to Kuhn’s ‘data gathering’ and ‘puzzle-solving’. The principal difference between the history of research on DVT and the histories of physics evaluated by Kuhn is that in the former, two rival ‘perspectives’ coexist during the same period of history, while in the latter, one ‘paradigm’ supersedes the other in a ‘scientific revolution’. For this reason, it may be injudicious to extrapolate Kuhn’s account of science to this aspect of biomedicine. There have been no ‘revolutions’ in the Kuhnian sense during the history of thrombosis research, though Harvey’s work on the circulation of the blood is certainly an example (Chapter 8).

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‘the fallacy of misplaced concreteness’. The fallacy occurs when abstractions are made concrete in people’s minds by converting ‘as if’ speculations or metaphors into statements of fact. This switch is clearly exemplified in the couplets thrombus/ thrombosis versus clot/clotting. The ancient word ‘clot’ has cognates in all Germanic languages; the modern English version derives from the Old English clott, denoting a clod of earth, an evocative metaphor for an inert, semi-solid mass. ‘Thrombus’ is a transliteration of the Greek qromboV = clot, which, we believe, originally signified a subcutaneous blood blister or haematoma. But although ‘thrombus’ appears in 17th-century texts, its modern technical use was specifically ordained by Virchow in the 1850s. The sharp distinction between these words indicated by Virchow needs to be maintained: a clot is said to form when blood semi-solidifies ex vivo; but a thrombus is formed only when blood semi-solidifies within a blood vessel in vivo. Given that the two words are obviously etymologically equivalent, some readers will consider our semantic insistence pedantic. We would argue that although the modern English words sarcophagus and carnivore are likewise etymologically equivalent (a Greek ‘flesh-eater’ versus a Latin one), substitution of one for the other could have bizarre or even dangerous consequences. The point is this: Virchow initially suggested that in vitro blood coagulation was a suitable analogue or exemplar for what he termed thrombosis – hence his nomenclature. He evaluated the analogy and found it wanting, as we shall explain in Chapter 6, but some of his successors mistreated his analogy as identity, read ‘is’ for ‘as if’, and thus caused clot/clotting to substitute for thrombus/thrombosis in biomedical discourse. The extent to which this ‘fallacy of misplaced concreteness’ has permeated our thinking will become clear when the following four sentences are examined. 1. 2. 3. 4.

A thrombus formed in the saphenous vein A clot formed in the saphenous vein A thrombus formed in the blood in the test tube. A clot formed in the blood in the test tube.

Only (3) will immediately strike most readers as semantically odd – which indeed it is, because semi-solidification of blood in vitro is not thrombosis and its product is not a thrombus. More significantly, (2) does not strike most readers as odd. This is because it has become commonplace to substitute ‘clot’ for ‘thrombus’ in such contexts – just as the plasma proteins involved in blood coagulation are infelicitously called ‘clotting factors’. It is simply not true that thrombus-formation in vivo is scientifically indistinguishable from clot-formation ex vivo, or that ipso facto a thrombus is effectively indistinguishable from a clot. Why then has this ‘fallacy of misplaced concreteness’ arisen, and why has it become so universally accepted? The essential point (Malone and Agutter 2006) is that whereas a thrombus results from the semi-solidification of flowing blood in a living body, a clot results from the semi-solidification of static blood in a non-living (ex vivo) environment. According to the ‘pathophysiological’ approach, the distinctions living/non-living and moving/static are fundamental.

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According to the ‘mechanistic’ approach, they may not be.3 ‘Mechanists’ can study biologically-derived material in vitro and judiciously extrapolate the findings to in vivo circumstances. ‘Pathophysiologists’ are dubious of such extrapolation. Thus, ‘mechanists’ see no serious semantic or philosophical problem in sentence (2), above, and the fact that few readers will immediately perceive the anomaly indicates the dominance and pervasiveness of the ‘mechanistic’ viewpoint in today’s biomedical world. This is not to say that the ‘mechanistic’ approach causes errors in thinking; it is to say that alone, deprived of an appropriate ‘pathophysiological’ complement, it may lead to errors in thinking. By the same token, the ‘pathophysiological’ approach alone is likely to lead to errors4 – in particular, errors of imprecision that follow from under-investigated processes. In the remainder of this chapter we shall concentrate on developments that followed the ‘Scientific Revolution’ in Europe, relying on primary sources where appropriate but also referring to secondary sources, notably Clendenning (1942), King (1963) and Porter (2001) for general information, Starr (1998) for the history of haematology, and Davies and McNicol (1983) and Browse et al. (1988a) for background information about thrombosis.

4.3

The Ancient World

Investigations into blood, blood-related diseases and what we now call the ‘circulation’ date back to antiquity. Anning (1957) and Dickson (2004) note that pathological haemostasis was described in China in about 2650 BC. From the many references to ‘drinking blood’ in ancient testaments, it could seem that the ancients had discovered techniques for anticoagulation. ‘Whipping’, which removes coagulable material, was perhaps the most likely method. The earliest known European writings to mention blood coagulation were those of Greek philosophers in the 4th and 3rd centuries BC. In particular, Plato (428–347 BC) asserted that the blood contained fibres that caused it to congeal when it cooled after leaving the warmth of the body, and his view was not refuted until the end of the 18th century. Hippocrates distinguished arteries from veins and may have recognised venous thrombosis in relation to venous ulcers. Diocles of Carystus, an early successor of Hippocrates, appears to have recognised a condition he called ‘inflammation of the veins’, but it is not known whether he meant phlebitis in the ‘infected’ sense, or what we now call thrombosis (i.e. ‘phlebitis’ in the sense used by Cruveilhier in the

3 During the course of the book we shall discuss other issues of terminology, and many of them bear the same relationship to the ‘rival’ approaches as the clot/thrombus distinction. 4 These errors do not, however, include ‘vitalism’ in the sense of evocation of a ‘vital force’. We shall discuss this point more fully in the following pages and the appendix. Mechanists (‘reductionists’) who regard proponents of the pathophysiological (‘holistic’) approach as ‘vitalists’ are guilty of sloppy thinking, and sometimes of ignorance.

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19th century). Studies of arteries and veins in Alexandria during the later centuries BC, notably by two pioneers of vascular surgery, Herophilos and Erasistratos, culminated in the work of Galen during the 2nd century AD. Galen used venesection to treat ulcers and varicose veins. His mainly accurate anatomy, and his mainly fanciful physiology, remained virtually unquestioned until the 16th century; we shall say more about this in Chapter 8. In 1452, Leonardo da Vinci produced superb anatomical drawings that included illustrations of superficial limb veins. These were important precedents for the revolution in anatomy that was to take place a century later in the school of Vesalius and his successors in Padua. Another major influence on mediaeval medicine was the encyclopaedic al-Tasrif (Art of Healing) of Abu’l-Qasim, written around AD 1000. Abu’l-Qasim, known in Europe as Albucasis, was the most famous surgeon of the Middle Ages, and his work influenced European medicine for half a millennium. Among other things, al-Tasrif contains what is perhaps the earliest clear description of haemophilia. Venous thrombosis was described in a European text around AD 1400 (Dickson 2004). However, the first recorded attempt to account for its aetiology was by Wiseman (1686). Wiseman, surgeon to Charles II, apparently attributed the phenomenon to ‘coagulation of the serum … or the obstruction of a vein …’, roughly identifying two of the elements of ‘Virchow’s triad’. He wrote in the aftermath of Harvey’s celebrated publication, and at the time of the early microscopists; an appropriate era in which to begin our historical survey.

4.4

‘Binary Oppositions’ in 17th–18th-Century Medicine

The two contrasting viewpoints we have mentioned have deep historical roots common to many areas of medicine, though they are not evident before the 17th century. General accounts of history often portray the past in terms of opposed pairs of metaphysical viewpoints5: Galenist versus Harveyan, humoralist versus solidist, vitalist versus mechanist, and so on. We shall follow this practice because it cannot

5 The once-fashionable structuralist Claude Lévi-Strauss held that the representation of experience as a set of binary oppositions – nature/culture, male/female, light/dark, etc. – is fundamental to the structure of the human mind, universal among preliterate societies and commonplace in developed cultures. Whatever the credibility of this judgment, it is difficult altogether to avoid the reduction of knowledge to such pairs of opposites, especially in outline accounts of the history of ideas. However, it is potentially misleading and can lend itself to the most vulgar rhetoric. An extreme ‘mechanist’ declaring ‘either you share my beliefs wholly or you’re a vitalist’ seeks to marginalise anyone who draws attention to the philosophical pitfalls of naïve reductionism; and as we shall see, exactly such aspersions have been cast on eminent figures from medical history by the less reflective among modern-day commentators. One is reminded of an American president seeking to preclude all public questioning of his aggressive foreign policy by declaring ‘either you’re with us or you’re with the terrorists’.

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be avoided without over-detailed analyses of particular historical events, which would distort the broad survey presented here. However, labels such as ‘solidist’ or ‘mechanist’ tell us very little about an individual or his work. They serve only to categorise general metaphysical standpoints, and sometimes they can mislead; in truth, the natural philosophy of the 17th and 18th centuries was rich and very varied.6 The overwhelming majority of 17th- and 18th-century researchers were individual amateurs, trained in the old schools of thought and therefore loaded with all its metaphysical prejudices, steeped in the need to refer all their discoveries in some way to religion, and as argumentative as any group of academics/scientists today, each individual convinced that he was right and everyone else partially or wholly wrong. The Scientific Revolution of the 16th and 17th centuries is mainly associated with the rise of mechanics, the new mathematical account of the physical world. However, the foundations of scientific medicine and biology were laid in the same period. There were clear differences between the disciplines. For example, the writings of Bacon, Galileo, Descartes and Gassendi explicitly rejected the Aristotelian account of the inanimate universe. But Aristotelianism was expunged from medicine and biology – if it has ever been completely expunged – not in a few confrontational decades but after a war of attrition that persisted into the 19th century. If there was a revolution in medicine during the ‘Scientific Revolution’ period, it was against Galen rather than Aristotle. Nevertheless, the 16th and 17th centuries witnessed a clear turning point in the history of western thought as a whole. Significantly, the seminal publications of Copernicus and Vesalius were published concurrently, and Harvey was a contemporary of Bacon and Descartes. Traditional learning and practices were being fundamentally challenged across the whole range of what we now dub ‘science’ from mechanics to medicine. Inevitably, therefore, all disciplines came under the critical metaphysical scrutiny that was integral to ‘natural philosophy’. As a result, medical writings of the 17th century reflect not only the well-known conflict between Harvey and his followers on the one hand and traditional Galenists on the other, but also a re-evaluation of the mediaeval belief that life was Godgiven, soul-dependent, essentially mystical and beyond the reach of natural philosophy.7 The famous thesis of Descartes, that animals may be regarded as machines, was applied equally to humans. His belief, the basis of iatrophysics or iatromathematics, stood in stark and deliberate contrast to tradition and attracted many followers. Later in the century, Robert Boyle (1627–1691) and others began to apply the new metaphysic to alchemical knowledge. Those early pioneers of

6 Our distinction between ‘mechanistic’ and ‘(patho)physiological’ approaches is ostensibly just as simplistic, but our reason for maintaining it will become increasingly evident as the book proceeds. 7 In the 17th century, and to an extent the 18th, the main thrust of natural philosophy was to support, not to oppose, religion and metaphysics. In the dozens of disputes among the leading thinkers of the day, the religious fitness of ideas formed the foreground, not the backdrop, to intellectual conflict. Nevertheless, the approach to acquisition and organisation of knowledge encapsulated in ‘natural philosophy’ was new and gradually came to predominate.

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chemistry were keenly interested in the modi operandi of organisms, particularly humans, and iatrochemistry appeared as an alternative challenge to medical tradition. Boyle’s most important medical work was the first attempt at a scientific analysis of the blood and can reasonably be considered the pioneering treatise on ‘animal chemistry’ (Boyle 1684). Historians write about opposition between iatrophysicists and iatrochemists, but both ‘groups’ shared an urge to drive the scalpel of natural philosophy into the concreted mass of mediaeval learning. Collectively, their writings constitute the wellspring of the metaphysical position in medicine and biology that came to be called mechanism,8 and it is difficult to define and sustain a clear distinction between them. In the 17th century, ‘mechanism’ denoted a way of testing and explaining phenomena that had come to be considered generally appropriate: the effort to account for all features of the observable world by mathematics. The early hope that all determinable aspects of the world would be explicated mathematically – parts, motion, time, space – had faded by the end of the century; indeed, had it not been for the invention of the calculus, the ideal of natural philosophy would probably have died in the cradle. At the beginning of the Enlightenment in the 18th century, the French philosophes began to theorise about natural religion, natural law and human institutions, and the term ‘mechanism’ took on a different and more specific sense, the sense pertinent to the ‘mechanism/vitalism’ debate (see previous footnote). This backdrop sense of ‘mechanism’ derived from Descartes’ view of animals as machines rather than ‘magic’. Its basic principle is that living matter can, and should, be described and explicated in the same language as non-living matter. The prolific writings of Boyle included not only forward-looking contributions to mechanics and chemistry, but also a defence of Aristotle’s analysis of the physical world in terms of ‘four causes’. The development of iatrochemistry in the work of Hoffmann (1660–1742) and Stahl (1660–1734) during the late 17th and early 18th centuries showed a similar admixture of Aristotelianism with natural philosophy. Hoffmann remained a ‘mechanist’ throughout his life and his work can fairly be regarded as a distant forerunner of modern biochemistry. As young men, he and Stahl were close friends and collaborators, sharing the same metaphysical commitment; but Stahl became increasingly convinced that organisms could not ultimately be described in exactly the same language as the non-living world, and the friendship cooled. Stahl’s writings about organisms evoked Aristotle’s entelechy; indeed, his thinking seems to have grown more rather than less Aristotelian, and less rather than more Cartesian, as he aged.9 Because the

8 Perhaps the first explicit enunciation of the ‘mechanist’ position in 18th-century biomedicine was L’Homme Machine (1738) by de la Mettrie, a disciple of Locke rather than Descartes. 9 Stahl is perhaps best remembered as the proponent of the phlogiston theory of combustion, which dominated chemistry for almost a century until the work of Priestley and Lavoisier rendered it valueless. ‘Phlogiston’ is an inherently Aristotelian concept; it denotes ‘the property of being combusted or combustible’ rather than a particular substance, though Stahl’s successors attempted to reify it. Stahl appears to have acquired the concept from a study of the works of Jean-Baptist van Helmont (1577–1644), who is sometimes considered the first theorist of chemistry.

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‘mechanism/vitalism’ debate is perhaps the best-known of the binary oppositions to which the history of biology and medicine is commonly reduced (Heim 1972), it is tempting to label Stahl a ‘vitalist’ (since he expressed caveats about ‘mechanism’). This would be anachronistic: the first clear statement of vitalism, i.e. the belief that living entities are distinguished from the non-living by a vital force (élan vital or Lebenskraft), did not appear until 1774 in the writings of Friedrich Casimir Medicus.10 It would also be inaccurate: there is no evidence that Stahl believed explicitly in any kind of ‘vital force’.

4.5

Harvey and the ‘Physiological’ Approach

We can generalise this assessment: not everyone who declined to adopt a radical ‘mechanist’ stance (in the Cartesian sense of ‘mechanist’) in the 17th and 18th centuries can be automatically labelled a ‘vitalist’. The point is moot in Harvey’s case. Exposed to the newly evolving metaphysic of natural philosophy from his formative years and a strong proponent of the Baconian approach to experiment, Harvey refuted Galen’s account of blood movement and laid the foundations of modern physiology (see Chapter 8); but he was not a ‘proto-mechanist’ like the iatrophysicists and iatrochemists. Yet to deem him a ‘vitalist’ would not merely be anachronistic, it would be absurd; it is hard to find a 17th-century writer less inclined towards mystical notions than Harvey. In refuting Galen, he distanced himself from mediaeval tradition no less sharply than those of his contemporaries whom we might term ‘proto-mechanist’. His surviving writings suggest a metaphysical stance akin to the approach we have dubbed ‘physiological’, ‘pathophysiological’ or ‘vital-materialist’. He exerted great influence during and after his lifetime. For example, the gifted vivisectionist Richard Lower (1631–1691), inspired by Harvey’s work, performed the first blood transfusion in western history in 1666, and 3 years later he gave an account of the anatomy and physiological action of the heart that is clearly ‘physiological’ in orientation (Lower 1669): a specifically living mechanism. This work included the first description of the ‘muscle pump’ (the ‘peripheral venous heart’) and its importance in maintaining venous return. Lower was acquainted with Wiseman, who had made the first attempt to account for the aetiology of DVT (see above). These observations suggest that the ‘(patho)physiological’ approach to biomedicine, like the ‘mechanistic’ alternative, had its ultimate roots in the intellectual ferment of the 17th century.

10 Friedrich Casimir Medicus (1736–1808), a celebrated botanist and an implacable opponent of Linnaeus, introduced the term Lebenskraft, or ‘vital force’, to animal chemistry. In 1796, Johannes Reil (1759–1813) included Lebenskraft as one of five types of force in Nature (Teich and Needham 1992).

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The contributions of the early microscopists during the second half of the 17th century are well known and can be summarised briefly. The lens-grinder Antonie van Leeuwenhoek (1632–1723) seems to have been the first to observe red blood cells. He wrote: ‘The red globules of the blood I reckon to be 25,000 times smaller than a grain of sand’ (van Leeuwenhoek 1674). This showed that blood is not a simple fluid, as previously supposed, because it contains formed elements. The discoveries and pioneering approach to research of Marcello Malpighi (1628–1694) aroused suspicion and hostility among jealous colleagues. His microscopic studies were informed by the natural-philosophical dictum ‘Be prepared to reason, but never go beyond the facts’. One of the earliest of these studies (Malpighi 1661) announced his famous discovery of blood flow in capillaries in mammalian lung tissue, which confirmed Harvey’s prediction. Malpighi described the ‘stagnation’ of blood in the smallest of these capillaries, an observation that would take on considerable significance in later years. Aside from their obvious importance in the history of the study of blood and the circulation, these pioneering microscopic studies illustrate how detailed observation as well as experiment played crucial parts in biomedical research. They also illustrate an important general point about the history of science: the progress of knowledge depends at least as much on new methods and techniques as on new ways of thinking. The discoveries of the early microscopists were, to a first approximation, ‘theory-neutral’. They invited different interpretations according to different metaphysical persuasions.11

4.6

The 18th Century: Solidism, Humoralism and the Work of Boerhaave

Most 18th-century physicians held that diseases arose in the solid tissues of the body, but they ignored the possible involvement of body fluids in pathology. Solidism became the fashionable reference framework, and changes in body fluids were considered symptoms rather than causes of disease. Nevertheless, the traditional beliefs rooted in the teachings and practices of Hippocrates and Galen continued to influence 17th- and 18th-century thoughts. Predominant among these

11

They were largely motivated by (a) the 17th-century fascination with light (manifest in Vermeer’s paintings, Wren’s architecture, the optical theories of Huygens and Newton, and the natural-philosophical insistence on the evidence of the senses – mainly vision – as the basis of all reliable knowledge of the world); and (b) the desire to discover the atomic, particulate basis of all matter, which was a postulate of mechanistic philosophy. But the early microscopists dramatically failed to find ‘atoms’ of living matter. Instead, they discovered that living things continued to show exquisite and complex beauty no matter how much they were magnified, testifying to the awesome magnificence of God’s creation. Religious inferences aside, these findings could be aligned with any of the various metaphysical stances taken by different natural philosophers; hence our claim about ‘theory-neutrality’.

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was humoralism, the belief that health depended on the maintenance of balance among the ‘four humours’. The most important of these humours, blood, was traditionally endowed with virtually mystical properties. Its life-preserving power was often ascribed to its very fluidity (Starr 1998). It conveyed the vital spirits on which the functions of organs such as liver, lungs and digestive tract depended. The persistence of this notion illustrates the slowness with which medicine divested itself of its Galenist past (see e.g. Spurgin 1827). Even the most ardent solidists accepted that blood was different from other body fluids, but the widespread use of purging, cupping, leeches and similar approaches to the treatment of disease reflected the dogma that a balance must be maintained among the humours. It therefore seems inevitable that studies of the blood attracted increasing attention as the 18th century wore on, and that these studies became progressively imbued with the metaphysic of natural philosophy. As chemistry gradually evolved into a recognisable discipline, so ‘animal chemistry’ began to loom large in the interests of investigators. The chemistry of the blood, pioneered by Boyle, became a dominant concern – as it still remains. Hermann Boerhaave (1668–1738) figures as the first professor of medicine since Hippocrates to teach at the bedside, thus instigating ‘modern’ teaching methods. He held three of the five professorships (medicine, botany and chemistry) in the University of Leyden for several years and single-handedly created Leyden’s international prestige as a centre of medical teaching. His influence – including his philosophical orientation, which was explicitly derived from the writings of Descartes, Boyle, Locke and Newton12 – spread within a generation to medical centres throughout Europe, particularly to Edinburgh, Vienna and various German cities (King 1963; Lindeborn 1968). His innovative discoveries, such as the purification of urea and the observation that water is a product of alcohol combustion, were balanced by an intimate knowledge and deep understanding of the classics (Burton 1746; Burton’s appendix lists all Boerhaave’s allegedly authentic works). Yet, like Newton, he was obsessively interested in alchemy and sought for years to transmute mercury into gold. In medicine he was a humoralist rather than a solidist, and was particularly interested in blood-related pathology.

12 From Boyle he no doubt derived his interest in chemistry, and specifically blood chemistry; from Locke (and, indirectly, Newton) he assimilated his empiricist inclination; and from Descartes his commitment to the ‘mechanistic’ ontology – perhaps a further indication that iatrophysics and iatrochemistry should be considered complementary rather than opposing approaches in 18th century biomedicine. The distinction between ‘empiricists’, typified by Locke, and ‘rationalists’, typified by Descartes, is anachronistic. Locke was just as metaphysical as Descartes, and Descartes just as ‘scientific’ as Galileo and Newton. If these men agreed about little else, they certainly agreed that experiment and observation were the indisputable basis for all reliable knowledge about the world, including our bodies. By the early 18th century, this principle had attained widespread official blessing and encouragement. Patronage, often extended by Church princes, wrought a great deal of influence on the thought of the age, and various societies arose in which ‘empiricism’ was deliberately cultivated. Few people questioned the stance of the natural philosophers, and those few were mostly confined to doctrinal institutions.

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Like his German contemporaries Stahl and Hoffmann, Boerhaave was a great systematist, but unlike Stahl he held that organisms were mechanisms, to be understood exclusively in the language of mechanics. He wrote: ‘The universal laws of nature, or affections of all bodies, depend on mechanical and physical principles, upon which alone their actions are explicable; the same laws are also true in the human body, for its matter appears to be universally the same with that of all other bodies’. Boerhaave stoutly defended Malpighi. His argument from Malpighi’s observation of blood stagnation in capillaries typified his mechanistic approach. He asserted that ‘inflammation’ (which we would now call ‘thrombosis’) is caused by a combination of blood stagnation in small capillaries and increased local blood velocity (due to vasodilatation): faster movement against greater resistance increases the pressure on the obstruction. The vein wall can also be pathogenic if it is too rigid (increasing pressure) or, alternatively, too fragile or lax (decreasing resistance), a notion that was soon to be developed by the pioneers of haemodynamics. As we observed earlier, ‘iatrophysics’ and ‘iatrochemistry’ are difficult to dissociate in much 18th-century thought. After Boerhaave, the medical world was roughly divided into practising physicians, who mostly adhered to the solidist metaphysic, and academic researchers, who increasingly emphasised the study of ‘animal chemistry’, espoused Boerhaave’s ‘new humoralism’ and regularly deplored the failure of physicians to study chemistry. William Cullen (1710–1790), professor of medicine at Edinburgh and an outstanding teacher of chemistry and medicine, argued that Boerhaave had neglected the influence of the nerves. He believed that morbidity in organs caused the nerves to increase blood flow to the affected parts, implying that blood is merely an agent acting under the influence of the nerves (Donovan 1975). Cullen’s strong influence on 18th-century medicine in Britain reinforced the tendency towards solidism among physicians. Nevertheless, Cullen’s friend and student George Fordyce, though a practicing physician, attempted chemical studies of blood and other body fluids, during which he identified ‘white globules’. Forward-looking though he was in these respects, Fordyce nevertheless continued to hold the residual Galenist belief that blood is produced by the digestion of food (Fordyce 1791). As we remarked earlier, ‘binary oppositions’ in the history of medicine are never absolute. Individuals and their ideas cannot be confined to metaphysical pigeonholes; and we see that notwithstanding Harvey’s influence, Galenism died a lingering death – if, indeed, it ever completely died (witness the continuing invocation of ‘stasis’ as a cause of DVT; see Chapter 8).

4.7

Hunter and Hewson

John Hunter (1728–1793) taught anatomy in London as assistant to his elder brother William, and when he was 40 he received the Diploma of the College of Surgeons. Experience and scientific contacts contributed further to his scholarship. Many of those with whom he fraternised were sceptical of the mechanistic

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philosophies of Hoffmann, Boerhaave and de la Mettrie.13 His practical experience in the Seven Years War as a naval surgeon treating military casualties made him expert on the subject of gunshot wounds and their treatment (Dobson 1969). Born a century after the publication of Harvey’s seminal work, Hunter appears to have assimilated Harvey’s ‘physiological approach’. That is to say, while he seems to have viewed the circulation of the blood as a haemodynamic mechanism, he nevertheless regarded it as a specifically living mechanism. His studies of blood coagulation and of what we now call ‘thrombosis’ led him to implicate the vein walls in the process, corresponding to what he dubbed ‘inflammation of the internal coats of the veins’ (Hunter 1793). He remarked (Hunter 1794)14 that ‘a dead body has all the composition a live one ever had’, which diametrically contradicted Boerhaave (see earlier quotation), and he posited that a failure of haemostasis (in vivo) may betoken either suspended function of ‘poorly re-spirited’ blood, or its non-function, i.e. blood death. Nowhere in his work did Hunter refer to or imply a ‘vital force’, so there is no justification for some commentators (e.g. Coley 2001) to label him15 ‘vitalist’. Hunter was cautious about the use of microscopes and admonished his readers not to mislead themselves by ‘what can be seen through magnifying glasses … globules etc.’ (Hunter 1794). But that was before the introduction of Lister’s achromatic lens and was probably a valid warning. However, his shrewd observation proved that the ‘red globules’ (erythrocytes) were not involved in blood coagulation. Observing the colourless or white blood of the Antarctic ice-fish, which is devoid of erythrocytes and of haemoglobin, he noted that it coagulated ex vivo no differently from red mammalian blood. Some of Hunter’s remarks seem curious to modern eyes. For instance, he taught that the formation of what we now call a ‘haemostatic plug’, together with the nature and contraction of ‘fiber’, was analogous to muscle and muscular contraction, or to web-spinning by spiders. He also devoted considerable time and attention to demonstrating that blood coagulation does not liberate heat. But these concerns were widespread during the late 18th and early 19th centuries. For example, Fourcroy (1787) suggested that blood coagulation might afford a mechanism for the formation of fibres in animal bodies,16 and several subsequent investigators reported that heat was produced during coagulation (e.g. Gordon 1814; Davy 1817). Hunter was addressing issues that his contemporaries regarded as pertinent.

13

De la Mettrie (1912); a translation of L’Homme Machine, first published in 1748. This was a posthumous publication, but its authenticity is not in doubt. Hunter’s use of the phrase ‘inflammation of the internal coats of veins’ is of great interest and we shall have more to say about it in a later chapter. 15 The whole thrust of Hunterian knowledge seeking was not directed to finding or establishing the ‘cause of life’. It had the more achievable object of investigating the effects of life by careful observation. So the accusation that Hunter was a ‘vitalist’ is nonsense. He was a physiological successor of Harvey. 16 Fourcroy may have invented the word ‘fibrin’. We have not seen the word in earlier writings. 14

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William Hewson (1739–1774) studied medicine at Newcastle and Edinburgh. A pupil and collaborator of William and John Hunter, he attended Fordyce’s lectures in London and was inspired to undertake chemical experiments on the blood. His most celebrated contribution was the discovery of a ‘coagulable lymph’, later to be known as fibrinogen; but importantly, he disproved the Boerhaavian idea that blood solidifies locally in vivo when it is not moving. His experiment was simple but convincing and the replicates were consistent. He doubly ligated the jugular vein of a living dog and observed that the blood remained liquid between the ligatures, where it was completely static. He wrote (Hewson 1771, p. 20): ‘From several experiments. … I found that … after being at rest for 10 minutes the blood remained fluid; nay, that after being at rest for three hours and a quarter, above two thirds of it were still fluid’. Hunter had observed that ‘the blood remains fluid in priapism’, an observation that could have preceded and possibly prompted Hewson’s experiment. Whatever the chronology of these studies, the persistent or acquired fluidity of blood in priapism, and the persistent fluidity of blood in a doubly ligated vein, combined to disprove Boerhaave’s ‘stasis hypothesis’. Therefore, stasis does not cause ‘inflammation of the internal coats of veins’ (thrombosis).17 If thrombi do not form in static blood, the only alternative is that they form in circulating blood. Hewson, who is sometimes dubbed the ‘father of haematology’, had thus demonstrated that what we now term ‘thrombosis’ is a different process from blood clotting ex vivo. There is a moral in the close collaboration between Hewson and Hunter: the ‘mechanist’ conducting animal chemistry and the ‘pathophysiologist’ insisting on the distinctiveness of living processes had, by working together, advanced knowledge. Thus, the unification of perspectives that we seek had an important precedent. But the underlying tendency of these viewpoints to diverge soon reasserted itself. Baillie (1793) stated that reduced blood flow after obstruction of the inferior vena cava leads to thrombosis. Later investigators insisted that the loss of ‘motion’ or ‘vitality’ caused blood to coagulate (e.g. Thackrah 1819; Stevens 1832). Maybe the notion that ‘stasis’ has a causal role in DVT was born, or re-born, in the work of this period18 rather than in Boerhaave’s writings per se; but we should not lose sight of its Galenist roots (Chapter 8).

17 As we might see the matter today, stasis may cause blood to die should it be sufficiently prolonged (i.e. should re-oxygenation of the blood cells cease in static conditions). Hunter (1794) wrote ‘It is not rest considered simply, but rest under certain circumstances, which appears to possess such a power.’ But the conclusion of Hewson and Hunter – that static blood in vivo does not coagulate – normally cannot be gainsaid. 18 As far as we can determine, ‘stasis’ was first related to venous thrombosis in the more modern literature by Homans in 1934; see Chapter 8.

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4.8

4 Historical Roots

Late 18th- and Early 19th-Century Studies of Blood Chemistry

From the time of Priestley and Lavoisier, new analytical techniques such as destructive distillation, the complete conversion of organic matter to carbon dioxide and water, began to lead to marked advances in animal chemistry. Salient among these were the contributions of Jöns Jacob Berzelius (1779–1848) and William Prout (1785–1850); in the following generation, the work of Wöhler and Liebig was prominent. As we observed earlier in this chapter, the growth of knowledge is often driven by new techniques. Analysis of blood remained high on the agenda of many animal chemists of this period, including both Berzelius (1812) and Prout (1819). The analysis of blood clots given by Berzelius directed the attention of many chemists to the coagulation process, though it is difficult to interpret in modern terms. The importance of blood chemistry for an understanding of pathology came to be recognized; Rees (1836) remarked: ‘The philosophical revival of a humoral pathology bids fair to render the analysis of diseased blood one of the most useful adjuncts to our medical knowledge’. The further evolution of chemical methodology sustained the impetus towards analysis of blood, towards understanding the coagulation mechanism and towards elucidating the mechanistic basis of blood-related diseases. We shall continue this story in the next chapter. It culminated in our modern understanding of coagulation and the consensus model of DVT aetiology. The benefit of the new chemical approach to what was to become ‘haematology’ is beyond question. The cost was the eventual divorce of this mechanistic program from work being undertaken in parallel from the ‘pathophysiological’ standpoint. Perhaps paradoxically, many of these early animal chemists did not accept the mechanistic metaphysic, at least not wholly; several of them were vitalists or included vitalistic elements in their writings. Some of the publications by Stevens (see above), and even Liebig, suggest vitalism. This makes it particularly misleading to equate proponents of the pathophysiological approach with ‘vitalism’ and proponents of the mechanistic approach with ‘mechanism’. The historical reality is more diverse.

4.9

The 18th-Century Pioneers of Haemostatics and Haemodynamics

Work informed by the ‘physiological’ approach during the 18th century significantly advanced our understanding of the physics of the circulation. These were presaged a few years after Harvey’s death in a remarkable study of the physics of muscle contraction, De Motu Animalium by Giovanni Borelli (1608–1679), which made mechanical sense of Harvey’s insight that the work of the heart is performed

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during systole, not during diastole as the Galenists had taught19 (see Chapters 2 and 3 in Foster 1901). An English parson, Stephen Hales (1677–1761), determined the heart capacity, stroke volume and cardiac output of a sheep by experiment. He made a wax cast of the chambers of the heart while it was still beating and calculated the cardiac output from the volume of the cast and the pulse rate. He also performed the first measurement of blood pressure, using the height of the column of blood in a glass tube inserted into an artery. His collected writings were published posthumously (Hales 1773). He considered his work to be an extension of Harvey’s, though it could reasonably have borne the label ‘iatrophysics’. Hales also investigated the reason why breathing stale air was dangerous, a study that was extended by Viktor Albrecht von Haller (1708–1777), a student of Boerhaave but also strongly influenced by Harvey. Haller (1786) clarified the relationship between respiration and blood flow. He also placed the relationship between heart action and blood flow on a quantitative basis. Thus, Hales and Haller laid the foundations of modern haemodynamics. There was no further major development in this field until Etienne-Jules Marey (1830–1904) invented the sphygmograph. When Lavoisier’s studies of respiration were published towards the end of the 18th century, the basis of our understanding of the relationship between respiration and circulation was also in place. (Lavoisier was executed during the Terror in 1794, as a member of the Farmers-General.) Granted the integral importance of blood circulation and ‘local flow’ in thrombosis, these haemodynamic studies and their modern-day counterparts (investigations of rheology) may be as relevant to the topic of this book as the mechanism of coagulation. We shall return to this theme in Chapters 8 and 9.

19

Borelli communicated regularly with Malpighi, as Foster (1901) observes.

Chapter 5

Coagulation and its Disorders: A History of Haematological Research

Abstract Studies of blood coagulation had originated among the ‘animal chemists’ of the 18th and early 19th centuries (Chapter 4), but were placed on a firmer footing in the 1830s by Andral’s pioneering compilation of medical knowledge and by Buchanan’s experimental studies. The history of blood coagulation research from the time of Andral and Buchanan to the present day can be arbitrarily divided into four phases. The third and fourth phases, covering the period between the Second World War and the present, were explored in Chapter 2. This chapter concerns the first two phases. Phase 1 extended from Buchanan’s publications to the maturation of Schmidt’s ‘classical hypothesis’: the action of thrombin was identified and the existence of prothrombin and antithrombins was inferred. These advances were underpinned by a philosophical movement; mechanistic materialism marginalised the role of cells in blood function and focused attention on the soluble components of the plasma. Phase 2, covering roughly the first half of the 20th century, saw the identification of prothrombin and an increasing number of coagulation factors, and the discoveries of heparin, vitamin K and dicoumarol. The mechanistic-materialist character of coagulation research became entrenched. At the end of the chapter, we discuss the origin of this philosophical movement in a schism that took place around 1847. This schism primarily involved du Bois Reymond, the leading protagonist of mechanistic materialism, and Virchow, the pioneer of cellular pathology and exponent of the ‘pathophysiological’ approach to biomedicine. Keywords Classical hypothesis of blood coagulation, haemophilia, in vitro studies, mechanistic materialism, thrombin

5.1

Introduction

Although Hewson has been dubbed the father of haematology, Gabriel Andral (1797–1876) founded the discipline.1 In his book on diseases of the blood, Andral (1843) introduced the terms anaemia and hyperaemia and named a range of 1 The word ‘haematology’ seems first to have been used by a Dutch physician, Thomas Schwencke, a disciple of Boerhaave, in the first half of the 18th century.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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pathological conditions including polycythaemia and septicaemia. More significantly for our purposes, he suggested that certain diseases might result from ‘excessive coagulation’. However, few of his successors2 took up the suggestion before the second half of the 20th century. This protracted near-silence is striking in view of the enormous influence of his five-volume Clinique Médicale, which long remained the encyclopaedia of French medicine. It implies that for more than 100 years after his pioneering publication, haematologists were mostly unconcerned by conditions of ‘excessive coagulation’. The history of haematology from Andral’s time to the present day can be divided into four unequal phases. The first phase began circa 1835 and encompassed the latter two thirds of the 19th century before culminating in Schmidt’s thrombin hypothesis. The concern throughout this phase was to explain the blood clotting process and thus, provisionally, the mechanism underpinning haemostatic plug formation. The second phase started in 1892 and lasted until the end of the Second World War. It was driven by the quest to explain and treat haemophilia, and among its achievements were the discovery of heparin, the growth of in vitro biochemical techniques, and the earliest ‘coagulation cascade’ concept. These two phases constitute the subject of this chapter. The third phase, from the Second World War to the early 1960s, saw the maturation of the ‘cascade’ concept as the basis for explaining bleeding diatheses. The fourth phase began in 1962–1963, when the consensus model of DVT took its lead from the elucidation of blood coagulation. We considered these two phases in Chapter 2. Phase I was marked by the schism in biomedical thought that divided the ‘mechanistic’ from the ‘pathophysiological’ (‘vital-materialist’) approaches. We shall discuss it in some detail here. The developments during phase II placed haematological research clearly on one side – the ‘mechanistic’ side – of the divide. Chapters 6 and 7 will deal with the parallel ‘pathophysiological’ strand in the investigation of DVT.

5.2

Phase 1: 1835–1893

In Chapter 4 we outlined the late 18th- and early 19th-century developments that brought new techniques of ‘animal chemistry’ to bear on the physiology and pathophysiology of blood coagulation. Although contemporaneous physicians viewed chemical research with suspicion, the new trend fostered an increasing interest in haemopathology, as Andral’s 1843 publication testifies. For example, Roupell (1833) described changes in the blood of cholera victims during his second

2 These few exceptions included Trusseau, Nygaard and Brown (see Chapter 3); there may have been others but we are not aware of them.

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Croonian lecture, writing: ‘it is to a more exact acquaintance with the chemical changes of the fluids in diseases that we are chiefly to look for the future advancement of physic as a science’. In France in particular, blood chemistry was placed on a more secure footing during the second quarter of the 19th century. However, interest in the chemistry of coagulation seems initially to have been a German concern, and later a predominantly British one.

5.2.1

Haemophilia and the Study of Coagulation

Cases of unnatural bleeding from slight injuries were first annotated in early 19th-century reports of haemorrhagic conditions afflicting sons whose mothers and sisters were unaffected. The term ‘haemophilia’ is attributed to Hopff in 1828 (Bulloch and Fildes 1911) and originally denoted excessive bleeding irrespective of aetiology. The notable hereditary haemorrhagic condition suffered by Queen Victoria’s male offspring, and carried by her female children to the royal families of Europe, probably fostered intensive investigation of haemophilia. It was recognised as a specific clinical entity3 by the early 20th century and the denotation of the word then became more restricted (Addis 1910; Bulloch and Fildes 1911). However, in the early 19th century, the phenomenon served to increase the interest of chemists in the coagulation process per se. Thackrah and others maintained that coagulation resulted from chemical changes caused by exposure of the blood to air (see Chapter 4); in other words, it was considered a negative phenomenon, a chemical process of ‘loss’ or ‘deterioration’.

5.2.2

Buchanan

The revolutionary idea that a specific component of the blood causes or positively initiates coagulation was first advanced by Buchanan (1835) when he was a junior surgeon at Glasgow Royal Infirmary. He published a second and more detailed paper (Buchanan 1845) when he was Professor of the Institutes of Medicine in Glasgow. Essentially, his experimental model was to mix blood and water in vitro and allow them to clot; he then strained the clot through a linen cloth and called the strained material ‘washed clot’. He shredded the ‘washed clot’ and added fragments

3 After this time, the word was applied only to cases in which the blood clotting time was prolonged, there was no obvious explanation for the phenomenon, and the sufferer was a male whose mother and sisters were clinically normal. Later, during what we have termed ‘phase 3’ of this history, increasing numbers of coagulation factors were recognised (Chapter 2) and the term became still more narrowly restricted. Advances in knowledge are generally associated with refinements of terminology.

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to various serous fluids. He observed the ‘clotting’ of hydrocoele fluid (which had not previously been known to ‘clot’) and inferred that the coagulant power of the blood inheres in the ‘colourless corpuscles’. ‘Colourless corpuscles/globules’ (leukocytes and, possibly, platelets) had been recognised by Fordyce in 1791 (see Chapter 4). However, microscopy had become much more trustworthy by 1845 than it had been in last decade of the 18th century, when Hunter had warned his readers against placing too much reliance ‘on images seen through magnifying glasses’. The introduction of J.J. Lister’s achromatic lens in 1827 had revolutionised microscopy (Davidson and Abrimovich 2002) and led directly to the conception of cell theory, which was being articulated in France and Germany4 when Buchanan was conducting his experiments. Donné perhaps observed platelets5 in 1842–1844 (see Bang 1982), but they had not been named by 1845. However, as happens in unprejudiced early writings, Buchanan (1845) made prescient comments, one of which might suggest that he saw platelets (without naming them): ‘On discovering the efficacy of ‘washed clot’ [fragments] in causing coagulation, I thought it probable that minute solid particles, which the microscope never fails to detect in blood serum, were the agents to which the coagulation of blood and hydrocoele ‘serums’ [mixed] ought to be ascribed’ [our emphasis]. Buchanan, no less than Andral, had combined clinical, microscopic and chemical observations to notable effect.

5.2.3

Buchanan’s Influence: The Impact of Mechanistic Materialism

In his remarkable paper ‘On the early stages of inflammation’ (1858) and his brilliant Croonian lecture ‘On the coagulation of the blood’ (1863), J.L. Lister both acknowledged Buchanan’s priority and noted the intrinsic difficulty of such work (‘… has engaged the best energies of very able men’). He repeated and confirmed

4 Most textbooks attribute cell theory to Matthias Schleiden in 1838 and Theodor Schwann in 1839, but the cell concept was established by François-Vincent Raspail (1794–1878) in 1827– 1828. Raspail asserted that all animal as well as plant tissues are made up of cells. He also believed that disease processes arose from pathology at the cellular level, and in this respect he anticipated Virchow by almost three decades; indeed, he is the first person known to have used the phrase omnis cellula e cellula, as early as 1825 – though this phrase may owe more than a little to Harvey’s omnis viva ex ovo. Schleiden and Schwann were aware of Raspail’s pioneering discoveries but held them in contempt: an illustration of the antipathy between French and German scientists that persisted throughout much of the 19th century. Famously, the philosopher Herder had advised German academics to ‘Spit the green slime of the Seine from their mouths’. 5 Several descriptions and alternative names of platelets were published during the following 30 years (Poole 1964). The greatly improved oil immersion microscopes available to e.g. Bizzozero (1882) clarified their role in haemostasis and thrombogenesis. Welch (1887) noted that platelet congregation preceded fibrin formation, and Aschoff (1924) concurred. We shall discuss this strand of history further in Chapter 7.

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Buchanan’s 1845 experiments and wrote: ‘… if a portion of ‘washed coagulum’ is put into hydrocoele serum, a web of fibrin is gradually spun around the coagulum’. (We have italicised the phrasing because it implies that the coagulum either preceded or perhaps coincided with secretion and spinning of the ‘web’.) These inferences were impressive at a time when the nature of proteins was unknown, enzymology was yet to be founded, and neither phagocytosis nor platelet function had yet been named. Lister recognised that coagulation is induced when blood comes into contact with a foreign surface, an observation also made by Brücke (1857). Buchanan’s pioneering work was acknowledged by his near-contemporaries Brücke (1857), Schmidt (1861, 1862) and Foster (1864) as well as Lister, but the following generation seemed to lose touch with it (see e.g. the historical survey by Bang 1982). It seems that the physiological – cellular – interpretations of Buchanan’s and Lister’s findings came to be overlooked, perhaps reflecting the controversial but highly influential mechanistic-materialist physiology advocated by du Bois-Reymond and his followers in 1847 (Cranefield 1957). The upshot was that while the pioneering work of Buchanan, Lister and fellow-thinkers had provisionally attributed fibrin formation and coagulation to living cell activity,6 Schmidt (1861, 1872), a committed mechanistic materialist, focused solely on the soluble chemical components of the plasma. Schmidt’s haematologist successors adopted his metaphysical stance. A few years later, the noted physician Sampson Gamgee (1879) felt it necessary to reproduce Buchanan’s 1845 paper in full in the Journal of Physiology, as though to highlight a forgotten story. His criticism of Schmidt was intriguing: ‘The very remarkable researches of the emeritus Professor of Physiology in the University of Glasgow – Dr Andrew Buchanan – on the Coagulation of the Blood have not received the acknowledgment or attention they deserved [our emphasis]. Most physiologists acquainted with the older scientific literature are aware that Buchanan pointed out 30 years before Professor Alexander Schmidt that the fluids of certain serous sacs – especially hydrocoele fluid – which do not coagulate spontaneously – may be made to yield a coagulum of fibrin by the addition of blood or serum’. Gamgee conceded that Schmidt’s ‘fibrin-ferment’ might be identical to the ‘rennetlike substance’ posited by Buchanan, but he remained doubtful about Schmidt’s position: ‘When in 1869 I delivered my first Physiology lectures in Edinburgh I gave an account of Buchanan’s and Schmidt’s researches, and pointed out that some of the facts of the former were irreconcilable with the doctrine [of the latter] that the coagulation of fibrin depends merely on the union of two individual factors previously existing in solution’. In a footnote, he quoted in German from Schmidt’s article to justify his interpretation of Schmidt’s conclusion: viz. that two soluble ‘proteids’, paraglobulin and fibrinogen, combine to produce fibrin.

6

Lister, like Thackrah (1819; see Chapter 4), concluded from his studies that the ‘loss of vitality’ of the vessel wall was crucial for coagulation in situ (i.e. thrombosis). A dead or seriously sick vascular intima behaved like a foreign surface and triggered the semi-solidification of the blood.

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Foster (1864) was similarly critical of Schmidt’s experiments. In a detailed review of four contemporary studies of coagulation he judged that Schmidt ‘had corroborated Buchanan’s findings but not his theories’ [our emphasis], and later wrote: ‘The fact is beyond cavil. But how is it to be explained?’ The ‘fact’ is that referred to above: dropping a little blood into hydrocoele fluid initiated local coagulation/clotting where the blood lay. Foster went on: ‘So far the researches of Dr Schmidt are very satisfactory. … Unfortunately they lack a happy and culminating issue. It is seldom if ever that coagulation can be obtained by mixing two weakly alkaline solutions of fibrinogen and globulin. The artificial factors seem to have lost energy during their preparation. The best coagulation is obtained working with natural factors … the next best with one natural the other artificial, and worst of all when both are artificial …’. Here, ‘artificial’ may be read as ‘(semi)-purified’. However, on p. 170, Foster wrote: ‘This hypothesis of Schmidt of course requires further investigation. In many points, which need not be entered on, it is unsatisfactory. … Brücke [a colleague of du Bois-Reymond from the 1840s and a fellow member of the Berlin Physical Society] is of the opposite opinion’. And later: ‘Granted that the process is such as has been described, how does it come to pass that globulin and fibrinogen do not unite to form fibrin within the living body?’ This last question was to exercise the best minds in the field for many years. Foster clearly reflected the philosophical conflicts arising from mechanistic materialism, which was characterised by the denunciation of other (past and contemporaneous) medical-biological viewpoints as ‘vitalist’. From the standpoint of the early 21st century, it is easy to underestimate the consequences of that philosophical ‘new beginning’ 160 years ago. Even Lister (1863) felt obliged to defend his Thackrah-like conclusion that ‘loss or lack of vital properties’ (either in the living body or in its dead or never-living environs) was the common factor leading blood to coagulate both in vitro and in vivo. He stated explicitly that he was not promoting belief in ‘vital forces’ but insisted, echoing Hunter, that ‘living matter has properties distinct from, albeit not inconsistent with, those that are traditionally addressed by physicists’. His point, and Foster’s, especially their use of the word ‘vital’, is readily misconstrued nowadays.7

7 A few years earlier, Pasteur had triumphed in his argument with Liebig about the nature of fermentation – another instance of 19th century Franco-German conflict. Liebig, though he had trifled with vitalist notions, insisted that fermentation was a purely non-living, chemical process; Pasteur retorted, and successfully proved, that it was a living process requiring intact organisms (yeasts). Lister’s view of blood coagulation was therefore ontologically similar to Pasteur’s view of fermentation; and it would seem the height of absurdity to infer that Pasteur believed in a ‘vital force’ – which notion, indeed, his subsequent experimental disproof of spontaneous generation did much to undermine.

5.2 Phase 1: 1835–1893

5.2.4

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The ‘Classical Hypothesis’ of Blood Coagulation

Such protestations availed little. Schmidt’s mechanistic conclusions were promulgated as the ‘classical theory (more correctly, hypothesis) of blood coagulation’ 20 years later (Schmidt 1892). He isolated fibrinogen from plasma by salt precipitation and ‘fibrin-ferment’ from serum by alcohol precipitation. He also found evidence suggesting the existence of antithrombins. ‘Fibrin-ferment’ caused the purified fibrinogen to coagulate and had a similar effect on hydrocoele fluid, but it could not be extracted from plasma. Since no ‘fibrin-ferment’ (later re-named ‘thrombin’) could be obtained from tissue extracts,8 the most plausible hypothesis seemed to be that it is formed during coagulation from a precursor in the plasma: Prothrombin ⎯?⎯ → Thrombin ⎯⎯⎯ → Fibrin Fibrinogen ⎯Thrombin Tissue extracts were presumed to contain a factor or factors that converted the then-hypothetical prothrombin to thrombin; this factor appeared to be alcoholsoluble and resistant to boiling. Not until the years immediately preceding the First World War (see Section 5.3) did further details emerge, but the philosophical orientation of the ‘classical hypothesis’ was clear from the outset. The mechanisticmaterialist perspective was explicit in the account of the thrombin hypothesis by Morawitz (1905). The findings that fibrinogen levels rise after trauma (Foster and Whipple 1922) and platelet levels rise after surgery (Evans 1929), concomitantly with an increased likelihood of DVT, helped to entrench this approach. Arthus and Pagès (1890) discovered that calcium was necessary for coagulation. It was quickly shown that it is not directly involved in fibrinogenesis, but none doubted that it was a cofactor in a biochemical reaction. The test tube-based, biochemical discipline that developed over the ensuing 50 years, and matured into the cascade model after the Second World War, was thus rooted in the mechanistic materialism of 1847. By the time Schmidt’s later papers were published, many physiologists had distanced themselves from the extreme ‘physicalist’ stance of du Bois Reymond and his colleagues (Cranefield 1957). However, the same metaphysic underpinned its successor, biochemistry, which aimed to reduce the phenomena of life to the language of chemistry instead of Newtonian mechanics. Both classical biochemistry and modern biochemically orientated haematology are rooted in du Bois-Reymond’s failed metaphysic.

8 In view of this history of discovery, the term ‘thrombin’ seems misconceived. Notwithstanding the commonly assumed desirability of Greek etymologies, the chemical preparations had been obtained from clots, not thrombi; so should ‘thrombin’ have been ‘clottin’? Quite possibly Schmidt and other mechanists were unaware of the basis of Virchow’s careful nomenclature, by then almost four decades old (see Chapter 6).

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5.3

5 Coagulation and its Disorders: A History of Haematological Research

Phase 2: 1893–1947

When the word ‘haemophilia’ was given its first clear clinical definition, Addis (1910) showed that addition of a small amount of normal plasma to haemophilic blood in vitro corrected its characteristically prolonged clotting time. Haemophilic blood was thus shown not to be deficient in either thrombin or fibrinogen and the hunt was on for the missing plasma component. Although the contemporaneous haematological literature emphasised this concern, it said little or nothing about thrombosis.

5.3.1

Prothrombin and its Conversion to Thrombin

Mellanby (1909) isolated the putative prothrombin from plasma, and Bordet and Delange (1914) found that it could be precipitated by calcium phosphate, leaving the plasma incapable of clotting. Nolf (1928) used the calcium phosphate technique to purify the precursor, and during the following two decades others refined the method. Eagle (1935) demonstrated that the conversion of the purified precursor to thrombin was stoichiometric. Seegers et al. (1945) achieved more complete purification, and evidence began to accumulate that the final step in prothrombin activation was proteolytic. Thus, ‘prothrombin’ was transmuted over a period of many years from hypothetical concept to chemical reality; but the problem of how and under what circumstances prothrombin is converted to thrombin remained a major challenge throughout this period. Few scientists doubted that the challenge would be met only by means of increasingly refined biochemical analysis. However, this proved a far from straightforward matter. Some investigators assumed that the still-mysterious ‘tissue factor’ acted directly on fibrinogen to produce fibrin; others adopted the complementary hypothesis, that prothrombin was secreted by the tissues. A prothrombin precursor, ‘serozyme’, was postulated to account for certain experimental findings (Bordet and Delange 1912, 1913), but no such precursor could be found. It was doubted whether thrombin alone could suffice to convert fibrinogen to fibrin. As late as the 1930s, some workers were persuaded that glycolysis and its products were directly involved in blood coagulation. Howell (1935) and Eagle (1938) reviewed these developments in detail. Intensive investigation of a complicated process invariably generates red herrings, and though history accords them scant regard, some are productive. The suggestion that thrombin activation is prevented in normal blood by heparin binding to prothrombin led to experiments in which plasma levels of circulating heparin were measured in healthy and ill subjects. Valuable information was gained even though the hypothesis was refuted (Hellsten 1942). Similarly, although the hypothetical ‘serozyme’ was also rejected, the demonstration en route that paraffin-coated glass does not initiate clotting enhanced our understanding of coagulation. False hypotheses make way for further hypotheses and further advances – provided only that they are experimentally refutable.

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Cramer and Pringle (1913) showed that the removal of all cells and cell breakdown products from mammalian blood in vitro delayed coagulation considerably. This finding seemed at first to herald a resurgence of interest in the role of cells in coagulation; in the event, however, it reinforced the drive to identify the biochemical composition of the hypothetical ‘tissue factor’. Bordet and Delange (1914) found the first evidence that the ‘tissue factor’ was, or contained, phospholipids. A year earlier, these same authors (Bordet and Delange 1913) had tried to explain the involvement of platelets in haemostasis9 by isolating a ‘thromboplastic’ factor from them. They believed the platelets served primarily, if not entirely, as a source of this factor – another false-butproductive idea.

5.3.2

Heparin, Vitamin K and the Dominance of in Vitro Studies

The divergence between the in vitro and in vivo approaches, and the growing preeminence of in vitro studies, was intensified after heparin was discovered (McLean 1916) and plasma proteins were implicated in its action (Howell and Holt 1918). During the period 1831–1918, prevention of clotting in vitro without chemically ‘killing’ (poisoning) the blood was achieved by (a) Whipping the blood and removing the ‘white material’; (b) Carefully (i.e. slightly) salting with a calcium chelator such as citrate or oxalate; (c) Cooling to close to 0°C. But the discovery of heparin greatly expanded the ambit of investigation. Not only did the mode of action of heparin require elucidation, leading to a highly productive line of inquiry into oral and systemic anticoagulants, but it was another major turning point in blood research. A new generation of laboratory bench experiments, using unsalted blood in test tubes, further demoted in vivo physiological studies and put progressively greater emphasis on in vitro approaches. It went virtually unnoticed that the burgeoning study of blood clotting in vitro deflected attention ever further from thrombosis in vivo. Regrettably, there was, and is, no Rosetta Stone that enables us to translate the results of in vitro clotting experiments into an explanation for in vivo thrombosis. The discoveries of streptokinase10 (Tillett and Garner 1933) and vitamin K (Dam et al. 1936) confirmed that chemical and biochemical evidence could inform our

9 See, for instance, the papers by Bizzozero and Welch cited earlier. Aschoff (1924, p. 278) noted that platelets remained mysterious in the 1920s: ‘But when it became practically certain that a thrombus takes origin exclusively from blood-platelets, uncertainty again arose, since the origin and characteristics of these platelets was veiled in obscurity ….’ We will discuss this work fully in Chapter 7. 10 It was to be a further two decades before the first definitive therapeutic use of thrombokinase was published: Tillett et al. (1955).

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understanding and treatment of clinical conditions. Quick (1937) explained the haemorrhagic tendencies of patients with obstructive jaundice in terms of defective fat absorption combined with vitamin K malabsorption and consequent haemostatic failure. Waddell et al. (1939) went on to explain haemorrhagic disease of the newborn in terms of poor vitamin K absorption, and Huebner and Link (1941) purified dicoumarol and identified its mode of action. These achievements inspired investigations into the mechanism of action of vitamin K. At about the same time, Murray et al. (1937) pioneered the clinical use of heparin as an anticoagulant. The scientific and clinical applications of these studies, and the therapeutic success of heparin and dicoumarol and its derivatives in thrombosis, acted to marginalise in vivo studies still further. New haemostatic factors were being identified all the time: the existence of the von Willebrand factor was inferred in the early 1930s (von Willebrand 1931), and Patek and Taylor (1937) partially purified ‘anti-haemophilia globulin’, later to be renamed factor VIII. ‘Antiplasmin’ was described by Christensen and MacLeod (1945). The idea of a ‘cascade model’ began to be explored (e.g. MacFarlane 1941). The biochemical approach to bleeding diatheses was thus entrenched before and during the Second World War. The paradox was that elucidation of the haemostatic mechanism, as it related to haemophilia, had effectively eliminated thrombosis as an issue for research among neophyte haematologists; yet as we saw in Chapters 2 and 3, post-War haematologists extrapolated their haemophilia-inspired thesis to explain thrombosis as well.

5.4

Philosophical and Semantic Considerations

Although mechanistic accounts of biological processes seek to be exclusively physico-chemical, their vocabulary is loaded with intentional terms. Examples include nouns such as ‘control’ and ‘recognition’, verbs such as ‘activate’ and ‘encode’, and adjectives such as ‘regulatory’ and ‘stimulatory’. Many of these words are borrowed from engineering, not physics or chemistry, and engineering concerns objects that are explicitly (by design) intentional.11 Any such word can be ‘translated’ without residue into a combination of physico-chemical and evolutionary definitions and propositions, so intentional language in biology and medicine is not irreducibly teleological (Agutter and Wheatley 1999). However, no one ‘translates’ such words at every usage. The adage ‘language creates thought’ presumably applies to workers in this field as it does to humans in general. Routine, and ipso facto unreflecting, use of intentional vocabulary carries implications for practical biology and medicine as well as for metaphysics. The account of coagulation that we accept today describes mechanisms in great detail but ignores the inherent intentionality of living cells. For example, current 11 Here, we echo philosophers of biology such as Mayr and Monod. We emphatically do not imply, or accept, the specious and insidious pseudo-arguments for ‘intelligent design’.

5.5 Reflective Anamnesis

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beliefs about the function of platelets in haemostasis make no explicit reference to their behaviour as living cells; we find only a flow chart of molecular components (Chapter 2, Fig. 2.1). The idea of the platelet as one of an ‘enormous number of centres of activity’, as Virchow phrased it, has been lost under a welter of biochemical detail. But if cells are the ‘fundamental units of life’, and living organisms are intentional, then a living cell is intentional; so if a platelet is a cell, a platelet is intentional. A mass of interacting biochemicals is not intentional. Thus, the mechanistic account lacks something. As Lister and other 19th-century workers perceived (see above), the something is ‘livingness’. Since we are discussing an aspect of the science of life, the lack is not trivial. No one would doubt that the details of blood coagulation elucidated over the past two centuries are profoundly relevant to understanding the formation and growth of thrombi. However, along with Hunter and Virchow, we contend that DVT results from a living process. Thrombi do not form in dead things (see Chapter 12). In so far as the consensus model of DVT is rooted in mechanistic haematology, and mechanistic haematology suppresses the notion of ‘livingness’, we infer that the consensus model is philosophically insufficient. To be more specific, the predominantly haematologist proponents of the mechanistic consensus during the past century seem, from a pathophysiological viewpoint: (a) To have been culpably uninformed about the differences between clots and thrombi that Virchow spelled out in his 1856 textbook – the ‘triad’ for which he was responsible, as distinct from the ‘triad’ retrospectively attributed to him; (b) To have relied excessively, and too often uncritically, on evidence from in vitro clotting; (c) Not to have been professionally fitted to discover histological, anatomical and physiological details; (d) To have marginalised Virchow’s discovery that thrombi form in circulating, not static, blood; (e) To have imagined that Virchow postulated ‘hypercoagulability’ as a contributing cause of venous thrombogenesis; (f) To have ignored (as irrelevant?) the microscopic structure of venous thrombi; (g) To have overlooked the finding that venous thrombi form in valve pockets and are anchored on the valve cusp leaflets. During the remainder of this book we shall attempt to address these shortcomings, without losing sight of the contribution to our understanding of DVT that accompanied the elucidation of the coagulation mechanism.

5.5

Reflective Anamnesis

This chapter has been concerned with a century’s growth of one strand of knowledge – the mechanistic, haematological strand – which would, after the Second World War, come to be related retrospectively to DVT. As we have seen, a cellular

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5 Coagulation and its Disorders: A History of Haematological Research

(potentially ‘physiological’ and ‘pathophysiological’) dimension to this strand was adumbrated in Buchanan’s studies, but was subsequently suppressed because of the academic dominance of mechanistic materialism, which is particularly apparent in the work of Schmidt and Morawitz. However, another centuries-old strand of thinking co-existed with the mechanistic materialist viewpoint, and was – and remains – directly relevant to the aetiology of DVT: the pathophysiological, ‘vital-materialist’ tradition, of which Virchow was the outstanding exemplar. These two conflicting ideologies constitute the 160-yearold bio-scientific schism that we hope this book will help to resolve. Early in the 19th century, while Andral was assembling his prescient work on haematology, Cruveilhier was pioneering the study of pathological anatomy. Later in the century, while Schmidt was articulating his (now ‘classical’) hypothesis of blood coagulation, Welch was undertaking his remarkable microscopic studies of thrombi. In the middle of the century, soon after Buchanan’s second paper appeared (1845), Virchow began his classical work on thrombosis and embolism (1846; published 1856). Chapter 6 will focus on the contributions of Cruveilhier and (especially) Virchow. Two anni mirabiles stand out in that century of biomedical progress. In 1857– 1858, Virchow published Die Cellularpathologie, Pasteur published the germ theory of disease, and Lister re-thought the subjects of ‘inflammation’ and ‘coagulation’ in two great papers. Together, these publications had a huge effect. They transformed knowledge, opinion and practice from a quasi-mediaeval state to what we may recognise as a ‘modern’ one. Everything that followed was changed utterly. The great biomedical schism of 1847–1848, to which we have adverted several times, had occurred 10 years previously. In the Berlin of the 1840s, Emil du Bois Reymond (1818–1896) recruited a group of like-minded young physiologists opposed to any suggestion of ‘vital force’. In its original form, their philosophy – mechanistic materialism – held that all valid facts about biology and medicine were amenable to experimental investigation and expressible in the language of physics.12 This was more than an abstract intellectual commitment. Members of the Berlin

12

It is important to understand the beliefs implicit in mechanistic materialism. The Berlin Physical Society, founded in 1845, was originally dedicated to explaining all aspects of life (including mental processes) in terms of Newtonian mechanics. Du Bois Reymond and his colleagues were particularly concerned to apply this approach to physiology. At the time, Dalton’s atomic theory was only 40 years old and modern understanding of chemical bonds did not exist. However, the notion of (thermal) atomic movement had been grasped; it was to underpin kinetic theory and, later, statistical mechanics. This random shivering of atoms occurs in non-living (and dead) as well as living objects – an idea that had in principle been grasped by Boerhaave (see Chapter 4). But there is an obvious, and massive, difference between the living and the dead, which is the motion or stillness of their body water. Convective water movement is co-extensive with life. The mechanistic materialists dismissed this ‘vitalist’ fact and sought to explain physiological processes in terms of principles that apply equally to dead and to living matter. It is therefore, fundamentally, a philosophy of dead or non-living material rather than a ‘philosophy of life’. In so far as classical biochemistry – and a fortiori biochemical haematology – is the metaphysical offspring of the Berlin Physical Society, the same inference applies to it. See the appendix for further discussion.

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Physical Society were, like Virchow, passionate and highly politicised, deeply involved in the nationalism that was burgeoning in the German states as the country approached unification. This may explain why their pronouncements tended to be polemical, even vitriolic. They were specifically concerned to exorcise ‘vitalist’ thought, which they associated with French influence, from chemistry and other sciences. However, their antagonism extended to any discourse that was not explicitly constrained by physico-chemical language, as mirrored in Lister’s defensive remarks quoted earlier. Although the original project of mechanistic materialism had failed within a generation (Cranefield 1957), its legacy had profound effects on the development of biology and medicine and its influence was to persist. A highly significant historical fact is that du Bois Reymond and his fellow mechanistic materialists invited Virchow to join their Berlin Physical Society. A no less significant fact is that he declined. Presumably he realised that were he to acquiesce, that would falsely align him with their metaphysic. Both Virchow and du Bois Reymond were political radicals, present at the barricades in Berlin during the political disturbances of 1848, but in respect of scientific philosophy they were poles apart – though both repudiated the notion of ‘vital force’. Both had been students of Müller,13 but while Virchow adhered to Müller’s philosophy throughout his working life, du Bois Reymond reacted against it. Virchow’s intent to promote his world-view was implicit in the Archiv für pathologisches Anatomie und Physiologie, und für klinische Medizin (subsequently known as Virchows Archiv), in which he encouraged publications on the frontiers of scientific medicine (Bartlett 1933). He founded it in the very year (1847) that the mechanistic materialists institutionalised their frame of reference within the Berlin Physical Society. Yet Virchow was aware that unless the two traditions of thought that he and du Bois Reymond represented were treated as complementary rather than mutually antagonistic, the progress of scientific medicine would be impaired. This is explicit in his preface to Die Cellularpathologie in ihrer Begründhug auf physiologische und pathologische Gewebenlehre (Cellular Pathology as based on Physiological and Pathological Histology = ‘pathophysiology’; Virchow 1858), a collection of lectures delivered in Berlin after his appointment to the Pathological Anatomy chair. In this preface he stated his intention: [T]o offer in a better arranged form than had hitherto been done, a view of the cellular nature of all vital processes, both physiological and pathological, animal and vegetable, so as distinctly to set forth what even the people have long been dimly conscious of, namely, the unity of life in all organised beings, in opposition to the one-sided humoral and neuristical (solidistic) tendencies that have been transmitted from the mythical days of

13 Johannes Peter Müller (1801–1858) attained his chair in Berlin in 1833. An outstanding teacher, he conducted extensive studies in many fields of biology and medicine: comparative anatomy and embryology, marine zoology, and – a particular influence on Virchow – general and microscopic pathology. Among his other achievements, he advanced new theories of colour vision and voice production, inspired by Goethe. His neurological studies led to the ‘doctrine of specific nerve energies’, i.e. the premise that each type of sensory nerve produces its own specific sensation.

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5 Coagulation and its Disorders: A History of Haematological Research antiquity to our own times, and at the same time to contrast with the equally one-sided interpretations of a grossly mechanical and chemical bias – the more delicate mechanism and chemistry of the cell. … I find … that the physicians of antiquity had not in all cases their senses shackled by traditional prejudices. … What should hinder me from avowing that the criticism of the learned has not always proved correct, that System has not always been Nature, and that a false interpretation does not impair the correctness of the fact?… Perhaps it is nowadays a merit to recognise historic rights, for it is indeed astonishing with what levity those very men, who herald forth every trifle, which they have stumbled upon, as a discovery, pass their judgement upon their predecessors. I uphold my own rights, and therefore I also recognise the rights of others. This is the principle I act upon in life, in politics and in science.

And in the first lecture of Die Cellularpathologie he resumed: According to my notions, the basis of both doctrines is an incomplete one; I do not say a false one, because it is really only false in its exclusiveness; it must be reduced within certain limits and we must remember that, besides vessels and blood, besides nerves and nervous centres, other things exist, which are not a mere theatre [Substrat] for the action of nerves and blood, upon which these play their pranks. [Our emphasis]

Chapter 6

Virchow and the Pathophysiological Tradition in the 19th Century

Abstract Chapter 5 traced the mechanistic materialist tradition of blood coagulation studies through the biomedical schism of 1847. This chapter follows the alternative (pathophysiological or ‘vital-materialist’) tradition from the 1830s to the late 19th century, focusing mainly on Virchow’s contribution to our understanding of venous thrombosis. The ‘doctrine of Cruveilhier’ is discussed and the use of the word ‘phlebitis’ is evaluated. Virchow’s life, times and philosophy are briefly reviewed, and we emphasise his familiarity with the work of Boerhaave and Hunter as well as Cruveilhier and the early 19th-century microscopists. Virchow effected a synthesis of these contributions to knowledge, demonstrated that pulmonary emboli originate by metastasis1 of distant venous thrombi, proved that thrombi and emboli form in moving not static blood, illustrated thrombi apparently anchored on the cusps of venous valves, distinguished clearly between thrombi and clots, and surmised that oxygen is required for thrombosis. We suggest that the ‘Virchow’s triad’ concept originated from a misapplication of his work, and we discuss his opposition to Cruveilhier and his philosophy of biology and medicine.

Keywords Cellular pathology, embolism, oxygen, phlebitis, venous valves

6.1

Introduction

Rudolf L. K. Virchow (1821–1902) passed his student years in Berlin during an exciting period in both European history and the history of biology. The 18thcentury Enlightenment fashion for ‘experimental philosophy’ had spread not only to the study of life, fostering inter alia the mechanism–vitalism debate, but also further into political and religious affairs (Merz 1928; Temkin 1946). The theory of ‘transformation’ (later to be dubbed ‘evolution’) was proposed in the early 1800s

1 This was Virchow’s term. The use of ‘metastasis’ has changed (become more restricted) since the 1850s.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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by Lamarck and Geoffroy St-Hilaire. It generated much debate in intellectual publications, with powerful political, religious and metaphysical overtones. New techniques and instruments such as J.J. Lister’s achromatic microscope brought a wealth of discoveries in their wake (Robb-Smith 1967).2 As we discussed in Chapter 5, major developments in medicine and haematology took place amid this ferment of ideas and methodological advances. Virchow’s work is characterised by energy and breadth of perspective. His subjects encompassed not only medicine and biology (to say nothing of his pioneering work in anthropology) but also social issues. He is now remembered mainly as the founder of cellular pathology, but in his lifetime he was noted for his liberal ideology and his concern with public health matters; his influence on German society in the late 19th century was considerable (Ackerknecht 1981). He was an avid social reformer, an implacable opponent of Bismarck; and in his later years, true to his principles, he refused elevation to the nobility, which had been offered in recognition of his fame and social standing. His public health concerns perhaps had their roots in his government-sponsored investigation of the typhus outbreak in Upper Silesia in 1848, but they were lifelong; on the Berlin City Council, from 1859, he urged reforms in public hygiene, meat inspection and hospital building. However, his conception of, and commitment to, cellular pathology during his Würtzburg years became the backbone of his contribution to medicine (Nuland 1988). Virchow’s starting points were the hypothesis that all pathological processes reflect malfunctions of cell physiology and the presumption that cells are living beings. Thus, like his predecessors Harvey and Hunter and his contemporary Lister, his stance in biology and medicine was very different from the ‘mechanism’ of Boerhaave and the mechanistic materialism of du Bois Reymond. Though many workers and thinkers in his intellectual environment (not least Schopenhauer) influenced his wide-ranging accomplishments, his principal contentions, as far as his studies of thrombosis and embolism were concerned, targeted the work of Jean Cruveilhier.

6.2

Cruveilhier

Cruveilhier’s ‘pathological anatomy’ dominated early 19th-century medicine. It was a major influence on Andral3 and ipso facto on the medical research community as a whole, and also an important influence on Raspail, whose pioneering ideas of cell theory and cellular pathology were mentioned in a footnote in Chapter 5.

2

J.J. Lister’s son, J.L. (later Lord) Lister, was in a uniquely privileged position during his boyhood: he had immediate access to the world’s best microscopes. He is said to have spent hours on beaches and cliffs during the 1830s and 1840s, microscope beside him. 3 Andral did not accept the dominance of ‘phlebitis’ on which Cruveilhier insisted. He saw that the blood and the solid organs of the body were complementary parts of an integrated whole, and he therefore considered that ‘haematopathology’ could not be sharply distinguished from general

6.2 Cruveilhier

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Cruveilhier (1791–1874) was a medical graduate of Montpellier, and was appointed Professor of Anatomy in Paris on the advice of the surgeon Guy Dupuytren in 1825, the world’s first chair in this subject. His knowledge of morbid anatomy was encyclopaedic and was encapsulated in his beautifully illustrated Anatomie Pathologique du Corps Humain (1829–1842), followed by his ‘Treatise on General Pathological Anatomy’ (1849–1864). His studies revealed that gastric acid was the cause of ulcers; he described a progressive muscular atrophy that is named in his honour (Cruveilhier’s disease); and he recognised congenital liver cirrhosis. He gave the first account of multiple sclerosis. He was particularly famed for the ‘doctrine of Cruveilhier’, the proposition ‘La phlébite domine toute la pathologie’ (Warren 1980), and he believed that what we now term ‘thrombosis’ resulted from injury to the venous endothelium (Duckworth 1913; Talbot 1970).

6.2.1

Previous Insights into Thromboembolism

By the time Cruveilhier wrote his treatise, it was well known that vein lumens could become occluded with what Hunter termed ‘coagulable lymph, pus and blood’ both in the immediate neighbourhood of surgical and traumatic wounds and at more distant sites (Abernethy 1793; Hunter 1794). Prior to Cruveilhier, however, there was confusion about the nature and cause of such occlusions. For example, Hull (1800) considered that the cause lay in ‘an inflammatory affection’ allowing ‘coagulating lymph’ to effuse into the affected tissues. The association of such occlusion with painful oedema was described independently by Bouillard (1823), Davis (1823), Lee (1829) and Lobstein (1833). It was generally associated with infection, though Ferriar (1810) had studied cases in which there was no obvious infection; both patients had been immobilised for long periods (The meaning of ‘infection’ in this context is not wholly clear.). Joy (1840) came close to identifying the cause of pulmonary embolism: ‘In many instances the puriform matter … is subsequently … deposited in distant points of the system … the lungs and liver, especially the former, are the organs into which these secondary purulent infiltrations most commonly take place … whenever purulent deposits of this kind take place, phlebitis and an altered state of the blood, arising therefrom, have always preceded and led to them’. Indeed, van Swieten (1776) had written that injurious substances introduced into the veins of dogs ‘render the blood grumous, which grumes flowing through the veins from a smaller to a larger capacity pass to the right ventricle … and thence to the lungs. … From such causes, therefore, may arise a peripneumony suddenly fatal’.4

pathology. Moreover, unlike Cruveilhier, he trusted the microscope (Hellsten 1942; Dreyfus 1957; Verso 1961). Cruveilhier was already 35 years old by the time the microscope became a reliable tool and attained no expertise in its use. 4 We are indebted to Professor Charles Warlow for drawing our attention to these early 19th century publications.

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6.2.2

Cruveilhier’s Contribution

Cruveilhier wrote [our emphases]: Phlebitis is the happy name given to inflammation of the veins by M Breschet, who gathered together the scattered facts known about the local effects of this inflammation in certain scientific notes. Misinterpreted for a long time, phlebitis was first studied by Hunter [1793], who in some ingenious experiments considered not only the local effects of inflammation of the internal membrane of the veins,5 but also its general effects; and tried to determine the way in which it influences the constitution. In a way, phlebitis dominates all pathology. It is the tie which unites the blind and almost instinctive humoralism of the ancients with the rational humoralism of the moderns. … It has also furnished the solution of a multitude of phenomena that defied all explanation and theory, because it gives opportunity for positive experiments. Phlebitis belongs to both surgery and medicine. … The death of most of those who succumb to wounds and surgical operations is the result of phlebitis. … According to my experiments (Nouv. Bib. Med. 1826, Recherches sur le siège immediat de l’inflammation), the capillary venous system is the source of all inflammation, as well as of all normal or morbid secretions. On the basis of its source, three kinds of phlebitis may be distinguished: (1) phlebitis of free veins [superficial VT], (2) phlebitis of veins contained in the depths of organs [DVT], and (3) capillary phlebitis In this article I shall examine: (1) the local phenomena of phlebitis, (2) consequential phenomena …

Later in this text (Cruveilhier trans. 1929), the author continued: Article 1: Local phenomena of phlebitis. The first effect of all phlebitis is coagulation of the blood and its adherence to the walls of the vessel/s. This coagulation with adherence may be observed in traumatic as well as spontaneous phlebitis. I have frequently produced it in my experiments on living animals – either by introducing a wooden splint into the veins, or by the injection of a chemically irritating substance. Stagnation of the venous blood, and ‘serosity’ [oedematous swelling?] in the corresponding parts, result from obstruction of the venous circulation in the inflamed vessel when the collateral veins are not sufficient for circulation. Painful oedema, phlegmasia alba dolens in women at childbirth may be considered a characteristic sign of phlebitis, just as after phlebotomy or in any other circumstance. Such oedema is caused by, and is proportional to, the trouble in the venous circulation.

5 Hunter had been puzzled by the observation that thrombi (as we now call them) appeared first as depositions of white material in the middle of the lumen, not at the vein wall, as was to be expected if the ‘pus’ entered from outside the vessel. Evidently Cruveilhier made the same observation. Although it is nowhere explicit in Hunter’s writings that thrombi are initiated on the valves, it seems highly likely that he made this interpretation, hence his nomenclature. Harvey had described the valves as ‘eminences’ on the ‘internal lining (tunicula intima)’ of the vein, and Hunter was unquestionably familiar with Harvey’s masterpiece in the Latin original. Hence, we suggest, Hunter’s nomenclature.

6.3 Other Formative Influences on Virchow

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Independently of oedema, external phlebitis is also characterised by the presence of a hard painful and easily defined cord, which exactly follows the course of the vein.

In Anatomie Pathologique du Corps Humain, his section on ‘Inflammation of the pulmonary artery’ contains the following passage: From the general consideration I wrote about above it follows that: (1) lobular pneumonia is the most frequent change (complication) following wounds and surgical operations; (2) this lobular pneumonia is nothing but capillary phlebitis; (3) this capillary phlebitis always coincides with some phlebitis more or less distant… (4) … but in the same way as one observes capillary phlebitis and phlebitis of large vessels in all parts of the body, so the lungs display in addition to the lobular inflammations (capillary phlebitis), inflammations of the principal divisions and even the trunk of the pulmonary artery – which carries venous blood and in consequence is to be equated with veins in other parts of the body.

Cruveilhier described a puerperal death from ‘multiple phlebitis’ and pulmonary ‘phlebitis’. He went on to describe the lung autopsy, which he illustrated with a plate: Making incisions in this lung I came across hard concretions filling the divisions of the pulmonary artery. This artery dissected with care showed a discoloured sanguinous concretion lightly adherent to the walls. This concretion divided in the manner of the artery, and penetrated to a certain number of ramifications thereof. It was evident that coagulation of the blood had started in the arterial trunk and extended to subdivisions. The clots in these small divisions were red and soft, whereas those in the main trunk were evidently old from their coherence and discolouration. I divided these clots and found in the centre of the main clot a collection of pus with all the characteristics of phlegmonous pus. Farther on the blood clot was discoloured, but solid. [Our emphasis]

The significance of these extracts will become clearer when they are compared with Virchow’s remarks in Die Cellularpathologie, but the main point is immediately evident: Cruveilhier was describing, under their previous names, precisely the same lesions that are now called ‘DVT’ and ‘pulmonary emboli’. By replacing ‘phlebitis’ with ‘vascular obstruction caused by thrombotic lesions’, we can recognise his insightful – if perhaps overstated – generalisation. What he called patches of ‘lobular pneumonia’ were (mainly) pulmonary infarctions consequent upon thrombosis. He recognised that when ‘old’ lesions are found in the pulmonary arteries, other lesions coexist elsewhere in the venous system. His observation that ‘pus’ (white material) appeared in the centre of the vessel lumen, not at the wall, echoed Hunter’s findings. We consider it highly significant. However, nothing in his work suggests that he anticipated Virchow’s working hypothesis that these lesions are embolic metastases.

6.3

Other Formative Influences on Virchow

Virchow graduated in 1843 and a year later he began pathological studies in Berlin as assistant to Robert Froriep. He engaged directly with the contemporaneous philosophical, conceptual and methodological developments in biology and

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medicine. He seems to have acquired his commitment to a cellular basis for pathology6 – and his concomitant opposition to Cruvelhier – from the outset of his postgraduate career. But the most distinctive feature of Virchow’s early work, notably his research on thrombosis and embolism, was the thoroughness with which he studied the contributions of his more remote predecessors, particularly Hunter. Froriep gave Virchow the task of examining the ‘doctrine of Cruveilhier’ when their collaboration began in 1844. We might imagine that the young Virchow acquired his highly critical view of the already-famous Cruveilhier from his mentor,7 and perhaps not least because Cruveilhier declined to use the microscope8 (Ackerknecht 1981). As we showed in Chapter 4, the idea that blood ‘stasis’ is a causal factor in thrombogenesis can be traced back to early 19th-century studies in ‘animal chemistry’, and beyond these to the work of Malpighi and Boerhaave. In the first half of the century, many students of ‘inflammation’ (thrombosis) such as Baillie, Bouchut and Davies, and no less a figure than Andral himself, accepted this causal connection (Lee 1842; Hellsten 1942; Anning 1957). Rokitansky (1852) reported that what would come to be called ‘venous thrombi’ arose at sites of injury or inflammation, or were associated with blood changes.9 Moreover, several microscopists of the period observed that blood stagnation was associated with margination of ‘colourless corpuscles’ on vascular endothelial surfaces (i.e. leukocytes, and what was then dubbed ‘leukocyte debris’ – later to be called platelets). These studies formed a significant part of Virchow’s intellectual environment; he was himself an accomplished microscopist. Of course, coagulated blood can neither circulate nor move, so it cannot be doubted that thrombosis causes stasis, but Virchow realised that the converse was less obvious. The conflict between Boerhaave and Hunter, he inferred, turned on the issue of whether thrombi form in static or in moving blood.

6 Virchow probably acquired the principle, if not the wording, of omnis cellula e cellula from the writings of John Goodsir (1814–1867), who had first identified the cell as the ‘centre of nutrition’; and through Goodsir, from Raspail – though he did not acknowledge the Frenchman. Significantly, too, Virchow dedicated his Cellular Pathology to ‘John Goodsir FRS, (Curator of the surgical museum and) Professor of Anatomy in Edinburgh, the earliest and most acute observer of celllife’. In view of geographical proximity, it seems at least plausible that Goodsir’s ideas about cells may also have influenced Buchanan – and, indeed, Lister, who became Syme’s assistant in Edinburgh in 1853 and later succeeded him as professor of surgery. 7 There is some evidence that Virchow had formed the belief that pulmonary emboli arise from distant venous ‘thrombi’ (as he later called them) as early as 1846, so perhaps the suggestion came from Froriep. See e.g. McLachlin et al. (1960). 8 In this we might see the shade of Hunter’s warning of its potential fallacies – though no doubt Hunter would have moderated his view in the light of the upgrading of microscopes after the introduction of Lister’s revolutionary achromatic lens. 9 There is evidence in Virchow (1856) that Virchow had used the word ‘thrombus’ in its modern sense as early as 1847; Rokitansky appears to have been one of the earliest adopters of this usage.

6.4 Resolving the Conflict: Virchow’s Synthesis

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To summarise: when Virchow was a student in the early 1840s, the following strands of thought pertinent to thromboembolism had been established: 1. Harvey had conceived and theoretically established the notion of circulatory flow that provided ‘spirits + pabulum’ to tissues. His approach was ‘physiological’ (‘vital-materialist’, leading to ‘patho-physiological’), recalling Aristotle’s perspective but without the same ontological presumptions. 2. Boerhaave, building on Malpighi’s observations and on Descartes’s accolade about Harvey, had proposed a mechanistic (chemical and haemodynamic) approach. From that perspective, the residual Galenic concept of ‘not-circulation’ – i.e. stasis – became the major premise of what we now term thrombogenesis. The early 19th century chemical studies mentioned in Chapter 4 seemed to have corroborated this position. 3. Hunter had reintroduced Harvey’s frame of reference to replace Boerhaave’s, and had conceived vessel wall ‘inflammation’ in response to ‘irritation’ as his major principle. He and Hewson had shown that what we now call ‘thrombi’ formed in circulating blood, not in static blood. They had also recognised that some components of the blood itself were causally significant. 4. Cruveilhier had observed that ‘phlebitis’ (later re-named ‘thrombosis’) was associated with injury to the vessel wall intima and had inferred a causal connection. He held that phlebitis is the root of all pathology, the ‘doctrine of Cruveilhier’. 5. Many microscopists had established that mechanical, chemical or electrical injury to the linings of blood vessels invariably led to their becoming congested with marginated blood/lymph ‘globules’, followed by the swarming together of the red and white blood constituents, resulting in ‘altered circulation’. Their work seemed in accord with Malpighi’s finding and the mechanistic perspective of Boerhaave, which presumed that stagnation of the blood causes ‘inflammation’. Virchow’s resolution of these conflicting strands of thought and evidence was a landmark in the history of medical science and the continual struggle to distinguish ‘cause’ from ‘effect’ in biology and medicine.

6.4

Resolving the Conflict: Virchow’s Synthesis

Virchow’s early papers About Fibrin and White Blood, followed by About the Obstruction of the Pulmonary Artery, were all published in Froriep’s Neue Notizen. Further Investigations about the Obstruction of the Pulmonary Artery and its Consequences was added in 1846 and was based on human case studies; he also published a classic paper on leukaemia (this term also being his invention) in 1845. Shortly afterwards, these investigations reappeared, with extensions, in Traube’s three Booklets; the 3rd Booklet remained unpublished and the findings appeared for the first time in the 1856 publication. By 1848, Virchow had shown that the semi-solid masses of blood that form, often

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fatally, in the pulmonary arteries are not produced in situ but have metastasised from the peripheral circulation. He seems to have considered this a refutation of Cruveilhier. When his service as Professor of Pathology at Würtzburg (1849–1856) terminated (a chair in Pathological Anatomy had been created for him in Berlin), he based his discussion in Gessamelte Abhandlungen zur wissenschaftlichen Medizin on these early papers (Virchow 1856). They were reprinted 1910 in Barth’s edition Klassiker de Medizin herausgegeben von Karl Sudhoff and translated into English by Bell and Matzdorff in 1998. His personal footnote, justifying the additional publication, appears on p. 109 of that translation: This paper (i.e. Traubes Beitrage Vol 2, pp. 13–109) was published in the year 1846 to this point. However, because Traube’s 3rd booklet of articles was never published, my treatise was still without an end – which I will now add for the first time.

The second and third Traube Booklets detailed thirty-four experiments: nos. 30–33 were dated to 1846 and nos. 27–29 to 1847. A ‘special series’ was started in 1851 and experiments 25 and 26 were dated 1853. In these experiments, Virchow introduced prepared plugs of India rubber, elderberry pith, lumps of muscle, fatty tissue, etc. into peripheral veins and demonstrated that they migrated via either cava to the pulmonary artery, where they blocked the circulation with various outcomes. The human case histories detailed in Booklet 3 were dated 1844–1855 and they all focused on the fact and consequences of clinical pulmonary emboli. (The words embolia and embolus were Virchow’s inventions.) Virchow (1856) wrote: ‘It is strange that, in the result … [interrupted bloodflow], which according to the prevailing assumptions would be the most significant, turned out to be the least important factors; indeed, in many cases they played so small a part that the animals used for the experiments were jumping about with the greatest zest immediately after release …’. This underlines his earlier remark about the ‘nature’ rather than the ‘fact’ of the blockage being relevant to embolism; but most importantly, the assertion was tantamount to declaring that blood stasis is not causal in the formation of emboli and thrombi. Because of the ‘great numbers of colourless corpuscles in the thrombus’, he concluded that thrombi may form when the circulatory flow is impaired or retarded (‘by compression of vessels, obstruction, varicosities, weakening of vessel walls … or lowering of venous pressure’), but not stopped. So many colourless corpuscles could not have been derived from the limited volume of blood that constitutes a particular thrombus. Therefore, the excess of white cells could be explained only by presuming that they are progressively sequestrated from the ‘whole’ blood volume passing that site over a much longer period of time – a brilliant inference. To put it simply, Hunter had been right, Boerhaave wrong: thrombi and emboli form in moving, not stationary, blood; ‘clots’ form in recently-shed, static, blood. It is often averred that Virchow said that ‘stasis is thrombogenic’. This claim might be based on the following quotation: ‘The formation of these extended clots [into the inferior vena cava] can also be explained by another cause. If the blood of a major vessel clots, for instance in the common iliac vein … an entire extremity is cut out of the circulation, then only the blood of one iliac vein will supply the

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inferior vena cava. And, especially in that part of the cava which is next to the obturated iliac, a layer of almost stagnating blood will develop. This situation is by all means able to initiate coagulation of the blood’ [our emphasis]. Virchow subsequently commented on the thrombogenic role of interrupted circulation in the pelvic vein of a typhoid patient: ‘This case was even more predisposed because of a special anomaly in the course of the veins. If there is a general or localised reduction in blood flow, we usually see spontaneous coagulation, most frequently in those veins that have anastomoses or that form a plexus, which means that they have a certain number of superfluous ducts …’ [our emphasis]. ‘Almost stagnating’ and ‘reduction in flow rate’ do not mean ‘static’. In short: nowhere did Virchow state, or imply, that ‘stasis’ is thrombogenic; quite the contrary.

6.5

Virchow on the Structure of a Thrombus

Virchow initially considered the clotting of blood ex vivo as a possible model for and analogue of thrombogenesis; a plausible hypothesis, but one that he rejected after critical histological studies. In the Gesammelte Abhandlungen we find his summary of the distinctive ‘triad’ of distinctions between in vivo thrombi and in vitro clots (pp. 514–515): 1. the structure of the thrombus is in layers [i.e. the lines of Zahn]10; 2. the fibrin content is denser; 3. the population of colourless corpuscles is denser, and to a striking degree; these corpuscles were present in the blood from the beginning, and were separated from it with the fibrin. … Thus there is evidence for spontaneous coagulation of the blood within the vessels, visible where the continuity of the vessels is interrupted, e.g. by a wound. This large segregation of colourless corpuscles is clearly associated with the retardation of the circulating blood.

These observations emphasise the inappropriateness of using the terms ‘clot’ and ‘clotting’ to denote, respectively, thrombus and thrombogenesis (Chapter 4); but there are numerous examples of this infelicity – and some examples, bizarrely, of the converse (e.g. Ardlie et al. 1967). Virchow’s studies of the morbid anatomy and histology of thrombi were remarkably detailed. Notwithstanding the important contributions of his many successors (notably the superb microscopic studies by Welch 1887, 1899), some of his observations seem not to have been replicated until the 1950s, and even then their probable significance was not widely recognised. Briefly: he showed that thrombi were always anchored to the vein wall; that as a thrombus grew

10

Although Zahn (1876) gave the first detailed description of these structures, they had been recognised by Lobstein (1833) and Bristowe (1856); but not until an early paper by Aschoff (1892) was the macroscopic appearance explicitly related to the successive deposition of platelets, leukocytes, fibrin and erythrocytes.

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(often to a remarkable length) from its fragile – almost single-point – anchor to the wall, it thickened to as much as half the cross-sectional area of the lumen, presenting a marked resistance to movement; that the combination of resistance and fragile anchoring was the likely cause of embolism; that there was a distinction between the mainly white Kopfteil close to the anchor and the mainly red Schwansteil at the outer, more newly formed end, with a neck or Halsteil between them11; and perhaps most importantly, that nearly all thrombi were anchored in the venous valve pockets (Virchow 1846; see Fig. 6.1). It goes without saying that blood clots formed ex vivo bear no resemblance to this structure, nor have they any specific anchorage to a substratum. Though Virchow depicted thrombi with white cores in the centre of the vein as Hunter and Cruveilhier had described them (i.e. not in contact with the vein walls), he was – as Hunter would have been were he still alive – greatly puzzled to see ‘pus’ located centrally in vessel lumens rather than adjacent to the walls. Virchow depicted streaks of white cell material running through the length of the thrombus and its linkages and stated that the thrombi are ‘seated on the valves’, though there is no valve in the illustration. Hence, it was not until we realised that thrombi are always initiated on valve cusps (Chapters 8 and 9) that we recognised the central significance of Hunter’s, Cruveillhier’s and Virchow’s observations: thrombi do indeed begin – literally – in the centre of a vein. Hunter’s perception that the white

Fig. 6.1 Virchow’s engraving of a venous thrombus showing anchoring in the valve pocket (Fig. 69 from Virchow 1858). Virchow’s legend to this figure read: ‘Thrombosis of saphenous vein. S. Saphenous vein, T. Thrombus:v, v1thrombi seated in the valves (valvular) in the process of softening, and connected by more recent and thinner portions of coagulum. C. Prolongation of the plug, projecting beyond the mouth of the vessel into the femoral vein C’. See text for explication

11

The terminology is due to Aschoff (1924), following Welch (1899), but the structure seems to have been observed first by Virchow. Virchow’s microscopic studies certainly provided the impetus for subsequent investigations, notably Welch’s; see Chapter 7.

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material of a thrombus is located in the centre of the lumen was therefore the first observation about the detailed pathology, but the histology was not unequivocally described until Virchow (1856).

6.6 Virchow on Oxygen and Thromboembolism Virchow (1856) recognised that thrombosis (and thromboembolism) is oxygendependent. He inquired whether the oxygen is already present in the circulating blood or must be supplied from outside the body. If it is introduced from outside the body there must be either (1) a breach in the vessel wall, or (2) absorption through exposed wounds, ulcers or various orifices. He reasoned against these possibilities. But if oxygen is already present in the circulating blood, carried by the ‘red corpuscles’, how is it liberated so as to cause coagulation? Of course, this work was conducted long before the structure and function of haemoglobin were understood; but it is interesting to see how Virchow wrestled with the problem. He rejected the hypothesis that there is a ‘spontaneous change in the interior constitution of the blood particles’ because this ‘could only be conceded if found still present in blood long stagnant’. He went on to suggest that ‘A more considerable change in the blood-vessels might be postulated, where molecular attraction between the blood and the vessel wall is seen to increase, especially when the blood comes into contact with porous foreign bodies. … A fibrolaminar clot … in a retarded blood-flow can initiate that linking of the fibrogenous substance with oxygen liberated from the blood corpuscles, and can organise about itself by attraction new fibrous material, which then takes effect as a new aggregation of contact bodies.’ He then proposed, once again echoing his predecessors, that ‘thickening and irregularity of the vessel lining may predispose to thrombus formation’. It is striking that Virchow recognised that the coagulation of blood, and the formation of thrombi and emboli, is oxygen-dependent. But he also knew that altered blood movement – and therefore, by implication, local hypoxaemia – is a causal factor in thromboembolism. (Incidentally, the word ischaemia is another of his additions to the medical vocabulary.) In this respect as in others, his contribution to the field was a milestone.

6.7

Virchow versus Cruveilhier

Virchow’s seemingly implacable antipathy to Cruveilhier could have been a reflection of the Francophobia characteristic of many 19th-century German scientists (see Chapter 5). There is no doubt that his work surpassed that of his eminent predecessor. He showed that pulmonary emboli arose from peripheral venous thrombi; he changed Cruveilhier’s nomenclature; he demonstrated that

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thrombosis required circulating blood and was oxygen dependent; and he described the structure of thrombi and their anchorage in venous valve pockets. But his denigration of the French pioneer of pathological anatomy seems undeserved. In Lecture X of Die Cellularpathologie, he wrote: ‘It was imagined by John Hunter that pus … was furnished as a product of secretion by the wall of the vessel. This doctrine, however, presented some difficulty, because it was soon pretty generally allowed that a primary purulent inflammation of the veins did not occur, but that, as was first distinctly shown by Cruveilhier, a clot of blood is always present. ‘Cruveilhier himself was so greatly surprised by this that he connected a theory with it which is beyond all medical comprehension. … The impossibility of explaining phlebitis seemed to him to be got over by raising coagulation into a general law, and by referring every inflammation to a phlebitis on a small scale (capillary phlebitis). … This manner of thinking, however, continued to be so entirely alien to that of the great majority of learned and unlearned physicians, that the separate conclusions propounded by Cruveilhier (adopted in medical science in part as drawn up by him) were altogether misunderstood. ‘Cruveilhier was right … that the so-called pus in the veins never, in the first instance, lies against the wall of the vein, but is always seen first in the centre of the previously existing clot which marks the start of the process. He imagined that the pus was secreted from the vessel wall, but did not remain there, but by means of ‘capillary attraction’ made its way into the centre of the clot. ‘This was a very singular theory, which can only be approximately comprehended by assuming, as was still the custom to do in Cruveilhier’s time, pus to be a simple fluid. But, apart from these extremely obscure interpretations, the fact remains … that before a trace of ‘inflammation’ is visible, we find a clot, and that shortly afterwards in the middle of this clot a mass displays itself which differs in appearance from the clot, whilst on the other hand it exhibits a greater or less resemblance to pus. ‘With this … starting point, I have endeavoured to clear up the doctrine of phlebitis, as far as lies in my power, by substituting, for the mysticism which pervaded Cruveilhier’s interpretation, a plain statement of the facts. ‘We do not know that inflammation, as such, has any necessary connection with coagula; on the contrary, it has turned out that the doctrine of stasis rests on manifold misinterpretations. … If, therefore, we leave inflammation on one side and confine our attention simply to the coagulation of the blood … it seems most convenient to comprehend the whole process under the term thrombosis. I have proposed to substitute this term for the different names – phlebitis, arteritis, etc., inasmuch as the affection really consists in a real coagulation of the blood at a certain fixed spot. [Our emphases]

It seems harsh to condemn Cruveilhier for failing to see the anchorage of thrombi on valve leaflets. And why should Cruveilhier’s thesis have been deemed ‘mystical’? Virchow’s discussions of ‘pus’ were no less ‘mystical’ when they were written – just when Pasteur’s epoch-making work on wine spoiling12 was being published. The phenomena of inflammation and pus were everyday realities calling for medical management – whatever their cause, whatever their name, and however they were to be explained (we shall discuss this important issue in more detail in Chapter 7). Moreover, although Virchow had indeed shown that ‘the doctrine of

12

In the same year (1857/1858) that Die Cellularpathologie was published, Pasteur published the work that would pull the carpet from under the old concept of pus, and Lister presented his paper on ‘The early stages of inflammation’. Hence our identification of this in Chapter 5 as an annus mirabilis.

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stasis rests on manifold misinterpretations’, it is not clear that Cruveilhier embraced this ‘doctrine’ unequivocally. Finally, we note that Virchow had ‘deemed it convenient’ to substitute ‘thrombosis’ for ‘phlebitis’, disguising the fact that Cruveilhier had proposed a plausible generalisation some years before this diatribe was written. Whatever Virchow’s motives, he certainly succeeded in damning Cruveilhier in the eyes of future generations. For example, Talbot (1970) wrote: ‘The study of inflammation … led [Cruveilhier] to the false belief that phlebitis and pyaemia were intimately related, and to the false conclusion that phlebitis dominated all pathology’ [our emphases]. That dismissal of Cruveilhier’s lifelong contribution to medicine sounds like biased, copycat history.

6.8

The Possible Source of ‘Virchow’s Triad’

The first indication of anything resembling ‘Virchow’s triad’ in Virchow’s own writings appears on pp. 293–294 of Klassiker de Medizin: In the final assessment, the mechanical as well as the chemical characteristics of the thrombus [in the pulmonary artery] were studied. The hard rough surface had caused grave damage to the interior wall of the lung artery … causing inflammation of the lung at least equivalent in gravity to the effects of the chemical action resulting from the organic substances introduced by accident into the lung. … Accordingly, the sequence of the possible stages and consequences of blockage may be classified and studied under three headings:(1) phenomena associated with irritation of the vessel and its vicinity; (2) phenomena of blood-coagulation; and (3) phenomena of interrupted blood-flow13.

This list of putative ‘phenomena’ ostensibly resembles the various outlines of ‘Virchow’s triad’ as introduced in Chapter 1. However, from the context, it is obvious that Virchow was not writing about thrombogenesis, but about emboli as ‘bungs’ in pulmonary arteries. He was determined to prove that what Cruveilhier had illustrated as spontaneous/in situ pulmonary ‘phlebitis’ did not arise de novo in the (narrowing) artery, but had come from a peripheral site. The original cause of those venous thrombi was of course a valid scientific question, but having only recently re-named the condition, he had not yet focused on the aetiology of thrombogenesis per se. This observation is by no means novel. Anning (1957) and Brinkhous (1969), and more recently Owen (2001) and Brotman et al. (2004), have all made the same point, and Dickson (2004) emphasised it in his excellent short review of the ‘Virchow’s triad’ epithet. The conclusion would seem to be confirmed in print: according to the second edition of Boyd’s textbook of pathology (1964), Virchow allegedly ascribed

13 In most of his writings, Virchow seems to have preferred the phrase ‘interrupted circulation’, referring specifically to a pulmonary artery obstructed by an embolus. We shall return to this semantic point in Chapters 8 and 9.

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thrombogenesis to the now-classical ‘causal triad’; but in its first edition (1932), Boyd stated that venous thrombosis resulted from vein wall damage caused by injury or inflammation, and made no mention of ‘stasis’ or ‘hypercoagulability’ – or of Virchow (Boyd 1932, 1964; Dickson 2004). The fact is that the elements of ‘Virchow’s triad’ were never clearly and exclusively listed until they were condensed for the first time in the late 1920s and early 1930s (McCartney 1927; Vance 1934). Even then, Virchow’s name was not linked to the compilation. This implies that ‘Virchow’s triad’, in the sense that would come to be used by proponents of the consensus model of DVT, was invented sometime between 1932 and 1962, 30–60 years after its supposed author’s death, and 76–106 years after the above quotation from Klassiker de Medizin was written. We have not been able to locate the first usage, but we are inclined to think that it dates from the early 1950s.

6.9

Reflective Anamnesis

In view of his enormous and wide-ranging contribution to scientific medicine, it is hardly surprising that we can fault some of Virchow’s ideas retrospectively. For instance, although he renamed ‘phlebitis’ as ‘thrombosis’, and knew about the margination of leukocytes, he denied the process of leukocyte migration. He believed, at least in the 1840s and 1850s, that platelets were leukocyte debris. And although he appreciated that oxygen is involved in thrombogenesis, his attempts to expound a relevant mechanism were garbled. Neither was he impressed or convinced by Pasteur’s germ theory of disease, though he did predict the discovery of bacterial toxins. No doubt, as for most of us, his opinions grew more inflexible and perhaps intolerant as he aged. His writing style became overblown and rhetorical, perhaps because of his increasing involvement in politics. But the magnitude of his scientific achievements renders these shortcomings insignificant by comparison. In the field of thromboembolism alone, he proved that Cruveilhier’s ‘pulmonary phlebitis’ results invariably from the metastasis of emboli produced as, and from, thrombi in peripheral veins. Although this now seems a commonplace, we have to appreciate the 10 years of outstanding experimental work that established it and disproved the conclusion of the great Cruveilhier. Virchow described the structure of thrombi in unprecedented detail, showing – inter alia – that they are always anchored tenuously but unequivocally to the venous endothelium (of inner valve pockets). He articulated a ‘triad’ of histological and macroscopic differences between ‘clot’ and ‘thrombus’, proved that oxygen is required for thrombus/embolus formation and that oxygen is supplied by the flowing blood, and taught that thrombi do not form in static blood. Among the terms that he added to our vocabulary, thrombus, embolus and ischaemia are particularly relevant to this book. His achievements in several other areas of medicine were of comparable significance. Although many of his opinions were not pertinent to the theme of this book, one was significant: he doubted that either the mechanistic or the pathophysiological/ vital-materialist approach alone would afford a satisfactory philosophical or meth-

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odological basis for scientific medicine. And although he was personally attuned to his own ‘pathophysiological’ viewpoint as indicated in Cellular Pathology, he argued strongly that the two contemporary philosophical positions were complementary, and that both were required for complete understanding. What he did not achieve was an explicit account of the aetiology of DVT. Yet his writings provided the basis and the inspiration for his successors to seek the causal factors in thrombogenesis, and several attempts were made during the 50 years or so following his death. However, not until the middle of the 20th century were such attempts ascribed, wrongly, to Virchow himself.

Chapter 7

The Pathophysiological Tradition after Virchow

Abstract The words ‘inflammation’, ‘phlebitis’ and ‘pus’ have denoted different entities and carried markedly different connotations at various times in history. These shifts of meaning can hinder 21st-century interpretation of some 18th- or 19th-century publications, and failure to take account of this difficulty has led to persistent misunderstandings. The continuing association of ‘phlebitis’ with venous thrombosis is particularly problematic. Discussion of the interpretation problem leads to an evaluation of the possible involvement of leukocytes as well as platelets in the aetiology of DVT and focuses attention on the origin of venous thrombi on the valve cusps. This discussion traces the continuation of the pathophysiological tradition into the 20th century.

Keywords Inflammation, leukocytes, platelets, phlebitis, pus

7.1 Problems of Nomenclature: ‘Phlebitis’ and ‘Inflammation’ Readers who have not studied the classical literature cited during the previous three chapters, but intend to peruse it, might misunderstand some of the terminology they encounter. The following comments are intended to forestall misinterpretation, and also to introduce other matters that we consider integral to the aetiology of DVT. Our initial remarks focus on the words ‘inflammation’, ‘phlebitis’ and ‘pus’. ‘Inflammation’ is a Platonic, metaphorical term. It is at least 2,000 years old; it was mentioned in the De Medicina of Celsus (c. 30 BC– AD 30), the leading authority on the subject for 1,700 years.1 The idea of ‘flame’ was invoked – fallaciously, as we now realise – to explain particular tissue disorders, including what we now call ‘thrombosis’. Nowadays, students are taught that inflammation is characterised by

1 De Medicina was republished during the Renaissance, ensuring that the influence of Celsus persisted after the end of the Middle Ages. Modern medical science may have inherited its concept of ‘inflammation’, indirectly, via Stahl’s phlogiston theory.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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four signs: rubor, calor, tumor and dolor (redness, heat, swelling and pain). This characterisation relates to observable manifestations, not to causes. Moreover, although it applies satisfactorily to the body surface, it hardly applies to internal organs or the insides of blood vessel walls, where pain is not referred, redness and heat are detectable only as increases in whole body temperature, and swelling can be identified only by scanning techniques or during exploratory surgery. Invariable concomitants of the inflammatory response such as phagocyte invasion and the release of various cytokines and complement factors are ‘hidden’ phenomena, not the macroscopic symptoms of patients or signs appreciable by examining physicians. In the late 16th and early 17th centuries, van Leeuwenhoek, Janssen, Kepler, Malpighi and Huygens studied ‘inflamed’ tissues by microscopy and challenged Celsus’s doctrine. Their findings enabled Hunter (1793) to define inflammation more precisely. He wrote: ‘Inflammation is to be considered only as a disturbed state of parts … it is not to be considered a disease, rather a salutary operation [‘healing’ in today’s terms] consequent on either violence or disease’. In other words, Hunter recognised that inflammation is a response to tissue injury; it is not a ‘cause’ of anything, and the term ‘inflammation’, though descriptive, has no aetiological implication. He then divided ‘inflammation’ into two categories: ‘adhesive’ and ‘suppurative’. These probably equate to ‘degenerative inflammation’ (e.g. arthritis) and ‘infection-related inflammation’ in today’s terminology, though some concepts that we now take for granted, such as ‘infection’, lay more than 60 years in the future when Hunter was writing. Virchow attributed the concept of ‘phlebitis’ to Hunter; Browse et al. (1988a,b) attributed the term ‘phlebothrombosis’ to the same source. But although Hunter (1793) referred repeatedly to ‘inflammation of the internal coats of veins’, he mentioned neither ‘phlebitis’ nor ‘phlebothrombosis’. The modern reader might assume that Hunter’s ‘inflammations of … veins’ meant suppurating wounds such as infected phlebotomy wounds, left to heal by second intention (without suturing) – what might now be dubbed ‘infected veins’ or ‘post-infusion thrombophlebitis’. The lesions to which he referred may not seem to have resembled unsuspected (silent), uninfected, autochthonous thrombosis. However, such a reading is wrong; the usage of ‘inflammation’ and its Greek equivalent changed dramatically between 1790 and the 1850s.2 Hunter, like Boerhaave before

2 The difficulty arises from a condition called by different names in different eras. We know from our own experience how the sense of words can change in 50 years – as they were seemingly changed by translation in the 50 years between 1793 and 1843. Moreover, though we can barely appreciate it now, a particular semantic problem grew up around ‘phlebitis’, which seems to have been conflated or equated with the ‘miasma’ concept, rife in the early 1840s. Virchow and others were perhaps fighting to rid the public of the delusion that ‘phlebitis = miasma’ was a ‘real’ medical entity, associated with a frightening, immaterial ‘force/thing’.

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him3 and Cruveilhier after him, meant what we now call ‘thrombosis’. What he saw were intravenous coagula. As we observed in Chapter 6, however, he was particularly puzzled by the location of the ‘pus’ in the middle of those coagula. Because contemporaneous teaching held that pus was ‘exuded’ from the vessel walls and tissues external to them into the veins, it should be found immediately adjacent to the vein wall, not in the middle of a coagulated red blood mass. Cruveilhier was similarly puzzled. Virchow, understanding Hunter’s exact meaning, translated ‘inflammation of the … veins’ literally; hence his attribution of the word ‘phlebitis’ to Hunter. Cruveilhier – and, apparently, Breschet – had interpreted the phrase as Virchow did; hence their use of ‘phlebitis’. In Die Cellularpathologie, Virchow (1858) gave the old, imprecise term ‘thrombus’ a new and exact meaning: a venous blockage formed in vivo by locally semi-solidified blood. He distinguished this ‘reactive blood semi-solidification’ process from the ‘inflammatory state’ of the vein wall that had previously been alleged to ‘cause’ it (biology has long struggled to distinguish between ‘cause’ and ‘effect’). This distinction alone constituted a remarkable advance in understanding (Lidell 1872).

7.1.1

‘Pus’

Why were thrombi interpreted as ‘inflammation’ by Boerhaave, Hunter and Cruveilhier? The answer is that in the pre-Pasteur era, pus betokened inflammation, and inflammation was manifest as pus. Although ‘pus’ and ‘purulent inflammation’ are irrevocably associated today with bacterial activity, that association was not to be established until the mid-19th century work of Pasteur and Lister (see e.g. Lister 1858, 1867). In Hunter’s day, any white amorphous material in the body was ‘pus’. Many readers may never have seen, or have forgotten, that thrombi always contain white streaks (see Fig. 7.1, taken from the first illustration in Hadfield 1950). These streaks were described by Hunter, recognised by Cruveilhier, cited by Virchow (1856, 1858) as ‘leukocyte debris’, and discussed by Zahn (1876). Even today, they are known as the ‘lines of Zahn’. For Hunter (1793), they were ‘pus’ or ‘purulent/suppurative’ material; hence ‘inflammation’. It is extremely difficult for the modern reader to grasp the mythical or magical overtones of ‘miasma’ in dissertation after dissertation on the subject of ‘pus’ in that era, and its cousins ‘pyaemia’, leukaemia, physiological leukocytosis, etc. To biologists before c.1860, pus was ‘laudable’ (an evaluative rather than an aetiological or descriptive adjective). It had nothing to do with infection. It is remarkable that 3 Hunter’s evident familiarity with Boerhaave’s hydrodynamic account of vein pathology might account for his own use of the term in this context. But ‘phlebitis’ translates as ‘vein inflammation’, not ‘inflammation of the internal (lining) coats of veins’. Since Hunter clearly observed that (what are now called) thrombi are rooted in the middle of the vein lumen, not on the walls, we think that the parts of the ‘lining coats’ to which he referred were probably the valve leaflets. As we noted in Chapter 6, Cruveilhier’s equivalent observation may be interpreted similarly. We shall return to this theme in Chapter 9.

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Fig. 7.1 (a) Copy of a drawing of a venous thrombus from Hadfield (1950). Copyright the Royal College of Surgeons of England. Reproduced with permission. (b) The same drawing with the presumed positions of the valves superimposed

Virchow (1858) distinguished the white material of the core of a thrombus as being ‘puriform but not purulent’, though he could not have been aware that the germ theory of disease was being created in Pasteur’s laboratory and appeared in print as he wrote. Like Hunter, he seems to have recognised the difference between infective and non-infective causes of leukocyte accumulation – though still without any concept of bacterial or other microbial infection. Hadfield’s line drawings precisely reflect Aschoff’s ‘Kopfteil, Halsteil and Schwanzteil’ of thrombi and the stages of thrombus development that they denote. However, the drawings are deficient both in terms of the hypothesis developed in

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this book and in terms of Harvey’s, and Virchow’s, explicit teaching and illustrations concerning the existence of valves in veins and their role in thrombus formation. Hadfield shows no valves or valve pockets. His thrombi all arise, wholly imaginatively, from ‘wrinkled’ endothelium/vessel wall – betokening abnormal endothelium, though whether or how or why it was abnormal, Hadfield could not know or guess. The point is clear when these illustrations are compared with Virchow’s engraving (Fig. 6.1); Virchow clearly recognised the role of valve cusps and pockets. It seems that in Hadfield’s time, the first half of the 20th century, the presence and significance of valves in our veins had come to be overlooked or ignored. This was an extraordinary ‘blind spot’: our concept of ‘the circulation’, and the historical escape from mediaeval Galenism, had depended on them entirely (Chapter 8). But only a year after Hadfield’s paper appeared, Paterson and the McLachlin brothers produced their paper on valve pocket thrombi (Chapter 9).

7.2

Leukocytes, Phagocytosis and Thrombosis

The source of ‘pus’ and its relationship to leukocytes was intensely debated during the 1840s, after the dawn of cell theory. The English microscopists Addison (1842, 1846) and Waller (1846) concluded that ‘pus globules’ and ‘leukocytes’ were identical, and that pus was normally formed by diapedesis of leukocytes from the blood vessels rather than the other way round. Remarkably, Virchow didactically denied this thesis. Having perceived the admixture of white material in the interstices of red venous thrombi, his logic suggested that it represented a venous clot associated with a focus of pus. This is why that generation of scientists had considered ‘clotted blood inside blood vessels’ to merit the suffix ‘-itis’. For them, the question was: should the vein wall or the coagulum itself be considered ‘inflamed’? Modern haematologists concentrate on the redness of thrombi and ignore the ‘white stuff’, but that focus requires explanation, since the white material in thrombi had fascinated some seven generations of pathologists between the time of Hunter and the 1960s. Aschoff’s belief remains pertinent: ‘in human beings the occurrence of fibrin coagulation is not the first stage of thrombosis … important changes in the morphological blood constituents [colourless elements] precede it. These last named changes must be explained before the mechanism of thrombosis can be understood’ (Aschoff 1924). In other words, while it is indeed essential to conceive how and by what agency fibrin is produced, the behaviour of leukocytes as well as platelets must also be integrated into the picture. Phagocytosis by leukocytes had not been described when Lister and Virchow wrote their seminal works. Both men, had they known of the phenomenon, may have seen it as an explanation for the attachment of viable ‘colourless corpuscles’ to endothelium deprived of ‘vital properties’, and of Virchow’s perspicacious observation that ‘the population of colourless corpuscles is so great in thrombi’. In a historical review of this field, Cameron (1952) stated that Virchow had noted the presence of red blood cells among lymph gland parenchymal cells in 1852.

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During the 1860s it was shown that particles introduced into the circulation were taken up by cellular elements and that white corpuscles exhibited chemotaxis (e.g. von Recklinghausen 1863). Preyer (1864) saw blood cells of Salamandra maculata taking up particles and red blood corpuscles. Langhans watched leukocytes removing extravasated blood cells (Lubnitsky 1885). Bizzozero (1882) recognised that macrophages could ingest ‘pus corpuscles’. Cameron therefore concluded that the essentials of what would become known as ‘phagocytosis’ were established by 1882. He quoted observers such as Panum, who in 1874 ‘… vaguely suggested that leucocytes might assist the destruction of bacteria, but made no convincing case for his conjecture’. However, he concluded: ‘But it is to Metchnikoff (1892) that we owe the essentially vital metaphysical concept that living, self-moving, cells may move towards and devour bacteria, thus playing an essential role in protecting the body against infection’ [our emphasis]. Metchnikoff demonstrated the process experimentally (see Metchnikoff 1893).4 He was a Russian contemporary of Pavlov, whose philosophy, like Virchow’s, was manifestly vital-materialist, devoted to the study of functional physiology in living animal bodies. The ingestion of dead autogenous or foreign living material by living cells thus became a general principle. The studies by Zahn (1876) on the involvement of frog leukocytes in intravenous coagulation must be understood in this context. Eberth and Schimmelbusch (1886) contended that the blood cells that Zahn considered to be (or called) ‘leukocytes’ were in fact platelets or ‘platelet analogues’; Löwit (1884, 1887) disagreed and maintained that they were indeed mammalian leukocyte analogues. This minor controversy5 may appear to have little relevance for modern understanding, but in fact it underpins an important debate and a continuing misconception. Zahn’s understandable presumption that the anatomy and physiology of ‘colourless corpuscles’ in frogs is analogous to those of ‘colourless corpuscles’ in warm-blooded animals seemed more tenable to many of his contemporaries than the allegation that they were different ‘units of life’. For example, Pitres (1876), who studied the phenomenon in warm-blooded animals, agreed with Zahn, Mantezagga, Lister and Virchow that leukocytes contribute to thrombogenesis. Microscopic study confirms that although the pale zones visible in cross-sections of thrombi

4 Virchow visited Metchnkioff in Sicily. Metchnikoff visited Claus, who suggested the term ‘phagocytosis’, and then went to work with Pasteur in Paris, where he published The Comparative Pathology of Inflammation in 1892. This book confirmed the reality of phagocytosis. Although Virchow no doubt found that the influence of his vital-materialist pathophysiology declined in his old age, his visit to Metchnikoff in Sicily may suggest that he was aware of the potential of phagocytosis to explain the margination and sequestration of leukocytes on sick or dying endothelium. However, that potential was not developed because haematology, dominated by the mechanistic-materialist perspective, began to focus on molecular rather than cellular accounts of blood coagulation (Chapter 5). 5 Between the time of Zahn’s publication and that of Eberth and Schimmelbusch, microscope technology had taken a further step forward. The Abbe apochromatic and oil immersion lenses were introduced by Zeiss. Many important advances in knowledge of cell structure followed within the next few years.

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consist largely of platelets, these zones are invariably bordered with leukocytes as well as fibrin. Only in the part of a thrombus that forms later (the Schwanzteil) does the ‘clot-like’ random association of erythrocytes and leukocytes suggest that they have become adventitiously entangled in the fibrin mesh. There is much more to this issue than the earlier confusion of ‘platelets’ with ‘leukocyte debris’.

7.3

Platelets

In 1861, two (or three) decades after cell theory was established, Max Schultze defined a cell as ‘an accumulation of living substance or protoplasm definitely delimited in space and possessing a cell membrane and nucleus’. He thought the membrane might be dispensable because the protoplasm was dense and viscous enough to sustain its own morphological integrity, but he did not seem to consider the nucleus ‘optional’. The nucleus had first been described in invertebrate blood cells by Hewson in 1777, then in chicken oocytes by Purkinje in 1830, in plant cells by Brown in 1833, and in neurons by Valentin in 1836. However, its significance remained unclear until around 1840, when Ehrenberg described nuclear division. Remak introduced the term ‘protoplasm’ around 1850, and he and Virchow showed that cell division begins with division of the nucleus.6 This paved the way for a critical question: if platelets have no nuclei, should they be considered ‘cells’? The question was pivotal because if cells are the minimal objects that can be considered ‘living’, then objects that are not quite cells (because they lack nuclei) may be considered not-living. The Virchowian, vital-materialist approach to explanation rested on cells as living entities. The mechanistic materialist approach, in both its original (physical) and later (biochemical) manifestations, rested on treating the components of organisms as objects governed by the same laws as inanimate matter. So the status of platelets – living entities or physico-chemical objects – played a key part in determining which approach was considered appropriate for both haematology and thrombology. The mere renaming of the blood cell/particle seen dimly by Buchanan in 1845 (Chapter 5) could not in itself be the basis of an explanatory theory; and microscopists could only make presumptions about the functional properties of what they saw. Platelets were clearly described, independently, by Bizzozero (1882) and Hayem7 (1882), though we have already mentioned the prior claim of Donné (1842) and possibly of Addison (1842). How their nature and function were to be understood rested on semantic and metaphysical presumptions.

6 These dates were obtained from Vladimir Matveev’s unpublished history of cell biology; Professor Matveev kindly furnished us with a copy. Among other things, the chronology once again illustrates the impact of Lister’s achromatic lens on the development of understanding of cell biology. 7 Hayem believed that platelets were precursors of erythrocytes but he also realised that they formed the initial haemostatic plug. The role of platelets in coagulation was confirmed by Lubnitzky (1885) and Eberth and Schimmelbusch (1886).

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Because mechanistic materialism had become dominant in physiology in the 1880s, and Schmidt’s thrombin hypothesis had gained ascendancy (Chapter 5), the physico-chemical rather than the cellular view of platelets came to be generally accepted. From this perspective, it was a short step to the ‘Sandbankbildung’ concept of Aschoff (1924). Platelets would in later decades be imagined to ‘silt’, ‘deposit’, ‘aggregate’, ‘agglutinate’ or ‘stick’ (as opposed to the vital-material ideas of ‘swim’, ‘congregate’, ‘swarm’, and other terms that connote living cells). Schwann’s ‘physical’ interpretation of cells8 would be applied uncritically by many readers: ‘Organised bodies of which the fundamental properties agree essentially with those of inorganic nature, working together blindly according to laws of necessity and devoid of any purpose’ [our emphases]. It is important to emphasise that this switch in perspective did not occur overnight, and that research into the coagulation mechanism was then in its infancy (Chapter 5). Basic questions remained unanswered, and the scope for speculative hypotheses was virtually unlimited. For example, Eberth and Schimmelbusch (1886) suggested that a thrombus may be imagined as a conglutination of (cellular) bodies that pre-existed in the blood rather than a coagulum of plasma proteins. ‘Conglutination’ was a gesture towards the idea of ‘living cells’. Significantly, this proposal was published in Virchows Archiv. Lubnitsky (1885) raised an important, closely related, issue: ‘Do platelets ‘transform into’ fibrin, or do they somehow ‘produce fibrin’ – i.e. from themselves, their own substance?’ Her question went to the heart of the contrast between the two metaphysical perspectives.9 Ought blood platelets to be perceived as living, functioning cells that do something, or as macromolecular lumps of material that are moved and ‘done to’? Despite these debates, the latter viewpoint gradually came to dominate, not only establishing a view of platelets that would persist for much of the succeeding century, but also leading to dismissal of the idea that leukocytes had any aetiological role in thrombosis. The discoveries of Virchow, Lister, Zahn, Eberth and Schimmelbusch, and many others were retrospectively attributed to unperceived admixtures of (relevant) platelets with (irrelevant) leukocytes.10 The later adoption of the synonym ‘thrombocyte’ for ‘platelet’ is ironic because of the suffix ‘-cyte’; the entire debate of the 1880s had turned on the question of whether platelets were or were not to be viewed

8 Two different ways of understanding ‘cells’, corresponding to the vital-materialist and mechanistic, were made explicit by Schwann in his pioneering publication on cell theory. Within a decade of this publication, du Bois Reymond and his colleagues had opted for the ‘physical’ (mechanistic) interpretation and Virchow for the ‘physiological’ (vital-materialist). 9 It might be wondered whether a woman declined to imagine one inanimate thing reproducing another (Schwann, with a male mind, could perhaps do so), rather than a living body reproducing a living body. The polymath Evelyn Fox Keller – theoretical physicist, molecular and developmental biologist, psychologist, historian of science and feminist – implied something of this kind, for example in her biography of Barbara McLintock (Fox Keller 1983; see also Lederman and Bartsch 2001). 10 Circulating assemblies of leukocytes and platelets are now known to be physiologically significant; see Chapter 12.

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as ‘cells’. The maturation of our modern view of platelets was reviewed by French (1967a) and French and Barcat (1968). Why did the investigators of the 1880s fail to recall the incontrovertible evidence that thrombi contain ‘great numbers of leukocytes and leukocyte debris’, stated explicitly by no less a figure than Virchow? The proposal that leukocytes are present only adventitiously, trapped in the fibrin web, is flatly inconsistent with the authoritative and oft-repeated observation that the core of a thrombus (or a nascent thrombus) consists almost exclusively of leukocytes and platelets – the erstwhile ‘pus’ – interspersed with fibrin. As far as we can judge, this failure to take cognizance of established knowledge can only be attributed to the metaphysical shift to mechanistic materialism that we discussed in Chapter 5.

7.4

The Persistence of the ‘Phlebitis’ Concept

Although the possible involvement of leukocytes in thrombosis was little discussed after the 1880s, there was no denying that they accumulate in thrombi, and the tradition of thought that had centred on ‘pus’ and ‘inflammation’ persisted; Virchow’s discoveries and nomenclature failed to put an end to it. The bastard term ‘thrombophlebitis’ was coined soon after and has survived: it is now the entry in the Index Medicus under which publications related to DVT are listed. The word ‘thrombophlebitis’ effectively repudiated the distinction so carefully drawn by Virchow; the implicit fusion of ‘-osis’ and ‘-itis’ generates an oxymoron. Because the phlebitis element of the term invokes the earlier concept of ‘pus’ in relation to thrombosis, and ‘pus’ in the post-Pasteur era implied infection, the neologism misled succeeding generations of surgeons and pathologists into continuing (or resurrecting) the erroneous notion of an infective cause for DVT. The semantic infelicity of the 18th and early 19th centuries continued to misguide them. Virchow (1898) was not inattentive to the shift of perspective concomitant with this coinage. The ageing genius was fully aware that all he had fought for was being overrun by the ‘other side’. In this valedictory address, he complained that the pathophysiological viewpoint that had informed his ‘cellular pathology’ and his explication of pulmonary embolism was being ‘revised’ and overturned – that pathology was reverting to ‘morbid anatomy’ and leaving Virchowian physiological pathology to wither on the vine. The distinction between the process that creates pathology or disease on the one hand, and the pathological anatomical end-point on the other, was yet again being blurred. Dickson (2004) surveyed various attempts made during the early 20th century to define the combination of elements required for thrombogenesis, emphasising that until the 1950s none of them explicitly referred to Virchow. For a time, some workers in the field sought to argue that DVT had two distinct aetiologies: one infectious, causing vein-wall inflammation and pain; one non-infectious and inherently asymptomatic. The latter was held to entail a higher pulmonary embolism risk than the former. This concept, which was first suggested by Welch (1899), is the root of the archaic and

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counterproductive distinction between ‘thrombophlebitis’ and ‘phlebothrombosis’. Loeb (1903), writing in the year after Virchow’s death and struggling to understand the relationship between thrombosis and normal haemostasis, suggested that the relevant causal factors in thrombosis were (1) the constitution of the blood, (2) the influence of surrounding tissue(s) upon ‘coagulable fluids’, and (3) the influence of foreign bodies such as bacteria and ligatures. Cordier (1905) was convinced that postoperative DVT invariably arose from sepsis and inflammation of the vein wall. A few years later, Bland-Sutton (1909) proposed four causal factors: (1) ‘mechanical disturbances in the blood’ (notably turbulence), (2) alterations in the blood, (3) endothelial lesions, and (4) sepsis. Both these authors believed that blood-borne infections, or at least infectious organisms, could be thrombogenic; but Bland-Sutton also included turbulence of blood flow, the role of which had apparently been mooted by Humphry11 in 1881. Slowing of the circulation, vascular injury, toxic effects (notably on the blood corpuscles) and bacteraemia, along with changes in coagulability, were listed in various combinations by Wilson (1912), Duckworth (1913) and Aschoff (1924). Aschoff had conducted extensive studies of outstanding quality during the First World War, which we shall discuss later. Well into the 1930s (before antibiotics became available), some authors continued to presume that infection had a pivotal role in thrombosis, along with trauma, dehydration, intimal injury and interrupted circulation (e.g. Bancroft and Stanley-Brown 1932). This was presumably because each new generation was baffled to explain why and how words such as thromboplebitis had been inducted into its lexicon. After the Second World War it became apparent that antibiotics did not decrease the incidence of DVT (Gibbs 1957), and the idea is seldom mentioned nowadays; thrombosis is only occasionally, and indirectly, associated with infection. But the term ‘thrombophlebitis’ survives, like a recurrent dream, though it is now supposed to denote only a swollen, hard, tender vein rather than bacterial colonisation of the vascular system (Ogston 1987). Its durability testifies to more than a century of misunderstanding of Virchow and to the long-sustained dominance of the mechanistic perspective.

7.5

Continuation of the Pathophysiological Perspective: Welch and Aschoff

Welch (1887) posed a new question: ‘What is the proper status of experimental thrombogenesis? Are its “experimentally produced thrombi” analogous to autochthonous thrombogenesis?’ His 1887 essay and his entry in Allbutt’s System of Medicine (Welch 1899) addressed these important issues and sourced much of the information

11

According to Dickson (2004), Humphry suffered venous thrombosis so he was personally motivated to elucidate its aetiology. He reasoned that eddies created in valve pockets might directly cause the deposition of fibrin. This focus on valve pockets reflects Virchow’s minute histological observations, but the point seems not to have been reiterated until the 1950s; see Chapter 9.

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we have given in this chapter. According to Welch, it was firmly established that experimental injury to the wall and endothelium of blood vessels is regularly (invariably) followed by thrombosis at or near the site of the vascular injury. His explicit conclusion was that autochthonous and experimental thrombi are microscopically and macroscopically the same lesion, despite the ostensibly different circumstances in which they are formed. This is striking in view of the clot-like character of the ‘experimental thrombi’ discussed in Chapter 3 (Ogston 1987). Naturally, he could not explain how experimental thrombosis (in deliberately and overtly injured veins) relates to clinical autochthonous thrombosis (in ostensibly uninjured human veins). Clinicians and pathologists evidently reserved judgment on his equation of (a) experimental thrombosis produced by artefactual injury to the endothelium with (b) autochthonous thrombosis (DVT) in which the mural endothelium is untraumatised and ‘uninjured’ (but the valve cusp endothelia are ignored). This equation was to become and to remain the Achilles heel of all models that induced experimental thrombi by injuring or killing blood vessel endothelia (see Chapter 3). Residual doubt was inevitable since it was observed that DVT is predominantly silent and that no injury to vein wall endothelium had been perceived by any observer. The issue was well discussed by French (1965, 1967b). This dichotomy was further reinforced in Welch’s 1899 definition of a thrombus as ‘a solid mass or plug formed in the living heart or vessels from constituents of the blood’. The terms ‘mass’ and ‘plug’ are interesting; it seems that Welch could not bring himself to use ‘clot’ in this context. Apparently, it had again become controversial whether a thrombus is analogous to an in vitro ‘clot’, and whether in vivo ‘white mass formation’ and in vitro clot formation can be considered the same lesion/entity. Welch’s opinion is apparent in his choice of vocabulary. In his classic Lectures on Pathology (1924), Ludwig Aschoff displayed a pathophysiological orientation that was occasionally mixed, but not integrated, with the mechanistic standpoint. For instance, he pictured a mechanistic explanation of thrombosis in terms of his sandbankbildung allegory. On the other hand, the metaphorical likeness he proposed between the microscopic appearance of the white thrombus and the skeleton of a coral placed him firmly in the Virchowian tradition. He considered this white structure to be the unique feature of thrombogenesis, rather than the redness-of-thrombi which he, like Hunter, recognised as incidental. He made his ‘vital property’ presumptions explicit in Lectures on Pathology. On p. 256 he wrote: ‘The vital question we can state with certainty: ‘Do thrombi arise in flowing or stationary blood stream’? As long as the view was that thrombi are built up from leukocytes, as Zahn pointed out, it had to be assumed that only in flowing blood could these masses be laid down. But when it became practically certain that thrombus originates almost exclusively from blood platelets, uncertainty again arose …’. The valve cusp hypoxia hypothesis (to be detailed in the following chapters) is in many ways inherent in Aschoff’s writings, as it is in those of Lister before him. Unfortunately, Aschoff chose to focus on the deaths of platelets rather on than their living response to the deaths of endothelial cells. He addressed his own rhetorical question, writing: ‘Why do platelets build themselves up into this peculiar framework?

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… To this important question I can give no final answer.’ However, in his next literary breath, he wrote: ‘Blood stream builds up lamellar system … from the sand-like multitudes of blood platelets … [from] … to-and-fro movements of waves of blood’. ‘Sand-like’ reiterates the mechanistic orientation; and ‘to-and-fro movement of waves of blood’ illustrates the extent to which ideas about thrombosis carried (and very likely still carry) mediaeval, pre-Harveian connotations (see Chapters 8 and 9, where we also discuss valve cusp movements). Aschoff went on to discuss the work of von Recklinghausen and Rehbok concerning eddies in the blood stream, though these thinkers had focused mainly on the observable effects of damming a stream and thus causing walze (helix motion), reverse flow and sandbankbildung – without considering valve pockets. Nor did Aschoff himself consider the valves; they are not mentioned in his glossary. Yet he concluded that: ‘All these experiments are … merely suggestive. We can take no account of the part played by living blood and the living vessel wall …’, again evoking Virchowian vital-materialism. Had he taken Lister’s work into account and asked himself about living blood passing dead vessel walls, his inferences may have been different. Later on in his discourse, on p. 264, he toyed inconclusively with that question, recognising that: ‘… defect in the heart’s action leads to a general slowing in the venous system’ and that ‘it is the retardation, not the stagnation, which must be reckoned the direct cause of the platelet conglutination thrombosis (Eberth and Schimmelbusch)’. On pp. 265–266 there are further hints of philosophical inconsistency: ‘We will now take up a third condition, which used to play a chief role in the teaching about thrombosis, but whose significance was greatly limited by Virchow. I refer to alteration in the vessel wall itself … Do platelets remain bound together only when the endothelium is damaged? … Covering this point of endothelial damage, the importance of which has always been accepted without question, and which has always been given great prominence in the literature, we know practically nothing …’ [our emphases]. On the following pages he approached even closer to the valve cusp hypoxia concept: ‘We must endeavour to get a clear idea of platelet adhesion… It is quite possible that the endothelium dies as a consequence of being covered by a layer of platelets … Or, the platelets, after lying on the wall for a time, may themselves die’. And he concluded: ‘If the slowing of the blood stream, and altered conditions for the platelets, are to figure as direct factors in thrombus formation, then we must consider indirect factors – changes in the wall, alterations in cardiac action, and loss of blood at operations’; in a word, underperfusion of tissues. We have quoted these extracts in extenso to highlight the unresolved admixture of vital-materialist and mechanistic thinking in Aschoff’s work. We shall return to his excellent First World War autopsy studies in a later chapter. Despite the mechanistic elements in his work, Aschoff (1866–1942) seems to have been the last pathologist to carry forward Virchow’s paradigm of disease (cellular malfunction = pathophysiology), a finger in the dyke to restrain the tide of mechanistic materialism that proceeded during the 20th century to wash away

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the ideas of Hunter, Virchow and Lister. We saw the results of this deluge in Chapters 2 and 3.

7.6

The Role of Leukocytes Reconsidered

How should thrombosis researchers have reacted to the validation of phagocytosis by Metchnikoff in 1892? Aschoff (1924) wrote, referring to Zahn’s markings and his own Kopfsteil/Halsteil distinction: ‘Along with the explanation of this marking stands or falls the whole problem of thrombus formation, so far as consideration of the majority of cases of autochthonous thrombosis goes’. This pronouncement was generally ignored for the reasons discussed earlier: the involvement of leukocytes in thrombosis was dismissed after the 1880s. In the early 20th century, the worker who came nearest to explicating thrombosis in terms of leukocyte and platelet sequestration and margination was Sandison (1931). Sandison had invented the rabbit-ear chamber, a device in which blood vessels are encouraged to grow between two plates of transparent material so that they may be studied as blood circulates through them. Experimenting on neovasculature in this chamber, he unintentionally and atraumatically produced neophyte ‘white thrombi’ in recently unperfused and then reperfused microvessels. Inadvertently, he racked the microscope objective down on to the glass coverslip over the thin sliver of tissue that had grown between it and a Perspex sheet. When he had racked it back up, thus removing the pressure on the chamber and the enclosed living tissues and allowing blood flow to be restored, he noted that passing leukocytes and platelets began to marginate spontaneously on the lately compressed and thus suffocated endothelium, and to congregate on the luminal surface of the vessel he was observing. He described his observation very beautifully (his Fig. 4 is reproduced here as Fig. 7.2). By calling the conjoined platelets and leukocytes ‘thrombi’ he seemed to realise its significance. Sandison wrote: ‘In another region a different kind of thrombus was seen to form (Fig. 4). This occurred in a wide capillary which was a direct connection between an arteriole and a venule – the thrombus forming on the venous side of the capillary. When first observed, two large, irregularly shaped cells were seen, sticking to the endothelium at one place in the lumen of the vessel and almost completely blocking the flow of blood. The nature of these cells was indeterminate because so many platelets adhered to them, they were granulated and one contained an erythrocyte’. [They were almost certainly macrophages; see Fig. 7.2.] ‘Gradually more platelets with no apparent change in their structure began to stick to two cells, until a long cylindrical mass formed and extended through the capillary and into the venule. It was not adherent to the endothelium at any point other than the two large cells. A few leukocytes adhered to this long body, but no erythrocytes showed any inclination to do so. A second mass formed and extended through part of the other capillary branch. These two formations broke up … leaving no trace – not even

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Fig. 7.2 Formation and disappearance of a platelet thrombus in capillaries that had been ‘pressed upon’. Copy of Fig. 4 from Sandison (1931). Sandison’s legend read: ‘The whole process covered a period of four and a half hours. Key: Plat. Throm. = platelet thrombus; P.L.M. = polymorphonuclear leucocytes; L.C. = two large cells in the blood stream, one containing an erythrocyte; N. C. = no circulation. X 310’. For Sandison’s explanation of ‘pressed upon’, see text. The inadvertent compression of the specimen would have caused instant and severe hypoxaemia in the tissue between the slide and coverslip lasting until the objective was ‘racked up’ again. Therefore, the changes seen were consequent on temporary underperfusion of living rabbit ear tissue and circulatory envelope and white blood cells

the ‘stickiness’ of any of the cells’. Although the author was explicit about the thrombus-like nature of the cellular association in the venule, his vocabulary (‘stick’, ‘adherent’, etc.) is notably mechanistic. Nevertheless, this work was largely ignored. It seems that thrombologists did not read the Anatomical Record. Sandison’s results show that temporary circulatory impairment is observably disturbing. If the circulating cells only ‘stick to’ injured endothelium, which is presumably inactive, the ‘large cells’, polymorphs and platelets must be the active elements. The long plugs that form evanescently while the flow is occluded by the intravascular platelet mass may either (i) stabilise should circumstances deteriorate further, stopping the vessel more permanently, or (ii) dissipate if matters improve. Clearly, living blood cells actively shut down the local circulation in the event of local (hypoxaemic) injury, but in the situation of the experiment this response is an ‘over-reaction’; beyond the next valve, the vein is actually uninjured. Even if the valve cusp were holed (cf. Fig. 9.3) the injury would not be disastrous, as it would be if (say) a retinal vessel were at risk of perforation. Bizzozero (1882) had observed the same phenomenon after crush injury to small mesenteric blood

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vessels and described the accumulation of both platelets and leukocytes at the site. According to Eberth and Schimmelbusch (1886) and Welch (1887), platelets congregate first at the site of injury, leukocytes later. Sandison made no such distinction, but was impressed by the formation of a ‘frond’ of massed platelets and leucocytes and ‘large cells’. He appeared to consider that the last-named were the cells that (primarily) ‘stuck to’ the endothelium in his preparation. Platelets then ‘stuck’ to these, forming long cylindrical platelet fronds of ‘white thrombus’. The leukocyte to platelet ratio in a thrombus should be in the order 1:50 if it simply reflects the relative numbers in the circulation (5,000 µl−1: 250,000 µl−1). Only if there is a significant departure from that ratio should we suspect a lesser or greater role in the production of thrombi. Of course, the migratory and chemotactic properties of leukocytes and their involvement in repair processes are now long-established (e.g. Movat et al. 1965; Lowenhaupt et al. 1973) and have become undergraduate textbook material.12 But there is still scepticism about their involvement in thrombosis. When the first account of the valve cusp hypoxia hypothesis was submitted to the Lancet in 1968–1969, there was complete antipathy to the notion that they played any part at all in the process. The Lancet’s rejection read: ‘It is widely accepted by all scientists working in the field that leukocytes play no part in thrombogenesis but are secondary arrivals caught up in a fibrin mesh’ [our emphasis], and added the rider: ‘Zahn’s view that they do so was a mistake based on his failure to consider that frog leukocytes are analogues of mammalian platelets’ – a reiteration of the 1880s contention that leukocytes are irrelevant to DVT. Five years later, Stewart et al. (1974) presented an SEM study of primary leukocyte activity in temporarily occluded veins, directly refuting the Lancet reviewer’s assertion. Remarkably, this was the first (published) claim since Zahn and Pitres 100 years earlier that leukocytes could be legitimately considered to play some part in thrombosis, and we shall discuss it further in a later chapter. In its introduction, the Stewart et al. article referred to work on the inflammatory process stretching back over a century, and did not reference any more recent studies in thrombosis science. By that time, however, the consensus model of DVT had been established as a by-product of the mechanistic trend of thought that had dominated haematology for a century.

12 Though platelets were still largely regarded as passive entities at this time; their active role as living entities, rather than as biochemical depots that are ‘acted on’, was established later. See Chapter 2.

Chapter 8

Interrupted Circulation: The ‘Stasis’ Hypothesis and the Significance of Venous Valves

Abstract The notion that ‘blood stasis’ is a causal or contributory factor in DVT is shown to be problematic: it has Galenic connotations. ‘Stasis’ in the literal English sense (absolute cessation of movement) entails local or organismic death; it has been shown experimentally to be anti-coagulatory and therefore antithrombogenic. The use of ‘stasis’ to denote ‘retarded flow’ or ‘interrupted flow’ is confusing not only for semantic and historical reasons, but also because it focuses attention on mean blood velocity in veins rather than on the pulsatility of blood movement. We contend that the (temporary) cessation of pulsatility in venous blood movement is instrumental in DVT. Since the aetiological hypothesis developed in this book focuses on the venous circulation and particularly on the venous valve pockets (VVP), we survey the history of the discovery of venous valves and the establishment of their function by Harvey, and explore the evolution of the ‘stasis’ concept against this background. We argue that sustained non-pulsatile (streamline) blood flow in veins results in hypoxaemia in the VVP, and that this initiates a potentially thrombogenic sequence of events.

Keywords Harvey’s circulatory hypothesis, ‘blood stasis’, venous valves, pulsatile flow, underperfusion

8.1

Introduction

It is commonly asserted that ‘stasis’ is a causal, potentiating or permissive factor in DVT (Chapter 1). This notion is in various ways problematic. For instance, as Rosendaal (2005) observed, thrombo-pathology affects both arteries with rapidly circulating blood and veins with more slowly circulating blood, so it is difficult to imagine that ‘stasis’ is a precondition for thrombosis in general. More significant is the covertly Galenic assumption that ‘flow’ and ‘stasis’ can both be accommodated within a single frame of reference (see below). Whereas ‘retarded flow’ is a relative term, ‘stasis’ is absolute. Since our knowledge is cast in words, scientific terminology requires that words say what they mean and mean what P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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they say.1 Meaningful explanations can only be created from meaningful combinations of closely defined words. No explanens of the DVT explicandum can claim to be soundly based unless it is married to the paradigm of circulation of the blood (Harvey 1628), the foundation of animal physiology for almost four centuries. The context of ‘blood flow’ in Harvey’s paradigm is invariably circulatory flow, so any convention that brackets ‘stasis’ with ‘flow’ (even ‘retarded flow’) appears to subvert the best-established premise of modern physiology. ‘Stasis’ may be conceived as coexisting with ‘flow’ only if it is spatially and temporally restricted, either by vasoconstriction in a single vessel, or to localised zones such as venous valve pocket (VVP) sinuses or soleal sinuses. Harvey’s great work, Exercitatio anatomico de mortu cordis et sanguinis in animalibus (DMCS), may be regarded as both symptomatic of, and instrumental in, the change from Galenism to the modern paradigm of physiology. Although it seems obvious to us that blood circulates, it was Harvey’s work alone that made it ‘obvious’. Of course, humans must have known from antiquity that stuck pigs, and humans put to the sword, bleed to death while blood spurts from their lacerated conduits, but it did not follow that the blood either moves in bulk or ‘circulates’. Even today, few people have observed blood circulating. The circulation is a received hypothesis because someone has observed and established it beyond reasonable doubt, but only cardiac surgeons experience ‘the circulation’ or its figure-of-eight double loop. It follows that we have no right to pillory Galenists for failing to suspect the existence of ‘the circulation’; we ourselves would not guess the real mode of blood motion if we had not been prompted by Harvey’s legacy. Nor should we be surprised that when it was first published, Harvey’s radical theory provoked dissent, as Harvey himself recorded (Harrison 1967).

8.2

The Maturation of the Circulation Hypothesis

Galenic physiology taught that blood flowed ‘to-and-fro’, which logically implies that there are transient nodes of stasis (zero flow rate) at the limits of the conduits; hence our assertion that the conceptual accommodation of ‘flow’ with ‘stasis’ is covertly Galenist. Harvey’s circulation hypothesis did not envisage such imagined nodes of stasis. However, as we shall discuss, it leads to a circulatory anomaly arising from the form and function of VVP. In the first part of DMCS, Harvey expressly disproved four major physiological premises received on Galenic authority and promulgated by Renaissance teachers

1

Scientific words, verbs in particular, are often metaphorical, or in some instances allegorical: an electron jumps orbit, an atom is excited, a computer programme is self-teaching, a cannon ball describes a parabolic curve, etc. But provided that the signification of a word in its context is clear to both writer/speaker and reader/listener – i.e. provided that it is closely defined – our normative proposition holds.

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(Harrison 1967; Porter 2001): (1) that venous and arterial bloods are different and separate2; (2) that the pulmonary vein is not a blood vessel but supplies pneuma/ spirits to the arterial blood and evacuates its ‘sooty vapours’; (3) that diastole is the active phase of the cardiac cycle, sucking blood out of the veins (arteries were held to agitate the blood by their massaging pulsatile action rather than to move blood from place to place); and (4) that venous blood passed from right to left heart through invisible pores in the interventricular septum.3 Harvey marshalled compelling evidence and reasoning against those traditional beliefs: anatomical studies, experiments on live frogs, the demonstration that the heart is muscular, forearm ligation studies. Furthermore, he calculated that the heart pumps more blood in an hour than is contained in the entire body. He was led to the radical inference that the blood does not merely flow but flows in only one direction: Since all things, both argument and ocular demonstration, show that the blood passes through the lungs and heart by the force of the ventricles, and is sent for distribution to all parts of the body, where it makes its way into the veins and porosities of the flesh, and then flows by the veins from the circumference on every side to the centre, from the lesser to the greater veins, and is by them finally discharged into the vena cava and right auricle of the heart, and this in such a quantity or in such a flux and reflux thither by the arteries, hither by the veins, as cannot possibly be supplied by the ingesta, and is much greater than can be required for mere purposes of nutrition; it is absolutely necessary to conclude that the blood in the animal body is impelled in a circle, and is in a state of ceaseless motion. [Our emphasis]

Only when radical scientific claims are timely can they result in a paradigm change, and Harvey’s claim was ‘timely’ in at least two respects. First, the general idea of ‘cycle’/‘circulation’ was becoming assimilated into the new worldview of natural philosophy when DMCS was conceived. Secondly, anomalies had accumulated in the Galenic paradigm during the previous century, and as Kuhn (1970) showed, accumulation of anomalies presages paradigm change. Prominent among these anomalies was the discovery of venous valves, which made functional sense for the first time in terms of Harvey’s theory.4 We focus on this discovery because, as we

2 It was taught that venous blood is created by the liver and nourishes the tissues, whereas arterial blood transports vital spirits to the body from the heart. (Readers should appreciate the age-old prominence of the ‘vital spirits’ concept in the Galenic hypothesis). 3 The inner eye of Galenists did not ‘see’ large volumes of blood moving rapidly through hearts, so they conceived ‘movement through the septum’ as ‘seepage’, not a torrent. It would be anachronistic to suppose that Galenists imagined the whole blood volume passing through the cardiac septum. Furthermore, Harvey’s own hypothesis implied ‘invisible pores in body tissues’, later to be called capillaries, with no more microscopic evidence than the Galenists had – and this time with a ‘torrent’ of blood passing through them. Thus, while the imagination of blood motion through the heart septum was wrong, it was not culpably or stupidly so. The ‘mind’s eye picture’ received and promulgated by any era dominates contemporaneous thought, and an imperfect picture – an imperfect metaphysical presumption – cannot but be associated with imperfect understanding. But it took over a century (approximately 1535–1650) to discard the old ideology and install the new. 4 Franklin (1937) relates that ‘Harvey told Robert Boyle … that it was a consideration of the venous valves that first induced him to think of a circulation’.

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shall argue, those valves (and particularly the VVP) are directly relevant to the aetiology of DVT. First, however, we shall consider the general 17th century idea of ‘circulation’.

8.3

Connections with the Revolution in Mechanics

The conceptual juggling by which Galenist minds sought to comprehend anomalies such as venous valves recall the complex pattern of deferents and epicycles by which Ptolemaic astronomers sought to accommodate new data before the Copernican model was finally accepted. The comparison, though inexact, is suggestive. Both Vesalius and Copernicus published their seminal works in 1543, signifying that the great changes in mechanics and medical science that followed were concurrent (Chapter 4) and not altogether independent. Galileo’s famous pronouncement about the ‘Book of Nature’ was echoed in Harvey’s ‘I profess to learn and teach anatomy not from books but from dissections … not from the tenets of Philosophers, but from the fabric of Nature’. Just as the revolution in mechanics that followed Copernicus5 reached its apogee in Newton’s Principia in 1687, so the revolution in anatomy and physiology that began with Leonardo and Vesalius reached its apogee in Harvey’s DCMS in 1628. These great works both evolved under the ancient, perhaps Platonic, image of ‘the cycle’ (Pagel 1957), and represented pinnacles of advancement in their respective fields. When Harvey became a student in Padua in 1597, the new astronomical ideas were controversial throughout academic Italy. He might have been shocked when Giodarno Bruno was burned at the stake in Rome in 1600 for – among other heresies – speaking about the ‘cycle’ of the planets. Moreover, Bruno had surmised that: ‘In us the blood continually and rapidly moves in a circle’ (Pagel 1951). Bruno had spent some years in England in the 1590s and met Queen Elizabeth, so Harvey, as a teenager in Caius during that decade, may already have been alerted to his controversial opinions. We may therefore speculate that Bruno’s death sentence indirectly advertised to Harvey the ‘metaphysical power’ of the ‘planetary circulation’. This may be suggested in the dedication of DMCS to Charles I, where the king is likened to both the sun at the centre of the universe and the heart at the centre of the body.6

5

Mechanics did not begin with the Copernican model. It was an intellectual attitude to the phenomenal world that emerged during the late scholastic era – in the wake of the writings of William of Okham, Jean Buridan and Nicole Oresme – and peaked in Galileo, Bacon, Descartes and their contemporaries. But because of its subsequent influence, Copernicus’s publication was a significant (and famous) landmark. 6 ‘To the heart is the beginning of life, the Sun of the Microcosm, as proportionally the Sun deserves to be called the heart of the world, by whose virtue, and pulsation, the blood is moved, perfected, made vegetable, and is defended from corruption, and mattering; and this familiar

8.4 The Discovery of Venous Valves

8.4

107

The Discovery of Venous Valves

Several attempts have been made to identify the discoverer of those ‘membranous portals’ – allegorically called ‘little doors’ because they open and close within veins in the manner of canal locks – that were so crucial to Harvey’s hypothesis and are equally crucial to the hypothesis developed in this book (Fig. 8.1). Franklin (1927), Friedenwald (1937), Leibowitz (1957) and Scultetus et al. (2001) surveyed the Renaissance literature to identify the discoverer. Franklin named Giambattista Canano of Ferrara c.1536, whose Portuguese teacher Amatus promulgated the concept, though with errors. He also mentioned Estienne in the 1530s, and recorded that Salomon Alberti, who published clear illustrations of venous valves in Germany in 1585, had specified Fabricius’s priority as their discoverer (Fig. 8.2). Scultetus et al. gave the palm to Jacobus Sylvius and Estienne, two Parisian claimants; though the relevant work by Sylvius was published posthumously (in 1555), and Franklin asserts that Sylvius appeared to have no knowledge of valves in 1541. It is intriguing that neither commentator noted Harvey’s own account in DMCS. Harvey preferred Fabricius as their discoverer but, in a scholarly rider to his own opinion, graciously acknowledged the opinion of Riolanus (Jean Riolan, his lifelong

Fig. 8.1 Venous valves, after Alberti (1585). Engravings reproduced from Franklin (1927). The two illustrations show the outside (a) and the inside (b) views of a segment of vein with the positions of the valves clearly visible. The labelling permits the two views to be related

household-god doth his duty to the whole body, by nourishing, cherishing, and vegetating, being the foundation of life, and author of all …’

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Fig. 8.2 Diagrams of venous valves. Reproductions of Figs. 1–5 in Franklin (1927) illustrating the structures of bicuspid venous valves. Morphological, functional and pathological details are discussed in Chapter 9

Galenist Parisian opponent.7) Geoffrey Keynes’s 1928 translation of DMCS, Chapter 13, p. 55, line 10, reads: ‘Fabricius, [or, as Riolanus would have it, Jacobus Sylvius] did first of any delineate those semilunary membranous portals …’. It could seem, therefore, that Scultetus et al. (2001) spoke for the Paris school whereas Franklin spoke for the schools of Padua and Ferrara. It may likewise be inferred (from the presumed exchange between Riolanus and Harvey in 1628) that it was already ‘well known’ in University of Paris circles that venous valves, as we now call them, had been observed by the Parisian professor of anatomy, Jacobus Sylvius, almost 100 years before Harvey claimed his teacher Fabricius as the discoverer. There is no doubt that Fabricius’s exquisite description of ‘membranous portals’ prompted Harvey to conceive and construct his hypothesis; because Fabricius believed that the valves did not close completely, he failed to identify

7 Jean Riolan was the doyen of Parisian surgery/medicine and Harvey’s contemporary. A scion of eminent medical teachers on both sides of his family, he was, in 1628, a 48-year-old, dyed-in-thewool Galenist who opposed Harvey’s initiative tooth-and-nail. Harvey was 50 when he published DMCS, and 70 when Riolanus wrote his critical counterblast in 1649, which Harvey felt compelled to answer in print; see Riolan’s Opuscula anatomica (1649) and Harvey’s retaliation, Exercitatio anatomica de circulatione sanguinis (1649). It is historically notable that Harvey quoted his ‘enemy’ twice in the 1628 magnum opus.

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their function.8 However, despite the numerous claimants for true originality (Friedenwald 1937) – Estienne, Sylvius, Canano, Amatus, Vesalius, Albertus, Fabricius and others9 – Sylvius may well have seen them in the 1530s. Sylvius was Vesalius’s professor, and a staunch Galenist. Perhaps he suppressed the inexplicable observation, which had no place in Galenic orthodoxy, so he did not mention them except in his posthumous work.10 On the other hand, Canano and Vesalius met several times and exchanged knowledge (Franklin 1927). News of the existence of valves may therefore have spread from both Sylvius and Canano to Vesalius, and from Vesalius to his contemporaries in Padua and Ferrara, and ultimately to Fabricius,11 who inspired both Albertus and Harvey. Whether Estienne made the discovery before or contemporaneously with Sylvius will probably remain uncertain, but the question seems unimportant today. It seems sufficient to know that the ‘discovery of valves in veins’ began in the 1530s and was dragged out over almost a century. Would that foresight were as clear as hindsight.

8.5

The Significance of Venous Valves

The prime significance of venous valves is that they begat the scientific revolution in medicine based on Harvey’s conversion of anatomical knowledge into physiological understanding. Unlike his Galenist teachers, Harvey did not ‘imagine’ his physiological concept; he inferred that blood circulates from his failure to pass a probe ‘caudad’ – the valves did not permit it. This showed that the centrifugal blood motion entertained by Galen was impossible, and Harvey went on to deduce that the orthodox teaching of the previous 1,500 years was necessarily invalid. That single observation stimulated his 28-year (1600–1628) endeavour to prove that the ‘to-fro blood movement’ concept and the centrifugal ‘seepage’ of venous blood of

8 We should not with hindsight imagine that Fabricius was culpably wrong. Unless the inner eye has been primed with an idea of bulk venous flow, there is no reason to imagine that the valves would necessarily close and open. 9 The original sources include C. Estienne (1545) De Dissectione Partium Corporis Humani, Paris; R. Colombo (1559) De Re Anatomica, Venice; S. Alberti (1585) De Valvulis Membraneis; H. Fabricius (1603) Venarium Ostiolis Patavii, Padua. 10 He berated Vesalius for doubting Galen in 1539, when he was 50 years old, and again in 1540. The work in which venous valves are mentioned, the Isogoge, was published in 1555, the year of his death. 11 Fabricius was born the year that Vesalius assumed his professorship in the University of Padua, 1537. When Vesalius was shipwrecked and died on Zante in 1562, his anatomy department was taken over by Fallopio, whom Fabricius succeeded in 1570. Though Fabricius described ‘valve pockets’ exquisitely, he remained a Galenist until he died in 1619 aged 78. According to Harvey (1628), ‘Fabricius did not know/understand the use of venous valves’. Neither did Harvey know that his hypothesis would be the nail in the coffin of Galen’s legacy, and a revolution in medical science.

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orthodox Galenism were erroneous, and that the basic tenets of contemporaneous medical science were consequently also erroneous. There were other anomalies in the Galenist corpus. The mechanics of blood movement had excited interest since classical times and the subject was addressed on several occasions during the Renaissance, not least by Leonardo da Vinci (see e.g. Skalak et al. 1981). Vesalius cracked the Galenist mould when he established that the inferior vena cava does not originate in the liver, and that there are no perforations in the interventricular septum. Others continued to chip away at the edifice: Servetus, Colombo and Cesalpino notably described the pulmonary transit and heart valve action (Foster 1901). Colombo’s realisation that the heart is a pump (i.e. not a suction drain) enabled Harvey to conceive that life is coextensive with constantly moving blood and, by implication, that ‘blood stasis’ is co-extensive with death.12 But the discovery of the valves was central to Harvey’s argument and to the paradigm shift that followed it.

8.5.1

The Venous Valves and DVT

This vignette from medical history is directly relevant to the foundation of our explanation of DVT. The explanens to be advanced is that ‘blood stasis’ and ‘blood motion’ come together sequentially and alternately in the VVP, whenever non-pulsatile luminal centripetal blood flow fails in particular circumstances to empty the VVP sinuses at (sufficiently) frequent intervals. Just as valves were central to – and in effect created – Harvey’s refutation of the received hypothesis of 16th century medical ideology, so they constitute the major premise of the valve cusp hypoxia hypothesis, becoming the explanens of the site and cause of DVT (the explicandum). As we shall show in the following chapters, thrombi originate after blood has been sequestered for a sufficient length of time in a VVP under non-pulsatile flow conditions. This circumscribed, temporary ‘stasis’ is not the only factor involved, but we shall argue that it is fundamental. DVT is a lesion usually initiated by normal VVP, in normal blood, when a failure or temporary suspension of muscular contraction in the limb(s) leads to temporary underperfusion of the pocket. Thus defined, the twinning of ‘stasis’ with ‘intermittently moving’ blood becomes easier to conceive: local stasis in VVP is physiological when it is fleeting and short-lived, but becomes pathological when greatly prolonged. There is no ‘permanent state’ definable as ‘stasis’ in a living body. Stasis in life can only be locally and temporally circumscribed. 12

This comment can be generalised: life is coextensive with continuous fluid movement through the organism as a whole, within tissues, between cells and within individual cells. Cessation of fluid movement indicates death. This point has been developed in a number of articles, e.g. Malone (1981), Wheatley (1985, 1999), Wheatley and Malone (1987), Agutter et al. (1995, 2000), Agutter and Wheatley (2000).

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Notwithstanding its Galenist connotations, however, ‘blood stasis’ became a recurring theme in medical history. It resurfaced early in the 18th century in Boerhaave’s work and, though criticised by Hunter and Hewson, it persisted in Cruveilhier’s thesis (which Virchow in Lecture 10 of Die Cellularpathologie described as ‘mystical’). By the 1960s, as we have seen, it had again become entrenched in the consensus model of DVT.

8.6

8.6.1

The Persistence and Misleading Character of the ‘Stasis’ Concept Inherent Difficulties in the ‘Stasis’ Dogma

It might be argued that if biomedical researchers agree to use the word ‘stasis’ to mean ‘retarded circulation’, its standard English connotation (i.e. ‘absolutely no movement whatever’) is irrelevant: once everyone in a particular scientific field has consented to a specialised language in which a common word is given a definite nonstandard meaning, semantic cavils become otiose. However, there are three important reasons for rejecting a ‘special context’ for the term ‘stasis’ in a private language. The first reason is metaphysical. As discussed above, ‘stasis’ flatly contradicts Harvey’s arguments against Galenism; and since Harvey’s contribution is now universally acknowledged to be the most permanent and notable in the history of physiology, it is inconsistent to laud DMCS while insidiously undermining and denying its most fundamental philosophical and scientific implications (and implicitly preserving the rejected central flaw in Galenic theory). The second reason is factual. It relates to the presumption that static/motionless blood in situ coagulates more readily, rapidly or intensely. On three occasions during the century 1770–1876, that premise was tested experimentally; and on each occasion it was demonstrated that blood stasis (in the standard English sense) does not cause thrombosis. Indeed, the opposite was proved. The discoveries of Hewson (1771) led Hunter to conclude that Boerhaave’s haemodynamic explanation of blood coagulation was invalid (Chapter 4). But such was (and is) the force of received opinions that subsequent generations found this conclusion incredible, just as Galenists had found Harvey’s ideas. A century later, Lister (1863) and Baumgarten (1876) repeated Hewson’s experiment independently, obtained precisely the same results, and confirmed that not until over three hours of stasis had elapsed did the blood apparently begin to semi-solidify – and then only very patchily. This showed that complete stasis of blood in doubly ligated veins (comparable to ‘pockets’ or ‘sinuses’ within veins) was anti-coagulatory, and presumably anti-thrombogenic. This left only altered circulation, not ‘static’ or stopped blood, as an unequivocally and manifestly thrombogenic factor. The third reason is conceptual. Ever since Harvey’s hypothesis was corroborated by Malpighi (cf. Cater and Silver 1961), it has been axiomatic that incessant movement

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of body fluids and circulation of the blood are coextensive with the living state in vertebrates. The natural corollary must be that cessation of fluid movement is co-extensive with local or total death. The infelicitous use of ‘stasis’ as a surrogate for ‘altered circulation’ therefore implies accordance with the extreme mechanistic position, which excludes the vital-materialist perspective and treats each component of the live body ‘as though it were dead’. The ‘stasis’ element in the consensus model of DVT signifies the exclusively mechanistic character of that model. Our central tenets in this debate are therefore: (a) that ‘almost stagnating’ is not ‘static’ and (b) that ‘local alteration of blood flow’ does not mean ‘stopped’. Conceptual opposites, however, can sometimes overlap.

8.6.2

The Survival of the ‘Stasis’ Dogma in the 19th Century

The survival of the ‘stasis’ dogma despite its rejection in the writings of Hewson, Hunter, Virchow, Lister and Baumgarten might owe something to the work of 19thcentury microscopists who saw blood moving and stopping within minute vessels. Cameron (1952) listed a number of these studies. Despite the limitations of the pre-Lister microscope, Gruithuisen (1811) recognised cessation of blood circulation (stasis) as a concomitant of ‘inflammation’. Thomson (1813) reported arterial constriction and drastic slowing of capillary flow in chemically poisoned tissues.13 A brilliant microscopic study by Dutrochet (1824) showed ‘colourless corpuscles’ marginating on and diapedesing through the endothelium during the pathogenesis of inflammation; Virchow didactically rejected ‘diapedesis’, though we recognise it today as an essential part of the response to injury. Dutrochet’s findings anticipated the observations of workers such as Travers (1844), who clearly described the stasis and margination of ‘colourless corpuscles’ (white cells) in blood vessels and their diapedesis through the endothelial boundary. Addison (1846) applied a crystal of poisonous salt externally to a frog’s web and found that the sequestration and margination of ‘lymph globules’ congested the small vessels to such an extent that ‘by the following morning the whole interior of the inflamed vessels appeared to be lined with lymph globules’, resulting in ‘stasis’. Waller (1846) gave experimental proof of leukocyte migration during inflammation and engorgement of vessels, accompanied by ‘stasis’ and reflux currents. Cohnheim (1889) repeated these various observations using greatly improved microscopes. Studies of this kind continued into the 20th century (e.g. Tait 1918; Sandison 1931). Such achievements helped to establish an association between thrombosis and leukocyte congregation and the cessation of blood movement, and they focused attention on the cellular as well as the soluble

13 Of course, it is well known that capillary blood movement is variable under normal physiological circumstances, not least because capillaries have smaller diameters than blood cells.

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components of the blood. By subsequently establishing the role of platelets in haemostasis, they presaged our modern account of coagulation (cf. Chapter 7).

8.6.3

‘Stasis’ and the Consensus Model of DVT Aetiology

It has long been known that patients paralysed and immobilised by stroke and spinal cord injuries, or otherwise immobilised, are at increased risk of DVT (e.g. Malpother 1880), especially in the lower limbs. Prolonged bed rest, for instance after surgery, is notorious in this regard (Wright and Osborn 1952; Gibbs 1957; McLachlin et al. 1960; Browse 1962a, b; Doran et al. 1964), and particularly after spinal anaesthesia or injury (Cerilli and Engell 1966; Merli et al. 1993) and after strokes (Warlow et al. 1972, 1976; Gibberd et al. 1976). By the 1930s, knowledge about the coagulation mechanism was accruing (Chapter 5) and it was becoming apparent that ‘stasis’ alone was insufficient to initiate thrombosis (McCartney 1927; Owre 1929). However, Belt (1934), Homans (1934) and Frykholm (1940) restated the principle that impairment of blood movement is involved in the chain of causation of DVT.14 Scott and Radakovich (1949), Massell and Kraus (1950), Ochsner et al. (1950) and others further indicted circulatory ‘stasis’ as a contributing factor (Ochsner et al. also mentioned ‘antithrombin levels’). Wright and Osborn (1952) provided experimental evidence that venous flow was reduced in recumbent limbs and probably promoted the development of positive approaches to early post-operative mobility, such as IPPC and elastic stocking enhancement of venous return. The general view seems to have been that ‘reduced blood flow’, and the resultant local ‘pooling’ of blood in the veins, facilitates interactions between blood constituents and the vessel wall or alters the balance between activated coagulation factors and inhibitors within the blood stream (Mammen 1992). Thus, by the 1960s, ‘venous stasis’ had come to be accepted as a contributing cause of thrombosis (e.g. Zweifach 1963) because – notwithstanding unequivocal evidence to the contrary – it was imagined to promote local coagulation. There were occasional doubters (Poller 1960a, b), but the consensus was established: Mustard et al. (1962), Wessler (1963) and others formulated the ‘stasis and hypercoagulability’ model of DVT discussed in earlier chapters.

14 Aschoff (1922, 1924) inferred from his studies of First World War victims that retardation of circulation contributed to thrombosis, but also believed that hypovolaemia resulting from wound haemorrhages contributed, contracting the circulating volume and the vascular bed. Unluckily, blood transfusion and volume replacement were then in their infancy. Aschoff’s detailed work and careful interpretations of his observations were well regarded by subsequent workers in the field, but his point about the thrombogenic potential of hypovolaemia elicited little further comment (cf. Richards 1944). Operative blood loss and the risks attendant on a depleted blood volume during and after surgical procedures have been underestimated by many physicians, but were re-emphasised by e.g. Le Quesne (1967) and Delikan (1972). Aschoff’s work will be discussed in detail in later chapters; it is curious that he made no mention of valves in his discussions of DVT – this may have contributed to the effective ‘suppression’ of Virchow’s discovery (1858) until Paterson and the McLachlin brothers ‘rediscovered’ it in the 1950s (see main text).

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8.6.4

8 Interrupted Circulation: The ‘Stasis’ Hypothesis and the Significance

Sevitt on the Aetiology of DVT

Sevitt’s deployment of the word ‘stasis’ in a leading article in Lancet (Sevitt 1961) indicates how deeply entrenched the usage had then become: That venous stasis, another member of Virchow’s Triad, also plays a part in the pathogenesis of thrombosis in the lower limbs and pelvis rests on strong, if circumstantial, evidence. Firstly, the blood may take minutes to pass along the thigh and leg veins when the limbs are supine, horizontal, and immobilised especially in elderly patients: secondly, venous thrombosis is particularly common in middle aged and elderly patients confined to bed after operation or injury, or for medical reasons – and, generally speaking, the longer the bed rest, the greater the frequency and extent of thrombosis: thirdly, at necropsy, the location of primary thrombi in valve pockets and at vein junctions is consistent with particularly stagnant pools in relatively stagnant channels. … Does stasis act mainly through local silting of platelets, leukocytes, and some red cells onto the endothelium or is the endothelium also abnormal? Histological examination of recent thrombi indicates that they are laid down on normal endothelium, but ordinary histological methods may be too crude to reveal subtle changes. Experimental work has shown that carbon particles and platelets adhere to vessel linings after endothelial injury, apparently at intercellular cement lines, and there is some evidence that local endothelial degeneration may follow anoxia of a segment of vein. Possibly endothelial anoxia follows prolonged venous stasis: and, if this be so, such anoxia is most likely where stasis is most evident – that is, at valve pockets and in venous saccules.

Sevitt’s reiteration of the likelihood that thrombi arise in VVP was highly significant. This had scarcely been discussed since Virchow15 first unequivocally illustrated valve pocket thrombi in Die Cellularpathologie (Chapter 6; Fig. 6.1). Likewise, remarkably few authors had posited that ‘anoxia’ caused by ‘stasis’/underperfusion could cause endothelial lesions. Finally, the suggestion that endothelial injury (a pathological lesion) might be ‘too subtle’ to be observable by ordinary methods was singularly perspicacious. (These points will be developed in the following chapters.) Sevitt also acknowledged the well-established relationship between impaired circulation and decubitus, though he was guarded about the evidence linking it to DVT. Notably, however, he used ‘stasis’ as a synonym of ‘impaired/interrupted circulation’, a semantic infelicity compounded in the oxymoron ‘relatively stagnant’. Several subsequent papers by Sevitt included superbly detailed illustrations of thrombi (Sevitt 1966, 1973, 1974a, b), which we shall discuss later. He believed that ‘stasis’ results primarily from dilatation of the veins caused by muscle pump paresis and inactivity, and that this must be exacerbated if arterial perfusion of the muscles is impaired, for example through congestive heart failure (Welch 1900; Belt 1934) or after surgery. However, we do not accept that ‘linear velocity’ is the main circulatory parameter of concern; we shall elaborate this claim shortly.

15 Paterson and McLachlin (1954) found that incipient thrombi were most abundant on the valve cusps. Dr. Simon Sevitt demonstrated venous valve thrombi in his dissections of venous valves in human leg veins at Birmingham Accident Hospital in 1958–1960 (PCM attended these). Sevitt had corresponded with J.C. Paterson, received copies of his joint papers with the McLachlin brothers, and subsequently taught that all venous thrombi originated in VVP.

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Sevitt and Gallagher (1961) identified six main sites of origin of deep venous thrombi: the iliac, common femoral, deep femoral, popliteal, posterior tibial and soleal veins and other intramuscular veins of the calf (see also Gibbs 1957). Very high incidences of DVT at necropsy, particularly in small veins in the calf, plantar and thigh adductor muscles, were also noted by Rössle (1937), Neumann (1938), Frykholm (1940) and Hunter et al. (1941, 1945). The inference that many thrombi have a peripheral origin was supported by pioneering phlebographic studies by Bauer (1940). Linear blood velocity is likely to be most markedly reduced in these vessels during prolonged bed rest and/or when arterial perfusion is impaired; but (more pertinently in our view) the veins listed by Sevitt and Gallagher are also the richest in valves.

8.6.5

The Consensus Model and the VVP as Sites of Thrombogenesis

In the ‘traditional’ form described earlier in the book, the consensus model holds that neither stasis nor oversensitivity of the coagulation cascade alone can suffice to induce thrombosis; the belief is that both conditions must be satisfied. Later debates about variants of the consensus model (e.g. Sixma 1980; Schaub et al. 1984) focused particularly on the association and relationship between the platelet plug and the vein wall (Chapter 2). Hume et al. (1970), having recognised that thrombi form in VVP (as shown by Virchow 1858), advanced a hypothesis that attributed thrombosis to a combination of ‘valve pocket anatomy’, ‘stasis’, ‘eddies’, ‘hypercoagulability’ and ‘consequent’ thrombin production. The ‘stasis and hypercoagulability’ allegory proposed by Hume et al. (1970) contended that blood cells16 ‘silt’ in VVP or venous saccules because low velocity eddy currents induce fibrinogenesis – either because the ‘silted platelets aggregate and activated coagulation factors are brought in from distant parts of the circulation’, or because ‘ADP is released from the accumulated erythrocytes and leukocytes’, or for both reasons. Later, Thomas (1987, 1988), one of the authors of this variant of the consensus model, explained localised thrombosis in VVP by proposing that ‘thrombin is generated in areas of retarded flow’, implying that VVP are zones of retarded flow and that ‘stasis’ is a ‘permissive factor’. The belief that ‘activated coagulation factors’ are present physiologically in the blood stream, making low-level fibrinogenesis continual, is hypothetical and questionable (Chapter 2); nor is it clear why accumulated blood corpuscles (suspended, not attached to the vein wall) should release ADP in effective quantities

16 This version of the ‘stasis and hypercoagulability’ model is interesting in that it recalls the ‘sludged blood’ papers by Knisely and co-workers (Knisely and Bloch 1950; Knisely 1951; Reneau et al. 1969). In Chapter 7 we criticised the notion that platelets and other cells ‘silt’ passively.

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or otherwise precipitate thrombosis. The Hume et al. account is therefore highly speculative17. Such products could conceivably accumulate if platelets are indeed actively sequestered in very slowly revolving blood vortices in VVP (Chapter 9). Also, activated coagulation factors might be less readily removed under conditions of reduced blood velocity. However, to make these speculations real, our ‘inner eye picture’ must detail how they might be thought ‘causal’ rather than ‘contributory’. They remain unproven. There has been no measurement of ADP release, or activated coagulation factors, or thrombin, in VVP. Moreover, were the blood temporarily trapped in the VVP to be ‘dead’ (because non-circulating cells/platelets are killed by prolonged local hypoxaemia), would thrombin be activated and would coagulation ensue? This suggestion is implausible: the findings of Hewson (1771), Lister (1863) and Baumgarten (1876) indicate that cells in this trapped blood must indeed be dead or dying.

8.7

Is the Current (Mis)Use of ‘Blood Stasis’ Equivalent to Virchow’s ‘Interrupted Circulation’?

The short answer is ‘no’. In his discussion of pulmonary obstruction by an embolised thrombus, Virchow wrote18: ‘This means that the list of possible consequences of the obstruction could be grouped into three categories …’, and went on to delineate the original ‘Virchow’s triad’ (Chapter 6). The 3rd of the ‘phenomena’ that he listed was ‘phenomena due to the interruption of the blood-stream’. This referred only to the cessation of blood flow in the obstructed pulmonary artery, and the description was unexceptionable: there can be no doubt that the blood stream of which he wrote was ‘interrupted’ – by embolisation of an already-formed thrombus. But this has nothing to do with the cause of thrombus formation in a peripheral vein, which takes place while the blood is still moving; we recall that ‘the doctrine of stasis rests on manifold misinterpretations’. The unthinking use of ‘Virchow’s triad’ as a general descriptor of DVT aetiology has therefore led to a serious semantic confusion.

8.7.1

Pulsatile Blood Movement in Veins

Harvey’s successors knew that the circulation is not a steady-rate flow. However, by concentrating on ‘flow rates’ for 400 years, they have played down its no less important intermittency or pulsatility. Physiologically normal blood movement is pulsatile – in a sense ‘interrupted’ – and we suggest that venous blood flow becomes pathogenic when it is not ‘interrupted’ in this sense. This key point in our argument must be explained fully. 17 18

Hume’s opinion changed within the following decade; see Hume (1985). See p. 110 of the 1998 translation of Thrombose und Embolie.

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Cardiac function involves systole and diastole, and blood flow responds to the cycle of pressure changes. However, a thoracic heart cannot (unaided) circulate blood around an organism more upright than an earthworm, snake or centipede. The thrust of cardiac contractions (vis-a-tergo) is greatly reduced by the time blood leaves the organs and enters the collecting venules, so a pumping19 action, which entails pulsatility, is provided by calf muscle contractions: the ‘peripheral venous heart’ (McLachlin and McLachlin 1958). Just as cardiac systole drives blood into the arterial vessels, so calf muscle contraction squeezes blood upwards against gravity through the veins of the lower limb; and just as cardiac diastole generates no blood movement along the arteries, so no blood movement along the veins is generated when the calf muscle relaxes (‘peripheral venous diastole’). Thus, normal flow in all blood vessels, both arteries and veins, is eternally pulsatile or ‘stop-start’. This is why we assert that physiologically normal circulation is ‘interrupted’, or at least ‘intermittent’: high-volume alternating with low-volume flow, or fast with slow flow or zero. Franklin (1937) addressed problems of venous flow and pressure, though most of the work he described was done on non-human animals, and in the horizontal rather than the erect position. (Quadrupeds, like humans, are prone to faint in the legs-down posture, or when centrifuged with the head central.) However, some of Franklin’s quoted sources are generalisable. For instance, Todd and Bowman (1856) wrote that ‘muscular movements likewise favour the venous circulation as is well shown during venesection – when the patient is instructed to move his fingers freely, the flow of venous blood is seen to increase immediately’. That was unequivocal evidence that ‘muscle pumping’ increases venous flow volume and rate. Rheological parameters of normal blood flow are important aspects of haemodynamics, but the essential intermittency and pulsatility of all blood flow, dependent on the vigour of systole and diastole in the (cardiac and calf muscle) pumping processes, are the significant determinants of normality. Consequently, prolonged non-pulsatile streamline venous flow – and a concomitant failure to achieve active sole compression by weightbearing – is abnormal and pathophysiological. In agreement with Schina et al. (1993), we argue that it is potentially thrombogenic. Thrombo-pathogenesis depends on the failure to drive blood back up from the feet to the heart.

8.7.2

Compression of Veins in the Soles of the Feet

When a foot is lifted from the ground, so that it is not supporting the body weight, the veins in the sole can fill with blood. When the next step is taken and the weight of the body is taken on that foot, blood must be squeezed from the veins of the sole into those atop the metatarsals and forefoot around the ankle, much as water can be

19 ‘Pumping’ is of course metaphorical. What is commonly called the ‘calf muscle pump’ is not literally a pump.

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wrung from a wet towel. Centrifugal reflux through venous valves is prevented by their normal functioning, but refluxing blood swirls into them. There is evidence that even the smallest veins have valves: the findings of Phillips et al. (2004) imply that the ‘canal-lock’ mechanism20 is ubiquitous in veins. When normal muscle bellies contract, they increase venous pressure and pulsatility by squeezing and emptying the muscles into the venules. Walking/motion pressures normally suffice to open the valve cusps against the weight of the blood column in the veins. When the distal pressure is less than central pressure and blood tends to reflux, competent valves close automatically and tightly and their sinuses billow. The pathological implications will be discussed in Chapter 11. von Recklinghausen (1883) found that the venous pressure in the feet in the standing position was below the theoretically expected hydrostatic pressure ‘on account of muscle movements that normal persons are unable to “prevent”’. Clearly, the pressure in foot veins is reduced by the venous valves. A series of closed venous valves at/above a particular point can mean that the venous pressure in a foot may be no more than a few centimetres (in the healthy non-varicose limb), i.e. the ‘weight’ of a very short blood column rather than the whole 4 ft in the average person. The closed valves support the blood column so that it does not ‘weigh upon’ the foot veins.21

8.7.3

Effects of Standing, Sitting and Lying on the Dynamics of Lower Limb Veins

Atzlet and Herbst (1923) showed that fluid and blood accumulation in the feet of healthy persons always increases during a day spent in the erect posture. The increase was pro rata when sitting for an hour, standing for an hour, sitting in a higher chair, and standing for 1, 2 or 3 h – at which it was maximal. Resting the feet up for half an hour reversed the leg swelling, and walking for an hour reduced the effect of the vertical posture. In short, gravity affects blood and water distribution in the body even in full normal health. In the erect position, the leg muscles work to return blood to the right heart by involuntary and insensible contractions akin to blinking of eyelids, sneezing, swallowing motions and limb fidgeting. VVP are probably minimally functional when subjects are recumbent in bed, though controlled observations have shown that subjects turn in their sleep 35 times

20 Harvey (1628) realised that the venous valve mechanism is akin in principle to that which lifts barges up flights of canal locks, as did those who first named the valves ‘portiunculi’. The scanning electron micrographs of Phillips et al. (2004) showed 2,376 valves in vessels no more than 300 µm in diameter in 410 cm3 of subcutaneous tissue, supporting the presumption that valves are a constant finding even in the smallest venules. 21 The variations likely in persons with varicose veins caused by venous pathology are unimportant in respect of DVT.

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a night on average and thus use leg and other muscles unconsciously every 15 min on average, presumably emptying their VVP when turning over. In sleep, as in active waking life, the same venous blood movement mechanism seems likely to obtain.

8.8

Towards the Valve Cusp Hypoxia Hypothesis: I – Altered Blood Movement

These considerations cast new light on the evidence cited in Section 8.6.3, previously interpreted in terms of the consensus-model view of ‘venous stasis and DVT’. We can now begin to examine our alternative model, the valve cusp hypoxia hypothesis (VCHH), and some of its implications. Whereas arterial blood flow is powerfully pulsatile, venous flow is only weakly so, but its pulsatility is fundamental to the maintenance of physiological normality. While slow arterial blood flow may underperfuse the whole body, we contend that steady non-pulsatile flow in the veins – irrespective of velocity – is thrombogenic. This pathological outcome may arise whenever valve cusps are no longer ‘flapped’ by pressure changes and the VVP are not emptied and refilled for a longer than ‘normal’ period. Non-pulsatile blood movement simply cannot perfuse VVP. This proposition is the first element of the VCHH: it describes a situation that may under certain circumstances initiate the thrombogenic process. A central contention of this book is that ‘slow blood flow’ in veins, alias ‘venous stasis’ and ‘retarded blood flow’, has distracted thinkers from the dynamics of the circulation as a whole. Specifically, it has led many in the field to overlook the pulsatile intermittency of the circulation. This pulsatility works the valve mechanisms of heart and veins in normality, contrasting with steady, non-pulsatile, streamline flow under pathological circumstances. In Chapter 9 we will focus on the structure, physiology and malfunctioning of venous valves and enlarge on the conceivable consequences of VVP underperfusion.

Chapter 9

Underperfusion of Valve Pockets and the Initiation of DVT

Abstract If the first stage in the aetiology of DVT is sustained underperfusion of the valve pockets (VVP) during extended periods of non-pulsatile flow, then the structure, function and pathology of venous valves are of crucial importance. The questions to be answered are: how might underperfusion of a VVP lead to thrombosis, and where in a valve would thrombus formation be initiated? This chapter reviews the accumulated evidence that VVP are indeed the sites of venous thrombogenesis, describes valve morphology and function, and considers how these may be changed under pathological conditions. At the end of the chapter we propose that hypoxaemia in long-underperfused VVP leads to endothelial hypoxia, specifically of the inner (parietalis) surface of the valve cusp(s), and that this may under certain conditions become the prelude to DVT. In the light of this proposal, we calculate the approximate time needed for non-pulsatile venous flow to become pathogenic.

Keywords Local hypoxaemia, venous valve pocket, valve cusp, parietalis endothelium, endothelial hypoxia

9.1

Introduction

Studies during the 1950s, 1960s and 1970s (Chapter 8) implied that non-perfusion of venous saccules and valve pockets (VVP) is causally related to DVT. However, it is historically inaccurate and semantically misleading to associate this claim with Virchow’s use of the phrase ‘interrupted circulation’. In Chapter 8 we emphasised that normal venous return to the right heart, particularly from the lower limbs, is inherently pulsatile under normal physiological conditions; i.e. ‘interrupted (or intermittent) circulation’ is the norm for veins as well as for arteries. Thus, streamline or continuous blood flow in veins is physiologically abnormal and, we suggested, is fundamental to the aetiology of DVT because it results in underperfusion or non-perfusion of the VVP. Following this line of reasoning, we now consider the structure, function and malfunction of the venous valves and their relationship to venous blood movement and to the causation of venous thrombosis. P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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Thrombi Originate in the Venous Valve Pockets

In the early 1950s, it was explicitly shown – for the first time since Virchow’s illustration (Figs. 6.1, 9.1) – that venous thrombi form almost exclusively1 in the VVP (McLachlin and Paterson 1951; Paterson and McLachlin 1954; Paterson 1969).2 These careful studies ‘categorically’ excluded ‘local injury to the vein wall, of an obvious type, [and] abnormality in those blood coagulation factors that were studied’ from the causes of thrombosis, leaving the authors to deduce that ‘venous stagnation’3 was primarily relevant (Fig. 9.2). Paterson and McLachlin (1954) wrote:

Fig. 9.1 Venous thrombi form in the valve pockets: (a) Fig. 2 from McLachlin and Paterson (1951). The original legend reads: ‘Case of multiple venous thrombi. The lower one is situated in a valve pocket at the upper end of the superficial femoral vein: the middle one on the left is situated in the proximal end of the profunda femoris vein, while the upper thrombus is lying in the common femoral vein by the junction of the long saphenous vein’

1

Although we have written ‘almost exclusively’, this may be over-cautious. In fact, we firmly believe that no thrombus forms other than in VVP. We cannot envisage DVT arising other than in the unique environmental circumstances of an ostial or parietal venous valve, because it is impossible to imagine so extraordinary a concatenation of ‘causes’ anywhere else in the venous system. Readers who cavil at this didactic statement might bear in mind the plethora of venous junctions with main veins, in which hundreds (rather than tens) of ‘single cusp’ ostial valves complement the more obvious, Harveian, parietal valves. 2 Angus McLachlin had previously worked with K.J. Franklin (see e.g. Franklin and McLachlin 1936), whose seminal studies on vein and valve morphology were mentioned in Chapter 8 and are further discussed below. Since Franklin had a thorough knowledge of the history of the field, it seems likely that McLachlin – and ipso facto his brothers and co-workers – could have become acquainted through this collaboration with Virchow’s demonstration that thrombi originate in VVP. 3 Unless otherwise specified, we shall use ‘velocity’ to denote the average linear speed of the blood through a short segment of vein over several pulse cycles. Over a prolonged interval (say, 1 min or more), this variable depends on the rate of input, which is influenced by the cardiac and respiratory cycles and by arterial function; and on the rate of throughput, which is influenced by skeletal muscular activity, right heart function, vena caval pressure, vein geometry and – to a limited extent – venous tone.

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Fig. 9.1 (b) Fig. 3 from McLachlin and Paterson (1951). The original legend reads: ‘A thrombus arising in a valve pocket at the upper end of the superficial femoral vein. The Lines of Zahn are clearly seen.* (Postmortem clot (rt.) for comparison.)’ The concept of ‘postmortem clot’ will be discussed in Chapter 13. These and the following micrographs are reproduced from Surg. Gynae. Obstet. 1951 and 1954 with permission from the American College of Surgeons

[V]enous thrombi … in their early stages … were found to arise, almost invariably, in valve pockets, a location that is assumed to be most susceptible to stagnation of blood. Larger and older thrombi do not show this peculiar localization; but this discrepancy can be easily explained. After originating in a valve pocket, a thrombus will propagate proximally until it eventually occludes the lumen of the vein. A column of stagnant blood will be produced by the occluding mass, and this should lead to propagation in the opposite direction. The thrombus will now overflow the valve pocket and extend distally, thus obscuring the site of primary deposition. If this be true, it explains why the origin of venous thrombi in valve pockets has not been stressed by previous workers in the field.

As previously noted, we would look askance at the overtones of the absolute term ‘stagnant’, take issue with the claim that thrombus propagation is likely in ‘a column of stagnant blood’ (see the Hewson, Lister and Baumgarten experiments discussed earlier), and recall that Virchow illustrated VVP thrombi in 1856–1858; but we cannot otherwise fault these elegant studies or the inferences drawn from them. Why do thrombi originate in VVP sites? Gibbs (1957) suggested that under conditions of low blood velocity, eddy currents and turbulence are set up around the VVP in the larger veins of the lower limb.4 In a total of 253 necropsy studies,

4 Both parietal and ostial valves are included in this statement (i.e. those within a vein and those at vein junctions; see following text). Von Recklinghausen (1883) observed that flow patterns in valve pockets were likely to differ from those in the mainstream and suggested implications for local tissue oxygenation. But this prescient observation attracted little notice for many decades.

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Fig. 9.2 Photographs of venous thrombi in valve pockets (a) Fig. 2 from Paterson and McLachlin (1954). The arrow indicates the valve cusp. (b) Fig. 3 from Paterson and McLachlin (1954). Several thrombi are visible in two adjacent veins

encompassing a wide range of ages and causes of death, he found thrombi in most of the larger leg veins; the soleal vein scored particularly highly (see also Sevitt 1973; Sevitt and Gallagher 1961). Thrombi were initiated as microscopic nidi in regions adjacent to the valve cusps, where their main constituents were leukocytes, platelets and fibrin (i.e. they began as ‘white thrombi’). The overall composition of a mature thrombus, notably its erythrocyte content, may therefore depend on its age and on the mean local blood velocity during its formation and development; but thrombus formation depends on blood movement within and near the VVP, and is initially associated with the local congregation of leukocytes. Sevitt (1973) listed a number of factors that he believed to have ‘thrombogenic potential’: (1) The silting into valve pockets and venous saccules of platelets, leukocytes and red cells.

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(2) The accumulation within the pockets and saccules of activated blood clotting factors either released locally or already arrived from a distance, by retarding their dilution [the reference here was to studies on the clot-like experimental ‘stasis thrombi’ mentioned in chapter 3]. (3) The local accumulation of ADP derived from the silted red cells and leucocytes. (4) Preventing the arrival from a distance of substances like anti-thrombin and plasma ADPase, which inhibit fibrin formation and ADP-induced platelet aggregation. (5) Possibly the production of hypoxia in endothelium lining valve pockets.

We reserved opinion on these suggestions in Chapter 8. ‘Silting’ of cells anywhere in the blood stream was rejected by Malone (1977), but the final item listed, VVP hypoxia as a consequence of underperfusion, seems to accord with the conjectures of von Recklinghausen (1883), Paterson and McLachlin (1954) and Gibbs (1957). It also accords with other early and mid-20th-century studies, intriguing but now largely forgotten, to be reviewed in Chapter 10. In a detailed study of 48 femoral VVP thrombi, Sevitt (1974a, b) described 90% of the valve cusps as ‘histologically normal’. However, critical re-inspection of his published micrographs reveals that the valve cusp parietalis endothelium was in some cases attenuated, potentially necrotic, dehisced/separated from the cusp basement membrane (Fig. 9.3; see also Fig. 10.2). Sevitt also described four cases of double thrombi, in three of which the second thrombus was initiated on one or other surface of the (presumed) first. The site of thrombus anchorage seemed to be in the depth of the VVP or attached to the ‘central’ cusp endothelial lining, as described by Virchow in 1858 (Fig. 6.1). Virchow’s striking observation remains consistent with the studies described above and with the clinical presentation of DVT. There are many more valves in large veins in the lower limb than elsewhere in the body. Also, unlike VVPs in the upper body, they do not empty automatically (by gravity); in the erect or seated position they have to be emptied (by skeletal muscular work). Lower limb VVP are therefore prone to malfunction and may be expected to be main sites of DVT; which, of course, they are. If autochthonous thrombi form in VVP or soleal valve sinuses, i.e. not on the mural lumen of the vein, then experimental thrombi produced by either gross or subtle injury to the venous intima in general (see Chapters 3 and 7) have limited value in helping us to understand their aetiology.

9.3

The Morphology and Pathology of Venous Valves

Harvey (1628) wrote: The celebrated Hieronymus Fabricius of Aquapendente, a most skilful anatomist, and venerable old man … first gave representations of these valves in the veins. … They are situated at different distances from one another, and diversely in different individuals; they are connate at the sides of the veins; they are directed upwards or towards the trunks of the veins; the two – for there are for the most part two together – regard each other, mutually touch, and are so ready to come into contact by their edges, that if anything attempt to pass from the trunks into the branches of the veins, or from the greater vessels into the less, they completely prevent it; they are farther so arranged that the horns of those that succeed are opposite the middle of the convexity of those that precede, and so on alternately. The discoverer of these valves did not rightly understand their use, nor have any succeeding

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Fig. 9.3 Micrograph showing hole in valve cusp, from Sevitt (1974a). The severe necrotic perforations manifest in this valve cusp accords with the thesis that thrombosis is essentially associated with degrees of valve cusp necrosis. Valve cusps are presumably not perforated readily even when the parietalis endothelium becomes necrotic, but should the basement membrane of a cusp be also killed by hypoxia and the detritus consumed/removed by viable white cells, the luminalis endothelium would then be unlikely to sustain the centrifugal blood pressure. The process of perforation could not have been witnessed, so the inference is that necrosis consequent on gross underperfusion caused DVT, cusp perforation, and a fatal pulmonary embolism. Determined post-mortem examination of the patient’s veins revealed this lesion. Probably the embolisation of the thrombus from this VVP (or another similarly ‘holed’) had caused the death. Copyright 1974 John Wiley and Sons. Reproduced with permission of Wiley-Liss Inc. on behalf or John Wiley and Sons Inc

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anatomists added anything to our knowledge: for their office is by no means explained when we are told that it is to hinder the blood, by its weight, from all flowing into inferior parts; for, the edges of the valves in jugular veins hang downwards, and are so contrived as to prevent blood rising upwards; the valves, in a word, do not invariably look upwards, but always towards the trunks of the veins, invariably towards the seat of the heart.

In the light of more recent studies, what can be added to this account of valve structure and function?

9.3.1

Venous Valve Morphology

The remark ‘nor have any succeeding anatomists added anything to our knowledge’ could, with minor reservations, have been echoed by Franklin (1927, 1937), or by Edwards and Edwards (1939) and Saphir and Lev (1952a), who provided further details. Typically, the vein cross-section near a valve is elliptical and slightly dilated. Also, the wall bounding the valve sinus ‘laterally’ is thinner than in the rest of the vein, rendering it more distensible (Franklin 1927, 1937; see also Edwards and Edwards 1939; Ackroyd et al. 1985; Stone and Stewart 1988; Qui et al. 1995). The valve cusps, in contrast, are fairly rigid; a parietal valve may have 1–5 cusps, most commonly two (Franklin 1927). The length of a cusp is typically about twice the diameter of the vein lumen. In the femoral vein, where the valves are large and relatively easy to study, each cusp comprises a collagen-elastin network continuous with the subendothelium of the intima, covered by a single layer of endothelial cells. Moreover, valve cusps are avascular, i.e. they are not perfused by vasa venarum. On the outer, distal surface (luminalis) of the cusp, the endothelial cells are elongated along the vessel axis. On the inner, proximal surface (parietalis) they are aligned transversely. Endothelium-lined crypts are distributed along both surfaces at irregular intervals. The cusp is thinnest at the unattached end, where the luminalis and parietalis meet, but the subendothelial matrix adds strength. The point of attachment of the cusp to the vein wall, the agger, is the thickest part (Fig. 9.4); here, the endothelium is folded. There is smooth muscle in the vein wall at the agger (Stone and Stewart 1988), but this seldom extends far into the cusp (Franklin 1927). In addition to the parietal valves within the veins, ostial valves are present at the confluences of tributaries with deep veins (Franklin 1927) (Fig. 9.5). An ostial valve is typically unicuspid. Its anatomy is unlike that of a parietal valve, being hidden within the tributary lumen; its agger cannot be seen from the main vein. Interestingly, parietal valves and vein confluences appear to be the sources of endothelial regeneration after a segment of vein is denuded of endothelium (Einarrson et al. 1984). There are subtle morphological differences among valves in different veins (Buss et al. 1979), but no known significant functional differences. The avascularity of the valve cusps was acknowledged without further comment by Sevitt (1974a), but we shall argue that the absence of capillaries from the cusps is uniquely thrombogenic.

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Fig. 9.4 Diagram of valve cusp after Saphir and Lev (1952a). (a) Schematic drawing of microscopic appearance of femoral vein valve giving the pertinent nomenclature. (b) Vein valve in a child; orcein preparation. (c) Section through a pallid, necrotic valve cusp showing a possible white thrombus within the VVP on each side of the cusp. Copyright American Medical Association (originally from Archives of Pathology). Reproduced with permission

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Fig. 9.5 Parietal and ostial valves; ‘open’ and ‘closed’. The key to these depictions is the relative ‘depth’ at which the agger of the valve is situated inside the tributary vein. That depth is as potentially obstructive (if filled or partly filled with thrombus) as the mechanism of the valve in preventing reflux

9.3.2

Valve Pathology: The Formation of Nascent Thrombi within VVP

Saphir and Lev (1952a) studied femoral valvulitis and found that ‘inflammatory injury’ to venous valves is common. Since the appearance of an ‘inflamed’ valve cusp represents margination and sequestration of leucocytes/platelets, it could be considered that an ‘inflamed valve’ is indistinguishable from a ‘white thrombus’ in

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its earliest (Kopfteil) phase (cf. Chapter 7). Valve cusps can become necrotic, e.g. after (or perhaps during the pathogenesis of) endocarditis or other heart diseases, and such necrosis appears to be thrombogenic. Leukocytes congregate around the injured cusp, and sometimes the connective material of the cusp is thickened and coarsened. These findings suggest a possible mechanism for the formation of

Fig. 9.6 From Stone and Stewart (1988). These photographs show the constancy of what Franklin termed ostial valves at the mouths of tributary veins, attached/ anchored within the tributary vein as it joins the main venous trunk concerned. Franklin’s parietal valves are the (predominantly) multi-cusp valves that intersect the main-vein lumen and hold up the blood column above them when the distal pressure below has fallen. In this illustration, a plastic was infused into the main vein lumen (and subsequently removed by a ‘corrosion’ technique). Very small protrusions of the solid (‘cured’) plastic are visible, retained by the ostial valve that had closed a few millimetres from the main vein orifice in each tributary (thus facilitating a valve thrombus at the junction, and manifest as a very short ‘stump’ of contained plastic). Copyright 1988 John Wiley and Sons. Reproduced with permission of Wiley-Liss Inc. on behalf or John Wiley and Sons Inc

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Fig. 9.6 (continued)

pre-thrombotic nidi, but they are not consistent with the contention (Sevitt 1974a) that only some 10% of venous thrombi were observed to originate on the cusps. This calls the exact site of nidus formation into question: is it the cusp itself (specifically, the luminalis or the parietalis endothelium), the neighbouring vein wall, or the sinus of the VVP? The studies of Saphir and Lev, Paterson and McLachlan, and Sevitt, led them to exclude the vein wall as the primary site of nidus thrombus formation. Leukocyte infiltration of cusps has subsequently been described in cases of venous insufficiency associated with injury to saphenous vein valves (Ono et al. 1998), and was beautifully demonstrated in scanning electron micrographs of the junctions between tributaries and jugular veins in dogs (Stewart et al. 1983, 1984; see Fig. 9.6). Notably, such infiltration is not immediately accompanied by fibrin deposition (cf. Aschoff 1924). Thrombosis can lead to the complete obliteration of venous valves in the lower extremities, leading to the major symptoms of post-thrombotic syndrome (Chapter 1; see below). In a subsequent paper, Saphir and Lev (1952b) reported that ageing is associated with increased collagen and decreased elastin below the parietalis endothelium, where the crypts tend to become smaller. Also, the elastic material below the luminalis endothelium thickens with increasing age. Generally, the connective tissue and

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muscle contents of the valve sinus decrease and fat deposits increase. We may speculate about the significance of these findings for the increased incidence of DVT in older people: the cusp becomes less flexible, impairing the efficiency of valve function. In addition, perhaps the lower resting venous tone in older patients decreases local blood velocity, exacerbating underperfusion by attenuating the intravascular pressure changes during pulsatile flow (see below). The weight of evidence therefore favours pro-thrombotic nidus formation on the valve leaflets; but if so, why did Sevitt, an eminent histopathologist, find so few thrombi anchored to cusp endothelia? We shall return to this question in Chapter 10. It is well-attested that thrombi can also arise in venous sinuses in the soleal and gastrocnemius muscles (for reviews see, e.g. Mammen 1992; Hirsh et al. 1986), and these structures do not usually have manifest valves (Dodd and Cockett 1976). However, Chapple and Wood (1980) made a telling observation about venous sinuses in the legs of the monkey Macaca fascicularis: they contain ‘flaps of endothelial tissue’. Similar flaps of endothelial tissue have been described in human venous sinuses (Cotton and Clark 1965). The mechanism we shall propose for the origin of pro-thrombotic nidi on the valve cusp parietalis endothelium could apply equally to the endothelial flaps of venous sinuses.5

9.3.3

Can the Venous Return Circulation and Valve Function be Correlated?

When a human is standing, or sitting erect (i.e. other than on a horizontal couch or bed), or suspended upright, all the venous valves close tightly under the weight of the column of blood in the inferior vena cava, femoral and calf venous systems (Chapter 8). The valves of tributary veins likewise close against the gravitational load. Franklin’s (1937) summary suggests that the actions of opposing muscle groups affecting blood movement are well-nigh impossible to illustrate diagrammatically, and extrapolation from animal experiments to human physiological actions is difficult. Thus, the accompanying diagrams (Fig. 9.5) are entirely imaginary in respect of the way that the opening and closing of parietal and ostial valves in main venous trunks are linked. A single diagram cannot capture all the voluntary and involuntary/insensible fibrillations, agonistic and antagonistic motions, etc. in normal standing or sitting persons, or the opening and closing of ostial valves when the tributary veins from working flexor muscles empty into a femoral vein during extensor inactivity and vice versa. It cannot be inferred that particular tributary valves in the main venous trunk lumen are necessarily open when others are closed.

5 This claim may not extend to the cerebral sinuses or the Circle of Willis, sites of thrombus formation on which there is a very extensive clinical literature.

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Some may be closed while others are pouring blood into the lumen, compensated either by opening of the cephalad valve or by alteration of vein-wall tone between the valves. Fig. 9.5 is a stylised image of what actually occurs.

9.4

The Valve Cycle and the Effects of Non-Pulsatile Flow

Lurie et al. (2002, 2003b) studied the movements of valves and the perivalvular region in healthy male and female subjects using ultrasound and Doppler scanning. They described a four-phase valve cycle: (1) an opening phase (about 0.25–0.30 s) during which the cusps move from the closed state towards, but not touching, the sinus walls (cf. Franklin 1927); (2) an equilibrium phase (about 0.6–0.7 s) during which the leading edges of the cusps remain suspended in the mainstream and oscillate with amplitudes of 0.1–1.6 mm; (3) a closing phase (about 0.35–0.45 s) during which the cusps move in synchrony to the centre of the vein lumen; (4) a closed phase (about 0.4–0.5 s). During the equilibrium phase, flow separation occurs at the leading edges of the cusps, reattaching at the wall of the sinus; part of the flow is directed into the sinus pocket and a vortex develops along the cusp. This vortex creates a pressure difference across the cusp, which dilates the thin-walled sinus (cf. Stone and Stewart 1988) and is instrumental in initiating the closing phase. The distance between the cusps during the equilibrium phase is about twothirds of the diameter of the adjacent lumen. Blood velocity through the gap is therefore greater than in neighbouring parts of the vein, decreasing the pressure in this region in accordance with Bernouille’s law.6 This contributes to valve closure, particularly during exercise when the mainstream velocity is greater. Thus, the function of a venous valve is not merely to prevent reflux (Harvey 1628) but also to accelerate the prograde movement of the blood. Little reflux is needed to close the valve completely (Qui et al. 1995). If the circulation is altered because the muscle pump temporarily ceases to operate or is impaired, e.g. in the deep veins of the lower limb during prolonged sitting, standing on sentry duty, or suspended for long periods, the pressure cephalad to certain valves in those veins will be greater than the pressure caudad. Therefore, the closed phase (phase 4) of the valve cycle will be indefinitely prolonged. As a result, the VVP may become hypoxaemic because the periodic evacuation and refilling associated with normal pulsatile blood movement will be temporarily abolished. Should the pressure caudad remain high enough to prevent valve closure, a suspension of muscle pump activity would replace the normal, physiological pulsatile 6 Bernouille’s law does not accurately describe the flow of viscous fluids, as Lurie et al. imply, particularly in thixotropic and heterogeneous fluids such as blood moving at variable velocity through distensible tubes; but the principle invoked here is valid. The same reservation applies to Pouseille’s law – though under most circumstances, the velocity profile of circulating blood approximates to the parabolic shape predicted by this law.

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movement of blood along the vein with slow, steady flow. In this situation, it may be supposed that the valve cycle described by Lurie and colleagues will be interrupted in a ‘half-open’ state; in effect, an indefinitely protracted phase 1 (or phase 3). The slow streamline flow through the lumen could maintain the vortices in the valve sinus, but because there is no complete opening (or closure), blood in the sinus will not be efficiently evacuated and replaced until muscle pump activity is restored. In principle, we may also imagine that a steady, prolonged high venous blood velocity would temporarily ‘lock’ the valve in its equilibrium phase (phase 2). Notwithstanding the oscillations or ‘fluttering’ of the cusp leaflets in the rapid streamline flow (the Venturi effect), the evacuation and refilling of the region of the valve sinus between the leaflet and the wall would again be inefficient. We consider inefficient turnover of the blood in the valve sinus to be instrumental in the aetiology of DVT. We therefore predict that venous thrombi could, in principle, develop if the venous blood velocity remained high but the flow was streamline for a substantial period. This prediction is obviously inconsistent with the idea that ‘stasis/stagnation’ in vein lumens is instrumental in thrombosis and therefore conflicts with the consensus model. It is experimentally testable.

9.5

Relevance of Venous Blood Rheology

If the evacuation and refilling of the sinuses under conditions of pulsatile venous blood movement is the primary consideration, then the rheology of venous blood would seem only marginally relevant to the aetiology of DVT. (Its pertinence to other circulatory and haematological lesions, linked and determined by cardiac output, vascular resistance, blood viscosity and vessel wall geometry, is not in doubt). The single point at issue relates to the intermittency of circulation as the key factor in filling and emptying VVP. We presume that this ‘reversal of flow direction’ usually happens in seconds or minutes; on such a time scale, the VVP could scarcely be injured by alterations of vessel bore, wall friction or blood viscosity. In short, injury to the valve cusp leaflets cannot possibly result from venodynamic or fluid mechanical changes, but it can be readily seen to result from muscular failure to evacuate and refill the valve sinus by blood pumping when, and for as long as, one phase of the valve cycle is indefinitely prolonged. In any case, the Navier–Stokes equations7 cannot be solved analytically when applied to blood flow, and numerical solutions are likely to be unreliable; the Maxwellian properties of blood have been thoroughly discussed (e.g. McMillan 7 If p is the local (instantaneous) pressure, t is time, u is the three-dimensional velocity vector, ρ is the density of the blood, the laminar viscosity coefficient is µ and the viscous sheer stress is f, then the principal Navier–Stokes equation states:

⭸u m 1 + ( u ⋅ ∇) u = − ∇p − ∇f + ∇ 2 u ⭸t r r

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and Utterback 1980) – indeed, hyperviscosity has been associated with DVT (Smith and La Celle 1982).8 Nevertheless, qualitative and commonsense inferences from the results of some rheological studies may throw some light on our subject. When the pseudo-shear rate (the ratio of mean blood velocity to vessel diameter) falls markedly below the physiological norm, the velocity profile ceases to be quasi-parabolic: a cell-free and consequently less viscous layer forms at the periphery (Bishop et al. 2001; see also Alonso et al. 1993). If this layer sufficed to insulate the vein wall from the circulating erythrocytes, the endothelium would tend to become hypoxic. Crucially, however, it is not known whether such a layer also forms within the VVP, and a high haematocrit or low total blood volume would presumably vitiate this effect. Nevertheless, the increase of blood viscosity under such circumstances would not only decrease the linear velocity, it may also attenuate the pressure differences associated with pulsatile flow and therefore make valve perfusion less efficient (see footnote 8). The closed phase (phase 4) of the valve cycle could be further prolonged. These effects may contribute to the increased incidence of DVT in cases of hypovolaemia or high haematocrit.

9.5.1

Relevance of the Vasa Venarum

Since the 1970s, the mechanisms of control of venous tone have been elucidated (Skalak et al. 1981), and fluid mechanical theory had been extended to variablevelocity flow through collapsible tubes (Kamm and Pedley 1989). An overview of vein physiology and pathology by Monos (1992) took account of these studies and of the effects of various endothelial secretions on the subintimal smooth muscle. There is considerable evidence that venous tone changes in response to hypoxaemia; smooth muscle activation is mediated through endothelial cell responses to hypoxia (Chapter 12). Although veins are better adapted to hypoxic conditions than arteries – for instance, their smooth muscles and endothelia are better equipped for anaerobic glycolysis and the accumulation of lactate (Malmqvist et al. 1991) – the response could in principle be physiologically significant. However, in many veins,

This equation is non-linear so it cannot be solved analytically unless simplifying assumptions can be made (e.g. m = 0), which are obviously invalid in the case of blood. Moreover, in a Maxwell (non-Newtonian) fluid such as blood, m (and hence j) is by definition a function of u; and when the flow is pulsatile, u is by definition a (sinusoidal) function of time. These considerations complicate the equation to such an extent that even established numerical approaches can hardly give reliable approximations to the solution, even if good experimental data are available – and measurement errors are of course inevitable. 8 An interesting possibility is that hyperviscosity has the same pathogenic effect as hypovolaemia, which as Aschoff (1924) observed is thrombogenic (Chapter 8); e.g. hypervolaemia consequent on dehydration necessarily entails hyperviscosity.

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not least the large deep veins of the lower limb, mild or moderate hypoxia apparently induces constriction, which should increase the local mean blood velocity. If mean velocity were relevant to DVT, therefore, hypoxia would presumably militate against thrombosis. There are exceptions – for instance, placental veins relax under partially hypoxic conditions and contract when re-oxygenated (Figueroa et al. 1993) – but hypoxia appears to cause constriction (ergo increased velocity) in the veins where DVT most commonly arises. This effect is probably slight. Although veins respond to locally produced smooth muscle agonists and antagonists (Stone and Stewart 1988), they are much less sensitive than arterioles, just as they are less sensitive to control via autonomic nerves. This suggests that hypoxic and other local effects on venous tone are minor. However, the vasa venarum may be more responsive, and this could help to maintain perfusion of the mural endothelium in hypoxaemic veins, but not of the valve cusp leaflets, which are avascular. Exactly such a response of the vasa venarum may be suggested by the dilatation of the agger capillary during thrombosis, as described by Edwards and Edwards (1936, 1939). Thus, increased blood flow through the vasa venarum by alteration of vessel tone during mural endothelial hypoxia may prevent pro-thrombotic changes. Venous thrombi arise on valve cusps, but as they develop they injure and even destroy the valves. This phenomenon was detailed in the Edwards and Edwards papers,9,10 and is illustrated in Fig. 9.3. Recent work on the molecular biology of the endothelium and its response to hypoxia (Chapter 12) may elucidate the mechanisms underlying these changes.

9 The nomenclature in this paper is of some interest. The authors used the word ‘thrombus’ in its standard Virchowian sense but denoted the process of thrombus formation by ‘phlebitis’ or ‘thrombophlebitis’. The persistence of these terms into the 20th century was discussed in Chapter 7. 10 The valve in which a thrombus originates, and valves affected by its subsequent growth, undergo morphological changes; the cusps ultimately fragment, detach or adhere to the wall of the lumen. The result, even after recanalisation of the thrombus, is a functionally (and often anatomically) valveless segment of vein, the principal characteristic of post-thrombotic syndrome. During this pathological process, the endothelial folds of the agger appear to be sites of fibroblast proliferation, collagen deposition and angiogenesis. The small capillary supplying this region of endothelium dilates and further capillary branches spread through the connective tissue forming within the angle of the valve. This angiogenesis is concurrent with the fragmentation of the valve cusp, which proceeds until only small pieces of elastic tissue are visible by microscopy, embedded in the growing thrombus. Alternatively, the cusp may be pushed against the vein wall, in which case the repairing connective tissue effects permanent adhesion. Therefore, even if one or both cusps do(es) not fragment, the valve becomes functionally useless. As the thrombus extends, the cusps of valves cephalad to the VVP of origin, including those at the junctions of tributary veins, become trapped and are destroyed or rendered functionless by a similar process of connective tissue formation and microangiogenesis. The newly formed capillaries appear to be critical for recanalisation but the lost valves cannot be replaced, except artificially; prosthetic valves have been designed to treat victims of this condition (e.g. Hill et al. 1985).

9.5 Relevance of Venous Blood Rheology

9.5.2

137

Flow Patterns within VVP

Karino (1986) and Karino and Motomiya (1984) used a novel method for visualising flow patterns in blood vessels and obtained a more complete description of vortex formation. In their experimental model, which simulated physiological conditions (though with cell concentrations much lower than normal), spiral secondary flows and recirculation zones formed in the VVPs, and sites of disturbed flow were observed where pathology is commonly found. Karino (1986) studied the flow patterns in VVPs in canine saphenous veins made transparent with methyl salicylate and discussed their implications for thrombosis. Cinematographed flow of inert particles and erythrocytes in trans-illuminated VVP revealed large stable paired vortices, located symmetrically about the plane bisecting the valve leaflets under quasi-physiological flow conditions; the primary vortex was driven by mainstream flow. Particles entering the VVPs from the mainstream described spiral orbits of gradually decreasing diameter until, after a considerable time, they rejoined the mainstream near their attachments to the vessel wall. More concentrated red cell suspensions revealed a smaller secondary vortex deep in each VVP, marked by a lower density of red cells than in the proximal pocket (where it was more or less equivalent to that in the mainstream). This secondary vortex (a) was driven by the primary vortex and rotated in the opposite direction at very low velocity, and (b) persisted for as long as non-pulsatile flow continued in the lumen. It did not rejoin the lumen until a pulse emptied and refilled the pocket. Karino and Motomiya (1984) demonstrated that the enduring slow secondary vortex deep in the VVP remained relatively erythrocyte-depleted. The implications were discussed by Karino and Goldsmith (1987): the slow vortices indicate reduced perfusion by blood from the mainstream. We believe these – admittedly artificial – reproductions of vortices of blood in VVP with unflapped (and stiffened) cusps provide an insight into pro-thrombotic nidus formation on the cusp endothelium.

9.5.3

Implications for the Formation of Pro-Thrombotic Nidi

When the valve cusp pathologies associated with so-called ‘venous valvulitis’ and ageing (Saphir and Lev 1952a, b) are reviewed in the light of these studies, further inferences about DVT may be drawn. The thickening of the cusp described by Saphir and Lev presumably attenuates the oscillations observed by Lurie et al. (2002, 2003b) in normal younger subjects during the equilibrium phase, with implications for Venturi effect, i.e. the stability of the vortices that are instrumental in initiating closure. This would clearly prejudice the valve cycle and ipso facto the emptying and refilling of the VVP. However, it does not explain why most thrombi originate within the valves, rather than elsewhere in a vein subjected to nonpulsatile blood movement for an extended period. All other things being equal, blood cells tend to congregate more at very low shear rates (Trowbridge 1982; Thurston 1994). Hence, when the circulation is first

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interrupted, cells are likely to accumulate in the VVP where the shear rate is negligible. Under otherwise normal physiological conditions, platelet masses generated ex vivo by ADP treatment and re-injected into experimental animals tend to congregate in the vortices in post-stenotic venous sinuses. If the blood velocity is low, these platelet masses grow larger (Machi et al. 1986); it was suggested the formation of such masses in VVPs in vivo could be relevant to thrombosis. On the face of it, this study could seem to support the model of Hume et al. (1970) described in Chapter 8, but we consider platelet massing in VVPs per se to be an effect, not the initial cause, of thrombogenic nidus formation (cf. Gibbs 1957; Sevitt 1974a). The secondary vortices – in which the erythrocyte content is in any case lower than the systemic norm (Karino and Goldstein 1987) – would indicate a further delay in the evacuation of oxygen-depleted erythrocytes and the influx of oxygenated ones, exacerbating any local hypoxaemia. Platelets in such regions would have an increased tendency to congregate on the vessel wall. This seems to us to be more pertinent to thrombosis.

9.6

Implications for Compression Prophylaxis

Irrespective of aetiological mechanism, the greatly increased risk and incidence of DVT attendant on sustained non-pulsatile venous blood flow affords a rational basis for prophylaxis by intermittent compression. This approach to prophylaxis, explored during the 1950s, was considered by Roberts and Cotton (1975) and its cost-effectiveness was assessed by Salzman and Davies (1980). Holford (1976), Scurr et al. (1977), Barnes et al. (1978), Hull et al. (1979, 1990), Nicolaides et al. (1980, 1983) and Dai et al. (1999) are among the many workers during the past three decades who have assessed its clinical efficacy. It seems especially effective for post-operative patients requiring prolonged bed rest. Most of these authors emphasised sequential compression to accelerate flow velocity; however, we maintain that maintenance of pulsatile flow is clinically far more important than boosting the linear blood velocity per se. Salzman et al. (1987) found that different types of compressor were similar in efficacy. Lurie et al. (2003a) showed that intermittent pneumatic compression significantly increases the venous flow volume and velocity, irrespective of whether the limbs are elevated, horizontal or dependent. The changes in segmental flow varied with the type of compression garment used and where it was applied (calf or foot), and there was individual variation among patients. Calf compression provided the maximal increases in volume flow and flow velocity through the deep veins. The efficacy and safety of mechanical prophylaxis for DVT after total hip replacement has been demonstrated by Pitto et al. (2004). We shall re-examine this topic in Chapter 11. This correspondence between clinical success on the one hand and aetiological theory on the other seems to confirm that local cessation of pulsatile blood flow is instrumental in DVT. Bizarrely, however, some workers in the field have interpreted the clinical success of sequential compression treatment in terms of

9.7 Towards the Valve Cusp Hypoxia Hypothesis: II-VVP Hypoxaemia

139

‘inhibition of thrombogenesis and promotion of fibrinolysis’ (e.g. Salzman et al. 1987). The consensus belief that ‘thrombosis is excessive or aberrant coagulation’ persists throughout much of the biomedical world, and seems to have become a conceptual straitjacket into which data meriting quite different interpretations are forced.

9.7

9.7.1

Towards the Valve Cusp Hypoxia Hypothesis: II-VVP Hypoxaemia Hypoxic Injury to the VVP Cusp Endothelium Is Potentially Thrombogenic: a Proposal

The outstanding papers of Lister (1858, 1863), discussed in previous chapters, showed that a wide range of chemical and physical irritants – croton oil, galvanic current, fixative or tissue poison, etc. – caused microvascular thrombosis. Lister presumed, and confirmed experimentally, that these substances caused suspension or loss of what he termed ‘vital properties’ in the vessel linings, and that margination and sequestration of ‘colourless corpuscles’ ensued, often obstructing circulation. In his 1863 Croonian lecture, he wrote: This view of the condition of intensely inflamed parts is exactly that to which I was led some years ago by a microscopic investigation, the results of which were detailed in a paper that received the honour of a place in the Philosophical Transactions. It was there shown, as I think I may venture to say, that the tissues generally are capable of being reduced under the action of irritants to a state quite distinct from death, but in which they are nevertheless temporarily deprived of all vital power, and that inflammatory congestion is due to the blood-corpuscles acquiring adhesiveness such as they have outside the body, in consequence of the irritated tissues acting towards them like ordinary solids … I cannot avoid expressing my satisfaction that this inquiry into the coagulation of the blood has furnished independent confirmation of my previous conclusions regarding the nature of inflammation. [Our emphases]

Indeed, no one now doubts that thrombosis follows gross injury to the venous intima: a subendothelium that has been injured along with its covering endothelium invites platelet activity, which initiates the coagulation cascade at that injury site. Wharton Jones (1851) described the phenomenon clearly and it became clinically useful. The modern surgical procedure of injecting sclerosant (endothelium-killing) agents into segments of dilated varicose veins irritates and kills the intima, thus inducing controlled occlusive thrombosis followed by auto-stenosis and cure of the varicose appearance. ‘Injecting varicose veins’, in effect, adapts the experimental work of two centuries to the cosmetic treatment of varicose disfigurement.11 However, as we have seen, Thomas and others doubted the relevance of the venous endothelium to thrombosis in the absence of visible injury, i.e. in cases of 11

According to Chapman (1864) the technique was first introduced by a French surgeon, M. Pravaz, using iron perchloride as sclerosant.

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9 Underperfusion of Valve Pockets and the Initiation of DVT

Fig. 9.7 Diagrams of thrombogenesis by massing on dead parietalis endothelium. 9.7a. Normal valve cusp movements and VVP blood flow patterns oxygenate the avascular valve cusps only from refluxing luminal venous blood, unlike the vein wall endothelium, which is oxygenated by the systemic arterial vascular system. Normal perfusion of pockets with fully oxygenated blood (erythrocytes) is therefore essential for keeping the parietalis endothelium alive. Hamer et al. (1981a,b) showed that the intra-pocket PO2 falls precipitously and progressively whenever pulsatile venous flow is replaced by streamline flow

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Fig. 9.7 (continued) (b) Temporary suspension of pulsatile venous flow. Normal cusp flapping ceases; in situ vortices then revolve slowly, driven by the centripetal luminal flow passing the mouths of VVP (Karino and Motomiya 1984; cf. Fig. 11.4). Trapped blood cells and parietalis endothelium then become progressively oxygen starved and non-functional and may die. Oxygen-starved, incapacitated, pre-necrotic endothelial cells are shown in a hypoxaemic VVP. No interaction is to be expected between non-viable blood cells/platelets and the non-viable parietalis endothelium (shown in hatched grey shading). The scene is now set for thrombogenesis to commence

‘spontaneous thrombosis’. In the light of Lister’s demonstration that thrombosis ensues when exogenous irritants are applied to the venous intima, we consider it reasonable to propose that autochthonous DVT is caused by an ‘endogenous irritant’, which affects the endothelium invisibly to direct observation or light microscopy. In view of the work reviewed in the foregoing sections, suffocating endothelial hypoxia resulting from hypoxaemia in underperfused VVP under conditions of sustained non-pulsatile flow is a plausible candidate (Fig. 9.7).

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Fig. 9.7 (continued) (c) Temporary restoration of pulsatile venous flow. If viable blood cells reflux into the lately hypoxaemic pocket during restored pulsatile flow, they may attack and marginate on the pre-necrotic or necrotic parietalis endothelium. A point will soon be reached when most or all of the oxygen in the VVP has been consumed and the PO2 is reduced to zero (cf. Fig. 11.3), at which level it will remain as long as the pocket remains unperfused. The phagocytic activity of the blood cells will soon cease

The extreme hypoxaemia necessary must be confined to a ‘local’ endothelium first and foremost (cf. Morrison et al. 1977): were such oxygen starvation widespread, the problem would be death of the whole body, not just death of the parietalis endothelium of an underperfused valve cusp. Prolonged hypoxaemia in the lumen of a vein is conceivable in many disorders but that would signal major, potentially fatal, cardio-respiratory failure. Our hypothesis asserts that DVT does not figure on the clinical radar until severe, prolonged, very localised hypoxaemia affects the metabolism of the ‘central’ endothelial surface of a cusp or cusps; that could indeed lead on to DVT in certain circumstances. Luminal hypoxaemia threatens the life of the whole person, whereas localised VVP hypoxaemia, causing hypoxic injury of parietalis endothelium, threatens necrosis of that endothelium and resultant thrombosis. The vascular endothelium is everywhere similar, albeit not identical in all parts (Chi et al. 2003); e.g. segments of saphenous vein are routinely and successfully used to replace segments of artery in bypass surgery (Golledge 2004). These observations lead to questions about (1) the detailed physiology of the endothelium and its response to hypoxia and (2) ways in which the VVP endothelium may be distinctive. We shall pursue these questions in the next three chapters.

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143

Fig. 9.7 (continued) (d) Recurrence of non-pulsatile venous blood flow. Viable phagocytes will scavenge their dead predecessors, but as hypoxaemia recurs, their activity must also cease in time. The solid grey spheres represent ‘normally’ oxygenated blood cells in the vessel lumen and the VVP. Round hatched dark grey discs in the hypoxaemic pockets are oxygen deprived; the oval hatched cells are oxygen-starved, defunct phagocytes. Therefore, a mass of combined blood and endothelial cells litters the defunct parietalis layer, built up over repeated periods of normal and abnormal flow. As emphasised in 9.7a, the mural endothelium is never oxygen-starved in life

9.7.2 VVP Hypoxaemia and Hypoxic Injury to the Parietalis Endothelium We propose that under non-pulsatile flow conditions, the valve cycle is interrupted; and irrespective of the phase in which it is interrupted, a volume of blood will remain sequestered in the valve sinus, neither evacuated nor replaced for as long as

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the non-pulsatility persists. We argue that this ‘focal stasis’ in VVP must result in local hypoxaemia and must predispose towards the formation of a pro-thrombotic white nidus (primarily a congregation of leukocytes and platelets interspersed with fibrin) on the oxygen-starved parietalis endothelium of the valve cusp leaflets. We shall enlarge on the mechanism of formation of this nidus in the next chapter, but for the present a key question is: how long a time is required for the ‘sequestered blood’ in the sinus to become sufficiently hypoxaemic for these pathogenic consequences to ensue? We can estimate this theoretically as follows. If the trapped blood has a volume of 0.1 ml (which corresponds to a sphere of radius ~3 mm, a reasonable estimate for the slightly dilated valve region of a 1 cm diameter vein), and the blood cell count is the same as that in the bulk venous blood, then it contains around 106 leukocytes. If about two-thirds of the trapped blood is surrounded by endothelium, then the area of endothelium is about 8 × 109 µm2; and presuming a mean diameter of 10 µm for the endothelial cells, this is equivalent to almost 3 × 107 cells. Thus, we can assume that the total number of respiring cells (essentially endothelial plus leukocytes) is 3 × 107. Equivalent calculations can obviously be made for valves in veins of any diameter. Let us suppose that the oxygen-consuming cells involved (i.e. the leukocytes in the trapped blood and the surrounding endothelial cells of the parietalis and the wall of the sinus) respire at the average rate of the body’s cells under BMR conditions. This corresponds to an oxygen consumption of about 1 mmol/min for the whole body, which in an adult human comprises some 1013 cells. Thus, our 3 × 107 cells will consume some 3 × 10−3 µmol of oxygen/min, or 0.18 mmol/h. Assuming that the erythrocyte content of our 0.1 ml of trapped blood is the same as that in the vein as a whole, and that the initial PO2 is the venous ‘norm’ of 40 mm Hg, the initial oxygen content is around 0.65 µmol. Given the estimated rate of oxygen consumption by the cells, this amounts (theoretically) to a supply for 3.5–4 h. However, we recall that the erythrocyte content of the trapped blood, and ipso facto the oxygen content, is substantially lower than the venous norm because erythrocytes are depleted in the secondary vortex. Theoretically, therefore, we would expect the oxygen supply to the cells to be completely exhausted in 3 h or less. Of course, the cells would show the injurious effects of hypoxia considerably earlier than this, when the oxygen supply was depleted by (say) 50–60%. Thus, we can estimate that VVP hypoxaemia is likely to become serious, i.e. injuring the local parietalis endothelium and the trapped blood cells, after around 1.5–3 h of non-pulsatile venous flow.12

12

We have not taken the capacity for anaerobic metabolism into account because we cannot judge how marked an effect it might have. As far as we are aware, there is no published evidential basis for a quantitative estimate of the ‘anaerobic capacity’ of the endothelium. More particularly, we do not know whether the valve cusp parietalis is ‘typical’ of vascular endothelia in this regard. It may not be: for instance, the absence of blood supply to the cusp strongly implies that lactate and other possible products of anaerobic metabolism cannot be removed efficiently and may therefore reach cytotoxic levels fairly quickly.

9.8 The Lesson of History

9.7.3

145

Testing the Predictions

Although much remains to be said about the effects of hypoxia on the venous endothelium, we have already articulated the valve cusp hypoxia hypothesis in sufficient detail to make specific, testable predictions: (a)During prolonged periods of non-pulsatile venous flow, the VVP become measurably hypoxaemic. Strong evidence for this was obtained by McLachlin et al. (1960) using a radiographic tracer, and hypoxaemia was directly demonstrated by Hamer et al. (1981a, b) using an oxygen electrode. When streamline flow had persisted for 2–3 h, no oxygen was measurable in the VVP blood; but a single skeletal muscle movement (or external application of pressure) returned the PO2 in the pocket to the venous blood norm. (b) Alternating long (1.5–3 h) periods of non-pulsatile flow with short interludes of pulsatile flow will cause the formation of coagula on the affected valves that are histologically indistinguishable from autochthonous venous thrombi. This was corroborated by Hamer and Malone (1984) using barbiturate-anaesthetised dogs that had no demonstrable thrombophilia. Prior to the formation of a histologically identifiable thrombus, a monolayer of leukocytes and platelets accumulated on the necrotic parietalis surface of the valve cusp (Malone and Morris 1978; I.A. Silver, personal communication). These experiments will be discussed in greater detail in Chapter 11, but their significance will already be apparent to the reader: the results are qualitatively and quantitatively consistent with the predictions of our hypothesis, but the consensus model cannot account for them.

9.8

The Lesson of History

Scientists who reflect on past eras in which our knowledge was laboriously created by trial and error must not forget that we too are finding our way by trial and error, and that our intellects are no less frail, no less prone to follow fads and no less prone to err than those of our predecessors. Locke (1689) looked upon the ideologies of 1535–1690 with forgiving eyes. He referred (p. 438) to the contemporary demise of age-old theories in the face of new ones, and reflected humorously on the invidious position of professors who were unfortunate enough to have their ignorance exposed for all to see (in both medicine and mechanics). Those errors, he said, were the consequences of received hypotheses, i.e. their teachers’ writings: Would it not be an insufferable thing for a learned professor, and that which his scarlet would blush at, to have his authority of forty years standing, wrought out of hard rock, Greek, and Latin, with no small expense of time and candle, and confirmed by general tradition and a reverend beard, in an instant overturned by an upstart novelist? … Can anyone expect that he should be made to confess that what he had taught his scholars thirty years ago was all error and mistake, and that he sold them hard words and ignorance at a very dear rate. What probabilities, I say, are likely to prevail in such a case?’

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Locke was probably referring to the then-recent publication of Newton’s Principia, but his comments could also have applied to the Galenic theory that Harvey had overturned 60 years previously. (Malpighi’s discoveries had corroborated the most critical prediction of Harvey’s circulatory hypothesis some three decades before Locke’s Essay was published.) Human certainties always remain open to question. Berkeley, Hume, Kant and others after Locke sought to define the elements of more reliable truth (see the appendix). However, science has always been a mix of sense and rubbish, right and wrong, which the glare of reputation and the fashions of convention often render contemporaneously indistinguishable. ‘Wrong’ teaching can be valuable: it stimulates some to identify the wrong and others to re-invent the right. Learned professors went to the stocks for having learned too well, and believed too loyally in, the misconceptions that their teachers had themselves learned. Now that a generation of teachers have suppressed the essence of Harvey’s wisdom for half a century, history could repeat itself – in all honesty and for the best of reasons.

Chapter 10

The Role of Endothelial Hypoxia in DVT

Abstract There is a mid-20th century literature on the relationship between venous endothelial hypoxia and thrombosis, as well as an abundance of more recent cell and molecular biological papers. Discussion of the more recent publications will be deferred until Chapter 12. In the present chapter, the implications of the older literature are discussed in relation to the proposal in Chapter 9: hypoxic death of the parietalis endothelium caused by VVP hypoxaemia during intermittent periods of non-pulsatile (streamline) flow is instrumental in DVT. Published micrographs of venous thrombi are re-evaluated to elaborate this proposal and illustrate the thrombogenic process stage-by-stage. Our account also relates to the induction of DVT-like changes by non-fatal carbon monoxide poisoning, the margination of leukocytes on hypoxic endothelia, Aschoff’s autopsies of First World War victims, and recent conflicting evidence about ‘traveller’s thrombosis’.

Keywords Vascular endothelial hypoxia, thrombus structure, carbon monoxide, cadaver blood, traveller’s thrombosis

10.1

Oxygen, the Venous Endothelium and Thrombosis

In Chapters 8 and 9 it was proposed that DVT may follow sustained non-pulsatile flow in under- or non-perfused venous valve pockets (VVP), which entails severe hypoxaemia in those pockets and, perhaps, suffocated hypoxic death of valve cusp parietalis endothelium. This hypothesis seems compatible with recent publications on vascular endothelial hypoxia (Chapter 12), extending into the vast literature on ischaemia-reperfusion injury. However, it is rooted in a considerably older literature that supports the central idea of this book: localised endothelial hypoxia is potentially thrombogenic. Hippocrates (c.460–377 BC) conceived that ‘an extremely subtle material agent is attracted into lungs, passes from lungs to blood, is distributed by the latter throughout the body, and all vital phenomena depend on the action of pneuma’ (see e.g. Porter 2001). Half a millennium later, Galen sought to unite the ideas and P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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observations of Hippocrates, Aristotle and the Alexandrians, and pointed to a constituent of air he called spirits,1 the nature of which he could not specify. During the 1,500 years that elapsed before Scheele, Priestley and Lavoisier isolated oxygen and defined its properties, Harvey (1628) recognised what we would now call the gas-transporting function of the circulation: ‘spirits … are transported by the circulation of the blood (to the tissues), and fulginous materials carried away’. Oxygen consumption in underperfused tissue, like fire in an enclosed space, depletes the tissue oxygen content and leads to tissue death. Haldane (1895) expressed the consequences succinctly: ‘Oxygen lack not only stops metabolism, it wrecks the machinery’. Barcroft (1920) was also specific about the physiological effects of an insufficient oxygen supply to living tissues. Anderson (1976) explained the pathology of tissue hypoxia as then understood: impaired oxidative phosphorylation, leading to impairment of ATP-dependent transport processes, hence osmotic imbalance, cell swelling, and necrosis and autolysis. The venous endothelium can survive moderately prolonged hypoxia (Jackson et al. 1988) by using anaerobic glycolysis to provide ATP (Malmqvist et al. 1991; Berna et al. 2001), but its physiology is altered and the anaerobic ‘safety-net’ may be good for only 1 or 2 h before death ensues2. Circulating blood cells are also susceptible to the effects of hypoxaemia with a comparable allowance for anaerobic functioning. This is a crucial point. Hypoxia evokes ‘emergency’ responses from the endothelium, changing the cell phenotype; if it is sufficiently intense or prolonged, the cells die, along with the blood cells sequestered in the VVP. This trapped blood stays fluid but inert, as the experiments of Hewson, Lister and Baumgarten (Chapter 8) showed. If and when pulsatile circulation recurs and introduces fresh, oxygenated blood into the pocket, it simultaneously reintroduces viable leukocytes and platelets. These living, oxygenated cells may respond physiologically to the possibly dead/necrotic endothelium lining the ‘central’ valve cusp, or perhaps to physiologically stressed though still (marginally) viable endothelium.3

10.2

Hypoxaemia, the Vascular Endothelium and Thrombosis

The first half of the 20th century saw several discussions of the vascular endothelium and the effects thereon of changes in perfusion (e.g. Clarke and Clarke 1935). A seminal paper by Frykholm (1940) made no explicit mention of oxygen, but was underpinned

1 It might be said that respiration is as old as the Colosseum, but pneumonia dates back to the Parthenon. 2 This may significantly over-estimate the anaerobic capacity of the avascular valve cusp endothelia; see footnote 12 in Chapter 9. 3 We emphasise necrosis rather than apoptosis. Endothelial cells can undergo apoptosis and this is associated with increased platelet anchoring (e.g. Bombeli et al. 1999), but we do not consider this to be relevant to the aetiology of DVT: hypoxia causes necrosis, not apoptosis, in most cell types.

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by the ‘suppressed major premise’ that the vitality of the endothelium might be impaired by ‘inadequate nutrition’, i.e. inadequate blood flow and local hypoxaemia. Frykholm wrote: ‘Physical endothelial injury can always give rise to thrombogenesis even without the association of other factors’, but even though this ‘has been verified in numerous animal experiments, and everyday in operative procedures, it has not been possible to accept such (artefactual) experimental injuries as an explanation of spontaneous (authochthonous) thrombosis in man’. Moreover: ‘When a patient is confined to bed the flow of arterial blood to the extremities diminishes in consequence of diminished need for such blood: the total quantity flowing per time-unit must diminish’. (His reference to ‘tissue need’ indicated his vital-materialist perspective.) Frykholm went on to hypothesise that ‘thin-walled’ veins probably collapse (1) in the horizontal position, relieved of the weight of the vertical column of blood, (2) under the weight of the horizontal leg – juxtaposing the anterior and posterior vein linings – or (3) if reduced vis-a-tergo reduces the throughput of blood. He envisaged the vein lumen reduced4 to ‘intima against intima, while a minimal stream of blood will percolate through a crack-like lumen in some part of the vessel …’ More interestingly, he discussed the reduction of venous pressure in the extremities of immobile or anaesthetised patients by arterial underperfusion, and the consequent lowering of vis-a-tergo in veins. He considered no other cause of venous hypotension, but his summary includes these points: (3) The vitality of the endothelial cells depends to a great extent on their contact with flowing blood, and, when the cells are deprived of this source of nutrition, disturbances of nutrition arise and the thrombosing process begin. (4) Injury to the intima is the most important factor in the pathogenesis of thrombosis. It can be counteracted by raising the head end of the bed so that the patient tends to slide downwards in the bed. Then the venous pressure in the extremities will rise so that the veins become distended with blood and the patient be forced to make active movements with his/her legs to restore position. Thus the veins which are especially threatened by thrombosis will be rhythmically emptied and distended. [Our emphases]

10.2.1

Association of DVT with Endothelial Hypoxia

The proposal in that final sentence was notable and in a sense original: Frykholm’s argument was underpinned by an idea that he did not specify elsewhere in his article. It suggested that the primary cause of DVT is a venous endothelial lesion caused by deprivation of oxygen and nutrients, which may be minimised or 4 This simple mechanical idea, that ‘external pressure on veins must narrow them fore and aft’, is untenable. It implies that anyone sitting in a chair, standing on the ground or lying in bed must be foreclosing some veins for possibly extended periods. But as we know, occlusion and exsanguination of limbs by tourniquet and high-pressure Esmarch bandaging ‘collapses’ veins in precisely the manner described by Frykholm, yet periods of less than 150 min of tourniquet application are not followed by thrombosis. Orthopaedic surgeons throughout the world regularly work in such ‘bloodless fields’ without mishap.

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mitigated by restoring and maintaining pulsatile blood flow. However, Frykholm did not analyse how the filling, emptying and re-filling of VVP (as emphasised here) would determine that ‘nutrition’. Approaching the subject from a different angle, the histology of thrombosis-like lesions in vessels in cases of CO poisoning, Drinker5 (1938) came to suspect that comparable hypoxaemia might be involved in the causation of DVT. He suggested this to O’Neill (1947), who pursued the possibility that the ‘vein wall nutrition’ is supplied by the vasa venarum rather than (or as well as) the blood circulating in the vessel lumen. O’Neill wrote: ‘No matter what the source of vein wall nutrition. … Oxygen, unlike foodstuffs, must be constantly supplied since storage facilities in tissues are practically non-existent.6’ Later, he observed: ‘Isolation without obstruction causes much more endothelial damage, but partial obstruction alone, though not as destructive of endothelium, is a slightly more frequent cause of thrombosis for the durations of these experiments’. Then he added: [I]t is not necessarily entailed that circulation through the vena [sic: vasa] venarum has been cut off. … But a variety of disorders may result in decreased oxygen supply to such vein walls: hypoxia from deep or prolonged anaesthesia or narcosis; interference with pulmonary ventilation; poor oxygen transportation as in cardiovascular diseases; occlusive arterial disease of extremities; or diminished blood flow to a resting or immobilised limb. … It is conceivable that, under such conditions, venous endothelium is caught in the middle, between two poor oxygen sources, whose total supplies are scarcely adequate to keep it intact. Add then stasis, which means allowing time, and a clot may promptly form. Such a theory … leads to a proposal that one should be able to produce experimental thrombi consistently in experimental animals by causing hypoxia plus obstruction of certain vein segments. [Our emphasis]7

Underperfusion of the vasa venarum is presumably synchronous with reduced venous velocity in most conceivable circumstances.8 Also, like Frykholm, O’Neill did not mention the valves or VVP, or the part likely to be played by intermittent/ pulsatile venous circulation. His work attracted some notice during the 1950s, when Samuels and Webster (1952) checked his findings and wrote: ‘From a biological 5 Philip Drinker was co-inventor of the famous iron-lung used to manage poliomyelitis. The apparatus had been previously designed to manage coal gas (carbon monoxide) poisoning, on which he was an authority. See following text. 6 This overlooks the anaerobic capacity of the venous endothelium; though in respect of the valve cusp parietalis the oversight might not be significant (see previous discussion). 7 That was in 1947; and although Samuels and Webster (1952) criticised O’Neill’s principles while confirming his findings (see following text), the theme had not been followed up when PCM thought to do so from an independent starting point in the mid-1960s, and did so experimentally in 1978–1984. 8 During extreme sporting exertion over a short period, the oxygen tension of venous blood leaving the active muscles may become extremely low. However, athletic activity is not usually complicated by venous thrombosis. Two considerations may be relevant here. First, the vasa venarum will remain perfused by normally oxygenated blood throughout the period of activity. Second, the valve pockets of the veins in and from the active muscle will be frequently and thoroughly emptied and refilled. We regard the second consideration as more important, for reasons explained in Chapters 8 and 9.

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point of view thrombosis is a means of maintaining the fluid level of the vascular compartment by plugging holes in it when they occur’. This phrase implies an overtly vital-materialist presumption, as appropriate in a surgical publication: i.e. a living, reactive vascular compartment may be presumed to respond purposefully to boundary lesions, a principle that recalls the argument of Lister (1863). However, the authors then argued for a need to curtail the spread of coagulation, since if unchecked it might be catastrophic. This view of thrombosis as a potential Sorcerer’s Apprentice was common amongst the thinkers of the era, whose experience of clotting in test tubes may have programmed them to expect the same in the vascular compartment. They were probably unaware of Lister’s magnificent and durably relevant 1863 Croonian Lecture. Later in their article, Samuels and Webster wrote: ‘It is commonly held that the endothelium of blood vessels draws its nutriment from the blood in contact with the surface,’ clearly dissociating themselves from O’Neill’s position, and went on: ‘JF O’Neill’s observations are confirmed, but the changes he saw are attributed to a foreign body reaction rather than to section of the vena [sic] venarum. … His thesis – that (underperfusional) injury to the veins of the lower limb plays an important part in the inception of thrombosis – is supported’. They made two other interesting points: (a) that heparin does not prevent thrombus formation on hypoxically injured endothelium, but limits thrombus development; (b) that vein distension during dissection decreases injury and thrombosis.

10.2.2

Endothelial Hypoxia and Thrombus Formation in VVP

These studies established empirically that hypoxic injury to the venous endothelium is thrombogenic, but they did not specify the relevant part of the endothelium. The central point is that since valve cusps are avascular, the parietalis endothelium of the cusp must necessarily obtain all its normal nutriment/oxygen from the blood within the VVP only. The luminalis endothelium may remain oxygenated by the (stillcirculating) luminal blood. Consequently, should the VVP not be regularly evacuated and refilled with a fresh charge of blood, the parietalis – specifically – must become oxygen-starved in proportion to the duration of the delay. If sufficiently prolonged, this must lead to death and local necrosis of that single layer of parietalis endothelium. Moreover, blood cells may be presumed sufficiently ‘alive’ and active to make the blood normally coagulable if the blood is still-circulating, still-living. Dead or dying blood within a long unperfused VVP will be incoagulable (Fig. 9.7). The painstaking study of Paterson and McLachlin (1954) was consistent with this account and their paper merits further discussion. They homed in on ‘perfusion’, perhaps in response to the Drinker-O’Neill findings. Again, however, they did not specifically indict a temporary cessation of pulsatile flow. Later, McLachlin et al. (1960) clearly demonstrated underperfusion of VVPs by a radiographic dye injection technique, which provided definitive evidence of VVP hypoxaemia without recourse to oximetry. Paterson (1969) subsequently stated that ‘initiation … of

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thrombi … is … never on the inner surface of the valve curtain’ – which is not true, though it is commonly inferred from micrographs (cf. Sevitt 1974a; see below). Concerning their examination of the venous wall endothelium close to in situ thrombi, Paterson and McLachlin commented: ‘Serial section of a whole thrombus is a formidable undertaking, and if anything less is done then a small precipitating lesion in the vein wall may easily be missed. We have therefore examined 21 tiny incipient valve pocket thrombi by complete serial section’. They did not describe the appearance of the valve cusps/ leaflets in detail, though Paterson (1969) referred to them again. They attributed leukocyte infiltration to ‘the thrombus interfering with the nutrition of the vein wall by blocking the normal process of imbibition, rather than to infection’. Crucially, however – unlike Frykholm, O’Neill or Sevitt (1974a, b) – they observed (re. their serial sections) that ‘of 21 tiny thrombi, 80% arose in valve pockets. The usual site of thrombus attachment was at or near the apex of the pocket where stasis of blood should be most extreme. Microscopically, thrombi were most organised at these points and of more recent structure as they extended up the pockets before emerging at the mouth… One can only speculate on the possible changes that occur at the apices of valve pockets and result in the initial thrombotic mass. Like other vascular channels, a vein depends on the imbibition of substances from the blood in its lumen for the nutrition of its inner (intimal) layers.’ [It is notable that these authors overlooked the probable significance of the vasa venarum.] ‘With venous stagnation, these substances, including oxygen, will be depleted; and it is reasonable to suggest that morphological changes of a highly intimate type occur in the lining endothelial cells and lead to primary thrombus deposition. We have tried to demonstrate such changes but our efforts have been unsuccessful …’. Their figure of 80% is striking because this study considered only the parietal (within-vein) valves; the other 20% of thrombi almost certainly arose in the ostial (vein-junction) valves,9 as implicit in the micrographs of Stewart et al. (1984) and Stone and Stewart (1988) shown in Fig. 9.6. However, Sevitt (1974a) wrote that he found less than 10% of thrombi associated with (i.e. in) VVP. The inconsistency is startling. In this context, it is instructive to re-examine Fig. 71 in Virchow (1858), reproduced below as Fig. 10.1.

9 In the context of counting VVP thrombus nidi, it must be emphasised that ostial VP thrombi may not appear to be associated with valves unless specifically ‘sought out’; and such ‘seeking out’ may itself destroy that evidence unless it is very carefully done; see main text and legend to Fig. 10.1. The diligence of post-mortem search and recognition of the pre-death dehiscence of unknown numbers of thrombi before death or during manipulation, and the hidden location of ostial thrombi that do not protrude into the main lumen (i.e. look like ‘residual blood’ in the tributary lumen), present constant challenges to thrombus enumeration and ‘capture’. For such reasons, to expect unequivocal and unbiased empirical/ observational results may be to ask the unattainable, even from such dedicated workers as Paterson and Sevitt.

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Fig. 10.1 Fig. 71 from Virchow (1858). This illustration is copied to draw attention to ostial thrombi, which are rarely illustrated but differ significantly from main vein parietal valve thrombi. C = femoral vein. Original legend: ‘Autochthonous and elongated thrombi. e, e1 Smallish, varicose, lateral branches (circumflex veins of thigh), filled with autochthonous thrombi, project beyond the orifices into the trunk of the femoral vein. t Elongated thrombus produced by concentrically apposed deposits from the blood. t1 Elongated thrombus, as it appears after fragments (emboli) have become detached from it’

10.2.3

The Significance of Ostial Valves

No histologist investigating DVT has reported evidence of endothelial injury on the vein wall, despite tedious serial sectioning of thrombus nidi and their environs; yet not all venous thrombi originate in parietal valves. That conundrum could be explained if some thrombi form in ostial valves and grow precisely as Virchow depicted in his engraving (Fig. 10.1; see also Figs. 9.5 and 9.6). Consequently, unless histopathologists serially section the mouths of tributaries, and plot the relationship to ostial valve cusps found in such sections, their failure to locate endothelial injury is to be expected. Virchow’s engraving (Fig. 10.1) suggests that different investigative procedures could reveal different proportions of parietal to ostial valve thrombi, and this might explain the extraordinary difference of opinion between Sevitt and Paterson. Moreover, as the engraving illustrated, the propensity of thrombi to dehisce and/or fracture to release emboli, and the tiny residual lesion barely visible in the tributary mouths, show that a search for the primary site of a fatal embolus could be vain, since it is marked only by the trivial little thrombus in, or poking out of, a femoral vein tributary. Such veins would have to be harvested and dissected very much more thoroughly, perhaps after isolation and glutaraldehyde fixation, if accurate knowledge of the true extent of thrombosis in leg veins and their valve-guarded tributaries were to be sought. Several other points arise from Fig. 10.1. 1. Virchow recognised that thrombi grow by ‘concentrically apposed deposits from the blood’, confirming that from the outset he recognised the critical distinction between ‘clot’ and ‘thrombus’: the analogy did not betoken identity.

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2. The engraving depicts a thrombus that has formed in and filled an ostial valve, after which the backed-up tributary lumen thrombus (distal to that valve) produces the extended blockage illustrated. 3. Virchow’s picture strikingly resembles the experimental dog thrombi produced by Hamer and Malone 1984, in which what appeared to be parietal valve thrombi blocked the vein lumens, but putative ‘thrombi’ also appeared in the mouths of tributary vessels in early stages of formation (see Fig. 11.5). 4. Only if both VVP in a double-cusp parietal venous valve are filled with occlusive thrombus may main venous trunk be grossly obstructed (a ‘double VVP’ thrombus). However, the ostial valves at the junctions of tributary veins with main veins are unicuspid, and a thrombus there may occlude their lumen-orifices completely. Virchow notes that such tributary veins are ‘varicose’. Ostial valve thrombi would be the prime cause of severe superficial venous varicosity, since the obstructed venous flow must find another return route. Thrombosis is potentially more dangerous in the unicuspid ostial VVP than in one of a pair of cusps in a parietal valve: the latter will leave the lumen partially patent, the former will not.

10.2.4

Interpreting Micrographs of Venous Thrombi: The Tendency of Thrombi to Embolise

Essentially, we suspect that both Paterson (1969) and Sevitt (1974a, b) – presumably led by consensus expectations – overlooked the likelihood of artefactual splits (fractures) between dead, often degenerate cusp parietalis and the dead layer of blood cells flimsily ‘attached’ to it. Layers of dead leukocytes/platelets inactivated on a necrotic endothelium are by definition a lifeless zone (see also Sevitt and Eeles 1967). Many of their micrographs show gaps between the parietalis of the leaflet and the thrombus. In others the parietalis had apparently dehisced from the leaflet basement membrane during histological preparation. Leukocytes and platelets in fresh, living, oxygenated blood in a VVP will attach themselves firmly to a necrotic parietalis endothelium and initiate repair, more or less as described by Ashford and Freiman (1967). However, if non-pulsatile flow is resumed, the blood in the VVP again starts to become hypoxaemic, prejudicing the ‘grip’ of the blood cells on the dead endothelium they have begun to remove (see also Chapter 12). Besides the very weak or fragile nature of the bond between the cusp and the nascent thrombus surface, it must be kept in mind that the VVP was, and may again become, filled (each refilling potentially restoring valve function, fully or fleetingly). If a thrombus has formed in a ‘half-closed/half-open’ valve, any resumption of centrifugal reflux into the pocket will tend to strip the valve curtain from the nascent thrombus and force the edges together once more, ‘ballooning’ the cusp off its surface. The new situation would be a partial or complete dehiscence of the valve curtain from the thrombus in situ. Alternatively, the parietalis – wholly or partly necrotic – could be stripped off on to the thrombus, leaving the central cusp surface denuded of endothelium. These concepts of in situ dehiscence are wholly consistent with the characteristic tendency of such thrombi to break free and embolise, and imply that all VVP

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Fig. 10.2 A photomicrograph of a longitudinal section through a thrombus in a venous valve pocket, It appears that the luminalis and basement membrane layer of the cusp has dehisced (been torn away) from the parietalis endothelial layer, which remains attached to the ‘face’ of the thrombus. This conjunction is only evident because the inner cusp layer has been stripped from the outer two at X at the base of the valve leaflet, a few millimetres up from the agger, clearly seen crossing the cusp-thrombus dehiscence gap, and then (with the eye of faith) running up the left face of the thrombus margin to the specimen boundary. (This micrograph and others reproduced below were kindly bequeathed to PCM by the late Dr. Simon Sevitt.)

thrombi have a predilection to dehisce from the moment they come into being. Were venous valves normally still and static, they could be imagined to remain so; however, once a semblance of normality is restored, they must be expected to flap open and closed like a flag in a high wind. They may therefore detach from a thrombus, depending on its size and on the relative strength of its attachments to the mural endothelium of the valve sinus and the flimsy valve cusp (Fig. 10.2). We shall return to this theme when we reassess the various published collections of postmortem thrombi (Chapter 13). Comparative electron microscopy of normal (uninjured) and pathological venous valves fixed rapidly in situ might be more informative, though it would

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always be difficult to exclude artefacts incurred either in vivo or during histological manipulation/preparation. However, there is a simple (presumptive) way of deciding whether the light-microscopic appearance of valve leaflet detachment from a thrombus occurred in vivo, or while awaiting post-mortem, or during preparation, fixing and staining. All endothelial dehiscences (‘elevations from’) living or newly dead blood-vessel wall are filled or contaminated by fresh blood and cells flowing into the ‘split’ zone between leaflets and thrombi or basement membrane and endothelium. We draw particular attention to the histological evidence published by Paterson (1969) and Sevitt (1974a, b), showing only one unequivocal example of a mass of blood in such a gap. This suggests that the remainder were histological artefacts, and that future studies in this field should aim to elucidate the finest detail of VVP thrombi fixed in situ to exclude such artefacts. Paterson and Sevitt were eminent histologists and pathologists with great experience in this field, but they may seem to have overlooked an apparently simple inference: because deep venous thrombi embolise anchorlessly to remote parts, it should be expected that sections will show dehiscence of thrombi from their first ‘moorings’. This may provide another explanation for Sevitt’s claim (Sevitt 1974a) that only 10% of thrombi appeared to be attached to valve cusps: if sustained hypoxaemia has rendered the endothelium and blood cells necrotic, dehiscence would seem certain to follow. Strikingly, Paterson (1969) placed less emphasis on the perfusion/hypoxia factor in thrombosis than he had in his earlier papers. His earlier association with surgical colleagues and their vital-materialist, physiological approach had perhaps yielded to the new consensus that emphasised ‘stasis and hypercoagulability’. Paterson said only that ‘conceivably – surface impregnations as used by O’Neill, Samuels and Webster, and Robertson et al., or even more sophisticated techniques, will reveal a lesion in the vein wall intima that will explain why platelets clump at one point in the valve pocket and not at another’. Like so many authors, his emphasis on vein wall injury made him oblivious to the significance of the valve cusp and its viability. Few publications of this period developed the perfusion/hypoxia theme: Lillehei et al. (1964) showed that ischaemic hypoxaemia proceeds to anoxia, but it is difficult to find others.

10.2.4.1

Re-Evaluation of the Micrographs of Sevitt (1974a)

The sequence of events that gave rise to the appearance of Fig. 10.3 (and are mirrored in some of Sevitt’s other micrographs) is fascinating to analyse; the following reconstruction, illustrated in Fig. 10.4, is of course speculative. Starting with a normal VVP, the first event was perhaps a period of more than 3 h during which the blood-filled, unemptied VVP sustained severe hypoxaemia that injured the parietalis endothelium (A in Fig. 10.3). The thrombus Th1 then formed by serial layering over a period of hours or perhaps days during random short restorations of VVP perfusion. Subsequently, the normal intermittency of blood flow in the vein was re-established and the intermittent pressure changes stripped off the

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Fig. 10.3 Fig. 13 from Sevitt (1974a). Dr. Sevitt published this histological slide as a ‘double thrombus’, which it is; but its complex structure is remarkable enough to merit theoretical analysis, suggested by the appearance in Fig 10.1 above. This raises questions about the interpretation of what seems unnoticed valve cusp pathology in Sevitt’s very valuable illustrations. The interpretation suggested in the text is inevitably suppositional. Its purpose is to encourage future studies, particularly in situ fixation of thrombi with their valves, in order to confirm scientifically that parietalis and ostial valve cusp endothelium is indeed the injured tissue that initiates thrombosis. From right to left, this image includes (VC) valve cusp, (Th2) a classically patterned thrombus, (PE) the parietalis endothelium in cross section, (Th1) a second classically patterned thrombus, (C) an equally classical un-patterned clot, (VW) vessel wall and endothelium. The upper two thirds of the valve cusp (VC) appears ‘thinner’ in this micrograph than in the lower third of the cusp. The cusp outline narrows from a few millimetres from its lower margin, and continues to do so all the way up from that point. We suggest that the upper two thirds of the cusp has lost its parietalis layer, which appears to be incorporated in the junction between Th1 and Th2. This might explain the attenuated ‘upper’ cusp outline. Copyright 1974 John Wiley and Sons Reproduced with permission

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Thrombus A forming on parietalis.

Thrombus B forming on the luminal side of the parietalis in the gap between it and the re-mobilised cusp.

Thrombus A and parietalis dehisced from basement membrane and luminalis.

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Fig. 10.4 A speculative reconstruction of the events leading to the appearance of the thrombus in Fig. 10.3. This series of diagrams illustrates the explanation given in the main text

cusp (A), which then ‘closed functionally’, thereby restoring valve competency (having torn the valve from its endothelial anchorage – see line C in Fig. 10.3). This re-establishment of normal valve cusp action was likewise temporary, and was followed by one or more periods of VVP non-perfusion, leading to the formation of a second thrombus, Th2. Th2 is structurally distinct from Th1; it seems to be layered longitudinally rather than transversely. We suggest that Th2 was formed considerably later than Th1 and that quasi-normal function was restored temporarily during the interval. Presumably Th1 attached to the vein wall F before it dehisced, permitting the gap E to fill suddenly with blood en masse that was not expelled by passive movement of the valve cusp (which acted only to create Th2 between VVP fillings and evacuations). The oblong coagulum OC is not a characteristic thrombus and probably represents the ‘falling away’ of the mass of blood that filled the gap left by Th1 when its weight caused it to dehisce from the mural endothelium. A final notable point is that the two older classical thrombi, Th1 and Th2, with the parietalis endothelium (PE) sandwiched between them, appear to be unanchored except to the oblong blood mass E. This mass is itself attached to the mural endothelium in the agger region, with the lateral aspect of Th1 seemingly attached to it at H. Conceivably the old dead Th1 was being ‘restrained’ from embolising by the ‘organising’ simple clot E. Fig. 10.5 shows more of Sevitt’s photographs of thrombi in VVP, and one photograph of a pulmonary embolus for comparison.

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Fig. 10.5 A compendium of photomicrographs bequeathed by Dr. Sevitt (a–c) Valve pocket thrombi; (d) pulmonary embolus. The detailed history of these photographs is not available, so only three general comments may be made. 1. They were obtained from patients almost certainly killed by pulmonary embolism arising from thrombi other than those illustrated. 2. The dehiscence between thrombus and either valve cusp or vessel wall is strikingly uncontaminated by any infusion of blood cells, suggesting that such dehiscence is a fixation or procedural artefact in most cases (see main text). 3. The massing of leukocytes/platelets on or close to the parietalis face of the valve cusps is undeniable

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Carbon Monoxide Poisoning and Anaemia

The role of oxygen-lack in the aetiology of DVT is singularly well illustrated by its known complication of carbon monoxide poisoning (in cases that survive for long enough). As we recounted earlier, O’Neill (1947) acknowledged Drinker’s (1938) suggestion, which was based on his finding of quasi-thrombi in (chronically) carbon monoxide-poisoned human blood vessels. When coal gas was widely used for illumination and heating, CO poisoning, accidental and suicidal, was common. Haldane (1895) established that its sole action10 was to reduce the oxygen carrying capacity of the blood, and thus to mimic a degree of acute anaemia proportional to the CO concentration in circulating oxygen-depleted erythrocytes (the PO2 in arterial blood plasma is normal, but the poisoned erythrocytes carry proportionately less oxygen). Because oxygen is stripped from the erythrocytes during transit through the perfused tissue, they become extremely oxygen-poor in the capillaries/venules (much below the normal venous value) whereas the plasma PO2 remains normal. Circulating ‘colourless corpuscles’ therefore have access to a normal supply of oxygen in the pulmonary capillaries and the systemic arteries, so they remain viable after they have passed through the peripheral capillaries into the severely hypoxaemic veins. Should they become attached to the mural endothelium, and if the atmospheric CO poisoning continues, these cells must die when their oxygen supply becomes exhausted. Drinker (1938) wrote (p. 124): ‘The incidence of widespread minute thrombi following carbon monoxide asphyxia is of particular moment… There are two schools of thought as to their cause – (i) that the blood becomes more coagulable and (ii) that thrombi follow minute injury to vessel walls. Neither contention can be proved, but in my opinion, the second is correct. … It seems probable then that thrombus formation is an expression of (hypoxic) damage to the vessel walls…’. This proposal prompted his suggestion that O’Neill investigate the role of endothelial oxygen-lack in thrombosis. Three years later, van Ottingen (1941) reviewed this well-researched field: According to Lechleitner (1933) injury of vascular endothelium [by CO] is quite common and may be observed in small and large vessels, and in the heart. Klebs (1865) assumed that the first reaction of blood vessels in CO poisoning was a functional change causing atonia of the vessel walls. A similar assumption was made by Hiller (1924) and Meyer (1926) who thought that stasis resulting from this dilatation was the cause of further degenerative changes. … Occurrence of thromboses and emboli as sequelae of CO poisoning is not exceptional: Berkhan observed a partly organised thrombus evidently originating from endothelial damage in a case of CO poisoning. Hedinger (1923) quoted Wachholz (1905) as being the first to report extensive thromboses as sequelae to CO poisoning, adding reports of two cases with extensive clotting in the heart and arteria pulmonalis possibly

10

With the benefit of hindsight, we might demur. Carbon monoxide inhibits a number of haemcontaining proteins, not least the cytochromes. But there is no doubt that powerful competition with oxygen for the binding sites on haemoglobin is the main toxic effect of CO in vertebrates.

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originating in varicose veins of the legs. Wiskowski (1921) saw extensive thrombosis in the lower right leg [of an 11 year old girl]. … Brack (1928) reported 22 cases of illuminating gas poisoning in which extensive capillary thromboses were scattered throughout the whole heart …. [Our emphasis]

Few authors since the Second World War have elaborated on the theme of carbon monoxide and vascular endothelial damage. Fowler (1954) described a case of leg gangrene following CO inhalation. An interesting paper by Fujita et al. (2001) showed that CO, inhaled in very low doses or produced endogenously by type 1 haem oxygenase, can suppress hypoxia-induced fibrin deposition in mammalian lungs,11 and this has led to proposals that low-dose CO may have therapeutic value in cases of vascular ischaemia and, inter alia, the prevention of thrombosis (e.g. Mishra et al. 2006). At present, however, we are considering the injurious effects of CO and will defer discussion of the hypothetical therapeutic mechanisms explored by Mishra et al. (2006).12 The occurrence of thrombosis during severe but non-fatal carbon monoxide poisoning suggests that severe anaemia could also be complicated by thrombosis. This is improbable, however, since the degree of hypoxaemia considered in VVP (above) would kill the person were it to occur throughout the body – as would the ‘equivalent’ anaemia. Furthermore, according to the available oxygen thesis13 (Nunn and Freeman 1964), anaemia of 50% may be compensated by an increased circulation rate of 200%, carrying the same amount of oxygen to the tissues provided the blood volume is normal. In a word, hypokinetic hypoxia consequent upon some form of circulatory impairment must be the ineluctable central cause of thrombosis. Anaemia and other defects of oxygen carriage may contribute to the whole picture, just as arterial hypoxaemia might (Jussila et al. 1977); but without underperfusion of a local endothelium within a VVP, thrombosis is likely to be a rare and improbable consequence. The logic (as above) is that anaemia and arterial hypoxaemia affect all tissues equally, so if they are severe enough to produce vascular obstruction in one vessel, then they are likely to produce it in all – and to kill the patient before localised cell death and a resultant thrombotic lesion become possible.

11 The mechanism appears to involve the activation of soluble guanylate cyclase, which inhibits the hypoxia-induced activation of plasminogen activator inhibitor-1. 12 Biphasic responses to toxins and other insults – protective at very low doses, deleterious at higher ones – are commonplace. There is a considerable literature on this phenomenon, dubbed ‘hormesis’, though it is not clear that any single mechanism is involved in all cases. 13 Available oxygen = perfusion rate × haemoglobin concentration × arterial pO2. This equation expresses theoretically how a minor reduction of each factor (to, say, 80% of normal) would yield a final available oxygen of 80% × 80% × 80% = 51.2% of normal.

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Endothelial Hypoxia and Leukocyte Margination

Endothelial lesions are characteristically followed by local margination of leukocytes and platelets (Chapter 7), whether the lesion be caused by experimental vessel wall injury, hypoxaemic endothelial injury or CO toxicity. This recalls the 19th century microscopic descriptions of ‘stasis’ in small vessels (Chapter 8). Because CO poisoning causes tissue and venous endothelial hypoxia, identical in effect to underperfusion, it causes leukocyte margination and diapedesis identical to that seen in non-perfused tissues (Thomas et al. 1983). Van Ottingen (1941; see above) continued: According to Altschul (192714) the vascular changes leading to thrombosis start with a mobilisation of leukocytes which are concentrated on the vascular walls, penetrate into and accumulate in the walls. Why this infiltration – what is its mechanism? Is it changes in the vessel walls … fatty degeneration of the vessel wall … or does parenchymatous change apply an attracting force to the white blood corpuscles?… It is undeniable that there is some connection between the observed infiltration and the CO-poisoning. … Then the apparent closing up of the vessels by the massing of white corpuscles, which, if a fatal outcome had not come so soon, might possibly have given rise to yet other phenomena (thrombosis, necrosis about the site) ….

It seems plausible to answer van Ottingen’s questions by proposing that sustained hypoxic or other injury to the endothelium provokes a phagocytic leukocyte response at the site. Sandison (1931) showed that leukocyte margination and ‘adhesion’ occurred very rapidly and occlusively after the induction of hypoxaemia, suggesting that it was triggered almost instantly by sudden total ischaemia; there was immediate recovery when normal flow in his rabbit ear chamber was restored (Chapter 7). Moreover, a series of papers by Stewart and her colleagues (e.g. Stewart 1975; Stewart et al. 1974, 1978, 1980) showed that plasma protein and leukocyte attachment to, and accumulation on, the endothelium may accompany/ coincide with trauma or surgery at distant sites in the body.15 Lost endothelium is rapidly replaced (within 48 h) by cellular proliferation unless it is very extensive or is impaired by fibrin deposition (Krupski et al. 1979). We infer that although endothelial hypoxia induces leukocyte infiltration, as shown by Gibbs (1957), such infiltration need not necessarily betoken necrosis. It may indicate that the attractant is dying rather than dead, or in the pre-necrotic phase. In thinking about hypoxic death (as such) it is important to keep in mind that

14 It was during this era (the 1920s) that the physiological effects of CO inhalation were extensively studied in Germany; cf. the citations in the earlier quotation from van Ottingen. 15 This will be discussed further in Chapter 11. Stewart and her colleagues suggested that the leukocytes inflict injury on the endothelium, and other authors have made similar suggestions. Of course, activated leukocytes are likely to remove endothelial cells that are already injured, but if they were to attack normal, uninjured cells then none of us would have any vascular endothelium at all (Chapter 12). Stewart and co-authors did not mention the very considerable ‘pooling’ of blood in the injured limb, one consequence of which would have been reduced perfusion of the damaged jugular vein and in particular its VVP.

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the living state is always ‘recoverable’ unless a point of no return has been passed; death from oxygen deprivation alone is rarely instantaneous. In short, the aetiology of DVT involves a sequence of events, which could make it naïve to imagine a simple/single linear causal chain.

10.5

Aschoff on the Coagulation of Cadaver Blood

Virchow (1856) wrote: ‘A fibrolaminar clot, a thread, a filament, in the retarded blood flow can initiate that linking of fibrogenous substance with oxygen from the blood corpuscles and can organise itself by attracting new fibrous material which then takes effect as a new aggregation of contact-bodies’ (see Chapter 6). Ludwig Aschoff was a pathophysiologist of Virchow’s school and seemed cognisant of Virchow’s imperfect speculation about the connection between oxygen and thrombosis. In a study to resolve whether thrombosis in cadavers is ante-mortem or post-mortem, Aschoff (1922) injected air/oxygen into the heart chambers of two subjects shortly after their deaths and found that the myocardial contractions restarted. These contractions continued rhythmically for 1 h before they stopped again. When he re-injected more air, the heart re-started, but not for long. Importantly, no thrombus formed. He drew no definitive conclusions from the experiment, but he seems to have expected the insufflation of oxygen to cause thrombosis: ‘I have tried to find an answer to this question [of intra-cardiac thrombosis] but without success, and wish to note only that it cannot be lack in oxygen [did he mean presence of oxygen?]. I have introduced a large amount of oxygen into the still entirely liquid blood of the right ventricle immediately after death without obtaining any coagulation, but the same blood coagulated immediately after I removed it from the heart’. Aschoff may have misinterpreted Virchow’s suggestion: Virchow said that coagulation/thrombosis depends on oxygen, not that it was caused by oxygen.16 He apparently shared Hunter’s view that coagulation of the blood is a living function; that dead blood can no more coagulate than a dead brain can think or a dead kidney can excrete urine. Yet elsewhere, his approach seems overtly mechanistic (Chapter 7). Aschoff (1922) presented 41 First World War autopsy records, including 11 cases of gassing on the battlefield. These soldiers had presumably died of gross respiratory (as opposed to circulatory) failure, with severe arterial and venous hypoxaemia. In none of his 11 cases was there any sign of coagulum, buffy coat or cruor in the heart or in any blood vessels, so there was no stratification. This retrospective evidence of incoagulable, thrombus-free blood confirmed that hypoxia does not ‘cause’ thrombosis. The blood in the dying or dead hypoxic vascular channels was itself dying from that same ‘universal’ hypoxaemia, so it could not coagulate and no thrombus

16 This is an obvious confusion: the central nervous and skeletal muscular activities involved in typing this footnote depend on oxygen but are not caused by oxygen.

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could form. Aschoff’s findings are obviously consistent with the observation (Hewson 1771; Lister 1863; Baumgarten 1876) that asphyxiated blood coagulates more slowly than normal and may stay fluid indefinitely. In any case, the blood of criminals executed by hanging (Morgagni 1769), and those killed by drowning and other ‘respiratory’ fatalities, conforms to this thesis: it always remains fluid after death. The remaining thirty autopsies in the series Aschoff reported in 1922 were on soldiers who were not gassed but had died from gun-shot, grenade shrapnel, carbon monoxide, pneumonia, sepsis, tuberculosis, fat embolism and other traumas. Nineteen of those 30 (non-gassed) bodies showed coagulum, buffy coat or cruor, in various degrees, in the heart and major vessels. Thus, 63% of general traumatic autopsies showed ‘clot formation’ in the heart and elsewhere. Aschoff underscored the frequency and normality of this finding by commenting about the ‘usual (i.e. coagulated) condition (of blood) in the heart’ (cf. Walton’s chapter in Curran and Harnden 1972). Of the 11 un-gassed soldiers who also had fluid/liquid blood at post-mortem, five had perhaps suffered other forms of acute terminal respiratory failure (two from tetanus – conceivably in terminal respiratory clonus, three from pneumonia – conceivably in terminal pulmonary arterial hypoxaemia). Four may have suffered instant deaths similar in effect to drowning/hanging, i.e. in acute respiratory failure. The remaining two were the subjects of the oxygen-insufflation experiments described above. The conclusion is inescapable: notwithstanding the key role of local hypoxaemia and endothelial hypoxia in thrombosis, oxygen (and the vital processes that depend on it) is a prerequisite for both clotting and thrombosis. Living blood cells are required, and blood cells die if they are oxygen-starved. The implications for post-mortem blood will be considered in Chapter 13.

10.6

Hypoxaemia and ‘Traveller’s Thrombosis’

The possibility of a link between oxygen-lack and DVT in passengers on long-haul flights (which had been known for decades but was very rare) came back to public notice when trans-oceanic jet flights attracted more and more mature travellers (though it has killed younger-than-average flyers). Investigators realised that all high-fliers are subject to mild hypobaric hypoxia (cabin pressure during commercial flights is set to that at 1,600–2,000 m above sea level), and although arterial pO2 varies among individuals, the average concentration is approximately 5% lower than at sea level (Muhm 2004). Bendz et al. (2000) suggested the lowered cabin O2 pressure could explain ‘traveller’s thrombosis’ and recruited conceptual support from the recent literature linking venous endothelial hypoxia with the activation of coagulation (Gertler et al. 1991) or inhibition of fibrinolysis (Gertler et al. 1993); see Chapter 12. The proposal has been widely debated (e.g. Crosby et al. 2003; Hodkinson et al. 2003). It is a consensus-

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model explanation, loosely related to the knowledge that voyages to high altitudes are thrombogenic, especially when accompanied by exercise (e.g. Bärtsch et al. 1989); though the hypoxia encountered in cases of ‘acute mountain sickness’ is many times more severe than that experienced in aircraft cabins and is accompanied by much more physical activity. ‘Acute mountain sickness’ has long been attributed to the stimulation of the coagulation cascade by severe hypoxia under conditions of strenuous exercise (Bärtsch et al. 1989). We seriously doubt such explanations.17 A thorough crossover study by Toft et al. (2006) showed that mild hypobaric hypoxia has no measurable effect on the likelihood of DVT under conditions simulating long-haul air travel if the subjects have no recognised ‘DVT risk factors’. However, very similar conditions are claimed to increase the incidence of thrombosis in subjects who have factor V Leiden or are using third-generation oral contraceptives (Schreijer et al. 2006), suggesting that mild hypobaric hypoxia might interact pathogenically with certain ‘risk factors’. A very interesting aspect of both these studies is that prolonged sitting with exposure to mild hypoxia did not affect circulating thrombin levels/activity; this result has occasioned some surprise (Bärtsch 2006), because it is clearly not what the consensus model would predict. The general view has been that prolonged sitting causes ‘stasis’ and that this results in pro-coagulant and anti-fibrolytic changes in the venous endothelium, which mild hypoxia may exacerbate. An alternative opinion, broadly consistent with the work we have surveyed during this and the two previous chapters, suggests that the primary effects of prolonged sitting may be related to venous blood dynamics (Sudoh et al. 2003). In Chapters 11–13 we suggest that prolonged sitting may involve a greater degree of non-pulsatile (streamline) venous return flow, related failure of ‘up-circulation’, and the hypoxic death of valve cusp parietalis endothelia. Fundamentally, it should never be considered that tissue oxygenation related to blood flow, as in ‘hypovolaemia’, affects all tissues uniformly. It is ‘basic physiology’ that arteriolar constriction under central sympathetic control rations the available blood to different destinations, and thus rations the available haemoglobin and available oxygen. It is therefore to be expected that in different bodily states of activity or indolence or illness, local areas may be ‘hypoxaemic’, ‘very hypoxaemic’ or dying or dead while other ‘more essential’ parts/organs are being assiduously kept alive. It is perhaps plausible that mild hypobaric hypoxia alters the venous PO2 sufficiently to decrease the time required for hypoxaemia in an unperfused VVP to become injurious. On its own, this might not increase the likelihood of DVT significantly; but with the addition of one or more ‘risk factors’ it could have a more critical effect. We shall discuss the issue of ‘risk factors’ further in Chapters 11 and 12.

17 More plausible explanations for the condition may lie in (a) the hypovolaemia consequent on severe dehydration, which Aschoff (1924) showed to be thrombogenic, (b) lying in cramped conditions in a tent while a blizzard rages outside, with the calf muscle pump inactive and no pressure on the soles of the feet. Severe dehydration and hypovolaemia seem unlikely to be involved in cases of ‘traveller’s thrombosis’.

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10.7 Overview: Articulating the Valve Cusp Hypoxia Hypothesis The literature surveyed in this chapter is generally consistent with the hypothesis that DVT may be initiated under conditions of suffocating parietalis endothelial hypoxia resulting from hypoxaemia in underperfused VVP during episodes of nonpulsatile flow. Such localised oxygen-starvation could result from a variety of pathological circumstances including impaired venous blood movement and pulsation, left heart dysfunction, acute non-fatal CO toxicity, anaemia and hypovolaemia. Experimental physical injury to the endothelium was demonstrated in the 19th century to be invariably thrombogenic. Severe local VVP hypoxia may cause thrombosis during prolonged sitting. A prerequisite for a venous thrombus to form is the recurrent introduction of ‘fresh’, viable (oxygenated) leukocytes into injured VVP. Leukocytes and platelets must be alive (recently arrived) if they are to congregate on injured sites forming prothrombotic nidi. Such ‘layered attachment’ may then in turn extend the original injury and expand the DVT lesion. In essence, this encapsulates the valve cusp hypoxia hypothesis (VCHH) of the aetiology of DVT. The VCHH will be explicated fully in Chapters 11 and 12 and its scientific and clinical implications will be considered.

Chapter 11

The Valve Cusp Hypoxia Hypothesis

Abstract This chapter draws together the experimental support for the valve cusp hypoxia hypothesis (VCHH), developed stage by stage in Chapters 8–10, and considers its scientific and clinical significance. The VCHH attributes the aetiology of DVT to (1) underperfusion of venous valve pockets (VVP), (2) consequent hypoxic necrosis of the VVP parietalis endothelium, and (3) active responses by viable leukocytes and platelets to the hypoxic or dead endothelium. Because these events necessarily occur in a reiterated succession, venous thrombogenesis is a gradual, sequential process. The VCHH thus re-deploys ancient and modern knowledge of valve cusp structure and function; it focuses on the precise site of prolonged blood ‘stasis’ in venous valve pockets, avoiding the old, imprecise, Galenic usage of ‘stasis’. The crux is why and how blood that entered a VVP ‘alive’ may have ‘died’ there if not replaced with fresh oxygenated blood for many hours. The VCHH is sharply distinct from the consensus (haematological) model in scientific character, medical implications and philosophical connotations, and these distinctions are briefly discussed.

Keywords Non-pulsatile flow, valve pocket perfusion, parietalis endothelium, hypoxia, leukocytes

11.1

Introduction

Since the end of the Second World War, the mechanistic consensus model of the aetiology of DVT invoked by haematologists and biochemists (Chapters 2 and 3) has marginalised the previously orthodox Virchowian, pathophysiological approach. We have challenged its dominance, arguing that it is misleading to regard DVT as a coagulation disorder, a perturbation of ‘the delicate balance of the haemostatic machinery’, and that the viewpoint of surgeons and pathologists such as Hunter, Lister, Welch and Aschoff is more productive: the condition is functio laesi. We have explored the roots of both schools of thought (Chapter 4), the mid-19th-century schism that divided them (Chapters 5 and 6), and the ensuing interpretations and misinterpretations of the elements P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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of ‘Virchow’s Triad’ – a phrase that we have shown to be a misnomer (Chapters 1 and 6). In Chapters 7–10 we examined these (mis)interpretations in detail, establishing the valve cusp hypoxia hypothesis (VCHH) as an alternative, pathophysiological account of the aetiology of DVT. The present chapter summarises the VCHH, compares its scientific and philosophical status with that of the consensus model, and considers some of its implications for biomedical research and clinical practice. PCM’s website (Malone 2003) applies its principles to traveller’s thrombosis. To establish a pathophysiological hypothesis (the VCHH) as an alternative to a mechanistic one (the consensus model) is to achieve only one of the aims of this book. It is also necessary to show that the VCHH can be reconciled satisfactorily and productively with mechanistically-orientated research (cf. Feinstein 1999). We believe that such a rapprochement can be achieved effectively in this and in other fields of medical investigation. We shall address that point at the end of Chapter 12 and in the appendix.

11.1.1

Criteria for an Aetiological Hypothesis

A hypothesis of the aetiology of DVT should satisfy two broad classes of criteria: those relating to medical-scientific hypotheses in general, and those relating to DVT in particular. The general criteria include: accounting coherently and consistently for established knowledge in the field; making specific, experimentally testable predictions; suggesting future research strategies; and providing a new understanding of prophylactic and therapeutic measures. The specific criteria include: (i) Explaining why all venous thrombi are initiated in parietal and ostial valve pockets; (ii) Relating the initiation of pro-thrombogenic nidi to recognised pathological changes in the valve cusp leaflets; (iii) Accounting for the known morphological features of a venous thrombus as discussed by Aschoff (1924); (iv) Elucidating the importance of pulsatile venous blood movement and the prophylactic success of intermittent compression; (v) Giving cogent reasons for the marked tendency of venous thrombi to embolise when the muscle pump becomes active again after immobility, or even when the local blood velocity remains low; (vi) Deducing the known ‘risk factors’ (Chapter 1 and O’Shaughnessy et al. 2005, 2007) from the principles of the hypothesis. The consensus model satisfies specific criteria (v) only trivially, (vi) only partially and (i)–(iv) not at all. These shortcomings may have arisen because the consensus model began as an attempt to explain and promote the success of anticoagulant therapy and prophylaxis (Chapters 1, 2 and 5) rather than to account for the aetiology of DVT per se, though it has dominated laboratory and clinical research in the field for half a century. More strikingly, perhaps, it fails on at least two of the general criteria:

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accounting coherently and consistently for established knowledge, and making experimentally testable predictions. Instead, it has marginalised a good deal of relevant information (the relevance of the VVP and pulsatile flow, the extraordinary structure of venous thrombi, etc.) and effectively denied some important findings (e.g. that static blood in vivo remains fluid). On the other hand, it has driven a great deal of productive research on thrombophilias and their interactions with other thrombogenic factors, and the continuing development of an array of clinically invaluable anticoagulants. If we are to replace the consensus model with a more adequate hypothesis, we must take care not to undermine these important achievements. A broad distinction between vital-materialist and mechanistic approaches was suggested in earlier chapters: the former concern the effects of life, the latter the cause or mechanisms of life. The VCHH is essentially ‘vital-materialist’. It considers the effects of living blood cells on dead/necrotic endothelial tissue to be the cause of DVT but it does not address ‘mechanisms’. The consensus model is essentially ‘mechanistic’. It considers the mechanism of the blood cell and coagulatory response to be the mechanism of DVT, but is metaphysically incapable of identifying ‘cause’. Hence the need for rapprochement or unification.

11.2

The General Aetiological Sequence: The ‘Trinity’

There is no doubt that the three elements of what is dubbed ‘Virchow’s triad’ (vein wall, blood flow and blood components)1 must enter into an aetiological account of DVT (cf. Brotman et al. 2004); though as we have seen (Chapter 6), Virchow discussed these three general ‘phenomena’ only in relation to the major pathological effect of DVT (pulmonary embolism), not its cause. After Virchow’s death, various attempts were made to list the relevant aetiological factors and some of these were considered to follow specific sequences: ‘stasis’, blood flow rate, hypercoagulability, fibrinogenesis, infection, inflammation, ‘phlebitis’, etc. (see Chapter 7). In the VCHH, the ‘elements of Virchow’s triad’ are combined to create a unique causal sequence. Of course, there are potentially six (= 3!) ways of ordering a set comprising three elements; our argument is that one of these six, dubbed ‘trinity’, provides an appropriate aetiological framework for venous thrombosis. This is, of course, conceptual repackaging, rearranging the general aetiological factors into a new and immutable causal order. The three putative consequences of obstruction by embolia described by Virchow (1856) were: (1) phenomena associated with the irritation of veins and their vicinity (vein wall change), (2) phenomena of blood coagulation (blood coagulation change), (3) phenomena of interrupted blood flow (blood movement change).

1 The three histological distinctions between thrombi and clots that Virchow highlighted (Chapter 6) would have been no less deserving of an eponymous label, but they seem to have been largely forgotten or overlooked.

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If these three general ‘phenomena’ are presumed to apply to the aetiology of DVT, then in principle they can be arranged in the sequences 1-2-3, 1-3-2, 2-1-3, 2-3-1, 3-1-2 and 3-2-1. ‘Trinity’ is the sequence 3-1-2. It is unique in that it is not merely a list but entrains its own causal chain: (3) Altered blood flow causes (1) ‘irritation (lesion) of’ (a particular part of) the vein endothelium, which causes (2) an in vivo blood-endothelial lesion in a VVP, which in some respects resembles (but is histologically distinct from: Virchow 1856) an in vitro clot. In more detail, this ‘new’ paradigm sequences: (a) Suspension of normal, intermittent, alternating blood flow (alias ‘interrupted flow’), replacing it by streamline non-pulsatile venous blood flow; which causes (b) Hypoxaemic (suffocated) parietalis endothelial lesions of VVP cusps; which cause (c) Active sequestration and margination of phagocytic leukocytes and platelets on the dead parietalis, which may lead to progressive thrombotic obstruction of the VVP sinus and, potentially, the vein lumen. Using this ‘trinity’ framework of the aetiology of DVT, we can now draw together the arguments of Chapters 7–10.

11.2.1

‘Interrupted Flow’ and Underperfusion of VVP

Virchow (1856) used the phrase ‘interrupted circulation’ to refer exclusively to pulmonary arterial obstruction and consequent arrest of the circulation, but this is an entirely different matter from normal ‘interrupted circulation’; i.e. alternating pulsatile flow in arteries or veins with unobstructed (thrombus-free) lumens (Chapter 8). In normal limb vessels, motor pressures fall when cardiac and/or venous valves close during extended diastolic periods, but the reduced rate and volume of venous return flow ‘pumping’ can be expected to normalise at the next ‘systole’. Pathophysiological malfunction of the venous return may arise in either erect/sitting or lying/horizontal circumstances, as discussed in detail in Chapter 8 and below: non-pulsatile streamline/ continuous flow supervenes, and this is pathogenic if sufficiently prolonged. Haemodynamic aspects of normal venous return are clearly of fundamental significance in the VCHH. The crucial role of the leg muscles was reviewed in detail by Franklin (1937); this work, discussed in Chapter 8, has never been superseded. The pathophysiological questions concern what happens when the ‘peripheral venous heart’ malfunctions in abnormal circumstances. We reasoned in Chapters 8 and 9 that the primary consequence is sustained underperfusion of the VVP sinuses, allowing the parietalis endothelium of the valve cusps to be suffocated by prolonged local hypoxaemia. The conclusion from these considerations is ideologically remarkable, an apparent paradox: ‘blood stasis’ and ‘blood circulation’ may coexist in a single vein

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because of the peculiar anatomical and physiological features of VVP and their cusps. Streamline blood flow circulating through a vein lumen for hours will nourish the mural and the cusp luminalis endothelia, but within less than a millimetre (the thickness of a typical valve cusp), the parietalis lining the pocket may die of hypoxia from zonal hypoxaemia. McLachlin et al. (1960) provided experimental evidence that ‘stagnant blood’ (they used the term ‘stasis’) co-exists alongside moving blood in these particular circumstances. An injected radiographic dye was visualised in what the authors presumed to be VVP of leg veins whenever the ‘peripheral venous heart’ pulsed less frequently and/or less forcefully than normal, i.e. when the ‘diastole’ of the calf-muscle pump was indefinitely prolonged by postponement of the next muscular ‘systole’. This resulted in the sequestering of the tracer in VVP as shown by x-ray photographs, implying underperfusion or non-perfusion of the valve cusps, which fluttered half-open and half-closed (Chapter 9). Thus, venous thrombi form in static conditions immediately adjacent to, but not within, moving blood.

11.2.2

The Specific Involvement of the Valve Cusp Parietalis Endothelium

The parietalis and luminalis epithelia of valve cusps are histologically similar (Chapter 9), but whereas the latter is bathed by oxygenated blood in the vein lumen under both pulsatile and streamline flow conditions, the former depends on pulsatile flow, which empties and refills the valve sinuses. Thus, whereas only the wholebody hypoxaemia of gross circulatory failure threatens the survival of the luminalis, the parietalis endothelium suffers oxygen lack each time the peripheral venous heart diastole is prolonged. Valve cusps cannot move actively, having little or no smooth muscle except at the agger, and are instead flapped passively by alternating venous blood pressures (the flowing blood). The mural epithelium of the valve sinus, like the vein wall, receives an oxygen supply from the arterial vasa venarum, so it cannot be oxygenstarved by VVP hypoxaemia during non-pulsatile flow. The upshot of this consideration, detailed in Chapter 9, is that the parietalis is uniquely susceptible to streamline flow, which kills it specifically each time the normal pulsatility of venous blood flow is suspended for a sufficient period. These considerations apply equally to both parietal and ostial valves, i.e. those within the vein and those at the junctions between main veins and tributaries (Franklin 1927, 1937). Thus, venous thrombi arise within venous valves, including those at tributary vein junctions, but not on or from the remainder of the vein wall. This conclusion is supported by illustrations in Virchow (1858), Paterson and McLachlin (1954) and Hamer and Malone (1984), reproduced in Figs. 10.1, 9.3 and 11.5, respectively, and is inferred from an illustration in Stone and Stewart (1988), reproduced in Fig. 9.6.

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11 The Valve Cusp Hypoxia Hypothesis

Blood Cell Congregation and Blood Coagulation

The assertions of Aschoff (1924) that ‘important changes in the morphological blood constituents’ preceded fibrinogenesis, and that ‘along with the explanation of this marking [Kopfteil, Halsteil, the ‘coralline’ structure of thrombi and the lines of Zahn] stands or falls the whole problem of … autochthonous thrombosis’ were discussed at length in Chapters 7 and 10. We also recalled that Hunter (1793) recognised that the core of what we now call venous thrombi was ‘pus’, and that Virchow (1856) identified ‘pus cells’, later called leukocytes, in normal blood (Chapters 6 and 7). We pointed out that far from being passively entrapped in the fibrin web of a forming thrombus, living leukocytes and platelets congregate on the dead or dying parietalis endothelium of hypoxaemic valve cusps and participate in forming fibrin webs during thrombogenesis (Chapter 7). When oxygenated venous blood (movement had been restored, even transiently, after VVP hypoxaemia) was brought into contact with the necrotic endothelium, white cells in that fresh blood congregated on that endothelium (Malone and Morris 1976, 1978). When similar hypoxaemic ‘dead blood’ in VVP was evacuated and replaced with fresh blood, the well-oxygenated, viable cells attacked the suffocated, dead or dying endothelium. The molecular mechanisms involved will be reviewed in Chapter 12. It is not necessary that all the parietalis be killed; in principle, even a single dead cell may suffice to elicit the leukocyte/platelet response. These living blood cells appear to be the active agents in the process;2 dead (parietalis or other) cells cannot be presumed to ‘do’ anything (Malone and Morris 1976, 1978). The effect was to create a single layer of marginated blood cells on the necrotic or pre-necrotic endothelium (see Fig 11.1a). The hypoxaemic sequence that led to death of the parietalis may then either cease, or be repeated once or many times. If it ceases, the attached leukocytes could perform their phagocytic task and initiate the replacement/repair of the dead parietalis. It is therefore by no means inevitable that a thrombogenic process is fated to proceed to disaster from each preliminary stage. If the interlude of non-pulsatile flow recurs once, the marginated layer of blood cells will also die of suffocating hypoxaemia, and at the next restoration of pulsatility this layer in turn will be covered by a further monolayer of fresh blood cells. Only if the hypoxaemic circumstances are serially repeated does the probability increase that successive layers of blood cells will be laid down and that a thrombus will result. In other words, a venous thrombus does not form instantaneously. DVT is not an event but a process that may take hours or days, and possibly represents several pro-thombogenic episodes mutually separated by similar periods – such as flying long-haul in one week and returning weeks or months later. An engraving by Virchow (1856), reproduced here as Fig. 11.1c, suggests that a blood coagulum in a vessel may comprise a thin white layer on the surface with a 90% red ‘clot’ beneath, with each layer a few microns thick rather than as shown in the bleeding cup. Moreover, as discussed in Chapter 7, platelets greatly outnumber leukocytes at the 2 We reviewed the debate about whether platelets should be regarded as ‘living cells’ or passive entities in Chapter 7. Our position is unequivocal: platelets are living, whatever their lifespan. In earlier chapters we have discussed at some length the semantic and philosophical implications of terminology that connotes either deadness and passivity or, in contrast, liveness and activity.

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Fig. 11.1 Thrombi form on valve cusps. (a) Dog vein valve cusp harbouring early thrombus (Hamer et al. 1981a, Fig. 2). Note the thin (single?) layer of blood cells (mainly WBC/platelets) marginated on the parietalis endothelium of a dog VVP cusp following streamline/non-pulsatile blood flow. Pulseless flow had been maintained in the vein lumen (blood flowing horizontally left to right) for 2 hours so that no exchange took place between the VVP concavity and the lumen throughout that period. The illustration shows viable blood cells that have formed the first layer of (potential) thrombus on the (concave) parietalis endothelium of a venous valve cusp, but there is no such deposition on the luminalis. A thrombus is considered to form when multiple layers (10s or 100s) marginate sequentially, as though the experimental sequence had persisted and repeated itself over several hours. (Luminalis endothelium faces left and caudad, parietalis faces right and cephalad, vein wall below horizontal, and blood flow is left to right). (b) Small thrombus, from Malone and Morris (1978). The platelets in the upper part of the micrograph are intact, replete with granules. Those in the lower part of the picture have secreted their granule contents and fibrin deposition is evident. Three leukocytes are visible in this image. Copyright Pathological Society of Great Britain and Ireland. Reproduced with permission granted by John Wiley and Sons on behalf of the PathSoc. (c) Engraving reproduced from Virchow (1856, Fig. 67; 1858, Fig. 60). Diagram of a bleeding-glass with coagulated ‘hyperinotic’ blood. a: the level of the liquor sanguinis; c: the cupshaped buffy-coat; l: the layer of lymph (cruor lymphaticus, crusta granulosa), with granular and mulberry-like accumulations of colourless corpuscles; r: the red clot

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Fig. 11.1 (continued)

site of valve cusp injury just as they do in the circulation (notwithstanding their apparently lower concentrations in the leg veins: Woldhuis et al. 1992). When platelets congregate on the dead parietalis, the coagulation process leading to fibrinogenesis may be initiated (Chapter 2). The resulting fibrin web may then begin to entrap red cells. From this, we may imagine that every ‘coagulum’ embodies a white/red alternation of layers (each only a few microns thick) so that every thrombus is composed of multiple, sequential, strata of white cells with thin backings of red cells: a venous thrombus is built up like a coal seam rather than as a ‘single clot in a basin’. Herein lies the answer to Aschoff’s conundrum and his illustration of thrombus histology: venous thrombi have layered, ‘coralline’, structures because of the way they are formed. Thus, while coagulation is undoubtedly an essential part of thrombosis, it is a consequence of earlier events and a concomitant of later ones, not the primary cause of DVT formation. This conclusion distinguishes the VCHH from the consensus model. A fortiori, although it is clear that thrombophilias increase the likelihood of clinically significant thromboembolism, and that anticoagulant and fibrinolytic treatments are clinically valuable, no thrombophilia or other disorder of coagulation can properly be regarded as a ‘cause’ of DVT (Chapter 3). In many (probably most) cases of DVT the blood is normally coagulable.3 The VCHH would predict that anticoagulants prevent the propagation rather than the inception of a thrombus, as observed empirically (Krupski et al. 1990). Moreover, aspirin – a platelet inhibitor – is said to be less effective than anticoagulants against DVT (Thomas 2001). Even in the presence of aspirin, platelets congregate 3 Since normal coagulation is rapid, it is unlikely that even a marked increase in ‘clotting rate’ would materially affect the formation of thrombi. It is obvious that more or less unlimited time is available for blood to thrombose when circumstances dictate.

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on the hypoxic or necrotic endothelium and thrombus nidi will form (mechanisms are discussed in Chapter 12). If this process progresses to the stage of protrusion from the VVP lumen, aspirin may prevent signalling to other platelets but will not prevent the association of circulating leukocyte–platelet assemblies with the growing mass of dead material (Chapter 12).

11.2.4

Pathological Consequences

The lethal part of the process must begin after the forming thrombus protrudes from the VVP. Prior to this point it is not life-threatening, though layers of blood cells may stiffen the cusp and lead to valve malfunction, impairing venous return and perhaps making varicosity more probable (‘post-thrombotic syndrome’); see Section 11.4.5. However, when an incipient thrombus protrudes from its VVP, it places a ‘dead’ lump of cellular coagulum in the way of the luminal blood flow (Fig. 11.2). If there is no subsequent interruption of normal pulsatile blood flow and normal oxygenation is maintained, more and more phagocytes and platelets may be recruited to enfold this dead mass in an ‘envelope’ of living cells. The mass will grow no

Fig. 11.2 Magnetic resonance imaging of a venous thrombus from O’Shaughnessy et al. (2005). This novel technique detects methaemoglobin in thrombi, and thus allows a thrombus/clot to be visualised without need for intravenous contrast (the endovascular methaemoglobin provides sufficient contrast). Femoral and popliteal thrombi are clearly visualised in these images. The conversion of oxyhaemoglobin to methaemoglobin signifies that the blood in thrombi so identified is dead/necrotic, thus confirming the central thesis of this chapter: that venous thrombosis manifests very local ‘tissue death’ extending to and involving the vascular envelope by massing of deceased leukocytes and platelets. Thrombi are to be thought of as the detritus of dead blood cell masses within the living vascular envelope. We are indebted to Dr. Peter Walton of Dendrite Clinical Systems Ltd., Bell St., Henley on Thames, for permission to reproduce this illustration

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larger, and indeed small protrusions from a VVP may be successfully removed by circulating phagocytes. However, if this does not happen, then the protuberance of even a tiny piece of thrombus from the mouth of a VVP becomes a potentially fatal development: it may continue to burgeon should successive covering layers of living cells die and become covered in turn by further fresh layers (Fig. 9.1b).

11.3 11.3.1

Experimental Support for the VCHH Polarographic Demonstration of VVP Hypoxaemia during Non-Pulsatile Flow

Hamer et al. (1981a, b) confirmed the central premise of the VCHH – that un-flapped/ un-agitated valves in paralysed, severely paretic, anaesthetised animals and patients must come to enfold hypoxaemic blood, and that if the initiating circumstances persist, such hypoxemia must inevitably progress to severe (cytotoxic) levels. In anaesthetised human subjects (in preparation for surgical treatment of their varicose veins), streamline flow in the leg veins was presumed when valve cusps were not seen to ‘flap’ normally when pulsatile flow was temporarily suspended. VVP hypoxaemia was demonstrated directly (Fig. 11.3) and was readily related to the perturbation of normal flow patterns within the pocket (Fig. 11.4). Intravenous (intra-pocket) polarography confirmed that the PO2 fell immediately, invariably and quite precipitously in VVP blood during such periods of streamline flow. It returned instantly to ‘normal’ each time a single pocket-emptying venous pulse occurred, or massaging external pressure was applied, thus proving that the PO2 within VVP depends on, and reacts to, sequential filling and emptying. A point of particular interest was that when the oxygen probe (with an inset terminal oxygen-measuring electrode) was advanced to impinge on the bottom of the pocket,4 no measurable oxygen was recorded. The implication was that in such an occluded position, no oxygen is being moved through the valve cusp intima (parietalis, lumenalis and basement membranes). Therefore, oxygen diffusion through the cusp is not measurable within the pockets (see Fig. 11.3). After 2 h of sustained non-pulsatile flow in the leg veins of anaesthetised dogs, blood cells had congregated in the VVP and on the parietalis, as predicted by the VCHH, shown in Fig. 11.1(a) and illustrated diagrammatically in Fig. 9.7.

11.3.2

Experimental Venous Thrombi Induced by a Non-Invasive Technique

Using a non-invasive procedure, Hamer and Malone (1984) generated experimental thrombi in the femoral veins of dogs, some of which occluded the deep femoral 4 See Fig. 9.2. The ‘bottom’ of the pocket is the ‘minimum of the parabola’ described by the cusp, not the point adjacent to the agger. Thus, the only endothelial cells close to or perhaps touching the electrode were those of the parietalis.

Fig. 11.3 Gradient of hypoxaemia in a valve pocket during non-pulsatile flow. (a) Fig. 1 from Hamer et al. (1981a) Copyright British Journal of Surgery Society Ltd. Reproduced with permission granted by John Wiley and Sons on behalf of BJSS Ltd. Diagrammatic longitudinal section of one side of a vein at the site of origin of a venous valve. The endothelium of the valve is designated ‘parietalis’ and ‘lumenalis’. Sites of PO2 measurements are shown in relation to the positions of the electrodes A–E. In the interest of clarity, position D has been represented nearer to the centre of the vein than was the case. The actual point of measurement was the centre of the tip of the probe. (b) Diagrammatic reconstruction of flow pattern around valve cusps related to PO2 values (Karino and Motomiya 1984). Copyright 1984; reproduced with permission from Elsevier Ltd

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Fig. 11.4 Venous valve flow pattern. (a) Streamline flow over a bicuspid valve. Longitudinal section of vein through a parietal valve. (b) The flow pattern. Both illustrations are from Karino and Motomiya (1984). Copyright 1984; reproduced with permission from Elsevier Ltd

trunk.5 These resembled autochthonous venous thrombi, being histologically distinct from the clot-like ‘experimental thrombi’ discussed in Chapter 3 (Fig. 11.5). The animals’ leg muscles were ‘paralysed’ by general anaesthesia (barbiturates), causing about 3 h of muscle relaxation. As the barbiturate was metabolised or excreted, the anaesthesia lightened and was succeeded by muscle clonus, jactitation and shivering. This was allowed to continue for about 10 minutes before the animal was ‘relaxed’ again with the anaesthetic, and followed by a further three hours of ‘muscular diastole’. The experiments were concluded after 6–8 h of anaesthesia. 5 An injected radio-opaque dye showed no flow through the vessel; the obstruction was confirmed by post-mortem dissection.

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Venous blood flow was uninterruptedly non-pulsatile during the periods of anaesthesia, but pulsatility was restored during the shivering interludes. No thrombi formed during single periods of anaesthesia, but typical venous thrombi had formed after alternate long non-pulsatile and short pulsatile periods of blood flow. This was consistent with the critical prediction of the VCHH (Hume 1985). Thomas et al. (1985) showed that patchy areas of exposed subendothelium produced by mechanical crushing of jugular veins became covered with activated platelets when the blood flow was restored, but no fibrin was formed after 5 min. When blood

Fig. 11.5 a. Experimental venous thrombus in dog femoral vein originating in an ostial valve at a tributary junction. The thrombus is propagating both proximally and distally (this illustration was rotated 180° in the original paper). Fig. 2 from Hamer and Malone (1984). Copyright the Royal College of Surgeons of England. Reproduced with permission

Fig. 11.5 (continued) b. Longitudinal section of an experimental dog thrombus in a parietal valve pocket of a femoral vein. Reconstruction from Fig. 4 in Hamer and Malone (1984). By courtesy of Dr Simon Sevitt. Re-evaluation of this image, twenty years after it was published, calls attention to the white cell massing opposite X. This appears to be a response to pre-necrosis of the parietalis endothelium and is the earliest ‘pre-thrombotic’ layer. No other inference as to the order of formation or artefactual ‘fracture’ can be suggested by hindsight

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183

flow was restored for an hour following the crush injury, white cells adhered to the injured sites, but electron microscopy revealed no visible fibrin; thrombi did not form even after a further 20 min period of ‘venous stasis’. Although the authors interpreted these results as showing that endothelial injury plays no part in the causation of DVT, they had in fact corroborated the VCHH by showing that leukocyte and platelet attachment to sites of vein wall injury precede – but do not directly result in – local coagulation. Had they repeated the experiment with a more extended period of ‘stasis’ followed by a further restoration of circulation, it may be predicted that coagula would have formed at the injured sites. However, such coagula would not have been identical to autochthonous venous thrombi because (1) the areas of exposed subendothelium were not (primarily) on the valve cusp parietalis (compare the studies of Stewart and colleagues discussed in Chapter 7), and (2) brief compression had not been proved to be as fatal to venous endothelium as prolonged suffocating hypoxaemia.

11.4

Clinical Implications

Were failure to perfuse the VVP of paretic or paralysed dependent limbs to be accepted as the root cause of DVT, the aetiological focus would shift from the consensus ‘altered blood coagulation’ of the past half century back to the ‘altered venous blood movement’ of previous centuries. The idea that weakening or failure of the ‘peripheral venous heart’ is related to DVT is not novel (McLachlin and McLachlin 1958), but the dominance of the consensus model marginalised it. It must be related specifically to the substitution of streamline for pulsatile venous blood flow. The VCHH concerns the regularity with which VVP are emptied and refilled. Frequently-emptied pockets will not contain incipient DVT, but pockets that remain unemptied/refilled for a sufficient duration may do so. As argued theoretically in Chapter 9 – and shown experimentally by Hamer et al. (1981) (Fig. 11.1a) – ‘sufficient duration’ means in the order of 1.5–3 h; but this figure is certain to be subject to individual variation (see below). The pathological outcome is most likely to affect anatomically perfect valves and may disable them (cf. Fig. 9.3). Hypoxaemia and thrombus formation occur in ‘normal’, ballooned and temporarily unperfused VVPs and are not to be expected in ‘pathologically disintegrated’ valves.

11.4.1

The Risks of Sleeping for Long Periods in the Sitting Position

People who have spent much longer than average sitting and sleeping (semi-erect), e.g. in deck-chairs, sofas, computer chairs and commercial airline seats, are more prone to suffer ‘idiopathic thromboembolism’ (Dalen 2003). However, because the aetiological sequence according to the VCHH is complex, only a minute proportion of ‘long-sitters’ may develop actual lesions, and then usually after a delay of days or weeks. Moreover, such instances cannot be predicted because different subjects respond in different ways to the same circumstances.

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The key factor in reducing venous return during such ‘long-sitting’ episodes is not lack of muscle contractions per se, but the failure of foot pressure on the ground to compress and evacuate blood from the soles. Once blood from the vessels in the feet is expressed from foot to ankle, local muscle contractions normally elevate it progressively against gravity to calf, thigh and heart. Without the priming force of weight borne on the soles of the feet, leg movements in situ are unlikely to have much effect; venous valves in the calf cannot be expected to ‘suck’ blood up from the foot. In the sitting position with only a bar on which to rest the feet, or feet not touching the floor, and certainly no more than the weight of the lower leg acting on the soles, the absence of compressive weight to initiate the ‘up-flow’ will probably be compounded by a concomitantly lowered muscle-pump action. The weight of the column of blood in the lowermost extremity is entirely opposed to the venous return. Some sitters, such as long-distance travellers, are fidgety while others display lassitude, and such differences may affect the efficiency of venous return. All circumstances promoting lassitude are likely to combine to increase the chances of clinically significant DVT and pulmonary embolic fatality. 11.4.1.1

Traveller’s Thrombosis Reconsidered

Since many older people are afraid of flight in general and high altitude transoceanic flight in particular, they may take hypnotics or lounge in an inebriated state for several hours – terrified, exhausted, drugged, or all three combined. That could be responsible for (or at least contribute to) DVT or pulmonary embolism on disembarkation. Such travellers could be inexperienced in the exigencies and difficulties of long distance air transport – sitting around in stations, cars, trains, buses and hotels on the way to major international hubs, transporting heavy luggage – and are particularly likely to suffer thromboembolic consequences.6 Thus, in addition to the distance and duration of such flights, the state of the passenger should be taken into account. All elderly, exhausted, terrified travellers, who are perhaps fearfully inebriated through excessive alcohol intake and/or may have used normal or unaccustomed doses of analgesics, tranquilisers, hypnotic medicaments, anti-nausea medication or neuroleptic drugs, should be considered at particular risk of prolonged relative insufficiency of their insensible ‘peripheral venous hearts’. Many commentators have noted that the leg/calf volume of long distance air passengers is frequently increased. Franklin (1937) remarked on such sequestration of body water in dependent legs, long before international flights figured in scientific concerns or papers. The semi-erect posture, inactive leg muscles and gravity are indisputably the main causes of limb swelling.7 6

The RAF (personal communication from Wing Commander John Aitken and William J. Coker, Occupational Health, RAF Innsworth), who transport thousands of young fit men thousands of miles, have no record of any traveller’s thrombosis case; though significant numbers of troops are not currently transported further than Afghanistan and Iraq. 7 Although the venous physiological mechanism described may well be indirectly associated with many forms of fluid retention in the legs, perhaps leading to oedema and swelling, that oedema – though very common and real – is not specifically or directly related to the cause of DVT. The two

11.4 Clinical Implications

11.4.1.2

185

Ethnic Differences in Susceptibility to DVT

It may be pertinent that Asian peoples, who sit or squat on the ground or on cushions at ground level, are noted to suffer DVT less frequently than Western people who routinely sit on chairs with seats at average adult knee height (Chapter 1). The ground-to-heart distance in a person sitting on the ground is hardly more than 2 ft in a tall person, as against 4–5 ft in the same person standing or sitting on a chair; so the height of the venous blood column, and therefore gravitational resistance to venous return, is at least halved. Squatting precludes ‘lounging’ and is wholly incompatible with sleeping. Neither can one be inebriated while sitting on a cushion or the ground because it is constantly necessary to maintain the upright posture by active muscle control; in any case, alcohol is prohibited in many such communities.

11.4.2

Simpson’s Cases

Simpson (1940) presented a famous case-series of elderly persons who, having slept overnight (~10 h) in deck-chairs on London Underground station platforms during the Blitz, died of pulmonary embolism in surprising numbers afterwards. He conjectured that the cross-bar beneath their knees obstructed the venous return blood flow from the legs. This was considered a reasonable supposition, and his solution – to replace deck chairs with bunk beds in the Underground – was brilliantly successful; his explanation was therefore generally accepted. However, we suggest that there was another ‘obstruction’ to venous return in persons sitting-sleeping exhausted in deck chairs: i.e. the normal (gravitational) weight of the column of blood between the feet and the heart aggravated the consequence of semi-erect (non-horizontal) sleep and the concurrent absence of foot-sole pressure. This suggestion could have led to a principle of wider applicability and provided a more cogent explanation of the pathophysiology of thrombosis: streamline venous return flow in exhausted (paretic) persons sitting ‘slightly upright’ in deck chairs, with the heart of the sleeper about 50 cm above the feet at ground level, caused VVP underperfusion by counteracting the venous blood return. In short, prolonged (more than about three hours) sleeping with lowered feet is very dangerous, since gravis gains the upper hand. The fatalities ceased immediately when deck-chairs were replaced by bunks. Though it is no longer possible to disprove the speculation that the cross-bars of deck chairs were indeed the causal factor, there was no experimental evidence in favour of Simpson’s ‘obstructive deck-chair crossbar’ hypothesis – except that his ‘prophylaxis’ worked. We suggest that the weight/resistance of their blood column must apply in principle to all people (Fig. 11.6), though we do not detract from Simpson’s perceptive observation or his successful prophylactic solution to the immediate problem.

conditions may or may not coincide. Perhaps the main difference between traveller’s thrombosis and traveller’s oedema caused by protracted sitting is that oedema manifests possibly widespread sequestration of body water in dependent limbs, whereas DVT is a single intra-VVP lesion. Oedema may of course also be a consequence of thrombosis, though not its cause.

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Fig. 11.6 Postures that may predispose to DVT. (a) Body erect or sitting upright, (b) body in deck chair, (c) body horizontal. The predictable circumstances might be: (a) A subject sitting for a long period, possibly sleeping, possibly drugged with a neuroleptic, hypnotic or alcohol excess (ground-to heart distance ~3 ft). (b) A subject sitting in a deck chair, as in Simpson’s cases (ground-to-heart distance ~1½–2 ft). (c) A body horizontal on an operating table, or in a bed having had a stroke, or suffering decubitus disability (ground-to-heart distance approximately zero). The preventable cause of DVT is most likely to be a complication of muscle-relaxant and/or prolonged anaesthesia. Other factors in such circumstances could be muscular paresis due to exhaustion, fright, age, or the above-mentioned causes of pathologically induced local paralysis

11.4 Clinical Implications

11.4.3

187

Anaesthesia

‘Muscle relaxation’ is a mainstay of modern anaesthetic technique for a significant class of operations. It has not specifically been identified as a cause of thrombosis, but according to the VCHH, any sustained inactivation of the limb muscles is potentially thrombogenic. The muscle relaxants8 used during certain surgical procedures block voluntary muscle contractions, and while the concomitant respiratory paralysis is compensated by the anaesthetist, the suppression of pulsatile venous flow in the limbs is not. Under such conditions, all venous valve cusps may remain in neutral positions, and the VVP will consequently be underperfused or unperfused for as long as the paralysis induced for the surgical procedure continues. Post-operative patients (with the muscle paralysis neutralised) do not usually recover immediately they leave the operation room, and may continue in a partially drugged sleep for another hour or two in bed. Limb muscles still do not contract completely normally and are inhibited by pain when full consciousness returns. All muscular pareses are likely to follow similar lines, so all must be considered to have the potential to kill the parietalis endothelium in isolated, cut-off valve sinuses after a sufficient duration of streamline, non-pulsatile underperfusion of VVP. It is clearly not as immediately necessary to maintain pulsatile flow in the limb veins as it is to maintain respiration. However, when the period of anaesthesia exceeds 2–3 h, VVP hypoxaemia may become complicated by the margination of a first layer of blood cells – the possible prelude to a delayed but dangerous sequel: many more such layers could form after as well as during the remainder of the operative procedure. Since VVP that empty by gravity cannot become sites in which thrombi originate or develop, a simple answer to possible thrombotic complication of muscle paralysis would be to tilt the table head down/feet up by 5° Trendelenburg/ anti-Trendelenburg at approximately hourly intervals throughout long operative procedures (i.e. more than 90 min). All literature references to Trendelenberg tilting relate to much greater tilts (10° or more); but all are for purposes unrelated to DVT and exceed the requirements for prophylaxis against this condition. For patients who come to surgery ‘off the street’, i.e. with no history of preoperative disablement (equivalent in duration to a flight from Europe to Australia), anti-coagulant prophylaxis should not be needed. For patients with a more chronic history, either a previous thrombotic episode or a period of decubitus might have already have initiated thrombosis; anticoagulant prophylaxis as well as actions to empty the VVP regularly would be indicated. Another issue pertinent to peri-operative anaesthesia, implicit in Aschoff’s realisation that hypovolaemia appears to be thrombogenic (see Chapter 10), is the case

8

Many safer alternatives to curare such as Pancuronium, Gallamine and Suxamethonium are now available and are widely used; the choice depends inter alia on the duration of muscle relaxation required. These drugs are not ‘coagulants’; the argument in the text is that they increase the likelihood of DVT only by paralysing the muscle pump, not by promoting coagulation or impairing fibrinolysis.

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for maintaining blood volume during surgery.9 Adequate VVP perfusion cannot be ensured unless this is done, so the principle of infusing a priming volume of a replacement fluid such as dextran (see Chapters 3 and 8) would seem a proper physiological precaution pending a subsequent clinical decision as to the indications or otherwise for whole blood replacement.

11.4.4

Crucifixion

We now offer a speculation that is, fortunately, unsupported by empirical evidence: the cause of death by crucifixion was ruthless extinction of the venous return. Special cruelties were not necessary; to ensure slow death, it sufficed to suspend victims from trees in the erect position, with the soles of their feet clear of the ground, either unsupported or with nails driven through their heels into the sides of the tree. The ancient practice inevitably prevented the blood that had been pumped down into the feet from being pumped back up from the lower limbs to the heart, since the feet no longer pressed against a resistant surface. Though history has emphasised the hammering of nails through hands in crucifixion, the more lethal, anti-physiological, detail may have been the hammering of nails through heel bones and ankles into the ‘uprights’, immobilising the legs. The severe pain entailed presumably inhibited the calf contractions and resulted in a progressively increasing fraction of the whole blood volume being pooled in the leg tissues, slowly starving the right heart of returning blood. Contemporaries variously reported that death was either very slow (many hours) or very fast (within 10 min). It cannot, of course, be proved that longer survival induced fatal DVT in the VVP of the unsupported (often immobilised) legs, but it seems plausible that pulmonary embolism contributed to, or actually caused, death in these cases. A modern analogue of those ancient barbarities may be ‘traveller’s thrombosis’: elderly travellers sitting slumped, perhaps sleeping, perhaps inebriated, ‘strung up’ nearly vertically in their seats for 12 or more hours and suffering DVT as a consequence.

11.4.5

Varicose Veins

Virchow (1858) accompanied the engraving that we reproduced in Fig. 10.1 by a striking comment: in the event of thrombi such as the one illustrated, the tributary

9 A Medical Tribune reporter quoted a comment by L.P. Le Quesne at the Lister Centenary 1967 under the heading ‘Safe surgery – Maintain circulating blood volume’: ‘… there are practically no circumstances when a patient’s safety is enhanced by undertaking surgery with a depleted blood volume. Critical lesion is a circulating blood volume deficit – usually of iatrogenic origin’. See the papers by Le Quesne (1967) and Delikan (1972) cited in footnote 14 of Chapter 8; also Malone (1988).

11.5 ‘Risk Factors’ for DVT Reconsideration in the Light of the VCHH

189

veins are varicose. Retrospective examination of this illustration and the accompanying text makes it clear that the occluding thrombus had formed in the ostial valve of a tributary vein and had subsequently protruded into the femoral vein lumen. Because ostial valves are unicuspid, a thrombus that forms in such a valve would be expected to occlude the tributary completely, so the tributary and its sub-tributaries will become distended. Thus, we can predict that ostial valve thrombi will usually, or perhaps always, result in varicose veins. More speculatively, we may propose the converse: that all or most cases of varicose veins are attributable to (probably silent) ostial thrombi. This speculation is consistent with recent accounts of chronic venous disease (Bergan et al. 2006).

11.5

‘Risk Factors’ for DVT Reconsidered in the Light of the VCHH

The major known ‘risk factors’ for DVT were listed in Chapter 1 and were evaluated e.g. by O’Shaughnessy et al. (2005, 2007). We noted (Chapter 3) that an aetiological hypothesis should explain why these factors increase the likelihood of DVT. However, a caveat must be entered: it would be both scientifically and philosophically erroneous to confuse factors that predispose towards a condition with causes of that condition. Combinations of the known ‘DVT risk factors’ certainly increase the likelihood of thromboembolism, but ad hoc circumstantial incidences are not a priori causes. DVT is not ‘multifactorial’. ‘Risk factors’, jointly or severally, figure because they exacerbate one or more stages in this aetiological process. Traumatic hypovolaemia and possibly high haematocrit may result in impaired perfusion and oxygenation of VVP (Chapter 9), and some autoimmune conditions lead to thrombophilias (Chapter 3). Oral contraceptives decrease antithrombin III levels by up to 60% (cf. Chapter 3), and may thus potentiate the risk of thrombus propagation.10 Some ethnic differences in susceptibility were considered earlier in this chapter (Section 11.4.1.2). All circumstances vitiating the ability of muscles to ‘oppose’ and overcome the adverse force of gravity are thrombogenic, as already argued in detail. Clearly, muscle weakness and absence of expulsive pressure on the soles of the feet will lead to sustained non-pulsatile venous blood flow, and if such periods are interrupted by short pulses of weak or recovering muscle activity, or external pressure/handling by nursing staff, then DVT may be encouraged. Cardiac failure is classically – notoriously – complicated by notable rates of DVT and embolism (Belt 1934;

10 It seems implausible that contraceptives would prolong venous valve evacuation. Perhaps hormone courses could change the living habits of the patients taking them, though this also seems unlikely. On the face of it, oral contraceptives seem far more likely to promote thrombus propagation than the inception of DVT, but the seemingly increased incidence is unexplained.

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Hadfield 195011). ‘Long term’ decubitus in the horizontal position, and any profound organic leg weakness, is prone to similar complications. In principle, this is comparable to the pathogenesis of ischaemia-reperfusion injury in arterial vessels. Cardiac failure of a certain degree necessarily reduces muscle blood flow and over a longer period may impair normal VVP perfusion. Ageing is generically associated with impaired bodily fitness. Increased inefficiency of valve cusp function and therefore an imperfect valve cycle (Chapter 9), as well as the expected onset of degenerative diseases, impaired mobility, pain and general deterioration of health after the age of 50, make hypoxaemic injury to the parietalis correspondingly more likely. A personal history of thromboembolic episodes increases the probability of progressive functional impairment in venous valves. Sustained periods of non-pulsatile flow may have injured valves in whatever segments of vein are involved and thrombi may have developed in some of these, destroying them in the process; others will have become functionally impaired because of the overlayering of blood cells on the parietalis. IV lines are considered to cause local injury to the endothelium and to lead to a ‘traumatic’ DVT. Speculatively: a catheter in a leg or arm vein could conceivably force/keep open a parietal valve through which it passes, and the commonly applied external splinting perhaps alters the balance of pulsatile and non-pulsatile blood flow. Smoking, which is often (O’Shaughnessy et al. 2005) though not universally considered a significant DVT risk factor, may aggravate VVP hypoxaemia, swaying the probabilities in otherwise less severe circulatory circumstances. Were the oxygen content of blood to be lowered further by cigarette CO, then the duration of non-pulsatile flow required to cause hypoxic injury to the endothelium could be shortened (cf. discussion of traveller’s thrombosis in Chapter 10). In the presence of other ‘risk factors’ such as a defined thrombophilia, the likelihood of DVT might increase significantly. In addition, there is evidence that smoking can reduce levels of factor V and activated protein C under certain conditions, notably late pregnancy (Kafkas et al. 2007). The relationship between other risk factors (cancers, infectious disorders and hormonal disturbances) and the VCHH will be discussed in Chapter 12, when the molecular biological aspects of DVT aetiology have been considered.

11.6

Prophylaxis

The VCHH is primarily concerned with the cause of DVT whereas biochemical studies address the mechanism (Chapter 12). Thus, the VCHH has extensive implications for prophylaxis against thrombi yet to happen, but none for treating those that have; biochemical studies are directed towards pharmaceutical therapy and prophylaxis. 11

Hadfield quotes the great cardiologist Paul White: ‘It is an astonishing and disconcerting fact that I and many others have been examining and treating patients for years without realising what we now know, namely, that P/E instead of being predominantly a surgical, or rather a post-operative, complication, is actually much more commonly a condition occurring in the practice of internal medicine, particularly in the heart itself’. This assessment had also been made by Belt (1934).

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The prevention of DVT requires only that VVP continue to be emptied regularly, whether the patient is in the erect, sitting or horizontal posture. Notwithstanding our theoretical argument in Chapter 9, no definite number can be ascribed a priori to the adverb ‘regularly’ save by intelligent surmise, because each case is individual. The principle is that hypoxaemia in a VVP must not persist to reach a danger level; this in turn entails an estimate of how long it takes for stationary blood in a VVP during streamline flow to cause hypoxic endothelial death of the parietalis endothelium. We suggest six approximations for the likely time span involved, but the figure cannot be considered as more than a guideline for any individual case: the PO2 in any particular vein or VVP is neither known nor controllable.

11.6.1

Our Theory-Based Estimate of 1.5–3 h

11.6.2

Lister’s Experience

Lister was asked to see a young patient who, following an injury, had had a tourniquet applied to an arm for a day. Custom at the time directed that such limbs should be amputated, but Lister, informed by his experimental studies, pondered whether to take this action. He opted to ‘save the limb’ and recounted how pleased he was a week or two later to find that the limb had recovered and had remained ‘useful’. Presumably, if blood vessels or their endothelial linings are deprived of oxygen for a single (long) period and normal circulation is then restored, the blood cells that re-circulate in those vessels (in the ‘first flush’) sequestrate and marginate to repair the injuries inflicted by hypoxaemia and prolonged anaerobic metabolism, and may thus ‘save them’; i.e. ‘Nature’ can save the day provided no further stoppage/tourniquet application follows. Lister subsequently (along with other pioneers) devised the practice of operating under a tourniquet ‘bloodless field’.

11.6.3

Normal Tourniquet Practice

In line with that pioneering experience, modern surgeons operating under tourniquet consider it permissible to do so for about 2.5 h. A precise time limit is not their main concern; they consider 2.5 h to be a guide-period. Their only insistence is that once a tourniquet has been removed to allow resumption of circulation, disaster would threaten were it reapplied for a further period. The safe period of tourniquet application (and, by extension, VVP cusp hypoxaemia) is not closely definable, but the number of applications is once only in any procedure. This concept accords with the VCHH in that the key consideration is not the duration of tourniquet application but whether it is a one-off or a repeated insult. Each added layer of blood cells sequestered on a parietalis endothelium adds to the

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preceding layer, and each additional hypoxic episode kills not only the parietalis endothelium but also the layers that follow and the layers that follow them. Though it cannot be stated with certainty, the critical period of VVP ‘stasis’ perhaps shortens with each new episode of non-pulsatile flow, because the accumulating cell layers diminish the effective volume of oxygenated blood that can be accommodated in each valve pocket.

11.6.4 Published Traveller’s Thrombosis Data The above conclusion seems to be supported by the finding (Lapostolle 2004) that the numbers of passengers affected by DVT rises exponentially with the time/ distance they have flown. It may be that the duration of VVP hypoxaemia required to cause the margination of the second and subsequent layers of blood cells on the parietalis shortens progressively with each successive cycle. The effect may depend not only on geometrical considerations (the volume of the VVP sinus must decrease as the layer of dead cells thickens, so the amount of available oxygen in the stagnated blood must decrease in proportion), but also on the capacity of the congregated blood cells for anaerobic metabolism, which may not equal that of the endothelial cells. These considerations suggest potential future lines of research.

11.6.5

Intermittent Positive Pressure Compression (IPPC) of Feet or Legs

The same considerations relate to the prophylactic use of IPPC. The principle is once again to empty ‘stale’, deoxygenated blood from VVP, mainly in the leg veins.12 So what might be the ideal frequencies, intervals and pressures at which the IPPC apparatus should be operated? Patients wearing such devices complain of the disturbing effect of compression. For some people, tight and aggressive13 squeezing

12 Jugular and arm veins do not (commonly) thrombose because they are emptied by gravity and do not have to pump against heavy columns of venous blood. Likewise, ambulant patients who are not taking sedatives, hypnotics or equivalents (including muscle relaxants) will naturally empty their own VVP without IPPC enhancement. However, operative and post-operative stupor, limb pain and equivalents may all impair or inhibit self-movement, and thus require routine mechanically-aided prophylactic IPPC assistance. 13 By ‘aggressive’ we mean the discomfort of a very rapid increase to quite high pressure of ‘tight’ inflatable stockings on feet or legs, intended speed up the venous return of blood from feet to heart by ‘milking’ it up the limb.

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of a limb is very uncomfortable or even painful, especially in the post-operative period; it is mentally tedious or disturbing to have it persist for days or weeks, confining to be tied to a machine incessantly, and exhausting to have sleep disturbed by intermittent nocturnal compression. Patients will appreciate the gentlest and least disturbing course of compression. Whenever possible, blood should flow downhill out of VVPs in bed-bound and horizontal patients. Those languishing for long periods in sedentary postures are always at risk of DVT and potentially require IPPC assistance with their venous return until they are no longer inhibited by weakness or recurrent pain and able to move themselves freely. The governing principle is that the selected rate of pressure increment and tightness of squeeze should not exceed the patient’s tolerance and comfort. Unbearably tight or too frequent compression has no theoretical advantage, and becomes adverse if patients decline to wear such ‘artificial peripheral venous hearts’. Rapid onset and build-up of pressure is not necessary: the gentlest possible pulse is as effective as the most powerful if it empties VVPs by ‘flapping’ the cusps – only the briefest of pressure changes is needed under the inflatable stocking. Should foot compression be used (i.e. the pulse applied to that smaller body area), a more ‘positive’ pulse might be desirable as there is probably an ideal ratio between the size of the compression sock area and the rate at which the squeeze pressure is applied. Patients could thus modify the frequency and force of IPPC to their liking within 30–90 min cycles. A key consequence of the VCHH is that there is absolutely no need to ‘pulse’ more often than once per hour, or even less frequently, to achieve full prophylactic effect, and no need to worry about traveller’s thrombosis if one is ‘shifting oneself’ or ‘shuffling feet’ in situ more than once in every two hours. Current thinking about IPCC is in need of updating. In Chapter 8 we argued that that venous flow rate is a secondary and almost certainly irrelevant issue, because VVP emptying and refilling depends not on flow rate but on pulsatility of flow. Even were the blood flow rate raised to an arterial level, but in streamline mode rather than pulsatile, the incidence of DVT might be little different; the cusps would probably lie flatter against the sinuses and the thrombi when they formed would perhaps be slimmer (Chapter 9). Gentle, low-frequency, compressivedecompressive cycles at very infrequent intervals (perhaps once per hour), which might be switched off and removed at night (if the lower end of the patient’s bed is elevated 5° all the time), would suffice to prevent DVT entirely in patients with no recent suggestive history of decubitus and no history of thrombotic episodes. In the event of such histories, anticoagulation would be a suitable prophylactic approach, and could be used in combination with regular VVP emptying. Thus, intermittent Trendelenburg tilting, possibly mechanized (if the numbers requiring reverse tilting at intervals exceed staff availability), could substitute for IPPC in patients horizontal during or after limb surgery. Permanent feet-up tilt of 2–5° leg elevation would achieve the same objective if the patient is not ambulant; the bed might be divided at hip level to allow elevation of the legs alone at 2–5° from the horizontal.

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The Animal Experiments of Hamer and Malone (1984)

These experiments were described in Section 11.3.2. With regard to the time spans likely to be causal in the initiation of thrombosis, they broadly indicated that two periods of about 3 hours paresis resulting in streamline luminal blood flow, interrupted by two or three periods of muscle clonus each lasting about 5 or 10 min, may cause unequivocal thrombus formation (Fig. 11.5).

11.7

Reflection

At the beginning of this chapter (Section 11.1.1) we stated that the consensus model of DVT fails to satisfy many of the criteria for a satisfactory aetiological hypothesis. In contrast, the VCHH accounts coherently and consistently for much of the established knowledge in the field and throws new light on aspects of prophylaxis. Its premises were validated experimentally and its predictions were corroborated during the 1980s (Section 11.3); other predictions suggest future lines of research, e.g. the suggestion that thrombi may be produced even if venous blood movement is rapid, provided that it is non-pulsatile for sustained periods (Chapter 9). More specifically, the VCHH relates this process to pathological changes in the valve cusp leaflets, accounts for the characteristic morphological features of venous thrombi, gives cogent reasons for the marked tendency of such thrombi to embolise (Chapter 10) and leads more or less immediately to explanations for many of the known ‘risk factors’. A fair objection to the VCHH as expounded here and in the preceding three chapters is that it takes little or no account of the plethora of cell and molecular biological research published during the last 2–3 decades. This apparent shortcoming needs to be addressed, not least because the endeavour may point to the rapprochement between approaches to biomedical investigation that we set out to achieve. In Chapter 12, therefore, we shall review the relevant cell and molecular biological literature in relation to the VCHH. We shall then consider the remaining known ‘risk factors’, and finally consider the issue of ‘unification or amalgamation of approaches’.

Chapter 12

Molecular Changes in the Hypoxic Endothelium

Abstract Gene expression patterns in endothelial cells (EC), presumably including those of the valve cusp parietalis, change in response to hypoxia and other challenges. These changes, the signalling networks involved and the consequences for cell phenotype have been elucidated in considerable detail, providing a mechanistic underpinning for our hypothesis (the VCHH) of the aetiology of DVT. In particular, they define mechanisms for (1) the increased congregation and anchoring of leukocytes and platelets on the hypoxic area, (2) the effects of activated neutrophils on the injured vascular endothelium, and (3) enhanced blood coagulation in the immediate neighbourhood. A significant part of the molecular-biological literature in this field concerns the effects of hypoxia on vasodynamics, which have limited relevance to the aetiology of DVT but may be pertinent to perfusion of the vessel wall via the vasa venarum. Satisfactory assimilation of these ‘mechanistic’ findings into the VCHH, as articulated in Chapter 11, exemplifies the reconciliation of approaches to medicine and biology outlined in the preface and discussed further in Chapters 4 and 5. We return to this fundamental point at the end of the present chapter and in the appendix.

Keywords Endothelial cell phenotype, elk-1/egr-1 signalling pathway, proteinaseactivated receptor, platelet activation, leukocytes

12.1

Endothelial Cell Physiology

The vascular endothelium is a confluent monolayer of thin, flattened, rhomboidal cells that normally prevents the escape of fluid from blood vessels other than capillaries. Endothelial cells (EC) produce enzymes and inhibitors that slowly and continuously remodel the subendothelial extracellular matrix (ECM), a structure consisting mainly of collagen, fibronectin and laminin supplemented with glycosaminoglycans, thrombospondin and von Willebrand factor (vWF) (Jaffe 1984). Although the vascular endothelium is not biochemically and physiologically homogeneous (Chi et al.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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2003), we may suppose that all vascular EC respond in broadly similar ways to similar stimuli. There is no evidence to suggest that the cells of the parietalis endothelium differ from other EC, except anatomically (see below).1 Healthy EC alter their biochemistry or that of the surrounding tissue in response to a wide range of stimuli (Davies and Hagen 1993). Some environmental changes, including hypoxaemia (cf. Morrison et al. 1977), can result in focal pathological sequelae such as DVT (see Mason et al. 1977 for review). Venous hypoxaemia can be produced locally by failure of venous return, with or without impaired arterial perfusion; or globally, as in hypokinetic hypoxia (e.g. Johnson and Rock 1988) due to cardiac insufficiency, or carbon monoxide or other poisoning. Being located at the interface between blood and solid tissue, EC are the first victims. The responses of EC to hypoxia have been intensively studied since the 1980s, and recent molecular-biological discoveries may lead to novel therapeutic approaches for vascular disorders (Pohlman and Harlan 2000). Aird (2002) suggested a complexitytheory approach to understanding vascular endothelial physiology, taking account of the flexibility and diversity of responses by healthy EC (Table 12.1) and the narrowing of their response range when they are injured, hypoxically or otherwise.

Table 12.1 Properties of endothelial cells potentially relevant to the control of coagulation and fibrinolysis Manufactured and secreted Binding sites on EC surface by EC Present on EC surface for High-molecular weight kininogena Tissue factor (TF) Factor Va Factor VIIIa Prostacycline Thromboxane A2e Nitric oxide (NO)e Carbon monoxide (CO)e Plasminogen activatorse Plasminogen activator inhibitors, notably PAI-1e

Tissue factor pathway inhibitor, TFPI (sequesters factors VIIa and X)b Protein Cc Protein Sc Thrombomodulinc Heparan sulphate (activates ATIII and TFPI)d ADPase (platelet inhibitor)e – – – –

High-molecular weight kininogen Factor VIII Factor IXa Factor X Factor XaTT – – – – –

Relevant references include: aJaffe et al. (1974) b Bajaj and Bajaj (1997) c Esmon and Fukudome (1995) d Nemerson (1992) e Simionescu (1988); Warren (1990); Ryan and Rubanyi (1992)

1 Most authors in this field ignore the role of the valve cusp parietalis as the specific sites of formation of prothrombotic nidi (Chapters 9–11); but as we have seen, other parts of the vascular (including venous) endothelium would seem irrelevant.

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12.2 The VCHH and the Molecular Responses of EC to Hypoxia Obviously, molecular biological studies throw no further light on the crucial issues of pulsatile/non-pulsatile flow or the morphology and function of valves, which are matters of macroscopic physiology. However, they have shown that venous EC exposed to hypoxaemic conditions develop markedly altered gene expression patterns and concomitant changes in phenotype (Shreeniwas et al. 1991; Faller 1999; Ten and Pinsky 2002), as illustrated in Fig. 12.1. These alterations may lead to angiogenesis, changes in vasomotor tone and endothelial permeability to neutrophils. More pertinently to thrombosis, they also enhance the local congregation and anchoring of leukocytes and platelets and lead to increased procoagulatory and decreased fibrinolytic activities. The mechanisms involved may be seen as articulating or complementing either the consensus model of DVT or the VCHH; but in respect of the VCHH, some caveats must be considered. Many molecular biological studies have been conducted on cultured human umbilical vein endothelial cells, which are a good model for research on this level but clearly do not allow anatomical distinctions to be made. Also, there is a fundamental difference between ‘changed phenotype’ and ‘necrotic tissue’, and it is clear from microscopic evidence (Chapter 10) that the parietalis endothelium dies before a frank thrombus forms. Moreover, ‘mechanism’ is not ‘cause’. The molecularbiological literature provides useful insights into the mechanism (the ‘how’) of DVT formation, but as we have established, the cause (the ‘why’) is the suffocating hypoxaemia of the valve cusp parietalis under conditions of intermittently nonpulsatile flow. An interesting point here is that a well-developed venous thrombus may largely occlude the vessel, potentially rendering the region cephalad of the blockage hypoxaemic. There seems a prima facie case for expecting the endothelium in this region to become hypoxic. Yet no new thrombi form on the vein wall cephalad of the growing one. It could be difficult to account for this observation if venous endothelial hypoxia in general caused DVT. If these caveats are kept in mind, the emerging picture of the molecular biology of endothelial hypoxia complements our understanding of the cause of DVT with a credible set of mechanisms. In our view, this amalgam exemplifies the value for both research and clinical practice of a rapprochement between the pathophysiological and mechanistic approaches to medicine and biology.

12.3

Phenotypic Changes in EC under Hypoxic Conditions

Endothelial hypoxia leads to increased expression of various isoforms of protein kinase C (PKC), receptors for platelet-derived and vascular endothelial growth factors (PDGF and VEGF), cytokines such as platelet activating factor (PAF) and interleukins IL-1, IL-6 and IL-8, tumour necrosis factor-α (TNF-α), and interstitial

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Fig. 12.1 Hypoxia-induced changes in endothelial cell phenotype. The gene expression pattern in vascular endothelial cells is altered in response to non-fatal hypoxia. Decreased ATP levels promote anaerobic glycolysis. Cytokines are secreted, ICAM-1 and P-selectin are expressed and thombomodulin is down-regulated; as a result, leukocytes and platelets are attracted and are enabled to anchor themselves to the affected area of endothelium. The actin cytoskeleton is disrupted, weakening cell–cell junctions, and matrix metalloproteinases remodel the extracellular matrix. These changes are believed to be instrumental in facilitating leukocyte diapedesis. Alterations in nitric oxide and eicosanoid production are generally vasodilatory, but we suggest that in cases of local venous endothelial hypoxia, these may be important only in increasing vessel wall perfusion through the vasa venarum

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adhesion molecule-1 (ICAM-1) and P-selectin (Pinsky et al. 1995; Karimova and Pinsky 2001). There is also increased synthesis of extracellular matrix (ECM) proteins, but nitric oxide synthetases (NOS), thrombomodulin and basic fibroblast growth factor (bFGF) are down-regulated (McQuillan et al. 1994). In cultured umbilical vein EC, the expression of nucleotide transporter-1 is also down-regulated (Casanello et al. 2005). The hypoxia associated with sickle cell disease apparently leads to redistribution of PECAM-1 (CD31), vWF and P-selectin on the luminal surfaces of these cells; expression of vWF and VEGF is increased and that of NOS decreased. Trampont et al. (2004) considered these to be general responses to venous endothelial hypoxia. Most of the best-known responses of mammalian tissues to hypoxia, such as increased production of erythropoietin, VEGF, glycolytic enzymes and NOS, are mediated by hypoxia-inducible factors (HIFs) (Wenger 2000). In particular, HIF-1 has a significant role in vascular repair processes (Agani and Semenza 1998), hypoxia-induced angiogenesis (Yamakawa et al. 2003) and neutrophil survival (Walmsley et al. 2005), which may be relevant to events in hypoxaemic VVP (see below). The regulatory (α) subunits of HIF-1 accumulate under hypoxic conditions and activate relevant transcriptional processes; the molecular mechanisms involved are well-established (e.g. Wenger 2000; Epstein et al. 2001; Lando et al. 2002; Hewitson et al. 2002; Berra et al. 2003; Willam et al. 2004; Zagorska and Dulak 2004; Yuan et al. 2005). However, thrombosis-related changes in hypoxic EC appear to involve not HIF-1 but a zinc-finger transcription factor (Gashler and Sukhatme 1995), early growth response-1 (Egr-1) (Yan et al. 1998, 1999a; Lo et al. 2001; Schalch et al. 2004). Egr-1 was first identified as a growth-promoting transcription factor expressed in epithelial cells, fibroblasts and lymphocytes. It has similar kinetics to immediateearly gene products and activates the transcription of many downstream genes (Sukhatme et al. 1988; Waters et al. 1990; Okada et al. 2001), notably procoagulant genes in both the endothelium and locally circulating leukocytes. It may also contribute to angiogenesis and neointimal hyperplasia during tissue repair (Ohtani et al. 2004). Expression of Egr-1 during renal ischaemia (Ouellette et al. 1990) suggested a possible role in tissue responses to hypoxia (cf. Huang and Adamson 1993). Its involvement in monocyte differentiation was suggested by Kharbanda et al. (1991) and within 5 years it had been implicated in endothelial responses to vascular injury (Khachigian et al. 1996). Many stimuli for Egr-1 induction in the endothelium have been identified, including shear stress (Schwachtgen et al. 1998) and, notably, hypoxia (Yan et al. 2000a).

12.3.1

Phenotypic Changes Consequent on Egr-1 Induction

Egr-1 up-regulates the expression of plasminogen activator inhibitor-1 (PAI-1) and TF (Houston et al. 1999). Concomitantly, the tissue plasminogen activator (tPA) is down-regulated; the receptors for tPA are proteoglycans on the EC surface. These

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changes tend towards fibrin production (Pinsky et al. 1998). Egr-1 also activates the transcription of genes associated with stress, including those that encode TNF-α, ICAM-1,2 CD44, PDGF, transforming growth factor-β(TGF-β), IL-1β and macrophage colony stimulating factor (M-CSF) (Yan et al. 2000a). Other examples include metalloproteinases that modify the subendothelial ECM, with possible implications for blood coagulation and leukocyte migration (Haas et al. 1999). Egr1 also mediates the induction of PDGF synthesis by EC exposed to fibroblast growth factor-1 (FGF-1) (Delbridge and Khachigian 1997). It is uncertain whether it is involved in the numerous responses of macrophages to hypoxia (increased metabolic activity and phagocytosis, altered morphology and cell surface markers and cytokine production) (Lewis et al. 1999), but it seems to be implicated in macrophage line differentiation (Nguyen et al. 1993). Interestingly, Egr-1 induces the formation of reactive oxygen species (ROS) in some cell lines (Bek et al. 2003). Some of these details are summarised in Fig. 12.2.

12.3.2

Elk-1 and SRF

A ternary complex between the serum response element (SRE: Qureshi et al. 1991), serum response factor (SRF) and the receptor tyrosine kinase elk-1 (Hipskind et al. 1994) appears to be required for Egr-1 expression. Hypoxia activates elk-1 via a PKC isoform and other receptor tyrosine kinases (Lo et al. 2001). The SRE is a 20 kb promoter region associated with immediate-early and actin genes (Mohun et al. 1987) as well as with Egr-1. SRF is a 67 kDa protein required for transcription of these genes (Treisman 1987). It is translocated to the nucleus after cAMP-dependent phosphorylation (Gauthier-Rouviere et al. 1995). Further phosphorylation (Prywes et al. 1988) by casein kinase II (Manak et al. 1990), calcium/calmodulin-dependent kinase or Ras (Miranti et al. 1995) is required for binding to the SRE. Yan et al. (1999b, 2000b) found strong evidence that Elk-1, a member of the ets oncogene superfamily (Rao et al. 1989), acts in concert with SRF to trigger Egr-1 transcription in monocytes exposed to hypoxia. The Elk-1 pathway involves the β isoform of PKC as well as Raf, mitogen-activated protein kinases (MAPK) and a MAPK kinase (Janknecht et al. 1993). Elk-1 is a known MAPK substrate (Yang et al. 1998). Fujita et al. (2004) showed that PKCβ implements the sequential functioning of the MAPKs ERK1/2 and JNK to activate Egr-1. Lo et al. (2001) reported similar findings but implicated the α rather than the β isoform of PKC.

2 ICAM-1 is believed to play a significant part in the effects of hypoxia. Interestingly, there is ‘cross-talk’ among these responses to Erg-1, leading to positive feedback. For instance, TNF-α induces transcription of the ICAM-1 gene through a pathway involving an isoform of protein kinase C (Rahman et al. 2000).

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Fig. 12.2 Gene expression through the Elk-1 and Egr-1 pathway. This scheme outlines the way in which the Elk-1/Egr-1 pathway is believed to alter EC phenotype in response to environmental changes, including hypoxia. The figure shows some examples of the products of genes transcriptionally activated by Egr-1. Key: ERK-1, extracellular signal receptor kinase-1; PAI-1, plasminogen activation inhibitor-1; ICAM-1, interstitial cell adhesion molecule-1; TF, tissue factor; M-CSF, macrophage colony-stimulating factor; PDGF, platelet-derived growth factor; TNF-α, tumour necrosis factor-α

Different combinations of MAPK appear to be involved in different cell types and it has proved difficult to dissect the signalling networks. At least three tyrosine kinase-related signalling pathways appear to be integrated at the ternary complex (Whitmarsh et al. 1995), and these pathways may be governed by the α and ε isoforms of protein kinase C (Soh et al. 1999). Mechtcheriakova et al. (2001) showed

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that VEGF3 mainly activates the ERK1/2 and p38 MAPKs in human EC, while TNF-α activates all three MAPK cascades as well as the classical inflammatory IκB/NFκB pathway. The MEK/ERK module of the MAP kinases seems to be the point at which the VEGF- and TNF-α-initiated signalling pathways converge; the former is highly PKC-dependent, the latter less so. Also, the induction of TF by VEGF via Egr-1 (Mechtcheriakova et al. 1999) is strongly PKC-dependent. Fig. 12.3 summarises a possible pathway of Egr-1 activation. Collectively, these findings demonstrate that EC respond to hypoxia via a welldefined signalling network. The responses include increased production of coagulation factors such as TF, and of factors involved in the congregation and anchoring of platelets and leukocytes (PAF, interleukins, vWF, ICAM-1 and P-selectin4); decreased production of coagulation inhibitors (e.g. thrombomodulin) and vasodilators (NO); and down-regulation of fibrinolysis (PAI is increased and tPA decreased). Although the valve cusp parietalis becomes necrotic before any visible thrombus is formed, these phenotypic changes must precede the local death of EC and are therefore likely to play a part in the mechanism by which DVT is initiated.

12.3.3

Other Regulators of Egr-1 Expression

Many other stimuli induce Erg-1 expression: granulocyte colony-stimulating factor (G-CSF) (Mora-Garcia and Sakamoto 2000), shear stress,5 mechanisms of vascular injury other than hypoxia (Khachigian et al. 1995, 1996) and FGF-2 (Santiago et al. 1999). Plasminogen and plasmin stimulate the production of FGF-1/2 through the MEK/ERK pathway (de Sousa et al. 2005). There also appears to be an Egr-1 pathway for the response to hyperoxia, which involves the EGF receptor, the MEK/ ERK pathway and other as yet unidentified signalling components (Jones and Agani 2003). A particularly significant Egr-1 activator is thrombin. Thrombin-mediated induction of Egr-1 is blocked by inhibitors of MEK1/2 but not by inhibitors of protein kinase C, phosphatidylinositol 3-kinase or p38 MAPK (Wu et al. 2002). Thrombin has a number of other effects on EC, several of which appear to be relevant to DVT (see below). There are also negative regulators of Erg-1 in EC. Shear stress-induced Egr-1 expression (Schwachtgen et al. 1998) is down-regulated by NO via the ERK

3

Apparently, VEGF levels are increased in hypoxic endothelial cells not because the gene for VEGF is more actively transcribed, but because the messenger RNA is stabilised. The mechanism has been investigated (Levy et al. 1998). 4 When platelets ‘roll’ on the endothelium, P-selectin is required (Frenette et al. 1995). 5 Shear stress also seems to activate the expression of NOS and of the lung Kruppel-like factor gene in the endothelium (Dekker et al. 2002); the latter inhibits PPAR-γ and promotes an inflammatory response. This seems much more relevant to arteries than veins, and not at all to the VVP.

Fig. 12.3 Ternary complex formation in response to endothelial cell hypoxia. The hypothetical pathway shown is based mainly on papers by Davis (1995), Whitmarsh et al. (1997), Müller et al. (1997), Soh et al. (1999), Lo et al. (2001), Berna et al. (2001) and Aley et al. (2005). According to this scheme, hypoxia causes ATP depletion, which liberates calcium from internal stores and increases calcium uptake from the extracellular medium. Phospholipase C is activated, and the diacylglycerols released from membrane lipids activate isoforms of protein kinase C, notably α and ε. These bind the mitogen-activated protein (MAP) kinase kinase kinases, Raf and RhoA. The former complex activates Mek (MAP kinase), which phosphorylates the MAP kinase ERK-1. The latter phosphorylates the serum response factor (SRF). ERK-1 is translocated to the nucleus and phosphorylates Elk-1. The phosphorylated forms of SRF and Elk-1 bind the serum response element in the gene promoter region to form the ternary complex that initiates translation of Egr-1 and other genes. Note that signals other than hypoxia, such as interleukin-1 (IL-1), growth factors, various toxins, shear stress and hypotonicity, can also induce ternary complex formation by activating ERK-1 and/or the other MAP kinases, JNK and p38 (Minami et al. 2003). Egr-1 may be activated by a similar pathway in other cells, including leukocytes, in response to hypoxia or to specific extracellular stimuli

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signalling pathway (Chiu et al. 1999). Peroxisome proliferator-activated receptor γ (PPAR-γ), a nuclear receptor that regulates inflammatory responses, appears to act in part by inhibiting Erg-1 induction (Okada et al. 2002). One effect is to suppress the attraction of monocytes to the hypoxic endothelium (Jiang et al. 1998; Ricote et al. 1998). Thus, PPAR-γ (or PPAR-γ analogues that are capable of entering cells) could have therapeutic value in cases of recurrent DVT. This may merit investigation.

12.4

Erg-1, Hypoxia and DVT

Among the products of genes up-regulated by Erg-1, TNF-α and IL-1β promote marked changes in cell phenotype (Esmon 1999). In particular, they further stimulate the production and intravascular secretion of TF. Thus, hypoxia increases TF expression in monocytes and subendothelial smooth muscle cells (Lawson et al. 1997) as well as EC (Solovey et al. 2004), with plausible implications for thrombosis as well as vascular development and haemostasis (Morrissey 2004). Under normal physiological circumstances, TF is restricted to the extravascular space, mainly on fibroblasts, which express it constitutively. However, hypoxia, sepsis6 and many cancers lead through the Egr-1/cytokine mechanism to increased circulating levels, perhaps contributing to thrombus propagation at sites of vascular injury (Balasubramanian et al. 2002). Intravascular TF also occupies proteinaseactivated receptors (PARs) on EC. PARs are 7-pass transmembrane receptors coupled to G-proteins (Coughlin 2000) and have fundamental roles in transforming EC phenotypes (Mackman 2004); see below. Other cytokines, especially IL-6, which is also induced in hypoxic EC (Yan et al. 1995), stimulate new platelet formation. New platelets are more sensitive to thrombin than mature ones and are therefore more likely to initiate local coagulation. Among the major anticoagulant mechanisms, the protein C pathway seems to be a target for cytokine action. In cultured EC, TNF-α, IL-1β and bacterial endotoxin all lead to a slow loss of thrombomodulin and protein C receptor (EPCR) from the cell surfaces. If this occurs in vivo, it may enhance any local tendency towards thrombosis.

12.5

Thrombin and the PARs

Thrombin is a multifunctional proteinase. It activates many of the blood coagulation factors (Chapter 2) and participates in the immune response, and it stimulates a variety of changes in vascular EC (Rydholm et al. 1998; Minami et al. 2004) by

6 Bacterial endotoxin (lipopolysaccharide) appears to activate the protein kinase C-Elk-1 pathway described above and to up-regulate TF, together with TNFα, in human monocytes (Guha et al. 2001). In view of this finding, it is interesting to recollect some of the early 20th century accounts of DVT aetiology, many of which cited bacterial infection among the causes (Chapter 7).

12.5 Thrombin and the PARs

205

binding to and cleaving PARs, particularly PAR-1 (see above). PAR-1 cleavage activates the G-protein and promotes interactions with other endothelial surface receptors (O’Brien et al. 2000). Other serine proteinases involved in blood coagulation, such as factor Xa, may also bind and cleave PARs under some circumstances (Camerer et al. 2000, 2002; Riewald and Ruf 2001); so does activated protein C, at least in cases of sepsis (Riewald et al. 2002). After PAR cleavage and G-protein activation, the thrombin signal is processed through several parallel pathways (Fig. 12.4). The EC respond inter alia by

Fig. 12.4 Effects of thrombin on EC phenotype. After PAR cleavage and G-protein activation, the thrombin signal is processed through several parallel pathways, resulting in wide-ranging phenotypic changes in the EC. Once leads through Akt and the 65 kDa isoform of NF-κB (Anrather et al. 1997) to transcription of the ICAM-1 gene (Rahman et al. 1999, 2002). A second operates through P13K and two isoforms of protein kinase C to NF-κB and GATA-2, which act in concert to switch on the VCAM-1 gene (Bassus et al. 2001; Minami and Aird 2001; Minami et al. 2003). A third activates DNA-binding protein-B, which up-regulates transcription of the PGDF and endothelial protein C receptor (EPCR) genes (Okazaki et al. 1992; Stenina et al. 2001). There is also the pathway that operates through MEK 1–2 and SRF to induce Egr-1 production (Wu et al. 2002), probably again via Elk-1 (Li et al. 2000). This is the most rapid of the thrombin-activated pathways, so thrombin is an important inducer of Egr-1-dependent changes in EC phenotype. Indeed, all the targets of these thrombin-activated pathways have ‘knock-on’ effects on other genes; several dozen genes in EC are up-regulated or down-regulated, directly or indirectly, as a result of thrombin binding to the surface PARs. Numerous interlocked signalling pathways are involved in this network of transcriptional changes, which involves feedback loops (e.g. some of the activated protein kinase isoforms inactivate the PARs: Yan et al. 1998a)

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12 Molecular Changes in the Hypoxic Endothelium

adopting different morphologies, and linkages with neighbouring cells and the ECM are modified. General consequences are believed to include effects on venous tone, coagulation, endothelial permeability to and anchoring of leukocytes, and in some instances cell proliferation. The impairment of endothelial barrier function appears to result from the disruption of the actin cytoskeleton, a consequence of the decreased cAMP levels incident on hypoxia (Ogawa et al. 1990; Bentley and Beavo 1992; Rabiet et al. 1996); though there can be no significant cell trafficking through the parietalis because the valve cusp is avascular. There are also effects on tone (via the vessel wall smooth muscle) but these cannot be directly relevant to DVT because (a) the valve cusp has no smooth muscle content and (b) as we discussed in Chapters 8–11, alterations in venous blood velocity per se play no significant part in the aetiology of DVT. However, there are potential implications for DVT. These include up-regulated expression of TF (Takeya et al. 2003), PAI-1 (Gelehrter and Sznycer-Laszuk 1986) and TAFI (Tobu et al. 2004), favouring fibrin formation; and increased prostacyclin (Houliston et al. 2002) and PAF (Lorant et al. 1991) production, leading to platelet activation. Also, the disruption of cell–cell and cell–subendothelial contacts may enhance the tendency of the parietalis endothelium to fragment and dehisce.

12.6

12.6.1

Interactions Between Platelets and the Hypoxic Endothelium Platelet Congregation and Implications for DVT

Platelets are not uniformly distributed in the veins; interestingly, they are less dense in such deep lower-limb vessels as the soleus and femoral vein, which are the most common sites of DVT (Woldhuis et al. 1992; see Chapter 8). Platelets interact with EC when the shear rate is sufficiently low (less than 100 s−1), as in the secondary vortices in VVP during streamline flow (Chapter 9). Weiss et al. (1986) investigated this process by intravital microscopy. They found that calcium secretion initiates the linkage of platelets to the endothelium within 15 s but does not make them congregate. The platelet numbers peak at about 25,000/mm2 s after roughly 1 min then progressively decrease. The grip of the platelets on the EC depends on von Willebrand factor (vWF), or perhaps fibrinogen, exposed on the luminal membranes (Savage et al. 1996), and on ICAM-1 and integrins (Bombeli et al. 1998); it is independent of P-selectin, PECAM-1 and PSGL-1. The platelet glycoprotein GPIbα is required (André et al. 2000) and other platelet glycoproteins might also be involved. EC undergoing apoptosis also bind platelets (Bombeli et al. 1999). Secretions from physiologically normal endothelium are said to modulate the early stages of thrombosis (Simionescu 1988; Warren 1990; Ryan and Rubanyi

12.6 Interactions Between Platelets and the Hypoxic Endothelium

207

1992). EC produce inhibitors of platelet recruitment and activation: NO, CO and a membrane-bound ADPase (CD39). The adenosine produced by the ADPase deactivates the platelets by elevating their cAMP levels. Like NO, CO activates a target cell guanyl cyclase. The increased cGMP inhibits platelet congregation and promotes the synthesis of endothelin-1 and PDGF (a paracrine effect by EC) (Morita and Kourembanas 1995). Under hypoxic conditions, however, NO synthesis is inhibited and endothelin is up-regulated (Eto et al. 2001). Since NO is a platelet inhibitor, platelet activation is enhanced. Several endothelial ion transporters are inhibited under hypoxic conditions; inter alia, intracellular calcium levels are increased and this induces the EC to release prostaglandins (Michiels et al. 1993). There are receptors for prostaglandins on EC and they also have mitogenic effects on the vascular smooth muscle (Kent et al. 1993). More prostacyclin (Houliston et al. 2002) and PAF-1 (Lorant et al. 1991) are released. As mentioned earlier, IL-6 secretion increases the supply of new platelets. Also, prostaglandins, adenine nucleotides and other intracellular components released from injured endothelium enhance platelet congregation (Mason et al. 1977). None of the many studies of the interactions between platelets and vascular walls has specifically considered the valve cusp leaflets. Nevertheless, the foregoing mechanisms may explain the association of fresh, viable platelets with the parietalis endothelium during reperfusion after a sustained episode of VVP hypoxaemia. Many of these mechanisms are likely to be insensitive to aspirin.7

12.6.2

Leukocyte–Platelet Complexes in the Circulation and their Association with the Hypoxic Endothelium

Many circulating monocytes and neutrophils are associated with platelets (Faint 1992). These assemblies are believed to be significant in, for instance, ischaemiareperfusion injury (IRI) (Massberg et al. 1998). P-selectin on the platelet surface engages with a leukocyte surface receptor, a glycoprotein. The resulting intracellular signalling within the leukocyte increases the expression of both TF and Mac-1 on its surface (Peters et al. 1999), and factor Xa and fibrinogen may bind to Mac-1 (Barnard et al. 2005). Thus, platelets and leukocytes associate with each other in the circulation. Their interactions with the vessel wall are governed by a multiplicity of controls. The changes in EC caused by hypoxia re-choreograph this system of controls and potentiate blood cell binding; in particular, to the hypoxic parietalis during reperfusion of VVP.

7 High concentrations of aspirin, however, inhibit leukocyte anchoring, though they do not affect NO production (Fricchione et al. 1998).

208

12.7

12 Molecular Changes in the Hypoxic Endothelium

Endothelial Hypoxia and the Congregation of Leukocytes

Interactions between leukocytes and blood vessel walls are promoted under hypoxic conditions (Ginis et al. 1993; Gonzalez and Wood 2001), consistent with the VCHH (Chapter 7). However, the signalling pathways involved8 are complex and have not been fully elucidated. Different parts of the vascular endothelium differ quantitatively, though not qualitatively, in their capacities for chemokine binding and association with leukocytes (Hillyer and Male 2005). Specific cell-adhesion and chemotactic molecules must be presented with precise timing and location (Geng 2003). Both large veins and post-capillary venules undergo inflammatory reactions when their endothelia are activated by e.g. TNF-α; in particular, the ‘adhesion molecules’ responsible for anchoring blood cells are markedly up-regulated (Eriksson et al. 2005). Three classes of adhesion molecules are involved in interactions between EC and leukocytes: selectins, which bind lymphocytes and neutrophils (Benvilacqua and Nelson 1993); immunoglobulins, which act as antigen-specific T and B lymphocyte receptors; and integrins, which are intimately involved in cell migration as well as platelet anchoring (Benvilacqua 1993; Smith 1993).9 More specifically: interleukin-1 (IL-1), TNF-α and TGF-β stimulate EC. Their effects are mediated through stimulation of one or more of the MAPKs – ERK, p38 and JNK/SAPK – and the transcription factor NF-κB (Kishimoto et al. 1994), and lead to increased expression of ICAM-1, VCAM-1, and the interleukins IL-1, IL-6 and IL-8. These cytokines upregulate TF (Li et al. 2006) and PAI-1 and down-regulate proteins C and S, so they tend to be pro-coagulatory. Therefore, leukocyte congregation/anchoring and the promotion of local coagulation are intimately related. Similarly, PAF, CD18/ CD11b (Mac) and ICAM-1 are involved in leukocyte congregation and activation (Arnould et al. 1993, 1994, 1995). Thrombin also stimulates leukocyte recruitment via the MAPK p38 (Marin et al. 2001; Kaur et al. 2003); it specifically enhances monocyte chemotaxis (Colotta et al. 1994). Neuropeptide Y increases leukocyte anchoring on some vessel walls (Sung et al. 1991).

8 Outside the VVP, neutrophils release proteinases and ROS that alter the ECM of the vein wall. Concomitantly, the endothelium releases mitogens that induce the subendothelial smooth muscle cell to dedifferentiate and proliferate (Michiels et al. 1994). Chronic repetition of these processes can eventually lead to alterations in the venous wall such as the ones observed in varicose veins. 9 During an immune response, leukocytes are recruited from the bloodstream to surrounding tissues (Grant 1965). This involves a transient ‘rolling’ interaction with the vascular endothelium followed by firm attachment to the vessel wall (Harlan 1985). The first stage involves interaction between selectins and membrane sialoproteins (Lawrence and Springer 1991), the second between leukocyte integrins and ICAM-1 on the EC. Over the vascular system as a whole, a balance develops between leukocytes entering the tissues by diapedesis and those returning to the circulation (Bienvenu et al. 1992).

12.7 Endothelial Hypoxia and the Congregation of Leukocytes

12.7.1

209

Monocytes and Macrophages

Monocytes/macrophages accumulate in areas that are likely to be hypoxic, presumably including the hypoxaemically injured valve cusp parietalis. Macrophages in such areas show increased metabolic activity and phagocytosis, altered morphology and cell surface markers, and cytokine production (Lewis et al. 1999). Hypoxic EC produce increased amounts of the monocyte chemoattractant protein JE/MCP-1 (Karakurum et al. 1994), which probably recruits monocytes and macrophages to the site as well as inducing TF expression in the local subendothelial smooth muscle (Schecter et al. 1997). The recruited cells secrete cytokines, notably IL-1 and IL-6 and also TNF-α (Signorelli et al. 2000). However, monocyte and macrophage chemotaxis appears to be inhibited by more prolonged (60–90 min) exposure to hypoxia; cells in increasingly hypoxaemic blood die slowly, as we have observed in earlier chapters. In addition, both calcineurin and angiotensin II promote the synthesis of MCP-1 in arterial smooth muscle cells (Chen et al. 1998; Satonaka et al. 2004) and may act similarly in the veins. Pathological concentrations of homocysteine induce MCP-1 production in macrophages by a mechanism involving NF-κB (Wang et al. 2001), which may be initiated by the activation of surface Fc receptors (Alonso et al. 2000). Fc receptors are stimulated by such ligands as C-reactive protein (Han et al. 2004). This effect of homocysteine might partly explain why homocysteinaemia increases the likelihood of DVT (cf. Heijer 2003; see Chapter 3). IL-1β promotes MCP-1 production by arterial and possibly venous EC (Takahashi et al. 1995). MCP-1 binds to a number of cytokine receptors that are coupled to G-proteins, notably CCR-2; occupation of these receptors leads to activation of ERK-1 and ERK-2 through a pathway involving protein kinase C, Ras and phosphoinositide-3-kinase (Jiménez-Sainz et al. 2003). Monocytes anchor themselves to endothelial cells via VCAM-1 (Imamoto et al. 2004). Indeed, both VCAM and ICAM-1 promote monocyte as well as neutrophil binding (Kaplanski et al. 1998). The attraction of monocytes to the hypoxic endothelium is inhibited by regulators of the inflammatory response (Jiang et al. 1998; Ricote et al. 1998). The only monocyte protein that seems to be transcriptionally up-regulated under hypoxic conditions or in response to TNF-α treatment is MAPK phosphatase 1 (MKP-1). This modulates MAPK activity and is implicated in the inhibition of chemotaxis and migration (Grimshaw and Balkwill 2001). Monocytes express more TF during hypoxia (Lawson et al. 1997), probably because the MAPK pathway is stimulated (Grimshaw and Balkwill 2001); TF interacts with factor VII in the plasma and also causes platelet congregation, albeit less efficiently than collagen (Barstad et al. 1995). Monocytes produce both kinds of plasminogen activation inhibitor, PAI-1 and PAI-2 (Castellote et al. 1990), and also TNF-α, which exerts autocrine stimulation of PAI-1 production via a protein kinase C pathway during macrophage differentiation (Lopez et al. 2000). In these ways, the activation of monocytes under hypoxic conditions may promote coagulation and/or inhibit

210

12 Molecular Changes in the Hypoxic Endothelium

fibrinolysis. It also enhances the phagocytosis of necrotic cells (Lewis et al. 1999; see above). The stimulatory effect of thrombin on macrophage recruitment (see above) implies a positive feedback effect once local coagulation has been initiated.

12.7.2

Neutrophils

Much has been written about the role of neutrophils in IRI (e.g. Thiangarajan et al. 1997; Carden and Granger 2000; Eltzschig and Collard 2004; Vinten-Johansen 2004). Although IRI concerns the heart and arteries, much information about it has been obtained from experiments on human umbilical vein endothelial cells. We can reasonably infer that many of the results apply to venous valve cusp EC, in particular their interactions with neutrophils. Under hypoxic conditions, EC release inflammatory mediators and growth factors (particularly PAF) that recruit neutrophils, promote their anchoring to the endothelium and activate them (Esmon 1999); this requires the induction of β-integrin (Kong et al. 2004). Prolonged hypoxia also enhances the expression of VEGF and PDGF. The key transcription factor in these responses appears to be HIF-1 (Michiels et al. 2000). Neutrophils gather and are activated in areas of low blood flow (Monos et al. 1995), such as VVP under non-pulsatile flow conditions. Under low shear stress conditions or in the presence of thrombin (or both), P-selectin is expressed quickly on the EC surface, E-selectin more slowly. These selectins anchor neutrophils and initiate surface rolling (Lawrence and Springer 1991); various neutrophil surface antigens are involved. Local haemodynamic fluctuations, endothelial activity and biochemical factors such as prostaglandins control the interaction of polymorphs with the endothelial surface; the interaction involves PAF and receptors on both the leukocyte and the EC (Arnould et al. 1992, 1994, 1995, 2001; Monos et al. 1995). Neutrophil anchoring and emigration10 are promoted by PAF (Kim et al. 1995), which is secreted in response to thrombin or ROS, and by leukotriene B4 (Shimizu et al. 1992). CD11b/CD18 (Mac) is very rapidly expressed on the neutrophil surface in response to a few cytokine molecules (Simon and Goldsmith 2002). PAF and VEGF promote the translocation of P-selectin to EC surfaces (Rollin et al. 2004), enhancing neutrophil anchoring (Wang et al. 1998) via the Mac complex

10 Neutrophil emigration appears to depend on the activation of endothelial ERK 1 and 2 (Stein et al. 2003), and to be promoted by NO (Scott Isenberg 2003). The leukocyte integrin CD11/CD18 (Mac) binds to ICAMs on activated EC and this halts the rolling movement. Expression of these Igs is regulated by cytokines and by shear stress (Bienvenu and Granger 1993). ICAM-1 is expressed on the bases as well as the apices of EC activated e.g. by TNF-α, and thus provides a pathway for leukocyte migration (Nagel et al. 1994; Yang et al. 2005). During migration, VEcadherin moves to opposite ends of the migration site and PECAM-1 opens to surround the migrating neutrophil (Su et al. 2002). These findings are pertinent to tissue injury in general, though they have no direct relevance to DVT; leukocytes cannot emigrate via valve cusps, which are avascular.

12.7 Endothelial Hypoxia and the Congregation of Leukocytes

211

(Imamoto et al. 2004). There are many known influences on these interactions. For instance, thrombin induces P-selectin and ICAM-1 expression by EC (see above) and this stabilises neutrophil anchoring (Sugama et al. 1992). Moreover, thrombomodulin inhibits the association of leukocytes with EC surfaces; the cleavage of thombomodulin by thrombin removes this inhibition (Conway et al. 2002). ROS and components of the complement system enhance ICAM-1-dependent neutrophil anchoring (Lehrer et al. 1988; Suzuki et al. 1991; Sellak et al. 1994); so does endothelin-1 (Anthoni et al. 2006). Stimulation of guanylate cyclase and NOS promotes the interaction, while increased cyclic-AMP levels are inhibitory (Schaefer et al. 1998). Interestingly, bacterial endotoxin also promotes neutrophil association with vein EC, probably via E-selectin (Fries et al. 1993), while antithrombin is inhibitory (Kaneider et al. 2003); Helicobacter pylori causes a fivefold increase in neutrophil anchoring to vein EC (Byrne et al. 2002). This recalls the long-debated involvement of bacterial infection in thrombosis. The findings may be relevant to thrombus formation during septicaemia. Thom et al. (2006) showed that neutrophils are activated by toxic but non-fatal levels of CO, recalling the work of Drinker (1938; see Chapters 10 and 11). The main inferences from this range of studies are: (1) if neutrophils or monocytes/ macrophages are activated at any (distant) site in the circulation, their capacity to congregate on an injured area of vascular endothelium is enhanced; (2) leukocytes are specifically attracted by factors produced and secreted by hypoxic EC; and (3) leukocytes themselves, particularly monocytes and macrophages, enhance local blood coagulation when they are activated.

12.7.3

Effects of Leukocytes on Injured Endothelium

12.7.3.1

Neutrophils and Hypoxic EC Interact

Neutrophils attack injured cells by secreting factors such as ROS and cationic proteins, an essential part of the phagocytic removal of injured tissue. Hughes et al. (2006) confirmed that neutrophils attack the endothelium after even moderate hypoxic insult, and this is significant in IRI.11 Schaub and Yamashita (1986) found that such injury involves lipoxygenation. Harlan et al. (1981) showed that neutrophils cause EC detachment by proteolysis of cell-cell adhesion proteins, notably fibronectin. The actin cytoskeleton is connected to the focal adhesions that link the cells to the ECM (integrins) and to each other (cadherins). These may also be attacked by neutrophil proteinases, perhaps complementing the fragmenting action of thrombin (see above) and promoting the dehiscence of the hypoxic parietalis

11 As we observed in Chapter 10, we cannot accept the apparent contention of Stewart and colleagues that leukocytes injure normal endothelium; but relatively slight, non-fatal injury to EC may conceivably elicit an aggressive response from living blood cells.

212

12 Molecular Changes in the Hypoxic Endothelium

endothelium. Although ECs have some protection against such attack (Rinaldo and Basford 1987), they also activate neutrophils by producing GM-CSF and PAF (Takahashi et al. 2001). In turn, activated neutrophils affect the EC phenotype, including expression of adhesion molecules, by secreting soluble mediators (Jacobi et al. 2006; see above). Nohe et al. (2005) showed that leukocyte anchoring correlates with tissue factor (TF) expression by the endothelium, again suggesting that leukocyte congregation may promote local coagulation.

12.7.3.2

Endothelial Proliferation

Parietal and ostial valves appear to be the sites of EC proliferation after vascular endothelium has been artificially removed (Einarrson et al. 1984). After 2–3 days, even extensive endothelial lesions in both arteries and veins are repaired by the migration of endotheliocytes from sites of proliferation (Krupski et al. 1979; Vialov and Miranov 1988). Haematopoietic progenitor cells (CD34+) recruited to the nascent endothelium have been implicated in stimulating this regenerative process (Grote et al. 2007). Hypoxia leads to increased endothelial proliferation, apparently because calcium influx into the EC is increased as the ATP levels fall, resulting in higher levels of ROS (Schaefer et al. 2006). The increased production of VEGF from hypoxic EC (see above) is clearly an important factor in proliferation; again, it enhances calcium uptake by the cells and stimulates NO production (Erdogan et al. 2005). The angiogenic response to hypoxia is presumably responsible for capillary development at the base of degenerating valve cusps concurrent with thrombus growth (Edwards and Edwards 1939; Chapter 9). When the valve cusp parietalis endothelium becomes necrotic after a single prolonged episode of non-pulsatile flow, it will be removed by the congregated phagocytes (neutrophils and macrophages) when pulsatility is restored. If there is no further period of hypoxaemia during the next 2–3 days, therefore, we may expect the endothelium to be regenerated by the foregoing mechanisms and no injury to persist at the site. This suggests that a tourniquet could safely be reapplied 3 days after it is removed; animal experiments to test this prediction can be conceived. Once again, only repeated episodes of non-pulsatile venous flow potentially lead to DVT.

12.8

The Endothelium and Coagulation

There is consensus agreement that physiologically normal EC control coagulation, promote anticoagulation, regulate fibrinolysis and produce thromboregulators (Eisenberg 1991; Furie and Furie 1992). Examples of the molecular components involved are summarised in Table 12.2. Collectively, these discoveries may help to explain why blood coagulation remains locally confined: blood coagulates only when and where the endothelium loses its structural and functional integrity or, perhaps, when the cell phenotype is markedly altered.

Endothelium-derived relaxing factor (EDRF) produced Prostaglandin and thromboxane production Endothelin-1 production15 and smooth muscle contraction

Increased ROS production; inhibition of endothelial surface ADPase Increased prostacyclin production and thromboxane release

Increased shear stress

Increased transmural pressure

Calcium channels

Calcium influx?

Membrane depolarisation

Membrane depolarisation and calcium influx Calcium release from intracellular stores Enhanced Ca influx, phospholipase activity, NO production and potassium influx

Membrane hyperpolarisation

Mechanism

Golledge (2004)

Brunkwall et al. (1989); Onohara et al. (1993)

Bevan and Joyce (1990); Marshenko and Sage (2000) Rubanyi (1991); Tsukahara et al. (1993) Henrion et al. (1992); Baron et al. (1993) Jones et al. (1998); Lerman et al. (1990); Masaki et al. (1991); Naruse and Sokabe (1993) Krotz et al. (2002)

Reference(s)

(continued)

Implications for platelet activity and vasodilatation Enhanced leukocyte binding; protein C pathway down-regulated

Platelet congregation

Increased synthesis of EDRF and prostacyclin

–

–

–

Possible implications for DVT

Other endothelium-derived constriction factors are released under hypoxic conditions or when the transmural pressure is high (Rubanyi and Vanhoutte 1985); these include some ROS and vasodilatory metabolites of arachidonic acid as well as endothelin-1 (Vanhoutte et al. 2005). 16 The increase in ICAM-1 expression is temporary; after a few hours, the newly synthesised adhesion molecule is cleaved by metalloproteinase-9 and solubilised (Sultan et al. 2004).

15

Increased blood flow rate

Increased blood flow rate

Increased intramural pressure

ICAM-116 and NO production upregulated; thrombomodulin expression decreased

Vasodilatation

Increased shear stress

Increased shear stress

Response

Responses of vascular endothelial cells to a range of stimulus conditions

Stimulus

Table 12.2

Vasoactive agents (bradykinin, acetylcholine, thrombin)

Increased intraluminal adenosine EDRF18

G-protein receptors, membrane phospholipases, altered inositol trisphosphate and intracellular Ca levels, NO synthetase activated/inhibited

Not certain

P2 purinergic receptors

Tone decreased; platelet congregation and anchoring inhibited Vasoconstriction or vasodilatation

Contractility diminished because response to noradrenalin, serotonin and prostaglandins inhibited Increased prostacyclin release

Hypoxia

P1 purinergic receptors

Prostacyclin synthesis inhibited; TxA2 production unchanged

Hypoxia

G proteins, phosphatidyl inositol turnover, PKC is forms activation, adenyl cclase activation, calcium influx Re-esterification of arachidonic acid inhibited; effects of ROS Locally lowered pH17 and increased PCO2

Mechanism

Increased cyclic AMP production

Vasoconstriction

Decreased radial pressure

Increased intraluminal ATP

Response

Stimulus

Table 12.2 (continued)

Implications for platelet activity and vasodilatation Implications for platelet activity and vasodilatation

Katusic and Vanhoutte (1989); Rubanyi (1991)

Choi et al. (2001); Mathie Implications for vessel tone et al. (1991); Hammer et al. (2003) Olsson and Pearson (1990); Stiles (1990) Glick et al. (1993); May Coagulation impaired et al. (1999) Eguchi and Katusic (2001) Effects on vessel dynamics

Johnson (1986); Arner and Hogestatt (1991)

Effects on smooth muscle myosin lead to vasoconstriction

Possible implications for DVT

Eguchi et al. (1997)

Reference(s)

Low levels promote eicosanoid production; high levels inhibit. Increased TNF-α? Vasodilatation

Vasodilatation

Reactive oxygen species

Carbon monoxide (CO)19

Guanyl cyclase and calciumdependent pathways activated Guanyl cyclase and calciumdependent pathways activated

Effects on cycloxygenase activities

Calcium uptake

Maines (1988); Morita and Kourembanas (1995); Motterlini et al. (1996)

Eguchi and Katusic (2001)

Eguchi et al. (1997); Eguchi and Katusic (2001) Valen et al. (1999)

Effect less marked in valve-rich regions of veins Implications for platelet activity and vasodilatation Platelet congregation inhibited Platelet congregation inhibited; PDGF and endothelin-1 produced

18

Lactate appears to make the vessel wall less sensitive to calcium: Soloviev and Basilyuk (1993). Two distinct, though similar, EDRFs are produced in arteries and veins, and they work through different signalling pathways (Wood et al. 1990; Yang et al. 1991). Arteries produce more ERDF and show more marked responses (Barker et al. 1994). 19 CO is produced by constitutive and inducible haem oxygenases. The latter are activated by NO, increased shear stress, oxidative stress and hypoxia, and are inhibited by calcium; see references in table.

17

Nitric oxide (NO)

Vasoconstriction

Bradykinin

216

12 Molecular Changes in the Hypoxic Endothelium

The intact endothelium also separates platelets and coagulation factors in the circulating blood from the subendothelial collagen and von Willebrand factor (vWF). Chapter 2 showed the potential significance of these aspects of endothelial physiology for thrombosis: perturbation of endothelial integrity, specifically on valve cusps, could result in local coagulation. In addition, the normal endothelium also secretes factors that may modulate the early stages of thrombosis, such as eiconsanoids (Table 12.2). Many of the factors mentioned in Table 12.2 also regulate the tone of certain vessels (see below). During this chapter we have shown that endothelial hypoxia promotes local coagulation by a number of different mechanisms. Increased quantities of coagulation factors such as TF are produced in both the injured intima and local monocytes; factors involved in the congregation and anchoring of platelets and leukocytes are up-regulated; levels of thrombomodulin and NO are decreased; fibrinolysis is down-regulated. Monocyte activation under hypoxic conditions appears to promote coagulation and inhibit fibrinolysis. Thrombin, produced when coagulation is initiated, further modifies the EC phenotype and also recruits macrophages.

12.9

The Endothelium and Vasomotor Tone

Generally, the vascular endothelium is profoundly involved in controlling vessel tone by secreting prostanoids, EDRF, ATP and other factors and by regulating sodium and calcium fluxes. Thus, it regulates mean blood velocity. In turn, these secretion processes are affected by changes in local blood velocity and shear stress, and the endothelium mediates the signalling.12 EC act as mechanosensors, transducing physical blood flow variables into biochemical signals to which the vessel wall responds (Davies and Tripathi 1993). Blood flow subjects them to shear stress and exerts pressure normal to the wall. These forces elicit the secretion of factors (Rongen et al. 1993) that inter alia affect the state of the smooth muscle and consequently the baseline tone. Veins are considerably less sensitive than arterioles in this regard but their EC appear to respond to much the same stimuli.13 According

12

Linear blood velocity is determined mainly by the biomechanical features of vessel walls (Fung and Liu 1992). Because veins are thin-walled they are more distensible than arteries at low transmural pressure, but the breaking pressure for some veins is surprisingly high; Archie and Green (1990) estimated it at 2,873 mm Hg (about 400 kPa, or 3.8 atm). The pressure–volume characteristics of the wall depend on the smooth muscle tone and – especially important over the physiological pressure range – the collagen and elastin contents. The wall behaves viscoelastically: haemodynamic changes induce either dilatation or constriction, depending on the smooth muscle tone (Monos et al. 1995). 13 Veins have a less abundant autonomic nerve supply but they are responsive to neurochemicals. In arterioles, acetylcholine (ACh) and perhaps other neurotransmitters, and bradykinin, promote the release of endothelium-derived hyperpolarisation factors (EDHFs) (He 2002), one of which has been identified as an epoxy-eicosotrienoic acid (Lundblad et al. 2005). EDHFs either activate the sodium–potassium pump or open potassium channels, so they act synergistically with NO and

12.9 The Endothelium and Vasomotor Tone

217

to Bevan and Joyce (1992, 1993) the main flow sensors are glycosaminoglycans that bind sodium and calcium ions, located not only on the plasma membranes of the endothelium, but also on the smooth muscle and in the ECM of the intima and media. Mechanical forces are also distributed to neighbouring cells and the ECM via the actin cytoskeleton. Since the aspect of blood flow that is pertinent to DVT is pulsatility or nonpulsatility and the concomitant perfusion or non-perfusion of the VVP, these aspects of flow rate in veins are of limited significance. In any case, valves produce less NO and are less responsive to calcium than other parts of the vein wall. Incipient necrosis of the parietalis EC will cause the release of intracellular ATP and potassium, potentially dilating the vein and decreasing the blood velocity,14 but in the light of the VCHH these factors are of minor significance. They may become relevant in a vein that is largely occluded by a mature thrombus, which markedly alters local blood flow characteristics. Also, the increased incidence of post-operative and spontaneous DVT and pulmonary embolism with age (Morrell and Dunnill 1968; Kakkar et al. 1970; Sigel et al. 1974) may be related (e.g.) to decreased vasopressin output, which presumably leads to a concomitant decrease in thromboxane production and thus in the contractile response of (e.g.) the saphenous vein. In addition, Schina et al. (1993) showed that the incidence of DVT correlates with the venous filling and ejection fraction and increases significantly with age-related changes in these parameters; but this could be interpreted as an age-related decrease in pulsatility of flow (Chapter 9). Effects on vessel wall perfusion via the vasa venarum are another matter. The mechanisms summarised in Table 12.1 could plausibly account for continuing efficient oxygenation of the intima in the event of luminal hypoxaemia. Some responses of EC to thrombin (see above) may be relevant in this way: e.g. NO synthesis is inhibited and endothelin is up-regulated (Kourembanas et al. 1991; Eto et al. 2001; Tobu et al. 2004), perhaps affecting the tone of the vasa venarum as well as enhancing platelet activation (Table 12.1). PPAR-γ, mentioned earlier as an inhibitor of Erg-1 induction, appears to counter thrombin-induced endothelin production (Delerive et al. 1999).

CO on vessel wall dynamics (Beny and Burnet 1988; see below). Acetylcholine relaxes arterioles but causes endothelium-dependent contraction of human veins, possibly because arterial smooth muscle is more directly or markedly affected, or perhaps because venous smooth muscle is less sensitive to endothelium-derived relaxation factor (EDRF) (Rubanyi and Vanhoutte 1988). Angiotensin II, ATP, bradykinin, histamine, serotonin and thrombin also induce EDRFmediated relaxation. Neuropeptide Y is another potent contractile agent found inter alia in the mesenteric, saphenous and uterine veins as well as arterioles (Fried and Samuelson 1991; Luu et al. 1992). NPY responds to nerve stimulation and is released from the sympathetic fibres along with noradrenalin, causing vasoconstriction; it also exerts a negative feedback on the synapse (Daly et al. 1988). 14 This presumably would not apply in the event of apoptosis in the endothelium, which seems to occur in cases of venous hypertension (Takase et al. 2004).

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12.10

12 Molecular Changes in the Hypoxic Endothelium

A Further Comment on ‘Risk Factors’

Cancers: Earlier, we reviewed evidence that many cancers lead through the Egr-1/ cytokine mechanism to increased circulating levels of TF. Egr-1 can also lead to angiogenesis and neointimal hyperplasia during tissue repair. C-reactive protein activates macrophages, enhancing the destruction of injured endothelium. VEGF enhances the regeneration of the parietalis endothelium, thus protecting against DVT. It may therefore be predicted that VEGF inhibitors used in cancer therapy increase the risk of venous thrombosis (Gupta and Zhang 2005). Septicaemia: Bacterial endotoxin causes slow loss of thrombomodulin and protein C receptor from the EC surfaces; it also promotes neutrophil binding to vein EC and enhances the production of E-selectin. For these reasons, bacterial infections – in particular those associated with septicaemia – may increase the likelihood of DVT. Septicaemia can be a sequel of inflammatory bowel disease, and this condition is sometimes noted as a distinct ‘risk factor’. Hormones: There is a very substantial literature on the relationships between hormone levels and thrombosis. The vast majority of this literature pertains to sex hormones used e.g. for oral contraception or hormone replacement therapy, and this matter was discussed briefly in Chapter 3. However, perturbed thyroid hormone levels also appear to increase the likelihood of DVT (as well as atherosclerosis); the mechanisms remain elusive (Squizzato et al. 2005), but protein C levels may be abnormally low (Nagumo et al. 2007). According to an intriguing recent paper, leptin increases TF production by peripheral monocytes, suggesting a possible reason why obesity is a ‘risk factor’ for DVT (Napoleone et al. 2007).

12.11

The Unification of Approaches

The molecular and cell biological literature reviewed in this chapter belongs to the mechanistic tradition, so most authors in the field have related their findings to the consensus model of DVT. In general, they have been concerned to show that certain insults to the vascular endothelium – including (particularly) hypoxia – tend to promote coagulation/fibrinogenesis or, alternatively, to down-regulate anticoagulatory factors such as thrombomodulin, or to impair fibrinolysis. Much of the motivation for this research has come from the need to elucidate the mechanisms of IRI and ameliorate its consequences for the patient; but many of the findings apply, explicitly or implicitly, to DVT as well. The importance and validity of this work are beyond question. But it is vital to remember that all these studies address mechanisms, not causes. Causes are addressed by the Virchowian, pathophysiological or vital-materialist approach that we adopted in establishing the VCHH (Chapters 8–11). The studies summarised in Chapter 11 are entirely different in character from those reviewed in Chapter 12, but they are complementary. Two points are clear. First, the VCHH as presented in Chapter 11 provides a context in which to place the mechanistic studies of EC hypoxia, so that both the primary cause and the

Fig. 12.5 The aetiology of DVT – summary of the VCHH. The flow diagram outlines a plausible aetiological scheme. When the parietalis endothelium of a valve cusp becomes hypoxic under conditions of sustained non-pulsatile flow, the elk-1/egr-1 pathway is initiated and the EC phenotype is changed. As a result, vasodilators are produced, perhaps increasing blood flow through the vasa venarum and helping to sustain the oxygenation of the mural endothelium. Thrombin is activated, enhancing the egr-1 pathway via PAR-1, and anti-coagulation factors such as thrombomodulin are inhibited. Also, cytokines are secreted that recruit leukocytes and platelets to the injured site when the hypoxaemic VVP is replenished with fresh blood by a brief episode of normal pulsatile flow. Continuation of the streamline flow leads to death/necrosis of the affected parietalis, and a (brief) restoration of pulsatility leads to the phagocytic removal of the dead cells. Signals such as VEGF (not shown in the diagram) may then help to stimulate endothelial regeneration and normal valve structure and function may be restored. In contrast, serial repetition of the non-pulsatile/pulsatile flow sequence leads to the deposition of multiple layers of live/dead blood cells on the injured valve cusp, interspersed with fibrin deposited in consequence of the locally activated coagulation cascade. Ultimately, this may lead to DVT; though such a pathological development may be prevented by full restoration of pulsatility, which could allow the fresh oxygenated phagocytes to remove the incipient thrombus. In that event, valve function may be impaired permanently and chronic venous disease may result

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mechanism of venous thrombosis can be understood. Neither approach alone is sufficient: to understand the aetiology of DVT or any other condition, both the pathophysiological cause and the molecular mechanisms must be comprehended together. Second, if biomedical scientists studying the potentially thrombogenic responses of EC interpret their work in relation to the consensus model, itself a product of the mechanistic approach, no such understanding can be achieved. Figure 12.5 is an outline summary of the ‘integrated’ picture of the aetiology of DVT that we have sought to establish. This scheme can no doubt be modified and elaborated, but its wider implication is clear: the biomedical schism that has persisted since the late 1840s can be bridged. The pathophysiological explanation of ‘why’ must logically precede, and provide a context for, the mechanistic account of ‘how’. We strongly believe that this general strategic principle can and should be applied to all areas of biomedical research.

Chapter 13

Cadaver Clots or Agonal Thrombi?

Abstract Post-mortem blood may be either (a) semi-solidified, resembling clots or thrombi or their combinations, or entirely ‘white’ thrombi; or (b), in occasional cases, wholly liquid and incoagulable. Because such variations have confused observers, we will suggest an explanation for them in terms of the valve cusp hypoxia hypothesis (VCHH). The central question is whether it can rightly be imagined that blood coagulates after death. This was intensely debated in the early 20th century but was then resolved ad hoc. To attempt a resolution, the forensic and judicial implications of our account of the states of post-mortem blood will be explored and some clinical inferences drawn. This discussion will highlight the essential value of an a priori account of DVT aetiology.

Keywords Agonal, cadaver, forensic, post-mortem

13.1

Can Blood Coagulate in a Cadaver?

Scientific/medical hypotheses are valuable only in so far as they lead to (a) new directions in therapy, prophylaxis or research and (b) solutions to persistent puzzles. We have already shown that the VCHH has potential applications in prophylaxis against DVT and thromboembolism, and we have outlined some of the novel lines of research to which it could lead. In this final chapter we aim to cast new light on a topic long characterised by confusion and uncertainty: the state of the blood in cadavers. It is well known that post-mortem blood may be largely coagulated, appearing clot-like, or may consist mainly of thrombus-like formations. In some cases it is almost entirely liquid. Morgagni (1769) observed that the blood became and remained incoagulable after sudden deaths, and the observation – corroborated by Hunter – has never been contradicted. What causes this variability? The question can have judicial as well as scientific implications, and debates about it have a long and tangled history.

P. C. Malone and P. S. Agutter, The Aetiology of Deep Venous Thrombosis. © 2008 Springer Science + Business Media B.V.

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The explanations offered in standard textbooks betray this uncertainty. Robbins (1962) differentiates cadaver thrombi from ‘post-mortem clots’ on the grounds that the former are ‘generally somewhat firm and friable’, show the typical ‘layering or lines of tangled fibrin’ and ‘have attachment to the underlying wall’. In contrast, the latter usually take the form of ‘a rubbery, gelatinous coagulum’, almost always ‘form a perfect cast of the vessel in which they arise’ and ‘have no attachment to their site of origin’. Robbins comments that ‘post-mortem clots’ ‘have a supernatant portion of coagulated clear plasma ‘chicken fat’ overlying a portion of darker hue where the red cells have settled’ [our emphasis]. Later, he claims that the blood separates in this way because ‘sufficient time often elapses after death for sedimentation of red cells to occur so that subsequent coagulation of the blood results in a plasma clot of the supernatant fluid, continuous with a mass of clot containing red blood cells’. He conjectures that the state of post-mortem blood is highly variable because ‘in many patients, activation of the fibrinolytic system prior to or immediately after death prevents postmortem clotting, while, in others, that does not occur and postmortem clots are abundant’ [our emphases]. This explanation seems odd: we know of no supporting evidence for it, and it is hard to see how any system can become ‘activated’ after death. Also, Robbins does not tell us why this alleged activation of fibrinolysis occurs so remarkably in some but not all cases. Perhaps significantly, this publication more or less coincided with the full articulation of the consensus model of DVT. Similarly, Walton, in Pathological Basis of Medicine (Curran and Harden 1972), gives an entirely reasonable account of thrombosis and embolisation but goes on to imply that blood coagulation after death is the norm. Neither of these authors seems to have been aware of the apparent contradiction between the notion of ‘general post-mortem coagulation’ and the findings of Hewson (1771), Lister (1863) and Baumgarten (1876), to which we have referred several times during the course of this book. Nor does either of them offer a persuasive explanation for the variable state of post-mortem blood.

13.2

An Early 20th-Century Debate

These standard textbooks seem to reiterate one side of a dispute that occurred early in the 20th century. Rost (1912, 1913), Ribbert (1916a, b), Marchand (1916) and Aschoff (1916) disagreed about the genesis, nature and pathological histology of the extensive coagula often found in the blood vessels of cadavers. Aschoff and Marchand favoured the now-orthodox viewpoint, whereas Rost and Ribbert took a now-heterodox line. Aschoff (1916) wrote: ‘For many years I have laid the greatest value on the demonstration of cadaver coagula in the body for judicial reasons and have emphasised the conclusions which can be drawn from them regarding the position of the cadaver … these cases can be explained by the laws of layers, which hold in the cavities of the cadavers as in a beaker’ [our emphases]. His view was that the

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position in which a body lies immediately after death (prone, supine, on a side, etc.) determines or significantly influences the position/stratification of the buffy coat, ‘cruor’, ‘hard thrombi’, liquid blood, yellow clots, white clots, long streamers and other coagulation formats found at post-mortem. In contrast, Ribbert (1916a), following Rost (1912, 1913), wrote: I begin with the macroscopic findings. … Aside from the relatively few cases in which the blood is fluid throughout, we find in the heart sometimes predominantly white coagula, sometimes chiefly cruor, and every variation in the relative quantities of the two. If we assume that the formation of solid masses occurs only after death, these marked differences are difficult to understand. For, even though the coagulability of the blood may vary somewhat, this cannot go so far that in some cases almost nothing but white clots, in others only cruor, is deposited, while, should the process be intravital, one need only assume an earlier beginning of the settling out when there are more abundant white coagula. … If the blood merely clotted’ [in situ, post-mortem] ‘in the heart, so much buffy coat could not possibly come into being.

Ribbert wrote this passage when research into the coagulation mechanism had scarcely progressed beyond the ‘classical hypothesis’ of Schmidt and Morawitz (Chapter 5). His thesis focused on Virchow’s 1856 observation and the triple inference to which we have referred in previous chapters: the fundamental difference between a thrombus and a mass of motionless blood clotted ex vivo is that the leukocytes in a clot can only have come from that volume of blood, whereas there are many millions in a thrombus, hence the pale or white streaks (lines of Zahn) visible macroscopically. As we have seen (Chapter 6), Virchow (1856) envisaged – explicitly – that this grossly ‘excessive’ leukocyte content can be explained only by presuming that those white cells were sequestered from a much greater volume of blood; in other words, that thrombi can form and develop only from and in circulating blood. Hence, thrombosis cannot occur after death, in the body or otherwise, because circulation has ceased. Thrombi cannot form in a cadaver. Ribbert (1916a) developed his theme as follows: In this way we can also understand the presence of fluid blood along with the solid masses: were post-mortem coagulation equivalent to what happens in a bleeding cup, the entire blood vascular content would become uniformly solid. … The white clot in the ventricles often continues into the pulmonary artery and the aorta, and, by pulling on it, one can ascertain that ever narrowing continuations go on into the ‘arterial’ branches. … It is ordinarily presumed that this is a manifestation of post-mortem coagulation: but, how could that be possible?… This finding is not compatible with postmortem coagulation; on the contrary, it suggests … that the formation of a white coagulum progresses farther and farther from the heart out into the arteries because the still flowing blood continues to add new material to the end of the thread in a longitudinal direction ….

He next described a cylindrical pulmonary artery thrombus, which he said ‘consisted almost entirely of leukocytes’, and remarked that ‘in some heart and vessel thrombi one finds so many leukocytes as to suggest they could represent the whole mass of circulating colourless corpuscles gathered together – leaving none circulating in the remaining blood’. This underscored the sequestration of circulating leukocytes in thrombotic masses and led him to the inevitable conclusion: such masses form in still-living, still-circulating blood, not in dead, stationary blood.

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It is tempting to wonder why white cells congregate in the circulating blood and swarm together in extremis – as though attempting, albeit failing, to repair or prevent the fatal outcome. Tellingly, Ribbert observed: ‘Especially characteristic of other locations … is the valve regions of femoral veins (and very likely other veins also, but I have carefully examined only this one)’. He went on to describe the thread-like, buffy-coat-like, deposit that originates in valves/lateral protuberances (and clearly in a heart valve pocket) and passes into cruor, similar to the masses found in the cardiac chambers. ‘From all these observations … it appears with great certainty that so-called cadaver coagula were in reality formed agonally, before death. … The agonal process therefore takes a kind of intermediate position between “in vitro clotting” and thrombosis, but has more of the peculiarities of the latter. We may speak of agonal thrombosis because it is a phenomenon that accompanies dying’ [our emphases]. Marchand and Aschoff, both of whom held positions of authority, dismissed Rost’s and Ribbert’s contentions. They sought to establish ad hoc that the gravitational settling of blood was the sole explanation for ‘layered’ post-mortem masses. The blood of cadavers, they implied, ‘settles’ as it does in the Erythrocyte Sedimentation Rate test. Their main intention seems to have been to support the judicial and forensic presumptions of pathologists. It is entirely tenable that any blood that stays fluid after death will indeed ‘settle’ in accordance with Aschoff’s ‘law of layers’. Ribbert, however, focused on a different question: why is liquid blood relatively rare at autopsy? We shall return to this question shortly.

13.3

Aschoff on ‘Post-Mortem Clots’

In this context, it is remarkable to see how Aschoff (1924) mixed the ‘mechanistic’ and ‘pathophysiological’ approaches without attempting to amalgamate or reconcile them. As we observed in Chapter 7, the conflict between these approaches led to inconsistencies. On the one hand, he agreed that thrombi do not form in stationary blood. He wrote (p. 258): ‘We have concluded, then, that the thrombus arising from the blood platelets has its origin in the circulating blood stream’, and went on to discuss the work of Eberth and Schimmelbusch (who visualised platelets marginating on vessel endothelium to create ‘bungs’ in bleeding vessels and to form the white Kopfteil followed by the red Schwantzteil; see Chapter 7). However, ‘the microscopic structure of the Schwanzteil of a thrombus … essentially resembles a post-mortem clot, and consists of an irregularly arranged mass of red and white corpuscles, blood platelets and fibrin’. Given this (indubitable) resemblance, how is an alleged ‘post-mortem clot’ to be distinguished from the Schwanzteil of an ante-mortem thrombus? Aschoff went on: ‘It must be admitted that here the leukocytes and blood platelets have a tendency to form themselves into masses, but there is no attempt at the formation of lamellae. The fibrin frequently shows striped thickenings running in

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definite directions. These may be the result of currents in the plasma, for, although the column of blood has been checked, it is still connected in manometer-like fashion with the rest of the vascular system …’ That makes the difficulty obvious. If a clot formed post-mortem, in no-longer-circulating blood, its histological appearance would have to involve mysterious ‘currents in the plasma’ and ‘manometer-like connections’. But what pressure differences would a manometer detect in stationary blood in a cadaver, and what could cause such ‘currents’? Not surprisingly, Aschoff was puzzled: ‘Why is a white thrombus built up from platelets in flowing streams, and why does red thrombus form when the blood flow has ceased?’ This might have led him to question his unspoken premise, but it did not. ‘… In considering the vital question of why a white thrombus occurs in the circulating blood, and an integral red thrombus-portion only when the stream is stationary … [the solution might be that] a red thrombus arises only when the lumen of a vessel is sufficiently closed by a white thrombus to bring the blood stream to a standstill in the peripheral portion of a vein. This stationary column of blood coagulates. But under the microscope this red thrombus, dense in the region of the Kopfteil, becomes more and more spongy as it extends, and is finally almost fluid in consistency, little different from post-mortem clotting’ [our emphasis]. ‘Sufficiently closed’ is an odd phrase; during life, surely, movement would come to a standstill only in a completely closed vessel. Aschoff then proceeded (pp. 268–270) to address the contradiction between his claims and the findings of Hewson, Lister and Baumgarten (though he mentioned only the last of these): [B]lood in a double-tied vessel does not clot – it stays liquid, but a “slowing” (eventually occluding) white thrombus does produce a clot cephalad to the initiating thrombus which then becomes an integral part of the whole thrombus. Why then does blood clot in dead vessels? … We know, as Baumgarten has stated, that blood remains fluid in a doubly ligated segment because the blood dies relatively slowly. … But it is quite evident that the two phenomena, agglutination of platelets/leukocytes and fibrin formation, do not signify the same thing and must be kept generically apart, since each can occur without the other. In the case of ordinary autochthonous thrombosis with which we have been dealing, the deposition and coagulation processes are closely related. The process of deposition brings about coagulation and is, so to speak, the indirect source of it. Then the question arises as to whether coagulation thrombosis can occur apart from the process of deposition, and what may be the indirect causes influencing them both. It is important in this connection to remember that whereas thrombosis by deposition can be brought about readily at any time, a primary coagulation thrombus is very difficult to induce. Stoppage of the blood stream alone does not suffice, as Baumgarten’s experiments have shown, for the ferment does not develop quickly enough [Our emphases].

The idea that fibrin can be formed in situ without the activation and congregation of platelets has been disproved (Chapter 2), and this alone undermines Aschoff’s reasoning. More fundamentally, Aschoff displays the philosophical inconsistency we mentioned earlier. Elsewhere in his 1924 Lectures, his orientation is emphatically Virchowian, but in this passage he espouses the mechanistic Schmidt-Morawitz thrombin hypothesis as then conceived (Chapter 5). The idea that persistently fluid blood ‘[does] not develop a “ferment” quickly enough’, implying that non-coagulation signifies some kind of chemical deficiency, may show how a working morbid

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pathologist (as distinct from a haematologist) felt obliged to express himself in the early 20th century.1 Remarkably, the autopsy studies on First World War victims (Aschoff 1922), discussed in Chapter 10, refuted this convoluted argument in favour of ‘post-mortem coagulation’.

13.4

The Debate Reconsidered in the Light of the VCHH

The VCHH predicts that neither clots, coagula nor thrombi can form in corpses after death. Every such structure found in blood vessels after death is a thrombus, either old or agonal, but not formed post-mortem. In short, we agree with Rost and Ribbert as opposed to Aschoff, Marchand and modern orthodoxy. But the question remains: why is the blood of some cadavers littered with coagula while others show a complete dearth? We must not lose sight of the obvious fact that ‘normal’, fresh blood shed into a receptacle coagulates rapidly en masse.

13.4.1

Death from Acute Respiratory Failure

According to the VCHH, nascent thrombi form when living leukocytes and platelets congregate on the dead (or severely hypoxically injured) parietalis endothelium of a valve cusp leaflet. Therefore, if death is the result of circumstances (acute respiratory failure) that kill the blood cells at least as quickly as the vascular endothelium, coagulation cannot occur and, in particular, thrombi cannot form. The blood will remain liquid. In persons who have drowned or have been executed by hanging or garrotting (i.e. died in acute respiratory failure), the whole blood volume is found at postmortem to have become ‘incoagulable’, in accordance with the statement by Morgagni (1769). Over the centuries, there have been pointers to the explanation. In particular, Virchow and Lister independently reasoned that oxygen (or carbon dioxide) somehow influences blood coagulation, though their explanations were unconvincing to their contemporaries.2 We can now explain it simply by noting that this mode of death causes immediate and gross arterial hypoxaemia. This kills the blood cells at the same time as the vascular endothelium, so no thrombi can form and (in the absence of living platelets) the blood cannot coagulate. 1 On the other hand, Aschoff (1924) may have had tongue in cheek, fighting a rearguard action on behalf of clinical pathologists. On p. 254 he remarks: ‘… in human beings the occurrence of fibrin coagulation is not the first stage of thrombosis … important changes in the morphological blood constituents precede it. These latter changes must be explained before the mechanism of thrombosis can be understood’. Nevertheless, this great pathologist failed to infer that ‘post-mortem clot’ is actually formed ante mortem, ín vivo, as Rost and Ribbert had reasoned. 2 Virchow correctly deduced that oxygen facilitates blood coagulation, though his account was garbled, whereas Lister wrongly interpreted ‘asphyxiated rabbit blood’ as representing the effects of an excess of CO2. See Chapter 6.

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Aschoff’s autopsy studies (Aschoff 1922) retrospectively corroborated this. The gassed soldiers presumably died asphyxiated, ‘arterial hypoxic’ deaths in acute progressive respiratory failure (Chapter 10). There was no sign whatever of coagulum, buffy coat or cruor in the heart or blood vessels of these soldiers, and consequently no stratification was observed anywhere in their vessels. This accords precisely with the evidence interpreted by Malone and Morris (1978) as proof that hypoxaemic blood loses the power to coagulate.

13.4.2

Death from Circulatory Failure

As Ribbert (1916a) pointed out, it is fairly rare for the blood to remain liquid postmortem. When the body dies from circulatory failure, both the blood cells and the vessel walls continue to consume the residual oxygen in that blood. This progressive de-oxygenation gradually ‘suffocates’ both blood and tissues, so that, over the course of (say) 3–4 h, gross hypoxaemia ensues. The white cells of normally oxygenated blood would instigate the repair or removal of dead or dying vascular endothelium, but we may not expect suffocated blood cells to perform this (or any other) service. The consequences of death by circulatory failure will depend on whether it is gradual, allowing (often massive) thrombi to form in the heart and major arteries, or sudden. If it is sudden, the vascular endothelium gradually dies, but only the leukocytes and platelets in its immediate vicinity will be available to congregate upon it; the circulation has ceased, so there is no further supply of ‘fresh’, stillliving, blood cells. The extent of such coagulation will depend on the numbers of leukocytes/platelets in the immediate neighbourhood that remain viable when the endothelium has died. Any coagulum that forms will not have the layered structure of a thrombus; it will be clot-like. However, if circulatory failure is gradual, the changes in endothelial cell phenotype resulting from hypoxia will become generalised throughout the vascular system: the deterioration of cardiac output will mean that the mural endothelium cannot be adequately oxygenated via the vasa venarum. In other words, the mechanisms discussed in Chapter 12 will no longer be confined to the venous valve cusp parietalis; potentially, they could take effect in all vascular endothelia. Still-viable blood cells throughout the circulation will continue to circulate over the dead or dying endothelia, and leukocytes will congregate. This will apply particularly to the right heart and pulmonary arteries, which are relatively hypoxaemic during life and would be expected to suffer hypoxic endothelial death more quickly than the rest vascular system. In these or similar circumstances, one might expect sequestration of perhaps all the white cells in the body, as Ribbert (1916a) suggested. As the coagula become more extensive, circulation is further impaired, exacerbating the endothelial hypoxia. The ‘thrombotic’ process therefore accelerates quasi-exponentially in the final vicious cycle of death. According to a recent paper by Porat et al. (2004), heart valve cusp endothelium shows a pro-coagulatory response to hypoxia. The mechanism underpinning the

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extensive agonal semi-solidification in the heart and major blood vessels described by Ribbert (1916a) may therefore be exactly analogous to that involved in venous valve pockets during the genesis of DVT; though the crucial receptor in the heart valves seems to be the receptor tyrosine kinase tie-1 (Porat et al. 2004), which has not been implicated in the venous endothelial response (Chapter 12). The particular tendency of agonal white thrombi to develop on the heart valves is consistent with these findings.

13.4.3

Summary: The Condition of the Blood Post-Mortem Depends on the Mode of Death

In essence, therefore, deaths fall into two main groups: primarily acute respiratory failure (acute hypoxaemic death), and primarily circulatory failure (acute underperfusion). The latter fall into two subgroups: slow/chronic gradual deaths versus sudden cardiac asystole. We suggest that this explains why pathologists find, respectively, (1) incoagulable blood, (2) ‘obvious’ ante-mortem thrombus, and (3) ‘obvious post-mortem clot’. Of course, there will be ‘intermediate’ cases. Combinations must occur in which either the respiratory failure is combined with sufficiently protracted circulatory failure, or circulatory failure is too sudden (as in catastrophic haemorrhages or exsanguinations) to allow time for coagulation to occur. If Aschoff (1922) had been able to collect more complete details of the mode of death in the last 11 cases of his series (see Chapter 10), we could have estimated whether there was an ‘agonal’ period in the hours or days before death and post-mortem examination. Aschoff’s key misconception seems to have been an unspoken presumption, contrary to what he actually stated, that a Schwanzteil thrombus forms in stationary blood. We maintain, on the contrary, that the Schwanzteil forms in moving – albeit slowly moving – blood. Then, of course, it fills the lumen with obstructive thrombus and reduces the volume flow to a negligible or even ‘stasis’ level. All coagulation, and all thrombosis, within blood vessels ceases after death and the ensuing progressive hypoxaemia. It is therefore a fiction to claim that blood can actively semi-solidify after death.

13.5

Consequences of Positing that All Thrombi Are Agonal

Forensic opinions are currently predicated on the disposition of the light and dark components in coagula/thrombi found at post-mortem. But as we have seen, all post-mortem intravascular coagula are formed ante-mortem (some old, some recent). Only with an unequivocal concept of how and when these lesions form may correct legal inferences be drawn and scientific forensic evidence prepared. If 60% of death-throes are accompanied by thrombosis, as our arguments suggest, then this thrombosis may have been the final instrument of death in that 60%,

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or to have contributed progressively thereto. This possibility has latent therapeutic implications; it is clear that thrombosis is potentially exponential, a fatal vicious cycle. But it behoves all pathologists not to mistake a (contributory) cause of death for a post-mortem phenomenon.

13.5.1

Possible Post-Mortem Changes

If a substantial part of the cadaver blood remains fluid and the post-mortem is performed within a day of death, there may conceivably be just enough residual oxygen within the vascular system to keep the blood cells ‘alive’ – helped, perhaps, by anaerobic metabolism. Blood re-exposed to atmospheric oxygen (say through an incision) before it is definitively dead may become ‘re-aerated’ and thus retain coagulability. Also, blood in superficial venous vessels may acquire some oxygen by diffusion through the skin of the dead body. Such blood would become progressively hypoxaemic and ‘sink’ to the lowest extent of the recumbent body within the dead blood vessels, displaying an ever weaker clotting response when released into a non-siliconed receptacle. If both aerobic and anaerobic metabolisms have ceased, however, coagulability cannot be revived.3 As Baumgarten (1876) expressed it: the blood, having ‘died slowly’, can no longer coagulate. Such a sequence can also be appreciated from Lister’s (1863) experiments. The shed blood of an asphyxiated rabbit was, he wrote, at first ‘dark’ and did not immediately coagulate; but on exposure to the atmosphere, the colour lightened and it began to coagulate ‘weakly’. Lister speculated that ‘CO2 was lost to the atmosphere’ – which of course it was, in parallel with re-oxygenation; but he did not make that connection. It seems logical to conclude that the oxygen in the blood of recently-deceased bodies continues to be consumed, and the blood becomes progressively hypoxaemic and thus dies gradually.

13.5.2

Summing up the Argument: Judicial Implications

1. (a) All coagula found in cadavers are either the Kopfteil or the Schwanzteil of agonal ante-mortem thrombi, as Ribbert contended. (b) There is no such entity as post-mortem thrombus, coagulum or clot. 2. Since thrombosis is a living, physiological (albeit pathophysiological) response of living blood to dying or dead vascular container/envelope, thrombi cannot form once the circulation stops, and liquid blood stopped in dead vessels will not clot therein. Nor will it coagulate if it is shed after being confined too long in dead vessels.

3 This also explains the transient restoration of heartbeat by instillation of oxygen into the two dead soldiers examined by Aschoff (1922); see Chapter 10.

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3. Consequently, the absence of thrombosis/coagulum formation from cadaver blood (i.e. the presence of exclusively fluid blood in post-mortem vessels) is positive and irrefutable proof that the subject died suddenly and acutely, from drowning, shooting, electrocution, head injury, execution by hanging/strangulation – and quite definitely not in circulatory failure. In effect, liquid blood at post-mortem is highly suggestive of death from unnatural causes. (It is not, however, proof of same without other definitive features or circumstances.) The blood that flows from such a vessel may: (a) Clot after it has been shed, because it is still alive; such blood is still ‘residually’ oxygenated for a period after the circulation has stopped. Both its PO2 and coagulation rate could give forensic information, such as the time elapsed since a sudden death; (b) Fail to clot after it has been shed. If this be soon after an observed death, it could suggest asphyxial blood death due to prolonged suffocation/respiratory failure; otherwise, it indicates a long interval between death and legal autopsy. Hypoxaemic/dead blood will not react either to a simultaneously dying vessel wall, or, if shed, to the surface of a receptacle. This suggests that the rate (i.e. residual efficiency) of coagulation of blood taken from a corpse, especially if death may have been recent, could give the time since death, independently of other indicators. 4. Aschoff’s belief that the stratification of blood in cadavers is judicially/medicolegally significant may be valid in limited circumstances, but not in any case where the blood is extensively thrombosed. Clearly, in sudden deaths from asphyxia, the blood will remain liquid and the erythrocytes will sediment as he said. This would be valuable for determining the precise position of a body after death, but such sedimentation of dying blood cells would have no relevance to the position of white coagula with abundant leukocytes/fibrin in the heart chambers and major vessels. In this respect, Ribbert and Rost on the one hand, and Aschoff and Marchand on the other, were arguing at cross-purposes. One party was describing the state of blood in slow deaths from progressive circulatory failure following debilitating or other illnesses; the other was (probably) correctly describing the medico-legal assessment of violent deaths in war or homicide in peacetime. The position of a body after death in a hospital bed or operating theatre has scant medico-legal significance.

13.6

Therapeutic and Prophylactic Implications

Ribbert would seem to have been anxious to develop the idea that dying may (often or always) be due directly to the thrombotic process extending throughout the vessels, obstructing the vascular channels and so further impairing blood flow therein. In this vicious cycle, more and more coagulation would obstruct the circulation of the remaining blood, thus accelerating the thrombotic process and

13.6 Therapeutic and Prophylactic Implications

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‘exponentially’ reducing the effective circulating blood volume. This possibility implies a prophylactic/therapeutic consideration in the management of terminal patients. Anticoagulating such patients might be considered, should a clinical probability suggest that their salvage might be worth (their) while. This notion is consistent with the reduced mortality from heart failure after anticoagulation.4 Aschoff (1922) recorded that the left ventricle is sometimes empty while the right ventricle is full of white or red clot/cruor. Perhaps the right heart (on the hypoxaemic side of the circulation) blocks the main venous trunks, right heart and pulmonary arterial tree by marginated and sequestrated leukocytes in the lung microvasculature, which is the source of the oxygen. Could the macroscopically observed thrombotic process in the pulmonary arterial tree be mirrored microscopically? This possibility, that widespread intravascular (arterial and venous) thrombosis is a potent cause of death, is readily appreciable and could explain the high percentage of cadavers in which pathologists observe ‘terminal’ intravascular thrombi (possibly the actual fatal lesions). Each cubic centimetre of blood that becomes fixed and no longer circulating is, by definition, lost to the circulation. Litres of blood could therefore accumulate in the non-circulating coagulum or thrombus, and progressively obstruct an inefficient heart with a widespread, ‘exponentially’ increasing intravascular mass. Such progressive sequestration of blood from ‘circulating’ to ‘fixed’ is plausible. It is obvious that a pulmonary embolus stops circulation in the whole or part of the pulmonary vascular bed, but there seems to have been a strange blindness to the equivalent possibility that non-embolic thrombi – non-embolic only because the weakening heart (vis a tergo) cannot detach them from their moorings – are perhaps the final killers. As there is no such entity as a post-mortem thrombus or clot, it follows logically that all cardiac mural thrombi are ante-mortem, as are venous valve thrombi. Should the heart beat weaken, a situation analogous to calf muscle pump failure must develop. In the event of auricular fibrillation, flutter or irregularity, perfusion of saccules and crevices may be impaired for long periods before atrial function is resumed. By extension, the VCHH predicts that if a minor infarction leads to mural necrosis of an auricle, the scene is set for thrombosis within a cardiac valve pocket (cf. Porat et al. 2004). A cusp thrombus might form, related to a part of the cardiac wall or to a trapped vortex of blood that becomes progressively hypoxaemic. These considerations favour the logic of anticoagulating coronary thromboses and auricular fibrillation. The other side of that coin, of course, is that anticoagulation, which blocks fibrinogenesis/coagulation, does not simultaneously block the cumulative sequestration of colourless corpuscles – ‘solid white thrombus formation’. To prevent this problem, other drugs might provide useful supplementary therapy.

4 This effect of anticoagulation is usually attributed to the encouragement of the arterial stenosis to lyse. Maybe it does lyse, but the prevention of embolic or obstructive, hypovolaemic death due to massive sequestration of blood in vessels all over the body could play a bigger role in salvage.

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By this unsuspected and somewhat circuitous route, it therefore transpires that Cruveilhier’s much derided epithet – that ‘thrombosis (“phlébite”) is the basis of all pathology’ – was a historically great generalisation. Once it is realised that Cruveilhier’s ‘phlébite’ and Virchow’s ‘thrombosis’ were one and the same phenomenon (by different names), we cannot but conclude that the generalisation deserved eternal recognition and not the contumely that Cruveilhier’s xenophobic critics poured on it. We therefore end by affirming that although we no longer use his term ‘la phlébite’, Cruveilhier’s dictum was invalidly expunged from the history of pathology. It is therefore incumbent on the next generation to retrace the received highways and byways of history and to implement the full restoration of Cruveilier to his proper position as the founding father of the science of ‘thrombosis (phlébite)’ – the original exponent of the concept that local and/or general thrombotic obstruction/occlusion is the kernel of the history of pathology.

Appendix: Science, Medicine and Philosophy

A.1

The Two Approaches to Medical Biology

We have argued that it is desirable, and possible, to bring together the ‘mechanistic’ and ‘pathophysiological’ (‘vital-materialist’) approaches to medical biology. However, because our account of DVT-related research has been largely historical, we have emphasised their divergence during the course of this book. In particular, we have stressed (1) the schism that separated the two approaches during the 1840s and (2) the overwhelming dominance of the mechanistic approach during the last half century. In this appendix we shall attempt to explain these two historical observations. We shall also explore why vital-materialism (the pathophysiological approach) is sometimes confused with vitalism. The biomedical schism originated in 19th-century Germany and cannot be understood without reference to its cultural context. Nineteenth century German science produced work of great and lasting value, but these achievements involved intense debate and controversy, which were underpinned by rival philosophical commitments. To explain the schism we must outline the nature and origin of these commitments and indicate their significance for science and for German culture as a whole. We shall take up this theme in section A.2. The more recent dominance of the mechanistic perspective is another matter, partly independent of the schism and its origins. The gold standard in modern biological and medical research is to describe or (purportedly) explain biological phenomena in terms of molecular genetics and protein chemistry: this is clearly a mechanistic enterprise in that it seeks to reduce the phenomena of life to subcellular and molecular processes. It has led to salient advances in many areas of biology and medicine. Examples in thrombosis research include the understanding of haemostasis and its pathological perturbations (Chapters 2 and 3), and an expanding knowledge of vascular endothelial cell biology and the changes produced by hypoxic or other injury (Chapter 12). Such achievements have reinforced the hegemony of the mechanistic approach, but they do not explain why this dominance came about in the first place. We shall consider this matter in Section A.4. In academic science today, to question the hegemony of ‘mechanism’ is to court an accusation of heresy, or eccentricity. (This has always been the case with monolithic

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standpoints in religion, politics, science and other aspects of culture.) By implication, the physician/pathologist/surgeon’s knowledge and experience are ranked lower than those of molecular biologists.1 Yet molecular biologists do not first encounter, for example, DVT in clinical practice and need no conception of cause in order to manage and treat such patients. Practitioners undoubtedly benefit from molecular biological understanding; but the converse is also true, or should be. The relative ‘downgrading’ of the clinician’s standpoint in the eyes of academic researchers seems, at least in part, to reflect the confusion between vital-materialism and vitalism. In Section A.3 we shall examine this confusion and attempt to resolve it.

A.2

The Philosophical Background to the Schism of the 1840s

Before the country was unified under Bismarck, Germany comprised dozens of principalities subject to the Habsburg crown. Early in the century, liberation from Empire and the prospect of self-government had fostered an intense nationalism. The French, until then accepted almost without question as cultural leaders in Europe, became a natural first target. The 19th century was littered with bitter precedence wrangles between German and French scientists, which we have exemplified elsewhere in this book. Nineteenth century German science was animated by the conviction that it was knocking on the doors of Ultimate Truths. The struggle to raise it to world leadership inspired all the rival schools of thought. The often bitter arguments among scientists of the period betrayed an impulse to use controversy to advance (German) science rather than to ‘destroy’ the other side (Veit-Brause 2001). This also applied to Geisteswissenschaften, which included history, literature, archaeology, technology2 and – crucially – philosophy.

1

For practical clinicians, particularly surgeons, an extra viewpoint is necessary in practice. Their vital-materialist approach is concerned more with ‘causes’ than with molecular mechanisms. From this perspective, the main concerns are the properties that objects have, and the effects they exert, because they are ‘living’. In other words, vital-materialists treat living processes as explanens while mechanists treat them as explicandum. The two approaches are therefore complementary in principle: neither is ‘more fundamental than’ or ‘superior to’ the other. Neither is the ‘sole source of truth’. For instance, blood is blood, whether in a test tube or in the vascular conduits in vivo. The test tube is a more convenient container than the body for molecular-level analysis, so in vitro studies have provided most of our knowledge about the biochemical events involved in clotting and their failures in bleeding diatheses. On the other hand, blood does what it does physiologically because it is moved around the circulatory system (whereas in test tubes it is static). For the clinician, this is a primary consideration. 2 Technology, which was still indistinguishable from science per se, may have tilted the balance decisively in favour of ‘mechanism’ (Gregory 1977). Methodical, reductive, mechanistic science became generally perceived as the great benefactor as well as the great explainer. Later in the 19th century, Haeckel expressed the general opinion that science would solve all mankind’s problems during the following two generations: Enlightenment optimism in new nationalistic garb. Kant

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At cost of some over-simplification, we can consider the biomedical schism between du Bois Reymond and Virchow to have been underpinned by the conflict between the philosophies, respectively, of Kant and Schopenhauer (or, more generally, between Kantianism and Naturphilosophie). To substantiate this claim, we shall now outline the backgrounds to Kant’s philosophy and Naturphilosophie and indicate how these accounts of (scientific) knowledge attained particular relevance for biological and medical research in the 1840s Germany. Their origins lie in earlier centuries.

A.2.1

The Aftermath of the Scientific Revolution

The fundamental transformation of European thought often ascribed to the publications of Copernicus and Vesalius in 1543 had been adumbrated in the nascent theory of impetus developed in Merton College, Oxford, in the 14th century and in the contributions of Buridan, Oresme, Nicholas of Cusa and other late mediaeval scholars (Hall 1954; Claggett 1959; Boas 1962; Butterfield 1965b). It had far-reaching cultural consequences. The three most venerated prophets of the succeeding age, Francis Bacon3 (1561–1624), Galileo Galilei (1564–1642) and René Descartes (1596–1650), articulated new ways of thinking and variously sought to replace the Aristotelian account of the cosmos (especially its concept of ‘motion’) that had dominated the mediaeval worldview (see e.g. Drake 1957; Cottingham 1992; Gaukroger 2001). The ‘Age of Reason’ that followed Newton’s lifetime (1642–1727) was marked by a metamorphosis not only in the way in which the natural world was to be understood, but in all aspects of culture from politics to literary style (Berlin 1956). Inter alia, there were profound changes in epistemology and ontology, which were to lead to the philosophies of Kant and his opponents and thus, indirectly, to the schism in medicine and biology.

A.2.2

The Empiricist Tradition

One of Newton’s few close friends, John Locke (1632–1700), derived his philosophy of knowledge (empiricism) from Bacon’s precepts for establishing reliable truths about the natural world, and from Gassendi’s revival of classical Epicureanism

stood behind this trend as an immensely powerful instigator. By the later part of the century, the ramifications of his philosophy of science had become practically incontestable. The Kantian monolith in German thought became inseparable from the urge towards national pre-eminence in technology, science and other areas of intellectual life. It may be worth noting that Nazi opposition to relativity theory and quantum mechanics resulted largely from the incompatibility between the new physics and Kantian philosophy; the Nazi intelligentsia adhered to the latter and consequently rejected the former. 3 In his Preface to the Encyclopaedia, d’Alembert dedicated the work to Bacon’s memory, stating that the Chancellor did not teach us what we know, but what we still have to know.

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(specifically, the belief in atomism4). The founders of the Royal Society committed themselves unanimously to the Baconian method, and Gassendi’s atomism became fashionably accepted, particularly in England (Dear 1985). The empiricists believed that true knowledge comes only from and through the evidence of the senses; the mind at birth is a tabula rasa on which sensory experience writes, and this is the source of all our ideas (Mackie 1976). Consistent patterns in the observed world are established in the mind by association of ideas (Locke’s phrase). Lockean empiricism became a major influence on the Philosophes of 18th-century France. In many respects it became the spirit of the Enlightenment, inspiring a new view of the Nature and of the human condition; it underpinned political movements5 as well as academic inquiry. It also made the scope of ‘science’ very wide. For example, Descartes had held that while the human body was ‘a machine’, sensation and thought betokened the soul6; but Locke had maintained that sensation, thought and emotions were of the body. His followers in the 18th and early 19th centuries were prepared to treat sensation and emotion as matters of physiology (Berlin 1956). The ‘soul’ was marginalised.

A.2.3

Hume: The Achilles’ Heel of Empiricism

David Hume (1711–1776) lived through the Enlightenment era. He inherited the Lockean tradition, but – paradoxically - undermined the certainties of that classical empiricism (Russell 1946; Kemp Smith 2005). What he wrote remains disconcerting today, but few of his contemporaries grasped its implications. Classical empiricism focused on inductive reasoning: the path to knowledge was considered to depend on generalisation from repeated instances of the same ‘association of ideas’. (In contrast, Descartes and other rationalists emphasised deductive reasoning, the logical inference of specific conclusions from general truths based on a priori axioms.) Hume pointed out that no possible number of instances of the same observation could justify a claim that the world was always so and will always be so. The possibility of black swans is not excluded because every swan we see is white. 4

Gassendi, P. (1658) Syntagma Philosophicum Epicurus, The Hague. There is an important historical distinction between atomism and ‘corpuscularianism’. Locke and his followers, most members of the Royal Society – and Newton – accepted Gassendi’s atomism. Descartes and others, including Leibniz, did not; they offered ‘liquid vortex’ models of the structure of the universe. The debate persisted for a century until Dalton effectively settled it in favour of atomism. 5 The American Declaration of Independence, for example, quotes Locke’s words wholesale. 6 Famously, Descartes held that bodies could be explained ultimately in terms of mechanics (see Chapter 4) but he became a ‘dualist’. There may seem a contradiction between his ‘dualism’ and his ‘mechanism’, but his personal history perhaps explains it. Before the early 1630s, Descartes the natural philosopher had espoused the mechanistic account of the universe and given no hint of dualism. His dualism – apparent in, e.g. the Discourse on Method of 1637 – appears to have been a reaction to his shock at the trial and house arrest of Galileo, after which he developed a paranoid fear of reprisals from the Church and lived the rest of his life like a hunted man.

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This simple argument had drastic implications: if knowledge grows by inductive generalisation from observations, but if such induction can never produce certainty, then (supposedly) reliable knowledge cannot be certain. In an age that had come to accept Newton’s account of the universe as Eternal Truth, and believed that Baconian empiricism had accurately represented Newton’s methodology, Hume’s exposure of the Achilles heel of empiricism was a devastating blow to ‘natural philosophy’. The implications were not only academic. The Bacon-Locke programme was seen as the means by which Paradise on Earth could be brought about by human endeavour. It had also inspired the trend towards political democracy manifested in the American War of Independence and the French Revolution. Hume had consequently created fundamental difficulties not only for scientific epistemology but also for the social and political optimism of the Enlightenment (Kemp Smith 2005).

A.2.4

Kant

Immanuel Kant (1724–1804) presumed that Newtonian theory was the absolute truth, but he also accepted Hume’s strictures on the limits of the alleged inductive process. He acknowledged the evident paradox: how could this juxtaposition allow observation statements to stand as exact knowledge? His proposed solution was instrumental in shaping his philosophy of knowledge. He divided the world into phenomena (what our perceptions supply to our minds) and noumena (the objective universe, ‘things-in-themselves’). Of the latter we are and must remain absolutely incognisant. Our perceptions filter our sense impressions and our minds impose order on phenomena; but we can neither confirm nor refute the existence and nature of noumena, or of God or spirit (Kemp Smith 1962). That was Kant’s attempt to ‘delineate the reach of knowledge in order to make room for faith’. According to Kant, our knowledge is in part a priori and therefore not inferred inductively from experience. He wrote7: When Galileo let his balls run down an inclined plane to test a gravity he had chosen himself; when Torricelli caused air to support a weight he had chosen beforehand… a light dawned on natural philosophers. They learnt that our reason can only understand what it creates according to its own design, and that we must coax from Nature the answers to our questions rather than cling to her apron strings. […] Observations made without prior plan and hypothesis cannot be connected to […] what our reason is looking for.

Nowadays, ‘observation statements are theory-laden’ is a familiar aphorism and it is orthodox to accept that observations and experiments are pre-loaded with theory. However, Kant’s ‘residual observer’ was ‘apart from’ the universe; a detached mind not subject to the laws of mechanics. This facilitated the intuition that the observer is ‘absolutely at rest’, which further entrenched the Newtonian/Kantian notions of absolute space and time – the metaphysical underpinning of classical mechanics 7

From the Critique of Pure Reason, translated by Kemp Smith.

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that Mach challenged in the closing years of the 19th century and Einstein replaced in 1905. Kant’s work had ontological as well as epistemological implications.

A.2.5

Naturphilosophie and its Influence on Philosophy and Science

Shortly after Kant’s major works were published, the tradition of ‘German Idealism’ flourished. One of its exponents, Friedrich Wilhelm Joseph von Schelling (1775–1854), took issue with Kant’s failure to explain how a free, knowing, non-determined subject (the observer) can arise from a Nature that is wholly governed by deterministic laws. Schelling attempted to resolve this problem by asserting that Nature – including ourselves as observers – constitutes one complete and self-forming unity (Bowie 1993), with an innate organising principle that struggles towards self-consciousness. Schelling’s Naturphilosophie was attacked and even ridiculed by most of his successors, but it came to exert considerable influence on philosophy and, indirectly, on science. The great polymath Johann Wolfgang von Goethe (1749–1832) insisted on both the unity of Nature and the unity between knowledge and feeling. His mature thought stood in self-conscious opposition to Kant (Viëtor 1950). On the one hand, he broadly agreed with Kant in abandoning empiricist epistemology; but on the other, he utterly rejected Kant’s dichotomy between Nature and Reason. Goethe’s philosophical antecedents included Schelling,8 and his approach to science emphasised the unification of outwardly dissimilar phenomena. His scientific ideas proved sterile,9 yet they may have contained the seeds of great syntheses such as Maxwell’s mathematical unification of the studies of electricity, magnetism and optics, and the achievements of complexity theorists today. His belief in the underlying unity of Nature inspired his enthusiasm for the theory of transformation10 (later dubbed ‘evolution’), proposed during the first decade of the 19th century by Geoffroy St Hilaire (1772–1844) and Lamarck (1744–1829) (Appel 1887; Burkhardt 1995). His stance on the confrontation between Geoffroy and Cuvier was later recognised and approved by Darwin. This confrontation was a matter of consuming interest for Goethe, hinging on whether ‘the idea’ or ‘the method’ would

8 Another major influence was Caspar Friedrich Wolff (1733–1794), considered to be a founder of epigenetic descriptive embryology. Wolff tried to explain the emergence of organisms by the actions of a ‘vis essentialis’, an organizing, formative force, very much in line with Schelling’s ideas. 9 Though Naturphilosophie, and particularly Goethe’s work, did influence a number of 19th-century scientists: see, e.g. Tyndall (1897); Ostwald (1910). 10 In Lamarck particularly, we find the idea of an ‘upward striving’ among organisms that is held to explain transformation and the increase in complexity of form over time. This notion clearly echoes the inherent spirit or organising principle of Nature characteristic of Naturphilosophie.

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triumph. Perhaps, in the last year of his life, he was comforted by the conviction that ‘the idea’ had won. Goethe was more a contributor of ideas than actual scientific accomplishments. He imparted no methodological impulse to the sciences in which he worked, but his conceptual influence was considerable. The Naturphilosophie he embraced offered an alternative to Kantian mechanism, which was already in the ascendant. His teachings encouraged an emphasis on the distinctiveness of living organisms in respect of their principles of organisation and their ‘purposiveness’. Crucially, these teachings were a major influence on Johannes Müller (see below).

A.2.6

Schopenhauer

Arthur Schopenhauer (1788–1860) studied Plato and Kant In his youth, and he associated and exchanged ideas with Goethe. He supported Goethe’s theory of colours against Newton’s.11 Fundamental to Schopenhauer’s philosophy was the metaphysical doctrine of the Will, which was a development from the ideas of Naturphilosophie. The ‘Will’ (i.e. the sense of self) is manifest in the continual ‘upward striving’ in Nature implicit in the theories of Lamarck and Geoffroy St Hilaire.12 Goethe stood behind this body of thought as a kind of benevolent eminence (cf. Nyhart 1995).

A.2.7

Significance for 19th-Century Physiology and Pathology

Naturphilosophie became a very strong influence on ‘organicism’ in biology – the emphasis on the organised forms of living things. It inspired Johannes Muller to adopt an ‘organicist’ approach to physiology, a viewpoint that was famously passed

11 Goethe’s Farbenlehre (Theory of Colours), written between 1805 and 1810, self-consciously sought to replace Newton’s objective explanation of the spectrum as a ‘thing-in-itself’ with a subjective account (Eastlake 1970). Newton had seen colours as components of white separable by a prism; but Goethe was concerned with the role of the observer’s eye in creating sensations of colour. The almost-contemporaneous resuscitation of Huygens’s wave theory of light by Young and Fresnel, an explicit contradiction of Newton’s corpuscular theory, might have influenced the Farbenlehre. The basis of Goethe’s theory was the polarity of black and white. Examined through a prism or a mist, black appeared as blue, which could be intensified to purple; white as yellow, which could be intensified to orange-red. Combinations of these generated other colours. The theory formed the basis of the artist’s colour-wheel and the concepts of harmonious and complementary colours. Each colour was associated with a particular emotional state; for instance, orange-red betokened ‘warmth and gladness’. But once again this theory, though it influenced artists such as Turner and probably the Pre-Raphaelites, proved scientifically sterile. 12 After his magnum opus was published he was delighted to discover that many of his ideas had been foreshadowed in the Upanishads.

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to his student, Virchow, and to Virchow’s student, the evolutionary theorist and taxonomist Ernst Haeckel. Later, Virchow was to be influenced by Schopenhauer and to grow suspicious of Kantian philosophy (Ackerknecht 1981; Nuland 1988). Elsewhere, the Kantian tendency in science predominated, and it found expression in the strongly mechanistic stance taken by other students of Müller, notably Emil du Bois Reymond. Hence our thesis is that the schism between du Bois Reymond and Virchow was underpinned by the conflict between Kantianism and Naturphilosophie.

A.2.8

The Opening of the Schism

Vital-mechanists’ primary concern is not the ‘cause of life’ but the effects of life – they are concerned with what living matter actually does because it is living (as opposed to non-living or dead).13 Under the influence of Naturphilosophie, 19th-century vital-mechanists became interested in principles of organisation and ‘harmony’ that were peculiar to biology. They were sceptical about extreme mechanistic views, just as Goethe and others were sceptical of Kant, but this did not commit them to vitalism, as is sometimes alleged (see Section A.2.9). This seems to have been Müller’s position,14 and probably, in his later years, Magendie’s. It was certainly Virchow’s position: for him, organisation and development were primary issues in biology and he deemed them proper objects of study in their own right, though he never suggested that they were in any way incompatible with physics. Consequently, he adopted a vital-materialist perspective akin to that of Harvey and Hunter, though the philosophical basis for that choice was different. In contrast, du Bois Reymond and his fellow mechanistic materialists held that all truths about Nature must be reducible to the language of Newtonian mechanics, in accordance with Kant’s teachings. Thus, it was presumed, physiology (and pathology) must be ultimately explained in a ‘mechanist’ framework of reference. Although this view was superseded within a generation because of the new understanding

13 Hunter’s remark that ‘a dead body has all the composition of a living one’ emphasised the functional rather than the causal and analytical aspects of life. 14 Müller was a powerful, inventive and communicative personality who espoused ‘the idea’ (in Goethe’s sense) and made a success of it. Like Goethe, he was concerned with objects as they presented themselves to sentient experience. Műller is sometimes deemed a ‘vitalist’, perhaps on account of the following sentence from his Elements of Physiology: ‘Though there appears to be something in the phenomena of living beings which cannot be explained by ordinary mechanical, physical or chemical laws, much may be so explained, and we may without fear push these explanations as far as we can, so long as we keep to the solid ground of observation and experiment.’ But this scarcely seems to express belief in a ‘vital force’. A more plausible interpretation of the word ‘phenomena’ here is Kantian, indicating no ontological commitment of a ‘vitalist’ kind.

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of chemistry,15 the underlying Kantian philosophy survived, as we suggested in Chapter 5. This begs the question: why did the chemical approach to biology remain intrinsically Kantian in orientation, allowing the commitments of the mechanistic materialists to be reborn in a new guise? We shall consider that question later (Section A.4).

A.2.9

The Origin and Development of Mechanistic Materialism

The extreme mechanistic materialist position had been adumbrated as early as 1841, when du Bois Reymond quoted Dutrochet with evident approval: ‘The more one advances in the knowledge of physiology, the more reasons one will have for ceasing to believe that the phenomena of life are essentially different from physical phenomena’. A year later, du Bois Reymond and Brücke had sworn to ‘validate the basic truth that in an organism, no other forces have any effect than the common physicochemical ones’. Elsewhere, we find: ‘a vital phenomenon can only be regarded as explained if it has been proven that it appears as the result of the material components of living organisms interacting according to the laws that those same components follow in their interactions outside of living systems’. Helmholtz had been recruited to this cause by the end of 1845, and Ludwig by the early part of 1847. The preface of du Bois-Reymond’s Researches on Animal Electricity (1848–1884) is virtually a mechanistic materialist manifesto,16 expressing the views of all four men, predicting the necessary and complete dissolution of physiology into physics. The experimental physiologist must proceed with physical and mathematical exactness, always seeking to explain biological phenomena in terms of mechanics. Du Bois Reymond defined ‘mechanics’ as the motion of particles of matter: ‘if only our methods sufficed, an analytical mechanics of the general life process would be possible. … All changes in the material world within our conception reduce to motions. Therefore even that process cannot be anything but motions …’ The mechanistic assertions of 1847 had three main facets: 1. They were inflexibly anti-vitalist, insisting on non-mystical causality for all living processes. 2. They fostered the use of observation and experiment. 3. They asserted that the attempt to reduce physiology to physics was practicable and potentially useful.

15

In the 17th century, Hoffmann had founded the tradition of iatromechanics, the inflexibly Cartesian belief that all chemical phenomena were reducible to classical mechanics irrespective of whether they were observed in living organisms or the non-living world (see chapter 4). Hence our earlier assertion that du Bois Reymond’s philosophy of science, explicitly Kantian though it was, can legitimately be traced to 17th century roots. 16 His philosophical views were also set out in a series of essays: The Limits of Natural Science (1872).

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The first two facets ensured that du Bois Reymond, Brücke, Helmholtz and Ludwig made invaluable contributions to physiology. In hindsight, the third facet served only to attract worldwide public attention to their ‘cause’ and to disseminate the spirit of anti-vitalism and experimentalism in physiology. These pioneers used physical methods and concepts and mathematical techniques to the limit of their powers, and to considerable effect; but their real contributions – especially those of Ludwig, the doyen of experimental physiologists – lay in specific factual discoveries in aspects of anatomy and histology and, later, chemistry, not in accounting for physiological processes in terms of classical mechanics. In effect, mechanistic materialism expunged ‘life’ from the vocabulary of biology. Thus, the distinction between ‘cause of life’ and ‘effect of life’ – so important for vital-materialists - had scant significance for du Bois Reymond and his colleagues. Their interest was in mechanisms; effects might be observed at the physiological, organismic level, but the received strategy of research was directed towards elucidating physical cause in each case.

A.2.9.1

Helmholtz

Helmholtz (1821–1894) was an academic ally of du Bois Reymond but he later came to adopt a position closer to British positivism17 than to German materialism. Though committed to reducing physiology to physics, and antagonistic to vitalism, he grew as philosophically opposed to Kant as to Schopenhauer. He repudiated Kant’s conclusion that time, space and causation were mental structures through which the world is comprehended, and returned to the empiricist view that all knowledge comes from the senses. His Handbook of Physiological Optics (1867) shows, as indeed do all his scientific works, a combination of philosophical insight, exact physiological investigation, mathematical precision and a deep grasp of physical principles. His theory of vision was far more precise than Young’s, and its exposition destroyed the influence of Goethe’s Farbenlehre and is (essentially) the account of colour vision accepted today.18

17

A school of epistemology associated with J. S. Mill (1806–1873) and his successors (Mill 2002) who held that the empiricists were essentially correct: everything that we know derives from what our senses tell us about the world. In view of Hume’s strictures, knowledge is always provisional; accumulating evidence increases the probability that our beliefs are true, though it can never give certainty. Mill also held that the progress of knowledge gives rise to, and is reflected in, a greater capacity to predict and control both the natural world and social behaviour; and that progress is inherent in both science and society. This attempt to restore Enlightenment optimism became popular in Victorian Britain, contaminating evolutionary theory (even after Darwin) with the notion of ‘inherent progressiveness’ (Bury 1920). Positivism attained an extreme form in the writings of Helmholtz’s contemporary and fellow-physicist Ernst Mach (1838–1916). According to Mach, the raw information acquired by the senses constitutes the whole of knowledge (Mach 1897). 18 There are alternative accounts. Hering, for example, proposed in 1878 that vision depends on three mechanisms responding to the opposed pairs light/dark, red/green and blue/yellow. His model had a sound psychophysical basis and was not incompatible with Helmholtz’s. Yet such

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A.3

243

Mechanism Versus Vitalism: The Distinctiveness of Vital-Materialism

Ancient and mediaeval thought was broadly ‘animistic’ in the sense that the world as a whole was considered to have a ‘soul’ or ‘spirit’ that accounted for its properties.19 Thomas Aquinas explicitly used life as a metaphor for the cosmos, imagining the whole of Nature as if it were alive. When Galileo and Descartes articulated the mechanistic account of (inanimate) Nature in the early 17th century, their writings signalled the end of this ‘universal animism’. It was inevitable that the status of living organisms would then come into question: were they to be explicated in the same mechanistic language as the inanimate world, or was a different language required? We surveyed some aspects of the ensuing debate in Chapter 4, particularly the difference that developed between Stahl and Hoffmann, and noted that the explicit idea of a ‘vital force’ (élan vital) was articulated by Friedrich Casimir Medicus in 1774. The ‘mechanism-vitalism’ debate in the strict sense therefore began in the 1770s.

A.3.1

The Mechanism-Vitalism Debate and its Implications

Both mechanists and vitalists in the 18th and 19th centuries were committed to ‘explaining the cause of life’, as opposed to ‘considering the effects of life’. The basic distinction between the two camps was simple: vitalists posited that a ‘vital force’ informed living as opposed to non-living matter; mechanists held that all components of living matter and all principles underpinning its behaviour were also components and principles of the non-living world. The debate between these two viewpoints is widely, if dubiously, held to have characterised the main conceptual and methodological dichotomy in biology and medicine during this period (Hoernlé 1920; Schubert-Soldern 1962; Heim 1972). The appeal of vitalism is readily appreciated. Biological entities are ‘endowed with purpose’ (Monod 1970), and ‘purpose’ (Aristotle’s entelechy) appears irreconcilable with Cartesian mechanism in which living activities were conceived as ‘machine-like’. Indeed, it was not reconciled with any strictly materialist ontology until late in the 19th century, when the implications of Darwinian theory became apparent. Moreover, common sense shows that the organisation of even the simplest organisms is qualitatively was the influence of Helmholtz that Hering’s model has subsequently received scant attention, and Goethe’s influence on scientific thought effectively ceased. This may be another reason why the vital-materialist frame of reference fell into relative disfavour, notwithstanding the eminence of such scientists as Virchow and Bernard. 19 Some phrases that remain current today are ‘semantic fossils’ of this belief: Mother Earth, Mother Nature, veins of ore, etc. The underlying principle of alchemy was the transfer of the spirit of one substance to another, effecting transmutation. The first description of the Earth’s magnetic field (by William Gilbert in 1600) equated the magnetic field with the soul of the world.

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distinct from that of any inanimate object. Mechanists could account for neither the purposiveness nor the organisation of living things without invoking the Creator. Vitalists could exclude God from their scientific arguments, but only at the cost of postulating a no less mysterious ‘vital force’. The vital force allegedly controlled the form and development of the organism and directed its activities, without any need for Divine intervention. By thus supplanting God, vitalism attracted the religious sceptic and, a fortiori, the political radical. Thus, although vitalism appears misguided to 21st-century eyes, it exerted a powerful influence in the 18th–19th centuries on political and religious disputes as well as biology and medicine. The religious overtones seem to have been deeply rooted in mediaeval thought (see above). The theory of transformation in its earliest form may have implied an acceptance of vitalism.20 The supposed ‘upward striving’ of organisms towards greater complexity seems to presume a ‘vital force’. Significantly, Lamarck was a political radical – a staunch Republican – and an anti-clerical religious sceptic. The English establishment banned his work for decades, fearful of its influence.21 Darwin encountered it only when he went to study medicine in Scotland. Post-Revolutionary France was a hotbed for the development of vitalist ideas. Many physiologists distanced themselves from the Cartesian interpretation of the body as a ‘mere machine’, and considered the laws of inanimate nature insufficient to explain all the manifestations of life. In making the body independent of the soul or spirit, those French physiologists deprived the soul of the position Descartes had residually assigned to it. Passions, instincts, thought and will were phenomena dependent upon our external and internal sensations and ipso facto on biological organisation. Locke and his followers would have approved. Geoffroy and Lamarck, no less than these contemporaneous French physiologists, implicitly removed the human species from the ‘special creation’ status in the Judeo-Christian corpus. The main objection of the mechanists to vitalism was not the ‘vital force’ concept per se, but the concomitant elimination of the spiritual distinction between humans and the rest of Nature. Physiologists22 such as Bichat retained his belief in the soul, but held that it must be sidelined in physiological research – that psychology should be considered a sub-discipline of physiology. Crucially, the same tradition that informed 19th century vital-materialism, Naturphilosophie, also inspired 18th and 19th century vitalism. Kantian mechanists were philosophically opposed to vital-materialism, as we have seen, but they were also scientifically opposed to vitalism. The enduring dominance of Kantian philosophy could therefore explain why so many vital-materialists, including Hunter,

20 However, Lamarck inculcated the use of the word ‘biology’ to denote the scientific study of life. Is it significant that he chose the Greek rather than the Latin word for ‘life’, and might this semantic device have been an attempt to dissociate him from the vitalism of many of his contemporaries? 21 The English political radicals of the 1830s and 1840s, the Chartists, were often accused of Lamarckian tendencies by the authorities. 22 Not all French physiologists of the era were vitalists. For example, Dutrochet’s pioneering work on osmosis and diffusion (or, in his terminology, endosmosis and exosmosis) illustrated that mechanistic doctrines were alive and well in France at the time of Bichat and Magendie (Hoernlé 1920; Schubert-Soldern 1962).

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Müller, Lister and even Harvey, have on occasions been accused of ‘vitalism’. This confusion reflects an inability to distinguish (1) a (legitimate) concern with the effects rather than the cause of life from (2) a (misguided) belief that there is a fundamental ontological difference between the living and the non-living.

A.3.2

Alternatives to Mechanism are Often Misrepresented

For mechanists, it became – and remains – easy to caricature alternative viewpoints in terms of a simple dichotomy, acceptable and unacceptable. Monod (1971) wrote ‘a few words…about the old quarrel between “reductionists” and “holists”. … According to these holist schools, which, phoenixlike, are reborn in every generation, the analytical attitude (“reductionist”) is doomed to fail in its attempts to reduce the properties of a very complex organization to the “sum” of the properties of its parts. It is a very stupid and misguided quarrel, which merely testifies to the holists’ total lack of understanding of scientific method …’ As examples of ‘holism’, Monod cited a collection of papers (Koestler and Smithies 1969) written by such distinguished exponents of scientific method as C. H. Waddington and Paul Weiss. Monod’s eminence is beyond question and his essay is scholarly and durable; but his dismissal of alternative perspectives, in all of which he perceived ‘vitalism’ or ‘animism’, exemplifies the attitude of many proponents of mechanism. In no way is the vital-materialist approach ‘vitalist’ or ‘animist’. Harvey, Hunter, Lister and Virchow countenanced neither an aetherial, non-physical ‘vital force’ in the animate world nor any ‘purposiveness’ in the material universe. Yet Monod was not alone in wrongly assuming that ‘anything that is not explicitly reductionist is vitalist or animist, and consequently unscientific’. In the 1976 Encyclopaedia Britannica article about Helmholtz, Johannes Müller’s approach to biology (from which the mechanistic materialist Helmholtz dissociated himself) is dubbed ‘vitalist’ (see footnote 14). Hunter and Lister have also been described as ‘vitalists’ by otherwise authoritative sources; they were not (Chapters 4–6). Labelling alternative scientific approaches ‘vitalist’ or ‘animist’ is akin to labelling unorthodox religious practices ‘heretical’. Justified or otherwise, the very label condemns, dismisses and ostracises the alternative viewpoint, its proponents and its practitioners without further consideration. One consequence is to create an aggressively Whiggish, value-laden reconstruction of the history of medicine and biology and to narrow the perspective and conceptual field of present-day researchers (Butterfield 1965a).

A.3.3

‘Extreme’ Mechanism: The 19th-Century German Materialists

Not until most of German biology (and physiology in particular) had distanced itself from Naturphilosophie did it begin to exert a significant influence on philosophy and popular thought. This trend seems to have begun in the late 1830s, and the

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explicitly materialistic bias of the younger generation of German physiologists at that time was to acquire political overtones, particularly manifest in the revolutionary movement of 1848. Just as many vitalists in France at the turn of the century had repudiated the independence of the soul from the body, so many German materialists began to question the notion that humans had spiritual attributes. In 1846, Carl Vogt (1817–1895) wrote the following notorious words (see Büchner 1864)23: ‘I believe that every scientist, if thinking at all consistently, will reach the opinion that all the faculties that we comprise under the name of “psychic actions” are nothing but functions of the cerebral substance, or, to express myself somewhat crudely, that thoughts have about the same relation to the brain as bile has to the liver or urine to the kidney’. Vogt scandalised his readers, but his sentiments were little different from those expressed by Bichat and Cabanis almost half a century earlier. Yet his biological frame of reference was wholly different: he was an outright mechanist, whereas Bichat and Cabanis had been vitalists. German physiologists opted to differ from their French predecessors in this fundamental respect and launched a veritable campaign against the assumption of a vital force. The first hint of this campaign appeared Schwann’s 1839 work that described the cellular nature of animals. Schwann was not entirely typical of the new generation of German materialists. For one thing, he was highly religious;24 his work carries a strong echo of Descartes in blending materialism with Deism. For another, he was influenced by Naturphilosophie. In a passage typical of his ‘split beliefs’ he wrote: ‘Either the organism is endowed with a force that forms it as a whole according to some idea, or … is subject to forces that act according to blind necessity, forces that are inherent in matter itself’. He sat on the fence but admitted that, if pushed, he would choose to fall on the mechanist side. However, if there is no vital force, how are we to account for the purposive character of organisms? This was not problematic for the deeply religious Schwann: like the older school of mechanists, he transferred purpose from biology to the world as a whole and attributed it to the Creator. Organisms were only quantitatively, not qualitatively, more purposeful than a mechanical system such as the Solar System. This solution to the problem of purpose was broadly adopted by several materialists during the following decade, despite its taint of Naturphilosophie. Lotze’s article of 1842 (Santayana 1971) attacked the assumption that there was anything specifically distinctive about living matter and upbraided the vitalists for their obscure use of the term ‘force’, but he too had recourse to Divine intervention to explain purposiveness. Even Vogt followed this path. For du Bois-Reymond and his followers, however, Schwann’s solution to the problem of purpose was unacceptable. Rather than offer any solution to this problem they dismissed it, even ridiculed it. Since in du Bois Reymond’s opinion the mechanistic doctrine had to prevail, problems such as organisation, development and purposiveness 23 Büchner’s popular compendium of the basis of (German) materialism gives an insight into contemporaneous views, though it is philosophically lightweight. 24 Schleiden, the alleged co-founder of cell theory, was such another, and in 1863 he published a vitriolic attack on the anti-religious trend of German science.

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were simply disregarded, treated as matters unworthy of serious scientific discussion. In this regard, they dissociated themselves from the more extreme materialists of their age. But they were in harmony with the extreme materialists in dismissing from consideration not only the problems of organisation, development and ‘purpose’, but also the vital-materialist concern with the ‘effects of life’. The confusion of vital-materialism with vitalism became entrenched.

A.4

The Modern Dominance of the Mechanistic Approach

The true legacy of 19th-century mechanistic materialism was the ‘reduction’ of biology (particularly physiology) not to principles of physics but to accumulated nuggets of hard experimental and observational fact, achieved by the deployment of physical and, later, organic-chemical techniques. This is illustrated in the influential book written by Fick’s most famous student, Jacques Loeb (Loeb 1912). During the 20th century, the premature efforts at biophysics that had characterised German physiology in the 1840s and 1850s gave way to the rise of biochemistry, which was to prove far more successful and productive.

A.4.1

The Metaphysical Dichotomy in Early 20th-Century Biology and Medicine

Nevertheless, there was a widespread conviction during the 1920s and 1930s that human physiology had ‘outgrown’ the mechanistic age. This view was far from universal; some writers still extolled the virtues of a physics-based approach to physiology. Thus, two kinds of physiologists were distinguished: those who believed that ‘vital’ processes should be investigated by the reductionist methods of physics and chemistry, and those who believed that the object of their science was the associated form and functioning of human body (cf. Thompson 1917; Cannon 1939; Eccles 1979; Faber 1987).25 In short, the two distinct, apparently incompatible, perspectives that had co-existed in the 19th century continued into the early 20th-century physiology. One was clearly mechanistic, though much transformed since the optimistic inception of mechanistic materialism. The other, associated with the majority of physiologists, was essentially vital-materialist.26

25 J.S. Haldane satirised the kind of biology that involved experiments on ‘fragments of frogs’ and wanted a return to a physiology that studied whole living bodies. 26 It was emphatically not vitalist; vitalism had become extinct by the end of the 19th century. Its last significant exponent, Hans Driesch, had used ‘entelechy’ to explain observations in experimental embryology that seemed at the time incompatible with the dictates of physics and chemistry, but can now be ‘explained’ by reference to the spatio-temporal distribution of chemical groups on cell surfaces.

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Thus, while the mechanistic tradition had faltered and been transmuted, ultimately finding its expression in biochemistry and allied disciplines, the vital-materialist tradition remained strong. During the second quarter of the 20th century it was usually labelled ‘holistic-materialist’ or ‘organicist’. It emphasised homeostasis, integration, adaptation, and organisation at the levels of cell, tissue, organ and whole organism. It was articulated in the work of Haldane, Barcroft and Cannon, but earlier in the century it had been, more or less, the view of the pioneering neurophysiologist Charles Sherrington, and of Christiaan Bohr, a one-time pupil of Ludwig. Strikingly, while academic physiologists of the time could align themselves with either the mechanistic or the vital-materialist traditions, clinical practitioners – particularly surgeons – were aligned almost exclusively with the latter. The practitioner must be primarily concerned with living tissues (as opposed to dead or necrotic tissues), and this emphasis necessarily relegates causal and analytical considerations to a subsidiary place. Not surprisingly, therefore, ‘organicism’ (also known as ‘surgical physiology’) came to dominate in the teaching of medical physiology in the first half of the 20th century.

A.4.2

Some Contributing Factors to the Hegemony of Mechanism

So why, and when, did the mechanistic approach attain its present hegemony? The vitalmaterialist perspective might have been marginalised and misrepresented in the 19th century because of its association with Naturphilosophie while the climate was predominantly Kantian, but many (perhaps most) early 20th century physiologists adopted it. The change seems to have occurred after the Second World War, when governments in developed countries poured money into academic scientific and medical research, and perceived ‘growth areas’ such as biochemistry were favoured. A number of highly able physicists, disaffected by the bombing of Hiroshima and Nagasaki, turned to biology; Francis Crick was an example. Such developments fostered the physico-chemical approach to biology and medicine, and ipso facto the mechanistic frame of reference. The consensus model of DVT and the implicit sidelining of Virchow’s legacy date from this post-War period. The rise of molecular biology during the later 1950s and early 1960s exacerbated the trend, and in this respect the landmark discoveries of Monod and Jacob and their colleagues at the Institut Pasteur were perhaps pivotal: molecular-level accounts of cellular control processes, specifically the control of gene expression and the control of metabolic enzyme activities. By implication, homeostasis, a key concept of ‘organicism’, was made to appear ‘reducible to’ the language of physics and chemistry, and this fostered the belief that the mechanistic approach to biology was ‘more fundamental than’ (and therefore presumably superior to) any alternative. This inference became steadily more entrenched when later developments in molecular and cell biology gave rise to the techniques of gene sequencing and manipulation that are staples of laboratory work today, and to the molecular-level

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elucidation of receptor-mediated control and cell–cell communication processes. Molecular biologists can now purport to explain what ‘organicists’ describe. The financing of research is another major influence. Molecular biology is expensive but it returns information that can be very profitable, for example, to the pharmaceutical industry, so it is handsomely supported. Vital-materialist research does not share this benefit; indeed, often the contrary. For instance, the consensus model of DVT has fostered the development of an ever-increasing range of anticoagulants and thrombolysins, the clinical value of which is beyond doubt. If slight tilting of hospital beds and the wider use of gentler intermittent pneumatic compression, as suggested by the VCHH, were to become more widely used in prophylaxis against DVT, and if it were to become clearer when to discontinue anticoagulant therapy (because the condition is presumed to be more than a solely ‘blood coagulation’ problem), the use of such drugs could be monitored in a revised framework.27

A.4.3

Molecular Motion Versus Bulk Transport: The ‘Newtonianism’ of Biochemistry

However, something more philosophically fundamental seems to be involved. Biology is distinct from the physical sciences inter alia because it is the study of objects that are in some respect self-moved. In particular, fluids are self-moved (i.e. undergo net ‘bulk’ flow). Incessant bulk water flow and a fortiori the transport of solutes is a ‘vital’ property, characteristic of living systems from the cellular to the whole-organism levels and inseparably responsible for the ‘effects of life’. Mechanistically orientated researchers frequently under-emphasise this characteristic, often presuming that biological fluids (especially within cells) are essentially ‘static’ and that diffusion is the primary (even sole) mechanism of transport. Elsewhere we have questioned the assumption that biological transport processes depend on ‘diffusion’.28 This point merits further examination. The basis of kinetic theory and statistical mechanics, from Kelvin through Maxwell and Boltzmann to Gibbs, is the interpretation of ‘heat’ as random molecular movement. That interpretation underpinned Einstein’s 1905 paper about Brownian motion, which was instrumental in establishing atomic theory in the face of the doubts expressed by Ostwald and Mach. Random molecular movements are ineluctable, except at absolute zero, and a fortiori occur inside organisms. No metabolic or other life process could occur otherwise. But this does not imply that the movements of fluids and solutes inside organisms depend on diffusion, at least over distances greater than a fraction of a micrometre. It has been estimated that any molecular (solute) movement inside a cell over a distance greater than 50–200 nm involves bulk-flow processes that are fundamentally different from Brownian

27 28

A possible exception here could be the development of PPAR-γ analogues, suggested in Chapter 12. These articles were cited in footnote 11 in Chapter 8.

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motion (Agutter and Wheatley 2000). Biological fluids, considered ‘in bulk’ (i.e. at the supramolecular level), can never be at rest during life, and their motion does not result from the random saccades of molecules, which are not extinguished by death. Du Bois Reymond stated explicitly that the ‘motion of particles’ was the basis of all true scientific explanation in biology (see Section A.2.9). Ludwig returned regularly throughout his working life to the problems of filtration and fluid movements in physiology. His most eminent student, Fick, authored the law of diffusion. So the idea that diffusion is fundamental to biological transport became entrenched through the work of the early mechanistic materialists.29 Subsequent developments in statistical mechanics, not to mention Einstein’s seminal paper, put the seal of authority on their notion (Agutter et al. 2000). Physics broke free of its Newtonian and Kantian shackles during the early part of the 20th century through the twin revolutions that gave rise to relativistic and quantum mechanics. Biology underwent no such revolution. Relativity theory (one axiom of which is that no observer can ever consider himself ‘at rest’) and quantum mechanics transformed the Newtonian account of the universe, not least in respect of its geometry, and our understanding of electromagnetics and radioactivity. But thermodynamics/statistical mechanics survived more or less intact through this period of revolution, and this entailed a Newtonian account of the mechanical behaviour of randomly moving molecules. Statistical mechanics is the theory-base of chemistry. Subsequent work showed that it is compatible, indeed reconcilable, with the relevant aspects of quantum mechanics (‘quantum chemistry’), but chemical kinetics today remains rooted in the fundamentally Newtonian work of Boltzmann and Maxwell. Chemistry began to acquire this theory-base just when the du Bois Reymond project of reducing all physiology to physics was beginning to falter (Cranefield 1957). As we have seen, a chemical rather than a physical basis for biological processes became the Holy Grail during the 20th century, and biochemistry and molecular biology became the cutting edge of progress. Underpinning this chemical approach to biology (most explicitly, its kinetic aspects) was the essentially Newtonian, statistical-mechanical, account of molecular movement. Thus, the physico-chemical approach to biology remained Newtonian, hence Kantian; and ipso facto as mechanistic as Descartes, or Hoffman, or Boerhaave, or du Bois Reymond, could have wished. So the hegemony of mechanism in biology today is not surprising. The mechanistic perspective cannot directly address the ‘self-moving’ characteristic of life, including the bulk flow of fluids within and among cells. The effect of this limitation is clearly perceptible in DVT research. The undeniable fact that

29 Of course, the notion of random particle (atomic) movements had a long and distinguished pedigree. Hobbes and others in the period preceding the foundation of the Royal Society had held that the material world comprises nothing but ‘atoms in a void’. This notion grew from the influential publications of Bacon and Gassendi that formed the background to Locke’s work. At the same time, it seems to reflect the spirit of Cartesian mechanism, though Descartes himself was not an atomist.

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thrombi invariably form in flowing blood was, in effect, deliberately disregarded for about half a century, allowing the consensus-model focus on ‘stasis and hypercoagulability’ to rule. Much valuable information about the risk factors for DVT has resulted, but the prophylactic and therapeutic measures inferred from the consensus model are limited and the explanation of predisposing factors is incomplete. The VCHH was motivated by these considerations.

A.5 Rapprochement between the Mechanistic and Vital-Materialist Approaches Neither the mechanistic nor the vital-materialist approach should be construed as ‘fact’. They are different metaphysical frameworks of reference in which to obtain, collate, and interpret evidence. They are complementary positions, not alternative or conflicting ones. To accommodate both perspectives simultaneously is to gain, as it were, a stereoscopic view of biology: cause and mechanism. To exclude one in favour of the other is to opt for monocular vision: cause or mechanism. Of course, mutual exclusion would accord with much of the venerable tradition of western thought: Thomist theology versus Cartesian deism, Stahl versus Hoffmann, the wars, revolutions and scientific controversies of the late 18th and the 19th centuries. But it is more profitable to focus on the present and the future of biomedical research. West and Brown (2005) made the following comment during their review of a well-known puzzle in physiology (the allometric scaling of basal metabolic rate): ‘Sciences typically cycle between periods of empiricism and theory, reductionism and holism. Empirical advances are . . . unified and synthesized by theoretical contributions. . . . Reductionist studies that discover components and processes at microscopic levels are given additional meaning by holistic studies that show how these phenomena contribute to the structure and function of large, complex systems at higher levels of organization. . . . Both are equally necessary for scientific progress’. This ‘cycle’ image seems to embrace science in general; but in biomedical investigation, the mechanistic and vital-materialist approaches (loosely equivalent to ‘reductionist’ and ‘holist’) often appear to exclude rather than complement one another. At each turn of the ‘typical cycle’ the ideas and discoveries of the previous generation can become buried, forgotten, considered outmoded and superseded, rather than enriched by new knowledge and new methods; or they can be misrepresented until reduced to near-parodies of their originators’ intentions. Our ideal is to accommodate both approaches. William of Ockham’s 650 years old ordinance – ‘It is vain to do with more what can be done with less’ – could seem to conflict with that ideal. Bertrand Russell (1946) commented: ‘. . . if everything in some science can be interpreted without assuming this or that hypothetical entity, there is no ground for assuming it. I have myself found this a most fruitful principle in logical analysis’. However, West and Brown (2005) did not imply that science thrives by accommodating two (divergent or opposed) theoretical accounts or methodologies concurrently. In other words, they did not argue that conceptual

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schisms – two traditions resting on different premises – are scientifically productive. If the two traditions in biomedical research cannot be reconciled, Ockham’s Razor bids us either to dispense with one set of premises, hence one or other tradition. If we wish to retain both we must endeavour to ‘amalgamate’ them. Scientists, like poets, use metaphor and analogy to create new images and achieve better viewpoints; the power of a scientific idea rests in the likeness it projects, just as the beauty of Shakespeare’s work resides in his poetically licensed metaphors. The ineluctable snag with metaphor and analogy is Whitehead’s ‘fallacy of misplaced concreteness’ – the substitution of ‘is’ in place of ‘as if’ (Chapter 4). The greatest confusion in the DVT literature has resulted from the widespread misperception of Virchow’s concept that thrombosis in vivo is ‘like’ – i.e. analogous to – clotting in vitro. Virchow’s analogy was highly plausible – a ‘thrombus’ is indeed more like a ‘clot’ than a ‘phlebitis’; but Virchow’s successors fell into the error of understanding him to have surmised that thrombosis is clotting, and that a thrombus is a clot (not just ‘like’ one). Correction of that fundamental error required unification, or at least harmonisation, between the metaphysically distinct but scientifically complementary positions of mechanism and vital-materialism – certainly in respect of DVT. What are the prospects for ending the imbalance between these two approaches more generally, and for enabling all biomedical workers now and in the future to perceive them as complementary rather than contradictory? In general, vital-materialists (mainly clinical practitioners) are willing to accept the findings of mechanistic research and where possible to exploit them; their difficulty is comprehension, since much of the modern biological literature looks to the outsider like a ‘private language’. On the other hand, mechanists remain dismissive of the vital-materialists; they tend to consider their work misdirected, antiquated, tainted with vitalism; and, mistakenly, they believe that its achievements have been subsumed in their own approach.30 There is therefore a pressing need to help mechanists to achieve a clearer and better-informed awareness of the past achievements of vital-materialist research and its legacy in biology and medicine today, and to recognise its continuing potential. As Feinstein pointed out, the needs are mainly educational.

30

A letter on this topic submitted to Arteriosclerosis, Thrombosis, and Vascular Biology in 2004 elicited interesting responses. One reviewer remarked that ‘The observations made by the author are convincing, the argument treated is interesting and the letter is well written’. The other wrote: ‘This letter espouses an unfortunate viewpoint for physician scientists interested in elucidating mechanisms of disease. The authors draw a dichotomy between biochemical/molecular/hematologic approaches and work to elucidate the pathophysiologic basis of disease (based on pathologic observations and rheology) … it is only through the integration of molecular approaches (with other research strategies) that targets of drug therapy can be determined as a basis for future therapies… data on the cellular response to hypoxia, in terms of transcriptional (HIF-1, HIF-2, Egr-1, NF-kB etc) and translational (expression of growth, proinflammatory and procoagulant cofactors) mechanisms, has [sic] exploded in recent years and has shown that equating hypoxia with cell death is simplistic …’ The letter did not, of course, seek to equate hypoxia with cell death, but it was not published. The second reviewer’s opinion exemplifies the attitude alluded to in the text.

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It is difficult in today’s climate to encourage students to pay attention to work done five years ago, let alone a century ago. They have been indoctrinated with the belief that new discoveries from the cutting edge of research are incomparably more important than work of the past. This raises barriers against appreciation of the vital-materialist approach; in any case it is a dangerous delusion. It produces graduates who are hardly aware of the rich tradition of observation, experiment and thought they inherit, and we disregard our professional heritage at our peril. Integration of relevant historical information into mainstream medical and biological education is urgently needed. The Select Committee of the House of Lords Committee considered the evidence available to them concerning ‘traveller’s thrombosis’; but their Lordships were so disgruntled at the paucity of reliable information and opinion that they deemed it ‘imperative’ that DVT should be explained. Along with this integration of history with teaching there must be a focus on improved thinking skills – i.e. on ‘philosophical’ treatment of scientific issues; unquestioning acceptance among today’s experts does not make words and ideas into immutable truths. Such fostering of critical debate should counter the dismissal of great past achievers and achievements as ‘vitalist’, ‘animist’ or otherwise ‘heretical’. It goes without saying that improved thinking skills will also pay dividends in both research and medical practice. This is a matter to be addressed in the longer run by universities and teaching hospitals; the present book has been accelerated by the need for an in depth consideration of the theoretical issues that will enter into such a conception. As for the evidence that vital-materialist work may contribute materially, or indispensably, to biology and to medical practice: books such as this may go some way towards providing it. To the best of our knowledge, no other such attempt exists in the biological or medical literature.

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Author Index

Abbe, E. 92 Abernethy, J. 73 Abu’l-Qasim 45 Ackroyd, J.S. 127 Addis, T. 59, 64 Addison, W. 91, 93, 112 Agani, F. 199, 202 Agirbasli, M. 25 Agutter, P.S. 9, 41, 43, 66, 110, 250 Ahlberg, A. 40 Aird, W.C. 196, 205 Alberti, S. 107, 109 Aley, P.K. 203 Alhenc-Gelos, M. 35 Alikhan, R. 2 Alonso, A. 209 Alonso, C. 135 Altschul, R. 164 Alving, B.M. 39 Amatus 107, 109 Anderson, F.A. Jr. 3 Anderson, J.R. 148 Andral, G. 32, 57, 60, 68, 72, 76 André, P. 206 Anning, S.T. 8, 44, 76, 83 Anrather, D. 205 Anthoni, C. 211 Appel, T. 238 Apperly, F.L. 15 Archie, J.P. Jr. 216 Ardlie, N.G. 79 Aristotle 46, 47, 77, 148, 243 Arkin, C.F. 35 Arner, M. 214 Arnould, T. 208, 210 Aronson, D. L. 34 Arthus, M. 63

Aschoff, L. 42, 60, 65, 79, 80, 91, 94, 96–99, 113, 131, 135, 164–166, 169, 170, 174, 222, 224–231 Asherton, R.A. 36 Ashford, T.P. 15, 154 Åstrup, T. 23, 24 Atzlet, E. 118

Bacon, F. 46, 48, 106, 235–237, 250 Badimon, L. 6 Baglin, T. 38–40 Baillie, M. 53, 76 Bajaj, S.M. 20, 196 Balasubramanian, V. 204 Bancroft, F.W. 96 Bang, N.U. 60, 61 Barcroft, J. 148, 248 Barker, J.E. 215 Barker, W.F. 15 Barnard, M.R. 207 Barnes, R.W. 138 Baron, A. 213 Barrett, W.D. 16 Barstad, R.M. 209 Bärtsch, P. 94, 166, 167 Bartlett, W. 69 Bassus, S. 205 Bauer, G. 115 Baumgarten, P. 111, 112, 116, 123, 148, 165, 222, 225, 229 Bek, M.J. 200 Belt, T.H. 113, 114, 189, 190 Bendz, B. 166 Bentley, J.K. 206 Benvilacqua, M.P. 208 Beny J.L. 217 Berg, W. 25

295

296 Bergentz, S-E. 40 Berlin, I. 235, 236 Berna, N. 148, 203 Bernard, C. 243 Berra, E. 199 Bertina, R.M. 35, 36 Berzelius, J.J. 54 Bevan, J.A. 213, 217 Bichat, M.F.X. 244, 246 Bienvenu, K. 208, 210 Biggs, R. 12, 13, 23 Bishop, J.J. 135 Bismarck, O. von 72, 234 Bizzozero, G. 60, 65, 92, 93, 100 Blackburn, M.N. 26 Bland-Sutton, J. 96 Blann, A.D. 6 Blomback, B. 22, 23 Boas, M. 235 Boerhaave, H. 8, 41, 42, 49–53, 55, 57, 68, 71, 72, 76–78, 88, 89, 111, 250 Bombeli, T. 148, 206 Bordet, J. 13, 64, 65 Borelli, G. 54, 55 Borgel, D. 35 Born, G.V. 19 Botti, R. E. 34 Bouchut, F-C. 76 Bouillard, M. 73 Bowie, A. 238 Bowman, W. 117 Boyd, W. 83, 84 Boyle, R. 46, 47, 50, 105 Brass, L.F. 17 Breschet, M. 74 Briet, E. 36 Brinkhous, K.M. 8, 12, 83 Bristowe, J.S. 79 Broekmans, A.W. 35 Brotman, D.J. 8, 83, 171 Brott, T. 5 Brown, G.E. 32 Brown, J.H. 251 Browse, N.L. 1, 13, 31, 34, 44 Broze, G.J. 20, 27, 88, 113 Brücke, E. von 241, 242 Brummel, K.E. 22 Brunkwall, J.S. 213 Bruno, G. 106 Buchanan, A. 57, 59–62, 68, 76, 93 Bulger, C.M. 5, 6 Bulloch, W. 59 Buridan, J. 106, 235 Burkhardt, R.W. 238

Author Index Burroughs, K.E. 4, 8 Bury, J.B. 242 Buss, H. 127 Butterfield, H. 235, 245 Byrne, M.F. 211

Cabanis, P.J.G. 246 Caen, J.P. 17 Camerer, E. 205 Cameron, G.R. 91, 92, 112 Canano, G. 107, 109 Cannegieter, S.C. 2 Cannon, W.B. 247, 248 Caprini, J.A. 39 Carden, D.L. 210 Carey, F. 17 Carson, J.L. 4 Casanello, P. 199 Casimir Medicus, F. 48, 243 Castellote, J.C. 209 Cater, D.B. 111 Celsus 87, 88 Cerilli, G.J. 113 Cesalpino, A. 110 Chandler, A.B. 34 Chapman, H.A. Jr. 24 Chapman, H.T. 139 Chapple, C.R. 132 Chen, X-L. 209 Chevallier, P. 19 Chi, J.T. 142, 195 Choi, J. 214 Chiu, J.J. 204 Christensen, L.R. 66 Cina, G. 3 Claggett, M. 235 Clarke, E.R. 148 Clarke, N. 16 Clendenning, L. 44 Cohnheim, J. 112 Coley, N.G. 52 Colman, R.W. 17, 19 Colombo, R. 109, 110 Colotta, F. 208 Comerota, A.J. 4, 5 Comp, P.C. 35 Conway, E.M. 211 Copernicus, N. 46, 106, 235 Corda, C. 36 Cordier, A.H. 96 Cottingham, J. 235 Cotton, L.T. 132, 138 Coughlin, S.R. 204

Author Index Cramer, W. 65 Crane, C. 14 Cranefield, P.F. 61, 63, 69, 250 Crosby, A. 166 Cruveilhier, J. 8, 44, 68, 71–84, 88, 89, 111, 232 Cullen, W. 51 Curran, R.C. 166, 222 Cushman, M. 2 Cuvier, G. 238

Dahlbäck, B. 35 Dai, G. 138 Dalton, J. 236 Daly, R.N. 217 Dam, H. 65 Darwin, C. 238, 242, 244 David-Ferreira, J.F. 17 Davies, G.C. 17 Davies, J.A. 5, 31, 44 Davies, M.G. 196 Davies, P.F. 216 Davis, D.D. 73 Davis, R.J. 203 Davy, J. 52 Day, T.K. 34 Dear, P. 236 Deitelzweig, S. 37 De la Mettrie, L. 47, 52 Delbridge, G.J. 200 Delerive, P. 217 Delikan, A.E. 113, 188 Demers, C. 35 De Nicola, P. 13, 15 De Sousa, L.P. 202 De Stefano, V. 35 Den Heijer, M. 2, 209 Denson, K.W.E. 13 Descartes, R. 42, 46, 47, 50, 77, 106, 235, 236, 243, 244, 246, 250 Dexter, L. 2 Deykin, D. 38 Diocles of Carystus 44 Dickson, B.C. 8, 44, 45, 83, 84, 95, 96 Dobson, J. 52 Dodd, H. 132 Donné, A. 60, 93 Donovan, A.L. 51 Doran, F.S. 113 Drake, S. 235 Dreyfus, C. 73 Drinker, C.K. 150, 151, 161, 162, 211

297 Du Bois Reymond, E.H. 41, 42, 57, 61, 62, 63, 68, 69, 72, 94, 235, 240–242, 246, 250 Duckworth, D. 73, 96 Dupuytren, G. 73 Dutrochet, R.J.H. 112, 241, 244

Eagle, H. 12, 64 Eastlake, C.L. 239 Eberth, J.C. 92–94, 98, 101, 224 Eccles, J.C. 247 Edwards, E.A. and J.E. 4, 127, 136, 212 Egeberg, O. 14, 35 Eguchi, D. 214, 215 Ehrenberg, C.G. 93 Ehrenforth. S. 38 Einarrson, E. 127, 212 Eisenberg, P.R. 212 Eisenmenger, W.J. 33 Eltzschig, H.K. 210 Ely, S.F. 37 Emonson, D.L. 2 Epstein, A.C.R. 199 Erdogan, A. 212 Eriksson, E.E. 208 Esmon, C.T. 20, 25, 35, 196, 204, 210 Estienne, J. 107, 109 Eto, M. 207, 217 Evans, W.H. 63

Faber, R.J. 247 Fabregues, F. 38 Fabricius, H. 107–109, 125 Faint, R.W. 207 Falcon, C.R. 36 Faller, D.V. 197 Fantl, P. 12 Fearnley, G.R. 23 Feinstein, A.R. 41, 170, 252 Ferriar, J. 73 Field, S.L. 37 Figueroa, R. 136 Fletcher, A.P. 19 Flute, P.T. 23 Forastiero, R.R. 36 Fordyce, G. 51, 60 Foster, D.P. 63 Foster, M. 55, 61, 62, 110 Fourcroy, A.F. 52 Fowkes, F.J.I. 1 Fowler, P.B.S. 162

298 Franklin, K.J. 105, 107–109, 117, 122, 127, 130, 133, 172, 173, 184 Freedman, M.D. 4 French, J.E. 6, 19, 34, 95, 97 Frenette, P.S. 202 Fricchione, G.L. 207 Frick, P.G. 19 Fried, G. 217 Friedenwald, H. 107, 109 Fries, J.W. 211 Froriep, R. 75–77 Frykholm, R. 113, 115, 148–150, 152 Fujikawa, K. 20 Fujita, T. 162, 200 Fulcher, C.A. 25 Fung, Y.C. 216 Furie, B. 212

Gaarder, A. 18 Gailani, D. 19 Galen 45, 46, 48, 49, 109, 147 Galileo 46, 50, 106, 235–237, 243 Gallerani, M. 38 Galli, M. 2 Gamgee, A. 61 Ganrot, P.O. 26 Gashler, A. 199 Gassendi, P. 46, 235, 236, 250 Gaukroger, S. 235 Gauthier-Rouviere, C. 200 Gay, J. 4 Geerts, W.H. 2 Gelehrter, T.D. 206 Geng, J.G. 208 Geoffroy St-Hilaire, E. 72, 238, 239, 244 Gerrard, J.M. 17 Gertler, J.P. 166 Girolami, A. 36 Gibberd, F.B. 113 Gibbs, N.M. 96, 113, 115, 123, 125, 138, 164, 249 Ginis, I. 208 Glick, M.R. 214 Godal, H.C. 35 Goethe, J.W. von 69, 238–240, 242, 243 Goldsmith, H.L. 137, 210 Golledge, J. 142, 143 Gomez, K. 37 Gonzalez, N.C. 208 Goodenough, L.T. 35 Goodsir, J. 76 Gordon, J. 52 Graham, J.B. 12

Author Index Grant, L. 208 Grant, P.J. 34 Gregory, F. 234 Green, R.A. 36 Griffin, J.H. 35 Grimshaw, M.J. 209 Grote, K. 212 Gruithuisen, F. v P. von 112 Grünberg, J.C. 35 Guha, M. 204 Guinto, E.R. 25 Gupta, K. 218

Haas, T.L. 200 Hadfield, G. 89–91, 190 Haeckel, E.H.P.A. 234, 240 Haldane, J.S. 148, 162, 247, 248 Hales, S. 55 Hall, A.R. 235 Haller, V.A. von 55 Hallwright, W.W. 15 Hamby, R.I. 6 Hamer J.D. 9, 140, 145, 154, 173, 175, 178, 179, 181–183, 194 Hammer, L.W. 214 Han, K.H. 209 Harlan, J.M. 196, 208, 211 Harrison, W.C. 104, 105 Harvey, W. 41, 46, 48, 52, 55, 72, 74, 77, 103–110, 118, 125, 133, 146, 148, 240, 245 Hayem, G. 93 He, G.W. 216 Heijboer, H. 35 Heijer, M. 2, 209 Heim, H. 48, 243 Hekman, C.M. 25 Heit, J.A. 2 Hellsten, W.O. 64, 73, 76 Helmholtz, H. von 241–243, 245 Henderson, R.R. 15 Henrion, D. 213 Hensen, A. 26 Herbst, R. 118 Herrmann, F.H. 18 Hewitson, K.S. 199 Hewson, W. 5, 39, 51, 53, 57, 77, 93, 111, 112, 116, 123, 148, 165, 222 Hill, R. 136 Hill, R.L. 15 Hillyer, P. 208 Hippocrates 44, 49, 50, 147, 148 Hipskind, R.A. 200

Author Index Hirsh, J. 5, 132 Hodkinson, P.D. 166 Hoernlé, R.F.A. 243, 244 Hoffmann, F. 47, 51, 52, 241, 243, 251 Holford, C.P. 138 Homans, J. 2, 53, 113 Houliston, R.A. 206, 207 Houston, P. 199 Howell, W.H. 64, 65 Hryszko, T. 25 Huang, R.P. 199 Huebner, C.F. 66 Hughes, S.F. 36, 211 Hull, J. 73 Hull, R. 138 Hume, D. 146, 236, 237 Hume, M. 34, 115, 116, 138, 181 Humphry, D. 96 Hung, D.T. 17 Hunt, P.S. 34 Hunter, J. 5, 8, 42, 51–53, 60, 62, 71–74, 76–78, 80, 82, 88–91, 97, 174, 221, 240, 244, 245 Hunter, W.C. 67, 99, 111, 112, 115, 169 Huygens, C. 49, 88

Imamoto, E. 209, 211 Inman, W.H.W. 38 Isaacson, S. 35

Jackson, R.M. 148 Jacobi, J. 212 Jacobson, B.F. 2 Jaffe, E.A. 17, 195, 196 Janknecht, R. 200 Jansen, H. 40 Janssen, H. 88 Jeremic, M. 36 Jesty, J. 20 Jiang, C. 204, 209 Jick, H. 2, 36 Jiménez-Sainz, M.C. 209 Johnson, A.J. 39 Johnson, P.C. 214 Johnson, T.S. 196 Jones, G.T. 213 Jones N. 202 Jussila, E. 163 Joy, W.B. 73 Joyce, E.H. 213, 217

299 Kafkas, S. 190 Kakkar, V.V. 217 Kamm, R.D. 135 Kamphuisen, P.W. 38 Kanaide, H. 23 Kaneider, N.C. 211 Kaplanski, G. 209 Karakurum, M. 209 Karimova, A. 199 Karino, T. 137, 138, 141, 179, 180 Katusic, Z.S. 214, 215 Kaufman, R.J. 36 Kaur, J. 208 Keeling, D. 34 Keeling, J. 38 Kemkes-Matthes, B. 36 Kemp Smith, N. 236, 237 Kent, K.C. 207 Kepler, J. 88 Khachigian, L.M. 199, 200, 202 Kharbanda, S. 199 Killewich, L.A. 4 Kim, F.J. 210 King, L.S. 44, 50, 106 Kinley, C.E. 13, 15 Kishimoto, T. 208 Klatsky, A.L. 2 Klein, P.D. 26 Kniffin, W.D. 2 Knisely, M.H. 115 Koller, F. 12 Kong, T. 210 Koster, T. 35 Koestler, A. 245 Kourembanas, S. 207, 215, 217 Krishnan, J.A. 8 Krotz, F. 213 Krupski, W. 164, 176, 212 Kuhn, T.S. 42, 105 Kyrle, P.A. 8

Lahav, J. 19 Lamarck, J-B. 72, 238, 239, 244 La Mettrie, J.O. de 47, 52 Lamphear, B.J. 18 Lando, D. 199 Lane, D.A. 35 Lapostolle, F. 2, 192 Lavoisier, A. 47, 54, 55, 148 Lawler, J.W. 19 Lawrence, M.B. 208, 210 Lawson, C.A. 204, 209 Lee, A.Y. 4

300 Lee, H. 18 Lee, R. 73, 76 Lehrer, R.I. 211 Leibowitz, J.O. 107 Lendrum, A.C. 23 Leonardo da Vinci 45, 110 Le Quesne, L.P. 113, 188 Lerman, A. 213 Leung, L.L. 19 Lev, M. 127–129, 131, 137 Levi, M. 38 Lévi-Strauss, C. 45 Levy, N.S. 202 Lewis, J.S. 200, 209, 210 Li, Q.J. 205 Li, X. 208 Lidell, J.A. 89 Lillehei, R.C. 156 Lindeborn, G.A. 50 Lister, J.L. 60–62, 67, 68, m 72, 76, 82, 88–92, 94, 97, 99, 111, 112, 116, 123, 139, 148, 151, 166, 169, 188, 191, 222, 225, 226, 229, 245 Lo, L.W. 199, 200, 203 Lobstein, J.F. 73, 74 Locke, J. 47, 50, 145, 146, 234, 236, 244 Loeb, J. 247 Loeb, L. 96 Long, E.R. 8 Lopez, S. 209 Lorant, D.E. 206, 207 Lotze, R.H. 246 Lowenhaupt, R.W. 19, 101 Lower, R. 48 Lo¢¢wit, 92 Lubnitsky, S. 92–94 Ludwig, K. 240, 242, 248, 250 Lundblad, M.S. 216 Lurie, F. 131, 134, 137, 138 Luu, T.N. 217

MacFarlane, R.G. 12, 16, 23, 66 Mach, E. 238, 242, 248 Machi, J. 138 Mackie, J. 236 Mackman, N. 204 Magendie, F. 244 Maines, M.D. 215 Makin, A. 6 Malmqvist, U. 135, 148 Malone, P.C. 9, 41, 42, 110, 125, 145, 154, 170, 173–175, 178, 180, 182, 194, 227

Author Index Malpighi, M. 49, 51, 55, 76, 77, 88, 111, 146 Malpother, E.D. 113 Mammen, E.F. 5, 6, 113, 132 Manak, J.R. 200 Mann, F.D. 12 Mann, K.G. 20 Mannucci, P.M. 25 Mantezagga, I. 92 Marchand, F. 222, 224, 226, 230 Marcus, A.J. 17 Marey, E-J. 55 Mariani, G. 20 Markwardt, F. 39 Marin, V. 208 Marshenko, S.M. 213 Martinelli, I. 2 Masaki, T. 213 Mason, R.G. 196, 207 Massberg, S. 207 Massell, T.B. 113 Mathie, R.T. 214 Matveev, V.V. 93 May, A.E. 214 Mayer, G.A. 39 Mayr, E. 66 Mazza, J.J. 34 McCartney, J.S. 84, 113 McConnell, D.J. 26 McGraw, R.A. 20 McLachlin, A.D. 4, 6, 76, 91, 113, 114, 117, 122, 123, 145, 151, 173, 183 McLachlin, J. 4, 6, 76, 91, 113, 114, 117, 122, 124, 125, 145, 151, 152, 173, 183 McLean, J. 65 McMillan, D.E. 134 McQuillan, L.P. 199 Mechtcheriakova, D. 201, 202 Medicus, F.C. 48, 95, 243 Meijers, J.C. 36 Mellanby, J. 64 Merli, G.J. 113 Merz, J.T. 71 Metchnikoff, E. 92, 99 Meyer, D. 17, 162 Michal, F. 17 Michiels, C. 207, 208, 210 Mill, J.S. 242 Minami, T. 203–205 Miranov, A.A. 212 Miranti, C.K. 200 Mishra, S. 163 Mitchell, J.R.A. 34 Mohun, T. 200

Author Index Monod, J. 66, 243, 245, 248 Monos, E. 135, 210, 216 Monto, R.W. 32, 33 Morange, P.E. 37 Mora-Garcia, P. 202 Morawitz, P. 13, 63, 68, 223 Morgagni, J.B. 165, 220, 226 Morita, T. 207, 215 Morrell, M.T. 217 Morrissey, J.H. 204 Morrison, A.D. 142, 196 Morssink, L.P. 38 Motterlini, R. 215 Movat, H.Z. 17, 101 Muhm, J.M. 166 Müller, J.M. 203, 239, 240, 245 Müller, J.P. 69 Murray, D.W.G. 39, 66 Mustard, J.F. 13, 14, 33, 113

Nachman, R.L. 17, 19 Nagel, T. 210 Nagumo, K. 218 Nagy, I. 35 Naka, M. 18 Napoleone, E. 218 Naruse, K. 213 Needham, D.M. 48 Negus, D. 40 Nemerson, Y. 20, 196 Neumann, R. 115 Newton, I. 49, 50, 63, 68, 106, 135, 146, 235–237, 239, 249, 250 Nguyen, H.Q. 200 Nicolaides, A.N. 4, 138 Niewiarowski, S. 25 Nohe, B. 212 Nolf, P. 13, 64 Nuland, S. 72, 240 Nunn, J.F. 163 Nygaard, K.K. 32, 58 Nylander, G. 5

O’Brien, P.J. 205 Ochsner, A. 13, 33, 113 Ogawa, S. 206 Ogston, D. 15, 34, 96, 97 Ohtani, K. 199 Okada, M. 199, 204 Okazaki, H. 205 Olsson, R.A. 214 O¢Neill, J. F. 150–152, 156, 161, 162

301 Ono, T. 131 Onohara, T. 213 Opstvedt, A. 17 Oresme, N. 106, 235 Ostwald, W. 238, 249 Ouchi, H. 23 Ouellette, A.J. 199 Owen, C.A. 8, 12, 14, 25, 83 Owen, J. 35 Owre, A. 113 Owren, P.A. 12, 14, 18, 19

Packham, M.J. 33 Pagel, W. 106 Panum, C. 92 Paramo, J.A. 25 Pasteur, L. 62, 68, 82, 84, 89, 90, 92, 93, 248 Patek, A.J. 66 Paterson, J.C. 91, 113, 114, 122–125, 131, 151–154, 156, 173 Pavlov, I. 92 Pavlovsky, A. 12 Payling Wright, H. 14 Peerschke, E.I. 119 Peters, M.J. 207 Peterson, C.W. 1, 4 Phillips, M.N. 23, 25, 118 Pinsky, D.J. 197, 199, 200 Pitney, W.R. 34 Pitres, A. 92, 101 Pitto, R.P. 138 Plato 44, 239 Plow, E.F. 25 Pohlman, T.H. 196 Poller, L. 113 Poole, J.C.F. 19, 34, 60 Poort, S.R. 36 Porat, R.M. 227, 228, 231 Porter, R. 44, 105, 147 Pravaz, M. 139 Prentice, C.R. 24 Preyer, W. 92 Priestley, J. 47, 54, 148 Prout, W. 54 Prywes, R. 200 Pulvertaft, R.J.V. 15 Purkinje, J.E. 93

Qui, Y. 17, 133 Quick, A.J. 12, 13, 66 Qureshi, S.A. 200

302 Rabiet, M.J. 206 Rahman, A. 200, 205 Ramzi, D.W. 8 Rand, J.H. 37 Rao, V.N. 200 Raspail, F-V. 60, 72, 76 Ratnoff, O.D. 19, 34 Rees, D.C. 35 Reil, J. 48 Reitsma, P.H. 34 Remak, R. 93 Reneau, D. 115 Ribbert, H. 222–224, 226–230 Richards, D.W. 113 Ricote, M.F. 204–209 Ridker, P.M. 39 Riewald, M. 205 Rijken, D.C. 24 Rinaldo, J.E. 212 Riolan, J. 107, 108 Robbins, K.C. 23 Robbins, S.L. 222 Robb-Smith, A.H.T. 72 Roberts, V.C. 138 Rokitansky, C. 76 Rollin, S. 210 Rongen, G.A. 216 Rosendaal, F.R. 2, 4, 34, 35, 38, 103 Rosing, J. 20 Rössle, R. 115 Rost, F. 222, 223, 226, 230 Roupell, G.L. 58 Rubanyi, G. 196, 223, 226, 230 Russell, B. 236, 251 Ryan, U.S. 196, 206 Rybak, M.E. 21 Rydholm, H.E. 204

Sainani, G.S. and R. 36 Salem, H.H. 25 Salzman, E.W. 5, 40, 138, 139 Samama, M.M. 2 Samuels, P.B. 150, 151, 156 Sandison, J.C. 99–101, 112, 164 Santayana, G. 246 Santiago, F. 202 Saphir, O. 127–129, 131, 137 Satonaka, H. 209 Savage, B. 266 Schaefer, C.A. 212 Schaefer, U. 211 Schafer, A.I. 35 Schalch, P. 199

Author Index Schaub, R.G. 115, 211 Schecter, A.D. 209 Scheele, S. 148 Schelling, F.W.A. von 238 Schleiden, M. 60, 246 Schmidt, A. 12, 13, 26, 57, 58, 61, 62, 68, 94, 223, 225 Schopenhauer, A. 72, 235, 239, 240, 242 Schreijer, A.J.M. 167 Schubert-Soldern, R. 243, 244 Schultze, M. 93 Schwachtgen, J.L. 199, 202 Schwann, T. 60, 94, 246 Schwarz, T. 2 Schwencke, T. 57 Scott, W.J. 113 Scott Isenberg, J. 210 Scultetus, A.H. 107, 108 Scurr, J.H. 2, 138 Seegers, W.H. 26, 64 Sellak, H. 211 Servetus, M. 110 Sevitt, S. 34, 39, 40, 114, 115, 124–127, 131, 132, 138, 152–157, 159, 160, 182 Sherrington, C. 248 Sherry, S. 4, 19 Shimizu, Y. 210 Shreeniwas, R. 197 Sigel, B. 217 Signorelli, S.S. 209 Silver, I.A. 111, 145 Silverberg, M. 19 Simionescu, M. 206 Simon, S.I. 210 Simpson, C.K. 185 Sise, H.S. 13, 33, 39 Sixma, J.J. 17, 115 Skalak, R. 110, 135 Skovby, F. 36 Smith, B.D. 135 Smith, C.W. 208 Smithies, J. 245 Soderqvist, T. 22 Soh, J.W. 201, 203 Soloviev, A.I. 215 Solovey, A. 204 Solum, N.O. 20 Soulier, J.P. 33 Spurgin, J. 50 Squizzato, A. 218 Stahl, G.E. 47, 48, 51, 243, 251 Stamatakis, J.D. 5 Stanton, J.R. 8, 15

Author Index Starr, D.P. 44, 50 Stein, B.N. 210 Stenina, O.I. 205 Stevens, W. 53, 54 Stewart, G.J. 101. 127, 130, 131, 133, 136, 152, 164, 173, 183, 211 Stiles, G.L. 214 Stone, E.A. 127, 130, 133, 136, 152, 173 Strachan, L. 34 Su, W.H. 210 Sudoh, M. 167 Sugama, Y. 25, 211 Sukhatme, V.P. 199 Sultan, S. 213 Sung, C.P. 208 Suttie, J.W. 20 Suzuki, M. 211 Swiatkiewicz, A. 36 Sylvius, J. 107–109 Syme, J. 76

Tait, J. 112 Takahashi, M. 209, 212 Takase, S. 217 Takeya, H. 206 Talbot, J.H. 73, 83 Teich, M. 48 Temkin, O. 71 Ten, V.S. 47 Thackrah, C.T. 53, 59, 61 Thaler, E. 35 Thiagarajan, P. 5, 8 Thom, S.R. 211 Thomas, D.P. 14–16, 34, 57, 115, 139, 163, 176, 181, 243 Thompson, D’Arcy W. 247 Thomson, W. 112 Thorneycroft, I.H. 34 Thorsen, S. 25 Thurston, G.B. 137 Tillett, W.S. 65 Tobu, M. 206, 207 Todd, A.S. 24 Todd, R.B. 47 Toft, W.D. 167 Tollefsen, D.M. 36 Torn, M. 40 Trampont, P. 199 Traube, P. 77, 78 Travers, B. 112 Treisman, R. 200 Trousseau, A. 32 Trowbridge, E.A. 137

303 Tsukahara, H. 213 Tyndall, J. 238

Valen, G. 215 Valentin, G.G. 93 Vance, B.M. 84 Van Dieijen, G. 20 Vandenbroucke, J.P. 38 Van Helmont, J. 47 Vanhoutte, P.M. 213, 214, 217 Van Hylckama Vlieg, A. 36 Van Leeuwenhoek, A. 49, 88 Van der Veer, J.B. 14 Van Ottingen, W.F. 162, 164 Van Swieten, Baron 73 Veit-Brause, I. 234 Verso, M.L. 73 Vesalius, A. 45, 46, 106, 109, 110, 235 Vialov, S.L. 212 Viëtor, K. 238 Vinten-Johansen, J. 210 Virchow, R.L.K. 5, 7, 8, 11, 14, 16, 31, 32, 41–43, 57, 60, 67–69, 71, 75–85, 87–89, 91–96, 98, 99, 111, 112, 115, 116, 123, 125, 152–154, 164, 165, 171–175, 188, 223, 226, 235, 240, 243, 245 Vogt, C.C. 246 Von Liebig, J. 54, 62 Von Recklinghausen, F. 92, 98, 118, 123, 125 Von Willebrand, E.A. 66

Waddell, W.W. 66 Wagner, R.H. 27 Waldmann, R. 19 Waldron, J.M. 33 Walker, F.J. 26 Waller, A. 91, 112 Walmsley, S.R. 199 Wang, G. 209 Wang, Y. 210 Ware, A.G. 12 Ware, J. 18 Warlow, C.P. 73, 113 Warren, J. 196, 206 Warren, R. 23, 33, 73 Waters, C.M. 199 Webster, D.R. 19 Webster, M.E. 19 Weiss, H.J. 206 Welch, W.H. 15, 16, 34, 42, 60, 65, 68, 79, 80, 95–97, 101, 114, 169

304 Wells, P.S. 40 Wenger, R.H. 199 Wessler, S. 13–15, 33, 34, 113 West, G.B. 251 Wharton Jones, T. 139 Wheatley, D.N. 66, 110, 250 White, J.G. 17, 19 White, R.H. 2 Whitehead, A.N. 42 Whitmarsh, A.J. 201, 203 Wilkins, R.W. 8 Willam, C. 199 William of Ockham 251, 252 Wilson, L.B. 96 Wiltzius, P. 23 Wiseman, R. 45, 48 Woldhuis, B. 23, 176, 206 Wood, K.S. 215 Wright, I.S. 23, 113 Wu, S-Q. 202, 205

Author Index Yamakawa, M. 199 Yan, S.-F. 199, 200, 204, 205 Yan, W. 199 Yang, L. 210 Yang, S-H. 200 Yang, S.T. 215 Yuan, G. 199

Zagorska, A. 199 Zahn, F.W. 79, 89, 92, 94, 97, 101, 123, 174, 223 Zeiss, C. 92 Zeng, X. 36 Zhang, J. 218 Zur, M. 20 Zweifach, B.W. 113

Subject Index

α2-antiplasmin 25 acetylcholine 214, 216, 217 achromatic lens, microscope 52, 60, 72, 76, 93 ACTH 33 actin 198, 200, 206, 211, 217 activate, -ation 5, 6, 12, 13, 15–18, 20, 23–25, 27, 28, 36, 64, 66, 135, 163, 166, 195, 201–210, 212, 214, 216, 217, 222, 225 activated protein C 35, 190, 205 - deficiency 35 acute 6, 42, 76, 162, 166, 167, 226–228 - mountain sickness 166 adenosine 207, 214 ADP 17–19, 115, 116, 125, 138 ADPase 23, 125, 196, 207, 213 adductor 115 adrenalin 17 aetiology 1–6, 8, 11, 14, 15, 31, 33, 36, 38, 39, 41, 45, 48, 54, 59, 68, 83, 85, 87, 96, 106, 113, 114, 116, 121, 125, 134, 148, 161, 164, 168–170, 172, 190, 195, 204, 206, 219–221 age, -ing 1, 50, 92, 95, 105, 124, 131, 137, 145, 186, 190, 217, 235, 237, 247 agger 127, 129, 136, 155, 159, 173, 178 aggregate, -ation 17, 81, 94, 115, 125, 165 agonal, -ly 221, 224, 226, 228, 229 agonist, -ic 132, 136 air travel 167 alchemy, -ical 46, 50, 243 alcohol 50, 63, 184–186 al-Tasrif 45 ambulant 192, 193 amputate, -ation 191 anaemia 57, 161–163, 167

anaerobic 135, 144, 148, 150, 191, 192, 198, 229 analgesic 184 analysis 46, 47, 54, 157, 234, 251 anatomy, -ical 45, 48, 51, 67–69, 72, 73, 75, 76, 78, 79, 82, 92, 95, 105, 106, 108, 109, 115, 125, 127, 136, 173, 183, 196, 197, 242 anchored, -ing 67, 71, 79, 80, 84, 130, 132, 148, 159, 195, 197, 202, 206–212, 214, 216 angiogenesis 136, 197, 199, 218 angiotensin II 209, 217 animal chemistry 41, 47, 48, 50, 51, 53, 54, 57, 58, 76 animal experiments 132, 149, 194, 212 animism 243, 245 anoxia 114, 156 antagonist, -ic 69, 132, 136, 242 ante-mortem 165, 224, 226, 228, 229, 231 antibiotic 14, 96 anticoagulant, - ation 7, 8, 11, 14, 17, 26, 31–34, 36, 38–40, 44, 65, 66, 170, 171, 176, 187, 193, 204, 212, 218, 231, 249 antigen 208, 210 antihaemophilic globulin 12, 28 antilupus antibody 37 antiphospholipid syndrome 36 antiplasmin 66 antithrombin III 26, 29, 35, 37 - deficiency of 36 anti-Trendelenburg 187 apoptosis 148, 206, 214 arachidonic acid 17, 213, 214 Aristotelian, -ism 46, 47, 235 artefact, -ual 34, 97, 149, 154, 156, 160, 182 arterial thrombosis 6, 7, 34–37, 39 arteriole 99, 136, 216, 217

305

306 artery, -ies 9, 17, 44, 45, 75, 77, 78, 83, 103, 105, 116, 117, 121, 135, 142, 162, 172, 202, 210, 212, 215, 216, 223, 227 asphyxia 162, 230 aspirin 17, 176, 177, 207 atomic theory 68, 249 atomism 236 ATP 148, 198, 203, 212, 214, 216, 217 auricle, -ular 105, 231 autochthonous 7, 34, 88, 96, 97, 99, 125, 141, 145, 153, 174, 180, 183, 225 autoimmune, -ity 2, 137, 189 autolysis 148 autonomic nerve 136, 216 autopsy 75, 98, 165, 224, 226, 227, 230 avascular, -ity 127, 136, 140, 148, 151, 206, 210

Baconian 48, 236, 237 bacteria 16, 92, 96 bacterial endotoxin 34, 204, 211, 218 barbiturate 145, 180 basement membrane 125, 126, 154–156, 158, 178 bed 7, 37, 113, 114, 118, 132, 149, 186, 187, 193, 230, 231 - rest 2, 14, 40, 113–115, 138 Bernouille’s law 133 biochemistry 11, 47, 63, 68, 169, 196, 247–250 biomedical 41, 43, 44, 58, 68, 71, 111, 139, 194, 220, 233, 235, 252 - research 41, 49, 170, 220, 251, 252 bleeding diatheses 12–14, 27, 37, 58, 66, 234 bleeding time 32 blood - cells 23, 49, 53, 91–93, 100, 112, 115, 137, 141–144, 148, 151, 154, 156, 160, 166, 171, 174, 175, 177, 178, 187, 190–192, 207, 208, 211, 219, 222, 226, 227, 229, 230 - dead 25, 174, 192, 219 - living 98, 100, 166, 171, 174, 211, 227, 229 - column, weight of 118, 185 - flow 4–6, 39, 42, 49, 51, 53, 55, 79, 96, 99, 104, 110, 112, 113, 116, 117, 119, 121, 134, 136, 138, 140, 143, 149, 150, 156, 167, 171, 172, 175, 177, 181, 183, 185, 189, 190, 193, 194, 210, 213, 216, 217, 219, 225, 230

Subject Index - interrupted 83, 171 - retarded 81, 119, 164 - non-pulsatile 103, 119, 164 - pulsatile 138, 150, 177 - streamline 103, 173, 175 - velocity 138 - volume 117, 138 - group 2, 36 - moving 105, 112, 113 - sequestered 144 - static 43, 53, 67, 71, 77, 78, 84, 171 - volume 40, 78, 105, 113, 135, 163, 188, 226, 231 blood group A 2, 36 buffy coat 165, 175, 223, 224, 227

cadaver blood 147, 164, 229, 230 calcineurin 209 calcium 17, 20, 28, 63–65, 200, 203, 206, 207, 212–217 calf 115, 117, 132, 138, 166, 173, 184, 188, 231 calmodulin 200 capillary, -ies 4, 49, 51, 74, 75, 82, 99, 100, 105, 112, 127, 136, 162, 195, 208, 212 carbon dioxide 54, 226 carbon monoxide 147, 150, 161–163, 165, 196, 215 cardiac - disease 2 - insufficiency 196 - output 55, 134, 227 - valve 231 cascade, coagulation 5, 6, 9, 11–28, 35, 58, 115, 139, 166, 219 casein kinase II 200 caudad 109, 133, 175 causal factor 6, 76, 81, 85, 96, 185 cause, causation 1–4, 6, 14, 38, 51–53, 67, 73, 77, 78, 80–83, 88, 90, 95, 98, 110, 113, 116, 138, 149, 150, 154, 162, 163, 165, 171, 172, 176, 183–188, 190, 197, 218, 220, 229, 231, 234, 240, 242, 243, 245, 251 cell - biology 93, 233, 248 - cultured 197 - phenotype 148, 195, 198, 204, 212, 227 - structure 92 - surface marker 200, 209 - theory 60, 72, 91, 93, 94, 246

Subject Index cellular pathology 57, 69, 71, 72, 76, 85, 95 centrifugal 109, 118, 126, 154 centripetal 110, 141 cephalad 133, 136, 175, 197, 225 chair 2, 69, 73, 78, 149, 183, 185, 186 channel 114, 152, 165, 213, 216, 230 chemistry 41, 47, 48, 50, 51, 53, 54, 58, 59, 63, 66, 69, 70, 76, 233, 241, 242, 247, 248, 250 chemoattractant protein 36, 209 chemotaxis, -tactic 17, 92, 101, 208, 209 Christmas disease 12, 28 chronic valve incompetence 1 chronic venous disease 4, 9, 189, 219 circulation 6, 14, 16, 23, 42, 44, 49, 52, 54, 55, 74, 77, 78, 91, 92, 96, 100, 101, 118, 121, 132–134, 137, 139, 148, 150, 163, 167, 172, 176, 183, 191, 207, 208, 211, 223, 227, 229, 230, 231 - interrupted 79, 83, 96, 103–118, 121, 172 circulatory 77, 78, 100, 103, 104, 113, 114, 134, 146, 163, 190, 234 - failure 165, 173, 227, 228, 230 cirrhosis 73 classical hypothesis of blood coagulation 57, 63, 68 clonus 166, 180, 194 clot – cadaver 221–232 - streamers 223 - white 223 - yellow 223 clotting 12, 17, 43, 53, 58, 60, 62, 64, 65, 67, 79, 125, 151, 162, 166, 176, 222, 224, 225, 229, 234, 252 - time 12, 32, 59, 64 coagulable 7, 9, 33, 37, 44, 53, 73, 96, 151, 162, 176 coagulation 4–9, 11–20, 23, 25–28, 32–38, 40, 41, 43–45, 52, 54, 55, 57–71, 74, 75, 79, 81–83, 91–94, 111, 113, 115, 116, 122, 139, 151, 164–166, 169, 171, 174, 176, 183, 187, 195, 196, 200, 202, 204–206, 208–212, 214, 216, 218, 219, 222, 223, 225–228, 230, 231, 249 - activated 5, 34, 113, 115, 116, 219 coagulation factors 12, 13, 16, 19, 20, 27, 34–36, 57, 59, 113, 115, 116, 122, 202, 204, 216, 219 - factor V 12, 13, 20, 35, 37, 38, 190 - factor VII 12, 13, 20, 35, 209 - factor VIII 12, 17, 20, 28, 35, 36, 66, 196

307 - factor IX 12, 20, 28, 36 - factor X 13, 20, 28, 196 - factor XI 19, 20, 28, 35, 36 - factor XII 19, 28 - factor XIII 23, 29, 36 coagulum, -a 61, 80, 82, 88, 91, 94, 145, 159, 165, 174, 176, 177, 183, 222–231 coal gas 150, 162 collagen 17, 19, 28, 127, 131, 136, 195, 209, 216 colour 69, 229, 239, 242 colourless corpuscles 60, 76, 78, 79, 91, 92, 112, 139, 162, 175, 223, 231 complement 26, 44, 88, 122, 211, 251 complexity 238, 244 compression 79, 100, 117, 138, 183, 192, 193 - intermittent pneumatic 138, 170, 249 - stockings 8 - treatment 138 connective 17, 19, 130, 131, 136 ‘consensus model’ of DVT 1, 8, 11–29, 31, 41, 54, 58, 67, 84, 101, 111–113, 194, 197, 218, 222, 248, 249 contact system (intrinsic system) 19–21, 26 contraction 5, 52, 54, 110, 117, 118, 165, 184, 187, 188, 213, 217 control 11, 16, 25, 26, 35, 66, 135, 136, 167, 185, 196, 210, 212, 242, 248, 249 convective water movement 68 C-reactive protein 209, 218 croton oil 139 crucifixion 188 cruor 165, 175, 223, 224, 227, 231 Cruveilhier’s disease 73 cultural 233–235 cyclic AMP (cAMP) 200, 206, 207, 211, 214 cyclic GMP (cGMP) 207 crypt 127, 131 current 139 cycle 105, 106, 117, 133–135, 137, 143, 190, 192, 227, 229, 230, 251 cycloxygenase 17, 215 cytokine 88, 197, 198, 200, 204, 208–210, 218, 219

dead, death 3, 25, 51, 52, 54, 61, 62, 67, 68, 74, 75, 84, 85, 92, 96–98, 103, 104, 106, 112, 116, 143, 148, 149, 151, 154, 156, 159, 164–167, 169, 171, 172, 174, 176, 177, 192, 219

308 decompression 193 decubitus 2, 40, 109, 110, 112, 114, 124, 126, 139, 142, 147, 153, 163, 186–188, 190, 191, 193, 202, 221–231, 240, 248, 250, 252 deep venous thrombosis (DVF) 1–9, 11–29, 31–42, 48, 51, 53, 54, 58, 63, 67, 68, 74, 75, 84, 85, 95–97, 101, 103, 104, 106, 110, 111, 113– 116, 118, 119, 121–172, 174, 176, 183–197, 202, 204, 206, 209, 210, 212–214, 217–222, 228, 233, 234, 248–253 degenerate, -ation 88, 114, 154, 162, 164, 190, 212 dehisce, -ence 125, 153–156, 158–160, 206, 211 dehydration 96, 135, 166 denuded of endothelium 127, 154 deposition 34, 74, 79, 96, 123, 131, 136, 152, 163, 164, 175, 219, 225 development 1, 9, 11, 39, 41, 44, 47, 55, 58, 64, 69, 72, 75, 90, 93, 113–124, 151, 171, 178, 204, 212, 219, 239–244, 246–250 dextran 40, 188 diabetes 4, 38, 39 diagnosis 4, 38, 39 diapedesis 91, 112, 163, 198, 208 diastole, -ic 55, 105, 117, 172, 173, 180 dicoumarol 39, 57, 66 differentiation 199, 200, 209 diffusion 178, 229, 244, 249, 250 dissect, -ion 75, 106, 109, 114, 151, 153, 180, 201 distal 4, 18, 127, 130, 154 distensible 127, 133, 216 D-dimer 4 doctrine of Cruveilhier 71, 73, 76 dog 53, 113, 145, 154, 175, 178, 181, 182 dogma 50, 111, 112 double thrombi 125, 157 drowning 166, 230 DVT, see deep venous thrombosis

early growth response-1 (egr-1) 195, 199–205, 218, 219, 252 economy class syndrome 2 eddy, -ies 96, 98, 115, 123 education 253 eicosanoids 17, 198, 215 elastic stocking 113 elastin 127, 131, 216

Subject Index elderly patient 114 elk-1 195, 200, 201, 203–205, 219 embolism, pulmonary 1, 4, 9, 15, 37, 73, 95, 126, 161, 171, 184, 185, 188, 217 embolus, -i 3, 4, 8, 71, 75, 76, 78, 81, 83, 84, 153, 159, 161, 162, 231 emotion 236 empirical 7, 14, 33, 40, 152, 188, 251 empiricism, -ist 50, 235–238, 242, 251 emptying of valve pockets 119, 134, 137, 150, 178, 193 encode, encoding 36, 66, 200 endocarditis 130 endothelial cell 17, 20, 25, 29, 36, 37, 97, 127, 135, 141, 143, 144, 148, 149, 152, 164, 178, 192, 195–198, 202, 203, 209, 210, 213, 227, 233 endothelial injury 1, 5, 6, 34, 114, 149, 153, 164, 183 endothelin-1 207, 211, 213, 215 endotheliocyte 212 endothelium, venous 11, 15, 73, 84, 139, 145, 147, 151, 163, 166, 167, 183, 228 endothelium-derived relaxation factor 213–217 Enlightenment 47, 71, 234, 236, 237, 242 Entelechy 47, 243, 247 enzyme 12, 16, 20, 36, 195, 199, 248 epistemology, -ical 235, 237, 238, 242 ERK-1 201, 203, 209, 210 erythrocyte 52, 79, 93, 99, 100, 115, 124, 135, 137, 138, 140, 144, 162, 230 - sedimentation rate 224 Esmarch bandaging 149 ethnic differences in susceptibility 185, 189 evacuation of blood from VVP 133, 134 evolution, -ary 6, 14, 41, 42, 44–46, 54, 66, 71, 103, 238, 240, 242 exercise 62, 133, 166 exogenous irritant 141 experiment, -al 7, 9, 13, 34, 48–50, 53, 55, 57, 59–62, 64, 65, 68, 71, 74, 78, 84, 96–98, 100, 105, 111–114, 123, 125, 132, 135, 137–139, 145, 148–150, 154, 163, 165–167, 173, 175, 178, 180–183, 185, 191, 194, 210, 212, 225, 229, 237, 240–242, 247, 253 - thrombi 15, 31, 34, 97, 125, 180 exsanguination 149, 228 extensor 132

Subject Index extracellular matrix (ECM) 195, 198–200, 206, 208, 211, 217 extravascular 204

factor V Leyden 35, 37, 38, 167 fallacy of misplaced concreteness 43, 252 femoral vein 5, 80, 12, 123 127, 128, 132, 153, 178, 181, 182, 189, 206, 224 ferment 48, 61, 63, 72, 225 fibre 44, 52, 217 fibrillation 132, 231 fibrin 4, 5, 15, 16, 21, 23–26, 28, 29, 32, 34, 52, 60–64, 77, 79, 91, 93–96, 101, 124, 125, 131, 144, 163, 164, 174–176, 181, 183, 200, 206, 219, 222, 224–226, 230 - cross-linking 16, 23, 25 - ferment 61, 63 fibrinogen 18, 19, 21–23, 25, 28, 53, 61–64, 206, 207 fibrinogenesis, -genic 11, 13, 16, 19–23, 25, 35, 63, 115, 171, 174, 176, 218, 231 - control of 16, 35 fibrinolysis, -lytic 4–6, 13, 16, 23–25, 34–36, 40, 139, 166, 176, 187, 196, 197, 202, 210, 212, 216, 218, 222 fibrinopeptides 23 fibroblast 136, 199, 204 fibroblast growth factor (FGF) 200, 202 - basic (bFGF) 199 fibronectin 17, 195, 211 fixative, - ing, -ion 139, 153, 156, 157, 160 flap of endothelial tissue 132 flap, -ping, of valve cusps 193 flexor 132 flow - pattern 179, 180 - secondary 137 - separation 133 fluid, -ity 12, 23, 49–51, 53, 59–63, 82, 96, 110, 112, 118, 133–135, 148, 151, 165, 166, 171, 184, 188, 195, 222–225, 229, 230, 249 flux 105 foot 117, 118, 138, 184, 185, 193 forensic 221, 224, 228, 230 fracture 5, 40, 153, 182 function 9, 19, 28, 33, 35, 42, 50, 52, 57, 61, 81, 93, 103, 194, 109, 117, 121, 122, 127, 132, 133, 135, 148, 154, 159, 165, 169, 190, 197, 206, 219, 231, 246, 251

309 Galenic, -ism 77, 103–105, 109, 111, 146, 169 gangrene 162 gastrocnemius 132 gene expression 195, 197, 198, 201, 248 general anaesthesia 2, 180 genetic 2, 233, 238 geometry, vessel wall 134 germ theory of disease 68. 84, 90 German science, 19th century 81, 233, 234 globule 49, 51, 52, 60, 77, 91, 112 glycolysis 64, 135, 148, 198 glycoprotein 18–21, 25, 28, 36, 206, 207 glycosaminoglycan 19, 115, 217 G-protein 17, 25, 204, 205, 209, 214 granulocyte - colony-stimulating factor (GCSF) 202 gravity 83, 117, 118, 125, 184, 187, 189, 192, 237 guanylate cyclase 163, 211

haem 163, 215 haematocrit 135, 189 haematology, -ical 7, 9, 14, 15, 40, 41, 44, 53, 54, 57, 58, 63, 64, 67, 68, 72, 92, 93, 101, 134, 169 haematuria 32 haemodynamic(s) 5, 41, 51, 52, 53, 55, 77, 111, 117, 172, 210, 216 haemoglobin 52, 81, 162, 163, 167 haemopathology 58 haemophilia 12–14, 28, 45, 57–59, 64, 66 haemorrhage 37, 113, 228 haemorrhagica 12 haemostasis 7, 14, 16, 19, 27, 33, 44, 52, 60, 65, 67, 96, 113, 204, 233 haemostatic plug 13, 23, 25, 28, 52, 58, 93 haem oxygenase 163, 215 hanging 17, 148, 165, 166, 226, 230 heart - failure 114, 231 - left 105, 167 - right 118, 121, 122, 227, 228 - valves 110, 224, 227, 228 heat 5, 52, 88, 249 hegemony of consensus model 7 heparan sulphate 26, 36 heparin 8, 26, 39, 40, 57, 58, 64–66, 151 heparin cofactor II 26, 36 hepatotoxicity 33 high molecular weight kininogen 19, 196 hip replacement 5, 138 Hirudo medicinalis 39

310 hirudin 39 histology, -ogical 15, 16, 67, 69, 79, 81, 84, 96, 114, 125, 145, 150, 154, 156, 157, 171–173, 176, 180, 222, 225, 242 histopathology, -ist 132, 152 historical exegesis 1, 9, 40 history 2, 5, 9, 12, 39, 42, 44–46, 48, 49, 51, 57–60, 62–64, 66, 68, 70, 71, 83, 87, 93, 103, 110, 111, 122, 145, 146, 160, 187, 188, 190, 193, 221, 232, 234, 236, 245, 253 - of haematology 44, 57, 70 holism, -ist 245, 251 homeostasis 248 horizontal 114, 117, 132, 138, 149,m 172, 175, 186, 190, 191, 193 hormesis 163 hormone, -al 189, 190, 218 House of Lords (UK) 2, 253 humoralism, -ist 45, 49–51, 54 hydrocoele fluid 60–63 hyperaemia 57 hypercoagulability 1, 4, 5, 8, 13, 14, 27, 31–40, 67, 84, 113, 115, 156, 171, 251 - acquired 2 hyperfibrinogenaemia 33, 35, 36 hyperhomocysteinaemia 36 hyperoxia 202 hypertension 217 hyperviscosity 135 hypnotic 184, 186, 192 hypobaric hypoxia 166, 167 hypokinetic hypoxia 163, 196 hypotension 149 hypothesis 9, 11–13, 31, 33, 34, 37, 38, 57, 58, 62–64, 68, 72, 75, 79, 81, 90, 94, 97, 101, 103–119, 139, 142, 143, 145–147, 149, 167–171, 185, 189, 194, 195, 221, 223, 225, 237 hypovolaemia 113, 135, 166, 167, 187, 189 hypoxaemia, -ic 9, 81, 100, 103, 114, 121, 133, 135, 136, 138–160, 162–167, 172–174, 178, 179, 183, 187, 190–192, 196, 197, 199, 207, 209, 212, 217, 219, 226–231 hypoxia, -ic 97, 98, 101, 110, 119, 121, 125, 126, 135, 136, 139, 141–145, 147–204, 206, 208–216, 218–221, 227, 233, 252 hypoxia-inducible factor (HIF) 199, 200, 252

Subject Index iatrochemistry 47, 48, 50, 51 iatromathematics 46 iatromechanics 241 iatrophysics 46–48, 50, 51, 55 ideology 72, 105, 110 idiopathic thromboembolism 183 iliac vein 78 immediate-early gene 200 immobilisation, -ed 16, 20, 73, 113, 114, 150, 170, 188 immune response 25, 204, 208 immunoglobulin 208 incidence of DVT 2, 5, 29, 36–38, 96, 132, 135, 138, 193 incoagulable 151, 165, 221, 226, 228 induction, -ive 147, 164, 199, 200, 202, 204, 210, 217, 236, 237 infection 2, 14, 37, 73, 88–90, 92, 95, 96, 152, 171, 204, 211, 218 inferior vena cava 53, 78, 79, 110, 132 inflammatory bowel disease 2, 218 inflammation 8, 44, 51–53, 60, 68, 74–77, 82–84, 87–89, 92, 95, 96, 112, 139, 171 infiltration 1, 2, 5, 73, 131, 152, 164 injured, injury 1, 2, 4–7, 9–17, 20, 23, 25, 34, 36, 37, 73, 76, 77, 84, 88, 96, 97, 100, 101, 112–114, 122, 125, 129–131, 134, 139, 142, 143, 147, 149, 151, 152, 155–157, 162, 164, 167, 168, 176, 183, 190, 191, 195, 196, 199, 202, 204, 207, 209–212, 216, 218, 219, 226, 230, 233 integrin 206, 208, 210, 211 intentional, -ity 66, 67, 99 intermittent pneumatic compression (IPPC) 113, 138, 192, 193, 249 interstitial adhesion molecule-1 (ICAM-1) 25, 198–202, 205, 206, 208–211, 213 interventricular septum 105, 110 intima, vascular 61 in vivo methods 17, 19, 65, 66 in vitro studies 57, 65, 234 intercellular 114 interleukin (IL) - 1 25, 197, 200, 203, 204, 208, 209 - 6 25, 197, 204, 207–209 - 8 36, 197, 208 intermittent circulation 121 intermittent claudication 32 internal organs 88 interrupted circulation 79, 83, 96, 103–119, 121, 152

Subject Index intima, venous 34, 125, 139, 141 - injury to 34, 125, 139 intravascular 13, 100, 132, 204, 228, 231 insensible 118, 132, 184 involuntary 118, 132 ion transporter 207 irritant 139, 141 ischaemia, - ic 81, 84, 156, 163, 164, 199 ischaemia-reperfusion injury (IRI) 9, 147, 190, 207, 210, 211, 218 isoform 197, 200, 201, 203, 205 isolation 150, 153 IV line 2, 190

jactitation 180 jaundice 66 JNK 200, 203, 208 judicial 221, 222, 224, 229, 230 jugular vein 53, 127, 131, 164, 181 junction - cell 198 - vein 114, 123, 152, 173

kallikrein 19, 26, 28 Kantian, -ism 235, 237, 239–241, 244, 248, 250 kinase 17, 197, 200–205, 209, 228 kinetic theory 68, 249

laboratory test, testing 31, 40 laminin 195 language 14, 2, 43, 47, 51, 63, 66, 68, 69, 111, 240, 243, 248, 252 laws of layers (judicial) 222, 224 layer, -ing 79, 98, 127, 135, 143, 151, 152, 154–157, 159, 168, 174–178, 182, 187, 191, 192, 219, 222, 224, 227 leech 39, 50 leptin 218 lesion 6, 12, 14–16, 75, 88, 96, 97, 110, 114, 126, 134, 149–153, 156, 163, 168, 172, 183, 185, 188, 212, 228, 231 leukaemia 77, 89 leukocyte - activation 101 - congregation 112, 124, 144, 208, 211, 212 - debris 76, 84, 89, 93

311 - infiltration 1, 4, 131, 152, 164 - migration 84, 112, 200, 210 - recruitment 208 life, ‘cause of’ 52, 240, 242, 243, 245 life, ‘effects of’ 52, 171, 240, 243, 247, 249 ligation 105 ligature 53, 96 limbs - dependent 138, 183, 185 - elevated 138 - horizontal 138 linear blood flow 115, 138, 216 lines of Zahn 79, 89, 123, 174, 223 lipopolysaccharide 204 lipoxygenation 211 liver 50, 73, 105, 110, 246 living 5, 17, 25, 43, 47–49, 52, 53, 61, 62, 66–68, 72, 74, 92–94, 97–101, 110, 112, 148, 151, 154, 156, 164–166, 171, 174, 177, 178, 189, 211, 223, 226, 229, 234, 239–241, 243–249 lumen, luminal 73–75, 80, 81, 89, 99, 110, 123, 125, 127, 130–134, 136, 137, 140–143, 149, 150, 152–155, 158, 172, 173, 175, 177–179, 189, 194, 199, 206, 214, 217, 225, 228 luminalis endothelium 126, 131, 151, 173, 175 lung 49, 50, 73, 75, 83, 105, 147, 150, 163, 202, 231 lymph 53, 73, 77, 91, 112, 175 lymphocyte 199, 208

Mac-1 207 macrophage 92, 99, 200, 209–212, 216, 218 - colony stimulating factor (MCSF) 200, 201 malabsorption 66 ‘manometer-like connections’ 225 MAPK phosphatase 1 209 margination 76, 77, 84, 92, 99, 112, 129, 139, 147, 163, 164, 176, 187, 192 materialism 57, 60, 62, 63, 68, 69, 72, 94, 95, 98, 233, 234, 241–244, 246, 247, 252 mathematical 46, 47, 238, 241, 242 Maxwellian 134 MCP-1 209 mechanics 46, 47, 51, 63, 68, 106, 110, 145, 235–237, 240–242, 249, 250

312 mechanism, -istic 7, 9, 11–13, 16, 23, 32, 37, 40, 41, 44, 46–52, 54, 55, 57, 58, 60–63, 66–72, 77, 84, 91–98, 100, 101, 112, 113, 118, 119, 129, 130, 132, 135, 136, 138, 144, 163–165, 169–171, 174, 177, 184, 190, 195, 197, 199, 202, 204, 207, 209, 212–214, 216–218, 220, 223–227, 233, 234, 236, 239–252 mechanistic materialism 57, 60–63, 68, 69, 71, 72, 92–95, 98, 240–242, 245, 247, 250 mechanosensor 216 media 217 megakaryocyte 17 membrane 16, 19, 23, 74, 93, 125, 126, 154–156, 158, 178, 203, 206–208, 213, 214, 217 - endothelial cell 16, 17 - platelet 17, 37 mesenteric vein 217 metabolism 36, 37, 142, 144, 148, 191, 192, 229 metalloproteinase 198, 200, 213 metaphor 43, 243, 252 metaphysic, -ical 45–51, 54, 61, 63, 66, 68, 69, 72, 92–95, 105, 106, 111, 171, 237, 239, 247, 251, 252 miasma 88, 89 microscope 52, 60, 72, 73, 76, 92, 99, 112, 225 - Abbe lens 92 - achromatic lens 52, 60, 72, 76, 93 - electron 155, 183 microscopy, -ist 41, 45, 49, 60, 71, 76, 77, 88, 91, 93, 112, 136, 141, 155, 183 - intravital 206 microvascular 139 mitogen 208 mitogen-activated protein kinase (MAPK) 200, 203 mobility 40, 113, 170, 190 molecular biology, -logical 136, 147, 190, 194–197, 234, 248–250 monocyte 36, 199, 200, 204, 207–209, 211, 216, 218 monolayer 145, 174, 195 morbid, -ity 1, 3, 4, 51, 73, 74, 79, 95, 225 morphology, -logical 4, 17, 23, 34, 91, 93, 108, 121, 122, 125, 127, 129, 136, 152, 170, 174, 194, 197, 200, 206, 209, 226 mortality 1, 3, 4, 38, 231

Subject Index motion 47, 53, 68, 98, 104, 105, 109, 110, 118, 235, 241, 249, 250 motor 172 movement 6, 8, 48, 51, 57, 68, 80, 81, 98, 103, 105, 109–113, 116–119, 121, 124, 132–134, 137, 140, 145, 149, 159, 167, 170, 171, 174, 183, 184, 192, 194, 210, 225, 236, 246, 249, 250 multifactorial 2, 189 multiple sclerosis 73 mural endothelium 97, 136, 143, 155, 159, 162, 219, 227 muscle 5, 52, 54, 78, 114, 115, 117–119, 127, 132, 145, 150, 172, 173, 180, 184, 185, 187, 189, 190, 194, 204, 206, 207, 231 - relaxant 186, 187, 192 - pump 6, 48, 114, 117, 133, 134, 166, 170, 173, 184 - smooth 127, 135, 136, 206–209, 213, 214, 216, 217 myocardial contraction 165 myosin 17, 214

narcosis 150 nationalism, -istic 69, 234 natural philosophy 41, 46–50, 105, 237 Nature 2, 45, 48, 51, 52, 61, 62, 69, 70, 73, 78, 93, 94, 99, 100, 106, 139, 148, 154, 191, 222, 233, 236–240, 243, 244, 246 Naturphilosophie 235, 238–240, 244–246, 248 Navier-Stokes equation 134 necropsy 114, 115 necrotic, -osis 39, 125, 126, 128, 130, 141, 142, 145, 148, 154, 156, 164, 171, 174, 177, 197, 202, 210, 212, 248 neointimal hyperplasia 199, 218 neoplasia 2 neuroleptic 184, 186 neuropeptide Y 208, 217 neutrophil 195, 197, 199, 207–212, 218 Newtonian mechanics 63, 68, 240, 250 NFκB 202, 205, 208, 209, 252 nidus, -i 124, 131, 132, 137, 138, 144, 152, 153, 168, 170, 177, 196 nitric oxide (NO) 17, 196, 198, 202, 207, 210, 212–217 nitric oxide synthetase (NOS) 199, 202, 211 non-perfusion 121, 159, 173, 217

Subject Index non-pulsatile 103, 110, 117, 119, 121, 133, 137, 138, 141, 143–145, 147, 154, 167, 169, 172–175, 178, 179, 181, 187, 189, 190, 192, 194, 197, 210, 212, 219 noumena 237 nucleotide transporter-1 199 nucleus, -i 93, 200, 203 nutrition, nutriment 76, 105, 49–152 Nygaard-Brown syndrome 32

obesity 218 obstruction 4, 45, 51, 53, 74, 75, 77, 78, 116, 150, 163, 171, 172, 180, 185, 232 occlusion 32, 73, 149, 232 Ockham’s Razor 252 oedema 4, 73–75, 184, 185 oil immersion 60, 92 omnis cellula e cellula 60, 76 omnis viva ex ovis 60 oncogene 200 ontology 50, 235, 243 oral anticoagulant 39 oral contraceptive 38, 167, 189, 218 organ 50, 51, 72–74, 88, 117, 167, 248 organicism, -ist 239, 248, 249 organisation 46, 239, 240, 243, 244, 246–248 organism 47, 51, 62, 67, 93, 96, 103, 110, 117, 238, 239, 241–244, 246, 248, 249 orthopaedic 149 oscillation 134, 137 osmotic 148 ostial valve 122, 123, 127, 129, 130, 132, 153–154, 157, 170, 173, 181, 189, 212 oximeter, -metry 151 oxygen 39, 71, 81, 82, 84, 138, 141–145, 147–152, 161–167, 173, 178, 190–192, 200, 215, 226, 227, 229, 231 oxygenated 138, 140, 143, 148, 150, 151, 154, 168, 169, 173, 174, 192, 219, 227, 230

p38 202, 203, 208 pain 88, 95, 187, 188, 190, 192, 193 paracrine 207 paradigm 12, 15, 42, 98, 104, 105, 110, 172 paralysis 2, 186, 187 paresis, -es 2, 114, 186, 187, 194

313 parietal valve 122, 127, 130, 153–154, 180, 182, 190 parietalis endothelium 9, 121, 125, 126, 131, 132, 140–144, 147, 151, 154–157, 159, 167, 169, 172–175, 182, 187, 191, 192, 196, 197, 206, 207, 212, 218, 219, 226 pathology, -ical, -ist 1, 3–6, 8, 9, 13–15, 19, 32, 37, 42, 44, 49, 50, 54, 57, 58, 60, 68, 69, 71–78, 81–83, 89, 91, 95, 97, 98, 103, 108, 110, 114, 118, 119, 121, 125, 129, 135–137, 148, 155–157, 167, 169–171, 175, 177, 183, 186, 194, 196, 209, 219, 222, 224, 226, 228, 229, 231, 233, 234, 239, 240 pathological anatomy 68, 69, 72, 78, 82 pathophysiology, -ical, -ist 9, 11, 38, 39, 41–44, 48, 53, 54, 57, 58, 63–69, 71, 84, 85, 87, 92, 95–98, 117, 165, 169, 170, 172, 185, 197, 218, 220, 224, 229, 233, 252 PECAM-1 (CD31) 6, 103, 115 perfusion, -ed 114, 115, 121, 127, 135–137, 140, 147, 148, 150, 151, 156, 159, 162–164, 169, 173, 188–190, 195, 196, 198 ‘peripheral venous heart’ 48, 117, 172, 173, 183, 184, 193 peri-operative 187 perivalvular 133 permissive factor 6, 103, 115 phagocyte, -itic 9, 25, 142, 143, 164, 172, 174, 177, 178, 211, 212, 219 - invasion 88 phagocytosis 16, 17, 91, 92, 99, 200, 209, 210 pharmaceutical 190, 249 phenomena 4, 9, 13, 45, 47, 59, 63, 74, 82, 83, 88, 91, 92, 100, 106, 116, 136, 139, 147, 163, 164, 171, 172, 224, 225, 229, 232, 233, 237, 238, 240, 241, 244, 251 phenotype, endothelial cell 195, 198, 227 philosopher 44, 49, 50, 60, 66, 106, 236, 237 philosophy, -ical 9, 38, 41, 42, 44–50, 54, 57, 62, 63, 66, 68, 69, 71, 75, 84, 85, 92, 98, 105, 111, 139, 169, 170, 174, 189, 225, 233–235, 237–242, 244, 245, 246, 249, 253 - of science 235, 241 phlebitis 8, 44, 71–75, 77, 82–84, 87–89, 95, 136, 171, 252

314 phlebographic 115 phlebothrombosis 88, 96 phlebotomy 74, 88 phosphatidylinositol 18, 20, 202 phosphatidyserine 18, 20 phospholipids 20, 36, 37, 65 phosphorylation 148, 200 physician 40, 49, 51, 57, 58, 61, 70, 82, 88, 113, 234, 252 physico-chemical 5, 66, 69, 93, 94, 248, 250 physics, -al 4, 42, 46, 54, 62, 66, 68, 69, 93, 94, 139, 149, 235, 240, 241, 242, 245, 247–250 physiology, -ical, -ist 15, 25, 33, 37, 38, 41, 42, 45, 46, 48, 52, 54, 58, 61, 63, 65, 67–69, 72, 77, 89, 92, 94, 95, 98, 104, 106, 109–112, 119, 121, 13, 133, 135, 137, 138, 142, 148, 156, 164, 167, 173, 184, 185, 188, 195–197, 204, 216, 229, 236, 239–242, 244–248, 250, 251 planet 106 plantar 115 plasma 16, 19, 20, 23, 25, 26, 28, 36, 57, 61, 63, 64, 125, 162, 209, 217, 222, 225 plasma protein 16, 19, 25, 26, 36, 43, 65, 94, 164 plasmin 23–25, 29, 202 plasminogen 23–25, 29, 36, 163, 196, 199, 201, 202, 209 platelet - activation 5, 16–18, 23, 195, 206, 207 - attachment to endothelium 16 - congregation 17, 60, 106, 107, 209, 213–215 - granules 17 - massing 138 - plug 16, 18–20, 23, 115 - receptors 17 platelet activating factor (PAF) 197, 202, 206–208, 210, 212 platelet derived growth factor (PDGF) 197, 200, 201, 207, 210, 215 pneuma 105, 147 pneumonia 75, 148, 165, 166 poisoning 65, 147, 150, 161–164, 196 politics, -ical 69–72, 84, 234–237, 244, 246 polycythaemia 58 polymerization 5 popliteal vein 115 population incidence 1, 35 positivism 242

Subject Index post-mortem 126, 152, 156, 165, 166, 180, 221–226, 228–231 post-operative 1, 113, 138, 187, 190, 192, 193, 217 post-thrombotic syndrome 1, 4, 131, 136, 177 Pouseille 133 PPAR-γ 202, 204, 217, 249 predict, -ion 13, 31–34, 37, 38, 49, 84, 133, 134, 145, 146, 167, 170, 171, 176, 178, 181, 183, 186, 189, 194, 212, 218, 226, 231, 241, 242 predisposing factor 1, 2, 6, 38, 251 prekallikrein 19, 28 pressure 4, 51, 55, 78, 99, 117–119, 122, 126, 130, 133–135, 145, 149, 156, 166, 172, 173, 178, 184, 185, 189, 192, 193, 213, 214, 216, 225 - intravascular 132 progenitor cell 212 proliferation 136, 164, 206, 212 prolonged inactivity, sitting 2, 133, 167, 168, 185 prophylaxis, -actic 7, 8, 32, 38–40, 138, 170, 185, 187, 190, 194, 221, 249 prostacyclin 37, 196, 206, 207, 213, 214 proteinase 19, 23, 28, 29, 204, 205, 208, 211, 213 proteinase-activated receptor (PAR) 195, 205, 219 protein C 25, 29, 35–37, 190, 204, 205, 213, 218 - activated (APC) 35, 37, 190, 205 - deficiency of 35 - resistance 35, 37 protein kinase C (PKC) 197, 200–205, 209, 214 protein S 26, 29, 35, 37 - deficiency of 35, 37 proteoglycan 199 proteolysis, -lytic 19, 64, 211 prothrombin 12, 13, 18, 20, 25, 27, 28, 36, 57, 63, 64 protracted bed rest 2 proximal 122, 123, 127, 137, 181 P-selectin 198, 199, 202, 206, 207, 210, 211 pseudo-shear rate 135 psychology 244 pulmonary artery 75, 77, 78, 83, 116, 223, 227 pulsatile, -ility 103, 105, 116, 17, 119, 121, 132, 135, 137, 138, 140–145, 147, 148, 150, 151, 154, 167, 170–173, 175, 177, 178, 181, 183, 187, 190, 193, 197, 219

Subject Index pump, -ing 6, 48, 110, 114, 117, 133, 134, 170, 172, 184, 187, 188, 192, 216 - calf muscle 117, 166, 173, 231 - heart 105 puriform 73, 90 purpose, -iveness 58, 94, 105, 157, 187, 230, 239, 243–247 purpura fulminans 35 purulent 73, 82, 89, 90 pus 73–75, 80, 82, 87–89, 91, 92, 95, 174

radiographic dye 151, 173 Raf 200, 203 Ras 200, 209 rationalism, -ist 50, 236 reactive oxygen species (ROS) 200, 208, 210–215 Reason 235, 238 recanalisation 4, 136 receptor 17, 19, 25, 195, 197, 199, 201, 202, 204, 205, 207–210, 214, 218, 228, 249 -tyrosine kinase 200, 228 recirculation zone 137 recognition 66, 72, 152, 232 recumbent 113, 118, 229 reductionism, -ist 44, 45, 245, 247, 251 refilling of valve pockets 137 reflux 6, 105, 112, 118, 129, 133, 140, 142, 154 regenerate, -ation 127, 212, 218, 219 regulate, -ation 16, 23, 32, 40, 202, 204, 208, 212, 216, 218 relaxant 186 relaxation 180, 187, 217 rennet-like 61 research 6, 8, 9, 11, 13–15, 41, 42, 49, 57, 58, 65, 66, 72, 76, 94, 170, 171, 192, 194, 197, 218, 220, 221, 223, 233, 235, 242, 244, 248–253 - strategy 170 resistance 35, 37, 51, 80, 134, 185 respiratory failure 142, 165, 166, 226–228, 230 respiration 55, 148, 187 re-unify, -ication 53, 69, 171, 194, 218, 238, 252 revolution 41, 42, 44–46, 106, 109, 235, 237, 250 revolutionary 59, 76, 244, 246 rheology, -logical 55, 117, 134, 135, 252

315 rigid 51, 127 ‘risk factors’ for DVT - age 1, 50, 92, 124, 131, 186, 190, 217, 237, 247 - autoimmune disorders 2, 37 - bed rest 2, 14, 40, 113–115, 138 - cardiac disease 2 - decubitus 2, 14, 40, 114, 186, 187, 190, 193 - endovascular irritation 2 - ethnicity 2, 185, 189 - general anaesthesia 2, 180 - infection 2, 14, 37, 73, 88–90, 92, 95, 96, 152, 171, 204, 211, 218 - inflammatory bowel disease 2, 218 - injury 1, 2, 4–7, 9, 12, 16, 17, 20, 23, 25, 34, 36, 37, 73, 76, 77, 84, 88, 96, 97, 100, 101, 112–114, 122, 125, 129, 131, 134, 139, 12, 143, 147, 149, 151, 153, 156, 162–164, 167, 168, 176, 183, 190, 191, 199, 202, 204, 207, 210–212, 230, 233 - neoplasias 2 - obesity 218 - oral contraceptives 38, 167, 189, 218 - paresis 2, 114, 186, 194 - pregnancy 38, 190 - sepsis 96, 165, 204, 205 - sex 218 - surgery 2, 9, 14, 45, 63, 74, 76, 88, 108, 113, 114, 142, 164, 187, 188, 193 - smoking 190 Royal Society 236, 250

salting 65 sand 49, 98 saphenous vein 43, 80, 122, 131, 137, 142, 217 scanning electron micrograph 118, 131 schism in biomedical thought 58 Scientific Revolution 41, 42, 44, 46, 209, 235 sclerosant 139 secretion 18, 19, 36, 61, 74, 82, 135, 204, 206, 207, 216 section 128, 151, 152, 155, 179, 180, 182 - serial 152 sedentary 193 semantic 11, 41–44, 66, 83, 88, 93, 95, 103, 111, 114, 116, 174, 243, 244 semi-solidified, -ication 5, 7, 17, 43, 61, 89, 111, 221, 228 sensation 69, 236, 239, 244

316 sense, -ory 5, 7, 12, 31, 32, 42, 44, 47, 48, 54, 69, 76, 84, 88, 103, 105, 111, 116, 136, 146, 149, 236, 237, 239, 240, 243 sepsis 96, 165, 205 septicaemia 58, 211, 218 sequential compression 138 sequestered blood 144 serotonin 17, 214, 217 serum 12, 34, 45, 60, 61, 63, 200, 203 serum response element (SRE) 200, 203 serum response factor (SRF) 200, 203, 205 settle, -ing 222–224, 236 shear rate 135, 137, 206 shivering 68, 180, 181 sickle cell disease 199 signalling 177, 207, 216 - network 195, 201, 202 - pathway 17, 25, 195, 201, 202, 204, 205, 208, 215 silicosis 33 silting 114, 124, 125 sitting, seated 2, 3, 80, 118, 125, 132, 133, 149, 167, 168, 172, 183–186, 188, 91 SLE 2, 37 sleep, -ing 118, 119, 183, 185–188, 193 sludged blood 115 sole of foot 185 soleal sinuses 5, 104, 125 - vein 115, 124 solidism, -ist 45, 46, 49–51, 69 soul 236, 243, 244, 246 stagnation of blood 49, 77, 123 stain, - ing 156 standing 72, 118, 132, 133, 145, 149, 185 stasis 1, 3–8, 13–15, 51, 53, 67–79, 82–84, 103, 104, 106, 108, 110–119, 125, 134, 144, 150, 152, 156, 162, 163, 167, 169, 171–173, 183, 192, 228, 251 statistical mechanics 68, 249, 250 stenosis 139, 231 stimulate, -atory 6, 17, 109, 146, 166, 202, 204, 208, 209, 211, 212, 217, 219 stratification 165, 223, 227, 230 streamline flow 119, 134, 140, 145, 147, 173, 178, 180, 191, 206, 219 streptokinase 65 structure 13, 23, 45, 67, 79–82, 84, 92, 97, 99, 108, 119, 121, 127, 132, 147, 152, 157, 169, 171, 174, 165, 195, 219, 224, 226, 227, 236, 242, 251

Subject Index subendothelium, -ial 17, 20, 28, 127, 139, 181, 195, 200, 206, 216 - connective tissue 17, 19 - matrix 127 - smooth muscle 204, 208, 209 superficial veins 45, 122, 123 superficial venous thrombosis 3 surgery, surgical 2, 5, 9, 14, 40, 45, 63, 73–76, 88, 108, 113, 114, 139, 142, 151, 156, 164, 178, 188, 193, 190 survival 112, 173, 188, 199 swelling 74, 88, 118, 148, 184 symptom 32, 49, 88, 131 systemic - circulation 234 systole, -ic 55, 117, 172, 173

teleology, -ogical 66 tenase complex 18, 20, 28, 37 therapeutic 7, 14, 65, 66, 163, 170, 196, 204, 229–231, 251 therapy 8, 14, 32, 33, 36–40, 170, 190, 218, 221, 231, 249, 252 thought 6, 9, 15, 23, 46, 49–51, 58, 60, 66, 68, 69, 77, 93, 95, 101, 105, 116, 150, 162, 169, 177, 234–236, 238, 239, 243–246, 251, 253 thrombectomy 8 thrombin 13, 17, 20, 21, 23–26, 28, 29, 57, 58, 63, 64, 94, 115, 116, 125, 167, 202, 204, 205, 208, 210, 211, 214, 216, 217, 219, 225 - activation of 64 - catalyses by 21 - control of activity 25, 26 - hypothesis 58, 63, 94, 225 thrombin-activated fibrinolysis inhibitor 24, 25, 36, 206 thrombocyte 94 thrombocytopaenia 36 thromboembolism 2, 29, 32, 33, 37, 38, 40, 73, 77, 81, 84, 176, 183, 189, 221 thrombogenic, -genesis 6, 40, 78, 79, 90, 103, 111, 113, 117, 119, 124, 139, 147, 151, 166, 168, 170, 171, 174, 187, 189, 220 thrombolysin, -lytic 8, 11, 40, 249 thrombolysis 4 thrombomodulin 23, 25, 29, 196, 199, 202, 204, 211, 213, 216, 218, 219

Subject Index thrombophilia - acquired 31, 32, 36, 37 - essentialis 32 - hereditary 35, 36, 38, 39 thrombophlebitis 88, 95, 96, 136 thrombolytic agents 8, 40 thrombospondin 19, 25, 195 thrombosis 1–9, 11–16, 31–40, 42–45, 51–53, 55, 61, 64–66, 68–71, 73, 75–77, 80–84, 87, 88, 91, 94–99, 101, 103, 111–116, 121, 122, 126, 131, 134, 136–139, 141, 142, 147–151, 153, 154, 156, 157, 162–168, 170, 171, 174, 176, 177, 184, 185, 187, 188, 190, 192–194, 197, 199, 204, 206, 211, 216, 211, 216, 218, 220, 222–226, 228–233, 252, 253 thromboxane A2 17, 196 thrombus, -i - agonal 221–232 - arterial 6, 7, 34–37, 39 - experimental 15, 31, 34, 46, 97, 125, 150, 178, 180 - deep venous 115, 156 thyroid hormone 218 tibial fracture 5 tibial vein 115 tissue extracts 12, 63 tissue factor (TF) 20, 28, 64, 65, 201, 212 - system (extrinsic system) 20 tissue factor pathway inhibitor (TFPI) 27, 29, 196 tissue plasminogen activator (tPA) 24–25, 199, 202 - inhibitor (PAI) 25, 196, 199, 201, 202, 206, 208, 209 tissue repair 23, 25, 199, 218 tourniquet 149, 191, 192 toxin, -ic 84, 96, 162, 163, 203, 211 tranquiliser 184 transcription, -al 199, 200, 205, 252 transcription factor 36, 199, 208, 210 transformation 71, 235, 238, 244 transforming growth factor-ß (TGF-ß) 200, 208 transfusion 48, 113 transit 110, 162 transplant 9 transport 105, 148, 184, 249, 250 trapped blood 116, 141, 144, 148 trauma 15, 63, 73, 74, 96, 164, 165, 190 - surgical 5

317 traveller’s thrombosis 1, 2, 147, 166, 170, 184, 185, 188, 190, 192, 193, 253 Trendelenburg 187, 193 tumour necrosis factor-α (TNF-α) 197, 201 turbulence 96, 123

ulcer, venous 44 umbilical vein 197, 199, 210 underperfusion, -ed 98, 100, 103, 110, 114, 119, 121, 125, 126, 132, 141, 142, 148, 151, 163, 167, 169, 172, 173, 185, 187, 228 ‘upward striving’ 238, 239, 244 urea 50

valve, venous 4, 71, 103, 105–110, 114, 118, 119, 121, 122, 129, 131–133, 154, 180, 184, 189, 190, 231 - closing 118, 132 - curtain 152, 154 - cusp 4, 34, 175, 187, 210, 227 - endothelium, luminalis 173 - endothelium, parietalis 125, 132, 147, 167, 169, 173, 212 - necrosis of 169, 212 - hypoxia hypothesis 97, 101, 110, 119, 139, 145, 167–195, 197, 198, 208, 217–219, 221, 226, 227, 231, 249, 251 - leaflet 67, 134, 136, 144, 170, 194, 207, 226 - cycle 133–135, 137, 143, 190 - closed phase 133, 135 - closing phase 133 - equilibrium phase 133, 134, 137 - opening phase 133 - discovery of 103, 104, 107 - function 132, 154, 219 - incompetence 1 - morphology 125, 127 - opening 132, 133 - pocket (VVP) 80, 82, 103, 104, 106, 110, 114–116, 118, 119, 121–126, 128, 129, 131, 133–145, 147, 148, 150–156, 159, 163, 164, 167–169, 171–178, 183, 185, 187–193, 199, 202, 206–208, 210, 217, 219, 228 - sinus 125, 127, 132, 134, 143, 155, 173, 187 valvulitis 129, 137

318 varicose veins 2, 45, 118, 139, 162, 178, 189, 208 vasa venarum 127, 135, 136, 150, 152, 173, 195, 198, 217, 219, 227 vascular endothelial growth factor (VEGF) 197, 199, 202, 210, 212, 218, 219 vascular repair 199 vascular surgery 195 vascular system, tree 14, 96, 140, 208, 225, 227, 229 vasoconstriction 6, 16–18, 104, 214, 215, 217 vasodilatation 5, 6, 19, 28, 51, 213–215 vasodynamics 195 vasopressin 217 VCAM-1 25, 205, 208, 209 VCHH, see valve cusp hypoxia vein - cross-section 127 - junction 114, 123, 152, 173 - peripheral 78, 84, 116 - tributary 129, 130, 132, 136, 154, 173, 189 - wall 4–6, 15, 16, 51, 52, 74, 79, 80, 84, 89, 91, 95–97, 115, 122, 131, 133, 135, 136, 140, 150, 152, 156, 159, 171, 173, 175, 183, 197, 208, 217 venesection 45, 117 venous - blood flow 116, 138, 143, 172, 173, 181, 183, 189 - insufficiency 131 - obstruction 4 - pressure 4, 78, 118, 149 - return 48, 113, 121, 132, 167, 172, 177, 184, 185, 188, 192, 193, 196 - saccule 114, 115, 121, 124 - stasis 1, 6, 15, 113, 114, 119, 183 - tone 122, 132, 135, 136, 206 - trunk 130, 132, 154, 231

Subject Index ventilation 150 ventricle 73, 105, 165, 223, 231 Venturi effect 134, 137 venule 99, 100, 117, 118, 162, 208 vertical 6, 118, 149 vessel - diameter 135 - lining 81, 114, 139 viable, -ility 91, 126, 141–143, 148, 156, 162, 168, 169, 174, 175, 207, 227 ‘Virchow’s triad’ 1, 4–6, 8, 14, 15, 31, 45, 71, 83, 114, 116, 170, 171 vis-a-tergo 117, 149, 231 viscosity 134, 135 vital - force 41, 44, 48, 52, 62, 68, 69, 240, 243–246 - spirits 50, 105 vitalism, -ist 44, 45, 47, 48, 52, 54, 62, 68, 69, 71 vital-materialism 98, 233, 234, 243, 244, 247, 252 vitamin K 20, 25, 57, 65, 66 vocabulary 66, 81, 84, 97, 100, 242 von Willebrand factor (vWF) 17, 28, 66, 195, 199, 202, 206, 216 vortex, -ices 116, 133, 134, 137, 138, 141, 144, 206, 231, 236

Warfarin 20, 39, 40 washed clot 59, 60 water 50, 54, 59, 68, 117, 118, 184, 185, 249 web 16, 23, 24, 52, 61, 95, 112, 174, 176 whipping of blood 44, 65 white thrombus, -i 97, 99, 101, 124, 128, 129, 221, 225, 228, 231 Will 239 wound 6, 16, 24, 79, 113