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Hougie 2004-journal of-thrombosis_and_haemostasis

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Hougie 2004-journal of-thrombosis_and_haemostasis

  1. 1. HISTORICAL SKETCH The waterfall-cascade and autoprothrombin hypotheses of blood coagulation: personal reflections from an observer C. HOUGIE Department of Pathology, University of California, San Diego, USA To cite this article: Hougie C. The waterfall-cascade and authoprothombin hypotheses of blood coagulation:personal reflections from an observer. J Thromb Haemost 2004; 2: 1225–33. The most cited and probably the two most important papers on blood coagulation of the 20th century were the independent reports on the Cascade or Waterfall hypothesis in 1964. The Cascade paper by R.G. Macfarlane appeared in Nature [1] and was shortly followed by the Waterfall report by Earl W. Davie & Oscar Ratnoff in Science [2]. As the papers are landmarks in the history of blood coagulation they have recently received much attention. Davie himself has written a brief history for the centenary issue of the Journal of Biological Chemistry [3] while Harold Roberts, in a paper [4] on the works and achievements of Oscar Ratnoff, has traced the evolution of the Waterfall theory using Ratnoff’s notes. I will focus more on aspects which were not considered or only briefly dwelt upon in these two earlier reviews. The Waterfall and Cascade reports were not only of interest to workers in hemostasis, but as they appeared in the two most prestigious journals widely read by scientists in all fields, had important impacts on research in other related fields of biology. The basic concept in both papers was that the formation of fibrinogen occurs in a series of progressive stepwise reactions in which the clotting factors act in pairs, one factor behaving as an enzyme and the other like a substrate (Fig. 1). It was postulated that each protein clotting factor with the exception of fibrinogen exists in plasma in an inactive precursor form and is sequentially converted to an active enzymatic form in the following order: Factor (F) XII, XI, IX, VIII, X, V and prothrombin and FXIII; the activated forms of the various factors was denoted by a lower case a. The initial triggering event was thought to be the activation of FXII which can occur by several mechanisms. The activated FXII or XIIa activates FXI to XIa and so on down the line with all the clotting factors except fibrinogen being converted to a form possessing enzymatic activity. In both schemes, phospholipid was an absolute requirement for two steps. The existence of feedback mechanisms was recognized and the great enhancement of both FV and VIII activities by thrombin was cited in both papers. Each of the reports recognized that inhibitors that bring the process to a halt have an important role. The extrinsic system was only very briefly alluded to in the text of the Waterfall but not as all in the Cascade paper. Macfarlane [1] observed that from one stage to the next in the Cascade, a greater amount of proenzyme is involved resulting in amplification. He estimated that in 100 mL of plasma, there was 15 mg of prothrombin, 1.8 mg of FX and 1.7 mg of FVIII and the final end product of the reaction converts as much as 250 mg of fibrinogen to fibrin in a few seconds. Macfarlane concluded from these numbers that the physiological need to link the initial minute physical stimulus of surface contact with the final explosive burst of thrombin has resulted in the evolution of a photochemical amplifier in which proenzymes are analogous to photomultipliers or transistors stages. If each enzyme activates 10 times its own weight of proenzyme during its time of operation, he calculated the overall gain in response to be a million-fold, a figure close to modern observations [4]. Macfarlane took the analogy to a photomultiplier even further. He suggested that the rapid disappearance of certain coagulant enzymes, and perhaps FVIII, almost coincidently with the appearance of fibrin were analogous to a circuit breaker actuated by the last in the series of operations which stops the whole process and prevents overrunning, a safeguard of this sort being a biological necessity if disastrous thrombosis is to be avoided. Apart from the concept of amplification, the two reports were virtually identical and the terms Waterfall and Cascade are used interchangeably. However the term Cascade is usually pre- ferred in that it connotes not only sequential events but also amplification. At the time of the two reports, Macfarlane and Ratnoff were two of the most distinguished clinical investigators in blood coagulation while Davie would soon become the foremost biochemist in the field. Robert Gywn Macfarlane (1907–87), a delightful, modest and an almost self-effacing figure, was the director of the Medical Research Council Blood Coagulation unit in Oxford from 1959 to 1967 where many major advances were made in the management of hemophilia (Fig. 2). With A.S. Douglas and Rosemary Biggs, he devised the thrombo- plastin generation test (TGT test) which provided an explan- ation for the function of FVIII and led to the discovery of FX. Correspondence: Professor C. Hougie, Emeritus Professor of Pathology, University of California, San Diego, CA, USA. Tel.: +1 858 459 1479, fax: +1 858 459 1988; e-mail: ch@san.rr.com Journal of Thrombosis and Haemostasis, 2: 1225–1233 Ó 2004 International Society on Thrombosis and Haemostasis
  2. 2. Following the first report of Paul Aggeler and his group of a male patient with a deficiency of plasma thromboplastin component (PTC), now designated FIX, Macfarlane and his associates reported a series of patients with this disorder. They showed the condition, which they named Christmas disease after the surname of their first patient with the disease, is inherited as a sex-linked recessive disease, and segregated it from the more common type of hemophilia due to FVIII deficiency. Macfarlane had also discovered the important coagulant properties of the Russell’s viper venom or Stypven shortly after graduating from medical school, and later with Peter Esnouf determined its mode of action. Macfarlane was an early pioneer in the field of fibrinolysis but his discoveries encompassed virtually the whole field of hematology. He was elected a Fellow of the Royal Society in 1957, one of the most coveted honors for a medical scientist in the UK. At the time of publication of the cascade paper, Macfarlane was at the pinnacle of his fame and the most distinguished and senior hematologist in the UK; he compares favorably with William Hewson (1732–1774) who was the first to perform meaningful scientific experiments in blood coagulation and is now consid- ered to be the father of hematology. Hewson was also a Fellow of the Royal Society. The life and achievements of Macfarlane has been the subject of a recent biography [5]. Davie & Ratnoff had been colleagues at Case Western Reserve University in Cleveland, Ohio, but at the time the Waterfall paper was written Davie had left Cleveland for Seattle, Washington. Following a hematology fellowship at Johns Hopkins, Oscar Ratnoff [4] took a staff appointment at Western Reserve in 1952 where he stayed for the rest of his career (Fig. 3). In 1955, with J Colopy, he discovered the Hageman factor (FXII) and had a major role in the elucidation of the early stages of blood clotting. Endowed with a very fertile and brilliant mind, he is a delightful companion with an exquisite sense of humor. Even at an early stage of his career, he was never afraid to stand up and challenge an erroneous viewpoint regardless of the eminence of the speaker. Ratnoff often remarks that one of his major contributions to hemostasis was to persuade Earl Davie to take an interest in blood coagulation, and this indeed can be listed as a major and significant accomplishment. Earl Davie received his PhD at the University of Washing- ton, Seattle, in 1954 under Hans Neurath, a leading authority in protein chemistry. His thesis involved a study of the transformation of trypsinogen to trypsin, and he made the important discovery that this occurred by the hitherto unrec- ognized process of limited proteolysis [6]. He then spent two productive postdoctoral years working in Fritz Lipmann’s laboratory at Harvard before being recruited in 1956 into the Department of Biochemistry at Case Western Reserve Uni- HAGEMAN F. (XII) P.T.A. (XI) CHRISTMAS F. (IX) ANTIHEMOPHILIC F. (VIII) STUART F. (X) PROACCELERIN (V) PROTHROMBIN (II) FIBRINOGEN (I) FIBRIN THROMBIN ACT. PROACCELERIN ACT. STUART F. ACT. ANTIHEMOPHILIC F. Ca++ Ca++ Ca++ phospholipid phospholipid ACT. CHRISTMAS F. P.T.A.ACT. ACT. HAGEMAN F. ? Fig. 1. Waterfall concept. The cascade was virtually identical. Fig. 2. Gywn Macfarlane. Courtesy of Paul Giangrande. Fig. 3. Oscar Ratnoff. 1226 C. Hougie Ó 2004 International Society on Thrombosis and Haemostasis
  3. 3. versity by Harland Wood, who was a giant in the field of intermediary metabolism. It was Wood who introduced him to Oscar Ratnoff. In 1962, after a fruitful collaboration with Ratnoff, Davie was persuaded by Hans Neurath to accept a faculty position at the University of Washington but he continued his collaboration with Oscar for a few more years. His laboratory soon began to play a dominant role in the isolation and characterization of most of the clotting factors and their eventual cloning. Earl Davie always seems relaxed and low-keyed but these appearances may be deceptive. I can recall several fishing trips I took with him in which Earl, lying flat on his back because of seasickness, with the fishing line loosely attached to one of his toes somehow always contrived to catch more and bigger fish than anyone else on the boat (Fig. 4). The collaboration of Ratnoff, a clinical investigator and Davie, an established protein biochemist with a fresh outlook on blood coagulation was a highly successful one and culminated in the Waterfall concept. However despite their distinction and connections, their paper ran into a major roadblock. At the time, I was a colleague of Earl at the University of Washington although in a different department, and I would frequently seek his advice. I happened to be in his laboratory several minutes after he had learnt that the Waterfall paper had been rejected by Science. The paper had received a severe panning from one of the reviewers. Davie informed me that Ratnoff who knew the editor of Science would appeal the decision. This was the first time in my career that I became aware that one could appeal an editorial decision which I had always thought to be final and irrevocable. I also happened to be in Davie’s laboratory when Macfarlane’s paper on the cascade appeared in Nature some weeks later. The remarkable similarity of the Waterfall with the Cascade was noted by Davie and added to his displeasure. Fortunately, shortly thereafter the editor of Science rescinded his decision and accepted the paper in its original form without any request for modification, but as a result publication was delayed several months. On the other hand, the paper of Macfarlane was published almost immediately and almost certainly without any peer review, such was his prestige in the UK. One of the major reasons for the importance of the papers was that they constituted the first major and direct challenge to Seegers’ unique view of blood coagulation, a fact that is now rarely alluded to. While Seegers’ supporters had become a dwindling number, they were influential and included most of the early pioneers with the notable exception of Armand Quick, the developer of the one-stage prothrombin time test, who was invariably at loggerheads with Seegers. Indeed, at the time, almost all biochemists outside the field and many within believed Seegers’ views were generally accepted. A towering and formidable figure with an aura of great authority, Seegers had been for many years the acclaimed leader of the past establishment. There were very few individ- uals who dared to argue or trifle with him; indeed he was rarely challenged, at least before 1964. He was revered by his colleagues who invariably remained loyal to him and his ideas, even after they had moved away from his orbit; there were few exceptions. Seegers dominated the blood clotting field in the USA from 1944 to1956. This is attested to by the perusal of the five conferences on blood clotting and allied problems held between 1948 and 1952 and sponsored by the Josiah Macy Jr. Foundation in New York [7]. All of the leading workers in the field were invited to attend; they were encouraged to actively participate, and their comments published with minimal editing. It can be seen from the published proceedings that Seegers was a prominent participant. Many of the investigators made a point of stating that the purified prothrombin used in their experiments had been supplied by Seegers. If the prothrombin had not been supplied by Seegers its purity would be invariably questioned. Seegers was the author of well over a thousand papers and his former colleagues, students and visitors to his laboratory numbered in the hundreds and included many individuals who were already distinguished in the field and occupied important positions all over the world ranging from Tokyo, Buenos Aires and Istanbul. His political influence was formidable since he was a highly sought after reviewer of papers on blood coagulation submitted to the major journals in the USA and was also a member of review panels for many granting agencies. Seegers (1910–96) obtained his PhD from the University of Iowa and in 1937 was recruited by H.P. Smith, the chairman of the Department of Pathology at that university. The two-stage prothrombin time test had been devised there by Warner, Brinkhous and Smith who had performed important studies on Vitamin K. As the result of their work, Iowa had become the major center of research in the world on blood coagulation. It was there that Seegers was indoctrinated into the field of blood coagulation and developed his interest in prothrombin and thrombin. When the Iowa group broke up at the outbreak of the second World War, Seegers was recruited by Parke Davis to work on thrombin and his purified bovine thrombin was successfully marketed and is probably still manufactured. In 1946, he moved on to Wayne State University in Michigan where he became the head and chairman of the Department of Physiological Chemistry.Fig. 4. Earl W. Davie with trophy king salmon caught in Alaska in 2003. Historical Sketch 1227 Ó 2004 International Society on Thrombosis and Haemostasis
  4. 4. Seegers had for many years maintained that he had purified bovine prothrombin to homogeneity completely free of any contaminating proteins [8]. This conclusion, which was based initially on the sedimentation characteristics using the ultracen- trifuge remained virtually unchallenged for many years. He regarded the absolute purity of his bovine prothrombin preparations an established fact and this was the basic tenet of all his work until the seventies. By 1962, he claimed that the additionalcriteriaofelectrophoresis,immunochemistryandend group analysis substantiated his claim of its homogeneity [8]. According to the concept of Seegers, which he held until the early seventies, all the activities associated with FVII, FIX and FX only appear when pure prothrombin is activated. There- fore, he reasoned they had to be derivatives of prothrombin, as he believed there was nothing else in the preparation. He considered prothrombin to be the only vitamin K-dependent clotting factor and he devised a new and unique nomenclature for the ÔderivativesÕ which he labeled autoprothrombins. Although a founding member of the International Committee for the Nomenclature of Clotting Factors, he was critical of all their decisions. He charged them with creating hypothetical factors and assigning them Roman numerals. Although these numerals were in current use and had been recommended by the committee, he refused to accept them [9,10]. His refusal was not willful, for if he had accepted the established nomenclature, this would have been an admission that the factors actually existed as discrete molecular entities or proenzymes rather than being mere derivatives of prothrombin. He was convinced that the new factors, other than FV, were merely a passing phase [11]: ÔWhen the peak of ‘‘attention getting’’ in my contemporary ‘‘factorphilic’’ world has been outgrown it will be doubtless easier for me to sell the relative importance of AcGÕ (AcG being factor V in modern terminology). Seegers [12] claimed that Ôthe beautiful concept of prothrombin derivatives is an original and revolutionary idea which was accurately conceived.Õ He believed there were no experimental or clinical findings that could not be explained by it, and placed the onus on those with the opposite viewpoint to prove it wrong. Statements, similar to the above that I have quoted verbatim, are replete in Seegers’ writings. While the reviewer who wrote the adverse critique of the Waterfall paper was anonymous, it expressed Seegers’ unique viewpoints. In any event, the attempt to block publication of the Waterfall paper failed and to add to the chagrin of the reviewer, the almost identical Cascade paper was published a few months later. Seegers’ reaction to the two reports was predictably both swift and vitriolic, and consistent with the negative review [10]. He wrote a blistering rebuttal of the hypothesis for a journal of which he was on the editorial board. This is a very unusual, although not unprecedented step in science, even more so as the paper was more than twice the combined length of the two papers he was attacking. Seegers wasted no time on niceties and was never one to mince words ÔI regard these presentations as being at the speculative levels of science created with the wild use of the imagination.Õ He found it against his sense of order to believe that six proteolytic enzymes were needed to obtain thrombin. He argued Ôthat would require six special sites in proteins where each enzyme does its work; each enzyme would not split the other five sites if located as single or multiple frequencies in the five other substrates.Õ He believed this to be biologically impossible. Seegers took exception to Macfarlane’s remarks that his prothrombin might contain some hidden contaminants responsible for the clotting of fibrinogen. But the main thrust of his argument was that the theory was totally incompatible with his own work in which he found no evidence for the existence of the precursor factors designated by the Roman numerals VII, IX and X. He disputed the existence of most of the steps in the cascade and wrote Ôfor they could only be accurate if it were possible to deny important conclusions stated in papers which are not at all considered. Evidently refutation of facts in these papers was not possible and a parochial kind of authority was promoted on the basis of ‘‘consistence’’ with most of the current investigationsÕ [10]. I interpret this as meaning that most of the steps outlined in the cascade or waterfall theories were not consistent with the ÔirrefutableÕ facts presented in papers, presumably those of Seegers, which were for that reason not cited in either of the two reports. Seegers maintained that prothrombin contained all the material required for its own activation and that it was in effect a molecular system. He believed: ÔWhoever reflects on this outline of prothrombin activation and is sincere with himself will have to recognize a conceptual formulation of beauty. It may well be that the open secret to the riddle of blood clotting is now there for anyone prepared to see it.Õ There were two substantive points buried in Seegers’ paper. He correctly pointed out that there are no enzymatic forms of FV or FVIII that will directly convert prothrombin to thrombin or FX to FXa, respectively. For many years, Seegers was one of the leading proponents of the two-stage prothrombin time test. He rarely used the one- stage prothrombin time test which he described as Ôexplosive chemistryÕ and deprecated all the conventional assay proce- dures based on one-stage systems. He regarded as invalid all assay procedures in which congenital deficient plasmas were used as a substrate [13]. As a result it was difficult for him to recognize a deficiency of any clotting factor other than FV, let alone assay it, providing a possible explanation for his failure to detect impurities in his prothrombin preparations. Seegers’s paper [10] was far more than a mere rebuttal; it was an uptodate compendium of his life’s work and philosophy. He evidently still clung to the lipid inhibitor theory of hemophilia first proposed by Tocantins, maintaining that this inhibitor had to be neutralized before platelet cofactor I (FVIII) could act, although the theory had long been abandoned by its originator. In the paper, he reiterates his belief that the most important discovery pertaining to prothrombin was Ôthe plainest, clearest, unique, outstanding and often confirmed demonstration of autocatalysis in prothrombin activation seen for the first time when prothrombin activated in 25% sodium citrate solutionÕ. SeegersÕs paper was entitled ÔBasic enzymology of blood coagulationÕ subtly implying that his theory was consistent 1228 C. Hougie Ó 2004 International Society on Thrombosis and Haemostasis
  5. 5. with basic enzymology while the cascade concept was not [10]. The paper was clearly hastily put together and does not appear to have been subjected to any peer review process. For example, in Seegers’ representation of the waterfall scheme, phospholipid appears to be a cofactor of the FIX to FIXa conversion instead of the subsequent stage. While this is clearly a typographical error of which there are several, there are at least two clear, but undoubtedly unintentional misstatements of the work of others. Macfarlane’s response [14] to Seegers’ paper appeared in a later issue of the same journal as Seegers’ paper. It was conciliatory in tone, and as one would expect coming from the pen of a modest but great scientist, carefully crafted and written in beautiful and lucid prose; he addressed each of Seegers’s objections point by point in carefully measured terms without hyperbole or dogma. He started off by pointing out that Ôby definition a hypothesis is speculative but ‘‘wild imagination’’ implies a charge of scientific irresponsibility which when publicly made by so influential a scientist as Dr Seegers could hardly go unchallenged.Õ His objective in writing his rebuttal was to indicate as briefly as possible the sources of evidence on which the cascade hypothesis was based so that those interested could judge the issue for themselves. He agreed that the evidence for certain steps such as the activation of FV to an active enzymatic form might be non-existent but he had included it for completeness. One of Seegers’ objections was that there was a lack of evidence that FX (presumably autoprothrombin III in Seegers’ terminology) played any role in intrinsic plasma coagulation. Seegers had cited some of Macfarlane’s own work as support- ing this contention, claiming that Macfarlane had shown that FX was not consumed or utilized during intrinsic coagulation. Actually Macfarlane and his colleagues had found that only a small amount of FX, rather than no FX was utilized; adding more FVIII to the system increased the amount of FX consumed. Macfarlane next pointed out that if one accepts the existence of multiple clotting factors, the next question to arise depends upon whether one thinks as a physiologist or a biochemist: ÔThe biochemist wants to know what the factors are; the physiologist wants to know what they do. It is recognized that, ultimately, physiological questions will be answered in terms of biochem- istry or biophysics, but at the onset of the investigation it is a matter of preference which line of approach is adopted. In practice it is usually the study of some physiological function which prompts the later detail investigation of its underlying mechanism in terms of molecular structure. The physiologically minded worker faced with seven or eight components of the functional whole is more concerned with the gross ways in which they interact than the details of their individual structure.Õ Macfarlane pointed out that an investigator would resent, and rightly, any suggestion that he should do no experiments and produce no new theories until each factor is purified and characterized. Seegers defined his prothrombin as a molecular system and relied on physical and biochemical characterization as the test of homogeneity despite the fact that several functions appear during its activation in addition to thrombin. Macfarlane remarked that a sample of 7S gamma globulins may appear to be homogeneous by physical and chemical criteria while an immunologist may show it to be a mixture of four different antibodies. Macfarlane pointed out that the body of evidence for the multiple factor theory of clotting was very large and drawn from real observations and experiments of independent work- ers in many countries during the preceding half century. He recognized that Ôsuch experiments create artifacts and can be open to more than one interpretation. However laboratory theory is brought into direct touch with physiological reality by the natural occurence in humans, and some animals of gross clotting defects which can be best explained and distinguished by postulating the separate deficiency of one or other of each of the plasma factors which have been assigned Roman numerals. Not only can these defects be diagnosed and quantitatively assessed on this basis but the use of concentrated preparations corresponding to the arbitrary laboratory definition of the missing factor correct the defect in vitro, and above all in vivo. No one who has seen the dramatic clinical effect of the accurate diagnosis and specific replacement therapy in cases of these potentially lethal conditions would doubt the existence of these factors unless he was given equally impressive evidence to the contrary.Õ Seegers appeared to have had no great interest in the study of patients with congenital deficiencies of a clotting factor, although he conceded such studies might be valuable. Macfarlane claimed that there was a large area of agreement between his picture of the clotting mechanisms and that of Seegers, however bleak the prospect may appear from the other direction. He suggested that if Seegers would be willing to refer to his Ôpurified prothrombinÕ as the Ôprothrombin complexÕ the gap between the two viewpoints would be narrowed. He proposed a possible compromise between the two viewpoints, but the effort is beyond even the ingenuity of Macfarlane; the result was clearly contrived and improbable. Perhaps the highlight of Macfarlane’s paper is its dramatic conclusion in which he depicts two shadows, one having the appearance of a wineglass and the other of a wine bottle. If only one of the two is seen by two observers and they are each told there is only one source of the two shadows, they might well get into an argument as to the source of the shadow even to the extent of accusing the other of wild imagination. In fact the two shadows were cast from a single object illuminated by two beams of light at right angles to each other. The Macfarlane rebuttal was perhaps too defensive and restrained for the evidence against the Seegers hypothesis was becoming overwhelming. Seegers’ views defied both common sense and the principles of basic enzymology for five compo- nents could hardly act simultaneously, and it was hard to conceive one proenzyme having so many different active sites and functions. Even Seegers [15] indirectly acknowledged this for he often referred to prothrombin as Ôa lively enzymeÕ. He even made the claim [16] that prothrombin had Ôa versatility as marvelous as that of any other unit in biology, such as, for Historical Sketch 1229 Ó 2004 International Society on Thrombosis and Haemostasis
  6. 6. example, the cellÕ. There was the compelling finding that prothrombin could be separated by Seitz filtration from FVII and FX. Moreover the levels of prothrombin, VII, IX and X did not decrease at the same rate when dicoumarol was given and also returned to normal at different rates when the drug was stopped. Surely if these factors were merely different functions of a single prothrombin molecule, each would have decreased and returned to normal at the same rate. Seegers was aware of these facts but he had no plausible explanation [17]. The Seegers’ theory did not fit the one protein–one gene hypothesis. Perhaps last but not least, both Soulier and Duckert, two of the most illustrious names in the field had each independently found and informed Seegers that a preparation of prothrombin purified by his method contained demon- strable levels of FVII, FIX and FX and also FV; one of these preparations had actually been provided by Seegers himself [8]. His response was that that the prothrombin preparations tested had not been sufficiently purified. Rapid improvements of analytical methods in biochemistry were being made at a rapid rate in the 1960s. Disk gel electrophoresis [18] was in general use in 1965; SDS polyacryl- amide gel electrophoresis was introduced shortly thereafter and by late 1969 was established as a reliable method of estimating the molecular weight of proteins. Immunochemical techniques were also well advanced in the early 1960s [19]. Any one of these procedures would have detected the presence of gross impurities in Seegers’ prothrombin and led him to abandon his theory. However it was not until the beginning of the 1970s when he could hardly have had a single supporter that he belatedly joined the mainstream. A contrite Seegers [20] proclaims ÔI started with a beautiful and time honored materialistic perspective and sincerely did my best to be objective just as if this could be possible. I believe my original orientation continues to retard scientific progress, accounts for heartaches and for a scientist being regarded circumspectly. It was eventually my good fortune to notice that idealism is compatible with progress and health and that subjectivity is the basis of all that is real.Õ It is only too easy in hindsight to be overly critical of someone who has been shown to be wrong and especially if he has conceded his mistakes. As Seegers’ views came under increasing fire from all quarters, the more important it must have seemed to him to vehemently defend them. He was still on the editorial boards of many of the journals devoted to blood coagulation and his papers, liberally sprinkled with philosophical comments, were being accepted and published, apparently without critical peer review. He undoubtedly felt it his duty to take every opportunity to present what he was sure was the correct viewpoint and prevent others from marching blindly down a blind alley. Accordingly he availed himself of every opportunity and venue that came his way to propagate what he believed to be the truth. Most scientists in his position and feeling as strongly as he did might well have done the same, though hardly with the same compulsive zeal. Seegers was a philosopher as well as a scientist and his papers are replete with obtuse philosophical quotations and state- ments. His scientific philosophy can be found in the book Living Consciously: The Science of Self which he coauthored with Dr John M. Dorsey, a psychiatrist [21]. When challenged in 1963 as to the purity of his prothrombin, Seegers [22] responded that he had written much on this, but in addition he would make it very plain that in his book with Dorsey they had stated: ÔI have never proved anything in all my life, and I am sure no one ever has. This is the way I look at it. It is an illusion that you can prove anything.Õ It is difficult to have a meaningful scientific discourse with anyone holding such a viewpoint and the response quoted above which he gave to a very fair and reasonable question may explain why he was so rarely challenged at meetings. A repeated theme seen in his writings is that Ôall my science is strictly private and my own subjective creation.Õ Indeed this is a fairly accurate description of his attitude to science. But in the modern age, dilettantism is seldom compatible with the objectivity so necessary in hard science. Seegers probably knew more of the history of blood coagulation than any of his contemporaries with the possible exception of C.A. Owen, a former colleague of his. But this did not prevent him from repeating some of the common errors of the past; his failure to distinguish a conclusion was one of these. Although outspoken in his views, Seegers was honest in his belief that the prothrombin he had prepared was truly homogeneous and contained no impurities. However he was stubborn in his refusal to accept any new evidence that became available and called for the abandonment of this belief, so in this sense the homogeneity of his prothrombin had become an obsession. It is not at all unusual in modern science for two or more groups working independently on the same problem to arrive at more or less identical conclusions at about the same time. So it was with the Cascade and Waterfall reports. The papers were actually submitted for publication within a few weeks of each other in 1964 but as the Waterfall paper was initially rejected its publication date was delayed and it appeared later than the Cascade paper. The concept was not entirely new and was known to both groups at least two years prior to the Science and Nature reports. Both Ratnoff and Macfarlane were participants at a meeting held in Stockholm in 1962 under the auspices of the Interna- tional Committee on Blood Clotting Factors. Macfarlane [23] presented some data on the activation of FX. The paper was very briefly commented on by Ratnoff [24] who spoke of Macfarlane’s presentation in glowing terms and then asked the rhetorical question: ÔWhy cannot a substrate be an enzyme?Õ He concluded by stating that he understood Ôclotting as a process in which one enzyme acts on a substrate to change it to an active enzyme which acts on the next substrate and so on down the line ad nauseam like the fleas.Õ I believe this to be the first published reference to the Waterfall hypothesis. Macfarlane agreed entirely with Ratnoff’s comments and stated that the sequence of substrate to enzyme was exactly what he visualized in the activation of FX by Russell’s viper venom. A figure of an almost identical version of the Waterfall reaction sequence 1230 C. Hougie Ó 2004 International Society on Thrombosis and Haemostasis
  7. 7. actually appeared in a paper by Ratnoff and Davie [25] in 1962. It was included in the discussion section of the paper and was based on previous work performed in the authors’ laboratories as well as new data presented in the paper. It is clear from this paper, and the Stockholm proceedings that the concept had by early 1962 fully crystallized in the mind of Ratnoff. This was confirmed by Roberts [4] from his examination of Ratnoff’s laboratory notes. I also have in my possession copies of two letters by Davie in which the Waterfall scheme is depicted. The first, to Fritz Lipmann, was written in March of 1962, while the second, written in June of the same year, was to Roger Lundblad. The Waterfall scheme was also shown on an application by Davie in 1962 for a clinical research project; I was listed as one of the investigators and received a copy. It was also known by Macfarlane as it appeared in 1962 in a textbook by Biggs and Macfarlane [25], although the substrate to enzyme chain reaction was not specifically outlined. In deriving the hypothesis, each of the two groups took advantage of the published data of the other and made the appropriate acknowledgments. Thus the first three papers cited by Macfarlane referred to work performed in Davie and Ratnoff’s laboratory while the first citation of Davie and Ratnoff was to the page in the book by Biggs and Macfarlane which contained the early diagram of the scheme. Both Macfarlane and Davie & Ratnoff are always assigned equal priority, and rightly so. However in a book published in 1993 on the life of Macfarlane, the author, Dr Alistair Robb-Smith [5], hints that there may have been plagiarism, although he was careful to point out that this was not Macfarlane’s opinion. He made this assertion on the sole grounds of the similarity of the two papers and the fact that the Waterfall report appeared in print later than the cascade; he believed that this was too much of a coincidence. This innuendo, as the facts above show, is of course totally untrue. In fact, Macfarlane and Davie & Ratnoff in their 1964 reports each cited the earlier 1962 contributions of the other as if to disclaim any priority; it is therefore clear that this question had not occurred to either group, just as one would expect from outstanding and established scientists. Needless to say, both Drs Ratnoff & Davie, who have always been models of fairness and propriety, were extremely and understandably distressed. The blood coagulation laboratories at Oxford and Cleveland were amongst the very few major centers investigating the various sequential reactions along conventional lines, and there was really only one logical explanation of the available experimental data. It was therefore almost inevitable that both laboratories would arrive at the same conclusions. Unlike Davie and Ratnoff who started at the top of the sequence and worked their way downwards, Macfarlane started at the bottom rungs and extrapolated upwards. Macfarlane is quoted [5] as stating that Ôas people tunneling from opposite sides of a mountain are supposed to do, we joined up with the work being carried out on the other side of the problem, mostly on the other side of the AtlanticÕ (referring to Ratnoff & Davie’s work). Macfarlane’s perspective also differs from that of Davie & Ratnoff in that he is more interested in the concept of amplification and less on the biochemistry of the interactions. He had long considered the sequential triggering of the various factors to be like an amplifier but it suddenly occured to him just prior to writing the paper that it was in fact an amplifier [5]. The idea of amplification in the electrical sense greatly appealed to Davie & Ratnoff, who considered the term Cascade more appropriate than Waterfall. There are several reasons why the impact of the hypothesis was so immediate and important. Prior to the publication of the Cascade and Waterfall papers in 1964, Seegers’ views were generally accepted by most biochemists, in and outside the field. But following publication of the two reports, Seegers’ views were supplanted by the new theory. The hypothesis acted as a unifying banner for workers in the field who now spoke in the same language and used well defined terms that could be understood by all. For the first time in years scientists in other fields who formerly shunned blood coagulation were attracted into the field. The theory became the standard and orthodox one and appeared in textbooks and reviews. Many theories of blood coagulation had been propounded in the 60 years following the Morawitz theory proposal at the turn of the 20th century but none had succeeded in replacing it as a practical working model. The Cascade hypothesis presented a composite of all the bare facts in a nutshell, providing an easy to understand and general concept more or less unique in biology. It filled in the vacuum that had been created when the Morawitz hypothesis became obsolescent in the early forties. Moreover the Cascade principle could be applied to other systems including that of complement, and Cascade became an accepted biological term. It is doubtful whether the theory would have met with the same general acclaim and immediate acceptance had it not been for the authority and distinction of all three authors and the fact that one was an established protein chemist. That the papers appeared in the two journals widely read by a broad spectrum of scientists also played an important role in its wide acceptance. Davie & Ratnoff in their paper pointed out that the sequence would require modification when new clotting factors were discovered and as further studied revealed the complexity of substances assumed to be pure. Macfarlane, in his rebuttal of Seegers, also conceded that there were obvious gaps and difficulties and that the hypothesis might be disproved in principle or in detail at any time, but he stressed that it provided opportunities for experimental testing which whatever the outcome was likely to provide new knowledge These guarded predictions proved to be correct and shortly after the reports appeared in print there was new evidence indicating modifica- tions were needed. Thus complex formation was demonstrated between FV and FXa and phospholipid in the presence of calcium [27] while a similar complex was found to occur at an earlier step involving FVIII and FIXa, phospholipid and calcium [28]. It is noteworthy that the former work was performed in the laboratory of Hanahan, a departmental colleague and former teacher of Davie and the latter work in Macfarlane’s laboratory. As the Cascade was a hypothesis, new findings such as these were merely incorporated into a modified Historical Sketch 1231 Ó 2004 International Society on Thrombosis and Haemostasis
  8. 8. and improved model. This process is still continuing to this day, 40 years later, attesting to the great utility of the concept. The Waterfall-Cascade concept was based on in vitro studies of blood from which the formed elements, often including platelets, had been removed. It remains a good model for the understanding of most tests used in clinical diagnostic work in which platelet poor plasma is usually used. But it had severe shortcomingswhenitwasappliedtoinvivoevents.Theseinclude the complete absence of a bleeding tendency in severe FXII deficiency and only mild bleeding in severe FXI deficiency; there was also the fact that the extrinsic or tissue pathway could not by-pass the intrinsic pathway. These facts were so obvious and well-known at the time that there was hardly any need to point them out. That the two processes of in vitro and in vivo clotting were different was known more than 200 years ago to William Hewson [29] who deduced that the endothelial lining did not merelyplayapassiveroleinphysiologicclotting.Butsuchmajor flaws in the theory were in a sense a strength for it stimulated effortstodiscovertiesbetweenthetwopathwaysandexplainthe clinical anomalies. This was to be an irresistible challenge to some investigators. My former colleagues in San Diego, Bjarne O¨ sterud and Sam Rapaport were amongst those who accepted the challenge. In 1976, they found that the tissue factor-factor VIIa- calcium-complex not only activated FX but also activated FIX thereby bypassing both FXII and FXI [30]. On their hypothesis, tissue factor initiates blood coagulation in vivo. The explanation why the activated FX did not completely by-pass the intrinsic pathway was later explained by the presence of a tissue factor pathway inhibitor. This was later isolated and characterized independently by both Rapaport and his group [31] and Broze [32]. In 1975 the discovery [33] of protein C which could not be detected by laboratory tests in use at the time and the finding that its activation required thrombin bound to thrombomod- ulin [34] on an endothelial cell was further evidence of a cellular component other than platelets and tissue factor-bearing cells being involved in in vivo coagulation. Harold Roberts and his group at Chapel Hill, NC [35] have recently devised an ingenuous system that appears to closely mimic the events that occur in vivo. Their experimental results establish that cellular elements play a key and pivotal role in vivo clotting and the mechanism involved is quite different from that seen in vitro clotting and depicted by the cascade hypothesis; their findings correlate remarkably closely with clinical observations. While the study of in vivo clotting is difficult, this is clearly the wave of the future but the days of the Waterfall or Cascade are far from over. Acknowledgments I wish to thank Earl Davie for sending me an advanced copy of his paper on the same topic which appeared in J Biol Chem, and also for sending me copies of his papers and letters on the topic. I am also indebted to my former colleague and good friend Ms. Kathy Donnelly who kindly read the manuscript and made many helpful suggestions. References 1 Macfarlane RG. An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier. Nature (Lond) 1964; 202: 498–9. 2 Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood clot- ting. Science 1964; 145: 1310–2. 3 Davie EW. A brief historical review of the waterfall/cascade of blood coagulation. J Biol Chem 2003; 278: 50819–32. 4 Roberts HR. Oscar Ratnoff and his contribution to the golden era of coagulation research. Br J Haematol 2003; 122: 180–92. 5 Robb-Smith A. Life and achievements of Professor Robert Gwyn Macfarlane FRS. 1993 London: Royal Society of Medicine Services Ltd. 6 Davie EW, Neurath H. Identification of the peptide split from try- psinogen during autocatalytic activation. Biochim Biophys Acta 1953; 11: 442. 7 Flynn JE (Ed) Blood clotting and allied problems. Trans 1st to-5th Conf of the Josiah Macy Jr Foundation 1948–52. New York, NY: J Macy. 8 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 25. 9 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 125. 10 Seegers WH. Basic enzymology of blood clotting. Thromb Diath Haemorrh 1965; 14: 213–25. 11 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 111. 12 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 127. 13 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 536. 14 Macfarlane RG. The basis of the cascade hypothesis of blood clotting. Thromb Diath Haemorrh 1965; 15: 591–602. 15 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 338. 16 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 611. 17 Seegers WH. Prothrombin. Cambridge, MA, USA: Harvard Uni- versity Press, 1962 p. 516–34. 18 Ornstein L. Disc electrophoresis-1. Background and theory. Ann NY Acad Sci 1964; 121: 321–49. 19 Denson KWE. The use of antibodies in the study of blood coagula- tion. Philadelphia PA, USA: FA Davis Co, 1967. 20 Seegers WH. Historical perspectives related to thrombin. In: Chemistry and Biology of Thrombin. Lundblad RL, Fenton JW, Mann KG eds. . Ann Arbor, MI, USA: Ann Arbor Science Pub- lishers, 1977. 21 Dorsey JN, Seegers WH. Living consciously. The science of Self. Detroit, MI, USA: Wayne University Press, 1959. 22 Seegers WH. Response to question. Thromb Diath Haemorrh 1963; 9 (Suppl. 2); 233. 23 Macfarlane RG. Observations on the interactions of factors VIII, IX and X. Thromb Diath Haemorrh ; 9 (Suppl. 2); 221–6. 24 Ratnoff OD. Comment on paper by Macfarlane. Thromb Diath Haemorrh ; 9 (Suppl. 2); 227. 25 Ratnoff OD, Davie EW. The activation of Christmas factor (factor IX) by activated plasma thromboplastin antecedent (activated Factor XI). Biochemistry 1962; 1: 677–85. 26 Biggs R, Macfarlane RG. Human Blood Coagulation and its Disorders, 3rd edn. Philadelphia PA, USA: FA Davis Co, 1962. 27 Papahadjopoulos D, Hanahan D. Observation on the interaction of phospholipids and certain clotting factors in prothrombin formation activator formation. Biochim Biophys Acta 1964; 90: 436–9. 28 Hougie C, Denson KWE, Biggs R. A study of the reaction product of factor VIII and factor IX by gel filtration. Thromb Diath Haemorrh 1967; 18: 211–22. 1232 C. Hougie Ó 2004 International Society on Thrombosis and Haemostasis
  9. 9. 29 Hewson W. An experimental Inquiry into the Properties of Blood, 2nd edn. London, UK: T Cadell, 1772. 30 Osterud B. Rapaport SI. Activation of factor IX by the reaction product of tissue factor and factor VII. additional pathway for initi- ating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260–4. 31 Sanders NL, Bajaj SP, Zivelin A, Rapaport SI. Inhibition of tissue factor/VIIa activity in plasma requires factor X and an additional plasma component. Blood 1985; 66: 204–12. 32 Broze GJ Jr. The rediscovery and isolation of TFPI. J Thromb Hae- most 2003; 1: 1671–5. 33 Stenflo J. A new vitamin dependent protein. Purification from bovine plasma and preliminary characterization. J Biol Chem 1976; 251: 355–63. 34 Esmon CT, Owen WG. Identification of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981; 78: 2249–52. 35 Roberts HR, Monroe DM, Oliver JA, Chang JY, Hoffman M. Newer concepts of blood coagulation. Haemophilia 1998; 4: 331–4. Historical Sketch 1233 Ó 2004 International Society on Thrombosis and Haemostasis

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