The discovery of factor V: a tricky clotting factor

Transcription

1 Journal of Thrombosis and Haemostasis, 1: REVIEW ARTICLE The discovery of factor V: a tricky clotting factor H. STORMORKEN Rikshospitalet, University of Oslo, Oslo, Norway Summary. The paper tells the story of how FV was discovered in 1943 and installed into the Morawitz theory, a dogma that had reigned the clotting field since 1905 without serious challenges. It is a witness to the fact, many times experienced throughout scientific history, that seminal achievements may arise from serendipity under awkward conditions. Under the worst of circumstances, only a brilliant mind, scientific curiosity and devotion, could the challenge Owren met in Mary s bleeding problem, lead to such a pivotal result. On top of establishing a new clotting factor, his work spurred an unprecedented activity in the field. The thorny road to the new factor s place and role in the clotting mechanism is depicted in some detail. But the factor turned out to be more capricious than its role in coagulation seemed to indicate. Thus, in recent times it has become clear that its platelet counterpart plays an additionally important role in hemostasis as a whole. The two polymorphisms of clinical importance discovered in platelet FV lead to a bleeding disorder, whereas one in plasma FV leads to a rather frequent venous thromboembolic state. Further surprises might therefore be expected from this chameleon of a factor reflecting the increasingly appreciated tendency that one biological compound appears in different roles. Keywords: factor V, coagulation, platelets, polymorphisms, discovery. Early observations on the clotting mechanism Correspondence: Professor Helge Stormorken, Institute for Research in Internal Medicine, Rikshospitalet, University of Oslo, 0027 Oslo, Norway. hestormo@frisurf.no Blood stanching has fascinated laymen and scholars in all cultures from the earliest times. However, no real explanation of the reactions leading to clot formation could be offered before chemistry progressed into the area of proteins, biochemistry, a process comparatively slower than the physical sciences. A starting point for modern development of knowledge in the field was provided by Arthus & Pagès [1] and Pekelharing [2] who showed that calcium ions were involved in clotting. Adding oxalate or other calcium binders to blood provided a means to keep blood fluid a basic condition for progress. A new step was the observation by Schmidt that tissue extract was active in initiating coagulation [3]. It was termed a zymoplastic substance, i.e. a substance that converted an hypothetic entity, prothrombin, into thrombin. Thrombin acted on another protein, fibrinogen, first to be reasonably purified by Hammarsten [4], to form fibrin the clot. Verifying these results and performing new experiments, in 1905, Morawitz formulated a theory by which the zymoplastic substance, renamed thrombokinase, converted prothrombin into thrombin in the presence of Ca 2þ [5]. It took many years of discussion before thrombin was established as an enzyme. It is a witness to how slowly knowledge in biochemistry progressed in the following decades, as compared with physics, that this theory sustained a longevity of 40 years. Or, did the medical profession not take advantage of the possibilities which after all existed? Serendipity opens a new era in clotting research The first part of the serendipity In 1943, a female from upcountry Norway, named Mary and aged 29 years, was admitted to medical department A, University Hospital, Rikshospitalet, Oslo, for the third time. She had been healthy for her first 3 years of life. Then she had a long-lasting nose-bleeding episode with unconsciousness for several hours. This led to loss of her vision, and she was admitted to the Eye Department of the same hospital. It is unknown whether anoxia or local bleeding was the cause, but total blindness of both eyes was established. After some time she regained tunnel vision of the right, but no improvement of the left eye. The following year she was admitted to the same ward because of continual nose bleeds, but was told that no help could be offered either for the blindness or bleeding problems. During the following 25 years she suffered easy bruising and numerous bleeding episodes of variable severity, usually staying at home, but sometimes cared for at the local hospital. The main reason for the new admittance was menstrual bleedings that had kept her anemic for years. Altogether, in her reproductive life, menstrual bleedings were the most significant. The other part of the serendipity It so happened that Paul Owren was assistant professor in the ward at that time (Fig. 1). He came in to specialize in internal medicine from a private practice in a small town in southeastern Norway. Owren had graduated from the Faculty of Medicine, University of Oslo in 1931, with scores that very nearly qualified for presentation to the King. He was a highly

2 Discovery of factor V 207 censured, all windows were blacked out at night for 5 years, traveling more than 30 km necessitated Fahrschein, trains were run on firewood, which also served as gasoline in containers mounted on the cars, misdemeanor was upgraded to war crime, the only university was closed in 1942, research literature, chemicals and equipment were more or less nonexistent, and communication with free countries was strictly forbidden. In this situation, Owren s extraordinary self-sufficiency, inventiveness and practical sense well appreciated in his lineage asserted themselves. Clinical information Fig. 1. Paul A. Owren. popular and busy practitioner a God to his patients. Besides the heavy load of medical tasks, he additionally became a devoted platinum fox farmer. This new fox mutation was very precious; in New York one pelt could be sold for dollars. However, the mutation suffered from low fertility. Bravely, he initiated studies on artificial insemination in such a small and ill-tempered animal, an area only being in its infancy in larger animals, like cows and sheep. As the first in the world, he succeeded, and made a good profit. This was a clear sign of his innovative mind that should later become so evident in his research career. Fortunately for clotting research, he had to give up fox farming. The fur market bottomed with the commencement of World War II, and he moved to Oslo to enter academic medicine in late Norway was not the place for doing research The conditions for research in Norway at that time were far from the best. The country was utterly poor after being occupied, first by Denmark for 400 years and, thereafter, by Sweden for the next 100 years until Norway became a free country in Then came World War I, with the following long-lasting depression, and in 1940 the country was occupied by German forces. When Owren started his studies on Mary s blood, nearly one-quarter of the inhabitants were German troops, and the regime was hard: all consumable items were either rationed or unavailable, radios were confiscated, newspapers stopped or Returning to Mary, she showed no signs or symptoms other than hemorrhages and poor vision, particularly no jaundice. Sedimentation rate was normal, as were hematological findings, except the usual responses to anemia. As the patient was female, classical hemophilia could not reasonably be the diagnosis, and joint bleedings were absent. On the other hand, bleeding time was high normal, and thus compatible with hemophilia. Both the parents, and seven sisters and one brother were free from bleeding tendency. Together with the information that she was healthy the first 3 years of her life, Owren believed that her disease was acquired. Only after some years did study of the family members reveal that some were carriers. Thus, factor (F)V deficiency was inherited as an autosomal recessive trait. Mary s blood under scrutiny The clotting method used in this country, as elsewhere, at this time was Quick s prothrombin time: mixing one volume of citrate plasma and one of thromboplastin suspension, incubate to 37 8C, add one volume of optimal calcium chloride, and starting the stopwatch simultaneously. With rabbit brain thromboplastin (tissue factor) the clotting time in normal blood is then s. Mary s plasma had a much longer clotting time, 70 s, which confirmed that hemophilia was not the diagnosis. Her fibrinogen was normal both in amount and function. According to Morawitz s theory and Quick s opinion, lack of prothrombin should therefore be the cause of her bleeding tendency. It was known that liver failure caused prolongation of the prothrombin time, and that prothrombin was synthesized by this organ. Owren therefore made meticulous studies of her liver functions. However, there were no abnormalities with any of the existing tests, as expected from the clinical information. Ingestion of high doses of vitamin K, recently discovered, was without effect. On the track The result of the prothrombin time indicated a prothrombin concentration less than 10%. Such a low prothrombin was only known to occur in grave liver disease or vitamin K deficiency, from which she did not suffer. On this background, the idea arose that perhaps the Quick test was not adequately reflecting

3 208 H. Stormorken the true defect in Mary s blood. Owren therefore ventured to challenge its reliability. If she lacked prothrombin, he reasoned, her prothrombin time should shorten in proportion to the amount of normal plasma added. Already at the first experiment he identified a puzzle: by serial dilutions of normal plasma added to Mary s plasma the clotting time normalized with only 10 15%, and also with plasma from a severe liver disease patient. Any further increase in normal plasma had no effect. Thus, lack of prothrombin alone could not be solely responsible for her defect. To substantiate this, he made plasma free of prothrombin by adsorbtion with BaSO 4 powder, or asbestos filters. The latter he came across incidentally as, due to lack of a refrigerator or freezer, his plasma samples deteriorated by bacterial growth. From his fellow bacteriologist, he obtained sterilization filters made from asbestos. But alas, after filtration the plasma was not only sterile, it also failed to clot. The procedure had removed the prothrombin (and as became clear years later, also other vitamin K-dependent factors). A new clotting factor revealed The prothrombin-free plasma provided the final proof that the lacking factor was different from prothrombin: addition of this plasma in the same dilutions as normal to Mary s plasma gave full normalization. Thus, in a few weeks he had substantiated a new clotting factor, at this stage different from prothrombin by not being adsorbable. He named it FV since Morawitz s theory comprised four factors. It was published in a lecture to the Norwegian Academy of Science in 1944, but was unknown to the outside world until after the war. Because of some similarities to hemophilia, he classified the disease as parahemophilia. Attempts to characterize the new entity Immediately after having substantiated the fact of a new, necessary factor in clotting, Owren set out to disclose its nature and its place in Morawitz s theory (Fig. 2). He considered his theory as basically correct, but incomplete. There were not many of the presently available tools for this purpose, either biochemical, biophysical or other. Nevertheless, and particularly because he had Mary s unique plasma at hand for assay of fractions, he established that it was a protein: labile to heat and ph changes, unable to pass through a dialysis membrane, and destroyed by proteolytic enzymes. It disappeared rapidly in serum, i.e. by being consumed, and/or inactivated by an inhibitor during clotting. The functional ph optimum was 7.4. Using acid precipitation, the most active fraction occurred between ph , and in the 33 50% fraction by ammonium sulfate precipitation. By removing prothrombin (and the Fig. 2. The Morawitz theory. Fig. 3. Owren s new theory 1947 (6). He was unsure at that time as to whether cofactor V and prothrombin could be identical because they were both adsorbable. He subsequently named FVI þ Ca prothrombinase the final prothrombin-converting enzyme an important fact related to the new theories formulated in 1964 (see below). unknown FVII, IX and X) in the starting plasma, he managed to prepare 11 mg of pure FV with an activity 100 times that of normal plasma. This provided an important weapon in forthcoming studies of the clotting mechanism. Furthermore, he could also produce the purest prothrombin to that date by using Mary s FV-free plasma as the starting point. Because his goal was to work with the purest possible reagents in order to get a true picture of the coagulation mechanism, he also improved on the preparation of fibrinogen. By preparing fibrinogen from BaSO 4 -adsorbed FV-deficient plasma, the annoying partial coagulation ( profibrin ) was avoided. This fibrinogen allowed an accurate definition of a thrombin unit for use in further studies. From many experiments, he also had hint of another factor being involved in clotting, called cofactor V, which later became known as proconvertin and finally FVII. He thought that this entity was activated by thrombokinase (thromboplastin) to what he named FVI. When united with FV, prothrombinase was formed, an enzymatic complex he assumed to be the final prothrombin-converting principle. Subsequently he realized that since FVI was a complex, the use of factor was incorrect. When his principle of using Roman numerals as designations for future new factors was adopted by the International Committee on Nomenclature of Blood Clotting Factors, FVI remained, but without content. By his achievements, the new look of the Morawitz s theory was as represented in Fig. 3. War is over Owren s discovery becomes known internationally After the armistice, the situation in Norway, as in many other European countries, was chaotic and it took time before Owren s results could be made known internationally. The process began in 1946 when he obtained a grant to go to the Lister Institute in London to search the existing literature a necessity before the final writing could be done. This was the most exiting travel in his life among so many to come. Luckily, he could assert that nobody else had investigated in the area of his findings. During his stay in London, he had the chance to present his work at the XVII International Congress of Physiology in Oxford, and then at the London Medical School, University College Hospital. He also met with the already renowned

4 Discovery of factor V 209 researchers in the field, R.G. Macfarlane and Rosemary Biggs, who immediately accepted his findings. In 1947, the full account was published as a doctoral thesis of 327 pages [6], and as a condensed version in the Lancet [7]. The work he had done at the Lister Institute on further purification and role of FV followed [8], in which he established that the labile activities in plasma observed by Quick [9], Fantl & Nance [10], and Ware et al. [11] were identical to FV. These publications came like a bombshell on the clotting community. An entirely unknown person had overturned ideas which had stood the test of time ever since From now on he was overrun with requests from countless countries to lecture, first and foremost about his new clotting factor, but also in general hematology and the use of anticoagulants in thrombo-embolic diseases. He won international recognition through a penetrating study of the pathogenesis of the hemolytic crisis [12], and by being an early promoter of the anticoagulation modality. Not everybody hailed his achievements Both A.J. Quick and W.H. Seegers had been in the game for many years when Owren s work was published, and they did not readily accept this newcomer. The two were not known for being the best of friends, and they were physically very different: Seegers was a giant, Quick a flyweight perhaps not an innocent reason for their antipathy. Sometimes their skirmishes became harsh, with glaring invectives flying through the air. Facing Seegers and Owren, Quick had hard times in defending the claim that his test was specific for prothrombin, and that his labile factor was part of prothrombin [9]. Seegers did not agree with Owren s theory, although he accepted the existence of FV. His group was even first to show that it (his accelerator globulin) was activated by thrombin [11]. However, the nomenclature and theory for coagulation of Seegers group deviated radically from the other two [13]. He held that prothrombin was the mother molecule for other clotting factors. Thus, there were rich possibilities for quarrel. The background for Seegers view was analysis of fractions which appeared from his pure prothrombin, which, when dissolved in strong citrate solutions, were converted into thrombin. In contrast to Quick, Seegers was an excellent biochemist. When the two camps Seegers and the rest of the clotting community came to a better understanding, it turned out that Seegers [13] had independently characterized FVII (autoprothrombin I), FIX (autoprothrombin II), FX (autoprothrombin III), and protein C (autoprothrombin IIa). Except for the latter, the factors were at this point all discovered from studies of bleeder patients. Both the fact that he did not refer his factors to such patients, and that his nomenclature was difficult to understand, his work was not adequately appreciated. Ironically, his successfully isolated factors were contaminants of his pure prothrombin. In 1953, he sent samples of his prothrombin to Owren and to Duckert in Basle for testing. Both found it to contain significant amounts of FVII and FV, as tested on authentic factor-deficient patient plasmas. Some are more difficult to convince than others Seegers did not accept these findings. His explanation was that the prothrombin of their patients was abnormal and falsely imitated a lack of the tested factors. Up until as late as in 1962 [13], he continued to fight for his view of the decisive importance of prothrombin. Thus, a considerable part of the more than 700 pages of his book is dedicated to this purpose. Other factors are only products of theories was his refrain, a refrain he might have bitterly regretted. It is as well to look after what nature itself has created, and there prothrombin is not converted in strong citrate solutions. What he accepted in 1953, when he sent Owren the joke headline depicted in Fig. 4, was an enigma, also to Owren. Nobody knows, however, whether it was a gag from some adversary. Like Seegers, stubbornly defending his theories for an unreasonably long period of time, Quick was no less stubborn in defending the specificity of his test for prothrombin. This was so in spite of Owren s clear demonstration to the contrary in 1947, and the subsequent work of many others, including the respected Chapel Hill and Oxford groups. Also, it was quite unnecessary as his prothrombin time from 1936 has been the dominating test for control of anticoagulant prophylaxis up until our time and will continue to spread his fame for many years to come. Ironically, however, this will not be for its specificity for prothrombin, but for its unspecificity, as the other vitamin K- dependent factors are more decisive for its usefulness. Only after many years did Quick hesitantly throw in the towel. In the meantime he had given up the idea that his labile factor was part of prothrombin. Thus the quarrel over these and other discrepancies went on for years. Such fights were more common than now-a-days the personalities were strong and more outspoken in insisting on their righteousness, sometimes in vulgar language, but also sometimes with a sophisticated, ironic or salty wit, as the following by Leo Vroman [14]: I shall continue below to use the term factor XI with the friendly understanding that I mean something that seems to be absent in certain patients under certain experimental conditions, and that it is either one protein, two proteins, one protein and a piece of prothrombin that somehow detaches itself, or a gang of hairy demons out to destroy the world of coagulationists. Even an error, if well defined, deserves a name, and factor XI is as good as any. Although Owren was not entirely free from such tendencies, he did not participate in the Quick Seegers fights, but had a reasonably good relationship with both particularly to Seegers whose wife was of Norwegian descent as well as to other researchers around the world. Where was the correct place of FV in the cascade? This question vacillated for years. As mentioned already, in 1947, Seegers group showed that small amounts of thrombin activated FV. Tage Astrup then recommended Owren to name FV with a more functional term: proaccelerin, the activated

5 210 H. Stormorken Fig. 4. The Joke headline sent to Owren in form accelerin. This would indicate both its mode of action, and that it circulated as an inactive molecule. As this fitted well with Owren s finding in his thesis, of an auto-catalytic formation of thrombin, it was readily adopted. Unknown to Owren, however, evidence of an auto-catalytic mechanism of the clotting mechanism had been published previously [15]. When it became clear that cofactor V was different from prothrombin, and was renamed proconvertin, he envisaged the two initial stages of the clotting mechanism, as illustrated in Fig. 5 [16]. This way of illustrating the clotting mechanism did not prove its dynamics. Henceforth, Owren built the thrombin feedback mechanism into his, often spectacular, schemes of the process. He also thought that there were other stations underway to appearance of thrombin. Figure 6 shows his comprehensive view in 1954 of the in vivo reactions leading to clot formation [17]. As can be seen, the extrinsic system is as of today, except that FX had not been discovered at that time. For the intrinsic system, FXI and FXII are not included, the former having been discovered recently, and the latter not yet known. The organization of the system in intermediates is evident, and Owren has a composite enzyme, prothrombinase, which is finalized by the addition of accelerin, as the prothrombinconverting principle. The acclerin was supposed to derive from proaccelerin through small amounts of thrombin produced by convertin, the immediate precursor of prothrombinase. Then thrombin formation proceeds into an auto-catalytic reaction. This stage is common to the intrinsic and extrinsic mechanisms, which Owren held were working intimately together in the

6 Discovery of factor V 211 Fig. 5. Owren s mechanism as of Proconvertin is identical with FVIII (16). body an idea that has revived in the modern scheme where tissue factor is considered the main initiator. He was not fond of the extrinsic intrinsic division, but accepted that it was didactically and practically useful. New theories Ten years later, two new mechanisms were proposed, the cascade/waterfall theories by Macfarlane [18] and by Davie & Ratnoff [19] in In these theories, the formation of thrombin was seen as continual proenzyme enzyme reactions, working as a biochemical amplifier system. The weak point in this theory was that two key factors, FVIII and FV, were never proven to be proenzymes. It was tempting, though, to believe so since FV could be activated by thrombin, and the same was shown to occur with FVIII in 1963 by Rapaport et al. [20]. Thus, at this stage there were two feedback mechanisms that could explain the explosive formation of thrombin. Both mechanisms were known when the theories were presented, but were not paid any attention. As seen from Fig. 7, FVIII was activated by IX a, and FV by X a, the feedback effect of thrombin not being taken into account. Importantly, FV a figured in the role of prothrombin converting enzyme. The latter is particularly difficult to understand because nothing in Owren s work or writings indicated this role for his baby. To the contrary, he had a composite enzyme, prothrombinase, as the thrombin-forming activity. It seems likely that if the proponents had read Owren s publications, a better theory would have resulted. However, there is no reference at all to his work in their papers, the reason for which is indeed intriguing. The role and place Owren gave FV 14 years before the cascade theories are still valid. Steadily new progress The new theories were seducing in their simplicity, but nature is rarely simple. A few years after it became clear that defined phospholipid micelles were crucial for ideal stereotactic attachment of the factors and cofactors (V, VIII). This implies not a continual but a stepwise mechanism alternating between liquid and solid phases. The amplifier function is mainly, if not entirely, based on the feedback activation by thrombin on FV and FVIII. Furthermore, another break in the cascade model was unraveled by Osterud & Rapaport [21] in that FIX may be Fig. 6. The 1954 version of Owren s coagulation mechanism (17).

7 212 H. Stormorken Fig. 8. P.A. Owren: cartoon from one of his brilliant lectures. Courtesy of Dr Rolf Gaertner. Fig. 7. The new theories. Left Macfarlane s, right Davie & Ratnoff s version [18,19]. activated directly by FVII a -TF a fact indirectly predicted by Josso & Prou-Wartelle [22]. This represented a prolog to the now dominating role allocated to the tissue factor. It is a question as to whether or not the cascade theories promoted progress of coagulation research. However, even a wrong theory my inspire to challenge and thus be fruitful. At this point of time, it seemed that the clinical role of FV a very rare deficiency causing prolonged blood coagulation and bleeding would finally be established. Not so, again new Marys, alert researchers, and other serendipities, testify to the contrary. In 1984, Tracy et al. [23] described one platelet FV-polymorphism and, in 2001, Weiss et al. [24] another, both causing a bleeding disorder in which platelet FV plays a different role than plasma FV. Furthermore, in 1994 it became clear that a rather frequent mutation, plasma FV Leiden, leads to an increased thrombotic tendency because it cannot be inactivated by protein Ca [25,26]. This proved an astonishing fact: a normal FV molecule, a procoagulant, is important to counter thrombo-embolism! Unexpectedly, FV has become a significant participant in the thrombo-hemorragic balance. It is wise to remember: surprises never end! The full impact and mechanisms of these new revelations remain to be learned [27]. Thus, Owren s discovery is still causing repercussions and new challenges for laboratories engaged in hemostasis research. Mary an internationally appreciated lady Soon after the discovery of FV was published, requests for samples of Mary s blood came in from all continents, notably Europe and USA, for comparison with their own pathological plasmas. Numerous times, Mary traveled the long distance to Rikshospitalet to give samples of her precious blood, always needing a relative s company due to her blindness. And she never objected or craved compensation. Can it benefit others with such a fate, there is no better pay for me was her attitude the greediness culture of today was alien to her. She was charming, cheerful and content, despite her serious handicaps (Fig. 9). Being one necessary part of the serendipity that led to a new deal in clotting research after 40 years without a breakthrough, she deserves to be remembered: Ten years after Owren s discovery, the number of clotting factors reached 13. Mary s life ended in March 2002, at the age of 88, from a traumatic bleeding in an ankle with subsequent intractable infection. Paradoxically, joint bleedings had been very rare in her long life.

8 Discovery of factor V 213 Fig. 9. Mary, as always with a cheerful smile. Professor Mannucci has accepted to investigate the DNA abnormality of Mary s FV, a task that I, regrettably, have ignored for too long. References 1 Arthus M, Pagès C. Nouvelle theorie chimique de la coagulation du sang. Arch Physiol Norm Pathol 1890; 5: Pekelharing CA. Untersuchung über das Fibrinferment. Internat Beitr Zschr Wissensch Med (Festschr Rudolf Virchow), 1891; 1: Schmidt A. Zur Blutlehre, Vogel, Leipzig, Hammarsten O. Weitere Beiträge zur Kenntnis der Fibrinbildung. Zeitschr Physiol Chem 1899; 28: Morawitz P. Die Chemie der Blutgerinnung. Ergebn Physiol 1905; 4: Owren PA. The coagulation of blood: investigations on a new clotting factor. JR Gundersen, Oslo, Dissertation 327 pp. (Also published as Suppl. 194 to Acta Med Scand 1947). 7 Owren PA. Parahaemophilia. Haemorrhagic diathesis due to absence of a previously unknown clotting factor. Lancet 1947; 1: Owren PA. The fifth coagulation factor (factor V). Preparation and properties. Biochem J 1948; 43: Quick AJ. On the constitution of prothrombin. Am J Physiol 1943/44; 140: Fantl P, Nance M. Acceleration of thrombin formation by a plasma component. Nature 1946; 158: Ware AG, Guest MM, Seegers WH. Plasma accelerator factor and purified prothrombin activation. Science 1947; 106: Owren PA. Congenital hemolytic jaundice. The pathogenesis of the hemolytic crisis. Blood 1948; 3: Seegers WH. Prothrombin. Monograph. Harward University Press, Harvard, Vroman L. Disc. In: Blood Clotting Enzymology, eds. W H Seegers. Academic Press, New York, 1967: Fisher A. Blutgerinnung als unbegrenzt Übertragbare Kettenreaktion. Biochem Ztschr 1935; 279: Owren PA. Proceedings of the International Congress of the Society of Hematology, Cambridge 1950, Grune & Stratton, New York, 1951: Owren PA. The present state of the converting and accelerator factors in prothrombin conversion. In: Koller TH, Merz WR, eds. Thrombosis and Embolism. I International Conference, Basle 1954, Benno Schwabe & Co, Basle, 1955: Macfarlane RG. An enzyme cascade in the blood clotting mechanism, and the function as a biochemical amplifier. Nature (Lond) 1964; 202: Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood clotting. Science 1964; 145: Rapaport SI, Shiffman S, Patch MJ, Ames SB. The importance of activation of antihemophilic globulin and proaccelerin by traces of thrombin in the generation of intrinsic prothrombinase activity. Blood 1963; 21: Osterud B, Rapaport SI. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: Josso F, Prou-Wartelle O. Interaction of tissue factor and F VII at the earliest phase of coagulation. Thromb Haemost Suppl. 1965; 17: Tracy PB, Giles AR, Mann KG, Eide LL, Hoogendoorn H, Rivard GE. (Quebec): a bleeding diathesis associated with a qualitative platelet factor V deficiency. Clin Invest 1984; 74: Weiss HJ, Lages B, Zheng S, Hayward CP. Platelet factor V New York: a defect in factor V distinct from that in factor V Quebec resulting in impaired prothrombinase generation. Am J Hemat 2001; 66: Dahlbäck B, Hildebrand B. Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V. Proc Natl Acad Sci USA 1993; 91: Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: Stormorken H. Paul A. Owren and the Golden Era of Haemostasis. Monograph 2003.