Portal de Periódicos da CAPES

Stuart Factor: discovery and designation as factor X

2003; Elsevier BV; Volume: 1; Issue: 5 Linguagem: Inglês

10.1046/j.1538-7836.2003.00259.x

1538-7933

John B. Graham,

Hemophilia Treatment and Research

Kenneth Brinkhous and I were colleagues for 50 years. In our last discussion before his final illness, probably in 1995 or 1996, he opined that the three most important products of our 50 years of effort in Chapel Hill had been the Partial Thromboplastin Time test, purification of factor (F)VIII from plasma, and the discovery of the Stuart Factor. The first two have been memorialized many times, but little has been said about Stuart Factor. The editors of the Journal of Thrombosis and Haemostasis are collecting stories about the discovery of the factors of the coagulation cascade and have asked me to write about Stuart Factor. I asked Dr Hougie to join me, but he is writing a book on the subject, and we settled for single authorship and mutual support. Readers will see why he was the vital partner in the Stuart enterprise. I shall try to describe succinctly how this key factor of the cascade was discovered and why it received the designation of factor X (Roman 10). But the story is complicated. It had been known since at least the 18th century that deficiency of a dietary substance, which we now know as vitamin C, results in scurvy. This was a major scourge during the days of sailing ships and probably was the greatest cause of severe bleeding [1Quick AJ. Bleeding, drugs, vitamins. Their impact on history 1976. Privately published.Google Scholar]. Scurvy causes deterioration of the walls of blood vessels, which can be alleviated by the eating of fresh vegetables, particularly citrus fruit. British sailors have long been nicknamed ‘Limeys’, because the British Navy has insisted since the 18th century that its vessels stock fresh vegetables and citrus fruit. During the 1920s and 1930s, a number of important discoveries were made about another hemostatic mechanism, the intrinsic process responsible for maintaining the fluidity of blood and avoiding hemorrhage. The hypothesis that I was taught as a student was formulated by Morawitz in 1904. Prothrombin→Thrombin in the presence of calcium and thromboplastin Fibrinogen→Fibrin in the presence of thrombin Fibrin causes blood to coagulate During the 1930s and 1940s, vitamin K was found to be essential for the production of prothrombin, and prothrombin was found to be synthesized in the liver. Armand Quick developed the very useful Prothrombin Time Test, and research workers at the University of Iowa—including Brinkhous—developed a two-stage quantitative assay for prothrombin. Patek, Stetson, and Taylor in Boston demonstrated that something was missing in the blood of hemophiliacs that could be replaced by the addition of normal blood. They postulated that normal blood contained an ‘antihemophilic factor.’ Thromboplastin was found in many tissues. Rat brain (Quick) and lung (the Iowa group) were the main sources. Karl Paul Link and his colleagues at the University of Wisconsin discovered that cows which ate fermented sweet clover bled excessively. This led to the discovery of the coumarins, chemicals that interfere with coagulation. One of these compounds was isolated, purified, synthesized, patented, named WARFARIN and the patent given to the Wisconsin Alumni Research Foundation. As COUMADIN, it is in worldwide clinical use today for preventing and treating thrombosis and is also an effective rat poison. A Nobel Prize was awarded in 1943 to Henrik Dam of Denmark and Edward Doisy of the USA, scientists who had made important discoveries in this field. Brinkhous told me that the Iowa group had been in the running for this prize, which had been awarded while he was away in military service. When I opportunistically entered the coagulation field in 1946, there was already a general feeling that the Morawitz hypothesis had serious shortcomings. This was accentuated by work done in Norway during World War II by Paul Owren. He had studied a paradox, a woman who seemed to have hemophilia, a condition thought to be limited to men. He showed that the principle missing in her blood was not the same as that described by Patek et al. in male hemophiliacs, and called the disorder ‘parahemophilia.’ Owren named the ‘new’ substance ‘accelerin’ and pointed out that it was, perforce, the fifth clotting factor. He observed that its activity could be enhanced by thrombin and suggested that enhanced factor V be designated factor VI. This was an unfortunate suggestion because, as we shall see, a later decision concerning Stuart Factor left a gap in the numerical sequence of ‘new’ factors. Owren's discovery opened Pandora's box and many additional factors were discovered during the 1950s, each independently several times and each discoverer giving a distinct name to his own discovery. They are listed Table 1 in the order of their appearance. The references to these discoveries are contained in the Bibliography of our original publications on Stuart Factor [2Hougie C. Barrow E.M. Graham JB. Stuart clotting defect I. Segregation of an hereditary hemorrhagic state from the heterogeneous group heretofore called ‘Stable Factor’ (SPCA, proconvertin, Factor VII).J Clin Invest. 1957; 36: 485-96Crossref PubMed Scopus (121) Google Scholar, 3Graham J.B. Barrow E.M. Hougie C. Stuart clotting defect II. Genetic aspects of a ‘new’ hemorrhagic state.J Clin Invest. 1957; 36: 497-503Crossref PubMed Scopus (47) Google Scholar].Table 1The key ‘factors’ involved in fibrin clotting with dates of discovery, names given by discoverers, corresponding disease states, and the numerals approved by the International Committee on Nomenclature of Clotting FactorsYear of discoveryName of factorCorresponding disease stateInternational nomenclature<1940FibrinogenAfibrinogenemiaF.-I<1940ProthrombinHypoprothrombinemiaF.-II<1940Antihemophilic FactorClassic hemophiliaF.-VIII1942–45Ac Globulin, proaccelerinParahemophiliaF.-V1948FSF, LLF, FibrinaseFibrinase (–)F.-XIII1949Spca, proconvertinSpca (–), hypoproconvertinemiaF.-VII1952PTC, Christmas FactorPTC (–), Christmas diseaseF.-IX1953PTAPTA (–)F.-XI19544th Thromboplastic ComponentTetartohemophilia1955Hageman FactorHageman trait (not a disease state)F.-XII1956Stuart FactorStuart Clotting DefectF.-XF.III(Thromboplastin) F.-IV(Ca††) F.-VI(Unassigned) Open table in a new tab F.III(Thromboplastin) F.-IV(Ca††) F.-VI(Unassigned) The ferment in the blood clotting field in the 1940s led the Macy Foundation to include blood clotting among the first set of disorders by which they hoped to stimulate progress. (The 13 disorders of the Macy's first set were the adrenal cortex, aging, biological antioxidants, blood clotting, blood pressure, connective tissue, consciousness, cybernetics, infancy and childhood, liver injury, metabolic interactions, nerve impulse, and renal function.) The foundation's ‘5-year plan’ was to pull together a group of experts in each field annually to discuss the nature of a specific field and to stimulate each other. The proceedings were published and copies made available at a reasonable price. Brinkhous was a member of the blood clotting discussion group, and I was an avid reader of the output. The Macy Conferences on Blood Clotting (1947–1952) gave Americans a great boost in the field of coagulation, since the Macy panel contained only three non-Americans: one Canadian and two Europeans. The ‘new’ clotting factors being discovered almost every year created great confusion among clinicians and teachers of medicine and produced a demand that the nomenclature be rationalized. A National Institutes of Health (NIH) grant was obtained to form a committee whose activities were based on the Macy model. The late Irving Wright, who had served as Chairman of the Macy committee and was a member of the Council of NIH's Heart Institute, shepherded the grant and became Chairman of a self-appointed ‘International Committee on the Nomenclature of Blood Clotting Factors.’ He co-opted onto it a number of former Macy members and became the ‘Godfather’ of the international effort. The idea of an international committee was first bruited about at a meeting in Switzerland in the mid 1950s, and the first meeting was held in Rome in 1958. Its initial decisions were to assign a Roman numeral to each of the clotting factors, and to have a working meeting in Montreux, Switzerland in July 1959. (The NIH grant assisted with the transportation and hotel expenses of the participants.) At these meetings, the discoverers of ‘new’ factors presented their data and the committee assessed the evidence. A vote was taken on the validity of a claim and an approved factor was assigned a Roman numeral. This process was followed until all claims had been judged. Figure 1 is a photograph of those who attended the 1959 meeting at the Palace Hotel in Montreux. Hougie, Stuart Douglas, and I are numbers 35, 36, and 37. Once the nomenclatural task had been completed, the committee was faced with a dilemma. What next? A minority felt that the committee should disband. The majority believed that since the meetings had been very useful, and since the workers in the field had become acquainted, some sort of effort should continue. The name was changed to the ‘International Committee on Haemostasis and Thrombosis’, a constitution was adopted, and a new NIH grant was obtained. The new committee eventually metamorphosed into a society: The International Society on Thrombosis and Haemostasis. Federal funding has continued and the leadership has always resided in Chapel Hill, first with Brinkhous, then Harold Roberts, and now Gilbert White. Researchers in the clotting field have been a diverse lot. Hemorrhage and thrombosis are clinical phenomena that complicate the therapy of all types of illness in patients of all ages. Thus internists, surgeons, pediatricians, gynecologists, etc., have been interested in solving the problems. Early on the research was done at the bedside or in the clinical laboratories. Biochemists soon became involved, e.g. Quick, Seegers, Link, etc., and many laboratories recruited in-house biochemists as full members of their research team (e.g. Wagner at UNC). There was an unspoken assumption that all problems should be reduced to their biochemical essence, and many of the successful clinicians in later years had obtained some sort of biochemical training. The inter-Society sessions on Blood Coagulation at the annual Federation Meetings in America became a preferred venue for presentation of the latest developments. Interestingly, all the ‘new’ factors listed in Table 1 were discovered or confirmed by study of exceptional patients. In a typical case, an unusual bleeder was discovered whose defective plasma could be shown to differ from that of all other known types of bleeders. The crucial tests were qualitative since no assays existed. Later on the plasmas of prototype patients were used as substrate for assaying specific factors under the assumption that the defective plasma contained little if any of the newly discovered ‘factor’ but had a full complement of all other factors. The biochemists were shocked by such assays since the chemical nature of the substances being measured and their physiological function were unknown. The ‘new’ substances were called ‘factors’ since there was no knowledge of their true nature. I was invited to give a paper at the annual meeting of the American Society of Human Genetics in September 1955 and outlined for the geneticists the essential facts of the case [4Graham JB. Biochemical genetics of blood coagulation.Am J Hum Genet. 1956; 8: 63-79PubMed Google Scholar]. Geneticists did not find the facts disturbing, since they saw that I was demonstrating ‘genetic complementation’, a phenomenon with which they were familiar from work in corn and fruit flies (Fig. 2). In the discussion I was asked how the multiplicity of factors interacted. My reply is shown schematically in Fig. 3. This scheme is a modification of the Morawitz hypothesis, which indicates that there are unknown interactions between a large number of distinguishable factors that trigger the clotting of blood. It required a decade of intense biochemical research before an overall answer was forthcoming. Macfarlane in the UK enunciated a ‘Coagulation Cascade’[5Macfarlane RG. An enzyme cascade in the blood clotting mechanism and its function as a biochemical amplifier.Nature. 1964; 202: 498-9Crossref PubMed Scopus (703) Google Scholar] and Ratnoff and Davie in the USA proposed a ‘Coagulation Waterfall’[6Davie E.W. Ratnoff OD. Waterfall sequence for intrinsic blood clotting.Science. 1964; 145: 1310-1Crossref PubMed Scopus (715) Google Scholar]. In his book, Dr Hougie will describe some of the hanky-panky that accompanied these essentially identical proposals from the two sides of the Atlantic. It is of interest that the Stuart Factor (now FX) was at the heart of both the ‘Cascade’ and the ‘Waterfall.’Figure 4 is a proposed explanation of the interactions, which was current in 1987 when I retired from the fray. Stuart Factor was discovered because workers at UNC re-investigated a purported example of ‘hypoproconvertinemia’ or SPCA deficiency. The discoverers of the Stuart Factor (Graham, Barrow, and Hougie) are shown in Fig. 5 together with Mr Rufus Stuart, proband of the Stuart kindred. At this time (summer of 1955), Cecil Hougie, a young Englishman, joined the University of NC. He had spent some time at Oxford in the laboratory of Biggs and Macfarlane and had learned to use their Thromboplastin Generation Test (TGT) and had used Russell's Viper venom (Stypven) on occasion. Macfarlane had suggested in Hougie's hearing that there seemed to be some heterogeneity amongst the subjects with SPCA or ‘hypoproconvertinemia’ seen at Oxford. This suggested the existence of two very similar factors. Hougie decided to investigate this possibility in NC and recruited me to help him. He had located an SPCA-deficient patient already studied by Drs Lewis and Ferguson [7Lewis J.H. Ferguson JH. Congenital hypoproconvertinemia.Proc Soc Exp Biol Med. 1953; 84: 651-4Crossref PubMed Scopus (6) Google Scholar]. I agreed to collaborate, because whether the man had SPCA deficiency or something else, a genetic study would be profitable. Hougie had made several trips to visit this person, but had not been able to obtain the material he needed. Together with my 10-year-old son, he and I visited Ashe County, North Carolina, during Thanksgiving weekend of 1955. We met Mr Rufus Stuart, the patient studied by Lewis and Ferguson, and convinced him to introduce us to his relatives. He was very friendly and guided us around the very rural and mountainous area. We drew blood from those we met, took a family history, and returned to Chapel Hill the next day. More details of our trips to Ashe County have been published elsewhere [2Hougie C. Barrow E.M. Graham JB. Stuart clotting defect I. Segregation of an hereditary hemorrhagic state from the heterogeneous group heretofore called ‘Stable Factor’ (SPCA, proconvertin, Factor VII).J Clin Invest. 1957; 36: 485-96Crossref PubMed Scopus (121) Google Scholar, 8Graham JB. Stuart Factor (Coagulation Factor X). A North Carolina saga.NC Med J. 1988; 49: 328-31PubMed Google Scholar] and the family tree is shown in Fig. 6. This pedigree chart [9Graham JB. Stuart clotting defect and Stuart factor.Thromb Diath Haemorrh. 1960; 4PubMed Google Scholar] shows that Mr Stuart's mother and father were related as aunt and nephew, a not unknown mating type in large families in isolated rural communities. We found that his blood clotted very slowly, and that a brother who had been seen earlier by Lewis, Fresh, and Ferguson had died since their visit. We found mild clotting defects in all his children and some of the relatives on both sides of his family. Clearly the mutant gene was autosomal and not completely recessive. It is ironic that the mutant gene was introduced into the Stuart family via the Blevins kindred, and the abnormality might more properly have been designated the Blevins Factor. But the cognomen Stuart is more euphonious, and the fashion in those days was to use the surname of the proband when naming a ‘new’ factor. We demonstrated conclusively that deficiency of Stuart Factor differed from SPCA deficiency by a plasma mixing experiment like those in Fig. 3. Ben Alexander did this for us in a blind experiment. He used plasma from his prototype SPCA-deficient patient to compare with a sample of Stuart's plasma. The two were very similar in almost all respects. Both had prolonged prothrombin times, but a normal prothrombin time was observed when they were mixed. Later Hougie showed that SPCA-deficient plasma appeared normal when tested with Stypven [10Hougie C. Effect of Russell's viper venom (Stypven) on Stuart clotting defect.Proc Soc Exp Biol Med. 1956; 98: 570-3Crossref Scopus (15) Google Scholar] and in the TGT [11Hougie C. Reactions of Stuart factor and Factor VII with brain and Factor V.Proc Soc Exp Biol Med. 1959; 101: 132-5Crossref PubMed Scopus (15) Google Scholar], while Stuart's plasma was abnormal in both tests. I established a warm friendship with Mr Stuart that lasted until his death in 1989. He was also very attached to Harold Roberts who was his medical doctor for many years and Philip Webster who was his dentist [8Graham JB. Stuart Factor (Coagulation Factor X). A North Carolina saga.NC Med J. 1988; 49: 328-31PubMed Google Scholar]. I was asked to participate in his funeral [12Graham JB. In memoriam: Rufus Stuart.NC Med J. 1989; 50: 298Google Scholar], which was a very moving experience. I do not think I have ever seen a patriarch who was more greatly loved by his family. Figure 7 is a photograph of Mr and Mrs Stuart. Their sweet natures and mutual affection are obvious. The collected Stuart Factor levels of the subjects studied are summarized in Table 2.Table 2The Stuart Factor levels of 14 members of the Stuart family. The levels of the factor in the three genotypes do not overlap and detection of carriership should be an easy matter among the grandchildrenPresumed genotypeNumber of persons% Stuart Factormean ± s.d.rangeHomozygous abnormal11–3Hetrozygous (carriers)836 ± 1021–52Homozygous normal597 ± 686–100+ Open table in a new tab We offered to supply Stuart's plasma to anyone wishing it, and at least 34 samples were sent out the first year. Twenty of the tested subjects proved to be examples of deficiency of the ‘stable factors’ (Stuart and SPCA deficiency). Nine were of Stuart Factor, seven were of SPCA deficiency and four were multiple deficiencies. Our data on Stuart Factor were presented to and accepted by the International Committee in Montreux in 1959. The question then became, ‘What numeral is it to be assigned?’ Fritz Koller approached me at lunch to ask if I would be willing to consider naming it Factor X. He had seen some sort of strange effect in the plasma of dicoumarolized rats and, I suppose, wanted part of the credit for discovery. It is to his credit that he never pursued this claim. I had no particular reason to object and agreed. Under the Parliamentary rules of the Committee, Brinkhous moved that the Stuart Factor be assigned the unassigned VI (Accelerin had been designated V and SPCA VII). The motion was seconded and discussed. Hougie and I were spectators, not members of the committee, and I felt that we could speak only when asked to do so. Leandro Tocantins of Jefferson Medical College in Philadelphia gave an impassioned speech against designating Stuart Factor Factor VI and in favor of naming it FX. He was particularly vehement against our mixing experiments and emphasized the superiority of experiments done in experimental animals. I had not informed Hougie of my conversation with Koller, and he was infuriated by Tocantins' remarks. He remembers that I gave him a kick under the table when he started to stand up and counter Tocantins' remarks. To make a long story short, Tocantins carried the day. This is unfortunate, because it left an inexplicable lacuna in the list of factors. Hougie and I are still mystified by Tocantins' heated rhetoric. The only reasons that occur to me are (i) this proud man felt that he had to beat Brinkhous on something, and (ii) he may have thought that I was supporting Brinkhous's motion and wanted also to pay me back for having destroyed his standing as a significant worker in the field. Perhaps I should digress and explain this. Tocantins had been the proponent of an alternative theory of the nature of hemophilia, which he had trumpeted on every occasion. The Chapel Hill group believed—as did almost everyone else—that Tocantins' theory that hemophilia resulted from excess of a lipid inhibitor was erroneous. Emily Barrow and I investigated this matter in experiments using hemophilic dogs and demonstrated that all his results could be explained as artefacts due to uncontrolled ionic strength. We presented our data at the 1955 Federation meeting in San Francisco and later that year at a meeting of the National Research Council. We published these data in the Journal of Experimental Medicine in 1957 [13Graham J.B. Barrow EM. Pathogenesis of hemophilia: an experimental analysis of the anticephalin hypothesis in hemophilic dogs.J Exp Med. 1957; 27: 273-92Crossref Scopus (5) Google Scholar]. Tocantins' theory was the chief basis for his standing in the field, and we had pulled the rug from under him. I felt sorry for him, but he had only himself to blame. Brinkhous was innocent. He was not even aware of what Barrow and I were up to until our work was completed. I suppose Tocantins thought he was killing two birds with one stone. (Later he was able to do me a bad turn by shortening the tenure of a grant that I had received from the Dental Research Institute, but that is another story.) The lesson I learned from my experience with Tocantins was best expressed by a friend who had been a big game hunter in Africa. He averred that ‘If you are hunting an elephant you had better kill it with the first shot, because if he is only wounded he will try to kill you’. Since the halcyon days of the discovery of new clotting factors and their naming, the field has changed enormously. The so-called factors are recognized as proteins, generally enzymes. Genetics and molecular biology have come into their own and clotting disorders are now studied using the latest genetic probes. The late William Dameshek, then editor of Blood, asked several authorities in 1953 to provide essays on ‘What is Hemophilia?’ Tocantins presented his usual eccentric view, and only I, Brinkhous' associate, gave a response in genetic terms [14Brinkhous K.M. Graham JB. Hemophilia and the hemophilioid states.Blood. 1954; 9: 254-7Crossref PubMed Google Scholar]. I am pleased in retrospect to see my prescience in 1954 about the nature of hemophilioid diseases. My remarks were made a decade after Beadle and Tatum had shown a one gene–one enzyme relationship in Neurospora[15Beadle G.W. Tatum EL. Genetic control of biochemical reactions in Neurospora.Proc Nat Acad Sci Wash. 1941; 27: 499-5Crossref PubMed Google Scholar] and the year after Watson and Crick had described the structure of DNA [16Watson J.D. Crick FHC. A structure for deoxyribonucleic acid.Nature. 1953; 171: 737-7Crossref PubMed Scopus (8527) Google Scholar]. I suspect that I was regarded as a wild eccentric by the blood clotters when I wrote, ‘The basic defect in all of these diseases appears to be failure of the body to synthesize certain plasma proteins necessary for the clotting process…a simpler and therefore, perhaps, less likely relationship between the mutant gene and the clotting factor is the possibility that each factor is itself a proenzyme or enzyme whose chemical structure is based directly on a genic model. Mutation or stereoisomeric rearrangements of the gene itself might result directly in a similar rearrangement of the enzyme molecule (clotting factor) causing it to lose specificity for its substrate.’ Fifty years later we take this for granted. The paper in which the above remarks were made was entitled ‘Haemophilia and the Haemophilioid States’ and its authors were Kenneth Brinkhous and myself. The order of names on the title page was decided by tossing a coin. The coin may have been a quarter (of a dollar) or a dime (10 cents). My annual salary to support myself, wife, and three children was $6000. Ken's salary to support himself, wife, and two sons was probably not more than $10 000. It is likely that we used the dime. Both of us were reared during the Great Depression, and we would not have risked the possibility of losing a quarter in a crack in the floor of old MacNider Hall.