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A Brief Historical Review of the Waterfall/Cascade of Blood Coagulation

2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês

10.1074/jbc.x300009200

1083-351X

Earl W. Davie,

Trauma, Hemostasis, Coagulopathy, Resuscitation

This article explores some of the events and people involved in unraveling the basic mechanisms leading to the clotting of blood. It also brings to focus the important role that my teachers and colleagues had on my career in research. When I entered college majoring in chemistry I had little idea that I would have an opportunity to become a professor of biochemistry at a major university. This was clearly the result of the excellent advice and encouragement that I received, particularly from my teachers early in my career. As a senior at the University of Washington, I took a course in biochemistry to complete my credit requirements for a B.S. in chemistry. The biochemistry course was taught by Donald Hanahan, an excellent lipid biochemist who understood the importance of a quantitative measurement. I was fascinated by the biochemistry class that Hanahan taught, as well as by a course dealing with the chemistry of natural products. When Hanahan invited me to work in his laboratory on a senior project, I got my first real experience of what it was like to do laboratory research and I enjoyed it. In the fall of 1950, I entered graduate school at the University of Washington and decided to do my thesis research with Hans Neurath to learn something about protein structure and function. Neurath was an excellent teacher, a distinguished protein chemist, and a leader in his field (Fig. 1). He also had high standards and was very demanding of his students. Neurath was originally trained as a colloid chemist at the University of Vienna. Following his postdoctoral training at the University of London in the chemistry department headed by Frederick Donnan, he immigrated to the United States in 1935, where his research interests shifted to protein structure. He then held positions at the University of Minnesota, Cornell University, and Duke University, and in 1950, he joined the University of Washington School of Medicine as chairman of a newly established Department of Biochemistry. His research interest then focused almost entirely on proteases, particularly pancreatic trypsin, chymotrypsin, and carboxypeptidase, because these proteins were available in sizable amounts and could be prepared in high purity. Much of his career then dealt with their structure and function, including their active sites, substrate specificity, their kinetics, amino acid sequence, interaction with inhibitors, and their mechanism of activation. Over the years, Neurath also became well known for his leadership in the publication of scientific literature, having founded and served as Editor-in-Chief of Biochemistry for 30 years. In 1990, he founded another new journal, Protein Science, as well as editing several excellent volumes, such as The Proteins initially with Kenneth Bailey and later with Robert Hill. For my Ph.D. thesis research, Neurath suggested that I should compare trypsinogen and trypsin to gain some insight as to the mechanism of zymogen activation. Little difference between the two proteins was anticipated because Cunningham and other postdoctoral fellows in Neurath's laboratory had shown that trypsinogen and trypsin had essentially identical molecular weights of 23,800 as measured in the ultracentrifuge (1Cunningham Jr., L.W. Tietze F. Green N.M. Neurath H. Molecular kinetic properties of trypsin and related proteins..Discuss. Faraday Soc. 1953; 13: 58-67Crossref Google Scholar). In my initial studies, I was unable to detect any difference between the two proteins at their carboxyl-terminal end. This suggested that no peptides or amino acids were removed or peptide bonds cleaved at the carboxyl end of trypsinogen during its conversion to trypsin. However, in France Rovery and co-workers (2Rovery M. Fabre C. Desnuelle P. Étude des extrémités n-terminales du trypsinogène et de la trypsine de boeuf..Biochim. Biophys. Acta. 1952; 9: 702Crossref PubMed Scopus (0) Google Scholar) found that trypsinogen contained an amino-terminal valine, whereas trypsin contained an amino-terminal isoleucine. Shortly thereafter, I isolated an acidic peptide (Val-(Asp)4-Lys) that was generated by limited proteolysis during the activation of trypsinogen (3Davie E.W. Neurath H. Identification of a peptide released during autocatalytic activation of trypsinogen..J. Biol. Chem. 1955; 212: 515-529Abstract Full Text PDF PubMed Google Scholar). Most importantly, the appearance of this peptide correlated exactly with the formation of the enzymatic activity of trypsin. This finding was consistent with the loss of a small amino-terminal valyl peptide from trypsinogen as well as a slight increase in the isoionic point of the protein during its conversion to trypsin (9.3-10.01). As a graduate student, I was very excited by the fact that we were the first to make this observation. Little did we realize at the time that "limited proteolysis" would be a common mechanism seen over and over again in biological systems. This mechanism included the activation of other proteases such as those participating in blood coagulation (4Davie E.W. Fujikawa K. Kisiel W. The coagulation cascade: initiation, maintenance, and regulation..Biochemistry. 1991; 30: 10363-10370Crossref PubMed Google Scholar), fibrinolysis (5Collen D. The plasminogen (fibrinolytic) system..Thromb. Haemostasis. 1999; 82: 259-270Crossref PubMed Google Scholar), and complement activation (6Kirkitadze M.D. Barlow P.N. Structure and flexibility of the multiple domain proteins that regulate complement activation..Immunol. Rev. 2001; 180: 146-161Crossref PubMed Scopus (0) Google Scholar). Furthermore, limited proteolysis is now known to occur in a wide range of biological reactions such as the processing of prohormones (7Steiner D.V. The proprotein convertases..Curr. Opin. Chem. Biol. 1998; 2: 31-39Crossref PubMed Google Scholar), the activation of cells via their protease-activated receptors (PARs) (8Coughlin S.R. Thrombin signaling and protease-activated receptors..Nature. 2000; 407: 258-264Crossref PubMed Scopus (0) Google Scholar), the cleavage of signal peptides from proteins destined for secretion (9Stroud R.M. Walter P. Signal sequence recognition and protein targeting..Curr. Opin. Struct. Biol. 1999; 9: 754-759Crossref PubMed Scopus (0) Google Scholar, 10Dalbey R.E. Von Heijne G. Signal peptidases in prokaryotes and eukaryotes—a new protease family..Trends Biochem. Sci. 1992; 17: 474-478Abstract Full Text PDF PubMed Google Scholar), and the cleavage of ubiquitin from proteins on their way to endosomes or proteosomes (11Wilkinson K.D. Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome..Semin. Cell Dev. Biol. 2000; 11: 141-148Crossref PubMed Scopus (0) Google Scholar). Additional examples are the removal of small or large fragments from proteins such as fibrinogen prior to polymerization (12Mosesson M.W. Siebenlist K. Meh D.A. The structure and biological features of fibrinogen and fibrin..Ann. N. Y. Acad. Sci. 2001; 936: 11-30Crossref PubMed Google Scholar) and the removal of small propeptides that are signals for protein modification such as carboxylation (13Furie B. Bouchard B.A. Furie B.C. Vitamin K-dependent biosynthesis of gamma-carboxyglutamic acid..Blood. 1999; 93: 1798-1808Crossref PubMed Google Scholar). Also, regulated intramembrane proteolysis (RIP) plays an important role in determining the level of cholesterol in membranes via the sterol regulatory element-binding proteins (SREBPs) (14Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans..Cell. 2000; 100: 191-198Abstract Full Text Full Text PDF Scopus (1079) Google Scholar). Other examples include signaling receptors such as Notch, a single transmembrane protein that is activated by proteolytic cleavage at three specific sites generating an intracellular domain of the protein (15Selkoe D. Kopan R. Notch and presenilin: regulated intramembrane proteolysis links development and degeneration..Annu. Rev. Neurosci. 2003; 26: 565-597Crossref PubMed Scopus (0) Google Scholar). Notch is then translocated to the nucleus where it binds to transcription factors that play a role in development. Thus, proteases have many functions in addition to the digestion of proteins in the gut. Being in the Neurath laboratory gave me an excellent chance to interact with his other graduate students and postdoctoral fellows. Individuals such as Leon Cunningham gave me considerable guidance during those early years. I also had the chance to meet and visit with many outstanding visitors and collaborators of Hans Neurath. Visitors such as Fred Sanger presented the amino acid sequence of the two chains of insulin, a milestone in amino acid sequence analysis (16Sanger F. Sequences, sequences, and sequences..Annu. Rev. Biochem. 1988; 57: 1-28Crossref PubMed Google Scholar). Another distinguished visitor was Bert Vallee from Boston (Fig. 2). His visit to Seattle led to the discovery of zinc in carboxypeptidase (17Vallee B.L. Neurath H. Carboxypeptidase, a zinc metalloenzyme..J. Biol. Chem. 1955; 217: 253-261Abstract Full Text PDF PubMed Google Scholar). Vallee received his early education at the University of Berne and then immigrated to the United States in 1938 where he entered New York University School of Medicine. In 1946, he moved to Boston for his studies at MIT and Harvard. Vallee developed a highly sensitive flame and spark emission spectrometer for the detection of trace metals in proteins, and this technique led to the development of the field of metalloproteins in his laboratory. Following the discovery of carboxypeptidase A as a zinc metalloenzyme, he then identified dozens of other zinc-containing enzymes employing atomic absorption spectroscopy (18Vallee B.L. Auld D.S. Zinc coordination, function, and structure of zinc enzymes and other proteins..Biochemistry. 1990; 29: 5647-5659Crossref PubMed Scopus (0) Google Scholar). These enzymes participate in lipid, protein, carbohydrate, and nucleic acid metabolism as well as gene transcription, cell division, and development. Zinc plays a structural as well as a catalytic or cocatalytic role in these proteins. When zinc is associated with DNA-binding proteins, it is bound to three distinct motifs referred to as zinc fingers, twists, and clusters (19Vallee B.L. Auld D.S. Functional zinc-binding motifs in enzymes and DNA-binding proteins..Faraday Discuss. 1992; 93: 47-65Crossref Google Scholar). More than 300 metalloenzymes have now been identified and include metallothionein, a small protein that Vallee found in horse kidney (20Vallee B.L. The function of metallothionein..Neurochem. Int. 1995; 27: 23-33Crossref PubMed Scopus (0) Google Scholar). This small protein has a unique structure, amino acid composition, and metal binding characteristics. Vallee made many other important contributions such as the discovery and role of angiogenin in blood vessel formation. These visitors made the Neurath laboratory a very stimulating place for a graduate student. When I received my Ph.D. in 1954, Neurath recommended Fritz Lipmann's laboratory in Boston for postdoctoral study (see "Hitler's Gift and the Era of Biosynthesis" by E. P. Kennedy (21Kennedy E.P. Hitler's gift and the era of biosynthesis..J. Biol. Chem. 2001; 276: 42619-42631Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)). Lipmann's laboratory was another very exciting place to work because Mary Ellen Jones and Leonard Spector were close to identifying carbamoyl phosphate as an intermediate in the formation of citrulline (22Jones M.E. Spector L. Lipmann F. Carbamoyl phosphate, the carbamoyl donor in enzymatic citrulline synthesis..J. Am. Chem. Soc. 1955; 77: 819-820Crossref Google Scholar), while Helmuth Hilz and Phil Robbins were characterizing "active sulfate" (3′-phosphoadenosyl 5′-phosphosulfate) (23Hilz H. Lipmann F. The enzymatic activation of sulfate..Proc. Natl. Acad. Sci. U. S. A. 1955; 41: 880-890Crossref PubMed Google Scholar, 24Robbins P.W. Lipmann F. Isolation and identification of active sulfate..J. Biol. Chem. 1957; 229: 837-851Abstract Full Text PDF PubMed Google Scholar), an intermediate employed in the biosynthesis of tyrosine sulfate in proteins and other molecules such as chondroitin sulfate. Lipmann suggested that I should try to isolate one of the amino acid carboxyl-activating enzymes that were thought to be involved in protein biosynthesis (25Hoagland M.B. Keller E.B. Zamicnik P.C. Enzymatic carboxyl activation of amino acids..J. Biol. Chem. 1956; 218: 345-358Abstract Full Text PDF PubMed Google Scholar). Hopefully a pure activating enzyme might provide an approach to finding the natural acceptor for this family of enzymes, something equivalent to the role that CoA played in the activation of acetate. At the time, the biological acceptor tRNA was not known. These studies were carried out with Victor Koningsberg, a postdoctoral fellow from The Netherlands. We employed beef pancreas as an enzyme source because this tissue was very active in protein biosynthesis. These studies led to the isolation and characterization of a tryptophanyl carboxyl-activating enzyme that catalyzed the exchange of pyrophosphate into ATP in the presence of tryptophan and the formation of tryptophan hydroxymate when hydroxylamine was added to the reaction mixture (26Davie E.W. Koningsberger V.V. Lipmann F. The isolation of a tryptophan-activating enzyme from pancreas..Arch. Biochem. Biophys. 1956; 65: 21-38Crossref PubMed Google Scholar). After two very enjoyable years in Lipmann's laboratory, I learned about a faculty position at Western Reserve University in Cleveland, Ohio in Harland Wood's Department of Biochemistry. Wood had built a first rate department with an outstanding reputation for its research in intermediary metabolism. The biochemistry faculty included distinguished scientists such as Mert Utter, Robert Greenberg, Warwick Sakami, and many others. Wood had talked to Lipmann about this position, and Lipmann had suggested me for the job. At the time, academic positions were rarely advertised as is presently done. Harland Wood was a very talented and productive scientist who was a giant in the field of intermediary metabolism (Fig. 3). He received his undergraduate training in chemistry and bacteriology at MacAlaster College in Minnesota. In 1931, he became a Ph.D. student in microbiology in the laboratory of Charles Werkman at Iowa State University. As a graduate student, he made the monumental discovery that carbon dioxide fixation occurred in heterotrophic bacteria (27Wood H.G. Werkman C.H. The utilization of CO2 in the dissimilation of glycerol by the propionic bacteria..Biochem. J. 1936; 30: 48-53Crossref PubMed Google Scholar). At the time, CO2 was thought to be an end product for all cells except for photosynthetic autotrophs. The fixation of CO2 was discovered long before its role was established in other important reactions such as a building block for purines, amino acids, and fatty acids. The reaction in which pyruvate and CO2 would generate oxalacetate in bacteria soon became known as the "Wood-Werkman reaction." In collaboration with Alfred Nier and co-workers at the University of Minnesota, Wood was able to show that 13CO2 was incorporated into succinate in bacteria (28Wood H.G. Werkman C.H. Hemingway A. Nier A.O. The position of carbon dioxide carbon in succinic acid synthesized by heterotrophic bacteria..J. Biol. Chem. 1941; 139: 377-381Abstract Full Text PDF Google Scholar). This was in complete agreement with the proposal that CO2 and pyruvate would generate oxalacetate followed by reduction to succinate. In 1946 Harland Wood moved to Western Reserve University where he was appointed chairman of the Department of Biochemistry. In Cleveland, he continued his studies with 13C to study metabolic pathways employing a mass spectrometer that he built from scratch (29Wood H.G. Then and now..Annu. Rev. Biochem. 1985; 54: 1-41Crossref PubMed Google Scholar). He then turned much of his efforts to enzyme isolation and characterization. One of his favorite enzymes was a biotin-containing transcarboxylase from Propionibacterium shermani (29Wood H.G. Then and now..Annu. Rev. Biochem. 1985; 54: 1-41Crossref PubMed Google Scholar). This very large protein (Mr ∼1,200,000) forms propionyl-CoA and oxalacetate from methylmalonyl-CoA and pyruvate. It consists of a hexameric central subunit attached to 12 biotinyl subunits and 6 dimeric outer subunits. Wood referred to this enzyme as the "Mickey Mouse" enzyme because of its appearance in the electron microscope and its multiple subunits. Wood was a wonderful boss and I admired him a great deal. He had phenomenal physical stamina and loved to work at the bench throughout his life. Furthermore, he had a strong passion for good science and was very supportive of the research programs of his faculty. He was also unusually frank and honest with his colleagues. When I first visited Cleveland for an interview for a faculty position, he informed me that there was only one tenure position in the department and that was his as Chairman. He assured me, however, that tenure wasn't too important anyway because if he and I didn't get along, one of us would have to leave and it wouldn't be him. This was not a threat, however—just a simple and honest fact of life. My initial studies in Cleveland dealt with the isolation and identification of aminoacyl adenylates that had been proposed as intermediates by Hoagland and co-workers (25Hoagland M.B. Keller E.B. Zamicnik P.C. Enzymatic carboxyl activation of amino acids..J. Biol. Chem. 1956; 218: 345-358Abstract Full Text PDF PubMed Google Scholar) and DeMoss and coworkers (30DeMoss J.A. Genuth S.M. Novelli G.D. The enzymatic activation of amino acids via their acyl-adenylate derivative..Proc. Natl. Acad. Sci. U. S. A. 1956; 42: 325-332Crossref PubMed Google Scholar) in the carboxyl activation of amino acids. These intermediates, however, had not been isolated from an enzymatic reaction. They were analogous, however, to adenylyl acetate suggested earlier by Berg in the formation of acetyl-CoA (31Berg P. Participation of adenyl-acetate in the acetate-activating system..J. Am. Chem. Soc. 1955; 77: 3163-3164Crossref Google Scholar). Experiments carried out in our laboratory with Henry Kingdon and Les Webster in Cleveland led to the isolation and characterization of adenylyl tryptophan and adenylyl serine and demonstrated that these elusive intermediates could be identified in reactions that included amino acid, ATP, Mg2+, activating enzyme, as well as pyrophosphatase to shift the equilibrium of the reaction toward the enzyme-bound intermediate (32Kingdon H.S. Webster Jr., L.T. Davie E.W. Enzymatic formation of adenyl tryptophan: isolation and identification..Proc. Natl. Acad. Sci. U. S. A. 1958; 44: 757-765Crossref PubMed Google Scholar, 33Webster Jr., L.T. Davie E.W. Purification and properties of a serine-activating enzyme from beef pancreas..J. Biol. Chem. 1960; 236: 91-96Google Scholar). In 1957, Wood introduced me to Oscar Ratnoff, a distinguished Professor of Medicine at Western Reserve University (Fig. 4). This introduction to Ratnoff started much of my career in a new direction, mainly research in blood coagulation. Ratnoff received his medical training from Columbia University College of Physicians and Surgeons and then worked with C. Lockard Conley and Robert Hartmann in the Department of Hematology at Johns Hopkins University in Baltimore (34Roberts H.R. Oscar Ratnoff: his contributions to the golden era of coagulation research..Br. J. Haematol. 2003; 122: 180-192Crossref PubMed Google Scholar). He joined the Department of Medicine at Western Reserve University in Cleveland in 1953 and soon developed an outstanding reputation in blood coagulation. Working on a project in collaboration with Ratnoff seemed like a good idea because he was well known for his great intellect, hard work, and an uncanny ability to relate a large pool of clinical information to basic research. Furthermore, I had developed a few skills in protein chemistry, and it was quite clear that they would be helpful to unravel some of the complex reactions leading to fibrin formation. One of the most unusual and remarkable properties of blood is its ability to solidify or clot. In humans, this physical change is initiated by tissue injury and destruction and involves plasma proteins, platelets, and tissue components. In invertebrates, the clotting reaction is primarily because of cell aggregation and agglutination. In higher organisms, however, the vascular pressures are high and this increases the risk of bleeding. Thus, the mechanisms for initiating and regulating blood coagulation in humans are far more complex and include the following three general processes: (a) the immediate contraction of blood vessels at the site of vascular injury, (b) formation of a platelet plug, and (c) the generation of a fibrin clot to stabilize the platelet plug. The latter reaction results from the interaction of tissue and plasma proteins in a series of reactions that occur primarily on the surface of the activated platelets and other cells. These cells provide phosphatidylserine, a membrane phospholipid that is essential for clotting and becomes exposed when cells are activated or damaged. Ratnoff had many interesting patients who had abnormal blood coagulation. One of Ratnoff's most unusual patients was John Hageman, who had a rather strange clotting abnormality in that his blood didn't clot when added to a glass test tube (35Ratnoff O.D. Colopy J.E. A familial hemorrhagic trait associated with a deficiency of a clot promoting fraction of plasma..J. Clin. Invest. 1955; 34: 602-613Crossref PubMed Google Scholar). This could be corrected, however, by the addition of a small amount of plasma or serum from normal individuals or from patients with other known coagulation disorders such as hemophilia. Surprisingly, however, John Hageman had not experienced any bleeding tendency. Ratnoff inquired as to whether I might be able to help him in the isolation of this plasma protein that he called Hageman factor. I suggested that column chromatography on DEAE or carboxymethylcellulose (CMC) might be useful because Peterson and Sober had just published a novel method by which one could separate plasma or serum proteins with these resins (36Peterson E.A. Sober H.A. Chromatography of proteins. I. Cellulose ion-exchange adsorbents..J. Am. Chem. Soc. 1956; 78: 751-755Crossref Google Scholar). Because these reagents were not available commercially, I prepared small amounts and started the purification of Hageman factor from normal pooled human plasma. In these studies, plasma was separated into 50 or 60 fractions, and these were given to Ratnoff for assay of their clot accelerating activity. A few days later he informed me that all the clotting activity was present in a few tubes that contained little or no detectable protein. It was clear from the earlier studies of Ratnoff and Rosenblum that Hageman factor (now called factor XII) was present in plasma in an inactive form and was activated in a test tube when bound to a glass surface or crushed glass or kaolin (37Ratnoff O.D. Rosenblum J.M. Role of Hageman factor in the initiation of clotting by glass: evidence that glass frees Hageman factor from inhibition..Am. J. Med. 1958; 25: 160-168Abstract Full Text PDF PubMed Google Scholar). This was consistent with the idea that Hageman factor activation could trigger fibrin formation in blood collected in a glass container. In contrast, blood collected in paraffin-lined or silicone-coated bottles would clot very slowly or not at all. After several purification steps, we were able to show that Hageman factor was a plasma protein capable of initiating blood coagulation in the test tube and did so in the absence of tissue extracts (38Ratnoff O.D. Davie E.W. The purification of activated Hageman factor (activated Factor XII)..Biochemistry. 1962; 1: 967-974Crossref PubMed Google Scholar). Thus, it was participating in the intrinsic pathway of blood coagulation in contrast to the extrinsic pathway that also required tissue extracts. Little was known, however, about the mechanism by which Hageman factor could activate the clotting process. Ratnoff and I then had a number of discussions about the early phases of blood coagulation because I had little knowledge of the field other than the conversion of prothrombin to thrombin by thrombokinase, and fibrinogen to fibrin by thrombin in reactions requiring calcium. This pathway had been proposed in the early 1900s by Morawitz, who described thrombokinase as a coagulant activity from platelets or damaged tissue (39Morawitz P. R. Hartmann Guenther P. The Chemistry of Blood Coagulation. Charles C. Thomas, Springfield, IL1958: 1-194Google Scholar). Howell (40Howell W.H. The nature and action of the thromboplastic (zymoplastic) substance of the tissues..Am. J. Physiol. 1912; 31: 1-21Crossref Google Scholar) called the clot-accelerating activity from tissue, thromboplastin, a complex that converted prothrombin to thrombin. Some investigators, however, thought the activity from tissue was because of phospholipid or lipoprotein. Purification was then carried out in a number of laboratories, including Chargaff and co-workers (41Chargaff E. Benedich A. Cohen S.S. The thromboplastic protein: structure properties, disintegration..J. Biol. Chem. 1944; 156: 161-178Abstract Full Text PDF Google Scholar), Williams and Norris (42Williams W.J. Norris D.G. Purification of a bovine plasma protein (factor VII) which is required for the activity of lung microsomes in blood coagulation..J. Biol. Chem. 1966; 241: 1847-1856Abstract Full Text PDF PubMed Google Scholar), and Nemerson and Pitlick (43Nemerson Y. Pitlick F.A. Purification and characterization of the protein component of tissue factor..Biochemistry. 1970; 9: 5100-5105Crossref PubMed Google Scholar) who identified thromboplastin as a combination of clotting activities. One was a protein now known as tissue factor present in most tissues except platelets and the other as phospholipid. Since then, tissue factor has been purified extensively from bovine (44Bach R. Nemerson Y. Konigsberg W. Purification and characterization of bovine tissue factor..J. Biol. Chem. 1981; 256: 8324-8331Abstract Full Text PDF PubMed Google Scholar) and human (45Broze G.J. Leykam J.E. Schwartz B.D. Miletich J.P. Purification of human brain tissue factor..J. Biol. Chem. 1985; 260: 10917-10920Abstract Full Text PDF PubMed Google Scholar) tissue. Over the years a number of clinicians identified many patients with different coagulation disorders. In 1936, Patek and Stetson (46Patek Jr., A.J. Stetson R.H. Hemophilia. I. The abnormal coagulation of the blood and its relation to the blood platelets..J. Clin. Invest. 1936; 15: 531-542Crossref PubMed Google Scholar) had found that patients with hemophilia were lacking a factor present in normal plasma. The following year it was partially enriched by Patek and Taylor who called it anti-hemophilic factor (AHF) or anti-hemophilic globulin (AHG) (47Patek A.J. Taylor F.H.L. Hemophilia. II. Some properties of a substance obtained from normal plasma effective in acceleration of the clotting of hemophilic blood..J. Clin. Invest. 1937; 16: 113-124Crossref PubMed Google Scholar). This deficiency now called hemophilia A (or factor VIII deficiency) is found almost exclusively in males and is one of the most common of the hereditary coagulation disorders. This clotting abnormality was corrected by the addition of a small sample of normal plasma to the hemophilic plasma restoring the generation of a fibrin clot in the presence of calcium. Hemophilia has been of considerable historical interest since the son of Tsar Nicholas II of Russia had hemophilia and his care occupied much of the time and effort of his parents distracting them from the political problems that were developing (48Massie R.K. Nicholas and Alexandra. Atheneum, New York1967Google Scholar). Consequently, hemophilia played a significant role in the Bolshevik Revolution in 1917 that led to the execution of Nicholas, his wife, and their children following the October revolution. In 1944, Robbins (49Robbins K.D. A study on the conversion of fibrinogen to fibrin..Am. J. Physiol. 1944; 142: 581-588Crossref Google Scholar) made the interesting observation that fibrin formed in the presence of a plasma protein and calcium became rather insoluble in urea. In further studies, Laki and Lorand (50Laki K. Lorand L. On the solubility of fibrin clots..Science. 1948; 108: 280Crossref PubMed Google Scholar) partially purified this plasma protein that became known as the Laki-Lorand factor, fibrin-stabilizing factor, and presently as factor XIII. Years later, Chen and Doolittle (51Chen R. Doolittle R F. Cross-linking sites in human and bovine fibrin..Biochemistry. 1971; 10: 4487-4491Crossref PubMed Scopus (0) Google Scholar) showed that activated factor XIII cross-links fibrin monomers by forming ϵ(γ-glutamyl) lysine bonds between two adjacent fibrin molecules. In 1947, shortly after World War II, Owren in Norway (52Owren P.A. The coagulation of blood. Investigations on a new clotting factor..Acta Med. Scand. 1947; 194: 521-549Google Scholar) described another hemorrhagic disease in a young woman lacking a plasma protein that was called proaccelerin. This disease, referred to as parahemophilia (factor V deficiency), was a rare disorder resulting in bruising and bleeding after minor lacerations or dental extraction. In 1949 Alexander and co-workers (53Alexander B. Goldstein R. Ladwehr G. Cook C.D. Coagulation serum prothrombin conversion accelerator (SPCA) deficiency: a hitherto unrecognized coagulation defect with hemorrhage rectified by serum and serum fraction..J. Clin. Invest.