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• W2045831710 abstract "I started my medical studies at Karolinska Institutet in Stockholm in 1949. I had actually planned to become a high school teacher and to that end had spent a semester studying literature and Nordic languages at the University of Uppsala. But money constraints led me to apply instead to study at Karolinska Institutet, where I was accepted and also obtained a welcome stipend covering housing and meals. I soon became quite interested in this new field and especially enjoyed the course in physiological chemistry at the department of chemistry. In 1951, the head of the department, Professor Erik Jorpes, allowed me to join his research group. In the meantime I had married a classmate, Margareta Wetter, and I told Professor Jorpes that Margareta was also interested in research work, so she was admitted too. Erik Jorpes suggested that we participate in studies on heparin, discovered by him as glycosaminoglycan in the 1930s. That involved work on the standardization of heparin, in vivo assays of heparin in sheep, and a method for fast assay of heparin during extracorporeal circulation. In order to neutralize thrombin, heparin needed a co-factor (now known as antithrombin) in plasma. We wanted to study this process and therefore needed purified fibrinogen. At that time the available fibrinogen preparations were either not sufficiently stable or too impure. This brings us close to the roots of the Fibrinogen Metamorphosis Tree (Fig. 1), which was to send out numerous branches as time went by. But the co-factor we had intended to study was more or less forgotten. We started by precipitating fraction 1 out of plasma according to Cohn's method 6 [1Cohn E.J. Strong L.E. Hughes Jr, W.L. Mulford D.J. Ashworth J.N. Melin M. Taylor H.L. Preparation and properties of serum and plasma proteins. VI. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids.J Am Chem Soc. 1946; 68: 459-75Crossref PubMed Google Scholar]. Fifty per cent of the protein in this fraction consisted of plasma proteins other than fibrinogen. As a preliminary for further purification we did not want to dissolve fraction 1 in buffer and thus risk activation of coagulation factors such as prothrombin. Extraction of such factors under conditions that prevented activation seemed to be an option. In line with studies by Sörensen's and Cohn's groups we considered the effects of dipolar ions, like the amino acid glycine, on the solubility of proteins [2Blombäck B. Blombäck M. Purification of human and bovine fibrinogen.Arkiv Kemi. 1956; 10: 415-43Google Scholar]. At low concentrations, glycine increases the solubility of certain proteins but decreases it at much higher concentrations. Experiments showed that separation of contaminants was highly effective in the presence of glycine; thus, high concentrations enhanced the salting out effect on fibrinogen while the contaminants were still very soluble. Extraction in slightly acid buffer containing 1 m glycine and 6.5% ethanol, at −3 °C, yielded a product, named fraction I-O, which was highly coagulable with thrombin and quite stable in solution [2Blombäck B. Blombäck M. Purification of human and bovine fibrinogen.Arkiv Kemi. 1956; 10: 415-43Google Scholar]. We initially believed that fraction I-O was more or less pure fibrinogen. However, we were startled to find that although most of the prothrombin had been removed, the fraction had practically the same factor (F) VIII content as the plasma volume used for fractionation. (We know now that the contaminant was the gigantic von Willebrand–FVIII complex and the low solubility is therefore not surprising.) This brings us to the first branch on the fibrinogen tree (Fig. 1). We had already used this fraction for treatment of fibrinogen deficiencies and now realized that it was also a good candidate for treatment of hemophilia A. It so happened that Dr Inga-Marie Nilsson, who had performed the testing of FVIII activity on our fibrinogen preparations, had at this time a 15-year-old girl on her ward at the University Hospital in Malmö, Sweden. This girl had had a severe hemorrhagic diathesis since childhood. When she started menstruation, the disease took a grave turn and large transfusions of blood were needed to arrest the bleedings. After a time, she suddenly developed severe reactions to the transfusions, which had to be abandoned. The coagulation status had shown a greatly prolonged bleeding time, delayed prothrombin consumption, somewhat prolonged clotting time but normal platelet count and function. Furthermore, FVIII activity was low. As fraction I-O had a high concentration of FVIII, it was decided to test its effect in this patient. To our surprise, the expected increase in plasma FVIII activity was accompanied by a normalization of bleeding time. Subsequently, under cover of fraction I-O, hysterectomy was successfully performed. It turned out that this girl was suffering from von Willebrand disease (VWD). This case was the beginning of the Bleeding Time Factor story, which will be extensively dealt with in a forthcoming review [3Blombäck B. Journey with bleeding time factor.in: Semenza G Wood EJ Selected Topics in the History of Biochemistry. Comprehensive Biochemistry. Elsevier Science, 2006Google Scholar]. We now found the effect on bleeding time in VWD was the same after removal of FVIII from fraction I-O by adsorption. Furthermore, fraction I-O prepared from blood of hemophilia A, missing FVIII, corrected the bleeding time in VWD. This and several other experiments convinced us that the bleeding diathesis in VWD was due to lack of a hitherto unknown factor that corrected bleeding time in VWD and at the same time normalized FVIII level [4Nilsson I.M. Blombäck M. Blombäck B. v. Willebrand's disease in Sweden. Its pathogenesis and treatment.Acta Med Scand. 1959; 164: 263-78Crossref PubMed Google Scholar]. We named it von Willebrand factor (VWF). Previously we and other authors had believed that the bleeding time prolongation and bleeding tendency in VWD was caused by deficiency of FVIII in combination with vascular disturbances. On the contrary our findings suggested that VWF effected bleeding time by acting on normal vessel wall and/or platelets and was necessary for cellular synthesis or secretion of FVIII, thereby explaining the FVIII deficiency in VWD [4Nilsson I.M. Blombäck M. Blombäck B. v. Willebrand's disease in Sweden. Its pathogenesis and treatment.Acta Med Scand. 1959; 164: 263-78Crossref PubMed Google Scholar]. Now followed a hectic time producing fraction I-O for treatment of hemophilia A and VWD in hospitals all over Sweden and in some cases also abroad. Starting in 1957 our laboratory turned out almost 2000 preparations of fraction I-O, each from between 2 and 6 L of plasma, before Kabi AB (Stockholm, Sweden) eventually took over production in 1967. The mysterious VWF continued to attract our attention for another 25 years, with little success. Then, in 1984, Birgit Hessel et al. [5Hessel B. Jörnvall H. Thorell L. Söderman S. Larsson U. Egberg N. Blombäck B. Holmgren A. Structure-function relationships of human factor VIII complex studied by thioredoxin dependent disulfide reduction.Thromb Res. 1984; 35: 637-51Abstract Full Text PDF PubMed Google Scholar] showed that it was simply a homopolymer of a single-chain protein to which FVIII was non-covalently attached. Our goal was to establish the complete primary structure of VWF in the same fashion that we had successfully used in our work on the structure of fibrinogen. However, other events were to lead the way. In a tour de force of elegant biochemistry the complete amino acid sequence of VWF was announced by Earl Davie and his group in 1985–1986 (references in [3Blombäck B. Journey with bleeding time factor.in: Semenza G Wood EJ Selected Topics in the History of Biochemistry. Comprehensive Biochemistry. Elsevier Science, 2006Google Scholar]). The intermezzo with VWF did not put a stop to our work on fibrinogen, which led to a highly purified product [2Blombäck B. Blombäck M. Purification of human and bovine fibrinogen.Arkiv Kemi. 1956; 10: 415-43Google Scholar]. The question that sprouted a new branch on the fibrinogen tree was: What is this protein's chemical nature? Luck now came to our aid. It so happened that Dr Ikuo Yamashina, a visiting scientist in Erik Jorpes’ department, had just come back from Pehr Edman's laboratory in Lund, where he had learned how to identify N-terminal amino acids in proteins qualitatively and quantitatively (he had used N-terminal analysis to follow the reaction catalyzed by enterokinase, a protein he had purified in Jorpes’ laboratory). Using a slightly modified method, we managed to show that fibrinogen from several animal species was composed of three pairs of polypeptide chains, strongly indicating a dimeric structure of the molecule (Fig. 2). Furthermore, two of the chains were cleaved by thrombin during fibrin formation [6Blombäck B. Yamashina I. On the N-terminal amino acids in fibrinogen and fibrin.Arkiv Kemi. 1958; 12: 299-319Google Scholar]. Other investigators had shown that thrombin acts as a proteolytic enzyme during this transformation and that in this process two peptides, named A and B, are released [7Bailey K. Bettelheim F.R. The clotting of fibrinogen. I. The liberation of peptide material.Biochim Biophys Acta. 1955; 18: 495-503Crossref PubMed Google Scholar]. We confirmed this and also showed that release of the A-peptide (FPA) appeared to be critical for subsequent fibrin formation, whereas the release of the B-peptide (FPB) from the Bβ-chain was not [8Blombäck B. Vestermark A. Isolation of fibrino-peptides by chromatography.Arkiv Kemi. 1958; 12: 173-82Google Scholar]. The next twig to appear on this branch of fibrinogen research was the clarification of amino acid sequences of fibrinopeptides from a variety of animal species and this in turn led to several sub-twigs. In these endeavors we were fortunate to co-operate with John Sjöquist from the University of Lund, a pupil of Pehr Edman and an expert on the identification of amino acid derivatives obtained in the Edman reaction. Another participant in these studies was Russel Doolittle from Cambridge University, MA, USA, and a visiting scientist in our laboratory. In a few years we established the amino acid sequences of fibrinopeptides from a large number of animal species (see [9Blombäck B. Selectional trends in the structure of fibrinogen of different species.Symp Zool Soc Lond. 1970; 27: 167-87Google Scholar]). Interestingly, all FPAs included an extremely well preserved sequence in the C-terminal part of the peptides, that is, the part close to the bond split by thrombin. This part of the sequence contained a phenylalanine residue at position 9 from the arginyl bond split by thrombin. The phenylalanine residue was apparently critical for the attachment of thrombin to the cleavage site. We discussed our findings with Göran Claesson and Lars Svendsen, organic chemists at Nobel Pharma AB in Gothenburg. What we had in mind was to find a thrombin inhibitor for clinical use. We speculated that the hydrophobic side chain of phenylalanine would somehow be brought into proximity with the susceptible arginyl residue and, by dwelling in a hydrophobic niche of the enzyme close to the active center, would activate the enzyme. For example, the nona-peptide sequence has an ordered structure that brings the penylalanine residue close to the C-terminal arginine, for instance by being part of an α-helical structure. We therefore decided to synthesize both the nona-peptide sequence and shorter variants of it, in which phenylalanine always occupied the N-terminal position. The nona-peptide ester did in fact inhibit thrombin action and this inhibition decreased as the peptide chain became shorter until the penylalanine came close to the arginine residue, when inhibition increased again and reached a maximum with the tripeptide, Phe-Val-Arg [10Blombäck B. Blombäck M. Olsson P. Svendsen L. Åberg G. Synthetic peptides with anticoagulant and vasodilating activity.Scand J Clin Lab Invest. 1969; 24: 59-64Google Scholar]. Our notion of phenylalanine in the vicinity of the thrombin-susceptible arginine residue was later supported by nuclear magnetic resonance (NMR) studies by Harold Scheraga's and Susan Lord's group [11Ni F. Konishi Y. Frazier R.B. Scheraga H.A. Lord S.T. High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the Aα chain of human fibrinogen.Biochemistry. 1989; 28: 3082-94Crossref PubMed Google Scholar]. However, contact was achieved, not by an α-helical structure but by folding to a hairpin-like structure. Crystallographic studies, by Wolfram Bode's group [12Stubbs M.T. Bode W. A player of many parts: the spotlight falls on thrombin's structure.Thromb Res. 1993; 69: 1-58Abstract Full Text PDF PubMed Scopus (443) Google Scholar], of complexes between FPA and α-thrombin showed phenylalanine nicely slotted into an apolar binding pocket of thrombin in proximity to the cleavable arginine, with the latter making contact with the catalytic triad of thrombin (Fig. 3). Desmond Hogg made an interesting study on the kinetics of the thrombin fibrinogen interaction. He found that prior to its attachment to the susceptible arginyl residue in FPA, thrombin appeared to be navigated into that position by interaction with nearby structures on the ‘fibrin’ side of the bond being cleaved, that is, Aα 34–51 [13Hogg D.H. Blombäck B. The mechanism of the fibrinogen-thrombin reaction.Thromb Res. 1978; 12: 953-64Abstract Full Text PDF PubMed Google Scholar]. In 1967 we applied for a patent on the tri-peptide, Phe-Val-Arg and variants of it, for use as inhibitors of thrombin. At that time, the pharmaceutical industry showed little interest in developing direct thrombin inhibitors. However, about 20 years later AstraZeneca caught onto our idea and after successful chemical modification presented the powerful oral thrombin inhibitor ximelagatran [14Gustafsson D. Bylund R. Antonsson T. Nilsson I. Nyström J.-.E. Eriksson U. Bredberg U. Teger-Nilsson A.-.C. A new oral anticoagulant: the 50-year challenge.Nat Rev Drug Discov. 2004; 3: 649-59Crossref PubMed Google Scholar], which proved successful in preventing thrombotic events after orthopedic operations and also showed promise as an anticoagulant for long-term treatment in cardiovascular diseases. However, its use is still ruled out by a risk of liver toxicity. Although the prospect of developing direct thrombin inhibitors was dim in 1967, the pharmaceutical industry was more keen to use the tripeptide, Phe-Val-Arg, as a base for developing substrates for measuring thrombin activity. This was simply achieved by linking a chromogenic group, p-nitro aniline, to the carboxylic function of the arginine residue in the tripeptide [15Svendsen L. Blombäck B. Blombäck M. Olsson P.I. Synthetic chromogenic substrates for determination of trypsin, thrombin and thrombin-like enzymes.Thromb Res. 1972; 1: 267-78Abstract Full Text PDF Scopus (0) Google Scholar]. The susceptibility of the substrate for thrombin was further enhanced by benzoylation of the free NH2-group of phenylalanine. Here was further support for our belief that hydrophobic interaction between thrombin and its substrate plays an important role in anchoring the enzyme. It may be of interest to mention that, when injected into dogs, the synthetic peptide derivative based on Phe-Val-Arg had a strong vasodilating effect [10Blombäck B. Blombäck M. Olsson P. Svendsen L. Åberg G. Synthetic peptides with anticoagulant and vasodilating activity.Scand J Clin Lab Invest. 1969; 24: 59-64Google Scholar]. As shown by Per Olsson, this was not caused by the peptide but by hydroxylamine, a by-product obtained during synthesis. One sub-branch of our fibrinogen studies was related to evolution. We compared fibrinopeptide's amino acid sequences in different animal species and asked whether the differences reflected an evolutionary process similar to that derived from studies of morphological characteristics of species. An esteemed researcher in these studies was Russell Doolittle, who spent about two years in our laboratory in the early 1960s. In addition to his interest in sequence technology, he spent much of the time on evolutionary implications of structural differences between fibrinopeptides. Surprisingly, the phylogeny predicted from variations in fibrinopeptide sequences showed good agreement with the classical scheme of evolution. Furthermore, the proposed amino acid substitutions that had occurred agreed up to 70–80% with a 1-base substitution in Nierenberg's RNA codon. There was one important misfit: a comparison of the reindeer sequence with those of sheep and goat indicated that the latter two branched off from a deer line rather than from the main bovine line in classical phylogeny. Pending firmer evidence, however, we favored the Linnean system based on morphological grounds [16Doolittle R.F. Blombäck B. Amino-acid sequence investigations of fibrinopeptides from various mammals: evolutionary implications.Nature (Lond). 1964; 202: 147-52Crossref PubMed Scopus (0) Google Scholar, 17Blombäck B. Blombäck M. Gröndahl N.J. Holmberg E. Structure of fibrinopeptides – its relation to enzyme specificity and phylogeny and classification of species.Arkiv Kemi. 1966; 25: 411-28Google Scholar]. Nevertheless, we attributed the good agreement to the fact that we had used a reliable reference point in our comparison of sequences, that is, the bond split by thrombin. The sequences around that bond we considered to be genetically equivalent in different species because of thrombin's narrow specificity. Further support for this was a study on cross-reactions between thrombin and fibrinogen from different animal species. With a given fibrinogen, there was little difference in the clotting activity of thrombins from various species [18Blombäck B. Teger-Nilsson A.-.C. On the thrombin–fibrinogen reaction in different species.Acta Chem Scand. 1965; 19: 751-3Crossref PubMed Google Scholar]. Let us return to the main trunk of the fibrinogen tree. N-terminal analysis had suggested that fibrinogen is a dimer of a three-chain protein (Fig. 2). Using light scattering, we found that fibrinogen had a molecular weight of about 350 000, which was close to the figure of 340 000 that other authors determined by ultracentrifugation. Agnes Henschen, working in our laboratory on her doctoral thesis, was able to isolate the three chains after oxidation of disulfide bonds, which apparently linked these chains [19Henschen A. Peptide chains in S-sulfo-fibrinogen and S-sulfo-fibrin: isolation methods and general properties.Arkiv Kemi. 1964; 22: 375-96Google Scholar]. Furthermore, the total molecular weight of the chains was about 170 000, in agreement with that for a half-molecule of dimeric fibrinogen. What held the monomers together was not known, neither was the detailed arrangement of disulfides. In fact, we did not know the amino acid sequence of fibrinogen chains beyond the fibrinopeptides. Our intention was to establish the complete sequence of the chains. At the time, we had Sadaaki Iwanaga in our laboratory, and he and Birgit Hessel were happy to follow up this research. Dr Iwanaga was a visiting scientist from Japan and spent several years with us in the 1960s. We first analyzed fragments of fibrinogen, somewhat longer than FPA and FPB, obtained by digestion with plasmin. These ‘mother peptides’, as Iwanaga used to call them, were identified by their release of FPA or FPB in the presence of thrombin. Much longer fragments of the chains were obtained when we used cyanogenbromide instead of plasmin for cleavage [20Blombäck B. Blombäck M. Henschen A. Hessel B. Iwanaga S. Woods K.R. N-terminal disulphide knot of human fibrinogen.Nature (Lond). 1968; 218: 130-4Crossref PubMed Scopus (0) Google Scholar]. In that case methionyl bonds were split. One fragment was shown to contain N-terminal fragments of all three chains of fibrinogen, linked by disulfide bonds. We named it the N-terminal disulfide knot (N-DSK), as it contained a high proportion of disulfides in fibrinogen. It was originally assumed that the fibrinogen molecule contained two non-linked N-DSKs, located at opposite ends of an elongated molecule. This proposition was in keeping with a birefringence study of fibrinogen [21Haschemeyer A.E.V. A polar intermediate in the conversion of fibrinogen to fibrinmonomer.Biochemistry. 1963; 2: 851-8Crossref PubMed Google Scholar] but it was ruled out when we found that the two N-DSKs are joined by symmetrical disulfides between Aα-chains and between γ-chains of the two N-DSKs. This finally proved that N-DSK and therefore also fibrinogen were true dimers [9Blombäck B. Selectional trends in the structure of fibrinogen of different species.Symp Zool Soc Lond. 1970; 27: 167-87Google Scholar, 22Blombäck B. Hessel B. Hogg D. Disulfide bridges in NH2-terminal part of human fibrinogen.Thromb Res. 1976; 8: 639-58Abstract Full Text PDF PubMed Google Scholar]. Dehydrated fibrinogen had been shown by Hall and Slayter [23Hall C.E. Slayter H.S. The fibrinogen molecule: its size, shape and mode of polymerization.J Biophys Biochem Cytol. 1959; 5: 11-7Crossref PubMed Google Scholar] to be a rod-like molecule, about 450Å long, and for the hydrated molecule, Bachmann et al. [24Bachmann L. Schmitt-Furmian W.W. Hammel R. Lederer K. Size and Shape of fibrinogen. I. Electron microscopy of the hydrated molecule.Macromol Chemie. 1975; 176: 2603-18Crossref Google Scholar] had depicted a more sausage-like structure. The fact that the two monomers of fibrinogen were joined at the N-terminal ends gave two realistic possibilities as regards the orientation of the chains in fibrinogen [25Blombäck B. Hogg D.H. Gårdlund B. Hessel B. Kudryk B. Fibrinogen and fibrin formation.Thromb Res. 1976; 8: 329-46Abstract Full Text PDF PubMed Google Scholar]. One was that the chains run parallel from the N-terminus to the C-terminus for about 450Å. However, low-angle X-ray diffraction studies of hydrated fibrinogen by Stryer and coworkers [26Stryer L. Cohen C. Langridge R. Axial period of fibrinogen and fibrin.Nature (Lond). 1963; 197: 793-4Crossref PubMed Scopus (0) Google Scholar] had shown axial repeats of 226Å. This made it more likely that, from where the monomers were linked by symmetrical disulfides, the chains run in opposite directions for 226Å towards the C-terminal ends. The two N = DSKs were thus linked at the very center of a molecule 450Å long. Bengt Gårdlund and Barbara Kowlska-Loth, the latter a visiting scientist from the University of Warsaw, were interested in finding out the location of fragments E and D, the main plasmin cleavage products, in the fibrinogen molecule. Fragment E was found to constitute part of dimeric N-DSK [27Kowalska-Loth B. Gårdlund B. Egberg N. Blombäck B. Plasmic degradation products of human fibrinogen. II Chemical and immunological relation between fragment E and N-DSK.Thromb Res. 1973; 2: 423-50Abstract Full Text PDF Google Scholar]. Fragment D, on the other hand, was a monomeric three-chain structure, most likely stretching out on either side of NDSK towards the C-terminal ends and together contributing roughly half of the disulfides in fibrinogen [28Gårdlund B. Kowalska-Loth B. Gröndahl N.J. Blombäck B. Plasmic degradation products of human fibrinogen. I. Isolation and characterization of fragments E and D and their relation to ‘disulfide knots’.Thromb Res. 1972; 1: 371-88Abstract Full Text PDF Google Scholar]. The amino acid sequences and secondary chain structure of N-DSK and related fragments took several years to complete. In addition to those mentioned here, many other excellent coworkers helped to make this endeavor a success: here can be mentioned the visiting scientists Desiré Collen, Jan Dyr, Yuji Inada, Eckart Irion, Masahiro Iwamoto, Franicek Kornalik, Bohdan Kudryk, Jane McDonald, Richard McDonald, Mayumi Makino, Gerard Margurie, Genesio Murano, Hubert Pirkle, Hannu Soumela, Munehiro Tomikawa, and Lyndal York. In parallel with our work, the sequences and secondary structure of the remainders of the three chains of fibrinogen were completed by Agnes Henschen's and Russell Doolittle's groups [29Henschen A. Lottspeich F. Kehl M. Southan C. Covalent structure of fibrinogen.Ann NY Acad Sci. 1983; 408: 28-43Crossref PubMed Google Scholar, 30Doolittle R.F. The structure and evolution of vertebrate fibrinogen.Ann NY Acad Sci. 1983; 408: 13-27Crossref PubMed Google Scholar]. The final goal, the crystal structure of fibrinogen, was eventually reached by Doolittle and colleagues and Cohen's groups [31Yang Z. Kollman J.M. Pandi L. Doolittle R.F. Crystal structure of native chicken fibrinogen at 2.7Å resolution.Biochemistry. 2001; 40: 12515-23Crossref PubMed Scopus (0) Google Scholar, 32Madrazo J. Brown J.H. Litvinovich S. Dominguez R. Yakovlev S. Medved L. Cohen C. Crystal structure of the central region of bovine fibrinogen (E5 fragment) at 1.4-Å resolution.Proc Natl Acad Sci USA. 2001; 98: 11967-72Crossref PubMed Scopus (0) Google Scholar]. What was the imperative for fibrin formation? Fibrinopeptide release was a primer, but what happens next? A new development gave us a clue. Margareta and I were invited as visiting scientists to The New York Blood Center in the fall of 1967. One day, Dr Eberhard Mammen phoned us from Wayne State University, Detroit, to say he was investigating a case of dysfibrinogenemia with severe bleeding symptoms. The fibrinogen level was normal but no clot was formed with thrombin. He asked whether we were interested in investigating this fibrinogen for the occurrence of structural abnormalities. Our first guess was that the culprit was fibrinopeptide A, but release of this peptide was normal and release of FPB also occurred albeit much delayed. The next guess was that structures following the FPA sequence had been perturbed. In order to address this we made what was called a ‘fingerprint’ (a two-dimensional electrophoresis-chromatography on thin-layer cellulose plates) of peptides obtained by tryptic digestion of the Aa chain of N-DSK. I remember the day of our first analysis. While the fingerprint was developing I went to the New York Hospital to give a lecture on fibrinogen structure. At the end of my talk I was asked whether I knew of innate changes in fibrinogen that might interfere with clotting. I confessed my ignorance but added that at that very moment we had an analysis running that might provide an answer. When we analyzed the fingerprint a few hours later we found to our delight that one spot in the dysfibrinogenemia sample had drastically moved; this was the tripeptide, Gly-Pro-Arg, which in normal fibrinogen follows the thrombin-susceptible arginyl bond (Fig. 4). We went to the nearest bar to celebrate with good New York dry martinis. A few days later, the result of the amino acid analysis of the new peptide showed that in the patient's fibrinogen, arginine at position Aα19 had been replaced by serine. So we headed for Detroit to report this and stopped on the way at Dr Oscar Ratnoff's laboratory in Cleveland to give some lectures, in which we of course mentioned the mutation we had found. At Detroit airport we met with Eberhard Mammen and his coworker Ananda Prasad (Fig. 5). We wrote a first draft of an article that, after some adjustments, was sent to Nature and quickly published [33Blombäck M. Blombäck B. Mammen E.F. Prasad A.S. Fibrinogen Detroit – a molecular defect in the N-terminal disulphide knot of human fibrinogen.Nature (Lond). 1968; 218: 134-7Crossref PubMed Scopus (0) Google Scholar]. A follow-up article came later [34Blombäck B. Blombäck M. Molecular defects and variants of fibrinogen.Nouv Rev Fr Hématol. 1970; 10: 671-8PubMed Google Scholar]. I believe this was the second mutation in a protein that could be coupled to defective function; the first, about 10 years earlier, was sickle-cell hemoglobin. Several years later Birgit Hessel identified another homozygous dysfibrinogenemia, fibrinogen Aarhus, where the arginine at position 19 had been replaced by glycine [35Blombäck B. Hessel B. Fields R. Procyk R. Fibrinogen Aarhus: An abnormal fibrinogen with Aα19 Arg–Gly substitution.in: Mosesson MW Amrani DL Siebenlist KR Fibrinogen 3. Biochemistry, Biological Functions, Gene Regulation and Expression. Elsevier Science Publishers, B.V. (Biomedical Division), 1988: 263-6Google Scholar].Figure 5Meeting at airport in Detroit. From left to right: Julia Murano, Birger Blombäck, Margareta Blombäck, Ananda Prasad, and Eberhard Mammen with his right forearm and wrist in plaster after fall on ice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It was now obvious that the Gly-Arg-Pro sequence, perturbed by the mutation Arg to Ser, was part of a domain (named A) in activated normal fibrinogen that in some way interacted with another domain (called a) in the polymerization process [36Blombäck B. Blombäck M. The molecular structure of fibrinogen.Ann NY Acad Sci. 1972; 202: 77-97Crossref PubMed Google Scholar]. The precise location of the domains was not yet clear. Alkjaersig and coworkers [37Alkjaersig N. Fletcher A.P. Sherry S. Pathogenesis of the coagulation defect developing during pathological plasma proteolytic (‘fibrinolytic’) states. II. The significance, mechanism and consequences of defective fibrin polymerization.J Clin Invest. 1962; 41: 917-34Crossref PubMed Google Scholar] had shown that fragment D, obtained by digestion of fibrinogen with plasmin, interfered with polymerization of ‘fibrin monomers’. Later" @default.
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• W2045831710 date "2006-08-01" @default.
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• W2045831710 title "Travels with fibrinogen" @default.
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