The primary focus of this review is the research that resulted in the discovery of a new factor (von Willebrand factor, VWF) in the coagulation of blood; the prime motor in initial haemostasis. Further research that lead to the identification of VWF as a multimeric protein that interacts with vessel wall and platelets is also considered, as is the regulation of this factor's activity in blood, an important aspect of both haemostasis and thrombosis.

The mechanisms involved in fibrin gel formation are reviewed. Furthermore, a new concept of the role of fibrinopeptide release in this process is presented.

Abstract: Coagulation of blood is such an evident phenomenon that its observation can be traced back to earliest historical times. The great philosophers and physicians of antiquity discussed and provided interesting explanations. However, it was not until the end of the seventeenth century that the structural component of the blood clot was described by Malpighi as a white fibrous substance. In the middle of the nineteenth century this was identified as a constituent of pathological thrombi and given the name fibrin. At about that time its precursor in blood, fibrinogen, was isolated in a highly purified form by Hammarsten who suggested that, preceding fibrin formation, activation of fibrinogen by thrombin occurred by limited proteolysis. The activation mechanism was eventually clarified in the 1950s. It was shown to proceed in two discrete steps, by removal of low molecular weight activation peptides. Ferry postulated, based on physicochemical observations, that the activated molecules aligned in a half‐staggered fashion to form polymers. The rapid post‐war development of biochemical technology permitted evaluation of the primary structure of fibrinogen. With that followed identification of molecular domains in the activated fibrinogen molecules that participate in polymer formation, crosslinking of polymeric structures, and domains for cellular attachment. Crystallization of fragments and, recently, of the entire molecule has confirmed and extended this knowledge. Lately, it has also been possible to obtain detailed information on the architecture of the fiber network in the fibrin gel. The gel structure is primarily determined by the initial rate of fibrinogen activation, but without infringement of this primary rule, several factors in blood may modulate the structure. Fibrinogen and fibrin play important roles in normal hemostasis, wound healing, and pathological processes, such as thrombosis and atherosclerosis.

I read with great interest a recent article on the transformation of fibrinogen in whole blood by Brummel et al. [ 1 Brummel K.E. Butenas S. Mann K.G. An integrated study of fibrinogen during blood coagulation. J Biol Chem. 1999; 274: 22862-22870 Crossref PubMed Scopus (118) Google Scholar ]. This communication contains a number of interesting observations and conclusions regarding reactions occurring during fibrin formation and crosslinking effected by release of fibrinopeptide A (FPA) and fibrinopeptide B (FPB), as well as the concomitant activation of factor XIII by thrombin and the fate of fibrinopeptides after their release. In whole blood the role of FPB release in activation and polymerization of fibrinogen appeared problematic, because at the time of almost complete FPA release and fibrin formation only a small fraction of FPB was released, and the subsequent release was attenuated and never reached completion [ 1 Brummel K.E. Butenas S. Mann K.G. An integrated study of fibrinogen during blood coagulation. J Biol Chem. 1999; 274: 22862-22870 Crossref PubMed Scopus (118) Google Scholar ]. The authors conclude that fibrin I (des FPA fibrin) is the preferred species formed during fibrinogen transformation in whole blood. In a study in 1978, we arrived at the same view as to the type of fibrin formed in whole blood [ 2 Blombäck B. Hessel B. Hogg D. Therkildsen L. A two-step fibrinogen-fibrin transition in blood coagulation. Nature. 1978; 257: 501-505 Crossref Google Scholar ]. In the following, I will restrict my discussion to the phenomenon of deficient FPB release which, under certain conditions, seems to be unique to whole blood but not to the isolated fibrinogen–thrombin system.

The idea to launch a new and unique journal in the field of thrombosis and hemostasis was born 30 years ago. It was the late Al Copley who came up with this idea and became the founder of the journal. He contacted the head of Pergamon Press, Robert Maxwell, and suggested that Pergamon Press serve as publisher of the journal. Maxwell was interested but requested that one additional Editor-in-Chief, together with Al Copley, take on the responsibility of editing the journal. In the early spring of 1971 Al Copley approached Birger Blombäck to ask whether he was willing to serve as Co-Editor-in-Chief, which he accepted. This was followed by long evening discussions on policy questions, which often took place in Al's 50 Central Park West apartment in New York City.

A thrombin-like enzyme has been identified in mosquitoes and partially purified. The enzyme preparation displays on SDS gel electrophoresis a major protein band of about 22 kDa and one minor of about 28 kDa. The enzyme preparation cleaves a synthetic substrate (S-2238) for thrombin with Km 113 μmol/L (as compared to 3 μmol/L for thrombin). The enzyme clots fibrinogen and plasma. During activation of fibrinogen, fibrinopeptide A (but scarcely fibrinopeptide B) is released. The enzyme is not inhibited by hirudin and activates (if at all) factor XIII differently from thrombin. Predominantly γ-dimers are formed in the cross linking process. As compared to thrombin a larger extent of activation is required to induce gelation (clotting) by the mosquito enzyme. At a given clotting time the enzyme produces tighter gel structures than thrombin. In its action the enzyme resembles the snake venom enzyme, batroxobin.

Previous studies, using cross-linked fibrin prepared from purified fibrinogen, showed low binding of a fibrin-specific monoclonal antibody designated T2G1 (Procyk et al., Blood 77:1469-75, 1991). In this study we investigated the binding of T2G1 and one other antibody to clots prepared from platelet poor plasma (PPP), platelet rich plasma (PRP) and whole blood. In contrast to our previous study, we used unlabelled antibodies and quantitated the level bound by ELISA, measuring antibody concentration in the non-adsorbed fraction. Antibody T2G1 bound 1.35+/-0.10 pmol/pmol fibrin (n=11) to whole blood columns, 1.64+/-0.18 (n=10) to PRP columns and 1.58+/-0.13 (n=8) to PPP columns. The binding of T2G1 to columns made from purified fibrinogen was 0.78+/-0.05 pmol/pmol fibrin (n=15). An antibody to a conformation-dependent epitope on Fragment D (Fd4-7B3) bound in comparable amounts to the different fibrins. Flow data show that whole blood columns, and also, but to a lesser extent those made with plasma, had a higher flow rate, permeability and fiber mass-length ratio than columns prepared from fibrinogen indicating a more coarse fibrin network. These data show that the presence of other proteins and blood cells, similar to what might occur in vivo, not only lead to an increase in the permeability of gels but also allow for better exposure of some epitopes.

Fibrinogen Tampere was found in a woman with severe thromboembolic disease. The thrombin induced clotting time of her plasma and purified fibrinogen was slightly prolonged. The activation of fibrinogen Tampere appeared to be normal but subsequent gelation was defective. We studied fibrin gels formed at different ionic strengths and at different fibrinogen and calcium concentrations by liquid permeation, turbidity, and 3D laser microscopy. Crosslinking was studied by SDS-gel electrophoresis. The gels formed from fibrinogen Tampere were at ionic strength above 0.2 much tighter and had lower fiber mass-length ratios than normal gels as judged by permeability and turbidity data. At ionic strength 0.15 and at different calcium concentrations analysis by permeability showed the same results for fibrinogen Tampere as for normal gels. Analysis by turbidity at ionic strength 0.15 suggested swelling of the fibers at low calcium concentrations. 3D microscopy revealed perturbed clot architecture under all conditions. In fibrin gels from fibrinogen Tampere, the gamma-chain crosslinking was normal but the crosslinking of alpha-chains was delayed at ionic strength 0.2 and also at lower ionic strengths on lowering the calcium concentration. The abnormal gelation may be due to a mutation in the fibrinogen molecule. Tendency to form tight fibrin gels and/or insufficient crosslinked fibrin matrix may be pathogenetic in this thrombotic disease.

The porosity, fiber dimension and architecture of fibrin gels formed in recalcified plasma on addition of thrombin are, within a certain range of thrombin concentrations, determined by the initial rate of fibrinogen activation. Furthermore, the initial network formed in this range creates the scaffold into which subsequently activated fibrinogen molecules are deposited. Change in thrombin concentration that occurs during gelation, as a result of indigenous thrombin generation in plasma, does not qualitatively alter this scaffold. The formation of the networks obeys a more complex rule when low amounts of thrombin are added or with recalcified plasma without added thrombin. These networks are tighter than would be expected from the initial rate of fibrinogen activation. In this case an extremely porous network is probably formed initially, followed by formation of a secondary, superimposed network of a less porous architectural quality. The latter structure appears to be governed by the rate of indigenous generation in plasma of thrombin-like enzymes in combination with the particular type of fibrinmonomers being produced. In addition our findings establish the rules for proper determination of gel structures in clinical plasma samples. The sequelae of a variety of clot structures that may be formed in vivo are discussed.

Congenital deficiency of antithrombin (AT) is associated with thrombotic events and AT consumption occurs in some severe disorders and after treatment with heparin. The aim of this study was to investigate whether variations in the level of plasma AT modify thrombin generation and the fibrin formation process after the intrinsic coagulation mechanism is triggered. Normal plasma was depleted of AT by immunoadsorption on CNBr-Sepharose coupled with the anti-AT-IgG fraction of antiserum. The AT-depleted plasma was reconstituted with AT (between 0.3 and 1.5 AT units per ml). Thrombin generation was measured as the development of thrombin-antithrombin complexes (TAT). The lag phase preceding fibrin formation depended on the concentration of AT. The short lag phase was seen in completely AT-depleted plasma and the long in plasma with 1.5 AT units per ml. TAT generation, determined in parallel consecutive samples, showed that the rate at which thrombin was generated was inverse to the AT concentration in plasma. The network structure of hydrated fibrin gels in the clotted plasma was studied by measuring the wavelength dependence of gel turbidity. The mass/length ratio value, -i.e. the thickness of fiber strands and porosity of the gel increased with increasing AT concentrations. It is concluded that plasma AT regulates the rate of prothrombin-thrombin conversion, the clotting time and the consequently network structure of the fibrin gel.

SummaryThe native fibrin gel structure formed in vitro from plasma samples was examined by liquid permeation of the hydrated fibrin gel networks in 18 men who had suffered a myocardial infarction before the age of 45 years and in 20 control subjects. Patients with an elevated plasma fibrinogen concentration had a considerably lower fibrin gel porosity (permeability coefficient, K s) compared with patients with a normal plasma fibrinogen level and with controls. The calculated fiber mass-length ratio of the fibrin gel networks was decreased in both patient groups. Gel porosity differed markedly between individuals at a given plasma fibrinogen concentration. Fairly strong inverse correlations were found between plasma orosomucoid level on the one hand and K s (r = –0.617, p <0.01) or fiber mass-length ratio (r = –0.499, p <0.05) on the other. The low density lipoprotein (LDL) cholesterol concentration also correlated inversely with K s (r = –0.471, p <0.05) and fiber mass-length ratio (r = –.522, p <0.05). Significant inverse relations, which were independent of plasma fibrinogen and lipoprotein concentrations, were detected between K s (r = –.519, p <0.05) and calculated fiber mass-length ratio (r = –.723, p <0.001) and number and severity of coronary artery stenoses determined by angiography. A proneness to formation of tight, rigid and space-filling fibrin network structures with small pores thus appears to be associated with premature coronary artery disease.

In the old German city of Dresden, Al started as a young man a career as playwright and actor with a local theater company. As the story goes, he encountered so many psychological conflicts between the members of the company and what was worse they all came to the warm and generous Al to get help. Perhaps, he said to himself, I ought to be a psychiatrist. The story shows AI's early interest in the arts. He was to become also a painter under the name (Mr) L. AIcopley, but that was to happen later in Kansas City, USA. For now he was to enter the medical school. Soon the physical and chemical sciences attracted his attention. This was in the thirties and protein chemistry was in its very beginning, sprouting out from the omnipotent Kolloid-Chemie. It was during studies with Edgar Wohlich at the Physiological Institute of the University of Wurzburg that he first came in contact with the field of blood clotting and haemostasis. He was, however, soon to leave Germany because of the oppressive political situation at that time. Most of his scientific contributions in haemostasis and thrombosis have been done in the U.S.A., France and England.

An ideal antithrombotic drug should inhibit thrombosis without affecting hemostasis.It should have a long half life.It should be absorbed after oral administration; it should be safe and it should have a wide therapeutic range.These criteria are discussed using new antithrombotics as examples.

Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A-alpha-442Cys-A-alpha-472Cys and 2-gamma-326Cys-gamma-339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A-alpha- and gamma-chains and the symmetrical disulfide bonds at gamma-8Cys, gamma-9Cys, and A-alpha-28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A-alpha-19-A-alpha-20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A-alpha-chain at A-alpha-19-A-alpha-20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma-326Cys-gamma-339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B-beta-15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.