B. Blombäck

THE MOLECULAR STRUCTURE OF FIBRINOGEN

The role of fibrinogen in the living organism is not easily narrowed down to the area of hemostasis, when we consider that low levels of fibrinogen can be tolerated without excessive bleeding i.e., in afibrinogenemia and defibrinated states. Fibrinogen may play a role not only in hemostasis, but also in fibroblast proliferation and defense mechanisms against infection. Comparative studies of different animal phyla have shown that aggregating proteins are also common in invertebrates in which cellular mechanisms primarily are responsible for hemostasis. A century has passed since Olof Hammarsten and Denis de Commercy performed their pioneering studies on thL isolation of fibrinogen and its transformation into fibrin. Since the turn of the century, we have gained considerable information regarding the physical and molecular properties of this protein. Several reviews have been written on the topic, and the reader is referred to these for further Despite all available information, today we can only speculate on how the fibrinogen units are arranged in the fibrin fiber, and which forces or bonds keep them together during the different phases of aggregation. This problem is of prime importance for us present here today, since the alignment of the fibrinogen units seems to determine the substrate specificity of factor XI11 in the cross-linking reaction.

Calcium and fibrin gel structure.

Calcium ions (Ca), when present in the gel forming system, were shown to influence liquid permeation of the gels formed, as judged from the Ks-values (permeability coefficients) of the final gels. On increasing Ca concentration, the Ks-values for Fibrin I and Fibrin II gels increase and a maximum is reached at about 10-20 mM for gels formed at ionic strengths between 0.18 and 0.24. For both gels, clotting times (Ct) were shortened on increasing Ca concentration and the shortening was accompanied by increase in Ks. Magnesium also shortened Ct but had no appreciable effect on Ks. The rate of activation of fibrinogen (release of FPA and FPB) was not much affected by Ca, but the activation required for gelation at Ct, decreased with increasing Ca concentration. After the gels were formed, the removal of Ca by EDTA did not change the flow properties. Our results showed that Ca is of importance for formation of the fibrin gel structure, but it may be of minor importance for preservation of the gel structure after its formation. There is a difference between Fibrin I and Fibrin II gels with regard to Ca dependence. The role of calcium in gelation as well as its physiological implications is discussed.

Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin.

In solutions containing fibrinogen and fibronectin, factor XIIIa catalyzes the formation of two types of crosslinked polymers: hybrid oligomers consisting of equimolar amounts of fibrinogen and fibronectin, and fibrinogen oligomers. The two types of oligomers are produced in amounts proportional to the starting concentration of fibronectin and fibrinogen in the reaction mixture. Increasing the fibronectin concentration relative to the fibrinogen concentration results in the production of more hybrid and less fibrinogen type oligomers. The lowest molecular weight hybrid oligomer, a dimer, is formed by ligation of one molecule of fibrinogen and fibronectin. The A alpha-chain of fibrinogen and one fibronectin subunit participate in the crosslinking. Larger size hybrid oligomers form by the joining of two hybrid dimers to each other via gamma-chain dimerization in the fibronectin moiety of the dimers. In fibrinogen oligomer formation, fibrinogen molecules are ligated by gamma-chain dimerization in a step-wise fashion producing fibrinogen dimers, trimers, tetramers, etc. without A alpha-chain crosslinking. The hybrid type and the fibrinogen type of oligomer grow in size and eventually become crosslinked to each other yielding large molecular weight complexes that interact to form a gel network.

Fibrinogen Aarhus — a new case of dysfibrinogenemia

Fibrinogen Aarhus was found in a woman with slightly prolonged whole blood clotting time. The thrombin induced clotting of plasma and purified fibrinogen was much prolonged. Kinetic analysis of FPA and FPB release revealed larger apparent Km and Vmax values for fibrinogen Aarhus than for normal fibrinogen. No clot formation of fibrinogen Aarhus was demonstrated in the presence of Batroxobin and the release of FPA was slower than normal. Upon addition of the clotting enzyme from Agkistrodon contortrix contortrix clotting did occur but the clotting time was much prolonged in comparison with normal fibrinogen. The turbidity of fibrin gels obtained from fibrinogen Aarhus was similar to normal fibrin gels at low thrombin concentrations. Increasing thrombin concentration resulted in appearance of degradation products in the fibrin gels from fibrinogen Aarhus and at the same time a relative increase in turbidity of the gels was observed. Possibly reasons for the slow release of fibrinopeptides, the delayed gelation, and susceptibility to degradation by thrombin are discussed.

Flow and antibody binding properties of hydrated fibrins prepared from plasma, platelet rich plasma and whole blood.

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.

Specificity of Thrombin and Its Action on Fibrinogen

In 1876 Olof Hammarsten suggested that the action of thrombin on fibrinogen was by limited proteolysis.’ Finn evidence of this was obtained about 70 years later? We know now that although thrombin has a narrow specificity of action, it can digest a large number of protein substrates in addition to fibrinogen. This is exemplified by the recent review on protein substrates for thrombin compiled by Muszbek and Laki.’ This list of substrates can be lengthened to include factor VIII: and immunoglobulins.’ The study of the cleavage points in these substrates shows that arginyl bonds are the preferred ones in all substrates. Furthermore, the presence of hydrophobic residues in P sites near the arginyl residue and usually small apolar residues in P’ positions, is evident. Otherwise, there are no similarities in the sequences preceding or succeeding the arginyl bond. It should, however, be stressed that a comparison of this kind is hampered by the lack of kinetic analyses of bond cleavage in most of the substrates. In fibrinogen, a peptide bond is split at a considerable rate in the Aaand BPchain, respectively, releasing fibrinopeptides A and B (FPA and FPB) from the NH,terminal end of the peptide chains. The release of FPA is by far the fastest? There is also a slow but complete release of a Gly-Pro-Arg peptide from the NH,-terminal of the a chain of fibrin.’ Kinetic analyses by several groups have shown that with regard to the A a chain, the structural elements responsible for binding of thrombin reside within the first 50 amino acids of the chain. The most important elements are confined to three regions of this structure, that is, Aa 1-23, 33-44, and 40-51, the last being of least significance.”’’ It has also been suggested that acidic residues in positions P’6-8 are of importance.” In the A a 1-23 segment, the interest became focused early in the study on a nonapeptide sequence preceding the COOH-terminal in FPA, because this sequence was shown to be selected during mammalian evolution.” In the BP chain, it is impossible at the present time to recognize a similarly preserved recognition sequence for thrombin. It should be pointed out that in purified fibrinogen-thrombin systems, the FPB is released at a very slow initial rate. Subsequent to cleavage of FPA and once polymerization has started, however, there is usually a dramatic increase in its rate of r e 1 e a ~ e . l ~ ~ ’ ~ Conformational changes in the substrate as a result of polymerization have been offered as an explanation for this phenomenon. A search for structures in FPA having affinity for thrombin was investigated in our laboratory some 20 years ago.12 Based on the phylogenetic studies, we considered the hydrophobic residues in the COOH-terminal part to be of considerable importance, and in particular, the phenylalanine residue at P,. It occurred to us that this residue was very likely part of an ordered structure that brought it near and in a fixed space

Effect of thrombin on the molecular weights of N-terminal fragments of human fibrinogen

Abstract Cyanogen bromide cleaves the human fibrinogen molecule to produce several fragments, one of which consists of amino terminal components of each of the three chains, α(“A”), β(B) and γ, which comprise the “N-terminal disulfide knot of fibrinogen”. Sedimentation equilibrium experiments indicate that it is a dimer which can be dissociated into monomeric chain components by reduction and alkylation of cystine residues. It has further been proven that the N-terminal disulfide knot and its α(“A”) and β(B) monomeric units as well retain their susceptibility to thrombin cleavage.

Measurement of fibrinogen in dog liver cell fractions by electroimmunoassay and radioimmunoassay

Dog liver was fractionated and the amount of fibrinogen, in the various cell fractions (organelles) involved in the secretory process, was determined both by the Laurell electroimmunoassay and by radioimmunoassay. An unequal distribution of fibrinogen was noted for each cell fraction after an appropriate correction was made. This involved the estimation of the level of contamination of each cell fraction by soluble (blood) fibrinogen. It was observed that significant amounts of added 125I-labeled fibrinogen could be found in each organelle following cell fractionation. This was especially true for the rough endoplasmic reticulum which contained as much as 31% of the added tracer. Both the smooth endoplasmic reticulum and the Golgi fractions contained much lower amounts of added tracer. Using this correction factor, it was found that the rough endoplasmic reticulum contains about 0.1-0.2 micrograms fibrinogen per mg organelle protein. On the other hand, the smooth endoplasmic reticulum and Golgi fractions contained much more fibrinogen. The fibrinogen concentration ranges found for the latter two organelles were 1.9-5.0 and 1.5-5.7 micrograms fibrinogen per mg organelle protein, respectively.

The Formation of the Fibrin Clot from Fibrinogen

The formation of fibrin from fibrinogen presents an excellent model of how a biological fibre develops. The initiation of the process leading ultimately to the formation of a fibrin thread is enzymatic in nature. The enzyme in this process is thrombin, which causes a limited proteolysis of the fibrinogen molecule.