Birgit Hessel

Human fibrinopeptides. Isolation, characterization and structure.

Abstract 1. 1. Four different fibrinopeptides, A, AP, Y and B, have been isolated from human fibrinogen by means of chromatography and precipitation procedures. 2. 2. The amino acid sequence has been determined with the phenylisothiocyanate degradation method and by means of fragmentation with proteolytic enzymes and partial acid hydrolysis. The A-, AP- and Y-peptides are similar in structure, the AP-peptide being an A-peptide phosphorylated at the serine residue in Position 3 from the N-terminal end and the Y-peptide being one amino acid residue shorter than the A-peptide from the N-terminal end. The B-peptide has been found to have pyroglutamic acid as N-terminal residue. 3. 3. The phosphorus in human fibrinogen is partly bound to an AP-peptide and partly to other structures of the fibrinogen molecule. 4. 4. On basis of the results the structure of human fibrinogen and the specificity of thrombin is discussed.

Fibrin in human plasma: Gel architectures governed by rate and nature of fibrinogen activation

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.

Native fibrin gel networks observed by 3D microscopy, permeation and turbidity

Native fully hydrated fibrin gels formed at different fibrinogen and thrombin concentrations and at different ionic strengths were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels were found to be composed of straight rod-like fiber elements that often came together at denser nodes. In gels formed at high fibrinogen concentrations, or with high amounts of thrombin, the spaces between the fibers decreased, indicating a decrease of gel porosity. The fiber strands were also shorter. Gel porosity decreased dramatically in gels formed at the high ionic strengths. Shorter fibers were observed and fiber swelling occurred at ionic strengths above 0.24. Quantitative parameters for gel porosity, fiber mass/length ratio and diameter were also derived by liquid permeation and turbidometric analyses of the gels. Permeation analysis showed that gel porosity (measured as Ks) decreased in gels formed at higher fibrin and thrombin concentrations in agreement with the porosity observed by microscopy. The turbidometric analysis showed good agreement with the permeation data for gels formed at various thrombin concentrations, but supported the permeation data more poorly in gels formed at different fibrinogen concentrations, especially above 2.5 mg/ml. Turbidometric analysis showed that the fiber mass/length ratio and diameter decreased in gels formed at ionic strength up to 0.24, as was seen in the permeation study. However, at higher ionic strengths swelling of the fibers was suggested from the gel turbidity data and this was also indicated by microscopy. These findings are discussed in relation to previous hydrodynamic and electron microscopic studies of fibrin gels.

Measurement in human blood of fibrinogen/fibrin fragments containing the Bβ 15–42 sequence

This paper describes a radioimmunoassay for the B beta 15-42 peptide derived from human fibrinogen or fibrin. Iodinated B beta 15-42 is bound by specific antiserum and binding can be completely inhibited by excess of the non-iodinated B beta 15-42, B beta 1-42 or B beta 1-118. These peptides cannot be distinguished in this assay. Furthermore, fibrinogen can also completely inhibit binding of iodinated B beta 15-42 peptide. Due to the cross-reaction with fibrinogen, clinical blood samples require a processing step prior to their use in this radioimmunoassay. Ethanol precipitation allows for fibrinogen removal and a near quantitative recovery of both added B beta 15-42 peptide as well as of the endogenous blood peptide(s) containing the B beta 15-42 sequence. The mean level of B beta 15-42 immunoreactive material in normal individuals was found to be 0.41 pmol/ml while that in one group of patients was 20-40 times this value.

Disulfide bridges in NH2-terminal part of human fibrinogen

Abstract The NH 2 -terminal portion of fibrinogen (N-DSK) obtained by cleavage with CNBr is a dimeric structure (M.W. 58,000). We have found that the half-molecules of N-DSK are held together by symmetrical disulfide bridges in its Aα- and γ-chains. Consequently the two half-molecules of fibrinogen are linked in the same way. In addition, N-DSK contains inter chain disulfide bridges within its half-molecules. The arrangement of these bridges is shown and the geometry in relation to function discussed.

Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization

Human fibrinogen was illuminated in the presence of methylene blue. The resulting photooxidized fibrinogen was devoid of polymerization activity and thrombin-induced coagulability. The initial rate of the thrombin catalysed release of fibrinopeptides from photooxidized fibrinogen was normal. It was shown that illumination of photooxidized fibrinogen and photooxidized fragment N-DSK caused the modification of histidine residues. Tryptophan residues were also modified. When fibrinogen was photooxidized immediately after the addition of thrombin, the capacity to polymerize was lost. The inhibition of polymerization was less marked when oxidation was initiated at the time when polymerization began or thereafter. Photooxidized fibrinogen acts as an inhibitor of the polymerization of fibrin monomers. Photooxidized fibrinogen has affinity for thrombin-activated fibrinogen-Sepharose and thrombin-activated fragment N-DSK-Sepharose. When the former conjugate is illuminated in the presence of methylene blue its affinity for fibrinogen is decreased. It is concluded that the fragment N-DSK domain of fibrinogen is affected by photooxidation.

Fibrinogen and fibrin formation

Abstract The present day concept of the primary structure of fibrinogen is outlined. Cleavage of the molecule with CNBr and plasmin has yielded a number of fragments which account for almost all of the structure of the chains (Aα, Bβ and γ) of the molecule. The three chains are linked together by disulfide bridges forming a structure with a unit weight of 170,000. This half-molecule is then joined to another identical half-molecule by means of symmetrical disulfides at the NH 2 -terminal ends. Consequently fibrinogen is a dimeric structure satisfying the formula (Aα, Bβ, γ) 2 . Fibrinogen is activated by thrombin-catalyzed hydrolysis, during which two peptides (fibrinopeptide A and B) are released from the NH 2 -terminal portion of the Aα-and Bβ-chains, respectively. Kinetic studies have shown, by and large, that in its proteolytic action, thrombin recognizes only the first 51 amino acid residues of the Aα-chain of fibrinogen. Recognition of the Bβ-chain with release of fibrinopeptide B appears to occur only after polymerization has commenced. There exist two functional domains in fibrinogen, the interaction of which are of importance for fibrin formation. One functional domain (A-A′) is located in the NH 2 -terminal portion of the molecule. This domain, which includes the fibrinopeptide structures, requires activation with thrombin in order to become functionable. The other domain (a-a′) is located in the carboxyterminal region of the molecule and is active already in fibrinogen as it circulates in blood. The ordered alignment of fibrinogen units in the fibrin fiber can be explained by the interaction between the NH 2 -terminal domain (A or A′) of one molecule with the carboxyterminal domain (a or a′) of another molecule.

FXIII induced gelation of human fibrinogen — An alternative thiol enhanced, thrombin independent pathway

Factor XIII induced gelation of human fibrinogen in the presence of calcium ions. At the end of this reaction between 95 and 100% of the fibrinogen was incorporated into the gel matrix. The gelation was dramatically enhanced by DTT. Cysteine and beta-mercaptoethanol also enhanced the reaction, but less efficiently. Thrombin activated factor XIII led to shortened gelation time and increased the rate of gelation. The reaction was inhibited by p-chloromercuribenzoate and iodoacetamide. Neither fibrinopeptide A, nor fibrinopeptide B were released during gelation, while quantitative release of FPA by thrombin was demonstrated from preformed gel matrices. SDS-PAGE showed the presence of gamma-dimers and alpha-polymers in the gel matrix. In the clot supernatants gamma-dimers were observed already before the gel point. We also observed that the clotting of fibrinogen by thrombin was perturbed by DTT. Preincubation of fibrinogen with calcium ions prevented this effect of DTT.

The proteolytic action of the snake venom enzymes arvin and reptilase on N-terminal chain-fragments of human fibrinogen.

Mammalian fibrinogens are built up by three pep tide chains, a(A), P(B) and 7 [l] . The N-terminal ends of these chains are linked together in a firm “disulfide knot” (DSK) by means of disulfide bridges [2] . When thrombin acts on fibrinogen small fibrinopeptides are split off from the N-terminal ends of the o(A) and /3(B) chains. The result of this proteolysis is fibrin. The peptides, which have a molecular weight of less than 2000, are fibrinopeptide A and its analogues AP and AY from the o(A) chain and fibrinopeptide B from the P(B) chain [3] . Fibrinopeptide AY has the same amino acid sequence as fibrinopeptide A minus the N-terminal alanine; AP is fibrinopeptide A with a phosphoserine residue replacing serine at position 3 from the N-terminal end. “A” refers to A+AP+AY. Polymerization starts when fibrinopeptide “A” is released. In its limited proteolytic action, thrombin rapidly hydrolyses the arginyl-glycine bonds binding the fibrlnopeptides to the rest of the molecule. However, some other arginyl or lysyl bonds can also be split. In this category is the arginyl-valyl bond, occurring 3 residues from the arginyl-glyceryl bond split when the A-peptide is released [2,4]. The tripeptide in question, Gly-Pro-Arg, (see fig. L), has been isol-

Native Fibrin Gel Networks and Factors Influencing their Formation in Health and Disease

Hydrated fibrin gels were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels from normal fibrinogen were found to be composed of straight rod-like fiber elements which sometimes originated from denser nodes. In gels formed at increasing thrombin or fibrinogen concentrations, the gel networks became tighter and the porosity decreased. The fiber strands also became shorter. Gel porosity of the network decreased dramatically in gels formed at increasing ionic strengths. Shortening of the fibers were observed and fiber swelling occurred at ionic strength above 0.24. Albumin and dextran, when present in the gel forming system, affected the formation of more porous structures with strands of larger mass-length ratio and fiber thickness. This type of gels were also formed in plasma. Albumin and lipoproteins may be among the determinants for the formation of this type of gel structure in plasma. Gels formed when factor XIIIa instead of thrombin was used as catalyst for gelation showed a completely different structure in which lumps of polymeric material were held together by a network of fine fiber strands. Our studies have also shown that the methodologies employed may be useful in studies of gel structures in certain dysfibrinogenemias as well as in other diseases. We give examples of two patients with abnormal fibrinogen and of patients with ischaemic heart disease.