Mechanism of fibrin coagulation based on selective, cation-driven, protofibril association

The cation sensitivity of linear and lateral assembly processes of thrombin- and reptilase-activated fibrinogen was examined. Analytic ultracentrifugation shows that the linear assembly of fibrin oligomers (protofibrils) is neither cation dependent nor sensitive to chelating agents. Protofibrils generated with thrombin–hirudin gelate with either 1–2 mM Ca(II) or 15–100 μM Zn(II). By contrast, protofibril B, generated with reptilase–diisopropylfluorophosphonate, gelates only with Ca(II) but is insensitive to Zn(II). These results indicate that the release of fibrinopeptides A and B (FPA and FPB) expose two types of lateral binding sites that are sensitive to Ca(II) and Zn(II) respectively. Transmission electron (TEM) micrographs of negatively stained gels indicate that the linear packing of the monomers within the fibrin- and cation-induced protofibrin fibers is essentially identical. Scanning electron (SEM) micrographs show that the Ca(II)-induced protofibrin B gel is similar to fibrin. In all, it seems that branching and gelation derive from two types of cation-sensitive, lateral associative processes. Based on these findings, a new paradigm for fibrin coagulation is proposed.     

Preparation of fibrin des-AA by thrombin

The active thrombin is formed in the blood stream when the blood coagulation system is activated. It attacks fibrinogen, splits off two fibrinopeptides A and fibrinogen is transformed into des-AA fibrin which is able to polymerize spontaneously forming protofibrils. At high thrombin concentration the enzyme splits off two fibrinopeptides B and des-AA fibrin units are transformed into des-AABB fibrin. These two forms of fibrin are widely used in the biological experiments. However des-AA fibrin is obtained usually from fibrinogen using the snake poisons (such as reptilase). Des-AA fibrin was obtained also by physiological enzyme thrombin, but that des-AA fibrin samples had the contamination of des-AABB fibrin. At the present paper we have described the method of the des-AA fibrin preparation by thrombin without any contamination of des-AABB fibrin.     

Differences in the Subunit Structure of Human Fibrin formed by the Action of Arvin,Reptilase and Thrombin

INTEREST has focused recently on the clinical use of proteolytic enzymes similar in properties to thrombin which can directly cleave fibrinogen. Potentially the most important are arvin, derived from the venom of Agkistrodon rhodostoma and reptilase, isolated from the venom of Bothrops atrox. These only release fibrinopeptide A from fibrinogen^1–3, whereas thrombin cleaves fibrinopeptides A and B from fibrinogen to form fibrin. Thrombin also activates fibrin stabilizing factor (FSF) which introduces amide bonds between the subunits of soluble fibrin⁴. FSF rapidly forms covalent links between pairs of γ(C)-chains giving γ(C)-dimers and in a slower reaction α(A)-chains are linked to produce high molecular weight polymers⁵. Although reptilase, like thrombin, activates FSF⁶, arvin apparently does not, which would explain why the fibrin formed by arvin seems to be more friable than that produced by thrombin or reptilase⁷.     

Effect of heparin on fibrinogen formation during experimental toxic syndrome of disseminated intravascular coagulation

The formation of circulation components of fibrinogen pool in toxigenic syndrome of disseminated intravascular blood coagulation (DBC-syndrome) caused by the Vipera lebetina turanica venom has been examined by the gel-filtration method. Simultaneously it has been studied what components of fibrinogen pool are removed in paracoagulation tests and with addition of the coagulating enzymes (thrombin, reptilase and the Echis multisguamatus venom) to the plasma. The preliminary heparinization of animals poisoned by Vipera lebetina turanica venom was found to prevent the fibrinogen formation mainly at the fibrin-monomer formation stage. Besides, the effect of polymerization inhibition was revealed in the plasma of such animals.     

Effect of fibronectin on fibrinogen conversion into fibrin

The effects of fibronectin on fibrinogen clotting induced by thrombin or reptilase and on fibrin monomer polymerization in a pure system in the absence of factor XIIIa were studied. It was shown that within a broad range of concentrations and molar ratios of the mixed proteins, fibronectin does not alter significantly the fibrinogen clotting time either under thrombin or under reptilase action. The effect of fibronectin on the fibrin self-assembly consists in a slight acceleration of this process, whose degree is directly dependent on the fibronectin/fibrin monomer molar ratio as well as on the absolute fibrin monomer content at a constant molar ratio. The stimulating effect of fibronectin is amplified by Ca2+. The experimental results suggest that fibronectin can noncovalently bind the fibrin monomer and/or intermediate polymers in the non-enzymatic phase of fibrinogen conversion to fibrin.     

Fibrin assembly in human plasma and fibrinogen/albumin mixtures

Magnetic birefringence is used to monitor the kinetics of thrombin-catalyzed fibrin polymerization in model systems of increasing complexity (i.e., fibrinogen solutions, fibrinogen/albumin mixtures, and plasma anticoagulated with citrate) and in plasma containing free calcium which is the physiological condition. The introduction of albumin into fibrinogen solutions shortens the lag period and enhances fiber thickness. The polymerization progress curves are sigmoidal at zero or low albumin concentrations, but at physiological and higher concentrations, they become hyperbola-like from the end of the lag period. High albumin concentration has thus induced a change in the assembly kinetics. The progress curves from plasma in which the cascade is dormant are also hyperbola-like although they round off more quickly because of antithrombin activity. In plasma containing free calcium, thrombin is endogenously produced, and the progress curves are nearly linear; hence, the assembly kinetics are very different from those of the model systems. The curves are not influenced by calcium-dependent cross-linking involving factor XIIIa. The progress curves are also linear when polymerization is induced with Russell's viper venom, which by directly activating factor X circumvents earlier steps in the cascade. This implies that linear polymerization is caused by events posterior to factor X activation and are thus likely to be largely dependent on the functioning of the prothrombinase complex. Addition of thrombin to plasma containing free calcium reduces the lag period. At low exogenous thrombin levels, the polymerization rate is increased, and the progress curves remain linear. However, at higher levels, the curves become more complicated and, paradoxically, full polymerization takes longer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrinogen and fibrin interaction with concanavalin a dimer and its influence on coagulation

Concanavalin A dimer interacts with fibrinogen and soluble fibrin at pH 5.2 Analysis of the binding data shows that there are in both cases four binding sites per molecule and that the dissociation constant does not change by removal of fibrinopeptides A and B. Ultracentrifugal studies shows that no aggregates of fibrinogen or fibrin are formed through concanavalin A binding and that up to four molecules of concanavalin A dimer can be bind to one molecule of fibrinogen or fibrin. These results imply that the four carbohydrate chains in the molecule are accessible to concanavalin A dimer. There is a diminution in the coagulation of fibrinogen by thrombin at low relative lectin concentrations and an increase at high concentrations. However, the lectin always favours the aggregation of fibrin monomers and does not have any inhibitory effect on the release of fibrinopeptides. We conclude that the electric charge in the neighbourhood of the carbohydrate in both chains, Bβ and γ plays an important role in the attraction between monomeric fibrin and fibrinogen-monomeric fibrin. The different effect of concanavalin A on the coagulation, depending on the relative concentration of the lectin, would be the result of the screening of this electric charge favouring either the interaction of fibrinogen-monomeric fibrin or the polymerization of monomeric fibrin.     

The proteolytic action of Arvin on human fibrinogen

1. Human fibrinogen was subjected to proteolysis by enzyme preparations (clinical Arvin and IRC-50 Arvin) from the venom of Agkistrodon rhodostoma. 2. IRC-50 Arvin releases three peptides from fibrinogen, and these were identified as fibrinopeptides AP, AY and A. 3. The less purified ;clinical' Arvin releases, in addition to fibrinopeptides AP, AY and A, small amounts of two heptapeptides derived from fibrinopeptides AP and A, probably because it contains another enzyme as well as Arvin. 4. No fibrinopeptide B is released by either Arvin preparation. 5. Thus, although Arvin is known to differ from ;reptilase' from Bothrops jararaca in that it does not activate the enzyme that cross-links fibrin (fibrin-stabilizing factor), it is identical with reptilase with respect to the peptides that it liberates from fibrinogen.     

Fibrin gels as biological filters and interfaces

We present data which show that fibrin gels are ordered network structures, the porosity of which is determined by the amount of thrombin being present during the activation step preceding gelation. On activation of fibrinogen, fibrinopeptides are released and simultaneously polymers are formed, obeying apparent first order kinetics. The pore size of the network is inversely related to the rate of polymer formation. It is proposed that at the time of gelation the polymers form an ordered lattice structure, which in the organism may serve as a biological interface.     

Fibrin aggregation before sol-gel transition.

Fibrinogen solutions (concentrations 2 mg/ml, 0.15-M Tris-NaCl buffer, pH 7.4) were incubated at 20 degrees C with quantities of reptilase or thrombin that were so small that the polymerization process could be followed for several hours by means of static and dynamic light scattering. The scattered intensity and its correlation function were recorded at scattering angles between 30 degrees and 150 degrees. The measured data were compared with model calculations based on the Flory-Stockmayer distribution, which predicts a sol-gel phase transition. This distribution is characterized by a parameter, lambda, that indicates the extent of aggregation. lambda = 0 corresponds to the monomeric solution, and lambda = 1 indicates the sol-gel transition. Good agreement was found for monomeric units of 75-nm length aggregating (a) end-to-end in the early stage (0 less than or equal to lambda less than or equal to 0.3), and (b) in a staggered overlap pattern for the progressing polymerization (0.3 less than or equal to lambda less than 1). Before the gel point was reached, no systemic difference was observed between the data obtained after activation with thrombin which releases both fibrinopeptides A and B from fibrinogen, and reptilase, which exclusively releases the fibrinopeptides A. This confirms that the release of the fibrinopeptides A is the essential prerequisite for the aggregation process.     

Assembly of fibrin. A light scattering study.

Using stopped flow light scattering, we show that assembly of fibrin following activation with non-rate-limiting amounts of thrombin or reptilase occurs in two steps, of which the first is end-to-end polymerization of fibrin monomers to protofibrils and the second is lateral association of protofibrils to fibers, in agreement with Ferry's original proposal. Polymerization is found to proceed as a bimolecular association of bifunctional monomers; the overall rate varies as the inverse first power of the concentration; end-to-end association of two monomers, of a monomer and an oligomer, and of two oligomers occurs with the same rate constant. The value of the rate constant is 8.2 C 10(5) M-1 s-1 in 0.5 M NaCl, is three times larger in 0.1 M NaCl (0.05 M Tris, pH 7.4), and is the same following activation by reptilase and by thrombin. The onset of growth of fibers from protofibrils takes 12 times longer in 0.5 than in 0.1 M salt, i.e. thick fibers ("coarse" gels) form from short protofibrils, and thin fibers ("fine" gels) form from longer protofibrils. Jumps of salt concentration at times when protofibrils, but not fibers, have formed result in immediate growth of thick fibers at low salt from long protofibrils formed at high salt. The rate of fiber growth in these experiments varies as the inverse first power of the concentration. 3the instant of gelation (formation of a network of fibers) falls in the later half of the time during which the scattering rises due to fiber growth; the rise of gel rigidity after gelation is found to continue beyond the end of this period. Jumps from low to high salt result in retention of whatever fibers have formed at low salt and a very small additional increase of the scattering due to further fiber growth at high salt. From a variety of evidence, we conclude that the properties of fibrin are determined by kinetics and not equilibria of assembly steps. Results obtained here agree with the following scheme of fibrin assembly: monomers polymerize to protofibrils; long protofibrils associate laterally to fibers; occasionally a long protofibril associates with two different fibers to form an interfiber connection; fiber growth does not reverse to yield stabler, more compact, structures and terminates in formation of a network of fibers. The typical delay of fiber growth is the time during which protofibrils form from monomers. Measurements at rate-limiting concentrations of thrombin have allowed estimation of turnover rates of fibrinopeptides that agree with kinetic parameters obtained with direct assay of fibrinopeptide. Release of fibrinopeptide B causes more rapid fiber formation. Addition of thrombin after activation by reptilase, at a time when protofibrils, but not fibers, have formed, is followed rapidly by fiber formation; this proves that thrombin readily removes fibrinopeptide B from protofibrils. On the basis of these new results and earlier work (in particular, Blomb?ck, B., Hessel, B., Hogg, D., and Therkildsen, L...     

The impact of delayed fibrinopeptide-A release on fibrin structure. Studies of an abnormal fibrinogen

The impact of delayed fibrinopeptide-A release on polymerization and structure of fibrin gels was studied utilizing a heterozygously transmitted variant fibrinogen. An arginine to histidine substitution at position 16 of the alpha chain of the abnormal fibrinogen delayed release of an abnormal fibrinopeptide-A (A) by thrombin and completely blocked release of A by reptilase. When clotted with thrombin, patient fibrin formed more slowly than normal fibrin, but clottability was normal and gel fiber mass/length ratios were decreased less than 10%. Gels formed with reptilase clotted slowly, demonstrated reduced clottability, but had normal fiber mass/length ratios. Reptilase clotted the normal but not the variant component of the patient fibrinogen. Thrombin-induced cleavage of fibrinopeptide-B prior to A occurred in these experiments, but polymerization of this species beyond trimers has been reported to be minimal under the conditions used. With time, A is removed by thrombin resulting in the slow production of normal fibrin monomer from the abnormal component. These monomers subsequently polymerize. The minimal change in gel fiber size caused by slow A release implies that fibrin fiber size is primarily a function of ionic environment and not of the sequence of peptide release.     

Clotting of fibrinogen. 2. Calorimetry of the reversal of the effect of calcium on clotting with thrombin and with ancrod

When clotting is effected by thrombin in the presence of calcium, the endotherm for the D nodules of fibrinogen broadens significantly and then becomes narrow again, while increasing in size. Clotting effected by the snake venom enzyme Ancrod, which releases only the A fibrinopeptides from the E nodule, shows only the broadening of the D endotherm. Accordingly, significant interactions of the D nodules of fibrinogen become possible only when the B fibrinopeptides of the E nodule are released on clotting. When calcium present during clotting is removed from the fibrin clot with ethylenediaminetetraacetic acid, the endotherm for the D nodules of fibrin shows nearly complete reversal if clotting was effected with Ancrod but appears to be divided into two endotherms if clotting was effected with thrombin. At neutral pH, new endotherms were observed for fibrinogen in the temperature range 105-140 degrees C.     

Fibronectin and fibrin gel structure

Plasma fibronectin is covalently incorporated into alpha-chains of fibrin gels in the presence of Factor XIII activated by thrombin (FXIIIaT) but not by Factor XIII activated by the snake venom enzyme batroxobin (FXIIIaB). FXIIIaB catalyzes introduction of gamma-gamma cross-links in fibrin but cross-linked alpha-chains are not formed. In the presence of FXIIIaT, fibrin gels formed by batroxobin incorporated fibronectin and the alpha-chains are cross-linked indicating that FXIIIaB has a different substrate specificity from FXIIIaT. In the presence of FXIIIaT the incorporation of fibronectin approaches 1 mol/340 kDa unit weight of fibrin. Fibronectin when present in a fibrinogen thrombin mixture containing FXIII does not influence the clotting time of the system nor the release of fibrinopeptides. Incorporation of fibronectin is not appreciable before the gel point. This indicates that the polymerization and gelation of fibrinogen is essentially not perturbed by the presence of fibronectin and that fibrin in the gel matrix rather than the fibrin polymers formed prior to gel point is the preferred structure for fibronectin incorporation. Incorporation of fibronectin into fibrin gels during formation leads to an increase in turbidity and a small decrease in Ks (permeability coefficient). This suggests that the width of the strands in the gel increases as a result of fibronectin incorporation. Fibronectin is also incorporated into preformed gels having completely cross-linked gamma- and alpha-chains perhaps indicating that the sites in fibrin involved in fibronectin incorporation are different from those involved in fibrin cross-linking. FXIIIaT appeared to be adsorbed to fibrin gel matrix in the presence but not in the absence of calcium ions.     

Specificity of hemorrhagic proteinase from Crotalus atrox (western diamondback rattlesnake) venom

Hemorrhagic proteinase, HTb, isolated from Crotalus atrox (western diamondback rattlesnake) venom was studied for its specificity. HTb showed fibrinogenase activity, hydrolyzing the A alpha chain of fibrinogen first, followed by the cleavage of the B beta chain. HTb is different from thrombin and did not produce a fibrin clot. The degradation products of fibrinogen were found to be different, indicating that the cleavage sites in the A alpha and B beta chains are different from those of thrombin. N-Benzoyl-Phe-Val-Arg-p-nitroanilide was not hydrolyzed by HTb, although this substrate was hydrolyzed by thrombin and reptilase.     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

Inhibition of the enzymatic activity of thrombin by concanavalin A

Concanavalin A, a carbohydrate lectin derived from the jack bean, prolongs the thrombin clotting time of human plasma or purified fibrinogen. Prolongation is due to delay in peptide release from fibrinogen. The rate of fibrin monomer polymerization is not affected. Hydrolysis of protamine sulfate by thrombin is inhibited by concanavalin A. All inhibitory effects are prevented by α-methyl-D-mannoside. Concanavalin A does not delay clotting of fibrinogen by reptilase (releases fibrinopeptide A only) or by Ancistrodon contortrix contortrix (releases fibrinopeptide B initially followed by a small amount of A). It is concluded that concanavalin A binds to a carbohydrate on the thrombin molecule thus inhibiting its enzymatic activity.     

The role of gamma A/gamma ' fibrinogen in plasma factor XIII activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.     

Polymerization and gelation of fibrinogen in D2O

The solution properties of fibrinogen and the thrombin-induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions. The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis-Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same. From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O. The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O. SDS-gel electrophoresis on fibrin samples, which were cross-linked by factor XIII, yielded results where the cross-linking of the gamma-chain appeared to be the same in D2O and H2O. The alpha-chain cross-linking was somewhat faster in D2O than in H2O. When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin. The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.