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.     

Differences in the clotting of lamprey fibrinogen by lamprey and bovine thrombins

1. Improved methods for the purification of lamprey thrombin and fibrinogen are presented. 2. Lamprey thrombin releases two fibrinopeptides from lamprey fibrinogen during the transformation into fibrin. Bovine thrombin releases only one of these, a peptide referred to as fibrinopeptide B. The differences in the by-products of fibrin formation are reflected in the different N-terminal amino acid compositions of the two types of fibrin. 3. The fibrinopeptide that is not removed from the lamprey fibrinogen by bovine thrombin can subsequently be released by treatment of that fibrin with lamprey thrombin. 4. Under the conditions used, lamprey thrombin releases both fibrinopeptides at about the same rate. 5. The differences in interaction among these pairs of related proteins are extreme manifestations of the phenomenon loosely referred to as `species specificity'.     

Interaction of a peptide inhibitor with two forms of monomeric fibrin differing in the degree of activation

The study is devoted to the interaction of peptide inhibitor of fibrin self-assemblage with two forms of fibrin monomer: deprived of fibrinopeptides A and preserving fibrinopeptides B (desAA-monomer) and fully activated (desAABB-monomer). It is shown that peptide inhibitor hinders the coagulative conversion of fibrinogen by thrombin, limiting the enzymic and nonenzymic stages of the process. The indispensible condition for the formation of inhibitor-monomeric fibrin associates is a preliminary modification of fibrinogen by thrombin.     

Molecular weight of human fibrinogen derived from phosphorus determinations

1. Fibrin clots obtained from diluted human plasma with bovine thrombin often contain amounts of phospholipids that cannot be diminished by further plasma dilution. 2. The ;cold insoluble residue' obtained during fibrinogen preparation has a higher phosphorus content than the purified fibrinogen. 3. Evidence showed that adsorption of phospholipids or phosphorus-containing fibrinopeptides on purified fibrinogen or fibrin was unlikely. 4. O-Phosphorylserine was detected in acid hydrolysates of human fibrin. 5. On the basis of phosphorus determinations the average molecular weight of human fibrinogen cannot be less than 342000 (304000-383000) for a group of ten donors, and 265000 for two other persons, assuming 1 phosphorus atom/molecule and incomplete splitting of the phosphorus-containing fibrinopeptide. Complete splitting of the phosphopeptide would require molecular weights twice as high. 6. Fibrinolysis was a possible cause of lower phosphorus contents found in isolated fibrinogen and fibrin from a donor who showed apprehension during blood collection and in a fibrinogen preparation that had been submitted to prolonged dialysis.     

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.     

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.     

The thrombin-fibrinogen interaction

The thrombin-catalyzed conversion of fibrinogen (F) to fibrin consists of three reversible steps, with thrombin (T) being involved in only the first step which is a limited proteolysis to release fibrinopeptides (FpA and FpB) from fibrinogen to produce fibrin monomer. In the second step, fibrin monomers form intermediate polymers through noncovalent interactions. In the third step, the intermediate polymers aggregate to form the fibrin clot. The molecular mechanisms of the first two steps are elucidated.     

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.     

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.     

Fibrin assembly after fibrinopeptide A release in model systems and human plasma studied with magnetic birefringence.

Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by the action of the snake-venom-derived enzymes reptilase and ancrod. A range of conditions was investigated. Fibrin polymerization in solutions of purified fibrinogen shows a distinct break near the gelation point. On addition of Ca2+ or albumin the lag period is shortened, fibre thickness is increased and the break in assembly almost vanishes, probably because both of these additives promote lateral aggregation. There are minor differences in the kinetics, depending on the venom enzyme used. The kinetics of fibrin assembly in model systems containing either Ca2+ or albumin and in human plasma with a largely dormant coagulation cascade are very similar. Therefore in the latter condition there is no significant alteration in the assembly process due to interaction between fibrin or the venom enzymes and any of the plasma proteins. When the cascade is activated, the polymerization progress curves have a character that resembles a combination of the reactions observed when the venom enzymes and endogenously generated thrombin separately induce coagulation, except for a region near gelation where, paradoxically, polymerization appears to be slower on activation. The low-angle neutron-diffraction patterns from oriented gels made with thrombin or reptilase are identical. Therefore at low resolution the packing of the monomers within fibres is the same when fibrinopeptide A only or both fibrinopeptides A and B are removed.     

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.     

Lamprey fibrinopeptide B is A glycopeptide

One of the peptides released from lamprey fibrinogen during its transformation into fibrin has been found to contain covalently bound carbohydrate. The peptide, which also contains tyrosine O-sulfate, corresponds to the mammalian fibrinopeptide B and is the amino-terminal segment of the lamprey fibrinogen β-chain. As noted previously, this peptide is the only one released when lamprey fibrinogen is coltted by mammalian thrombin. Of the more than fifty sets of fibrinopeptides characterized from various species, this is the first one found to contain carbohydrate.     

Structural basis for sequential cleavage of fibrinopeptides upon fibrin assembly

Nonsubstrate interaction of thrombin with fibrinogen promotes sequential cleavage of fibrinopeptides A and B (fpA and fpB, respectively) from the latter, resulting in its conversion into fibrin. The recently established crystal structure of human thrombin in complex with the central part of human fibrin clarified the mechanism of this interaction. Here, we reveal new details of the structure and present the results of molecular modeling of the fpA- and fpB-containing portions of the Aalpha and Bbeta chains, not identified in the complex, in both fibrinogen and protofibrils. The analysis of the results reveals that in fibrinogen the fpA-containing portions are in a more favorable position to bind in the active site cleft of bound thrombin. Surface plasmon resonance experiments establish that the fpB-containing portions interact with the fibrin-derived dimeric D-D fragment, suggesting that in protofibrils they bind to the newly formed DD regions bringing fpB into the vicinity of bound thrombin. These findings provide a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils and/or fibrils at the second stage.     

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional structural studies on fragments of fibrinogen and fibrin

Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant γC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from 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.     

Examining thrombin hydrolysis of the factor XIII activation peptide segment leads to a proposal for explaining the cardioprotective effects observed with the factor XIII V34L mutation

In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a(2) dimer by cleaving the Factor XIII activation peptide segment at the Arg(37)-Gly(38) peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K(m), k(cat), and k(cat)/K(m) were determined for thrombin hydrolysis of fibrinogen Aalpha-(7-20), Factor XIII activation peptide-(28-41), and Factor XIII activation peptide-(28-41) with a Val(34) to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28-41) exhibits a 10-fold lower k(cat)/K(m) value than fibrinogen Aalpha-(7-20). With the Factor XIII V34L mutation, decreases in K(m) and increases in k(cat) produce a 6-fold increase in k(cat)/K(m) relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.     

Effects of fibrinopeptide cleavage on the plasmic degradation pathways of human cross-linked fibrin

The presence of fibrinopeptide B in human fibrin has a significant effect on plasmic degradation pathways of cross-linked clots. Two types of fibrin were obtained from fibrinogen by incubation either with thrombin, to remove both fibrinopeptides A and B, or with batroxobin, to cleave fibrinopepitde A only. Fibrins obtained after various incubation times were characterized by the determination of the NH2-terminal amino acids, the content of fibrinopeptides, and the extent of cross-linking. The fibrins were digested by plasmin and were analyzed by polyacrylamide gel electrophoresis. The presence and concentration of the (DD)E complex, as well as fragments E1 and E2, in the digests were dependent upon the loss of fibrinopeptide B from cross-linked fibrin. These degradation products, and also fragment DD, appear to be useful molecular markers of fibrinolysis.     

蕲蛇酶抗栓作用机理的初步分析

动物实验结果示,蕲蛇酶能裂解纤维蛋白原成为可溶性纤维蛋白,降低血中纤维蛋白原浓度,抑制血小板聚集,对抗在酶诱导的血浆凝块订的血浆凝块回缩,因而发挥防血栓形成作用。对纤维蛋白平板试验无直接溶纤作用,但能增加实验动物血中ｔ－ＰＡ活性。可能通过促使血管内皮细胞释放ｔ－ＰＡ而发挥溶栓作用。     

Soluble fibrin-fibrinogen complexes as intermediates in fibrin gel formation

Oligomer formation in fibrinogen solutions following addition of thrombin was studied by addition of thrombin inhibitor at various times subsequent to thrombin, followed by size-exclusion chromatography (SEC) on a high-performance SEC column capable of resolving species of molecular weights less than or equal to 10(6). Peaks corresponding to species with 1, 2, 3, and 4 or more times the molecular weight of fibrinogen were detected and quantified via nonlinear least-squares curve-fitting procedures. The evolution of each of these peaks with time is well accounted for by a kinetic model in which the predominant component of each oligomeric molecular weight species is a linear complex of fibrinogen and fibrin. The observed predominance of trimeric over dimeric oligomers even at short times suggests that the thrombin-catalyzed release of the two A fibrinopeptides from a single molecule of fibrinogen is highly cooperative.