A unique property of a plasma proteoglycan, the C1q inhibitor. An anticoagulant state resulting from its binding to fibrinogen.

The C1q inhibitor, C1qI, an approximately 30-kD circulating chondroitin-4 sulfate proteoglycan, displayed concentration-dependent prolongation of plasma and fibrinogen solution clotting times. Under factor XIIIa catalyzed cross-linking conditions and maximum C1qI concentrations, minor amounts of clot formed displaying complete gamma-gamma dimer formation but virtually no alpha-polymer formation. The anticoagulant effect was undiminished by its binding to C1q, by increased ionic strength, and by CaCl2, but was abolished by incubation of C1qI with chondroitinase ABC. 125I-labeled C1qI bound to immobilized fibrinogen, fibrin monomer, fibrinogen plasmic fragments D1 and E, and fibrin polymers. Occupancy on the E domain required uncleaved fibrinopeptides together with another structure(s), and it did not decrease binding of thrombin to fibrinogen. Occupancy on the D domain did not decrease the fibrinogen binding to fibrin monomer. We conclude that the E domain occupancy impaired fibrinopeptide cleavage, and occupancy on the D domain impaired polymerization, both steric hindrance effects. C1qI binding to fibrinogen explains at least in part the well-known fibrin(ogen) presence in immune complex-related lesions, and the fibrinogen presence in vascular basement membranes and atheromata. We postulate that fibrin binding by resident basement membrane proteoglycans provides dense anchoring of thrombus, substantially enhancing its hemostatic function.     

Interaction of histidine-rich glycoprotein with fibrinogen and fibrin.

Histidine-rich glycoprotein (HRGP) is a human plasma and platelet protein of apparently diverse biological functions. In this study a new interaction for HRGP is described. HRGP specifically interacts with fibrinogen as demonstrated by two independent systems. Using an enzyme-linked immunosorbent assay it was demonstrated that HRGP bound to adsorbed fibrinogen in a concentration-dependent and saturable manner, with an apparent dissociation constant (Kd) of 6.7 nM. The binding was specific, reversible, and not mediated by a conformationally altered adsorbed fibrinogen molecule. The interaction was divalent cation-dependent and ionic in nature. The HRGP-fibrinogen interaction was also demonstrated using rocket immunoelectrophoresis. The HRGP-fibrinogen interaction had an effect on the kinetics of conversion of fibrinogen to fibrin as demonstrated by a prolongation of the thrombin time. HRGP also became incorporated into fibrin clots in a concentration-dependent and saturable manner, with an apparent Kd of 0.25 microM. The incorporation of HRGP into fibrin clots occurred in a plasma milieu as demonstrated by the direct incorporation of radiolabeled HRGP into plasma clots and by a significant decrease in serum HRGP levels as compared with plasma levels. HRGP prolonged the lag time phase of fibrin gel formation, and decreased the rate of turbidity rise, as well as the final absorbance of fibrin gels. Since the extent of fibrin polymerization was not influenced by the presence of HRGP, these data suggest that fibrin is distributed over more, but thinner, fibrils in the presence of HRGP. In addition to its potential effect on fibrin polymerization, the HRGP-fibrin interaction may play a role in the cell-cell interactions of platelets and macrophages.     

Exosites in the substrate specificity of blood coagulation reactions

Summary.  The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand–exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa•factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.     

Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors.

Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release mediated by fluid-phase thrombin with their activity against the clot-bound enzyme. Incubation of thrombin with citrated plasma results in concentration-dependent FPA generation, which reaches a plateau within minutes. In contrast, there is progressive FPA generation when fibrin clots are incubated with citrated plasma. Heparin, hirudin, hirudin dodecapeptide (hirugen), and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) produce concentration-dependent inhibition of FPA release mediated by fluid-phase thrombin. However, heparin is much less effective at inhibiting thrombin bound to fibrin because a 20-fold higher concentration is necessary to block 70% of the activity of the clot-bound enzyme than is required for equivalent inhibition of fluid-phase thrombin (2.0 and 0.1 U/ml, respectively). In contrast, hirugen and PPACK are equally effective inhibitors of fluid- and solid-phase thrombin, while hirudin is only 50% as effective against the clot-bound enzyme. None of the inhibitors displace bound 125I-labeled thrombin from the clot. These studies indicate that (a) clot-bound thrombin is relatively protected from inhibition by heparin, possibly because the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin, and (b) clot-bound thrombin is susceptible to inactivation by antithrombin III-independent inhibitors because the sites of their interaction are not masked by thrombin binding to fibrin. For these reasons, antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings.     

Functional evaluation of an inherited abnormal fibrinogen: fibrinogen “Baltimore”

The rate of clotting and the rate of development and degree of turbidity after addition of thrombin to plasma or purified fibrinogen from a patient with fibrinogen Baltimore was delayed when compared with normal, especially in the presence of low concentrations of thrombin. Optimal coagulation and development of translucent, rather than opaque, clots occurred at a lower pH with the abnormal fibrinogen than with normal. Development of turbidity during clotting of the abnormal plasma or fibrinogen was less than normal at each pH tested, but was maximal in both at approximately pH 6.4. The physical quality of clots formed from fibrinogen Baltimore was abnormal, as demonstrated by a decreased amplitude on thromboelastography. The morphologic appearance of fibrin strands formed from fibrinogen Baltimore by thrombin at pH 7.4 was abnormal when examined by phase contrast or electron microscopy, but those formed by thrombin at pH 6.4 or by thrombin and calcium chloride were similar to, though less compact, than normal fibrin. The periodicity of fibrin formed from fibrinogen Baltimore was similar to normal and was 231-233 A.A study of the release of the fibrinopeptides from the patient's fibrinogen and its chromatographic subfractions verified the existence of both a normally behaving and a defective form of fibrinogen in the patient's plasma. The defective form differed from normal in three functionally different ways: (a) the rate of release of fibrinopeptides A and AP was slower than normal; (b) no visible clot formation accompanied either partial or complete release of the fibrinopeptides from the defective form in 0.3 M NaCl at pH 7.4; and (c) the defective component possessed a high proportion of phosphorylated, relative to nonphosphorylated, fibrinopeptide A, while the coagulable component contained very little of the phosphorylated peptide (AP). The high phosphate content of the defective component did not appear to be the cause of the abnormality, but may be the result of an associated metabolic or genetic phenomenon.     

Rapid dissociation of platelet-rich fibrin clots in vitro by a combination of plasminogen activators and antiplatelet agents.

Thrombin promotes the formation of arterial thrombi by converting fibrinogen to fibrin and by causing platelets to aggregate. We have examined the combined effects of plasminogen activators and inhibitors of platelet aggregation on the lysis of platelet-rich fibrin clots formed by alpha-thrombin in citrated platelet-rich plasma. The extent of platelet aggregation and clot formation were measured by recording light transmission in an aggregometer. Immediately after the formation of platelet-rich fibrin clots, addition of 2,000 U/ml streptokinase or 50 micrograms/ml recombinant tissue-type plasminogen activator alone resulted in the degradation of polymerized fibrin and the release of trapped platelet aggregates without causing significant platelet deaggregation. Preincubation of the platelet-rich plasma with 20 microM indomethacin for 1 min before thrombin stimulation or simultaneous addition of prostaglandin E1 (10 microM) with the plasminogen activators after thrombin stimulation resulted in spontaneous platelet deaggregation. Because platelet aggregation is, in part, mediated by the binding of Arg-Gly-Asp-containing adhesive proteins to activated platelets, the effect of Arg-Gly-Asp peptides on platelet deaggregation was examined. By itself, Gly-Arg-Gly-Asp-Ser-Pro specifically caused dose- and time-dependent deaggregation of platelet aggregates formed by ADP or by thrombin in the presence of 1 mM Gly-Pro-Arg-Pro, but had no effect on the dissociation of thrombin-induced platelet-rich fibrin clots. In combination with streptokinase or recombinant tissue-type plasminogen activator, Gly-Arg-Gly-Asp-Ser-Pro enhanced the rate of lysis of platelet-rich fibrin clots. The control Gly-Arg-Gly-Glu-Ser-Pro peptide was completely ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

"Fibrinogen Tokyo II". An abnormal fibrinogen with an impaired polymerization site on the aligned DD domain of fibrin molecules.

A hereditary dysfibrinogenemia associated with defective aggregation of fibrin monomers was found in a 39-yr-old female and in the members of her immediate family, who had all been asymptomatic. The abnormality was probably due to an impaired polymerization site exposed in the DD domain of two adjacent fibrin molecules, because plasmic fragment DD derived from the propositus' cross-linked fibrin bound far less tightly to insolubilized normal fragment E than that from the normal one. Its complementary polymerization site in the E domain of fibrin, which was exposed by thrombin cleavage, and the polymerization site in the D domain of fibrinogen, which was available without activation by thrombin, were both found to be normal. More anodal migration of the abnormal fragment DD than the normal one, as shown by immunoelectrophoresis, seemed to support our concept that the mutation most likely resides in the D domain of the abnormal fibrinogen molecule at or near a region closely related to the polymerization site that is exposed when two fibrin molecules are linearly aligned. The work of others on the polymerization of normal fibrin with different techniques yielded results consistent with our conclusions. We tentatively designate this type of abnormal fibrinogen "fibrinogen Tokyo II," but its possible identity with other abnormalities of fibrinogen reported heretofore is not excluded.     

Enhanced binding of fibrinogen by the subendothelium after treatment of the rabbit aorta with thrombin

Damage to the endothelial surface of an artery invokes a hemostatic response that causes platelet deposition and activation of the coagulation and fibrinolytic pathways on the exposed subendothelial surface. Plasma fibrinogen is rapidly adsorbed at the site of injury. To gain insight into fibrinogen uptake, undamaged and de-endothelialized rabbit thoracic aortas were pretreated with various concentrations of thrombin and then incubated with fibrinogen labeled with iodine 125 in vitro. Uptake of fibrinogen by the subendothelium was not affected by low thrombin concentrations (less than 10 nmol/L), probably because of the antithrombin capacity of the vessel wall to inactivate any thrombin adsorbed. Over the thrombin concentration range of 10 to 90 nmol/L. fibrinogen binding increased linearly as binding of thrombin labeled with iodine 131 increased. In contrast, treatment of the subendothelium with enzymatically inactive thrombin did not enhance fibrinogen binding. Fibrinogen binding was inhibited by exposing the thrombin-treated subendothelium to hirudin or phenylalanyl-prolyl-arginyl-chloromethyl ketone. High thrombin concentrations (greater than 100 nmol/L) caused either a steadily decreasing uptake of fibrinogen with low fibrinogen concentrations or fibrin coagulation on the subendothelial surface from a high fibrinogen concentration. Glycyl-prolyl-arginyl-proline (0.1 mg/ml), a selective inhibitor of fibrin polymerization, inhibited 72% to 78% of fibrinogen uptake by the thrombin-treated subendothelium. Fibrinogen uptake was Ca2(+)-dependent, but ethylenediaminetetraacetic acid (10 mmol/L) did not displace subendothelium-bound fibrinogen. Plasmin effectively removed at least 75% of bound fibrinogen, indicating an extracellular location for the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibrin induces release of von Willebrand factor from endothelial cells.

Addition of fibrinogen to human umbilical vein endothelial cells in culture resulted in release of von Willebrand factor (vWf) from Weibel-Palade bodies that was temporally related to formation of fibrin in the medium. Whereas no release occurred before gelation, the formation of fibrin was associated with disappearance of Weibel-Palade bodies and development of extracellular patches of immunofluorescence typical of vWf release. Release also occurred within 10 min of exposure to preformed fibrin but did not occur after exposure to washed red cells, clot liquor, or structurally different fibrin prepared with reptilase. Metabolically labeled vWf was immunopurified from the medium after release by fibrin and shown to consist of highly processed protein lacking pro-vWf subunits. The contribution of residual thrombin to release stimulated by fibrin was minimized by preparing fibrin clots with nonstimulatory concentrations of thrombin and by inhibiting residual thrombin with hirudin or heating. We conclude that fibrin formed at sites of vessel injury may function as a physiologic secretagogue for endothelial cells causing rapid release of stored vWf.     

Fibrin polymerization and its regulatory role in hemostasis

Proteolytic conversion of fibrinogen to fibrin results in self-assembly to form a three-dimensional clot matrix that subsequently becomes cross-linked by fXIIIa to form the central structural element of the in vivo thrombus. The process of fibrin formation and assembly leads to new properties that serve to regulate the rate and extent of clotting, cross-linking, and fibrinolysis. These are brought about by the ability of fibrin (1) to bind thrombin at a noncatalytic site, thus limiting its diffusability but at the same time preserving its catalytic potential; (2) to bind fXIII, regulate its activation to fXIIIa, and limit further activation of fXIII once fibrin cross-linking has occurred; and (3) to bind alpha 2-PI, tPA, and plasminogen and regulate the initiation and propagation of fibrinolysis. Additional interactions not covered in this review between fibrin(ogen), and other plasma proteins, cells or matrix components suggest other functions for fibrin that, along with those discussed above, define a critical role in modulating hemostasis, inflammation, and the wound healing process.     

Fibrinogen logroño. A new case of congenital dysfibrinogenemia

Summary An abnormal fibrinogen was discovered in a 9-year-old male subject without history of hemorrhagic diathesis. Coagulation time, prothrombin time and reptilase time were prolonged. The thrombin time was corrected using increasing concentrations of normal plasma and bovine thrombin; there was 2 partial correction at pH 6.5 and ionic strength 0.05. A study of the family showed that the mother and a brother of the propositus presented the same abnormalities. Analysis of the purified fibrinogen showed normal fibrinopeptide release and normal levels of sialic acid and hexosamines. However, coagulation index, polymerization of fibrin monomers, isoelectric point and sedimentation coefficient were abnormal. In view of the abnormalities described and by comparison with the data reported in the literature, we believe that this should be considered a new variant of the fibrinogen molecule and we have designated it ‘fibrinogen Logro?o’.     

Thrombin generation, fibrin clot formation and hemostasis.

Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during clot formation dictates the ultimate fibrin structure. In most prior studies of fibrin structure, clotting was initiated by adding thrombin to a solution of fibrinogen; however, clot formation in vivo occurs in an environment in which the concentration of free thrombin changes over the reaction course. These changes depend on local cellular properties and available concentrations of pro- and anti-coagulants. Recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots that are associated with an increased risk of bleeding or thrombosis. Further studies of fibrin formation during in situ thrombin generation are needed to understand fibrin clot formation in vivo.     

Fibrinogen γ' chain binds thrombin exosite II

Summary.  A high-affinity thrombin-binding site in an alternately processed fibrinogen variant, the γA/γ' isoform, is characterized in this report. The binding site has been shown to be situated between γ' 414 and 427, and Tyr418 and 422 in this part of the γ' chain are known to be sulfated. A synthetic peptide corresponding to the γ' chain carboxyl terminus is shown to bind thrombin with a K _d = 0.63 ± 0.16 µmol L⁻¹. Maximum binding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the γ' peptide via exosite II. Thus, thrombin binding to the γ' chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin-bound thrombin can retain enzymatic activity, and why fibrin-bound thrombin is heparin-resistant.     

Regulation of fibrinogen biosynthesis: effect of fibrin degradation products, low-molecular-weight peptides of fibrinogenolysis, and fibrinopeptides A and B.

Fibrinogen synthesis in rabbits was evaluated following intravenous infusions of stage 3 degradation products of homologous fibrinogen or fibrin, prepared in vitro. Fibrinogen production was measured by determining the rate of appearance of 75SeM into circulating fibrinogen. Fibrinogen synthesis increased threefold after the administration of stage 3 FDP (D and E), dialyzed to remove LMW digestion fragments, In contrast, the fdp obtained by plasminolysis of crosslinked thrombin clots or of noncrosslinked ancrod or thrombin clots failed to enhance basal fibrinogen production. Accelerated fibrinogen production was not accompanied by alterations in haptoglobin concentration or by increased incorporation of 75SeM into haptoglobin. Fibrinogen synthesis was not increased after infusions of FPA and FPB.     

Defective alpha-polymerization in the conversion of fibrinogen Baltimore to fibrin.

The subunit structure of fibrinogen Baltimore and fibrin formed from this inherited dysfibrinogenemia was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The molecular weights of the alpha-, b- and gamma-chains of fibrinogen Baltimore were found to be identical to those of normal fibrinogen. Noncross-linked fibrin formed from both purified fibrinogen Baltimore as well as normal fibrinogen contained two alpha-monomers (alpha1 and alpha2). alpha2 was presumed to be alpha-monomer from which fibrinopeptide A had been released. The evolution of alpha2 during clotting of fibrinogen Baltimore was delayed and appeared to be quantitatively reduced when compared to normal. Crosslinked fibrin formed from fibrinogen Baltimore possessed an abnormal subunit structure. alpha-polymers were not generated in thrombin-induced, factor XIII-rich clots of fibrinogen Baltimore under conditions of pH and calcium concentration suitable for complete alpha-polymerization in normal fibrin. If clotting was carried out with calcium concentrations twice that required for normal clots or at pH 6.4, fibrin from fibrinogen Baltimore was completely cross-linked. These structural analyses of fibrin formed from fibrinogen Baltimore substantiate earlier findings that indicate a defect in the alpha-chain of this dysfibrinogenemia.     

Inhibition of human platelet aggregation by monovalent antifibrinogen antibody fragments.

Monovalent goat antibody fragments (Fab) that were monospecific for human fibrinogen were isolated by affinity chromatography on fibrinogen-Sepharose and used as a direct probe for the involvement of fibrinogen in platelet aggregation and the release reaction. The antifibrinogen Fab inhibited aggregation of washed human platelets induced by thrombin (0.1-10 U/ml) by 50-95%, but had no effect on (14-C)-serotinin release and only a slight inhibitory effect on 125-I-thrombin binding to platelets. Inhibition of aggregation was not observed with nonimmune goat Fab or rabbit antihuman albumie bound tightly at saturation to surface fibrinogen molecules. After washing the platelets once to remove unbound Fab, aggregation by subsequently added thrombin was no longer inhibited. The antifibrinogen Fab inhibited the clotting of fibrinogen by thrombin but did not effect the rate of fibrinopeptide A release, indicating that the Fab inhibits clotting by interfering with the polymerization of fibrin monomers. Our experiments suggest that fibrinogen released from platelets is directly involved in thrombin-induced aggregation of washed platelets, perhaps through polymerization of fibrin monomers generated by proteolytic cleavage of released fibrinogen.     

Molecular basis of fibrinogen Naples associated with defective thrombin binding and thrombophilia. Homozygous substitution of B beta 68 Ala----Thr.

In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G----A) in the B beta chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the B beta chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human alpha-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound less than 10% of active site inhibited alpha-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound approximately 50% of alpha-thrombin. These results suggest that the mutation of B beta Ala 68----Thr affects the binding of alpha-thrombin to fibrin, and that defective binding results in a decreased release of FpA and FpB in both homozygous and heterozygous abnormal fibrinogens.     

Fibrinogen γ chain functions

Summary.  This review covers the functional features of the fibrinogen γ chains including their participation in fibrin polymerization and cross-linking, their role in the initiation of fibrinolysis, their binding and regulation of factor XIII activity, their interactions with platelets and other cells, and their role in mediating thrombin binding to fibrin, a thrombin inhibitory function termed 'antithrombin I'.     

Cleavage of blood coagulation factor XIII and fibrinogen by thrombin during in vitro clotting.

Thrombin cleavage of blood coagulation Factor XIII (a2b2) and fibrinogen was studied during in vitro clotting to determine the physiologic sequence of these events. First, the time course of fibrin formation and cleavage of Factor XIII was measured in platelet-rich plasma. Cleavage of fibrinogen was measured by using a radioimmunoassay for fibrinopeptide A. Conversion of trace amounts of radioiodinated a-chains of 125I-Factor XIII to thrombin-modified a-chains was measured in unreduced 10% sodium dodecyl sulfate-polyacrylamide gels. During spontaneous clotting, a similar percentage of 125I-Factor XIII and fibrinogen was cleaved at each time point. Visible gelation of polymerized fibrin monomer occurred when 24 +/- 8% of fibrinogen was cleaved and 21 +/- 6% of Factor XIII was converted to Factor XIII'. Thrombin cleavage of Factor XIII and fibrinogen was also studied in platelet-poor plasma to which thrombin was added. In order to measure Factor XIIIa activity, fibrin polymerization was completely inhibited by the addition of Gly-Pro-Arg-Pro. Factor XIIIa formation was measured by the incorporation of [3H]putrescine into casein. The concentration of added thrombin required to cleave 50% of fibrinogen and Factor XIII was 0.65 U/ml and 0.35 U/ml, respectively. The rate of cleavage of fibrinogen by thrombin was 43-fold greater than cleavage of Factor XIII. Lower Gly-Pro-Arg-Pro concentrations were used to determine the effects of incompletely inhibiting fibrin polymerization on cleavage of Factor XIII and fibrinogen. Thrombin cleavage of Factor XIII but not fibrinogen was dependent on the extent of fibrin polymerization. The more marked the degree of inhibition of fibrin polymerization, the slower the rate of Factor XIIIa formation. Thus, in platelet-rich plasma, thrombin cleavage of Factor XIII and fibrinogen are closely related events during spontaneous clotting. Furthermore, cleavage of Factor XIII during clotting is enhanced by fibrin polymerization in platelet-poor plasma.     

Comparison of thrombin-catalyzed fibrin polymerization and factor XIIIa-catalyzed cross-linking of fibrin among three recombinant variant fibrinogens, gamma 275C, gamma 275H, and gamma 275A.

BACKGROUND AND OBJECTIVES: We have previously reported that recombinant gamma 275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild-type, gamma 275Arg, and plasma fibrinogen from a heterozygous proband. Since gamma Arg275His mutations have also been reported in 10 families, we synthesized recombinant gamma 275His fibrinogen and gamma 275Ala fibrinogen (as a control) and analyzed and compared them with gamma 275Cys and gamma 275Arg. METHODS: A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen A alpha- and B beta-chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin-catalyzed fibrin polymerization and factor XIIIa (FXIIIa)-catalyzed gamma-gamma dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles. RESULTS: By comparison with both gamma 275His and gamma 275Ala fibrinogens, recombinant gamma 275Cys fibrinogen exhibited a more impaired gamma-gamma dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of gamma 275Arg and gamma 275Cys fibrinogens or gamma 275Arg and gamma 275His fibrinogens, thrombin-catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with gamma 275Cys or gamma 275His alone). CONCLUSION: These results strongly suggest that an amino acid substitution of gamma 275Arg alone disrupts D:D interactions in thrombin-catalyzed fibrin polymerization and the formation of fibrin bundles and fibrin clots. Moreover, the existence of a subsequent disulfide-linked Cys in gamma 275C fibrinogen augments the impairment caused by a His or Ala substitution.