Summary. Background: Fibrin polymerization is mediated by interactions between knobs ÔAÕ and ÔBÕ exposed by thrombin cleavage, and holes ÔaÕ and ÔbÕ always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous. Objectives: To determine whether A:b or B:b interactions have a role in thrombin-catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes ÔaÕ: c364Ala, c364His or c364Val. Methods: We examined thrombin-and reptilase-catalyzed fibrinopeptide release by high-performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa-catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis. Results: Thrombin-catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin-catalyzed polymerization; polymerization of c364Val and c364His were more delayed than c364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs ÔAÕ. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose-dependently by the addition of either Gly-Pro-Arg-Pro (GPRP) or Gly-His-Arg-Pro (GHRP), peptides that specifically block holes ÔaÕ and ÔbÕ, respectively. FXIIIa-catalyzed crosslinking between c-chains was markedly delayed for all the variants. Conclusion: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes ÔaÕ. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.
Summary. We found two heterozygous dysfibrinogenemias, designated fibrinogen Kosai and fibrinogen Ogasa. Kosai was associated with arteriosclerosis obliterans but Ogasa showed no bleeding or thrombotic tendencies. The plasma fibrinogen concentrations from the two propositi (Ogasa and Kosai) were much lower when determined by the thrombin-time method (0.94 and 1.06 g L À1, respectively) than when determined by the immunological method (2.87 and 2.72 g L À1 , respectively). We performed DNA sequencing and functional analyses to clarify the relationship between the structural and functional abnormalities. Genetic analysis of PCR-amplified DNA from the propositi identified the heterozygous substitution Bb15Gly!Cys (GGT!TGT). Western blotting analysis of purified fibrinogen revealed the existence of albumin-fibrinogen complexes. Functional analyses indicated that compared with the normal control, the propositi's fibrinogen released only half the normal amount of fibrinopeptide B and showed markedly impaired polymerization. In addition, the observation of thinner fibers in fibrin clots (by scanning electron microscopy) indicated markedly defective lateral aggregation in the variant fibrinogens. The impaired functions may be due to the substitution of Cys for Bbo15Gly plus the existence of some additional disulfide-bonded forms.
Summary. Background and objectives: Analysis of dysfibrinogens has improved our understanding of molecular defects and their effects on the function of intact fibrinogen. To eliminate the influence of plasma heterozygous molecules, we synthesized and analyzed recombinant‐variant fibrinogens. Methods: We synthesized two recombinant‐variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bβ‐chain: namely, Bβ15Cys and Bβ15Ala. Results: Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bβ15Cys fibrinogen. For Bβ15Cys fibrinogen, functional analysis indicated (a) no thrombin‐catalyzed fibrinopeptide B (FPB) release and (b) markedly impaired lateral aggregation in thrombin‐ and reptilase‐catalyzed fibrin polymerizations. For Bβ15Ala fibrinogen, such analysis indicated slight impairments of both thrombin‐catalyzed FPB release and lateral aggregation in thrombin‐catalyzed fibrin polymerization, but nearly normal lateral aggregation in reptilase‐catalyzed fibrin polymerization. These impaired lateral aggregations were accompanied by thinner fibrin fiber diameters (determined by scanning electron microscopy of the corresponding fibrin clots). Conclusion: We conclude that a region adjacent to Bβ15Gly plays important roles in lateral aggregation not only in desA fibrin polymerization, but also in desAB fibrin polymerization, and we speculate that the marked functional differences between Bβ15A and Bβ15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bβ15C fibrinogen, but also to the existence of disulfide‐bonded forms. Finally, our data indicate that the Bβ15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.
To cite this article: Hirota-Kawadobora M, Terasawa F, Suzuki T, Tozuka M, Sano K, Okumura N. Comparison of thrombin-catalyzed fibrin polymerization and Factor XIIIa-catalyzed cross-linking of fibrin among three recombinant variant fibrinogens, c275C, c275H, and c275A. Summary. Background and objectives: We have previously reported that recombinant c275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild-type, c275Arg, and plasma fibrinogen from a heterozygous proband. Since cArg275His mutations have also been reported in 10 families, we synthesized recombinant c275His fibrinogen and c275Ala fibrinogen (as a control) and analyzed and compared them with c275Cys and c275Arg. Methods: A variant c-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen Aa-and Bb-chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin-catalyzed fibrin polymerization and factor XIIIa (FXIIIa)-catalyzed c-c dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles. Results: By comparison with both c275His and c275Ala fibrinogens, recombinant c275Cys fibrinogen exhibited a more impaired c-c dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of c275Arg and c275Cys fibrinogens or c275Arg and c275His fibrinogens, thrombin-catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with c275Cys or c275His alone). Conclusion: These results strongly suggest that an amino acid substitution of c275Arg 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 c275C fibrinogen augments the impairment caused by a His or Ala substitution.
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