Hirota-Kawadobora M, Kani S, Terasawa F, Fujihara N, Yamauchi K, Tozuka M, Okumura N
Department of Pathology, Shinshu University School of Medicine, Matsumoto, Japan.
J Thromb Haemost. 2005 May;3(5):983-90. doi: 10.1111/j.1538-7836.2005.01294.x.
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.
We synthesized two recombinant-variant fibrinogens with a single amino acid substitution at the 15Gly residue in the Bbeta-chain: namely, Bbeta15Cys and Bbeta15Ala.
Western blotting analysis of purified fibrinogen revealed the existence of a small amount of a dimeric form only for Bbeta15Cys fibrinogen. For Bbeta15Cys 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 Bbeta15Ala 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).
We conclude that a region adjacent to Bbeta15Gly 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 Bbeta15A and Bbeta15C fibrinogens in FPB release and fibrin polymerization might not only be due to the presence of a substituted cysteine residue in Bbeta15C fibrinogen, but also to the existence of disulfide-bonded forms. Finally, our data indicate that the Bbeta15Gly residue plays important roles in FPB release and lateral aggregation of protofibrils.
对异常纤维蛋白原的分析增进了我们对分子缺陷及其对完整纤维蛋白原功能影响的理解。为消除血浆杂合分子的影响，我们合成并分析了重组变体纤维蛋白原。
我们合成了两种在Bβ链15Gly残基处有单个氨基酸取代的重组变体纤维蛋白原，即Bβ15Cys和Bβ15Ala。
对纯化纤维蛋白原的蛋白质印迹分析表明，仅Bβ15Cys纤维蛋白原有少量二聚体形式存在。对于Bβ15Cys纤维蛋白原，功能分析表明：（a）无凝血酶催化的纤维蛋白肽B（FPB）释放；（b）在凝血酶和蛇毒凝血酶催化的纤维蛋白聚合反应中，侧向聚集明显受损。对于Bβ15Ala纤维蛋白原，此类分析表明在凝血酶催化的纤维蛋白聚合反应中，凝血酶催化的FPB释放和侧向聚集均有轻微受损，但在蛇毒凝血酶催化的纤维蛋白聚合反应中，侧向聚集接近正常。这些受损的侧向聚集伴随着更细的纤维蛋白纤维直径（通过对相应纤维蛋白凝块的扫描电子显微镜检查确定）。
我们得出结论，Bβ15Gly附近的区域不仅在desA纤维蛋白聚合中，而且在desAB纤维蛋白聚合中对侧向聚集起重要作用，并且我们推测Bβ15A和Bβ15C纤维蛋白原在FPB释放和纤维蛋白聚合方面的显著功能差异可能不仅归因于Bβ15C纤维蛋白原中存在取代的半胱氨酸残基，还归因于二硫键结合形式 的存在。最后，我们的数据表明Bβ15Gly残基在原纤维的FPB释放和侧向聚集中起重要作用。
