Studies on the pathogenesis and pathophysiology of thromboembolisms in the field of surgery. Development of novel techniques for analyzing the regulatory systems of blood coagulation.

1.异常纤维蛋白原的结构分析：我们分析了本研究资助期间从多个机构和全国各地新提交给我们的10种异常纤维蛋白原。已鉴定的突变包括2个Arg-275到His，4个Arg-275到Cys，1个Met-310到Thr，并伴有N-糖基化的Asn-308，Asp-330到Tyr和Arg-375到Gly替换。所有这些突变都可以通过编码相应氨基酸的密码子中的单个碱基交换来解释。伽马链的这一特定区域似乎构成了纤维蛋白聚合所需的关键结构，它们仅通过单一氨基酸的替换就应该导致纤维蛋白凝块的严重改变。除了γ-Arg-275对其进行替换外，所有在伽马链的羧基末端附近发现的突变都是新发现的结构错位。O…目前正在进行基因分析研究。在这些结构分析中，我们成功地应用了几种识别纤维蛋白原不同结构的单抗，这将在后面的项目3.2中说明。凝血和纤溶对培养的人内皮细胞(EC)的影响：我们已经提供了一系列证据，证明通常由肝细胞产生的蛋白C也是由EC在鉴定蛋白C分子和来自培养的EC的蛋白C的信使RNA的基础上合成的。这一发现到目前为止还没有报道，因此需要进一步的研究来更准确地阐明EC介导的活体血栓形成的调节。针对凝血和纤溶相关物质的单抗：我们已经制备并鉴定了各种针对纤维蛋白原、凝血因子IX、X和XIII、纤溶酶原激活物抑制物和凝血酶-抗凝血酶复合体的单抗。一种识别纤维蛋白原的γ86-302残基的抗体和另一种识别血浆片段D的NH_2末端构象的抗体被成功地用于异常纤维蛋白原的结构分析。针对其他分子的抗体也被引入到与血栓形成及其调节相关的分子相互作用的分析中。较少

1. Structure analysis of abnormal fibrinogens: We have analyzed 10 abnormal fibrinogens newly referred to us from several institutions an over the country during the term covered by this research grant-in- aid. The mutations identified included two Arg-275 to His; four Arg-275 to Cys; one each of Met-310 to Thr accompanied by N-glycosylated Asn-308; Asp-330 to Tyr and Arg-375 to Gly substitutions. All these mutations could be accounted for by a single base exchange in the codon encoding respective amino acids.This particular region of the gamma chain seems to constitute critical structures required for fibrin polymerization, and their perturbation solely by a single amino acid replacement should have resulted in severely altered fibrin clot formation. Except the gamma Arg-275 to His substitution, all the mutations identified near the carboxy-terminal region of the gamma chain were found to be newly elucidated structural derangements. Gene analysis studies are currently in progress in o … More ur laboratory.In these structure analyses, we successfully applied several monoclonal antibodies recognizing various structures of fibrinogen as will be stated later in item 3.2. Blood coagulation and fibrinolysis which proceed on the cultured human endothelial cells(EC): We have provided lines of evidence that protein C, normally produced by hepatocytes, was also synthesized by the EC's on the basis of identification of protein C molecules as well as messenger RNA for protein C derived from the cultured EC's. This finding has not been reported heretofore, and thus further studies are necessary to more precisely elucidate the EC-mediated regulation of thrombus formation in vivo.3. Monoclonal antibodies raised against substances related to blood coagulation and fibrinolysis: We have prepared and characterized various monoclonal antibodies against fibrinogen, factors IX, X and XIII, plasminogen activator inhibitor and the thrombin-antithrombin complex. An antibody that recognizes the gamma 86-302 residue peptide of fibrinogen and another one that recognizes the NH_2-terminal conformation of plasmic fragment D were successfully utilized for the structure analysis of abnormal fibrinogens. Antibodies raised against other molecules have also been introduced into the analyses of molecular interactions relevant to thrombus formation and its regulation. Less