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.

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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