Pathogenesis and pathophysiology of thromboembolic diseases - analysis of the mechanisms of blood coagulation and its regulation at the molecular and gene levels.

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1. Analysis of genetic abnormalities of plasma proteins related to blood coagulation and its regulation. As reported in the prelimary repost-last year, we have accomplished gene analyses on four abnormal fibrinogens with a point mutation in the gamma chain(see Ref. 16). We have also identified new types of point mutations in the Aa chain of two abnormal fibrinogens with impaired fibrin clot formation referred to us from Venezuela, fibrinogen Caracas II(Ref. 11), and Peru, fibrinogen Lima(Ref. 17). Very interestingly, both of these mutant fibrinogens were found to be linked with biantennary oligosaccharides, most of them having been disialylated, due to newly created glycosylation sequences of Asn-X-Thr/Ser by respective point mutations. Because of these structural alterations, they failed to form solid gels as repidly as normal molecules. Nevertheless, they both enhanced t-PA-catalyzed plasminogen activation in a normal fashion, indicating that the initial two-stranded fibrin protofibr … More ils had been normally constructed. Besides these, papers on three other types of mutations identified in fibrinogens Kyoto II, Ise and Osaka IV have been published in Refs. 12, 14, and 15, respectively. In fibrinogen Osaka IV, clinical aspects relevant to surgery have been discussed. By closely relating the structural alterations with the functional abnormalities observed in these abnormal molecules, we were able to Orovide lines of new evidence regarding the mechanisms of fibrin formation at the molecular level.In the study supported by this grant-in-aid, we have identified a new type of structural alteration of arginine-15(CGG)to glycine(iGG), in the so-called Gla region of an abnormal protein C, protein C Yonago, by utilizing polymerase chain reaction. This region has been shown to be critical for eliciting calcium-dependent conformations required for the interaction with phospholipids for activation to an enzyme, activated protein C(Ref. 18). This work has been conducted in collaboration with Prof. Nakamura, Tottori university school of medicine.2. Studies on the interaction between the vascular endothelial cells and proteins related to blood coagulation : We have provided evidence that protein C, which has been believed to be synthesized solely in the hepatocytes, was synthesized in the cultured vascular endothelial cells in the presence of vitamin K. This conclusion was derived from time-dependent increase of protein C molecule and its MRNA(Ref. 13). This new information may imply the presence of a mechanism of the regulation of thrombus formation at the loci where no blood supply is guaranteed owing to the cessation of blood circulation. For these experiments, a variety of polyclonal and monoclonal antibodies prepared and characterized in this study supported by this grant-in-aid have been successfully utilized. Less