Etiology and pathophysiology of thrombosis : A molecular biological aproach to elucidate disturbed mechanisms of blood coagulation and its inhibition.

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1.Structure analysis of hereditary dysfibrinogens. Two hitherto unknown amino acid substitutions have been identified, i.e., gammaGly-268 to Glu in a homozygous dysfibrinogen Kurashiki I (Blood, in press) and gammaArg-275 to Ser in a heterozygous dysfibrinogen Kamogawa (manuscript in preparation) by amino acid sequence and gene analyzes. Both dysfibrinogens have been found to have a dysfunctional D : D self-association site, a recently proposed fibrin polymerization site and reported to be defective in fibrinogen Tokyo II (J.Clin.Invest.90 : 1053,1995). Thus, both dysfibrinogens manifest impaired fibrin protofibril formation. Although not an entirely new type of amino acid substitution, an AalphaArg-19 to Gly substitution has been identified in fibrinogen Kumamoto associated with thrombosis. Since affinity for thrombin is significantly low in Kumamoto fibrin as compared with normal fibrin, the observed defective localization of thrombin onto fibrin clots appears to be related to the oc … More currence of thrombosis (manuscript in preparation). Besides these, eight dysfibrinogen samples have been under investigation including one each from Israel, Germany and Venezuela, and five from inside of our country.2.Preparation of anti-fibrinogen monoclonal antibodies (mAb's) and their application to the study of basic and clinical aspects of fibrinogen. Three unique mAb's have been produced. (1) A mAB recognizing the calcium-dependent structure of the D domain of fibrinogen has been established. Utilizing this mAb as a ligand for immuno-affinity chromatography, fibrinogen has been highly purified even from limited amounts of plasma samples of abnormal fibrinogens including fibrinogen Marburg (Thromb.Haemostas.73 : 662-667,1995). (2) One mAb was found to completely inhibit factor XIIIa-catalyzed crosslinking of the fibrin gamma-chains as well as fibrin monomer polymerization. This mAb served as powerful tool to study the possible involvement of the gamma-chain crosslinking in fibrin polymerization (in preparation). (3) Another one is a mAb that recognizes a specific conformation of the E domain of fibrin monomer elicited upon its binding with fibrinogen. This mAb has been successfully utilized for the study of the mechanisms of fibrin monomer polymerization and their relevance to the occurrence of thrombosis (Blood, in press).Localization of vitronectin-and fibronectin-receptors on cultured human glioma cells. On the surface of glioma cells cultured on vitronectin and fibronectin, distribution of respective integrins was found to vary upon contact with and during culture on the adhesion molecules. Participation of high-molecular weight kininogen in the attachment and spreading of cells on the ligands has also been studied (J.Biol.Chem., in press). Less