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

点击翻译按钮获取中文摘要

I.Analyses of hereditary dysfibnnogens : During the three year-term of studies supported by the Grant-in-Aid for Scientific Research, No. 08407034, we were able to analyze more than 10 abnormal fibrinogen molecules, some of which had been associated with clinical manifestations of either bleeding or thrombosis, or both. Fibrinogen (Fbg) Caracas II with a unique Aalpha Ser-434 to Asn substitution, to which an extra oligosaccharide is linked (J Biol Chem 266 : 11575-11581, 1991) was further studied by electron microscopy (EM). The Caracas II fibrin fibers are found to be loosely associated and fibrin gels appeared to consist of irregularly aligned fibrin bundles with scattering large caves and pores. The permeability experiment showed a high permeability rate as compared with normal fibrin gels (J Biol Chem 271 : 4946-4953, 1996). On the other hand, Fbg Marburg (truncation of 150 amino acids in the Aalpha-chain and partly linked with serum albumin) associated with severe postoperative bl … More eeding accompanied by successive recurrent thrombo-embolic complications was found to partly linked with one or two serum albumin molecules by EM as anticipated by SDS-PAGE.The fibrin gels are composed of extremely thin and highly branched fibrin fibers, forming extraordinarily compact gels. These fibrin clots account for thrombo-embolic complications together with bleeding due to delayed fibrin clot formation. Part of these data was published in BLOOD (91 : 3282-3288,1998), and the remainder is now in preparation for publication. Four other abnormal Fbg's were also characterized and published (Kurashiki : gamma Gly-268 to Glu, Blood 87 : 4686-4694, 1996 ; Kumamoto : Aalpha Arg-19 to Gly, Jpn J Thromb Hemost 8 : 382-392, 1997 ; Kamogawa : gammaArg-275 to Ser, Thromb Haemostas, in press ; Niigata : Bbeta Asn-160 to Ser with an extra oligosaccharide N-linked to Bbeta Asn-158 ; Blood, in press). In three other dysfibrinogens, we have also identified new types of point mutations, Fbg's Pretoria (gamma Cys-139 to Tyr) ; Tokyo V (gammaAla-327 to Thr) and Osaka VI (12 amino acid extension due to the stop codon TAA to AAA for Lys). Further analyses on these molecules are going on, and will be submitted for publication.II.Molecular mechanisms of fibrin (Fbn) to function as adhesion molecule : Fbg functions as adhesion molecule only after transition to fibrin. When human fibroblasts were cultured on a fibrin monolayer, the bi-integrin as well as the already known beta3-integrin was expressed on the cell surface, and spreading of cells progressed in an RGD-dependent manner (J Biol Chem 272 : 8824-8829, 1997). When human glioma cells were cultured, a two-step mode of spreading via the beta1 -integrin was noted : firstly with fibrin (Aalpha RGD 572-574) and then with the autologous fibronectin secreted and incorporated in the extra-cellular matrix. These findings are now in the status of"in press" in Thrombosis Research. Furthermore, plasma kininogen was found to behave not only as an adhesion molecule but also as an inhibitor. These opposing effects were characterized and published (J Biochem 124 : 473-484, 1998). Less