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

1 .遗传性纤维蛋白异常原的分析：在科研资助项目（08407034）的三年研究中，我们分析了10多个异常纤维蛋白原分子，其中一些异常纤维蛋白原分子与出血或血栓形成的临床表现有关，或者两者都有。纤维蛋白原（Fibrinogen, Fbg） Caracas II具有独特的α α Ser-434到Asn取代，其上有一个额外的低聚糖连接（J生物化学266:1175 - 1181,1991）。Caracas II型纤维蛋白纤维被发现松散结合，纤维蛋白凝胶似乎由不规则排列的纤维蛋白束组成，散布着大的洞和孔。渗透性实验表明，与普通纤维蛋白凝胶相比，纤维蛋白凝胶具有较高的渗透性（J .生物化学学报，2011,31:446 - 453,1996）。另一方面，Fbg马尔堡（α -链中截断150个氨基酸，与血清白蛋白部分连接）与严重的术后bl相关。更多的出血伴随着连续的复发性血栓栓塞并发症，EM发现与SDS-PAGE预测的一个或两个血清白蛋白分子部分连接。纤维蛋白凝胶由极薄且高度分枝的纤维蛋白纤维组成，形成非常致密的凝胶。这些纤维蛋白凝块可导致血栓栓塞并发症以及纤维蛋白凝块延迟形成引起的出血。这些数据的一部分发表在《BLOOD》杂志上（91:3282-3288,1998），其余的正在准备发表。另外四种异常Fbg也被鉴定并发表（Kurashiki: gamma Gly-268 to Glu, Blood 87: 4686-4694, 1996; Kumamoto: α α Arg-19 to Gly, Jpn .血栓血液学杂志8:382-392,1997;Kamogawa: gamma arg -275 to Ser, Thromb Haemostas, in press； Niigata: Bbeta Asn-160 to Ser与一个额外的寡糖n连接到Bbeta Asn-158； Blood, in press）。在其他三种异常纤维蛋白原中，我们也发现了新的点突变类型，Fbg的Pretoria (γ Cys-139 to Tyr)；东京V （γ - ala -327到Thr）和大阪VI（由于终止密码子TAA到赖氨酸的AAA而延长了12个氨基酸）。纤维蛋白（Fbn）作为粘附分子的分子机制：纤维蛋白只有在转化为纤维蛋白后才能发挥粘附分子的作用。当人成纤维细胞在纤维蛋白单层上培养时，bi-整合素和已知的β -整合素在细胞表面表达，细胞的扩散以rgd依赖的方式进行（J .生物化学学报，2007,29:824- 829,1997）。当培养人类胶质瘤细胞时，发现通过β 1 -整合素的两步扩散模式：首先是纤维蛋白（α α RGD 572-574），然后是分泌的自体纤维连接蛋白并结合到细胞外基质中。这些发现目前在血栓形成研究中处于“出版”状态。此外，还发现血浆激肽原不仅是一种粘附分子，而且是一种抑制剂。这些相反的效应被描述并发表（J Biochem 124: 473-484, 1998）。少

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