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

1.凝血相关血浆蛋白遗传异常及其调控分析正如去年初步报道的那样，我们已经完成了对四种在伽马链上有点突变的异常纤维蛋白原的基因分析(见参考文献。16)。我们还发现了两种异常纤维蛋白原AA链上的新类型的点突变，这些异常纤维蛋白原来自委内瑞拉，纤维蛋白原加拉加斯II(参考文献2)。11)和秘鲁，纤维蛋白原利马(参考文献11)17)。非常有趣的是，这两个突变的纤维蛋白原都被发现与双天线寡糖相连，其中大多数已经被二唾液酸化，这是由于各自的点突变产生了新的ASN-X-Thr/Ser糖基化序列。由于这些结构变化，它们不能像正常分子那样迅速形成固体凝胶。然而，它们都以正常的方式增强了t-PA催化的纤溶酶原激活，表明最初的双链纤维蛋白原…更多的ILS已经正常建造。此外，有关纤维蛋白原京都II、Ise和大阪IV中发现的其他三种突变的论文已发表在参考文献中。分别为12、14和15。在纤维蛋白原大阪IV中，已经讨论了与手术相关的临床方面。通过在这些异常分子中观察到的结构变化与功能异常的密切联系，我们能够在分子水平上获得关于纤维蛋白形成机制的新证据。在这项助学金的支持下，我们利用聚合酶链式反应在异常蛋白C Yonago的所谓GLA区域发现了一种新型的精氨酸-15(CGG)到甘氨酸(IgG)的结构变化。这一区域已被证明是关键的引发钙依赖的构象需要与磷脂相互作用激活到一种酶，激活蛋白C(参考。18)。这项工作是与鸟取大学医学部的中村教授合作进行的。血管内皮细胞与凝血相关蛋白相互作用的研究：我们提供的证据表明，在维生素K存在的情况下，培养的血管内皮细胞合成了被认为仅在肝细胞中合成的蛋白C。这一结论是由于蛋白C分子及其mRNA的时间依赖性增加。13)。这一新信息可能暗示在由于血液循环停止而不能保证血液供应的部位存在调节血栓形成的机制。对于这些实验，成功地利用了本研究中由该赠款支持的各种多克隆和单克隆抗体。较少

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