Studies on the etiology and pathophysiology of thrombosis : molecular biological approaches to the perturbed blood coagulation and its regulation.

1.遗传性凝血纤溶异常分子分析。1)遗传性纤维蛋白原异常血症：在1992 - 1993年期间，我们完成了对两种纤维蛋白原异常的研究，纤维蛋白原（Fbg）不莱梅（A α Gly-17至瓦尔）和Fbg Mitaka II（A α Glu-11至Gly）。在一名14岁德国男孩因手术出血和伤口愈合延迟而发现Fbg不莱梅，并将血样送到我们实验室进行结构分析。A α Gly-17替换为瓦尔提供了支持证据，即纤维蛋白α链的氨基末端三残基肽Gly-Pro-Arg构成了暴露于纤维蛋白原中心结构域的聚合位点"A"。通过利用具有正常或不莱梅型序列或其相关序列的合成肽，我们已经证明纤维蛋白α链氨基末端残基的游离氨基对于"A"位点的功能是最关键的，因为Gly替换为Ala或瓦尔仅表现出微小的功能。 关于我们 对其他氨基酸的作用未能显示聚合促进活性（公开号15）。Fbg Mitaka II的特征在于与凝血酶的结合缺陷。因此，Aalpha Glu-11似乎在与凝血酶的结合中起关键作用。实际上，最近X射线晶体学显示，该氨基酸的侧链羧基与凝血酶的Arg-173的侧链胍基形成盐桥，并且还稳定II型β-转角，该转角是纤维蛋白原装配到凝血酶的酶口袋中所必需的。我们的数据支持这一假设，并在血栓形成的研究中提供了重要的意义（出版物编号13和15）。2)在轻度血友病患者中发现的异常因子IX：通过基因分析，我们鉴定了编码因子IX的瓦尔-182的核苷酸20525处的T到C突变。该位点靠近切割位点2。细胞粘附研究：高分子量激肽原转化为双链分子（HKa）后，具有促进细胞粘附的活性。我们一直在分析这种现象的分子基础，利用电池的合成肽和单克隆抗体的支持下，这项研究fund.We还确定了特定的整合素培养的胶质瘤细胞接触玻连蛋白和纤维蛋白，这是正常情况下，但暴露在接触这些粘附分子。有趣的是，这些整联蛋白似乎随着细胞形状的变化而在细胞表面迁移（提交给Brain Research）。少

1. Analyzes of hereditary abnormal molecules of blood coagulation and fibrinolysis. 1) Hereditary dysfibrinogenemias : During 1992-1993, we have completed a study on two dysfibrinogens, fibrinogen (Fbg) Bremen (Aalpha Gly-17 to Val) and Fbg Mitaka II(Aalpha Glu-11 to Gly). Fbg Bremen had been found in a 14-year-old German boy with surgical bleeding and delayd wound healing, and the blood sample was sent to our laboratory for structure analysis. The Aalpha Gly-17 to Val substitution provided supporting evidence that the amino terminal three-residue peptide of fibrin alpha-chain, Gly-Pro-Arg, constitutes a polymerization site, "A" exposed onto the central domain of fibrinogen. By utilizing synthetic peptides with a normal or a Bremen type sequence, or their related sequences, we have shown that the free amino group of the amino-terminal residue of fibrin alpha-chain is most critical for the function of the "A" site, since the replacement of Gly to Ala or Val manifested only a minute func … More tional disturbance, whereas that to other amino acids failed to manifest polymerization-promoting activity (Publication No. 15). Fbg Mitaka II was characterized by defective binding with thrombin. Thus Aalpha Glu-11 appears to play a crucial role in the binding with thrombin. indeed, the side-chain carboxy group of this amino acid has recently been shown by X-ray crystallography to form a salt bridge with the side-chain guanidino group of Arg-173 of thrombin, and also to stabilize the type II beta-turn which is mandatory for fibrinogen to be fitted into the enzyme pocket of thrombin. Our data support this hypothesis, and provide important implications in the study of thrombus formation (publications No. 13 and 15). 2) An abnormal factor IX Tokyo I found in a mild hemophiliac : By gene analysis, we have identified a T to C mutation at nucleotide 20525 coding for Val-182 of factor IX.Thus the patient's factor IX must have an ala-182 substitute. This site is close to the cleavage site by2. Study on the cell adhesion : High-molecular-weight kininogen was found to manifest cell attachment promoting activity when converted to a two-chain molecule (HKa). We have been analyzing the molecular basis for this phenomenon by utilyzing a battery of synthetic peptides and monoclonal antibodies produced by the support of this research fund. We have also identified specific integrins on cultured glioma cells upon contact with vitronectin and fibrinonectin, which are normally absent but exposed upon contact with these adhesion molecules. Interestingly these integrins appear to migrate on the cell surface in accordance with the cell shape changes (submitted to Brain Research). Less