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

1. 凝血、纤溶遗传异常分子分析。1)遗传性纤维蛋白异常基因：在1992-1993年期间，我们完成了两种异常纤维蛋白原的研究，纤维蛋白原（Fbg） Bremen （α - Gly-17至Val）和Fbg Mitaka II（α - Glu-11至Gly）。Fbg Bremen曾在一名14岁的德国男孩身上发现手术出血和伤口愈合延迟，并将血液样本送到我们的实验室进行结构分析。α - Gly-17到Val的取代为纤维蛋白α链氨基末端三残基肽Gly-Pro-Arg构成了一个暴露在纤维蛋白原中心结构域的聚合位点“a”提供了支持证据。通过使用具有正常或不莱梅型序列的合成肽，或其相关序列，我们已经表明，纤维蛋白α链氨基末端残基的游离氨基对“a”位点的功能最关键，因为Gly被Ala或Val取代只表现出一分钟的功能紊乱，而其他氨基酸的替换未能表现出促进聚合的活性（出版物15）。Fbg Mitaka II的特点是与凝血酶结合缺陷。因此，α - Glu-11似乎在与凝血酶的结合中起着至关重要的作用。事实上，该氨基酸的侧链羧基最近已通过x射线晶体学显示与凝血酶Arg-173的侧链胍基形成盐桥，并稳定II型β -turn，这是纤维蛋白原被装入凝血酶袋所必需的。我们的数据支持这一假设，并为血栓形成的研究提供了重要的启示（出版物编号13和15）。2)在轻度血友病患者中发现异常因子IX Tokyo I：通过基因分析，我们在编码因子IX的Val-182的核苷酸20525处发现了T到C突变。因此病人的ala - 182 IX因子必须有一个替代品。该位点靠近卵裂位点2。细胞黏附的研究：发现高分子量激肽原转化为双链分子（HKa）时具有促进细胞黏附的活性。我们一直在分析这一现象的分子基础utilyzing电池生产的合成肽和单克隆抗体研究基金的支持。我们还在培养的胶质瘤细胞上发现了与玻璃体连接蛋白和纤维蛋白连接蛋白接触的特异性整合素，这些整合素通常不存在，但在与这些粘附分子接触时暴露出来。有趣的是，这些整合素似乎根据细胞形状的变化在细胞表面迁移（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