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

血栓栓塞性疾病的发病机制和病理生理学——从分子和基因水平分析血液凝固机制及其调控。

• 批准号: 02454311
• 负责人: MATSUDA Michio
• 金额: $ 4.42万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454311/
• 关键词: Molecular abnormality,; Abnormal fibrinogen,; Protein C,; Monoclonal antibodies,; Thromboembolic disease,; Thrombus formation; Vascular endothelial cells,; Regulation of thrombus formation; 血栓症; 遺伝性分子異常症; 異常プロテインC; アミノ酸置換; 遺伝子解析

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

1. 血浆凝血相关蛋白基因异常分析及其调控。正如在去年的初步报告中所报道的，我们已经完成了对四种异常纤维蛋白原的基因分析，这些纤维蛋白原在γ链上存在点突变（见参考文献16）。我们还发现了两种异常纤维蛋白原的Aa链上的新型点突变，这些突变与纤维蛋白凝块形成受损有关，这些纤维蛋白原来自委内瑞拉。11)，秘鲁，纤维蛋白原利马（参考文献）。17)。非常有趣的是，这两种突变纤维蛋白原都被发现与双天线寡糖相连，其中大多数已被二化，这是由于各自的点突变新产生的Asn-X-Thr/Ser的糖基化序列。由于这些结构上的改变，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催化的纤溶酶原的激活，这表明最初的两股纤维蛋白原纤维已经正常构建。除此之外，在纤维蛋白原Kyoto II、Ise和Osaka IV中发现的其他三种类型的突变的论文分别发表在参考文献12、14和15中。在大阪纤维蛋白原IV中，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改变与功能异常密切相关，我们能够在分子水平上提供关于纤维蛋白形成机制的新证据。在这项资助资助的研究中，我们利用聚合酶链反应在异常蛋白C - Yonago的所谓Gla区发现了一种新型的精氨酸-15（CGG）到甘氨酸（iGG）的结构改变。该区域已被证明是引发与磷脂相互作用所需的钙依赖性构象的关键，以激活酶，活化蛋白C（参考文献）。18)。这项工作是与鸟取大学医学院Nakamura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，一直被认为只在肝细胞中合成的蛋白C，在维生素k存在的情况下，在培养的血管内皮细胞中合成了蛋白C分子及其MRNA的时间依赖性增加(Ref。13)。这一新的信息可能意味着在血液循环停止而没有血液供应保证的位点存在一种调节血栓形成的机制。在这些实验中，本研究中制备并鉴定的多种多克隆和单克隆抗体已成功应用于本研究。少

项目成果

Hisato MAEKAWA: "An Aα Ser-434 to N-glycosylated Asn substitution in a dysfibrinogen,fibrinogen Caracas II,characterized by impaired fibrin gel formation." J.Biol.Chem.266. 11575-11581 (1991)

Hisato MAEKAWA：“纤维蛋白原 Caracas II 中的 Aα Ser-434 被 N-糖基化 Asn 取代，其特征是纤维蛋白凝胶形成受损。”J.Biol.Chem.266（1991）。

Teruko Sugo: "Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (Des-gamma-carboxy) prothrombin." J. Biochem.108. 382-387 (1990)

Teruko Sugo：“对凝血酶原中的 γ-羧基谷氨酸残基进行化学修饰会引发与异常（脱γ-羧基）凝血酶原相似的构象。”

Kensuke YAMAZUMI: "Fibrinogen Osaka IV:A congenital dysfibrinogenemia found in a patient originally reported in reration to surgery,now defined to have an A_α arginineー16 to histidine substitution." Jpn.J.Surg.

Kensuke YAMAZUMI：“纤维蛋白原 Osaka IV：最初报道的与手术有关的患者发现的先天性纤维蛋白原异常血症，现在被定义为 A_α 精氨酸 16 被组氨酸取代。”

Shinji ASAKURA: "Fibrinogen Sapporo:Disfibrinogenemia characterized by the replacement of Aα arginine-16 by histidine resulting the delayed release of fibrinopeptide A by thrombin." Acta Haematol.Jpn.52. 130-140 (1989)

Shinji Asakura：“纤维蛋白原札幌：去纤维蛋白原血症的特征是 Aα 精氨酸-16 被组氨酸取代，导致凝血酶延迟释放纤维蛋白肽 A。Acta Haematol.Jpn.52 (1989)。”

Shinji ASAKURA: "Hydrophobic residues 382-386 of antithrombin III,Ala-Ala-Ala-Ser-Thr,serve as the epitope for an antibody which facilitates hydrolysis of the inhibitor by thrombin." J.Biol.Chem.265. 5135-5138 (1990)

Shinji ASAKURA：“抗凝血酶 III 的疏水残基 382-386，Ala-Ala-Ala-Ser-Thr，充当抗体的表位，促进凝血酶水解抑制剂。”