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

血栓形成的病因学和病理生理学：一种分子生物学方法，用于阐明凝血及其抑制的紊乱机制。

• 批准号: 06404043
• 负责人: MATSUDA Michio
• 金额: $ 11.2万
• 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 (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06404043/
• 关键词: Fibrinogen; Hereditary dysfibrinogen; Fibrin polymerization; Crosslinking; Calcium-dependent structure; Monoclonal antibody; Cell attachment; Adhesion molecule; モノクローナル抗体; 遺伝性フィブリノゲン異常症; 出血; 血栓; 創傷治癒不全; 接着分子のレセプター

1.Structure analysis of hereditary dysfibrinogens. Two hitherto unknown amino acid substitutions have been identified, i.e., gammaGly-268 to Glu in a homozygous dysfibrinogen Kurashiki I (Blood, in press) and gammaArg-275 to Ser in a heterozygous dysfibrinogen Kamogawa (manuscript in preparation) by amino acid sequence and gene analyzes. Both dysfibrinogens have been found to have a dysfunctional D : D self-association site, a recently proposed fibrin polymerization site and reported to be defective in fibrinogen Tokyo II (J.Clin.Invest.90 : 1053,1995). Thus, both dysfibrinogens manifest impaired fibrin protofibril formation. Although not an entirely new type of amino acid substitution, an AalphaArg-19 to Gly substitution has been identified in fibrinogen Kumamoto associated with thrombosis. Since affinity for thrombin is significantly low in Kumamoto fibrin as compared with normal fibrin, the observed defective localization of thrombin onto fibrin clots appears to be related to the oc … More currence of thrombosis (manuscript in preparation). Besides these, eight dysfibrinogen samples have been under investigation including one each from Israel, Germany and Venezuela, and five from inside of our country.2.Preparation of anti-fibrinogen monoclonal antibodies (mAb's) and their application to the study of basic and clinical aspects of fibrinogen. Three unique mAb's have been produced. (1) A mAB recognizing the calcium-dependent structure of the D domain of fibrinogen has been established. Utilizing this mAb as a ligand for immuno-affinity chromatography, fibrinogen has been highly purified even from limited amounts of plasma samples of abnormal fibrinogens including fibrinogen Marburg (Thromb.Haemostas.73 : 662-667,1995). (2) One mAb was found to completely inhibit factor XIIIa-catalyzed crosslinking of the fibrin gamma-chains as well as fibrin monomer polymerization. This mAb served as powerful tool to study the possible involvement of the gamma-chain crosslinking in fibrin polymerization (in preparation). (3) Another one is a mAb that recognizes a specific conformation of the E domain of fibrin monomer elicited upon its binding with fibrinogen. This mAb has been successfully utilized for the study of the mechanisms of fibrin monomer polymerization and their relevance to the occurrence of thrombosis (Blood, in press).Localization of vitronectin-and fibronectin-receptors on cultured human glioma cells. On the surface of glioma cells cultured on vitronectin and fibronectin, distribution of respective integrins was found to vary upon contact with and during culture on the adhesion molecules. Participation of high-molecular weight kininogen in the attachment and spreading of cells on the ligands has also been studied (J.Biol.Chem., in press). Less

1.遗传性纤维蛋白原异常的结构分析。已经鉴定了两个迄今未知的氨基酸取代，即，通过氨基酸序列和基因分析，在纯合子纤维蛋白原异常仓敷I（Blood，出版中）中γ Gly-268变为Glu，在杂合子纤维蛋白原异常Kamogawa（手稿在准备中）中γ Arg-275变为Ser。已发现两种异常纤维蛋白原都具有功能障碍的D：D自结合位点，这是最近提出的纤维蛋白聚合位点，并且据报道在纤维蛋白原Tokyo II中是有缺陷的（J.Clin.Invest.90：1053，1995）。因此，两种纤维蛋白原障碍都表现为纤维蛋白原纤维形成受损。虽然不是一种全新类型的氨基酸取代，但在熊本纤维蛋白原中已鉴定出与血栓形成相关的A α Arg-19至Gly取代。由于与正常纤维蛋白相比，熊本纤维蛋白对凝血酶的亲和力明显较低，因此观察到的凝血酶在纤维蛋白凝块上的定位缺陷似乎与凝血酶的亲和力有关。 ...更多信息 血栓形成的存在（手稿正在编写中）。除此之外，还有8个纤维蛋白原异常样本正在接受调查，其中以色列、德国和委内瑞拉各1个，我国境内5个。2.抗纤维蛋白原单克隆抗体（mAb）的制备及其在纤维蛋白原基础和临床研究中的应用。已经产生了三种独特的mAb。(1)已经建立了识别纤维蛋白原D结构域的钙依赖性结构的单克隆抗体。利用这种单克隆抗体作为免疫亲和层析的配体，甚至从包括纤维蛋白原马尔堡（Marburg）在内的少量异常纤维蛋白原的血浆样品中高度纯化了纤维蛋白原（Thromb.Haemostas.73：662- 667，1995）。(2)发现一种mAb完全抑制因子XIIIa催化的纤维蛋白γ链交联以及纤维蛋白单体聚合。该mAb作为研究γ链交联可能参与纤维蛋白聚合（制备中）的有力工具。(3)另一种是识别纤维蛋白单体的E结构域的特异性构象的mAb，所述E结构域在其与纤维蛋白原结合时引起。该mAb已成功用于研究纤维蛋白单体聚合的机制及其与血栓形成发生的相关性（Blood，in press）.Localization of vitronectin-and fibronectin-receptors on cultured human glioma cells.在玻连蛋白和纤连蛋白上培养的胶质瘤细胞的表面上，发现在与粘附分子接触时和在粘附分子上培养期间，各个整合素的分布不同。还研究了高分子量激肽原参与细胞在配体上的附着和铺展（J.Biol.Chem.，印刷中）。少

项目成果

Takebe, M., Soe, G., Kohno, I., Sugo, T.and Matsuda, M.: "Calcium-dependent monoclonal antibody against human fibrinogen : preparation, characterization, and application to fibrinogen purification" Thromb.Haemost.73 (4). 662-667 (1995)

Takebe, M.、Soe, G.、Kohno, I.、Sugo, T. 和 Matsuda, M.：“针对人纤维蛋白原的钙依赖性单克隆抗体：制备、表征和在纤维蛋白原纯化中的应用” Thromb.Haemost.73

Soe, G., Kohno, I., Inuzuka, K.and Matsuda, M.: "A rapid latex immunoassay for the detection of plasmin-alpha2-plasmin inhibitor complex : Utilization of two monoclonal antibodies differentially recognizing respective components of the complex." Blood Coa

Soe, G.、Kohno, I.、Inuzuka, K. 和 Matsuda, M.：“用于检测纤溶酶-α2-纤溶酶抑制剂复合物的快速乳胶免疫测定法：利用两种单克隆抗体差异识别复合物的各个成分。

Sugo, T.: "Calcium ion binding to vitamin K-dependent clotting factors." Jpn.J.Thromb.Hemost.6 (4). 252-264 (1995)

Sugo, T.：“钙离子与维生素 K 依赖性凝血因子结合。”

Kazuki Niwa: "AγGly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki l." Blood. (in press).

Kazuki Niwa：“AγGly-268 替换为 Glu 是导致纯合异常纤维蛋白原 Kurashiki l 血液中纤维蛋白组装受损的原因。”

Nobuhiko Yoshida: "Effect calcium on the mobility of γ-chain from fibrinogen Osaka V on sodium dodecyl sulfate-polyacrylamide gel electrophoresis." Thromb.Res.73(1). 79-82 (1994)

Nobuhiko Yoshida：“在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上影响钙对纤维蛋白原 Osaka V 的 γ 链的迁移率。”Thromb.Res.73(1) (1994)。