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: 08407034
• 负责人: MATSUDA Michio
• 金额: $ 15.23万
• 依托单位: Jichi Medical School, School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08407034/
• 关键词: hereditary dysfibrinogen; thromboembolism; electron microscopy; fibrin; adhesion molecule; beta1-integrin; kininogen; キニノゲン; フィブリノゲン; 血栓症; 出血性素因; インテグリン; 腫瘍の転移; 分子異常; フィブリンの電顕像; フィブリン重合; 一過性虚血発作; 接着分子受容体; RGD配列

I.Analyses of hereditary dysfibnnogens : During the three year-term of studies supported by the Grant-in-Aid for Scientific Research, No. 08407034, we were able to analyze more than 10 abnormal fibrinogen molecules, some of which had been associated with clinical manifestations of either bleeding or thrombosis, or both. Fibrinogen (Fbg) Caracas II with a unique Aalpha Ser-434 to Asn substitution, to which an extra oligosaccharide is linked (J Biol Chem 266 : 11575-11581, 1991) was further studied by electron microscopy (EM). The Caracas II fibrin fibers are found to be loosely associated and fibrin gels appeared to consist of irregularly aligned fibrin bundles with scattering large caves and pores. The permeability experiment showed a high permeability rate as compared with normal fibrin gels (J Biol Chem 271 : 4946-4953, 1996). On the other hand, Fbg Marburg (truncation of 150 amino acids in the Aalpha-chain and partly linked with serum albumin) associated with severe postoperative bl … More eeding accompanied by successive recurrent thrombo-embolic complications was found to partly linked with one or two serum albumin molecules by EM as anticipated by SDS-PAGE.The fibrin gels are composed of extremely thin and highly branched fibrin fibers, forming extraordinarily compact gels. These fibrin clots account for thrombo-embolic complications together with bleeding due to delayed fibrin clot formation. Part of these data was published in BLOOD (91 : 3282-3288,1998), and the remainder is now in preparation for publication. Four other abnormal Fbg's were also characterized and published (Kurashiki : gamma Gly-268 to Glu, Blood 87 : 4686-4694, 1996 ; Kumamoto : Aalpha Arg-19 to Gly, Jpn J Thromb Hemost 8 : 382-392, 1997 ; Kamogawa : gammaArg-275 to Ser, Thromb Haemostas, in press ; Niigata : Bbeta Asn-160 to Ser with an extra oligosaccharide N-linked to Bbeta Asn-158 ; Blood, in press). In three other dysfibrinogens, we have also identified new types of point mutations, Fbg's Pretoria (gamma Cys-139 to Tyr) ; Tokyo V (gammaAla-327 to Thr) and Osaka VI (12 amino acid extension due to the stop codon TAA to AAA for Lys). Further analyses on these molecules are going on, and will be submitted for publication.II.Molecular mechanisms of fibrin (Fbn) to function as adhesion molecule : Fbg functions as adhesion molecule only after transition to fibrin. When human fibroblasts were cultured on a fibrin monolayer, the bi-integrin as well as the already known beta3-integrin was expressed on the cell surface, and spreading of cells progressed in an RGD-dependent manner (J Biol Chem 272 : 8824-8829, 1997). When human glioma cells were cultured, a two-step mode of spreading via the beta1 -integrin was noted : firstly with fibrin (Aalpha RGD 572-574) and then with the autologous fibronectin secreted and incorporated in the extra-cellular matrix. These findings are now in the status of"in press" in Thrombosis Research. Furthermore, plasma kininogen was found to behave not only as an adhesion molecule but also as an inhibitor. These opposing effects were characterized and published (J Biochem 124 : 473-484, 1998). Less

I.遗传性纤维蛋白原异常的分析：在科学研究补助金（第08407034号）支持的三年研究期间，我们分析了10多种异常纤维蛋白原分子，其中一些与出血或血栓形成或两者的临床表现有关。用电子显微镜（EM）进一步研究了纤维蛋白原（Fbg）加拉加斯II，其具有独特的A α Ser-434到Asn的取代，并与额外的寡糖连接（J Biol Chem 266：11575-11581，1991）。发现加拉加斯II纤维蛋白纤维松散相关，纤维蛋白凝胶似乎由不规则排列的纤维蛋白束组成，并有分散的大洞穴和孔隙。渗透性实验表明，与正常的纤维蛋白凝胶相比，渗透率高（J Biol Chem 271：4946-4953，1996）。另一方面，Fbg马尔堡（A α链150个氨基酸的截短，部分与血清白蛋白连接）与严重的术后出血有关。 ...更多信息 电镜观察发现，伴随血栓栓塞并发症反复发作的纤维蛋白凝胶与一个或两个血清白蛋白分子部分相连，纤维蛋白凝胶由极细的高度分支的纤维蛋白纤维组成，形成非常致密的凝胶。这些纤维蛋白凝块导致血栓栓塞并发症以及由于延迟纤维蛋白凝块形成引起的出血。这些数据的一部分发表在《血液》（91：3282- 3288，1998）上，其余的正在准备发表。其他四个异常的纤维蛋白原的特点和出版（仓敷：γ Gly-268 to Glu，Blood 87：4686-4694，1996 ;熊本：A α Arg-19 to Gly，Jpn J Thromb Hemost 8：382-392，1997 ; Kamogawa：γ Arg-275 to Ser，Thromb Haemostas，in press ;新泻：Bbeta Asn-160到Ser，具有额外的N-连接到Bbeta Asn-158的寡糖;血液，出版中）。在其他三种纤维蛋白原异常中，我们也发现了新类型的点突变，Fbg的比勒陀利亚（γ Cys-139变为Tyr）;东京V（γ Ala-327变为Thr）和大坂VI（由于终止密码子TAA变为Lys的AAA而导致的12个氨基酸延伸）。对这些分子的进一步分析正在进行中，并将提交出版。II.纤维蛋白（Fbn）作为粘附分子的分子机制：Fbg只有在转变为纤维蛋白后才作为粘附分子发挥作用。当在纤维蛋白单层上培养人成纤维细胞时，双整联蛋白以及已知的β 3-整联蛋白在细胞表面上表达，并且细胞的扩散以依赖于RGD的方式进行（J Biol Chem 272：8824-8829，1997）。当培养人神经胶质瘤细胞时，注意到通过β 1-整联蛋白扩散的两步模式：首先是纤维蛋白（A α RGD 572-574），然后是分泌并掺入细胞外基质的自体纤连蛋白。这些发现目前在血栓形成研究中处于“印刷”状态。此外，血浆激肽原被发现不仅表现为粘附分子，但也作为一种抑制剂。这些相反的作用被表征并发表（J Biochem 124：473-484，1998）。少

项目成果

YASUDA, Toyotoshi: "Fibrinolytic components in nasal mucosa and nasal secretion." Histochem. Cell Biol.110. 449-455 (1998)

安田丰俊：“鼻粘膜和鼻分泌物中的纤溶成分。”

MUTO, Terukazu: "Factor XIII supplement therapy-effects on disturbances of wound healing." Med.Progr.10. 16-19 (1997)

MUTO、Terukazu：“因子 XIII 补充疗法 - 对伤口愈合障碍的影响。”

SUGO, Teruko: "Factor XIIIa-cross-linking of the Marburg Fibrin : Formation of αm・γ n-heteromultimers and the α-chain-linked albumin・γ complex, and disturbed protofibril assembly resulting in acquisition of plasmin-resistance relevant to thrombophilia." B

SUGO，Teruko：“马尔堡纤维蛋白的 XIIIa 因子交联：αm·γ n-异多聚体和 α-链连接的白蛋白·γ 复合物的形成，以及原纤维组装的紊乱，导致获得与血栓形成倾向相关的纤溶酶抗性.“B

Funayama H,Sakata Y,Kitagawa S,Ikeda Y,Takahashi M,Masuyama J,Mimuro J,Matsuda M,Shimada K: "Monocytes modulates the fibrinolytic balance of endothelial cells." Thromb Res. 85. 377-385 (1997)

Funayama H，Sakata Y，Kitakawa S，Ikeda Y，Takahashi M，Masuyama J，Mimuro J，Matsuda M，Shimada K：“单核细胞调节内皮细胞的纤溶平衡。”

TAMAGUCHI,Shu-ichi: "Fibrinogen Kumamoto with an Aα Arg-19 to Gly substitution has reduced affinity for thrombin : Possible relevance to thrombosis." Jpn.J.Thromb.Hemost.8(5). 382-392 (1997)

TAMAGUCHI, Shu-ichi：“将 Aα Arg-19 替换为 Gly 的纤维蛋白原降低了对凝血酶的亲和力：可能与血栓形成相关。”Jpn.J.Thromb.Hemost.8(5) (1997)。