Activating mechanisms of platelet GPIIb-IIIa complex by the interaction of intracellular proteins

细胞内蛋白相互作用激活血小板 GPIIb-IIIa 复合物的机制

• 批准号: 11671001
• 负责人: FUJIMOTO Tetsuro
• 金额: $ 2.05万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671001/
• 关键词: platelets; GPIIb-IIIa complex; affinity modulation; β3-endonexin; CD98; IAP; two hybrid system; FBLP-1; 細胞内ドメイン; フィラミン; インテグリン; GP IIb-IIIa複合体; 情報伝達; MAPキナーゼ; GPIIb-IIIの複合体; affinity modulation; MAP-キナーゼ

In order to determine the activating mechanisms of platelet GPIIb-IIIa complex, the role of several associated proteins was analyzed. β3-endonexin activated GPIIb-IIIa complex. However it did not effectively localize near the plasma membrane since it was a nuclear protein. CD98 activated GPIIb-IIIa complex during platelet and cell adhesion. IAP strongly activated GPIIb-IIIa complex and induced soluble ligand bindings only when it interacted with C-terminal domain of thrombospondin. Several cytoskeletal proteins including talin and filamin influenced the function of GPIIb-IIIa complex. To know these downstream pathways, proteins that bind to filamins were identified by yeast two hybrid screening. One clone was a novel cDNA, which encoded 375 amino acids and 45kDa protein. Deduced amino acid sequence showed that it contained a proline-rich domain at its N-terminal half and two LIM domains at C-terminus. We therefore, named it as FBLP-1 (Filamin Binding LIM Protein-1). Subcellular localization analysis showed that FBLP-1 co-localized with stress fibers, which stretched from focal adhesions containing GPIIb-IIIa complex. The cells, which over expressed FBLP-1, spread more effectively than the cells which did not express FBLP-1, suggesting that FBLP-1 plays a significant role on platelet adhesion. Further investigation of these associated proteins would clarify the activating mechanisms of platelet GPIIb-IIIa complex.

为了确定血小板GPIIb-IIIa复合体的激活机制，分析了几种相关蛋白的作用。β3-内切蛋白激活了GPIIb-IIIa复合体。然而，由于它是一种核蛋白，所以不能有效地定位在质膜附近。CD98在血小板与细胞黏附过程中激活了GPIIb-IIIa复合体。IAP仅在与凝血酶原蛋白C端区相互作用时，才能强烈激活GPIIb-IIIa复合体，并诱导可溶性配体结合。几种细胞骨架蛋白，包括Talin和细丝蛋白，影响了GPIIb-IIIa复合体的功能。为了了解这些下游途径，通过酵母双杂交筛选鉴定了与丝蛋白结合的蛋白质。其中一个克隆为新的cDNA，编码375个氨基酸和45 kDa的蛋白质。推导的氨基酸序列表明，它在N-末端含有一个富含Pro的结构域，在C-末端含有两个LIM结构域。因此，我们将其命名为FBLP-1(微丝结合LIM蛋白-1)。亚细胞定位分析表明，FBLP-1与含有GPIIb-IIIa复合体的局灶性粘连中的应力纤维共定位。高表达FBLP-1的细胞比不表达FBLP-1的细胞更有效地扩散，提示FBLP-1在血小板黏附中起重要作用。对这些相关蛋白的进一步研究将有助于阐明血小板GPIIb-IIIa复合体的激活机制。

项目成果

Kawano, H. et al.: "Down-regulation and redistribution of GPV/GPVf2, a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin-stimulated human platelets"Br. J. Haematol.. 104. 55-63 (1999)

Kawano, H. 等人：“GPV/GPVf2（血管性血友病因子受体（GPIb/IX/V 复合物）的一个亚基）在凝血酶刺激的人血小板表面膜上的下调和重新分布”Br。

Kawano H., et al.: "Down-regulation and redistribution of GPV/GVPf2 a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin-stimulated human plateles."Br. J. Haematol.. 104. 55-63 (1999)

Kawano H.等人：“在凝血酶刺激的人血小板表面膜上，冯维勒布兰德因子受体（GPIb/IX/V复合物）的亚基GPV/GVPf2的下调和重新分布。”Br。

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