Expression cloning of an intracellular activating factor for platelet GPIIb-IIIa complex

血小板 GPIIb-IIIa 复合物细胞内激活因子的表达克隆

• 批准号: 08671236
• 负责人: FUJIMOTO Tetsuro
• 金额: $ 1.47万
• 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671236/
• 关键词: platelets; GPIIb-IIIa complex; affinity modulation; cytoplasmic domain; expression cloning; beta 3-endonexin; fibrinogen; GPIIb-IIIa複合体

We have tried to identify an intracellular activating factor for the platelet GPIIb-IIIa complex by expression cloning strategy. The cDNA library was made from megakaryocytic CMK cells which were treated by thrombopoietin. The cDNA were ligated to the expression vector, pBK-EF or pREP,which were then transfected into CHO or Namalwa cells, expressing the GPIIb-IIIa complex stablly. The cells were incubated with FITC-labeled purified fibrinogen, and the cells which had the high-affinity-state of GPIIb-IIIa were collected by cell sorting. Plasmids were recollected by Hirt's supernatant from these cells, and then transfected again to the cells. After the several rounds of the secreening, the intensity of fluorescence of the transfected cells did not change markedly. We performed nucleotide sequencing of the several recovered clones, and identified one interesting clone which was was revealed as a novel alternative spliced form beta 3-endonexin, which was recently reported as an associated molecule with the cytoplasmic domain of GPIIIa. When this clone was transfected, the increase of PAC-1 binding, which is an antibody for the active form of GPIIb-IIIa, was observed. Simultaneously, we purified small amount of mRNA from bone marrow megakaryocytes and obtained the cDNA by solid-phase RT-PCR.The cDNA was used for another cloning rounds of the same strategy. However, positive clones were not finally obtained. The activating mechanism of GPIIb-IIIa complex may not simple, and several molecules might be involved in these signal transducing pathway.

我们试图通过表达克隆策略鉴定血小板GPIIb-IIIa复合物的细胞内活化因子。cDNA文库由血小板生成素处理的巨核细胞CMK细胞制备。将cDNA与表达载体pBK EF或pREP连接，转染CHO或Namalwa细胞，稳定表达GPIIb IIIa复合物。将细胞与FITC标记的纯化纤维蛋白原孵育，并通过细胞分选收集具有GPIIb-IIIa高亲和力状态的细胞。用Hirt的上清液从这些细胞中回收质粒，然后再次转染到细胞中。经几轮分泌后，转染细胞的荧光强度无明显变化。我们对几个回收的克隆进行了核苷酸测序，并鉴定了一个有趣的克隆，该克隆被揭示为一种新的可变剪接形式β 3-内切蛋白，其最近被报道为与GPIIIa的胞质结构域相关的分子。当转染该克隆时，观察到PAC-1结合的增加，PAC-1是GPIIb-IIIa活性形式的抗体。同时，从骨髓巨核细胞中提取少量mRNA，用固相RT-PCR方法获得cDNA，并将其用于相同策略的第二轮克隆。然而，最终没有获得阳性克隆。GPIIb-IIIa复合物的激活机制可能并不简单，可能有多个分子参与了这些信号转导途径。

项目成果

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