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

我们尝试通过表达克隆策略鉴定血小板 GPIIb-IIIa 复合物的细胞内激活因子。 cDNA文库由经过血小板生成素处理的巨核细胞CMK细胞制成。将cDNA连接到表达载体pBK-EF或pREP上，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一起孵育，并通过细胞分选收集具有高亲和力状态的GPIIb-IIIa的细胞。通过Hirt上清液从这些细胞中重新收集质粒，然后再次转染至细胞中。经过几轮筛选后，转染细胞的荧光强度没有明显变化。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，它被揭示为一种新的选择性剪接形式 β3-内毒素，最近报道它是与 GPIIIa 细胞质结构域相关的分子。当该克隆被转染时，观察到 PAC-1 结合的增加，PAC-1 是 GPIIb-IIIa 活性形式的抗体。同时，我们从骨髓巨核细胞中纯化了少量mRNA，并通过固相RT-PCR获得了cDNA。该cDNA用于相同策略的另一轮克隆。但最终并未获得阳性克隆。 GPIIb-IIIa复合物的激活机制可能并不简单，这些信号转导通路中可能涉及多个分子。

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.