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

我们试图通过表达克隆策略来寻找一种针对血小板GPIIb-IIIa复合体的细胞内激活因子。以巨核细胞系CMK细胞为材料，经促血小板生成素处理后，构建cDNA文库。将其连接到表达载体pBK-EF或PREP上，分别转入CHO或Namalwa细胞，稳定表达GPIIb-IIIa复合体。将细胞与FITC标记的纯化纤维蛋白原孵育，通过细胞分选获得具有高亲和力的GPIIb-IIIa细胞。用Hirt‘s上清液从这些细胞中回收质粒，然后再将其导入细胞。几轮分泌后，转基因细胞的荧光强度没有明显变化。我们对回收的几个克隆进行了核苷酸序列分析，发现了一个有趣的克隆，它被发现是一种新的选择性剪接形式β3-endonexin，它是最近被报道的与GPIIIa的细胞质结构域相关的分子。当该克隆被转染时，观察到抗GPIIb-IIIa活性形式的抗体PAC-1的结合增加。同时，我们从骨髓巨核细胞中提纯了少量的mRNA，并通过固相RT-PCR获得了该基因，并将其用于另一轮相同策略的克隆。然而，最终没有获得阳性克隆。GPIIb-IIIa复合体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。

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.