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

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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.