Interaction of a SNARE-Associated Protein, Snapin with the Cytoplasmic Domain of the Platelet Membrane Glycoprotein V.

Glycoprotein V (GPV) is a subunit of the platelet GPIb-IX-V receptor for von Willebrand factor (vWF) and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in platelet activation is still unknown. It is reported that the GPV knockout mouse did not suffer from a bleeding syndrome, and the GPV-deficient platelets showed normal GPIb expression and vWF-mediated adhesion. One report showed an increased sensitivity of these platelets to activation by low concentrations of thrombin, suggesting that GPV may act as a negative modulator of the thrombin response. Another showed that GPV-deficient platelets exhibited defective adhesion to a collagen-coated surface, suggesting GPV binds to collagen and participates in platelet adhesion and aggregation responses to this agonist. To define the signaling pathway through GPV, we searched for proteins that bind to the GPV. We preformed a yeast two-hybrid screening in a human placenta library, using the sequence for GPV cytoplasmic domain as a bait. As a result, we isolated 2 independent positive clones, and the both clones encoded a partial and a full length sequence of the same protein, named snapin (SNAPAP), which was previously reported as one of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)-associated proteins. By RT-PCR analysis, snapin mRNA was detected in megakaryocyte cell lines and peripheral blood platelets. When the snapin was expressed as a GFP-fusin protein in K562 cells, which expressed intrinsic GPIb-IX-V complex, the protein localized at perinuclear region and cytosol. By immunoprecipitation-western blotting study, GPV was co-immunoprecipitated with GFP-snapin from the lysates of these cells by anti-GFP antibody, whereas GPV was not detected in the precipitates by a control antibody. Previous reports showed that snapin is a binding protein of SNAP-25 (synaptosome-associated protein-25) and SNAP-23, and influences the release response of neurons and chromaffin cells by enhancing the synaptotagmin binding to the SNARE complex. Therefore, the novel interaction between GPV and snapin, identified in this study, might be involved in the intracellular membrane-fusion events and the granule release reaction during the platelet activation.