Involvement of SNARE proteins in thrombin-induced platelet aggregation: Evidence for the relevance of Ca2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca2+ mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca2+ entry, the main mechanism for Ca2+ influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca2+ entry for full activation. Depletion of the intracellular Ca2+ stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca2+ concentration reached a critical level (around 1.5 μM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca2+ mobilization in human platelets.