VAMP-7 Interacts With The Actin Cytoskeleton To Control Platelet Spreading and Mediate α-Granule Exocytosis During Thrombus Formation

Platelet granule secretion is important not only for hemostasis and thrombosis, but also for a variety of physiological processes including inflammation, angiogenesis and malignancy. Vesicle Associated Membrane Proteins (VAMPs) are a group of v-SNARE proteins resident on the platelet granule surface that participate in granule secretion. Platelets contain several VAMP isoforms including VAMP-2, VAMP-3, VAMP-7, and VAMP-8. VAMP-7 is unique in that it contains an N-terminal profilin-like longin domain. Previous work by our group demonstrated spatial segregation of granules expressing different VAMPs during platelet spreading. Granules expressing VAMP-3 and VAMP-8 localized to the granulomere of spreading platelets, while those expressing VAMP-7 moved towards the periphery. Based on this observation, we proposed that VAMP-7⁺ granules move to the periphery of the spreading platelet to add membrane to growing actin structures. To assess this hypothesis, platelets from VAMP-7 null mice were used to analyze the role of VAMP-7 in platelet spreading, aggregation and secretion. VAMP-7 null platelets were normal in size, shape, and number. When compared to wild-type platelets, VAMP-7 null platelets did not show any defects in aggregation upon exposure to increasing doses of the PAR4 agonist peptide, AYPGKF, or collagen. In contrast, the surface area of VAMP-7 null platelets following 15 min of spreading on poly-L-lysine was only 51% that of wild-type of platelets (P < 0.05). To assess mechanisms of the movement of VAMP-7 to the platelet periphery, the association of VAMP-7 to the Triton X-100-insoluble platelet cytoskeleton was evaluated and results showed that VAMP-7 associated with the actin cytoskeleton. Moreover, VAMP-7 null platelets showed impaired P-selectin surface expression and PF4 secretion at low concentrations of AYPGKF. TIMP-2 and VEGF localize to VAMP-7 expressing granules in the periphery of spread platelets. We therefore evaluated the secretion of TIMP-2 and VEGF from VAMP-7 null platelets. Secretion of TIMP-2 and VEGF was reduced even at saturating doses of agonist (300 mM AYPGKF). To examine the role of VAMP-7 in a-granule exocytosis during platelet activation in vivo, PF4 release was monitored following laser-induced injury of cremaster arterioles. Platelet accumulation at sites of laser injury was identical in wild-type and VAMP-7 null mice. In wild-type mice, PF4 was secreted by activated platelets and bound back to activated endothelium and platelets producing a localized concentration of PF4 that accumulated over 15 min following injury. PF4 release from platelets lacking VAMP-7 was decreased to 47% of that of control. These results demonstrate that VAMP-7 interacts with the actin cytoskeleton and functions selectively in a-granule exocytosis. VAMP-7 associates with the actin cytoskeleton and functions during platelet spreading, adding further support to the premise that membrane fusion occurring during granule secretion is an essential component of normal platelet spreading. This VAMP-7 mediated, actin-dependent mechanism of secretion is not important for platelet thrombus formation, but rather functions in the release of particular granular contents, such as PF4, at sites of vascular injury.