PreDoctoral Fellowship for Joshi: The Role of Vesicle- Associated Membrane Proteins (VAMPs) in Differential Platelet Exocytosis and Granule Biogenesis

Platelets play a key role in the vasculature, by regulating hemostasis and thrombosis. Upon vascular injury, platelets adhere to the injured site and release their granular contents from dense granules, alpha granules, and lysosomes. This release is mediated by the Soluble N- ethylmaleimide sensitive factor attachment protein receptors (SNARE) complex. Previously, we showed that a v-SNARE, VAMP-8, is the primary v-SNARE and VAMP-2 and -3 play ancillary roles. This heterogeneous usage of VAMPs may mediate subtle fusion processes that contribute to distinct spatial and temporal release of cargo contents influencing vascular environment. Though crucial for understanding of cardiovascular environment, the heterogeneity in VAMP usage has not been fully investigated. Preliminary data suggest that VAMP-2/3-/- platelets have a mild secretion defect and VAMP-2/3/8-/- platelets have a robust secretion deficiency. Moreover, both endocytosed and de novo synthesized granule cargo levels are affected in these strains. However, it is not clear how the genetic ablation of one or more VAMPs changes the dynamics of SNARE- mediated fusion, granule cargo biogenesis and trafficking in platelets. Here we propose to characterize the roles of platelet VAMPs by examining their localization, their interaction with other SNAREs, and their importance to each of the three granule release events. To achieve these goals, we will use secretion assays, lumi-aggregometry, FACS, electron microscopy, antibody array analysis, and various in vivo assays for platelet cargo proteins. When completed, these studies will define the specific roles of each VAMP in platelet secretion, in granule biogenesis and ultimately in platelet function.