Molecular Mechanism of Platelet Exocytosis

Platelet exocytosis is integral to hemostasis and vascular integrity. Conversely, inhibiting exocytosis is an important target for preventing improper thrombosis, which can ultimately lead to strokes and heart attacks. Understanding the molecular events required for platelet exocytosis will broaden our ability to control platelet activity. In the initial funding period it was proposed that membrane proteins in granule and open canalicular system membranes (OCS) mediate platelet secretion. From that work, it is now clear that specific Soluble NSF Attachment Protein Receptors (v- and t-SNAREs) are required for membrane fusion in each of the three, platelet exocytosis events. The original hypothesis is now expanded to address how this platelet exocytosis machinery is regulated. The hypothesis of this proposal is that exocytosis by stimulated platelets is governed by specific proteins that control SNARE-SNARE interactions. Several potential SNARE regulators have been identified in platelets. Using the in vitro platelet exocytosis assay (developed during the previous period). it is now possible to determine how these potential regulators function during platelet activation. Two specific aims are proposed: 1. To determine the role that v-SNARE proteins play in platelet exocytosis. The ultimate goal is to produce a complete description of the SNARE proteins required for platelet exocytosis. While several platelet t-SNAREs have been identified and functionally defined, the platelet v-SNAREs are yet to be characterized. This aim focuses on the role of platelet v-SNAREs. These studies together with previous work will yield a comprehensive picture of all the molecular machinery required for the final step of the release reaction. 2. To determine the role of t-SNARE regulatory proteins in the platelet release reaction. The goal is to determine how SNARE interactions are controlled during platelet exocytosis. The in vitro release assay will be used to determine which t-SNARE regulators are functional in platelets and how their activity affects SNARE complex formation and function. The studies of this aim will begin to elucidate the proteins and molecular interactions that are required to promote membrane fusion in activated platelets.