Fellowship/Ren: Regulation of Platelet Exocytosis by Munc 13-4.

Central to hemostasis is the release of cargo from three platelet stores: dense-core granules, alpha granules, and lysosome. Exocytosis from these granules is mediated by integral membrane proteins called SNAREs (vand t-SNAREs) present in the granule and target membranes. These proteins form trans-bilayer fusion complexes that facilitate membrane fusion. Formation of these complexes is tightly controlled by SNARE binding proteins that regulate SNARE-SNARE binding and prime the machinery for fusion. Munc18s are thought to act as syntaxin (t-SNARE) chaperones and Munc13s are thought to control t-SNAREs and to promote the fusion competence of secretory granules. Our initial results suggest that Munc13-4 regulates platelet secretion, possibly by effecting interactions with platelet syntaxins. To understand how platelet secretion is regulated we will seek to understand the role of Munc13-4 in platelets. The guiding hypothesis of this work is that Munc13-4 plays a regulatory role in platelet exocytosis by controlling syntaxins' availability to bind other SNAREs. Munc13-4 primes the machinery to promote fusion competence in the platelet granules. We will use three specific aims to address this hypothesis. 1) To characterize the effects of Munc13-4 on platelet secretion in vivo. 2) To determine whether Munc13-4 has a priming function in platelet secretion. 3) To determine if Munc13-4 plays a role in syntaxin/Munc18 complex dissociation. To complete these aims we will study whether Munc13-4 affects syntaxin/Munc18, and syntaxin/SNAP-23NAMP complex formation. We will determine whether platelets contain a "primed" or ready releasable pool of granules and study Munc13-4's effect on this pool. Lastly, we will use recombinant syntaxin/Munc18 complexes to determine whether Munc13-4 and/or other platelet proteins playa role in their dissociation. Completion of these aims will yield great insight into the mechanisms by which platelet secretion is regulated and point to potential therapeutic targets that can be use to ameliorate platelet function and correct platelet dysfunction.