Molecular Mechanism of Platelet Dense Granule Biogenesis

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DESCRIPTION (provided by applicant): Platelets play pivotal roles in both hemostasis and thrombosis. Platelet activation triggers secretion and the release of content from dense granules, a-granules, and lysosomes that in turn leads to the recruitment and aggregation of additional platelets and white cells. While impaired platelet function has been associated with disorders that manifest with moderate to severe mucocutaneous bleeding, excessive platelet aggregation is a major cause of morbidity and mortality due to its effect in myocardial infarction and stroke. In spite of the relevance of platelet dense granules for human health, little is known about their biogenesis. Therefore, our goal is to understand the molecular mechanism responsible for the biogenesis of platelet dense granules. Dense granules belong to a group of lysosome-related organelles (LROs). Formation of LROs involves two parallel protein transport pathways defined by Adaptor Protein-3 (AP-3) and Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2). AP-3 is an adaptor that selects proteins with specific targeting signals in early endosomes and packages them into vesicles for transport to LROs. BLOC-2 also localizes to early endosomes but its function is unknown. We have recently obtained preliminary evidence suggesting that BLOC-2 has adaptor-like properties but with the ability to bind new targeting signals in dense granule proteins, different from the signals recognized by AP-3. Moreover, we obtained substantial preliminary results indicating that five proteins are fundamental components and new players in the pathways to dense granules: two "molecular switches", two novel proteins containing vesicle scission domains, and a molecular motor. These findings have opened new avenues to study the biogenesis of platelet dense granules. We propose to: (1) establish new in vitro and in vivo systems to study the biology of dense granules, (2) test the hypothesis that new dense granule targeting signals exist in dense granule proteins and that BLOC-2 is an adaptor that recognizes these signals and packages the corresponding proteins into vesicles destined for dense granules; (3) test the hypothesis that tissue specific "molecular switch" proteins recruit AP-3, BLOC-2, and other ubiquitous components to endosomal membranes to specifically direct transport to dense granules; (4) test the hypothesis that new vesicle scission and molecular motor proteins mediate the formation and transport of vesicles loaded with dense granule membrane proteins to dense granules; and (5) test the possibility that numerous patients that present in the clinic with platelet type bleeding disease of unknown etiology may have deficiencies in these new molecular switches, scission, and molecular motor proteins involved in dense granule biogenesis.