PROJECT SUMMARY Platelets play central roles in hemostasis and thrombosis. Platelet activation triggers secretion and release of contents from α-granules, δ-granules and lysosomes that in turn leads to the recruitment and aggregation of additional platelets and a myriad of physiological responses. While impaired platelet function is associated with disorders that manifest with moderate to severe bleeding, excessive platelet aggregation is a major cause of morbidity and mortality due to its effect in cardiovascular disease and stroke. Alpha-granules are crucial to these platelet functions both in health and disease. However, in spite of the relevance of platelet α-granules for human health, remarkably little is known about their biogenesis. Therefore, our goal is to understand the pathways and molecular mechanism responsible for the biogenesis of platelet α-granules. Mutations in VPS33B, VPS16B and NBEAL2 cause the α-granule deficiency and bleeding manifestations observed in patients suffering Arthrogryposis, Renal Dysfunction and Cholestasis (ARC) syndrome and Gray Platelet syndrome (GPS). However, the mechanism of action of these proteins in α-granule biogenesis is a mystery. Consequently, a major objective of this proposal is to address the function of VPS33B, VPS16B and NBEAL2 at the cellular and molecular level. Platelet α-granules are produced in the megakaryocyte, the platelet precursor cell. In addition to soluble proteins taken up by endocytosis, α-granules contain hundreds of proteins synthesized by the megakaryocyte. The pathways taken by megakaryocyte-synthesized proteins to reach the α-granule are unknown. In this proposal, we show that the sorting endosome is a fundamental precursor organelle in α-granule formation that is used by megakaryocyte-synthesized proteins. This implies a more complex biogenesis mechanism than previously anticipated. In this application we build on our novel findings, hypothesizing that fundamental components of the α-granule biogenesis machinery work at the megakaryocyte sorting endosome by regulating vesicular trafficking. We propose two specific aims. Aim 1 will determine the transport pathways followed by newly synthesized α-granule soluble and membrane proteins using an innovative method to synchronize and evaluate transport of proteins to α-granules in real time. We will test the hypothesis that there are multiple, separate pathways followed by megakaryocyte-synthesized α-granule proteins and test whether α-granules segregate into distinct populations. Aim 2 will define the molecular mechanism of platelet α-granule biogenesis by: (i) testing the hypothesis that VPS33B and VPS16B regulate the SNARE-mediated fusion of Golgi-derived vesicles containing α-granule cargo with sorting endosomes; (ii) addressing the function of novel components of the transport machinery identified here, including a new complex that coordinates α-granule cargo traffic through sorting endosomes; (iii) testing the hypothesis ...