Molecular Mechanisms of Platelet Alpha Granule Biogenesis

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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 that NBEAL2 mediates the exit of α-granule cargo from sorting endosomes in association with actin and Vac14. This research will transform our view of the platelet α-granule field by bringing about a highly mechanistic understanding of the biogenesis process. Our work will yield insights into the bleeding and myelofibrosis manifestations observed in ARC and GPS patients. Ultimately, this knowledge will help design new strategies for the treatment of bleeding and thrombotic disorders and other diseases in which α-granules have emerging roles including angiogenesis and cancer.