Molecular Mechanisms of Platelet Alpha Granule Biogenesis

项目总结 血小板在止血和血栓形成中起着核心作用。血小板活化触发内容物的分泌和释放 来自α颗粒、δ颗粒和溶酶体，进而导致额外的血小板和 无数的生理反应。而血小板功能受损与表现为 中到重度出血，血小板过度聚集是发病率和死亡率的主要原因，因为它影响 心血管疾病和中风。在健康和疾病中，阿尔法颗粒对这些血小板功能都是至关重要的。然而， 尽管血小板α颗粒与人类健康相关，但人们对其生物发生机制知之甚少。因此， 我们的目标是了解血小板α颗粒生物发生的途径和分子机制。 VPS33B、VPS16B和NBEAL2基因突变导致α颗粒缺陷和出血表现 患有关节紊乱、肾功能不全和胆汁淤积(ARC)综合征和灰色血小板综合征(GPS)的患者。 然而，这些蛋白质在α颗粒生物发生中的作用机制仍然是一个谜。因此，一个主要目标是 VPS33B、VPS16B和NBEAL2在细胞和分子水平上的功能。 血小板α颗粒产生于巨核细胞，即血小板前体细胞。除了服用的可溶性蛋白质外 通过内吞作用，α颗粒含有数以百计的由巨核细胞合成的蛋白质。他走过的小路 巨核细胞合成的蛋白质到达α颗粒是未知的。在这个提案中，我们展示了排序 内小体是巨核细胞合成的蛋白质所使用的α颗粒形成的基本前体细胞器。 这意味着一种比先前预期更复杂的生物发生机制。在这个应用程序中，我们以我们的小说为基础 发现，假设α颗粒生物发生机制的基本组件在巨核细胞中起作用 通过调节囊泡运输来分选内体。我们提出了两个具体目标。目标一号将决定运输 利用一种创新的方法研究新合成的α颗粒可溶性蛋白和膜蛋白所遵循的途径 实时同步和评估蛋白质到α颗粒的运输。我们将检验这一假设，即存在多个， 分离巨核细胞合成的α颗粒蛋白所遵循的途径，并测试α颗粒是否分离到 不同的种群。目的2将通过以下方法确定血小板α-颗粒生物发生的分子机制：(I)检测 VPS33B和VPS16B调控含α颗粒高尔基体小泡SNARE融合的假说 具有分拣内体的货物；(2)处理此处确定的运输机械的新部件的功能， 包括一个新的复合体，通过分选内含体来协调α-Granule货物运输；(Iii)检验假设 NbEAL2与肌动蛋白和Vac14结合，介导α颗粒货物从分选内小体中退出。 这项研究将改变我们对血小板α颗粒领域的看法，带来一种高度机械性的 对生物发生过程的理解。我们的工作将对出血和骨髓纤维化的表现有深入的了解 在ARC和GPS患者中观察到。最终，这些知识将有助于设计治疗出血的新策略。 以及血栓性疾病和其他疾病，α颗粒在其中具有新的作用，包括血管生成和癌症。

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.