Molecular Mechanisms of Platelet Alpha Granule Biogenesis

血小板α颗粒生物发生的分子机制

• 批准号: 10319019
• 负责人: Santiago Mauro Di Pietro
• 金额: $ 38万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-15 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319019
• 关键词: Actins; Address; Alpha Granule; Arthrogryposis; Biochemical; Biogenesis; Blood; Blood Coagulation Disorders; Blood Platelets; Bone Marrow; CRISPR/Cas technology; Cardiovascular Diseases; Carrier Proteins; Cell Culture Techniques; Cells; Cellular biology; Child; Cholestasis; Collaborations; Complex; Cytoplasmic Granules; Data Set; Development; Disease; Endocytosis; Endoplasmic Reticulum; Endosomes; Gene Targeting; Genes; Genetic; Genetic Diseases; Goals; Golgi Apparatus; Health; Hemorrhage; Hemostatic Agents; Hemostatic function; Hospitals; Human; Human Genetics; Impairment; Inflammation; Knowledge; Lead; Letters; Lung; Lysosomes; Malignant Neoplasms; Mediating; Megakaryocytes; Membrane Proteins; Methods; Molecular; Morbidity - disease rate; Mutate; Mutation; Myelofibrosis; Organelles; Pathway interactions; Patients; Physiological; Platelet Activation; Platelet aggregation; Play; Population; Positioning Attribute; Prevention; Process; Proteins; Publications; Research; Role; SNAP receptor; Site; Sorting - Cell Movement; Stroke; Syndrome; System; Testing; Thrombosis; Time; Universities; Vesicle; Washington; Work; angiogenesis; biochemical tools; design; experimental study; innovation; insight; kidney dysfunction; mortality; mouse model; novel; novel therapeutic intervention; platelet function; polymerization; population based; precursor cell; recruit; response; stem cells; syntaxin; time use; trafficking; transcriptome sequencing; treatment strategy; wound healing

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.

项目摘要 血小板在止血和血栓形成中起核心作用。血小板活化触发内容物的分泌和释放 从α-颗粒、δ-颗粒和溶酶体，这反过来又导致额外血小板的募集和聚集， 无数的生理反应虽然受损的血小板功能与表现为 中度至重度出血，过度血小板聚集是发病率和死亡率的主要原因，这是由于其在 心血管疾病和中风。α-颗粒对健康和疾病中的这些血小板功能至关重要。然而，在这方面， 尽管血小板α-颗粒与人类健康相关，但对其生物起源知之甚少。因此，我们认为， 我们的目标是了解血小板α颗粒生物合成的途径和分子机制。 VPS 33 B、VPS 16 B和NBEAL 2的突变导致了α-颗粒缺陷和在100例患者中观察到的出血表现。 患有关节弯曲、肾功能不全和胆汁淤积（ARC）综合征和灰血小板综合征（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 本研究的目的是在细胞和分子水平上研究VPS 33 B、VPS 16 B和NBEAL 2的功能。 血小板α颗粒在巨核细胞（血小板前体细胞）中产生。除了可溶性蛋白质外， α-颗粒内含有巨核细胞合成的数百种蛋白质。所采取的途径 巨核细胞合成的蛋白质到达α-颗粒是未知的。在这个建议中，我们表明，排序 内体是巨核细胞合成蛋白质所使用的α颗粒形成中的基本前体细胞器。 这意味着一个比以前预期的更复杂的生物发生机制。在这个应用程序中，我们建立在我们的小说 研究结果，假设α颗粒生物发生机制的基本组成部分在巨核细胞中起作用， 通过调节囊泡运输来分选内体。我们提出两个具体目标。目标1将决定运输 新合成的α-颗粒可溶性和膜蛋白的途径， 真实的时间同步和评估蛋白质到α-颗粒的运输。我们将检验存在多个， 分离巨核细胞合成α颗粒蛋白的途径，并测试α颗粒是否分离成 不同的人群。目的2将通过以下方法阐明血小板α-颗粒生物合成的分子机制：（i）检测血小板α-颗粒的生物学活性， VPS 33 B和VPS 16 B调节SNARE介导的含有α-颗粒的高尔基体衍生囊泡的融合的假设 具有分选内体的货物;（ii）解决这里确定的运输机器的新组件的功能， 包括一个新的复合物，通过分类内体协调α颗粒货物运输;（iii）测试假设 NBEAL 2与肌动蛋白和Vac 14一起介导α-颗粒货物从分选内体中退出。 这项研究将改变我们对血小板α颗粒场的看法， 了解生物发生过程。我们的工作将有助于深入了解出血和骨髓纤维化的表现 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，其中α-颗粒具有新的作用，包括血管生成和癌症。