Platelet granule biogenesis in health and disease

描述(申请人提供)：活化的血小板分泌大量的可溶性介质，这些介质在正常的血液凝块形成、炎症、血管生成和其他生理反应中发挥关键作用。在静息状态的血小板中，大多数这些介质至少储存在两种不同的分泌颗粒中：蛋白质介质如纤维蛋白原和von Willebrand因子储存在1颗粒中，而ADP、ATP、钙和5-羟色胺储存在致密颗粒中。这些颗粒在巨核细胞中产生，并隔离形成血小板。这两种类型的颗粒被认为与溶酶体有一些共同的特征，但它们是如何形成的，它们的内容如何与巨核细胞中传统的溶酶体内容具体分离尚不清楚。任何一类颗粒的形成或其中特定成分的储存存在缺陷，都会导致长期出血和异常的血小板聚集。特别是，在Hermansky-Pudlak综合征(HPS)患者中，其他几种细胞类型中的致密颗粒和组织特异性溶酶体相关细胞器，包括色素细胞中的黑素体，都是畸形的。HPS是一组至少有八个基因突变的疾病。我们在了解几个HPS相关基因的产物如何在蛋白质从内小体运输到黑素体的过程中取得了相当大的进展，并表征了与其中一些基因产物相互作用的细胞质分选信号，并介导了整膜蛋白对黑素体的特异性分选。在这个提案中，我们将利用我们对黑素细胞的知识，两种独特的巨核细胞培养系统的可用性，以及最近发现的致密颗粒上的整膜蛋白来检验这样的假设，即类似的途径调节蛋白质分选到黑素细胞中的黑素小体和巨核细胞中的致密颗粒，并且这些途径不同于调节蛋白质分选到1颗粒的途径。我们还将测试这样一种假设，即适当地将蛋白质定位于致密颗粒需要HPS相关的基因产物。我们的具体目标是：1.检测黑素细胞中致密颗粒蛋白是否定位于黑素小体，以及黑素小体蛋白是否定位于巨核细胞中致密颗粒。2.测试细胞质靶向信号是否像黑素体蛋白那样是将SLC35D3靶向致密颗粒或其前体所必需的和/或充分的。3.检测在缺乏不同HPS相关基因产物的巨核细胞中，致密颗粒蛋白是否差异地错位定位于内体。 公共卫生相关性：为了在组织损伤期间形成血栓，必须激活血小板，以释放被称为1和致密颗粒的特殊颗粒中的一些成分。这些颗粒的形成是一个临床上重要的过程，因为它们的畸形是许多遗传性和获得性出血性疾病的基础，但人们对它们是如何形成和积累其内容物知之甚少。在这个项目中，我们将利用我们对色素细胞中不同但相关细胞器的形成的理解来确定血小板颗粒是如何形成的，以及在某些遗传性出血疾病中出了什么问题。

DESCRIPTION (provided by applicant): Activated platelets secrete a host of soluble mediators that play critical roles in proper blood clot formation, inflammation, angiogenesis, and other physiological responses. In resting platelets, most of these mediators are stored within at least two distinct classes of secretory granules: protein mediators such as fibrinogen and von Willebrand factor are stored in 1 granules, whereas ADP, ATP, calcium and serotonin are stored in dense granules. These granules are generated within megakaryocytes and sequestered into forming platelets. Both types of granules are thought to share some features with lysosomes, but how they are formed and how their contents are specifically segregated from traditional lysosomal contents within megakaryocytes is not known. Defects in the formation of either class of granule, or in the storage of specific components within them, result in prolonged bleeding and abnormal platelet aggregation. In particular, dense granules and tissue-specific lysosome-related organelles in several other cell types, including melanosomes in pigment cells, are malformed in patients with Hermansky-Pudlak Syndrome (HPS), a group of disorders with mutations in any of at least eight genes. We have made considerable progress in understanding how the products of several HPS-associated genes function in trafficking of proteins from endosomes to melanosomes, and have characterized cytoplasmic sorting signals that interact with some of these gene products and mediate sorting of integral membrane proteins specifically to melanosomes. In this proposal, we will exploit our knowledge of the melanocyte, the availability of two unique megakaryocyte culture systems, and a recently discovered integral membrane protein on dense granules to test the hypothesis that similar pathways regulate protein sorting to melanosomes in melanocytes and to dense granules in megakaryocytes, and that these pathways are distinct from those that regulate protein sorting to 1 granules. We will also test the hypothesis that proper targeting of proteins specifically to dense granules requires HPS-associated gene products. Our Specific Aims are: 1. To test whether dense granule proteins localize to melanosomes in melanocytes and whether melanosomal proteins localize to dense granules in megakaryocytes. 2. To test whether cytoplasmic targeting signals like those on melanosomal proteins are necessary and/or sufficient to target SLC35D3 to dense granules or their precursors. 3. To test whether dense granule proteins are differentially mislocalized to endosomes in megakaryocytes that lack different HPS-associated gene products. PUBLIC HEALTH RELEVANCE: In order for blood clots to form during tissue damage, platelets must be activated to release a number of components from specialized granules called 1 and dense granules. Formation of these granules is a clinically important process because their malformation underlies a number of genetic and acquired bleeding disorders, but very little is known about how they form and accumulate their contents. In this project, we will exploit our understanding of the formation of a different but related organelle in pigment cells to determine how the platelet granules are formed and what goes wrong in certain heritable bleeding disorders.