Platelet granule formation and function in health and disease

说明(申请人提供)：血小板在止血、炎症、血管生成和其他过程中发挥基本功能。这些功能中的许多都需要从三种类型的储存室-颗粒、致密颗粒和溶酶体-调节分泌。许多出血性和血栓性疾病是由颗粒形成和/或内容物释放的失调引起的。特别是，Hermansky-Pudlak综合征(HPS)的出血素质是由于缺乏可检测到的血小板致密颗粒以及随之而来的激活依赖的钙和ADP释放的缺陷导致的，而激活依赖的钙和ADP通常促进血栓形成。尽管血小板颗粒很重要，但其在巨核细胞(MK)和/或其衍生的血小板中的形成和膜动力学的基本机制尚不清楚，而且在9个特征的HPS变异体中存在缺陷的基因调控的途径也不清楚。在其他类型的细胞中，HPS相关基因调节囊泡运输过程，所需的囊泡运输过程需要将驻留的完整膜蛋白从早期内小体运送到与溶酶体相关的细胞器--细胞类型特定的细胞内储存室，如？和致密颗粒。我们和我们的合作者最近确定了两种完整的膜蛋白-SLC35D3和VMAT2-作为致密颗粒特定的候选货物，可以作为研究致密颗粒生物发生的标志物。令人惊讶的是，这两种货物都定位于野生型和HPS模型小鼠的MK的早期内吞体内。然而，SLC35D3在相同的HPS模型的血小板中是不稳定的。基于这些和其他数据，我们假设在血小板从巨噬细胞分化的后期，致密的颗粒完整膜蛋白从早期的内小体输送到未成熟的致密颗粒。我们进一步假设致密颗粒膜不是静态的，而是在活性血小板中不断重塑，可能为向血小板致密颗粒输送药物提供了一条途径。最后，初步数据表明，激活诱导的内容释放不仅来自致密的颗粒，而且还来自HPS模型血小板的颗粒和溶酶体，无论是体内还是体外。由于内容物释放需要颗粒膜与质膜融合，我们假设HPS模型中的这种分泌缺陷反映了融合机制向颗粒和溶酶体的货物输送受损。我们将在以下特定目标中检验这些假说：1.评估HPS相关蛋白是否调节候选致密颗粒膜蛋白在血小板中的动态定位。2.评估HPS是否通过不同前体细胞器的融合影响致密颗粒的成熟。3.检测HPS亚型对血小板颗粒和溶酶体分泌的影响。

DESCRIPTION (provided by applicant): Platelets fulfill essential functions in hemostasis, inflammation, angiogenesis, and other processes. Many of these functions require regulated secretion from three types of storage compartments - ¿ granules, dense granules, and lysosomes. Many hemorrhagic and thrombotic disorders are caused by dysregulation of granule formation and/or content release. In particular, bleeding diathesis in Hermansky-Pudlak syndrome (HPS) results from an absence of detectable platelet dense granules and consequent defects in activation-dependent release of calcium and ADP, which normally promote thrombus formation. Despite the importance of platelet granules, the mechanisms underlying their formation and membrane dynamics within megakaryocytes (MKs) and/or their derived platelets are largely uncharacterized, and the pathways regulated by the genes that are defective in the nine characterized HPS variants are not known. In other cell types, HPS-associated genes regulate vesicular transport processes required to deliver resident integral membrane proteins from early endosomes to lysosome-related organelles - cell type-specific intracellular storage compartments like ¿ and dense granules. We and our collaborators have recently identified two integral membrane proteins - SLC35D3 and VMAT2 - as candidate dense granule-specific cargoes that can serve as markers to study dense granule biogenesis. Surprisingly, these two cargoes localize to early endosomes in MKs from both wild-type and HPS model mice. Nevertheless, SLC35D3 is destabilized in platelets from the same HPS models. Based on these and other data, we hypothesize that dense granule integral membrane proteins are delivered from early endosomes to immature dense granules at a late stage of differentiation of platelets from MKs. We further hypothesize that dense granule membranes are not static but are constantly remodeled in active platelets, perhaps providing an avenue for delivery of pharmacological agents to platelet dense granules. Finally, preliminary data suggest that activation-induced content release is impaired not only from dense granules but also from ¿ granules and lysosomes in HPS model platelets, both in vivo and ex vivo. Since content release requires fusion of the granule membrane with the plasma membrane, we hypothesize that this secretory defect in HPS models reflects impaired cargo delivery of the fusion machinery to ¿ granules and lysosomes. We will test these hypotheses in the following Specific Aims: 1. To assess whether HPS-associated proteins regulate the dynamic localization of candidate dense granule membrane proteins in platelets. 2. To assess whether HPS impacts maturation of dense granules by fusion of distinct precursor organelles. 3. To test whether and how HPS subtypes impact ¿-granule and lysosome secretion from platelets.