Molecular Mechanism of Platelet Dense Granule Biogenesis

描述（由申请人提供）：血小板在止血和血栓形成中起关键作用。血小板活化触发致密颗粒、a-颗粒和溶酶体的分泌和释放，进而导致额外的血小板和白细胞的募集和聚集。虽然血小板功能受损与表现为中度至重度粘膜皮肤出血的疾病有关，但由于血小板过度聚集在心肌梗死和中风中的作用，它是发病率和死亡率的主要原因。尽管血小板致密颗粒与人类健康相关，但对其生物成因知之甚少。因此，我们的目标是了解血小板致密颗粒生物发生的分子机制。致密颗粒属于一类溶酶体相关细胞器（LROs）。LROs的形成涉及两个平行的蛋白质运输途径，即Adaptor protein -3 （AP-3）和Biogenesis of lysosomes related Organelles Complex-2 （block -2）。AP-3是一种适配器，它在早期核内体中选择具有特定靶向信号的蛋白质，并将其包装成囊泡运输到LROs。block -2也定位于早期核内体，但其功能尚不清楚。我们最近获得的初步证据表明，block -2具有适配器样特性，但具有结合致密颗粒蛋白中新的靶向信号的能力，与AP-3识别的信号不同。此外，我们获得了大量的初步结果，表明五种蛋白质是致密颗粒形成途径的基本成分和新参与者：两种“分子开关”，两种含有囊泡断裂结构域的新蛋白质和一种分子马达。这些发现为研究血小板致密颗粒的生物发生开辟了新的途径。我们建议：(1)建立新的体外和体内系统来研究致密颗粒的生物学特性；(2)验证致密颗粒蛋白中存在新的致密颗粒靶向信号的假设，并且block -2是识别这些信号并将相应的蛋白质包装到致密颗粒囊泡中的接头；(3)验证组织特异性“分子开关”蛋白将AP-3、block -2和其他普遍存在的成分招募到内体膜上，特异性地直接运输到致密颗粒的假设；(4)验证新囊泡断裂和分子运动蛋白介导装载致密颗粒膜蛋白的囊泡形成和向致密颗粒转运的假说；(5)检验临床出现的众多病因不明的血小板型出血性疾病患者可能缺乏这些参与致密颗粒生物发生的新分子开关、断裂和分子运动蛋白的可能性。

DESCRIPTION (provided by applicant): Platelets play pivotal roles in both hemostasis and thrombosis. Platelet activation triggers secretion and the release of content from dense granules, a-granules, and lysosomes that in turn leads to the recruitment and aggregation of additional platelets and white cells. While impaired platelet function has been associated with disorders that manifest with moderate to severe mucocutaneous bleeding, excessive platelet aggregation is a major cause of morbidity and mortality due to its effect in myocardial infarction and stroke. In spite of the relevance of platelet dense granules for human health, little is known about their biogenesis. Therefore, our goal is to understand the molecular mechanism responsible for the biogenesis of platelet dense granules. Dense granules belong to a group of lysosome-related organelles (LROs). Formation of LROs involves two parallel protein transport pathways defined by Adaptor Protein-3 (AP-3) and Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2). AP-3 is an adaptor that selects proteins with specific targeting signals in early endosomes and packages them into vesicles for transport to LROs. BLOC-2 also localizes to early endosomes but its function is unknown. We have recently obtained preliminary evidence suggesting that BLOC-2 has adaptor-like properties but with the ability to bind new targeting signals in dense granule proteins, different from the signals recognized by AP-3. Moreover, we obtained substantial preliminary results indicating that five proteins are fundamental components and new players in the pathways to dense granules: two "molecular switches", two novel proteins containing vesicle scission domains, and a molecular motor. These findings have opened new avenues to study the biogenesis of platelet dense granules. We propose to: (1) establish new in vitro and in vivo systems to study the biology of dense granules, (2) test the hypothesis that new dense granule targeting signals exist in dense granule proteins and that BLOC-2 is an adaptor that recognizes these signals and packages the corresponding proteins into vesicles destined for dense granules; (3) test the hypothesis that tissue specific "molecular switch" proteins recruit AP-3, BLOC-2, and other ubiquitous components to endosomal membranes to specifically direct transport to dense granules; (4) test the hypothesis that new vesicle scission and molecular motor proteins mediate the formation and transport of vesicles loaded with dense granule membrane proteins to dense granules; and (5) test the possibility that numerous patients that present in the clinic with platelet type bleeding disease of unknown etiology may have deficiencies in these new molecular switches, scission, and molecular motor proteins involved in dense granule biogenesis.