Molecular Mechanism of Platelet Dense Granule Biogenesis

描述（由申请方提供）：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、α-颗粒和溶酶体的分泌和内容物释放，进而导致额外血小板和白色细胞的募集和聚集。虽然受损的血小板功能与表现为中度至重度粘膜皮肤出血的疾病相关，但由于其在心肌梗死和中风中的作用，过度血小板聚集是发病和死亡的主要原因。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，我们的目标是了解负责血小板致密颗粒的生物发生的分子机制。 致密颗粒属于一组溶酶体相关细胞器（LRO）。LRO的形成涉及两条平行的蛋白质转运途径，即衔接蛋白-3（AP-3）和溶酶体相关细胞器复合物生物发生-2（BLOC-2）。AP-3是一种衔接子，它选择早期内体中具有特异性靶向信号的蛋白质，并将其包装到囊泡中以转运到LRO。BLOC-2也定位于早期内体，但其功能尚不清楚。我们最近获得的初步证据表明，BLOC-2具有类似接头的特性，但能够结合致密颗粒蛋白中的新靶向信号，不同于AP-3识别的信号。此外，我们获得了大量的初步结果表明，五个蛋白质的基本组成部分和新的球员在致密颗粒的途径：两个“分子开关”，两个新的蛋白质含有囊泡断裂域，和一个分子马达。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，（2）测试以下假设：致密颗粒蛋白中存在新的致密颗粒靶向信号，BLOC-2是识别这些信号并将相应的蛋白包装到预定用于致密颗粒的囊泡中的适配器;（3）检验组织特异性“分子开关”蛋白将AP-3、BLOC-2和其他普遍存在的组分募集到内体膜以特异性地引导转运到致密颗粒的假设;（4）验证新的囊泡断裂和分子马达蛋白介导装载致密颗粒膜蛋白的囊泡形成和转运到致密颗粒的假设;以及（5）测试在临床上出现的许多病因不明的血小板型出血性疾病患者可能存在以下缺陷的可能性：这些新的分子开关、断裂和分子马达蛋白参与致密颗粒的生物发生。

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.