Platelet granule exocytosis and thrombus formation

描述（由申请人提供）：颗粒胞吐是血小板的基本功能，不仅有助于止血和血栓形成，还有助于炎症、伤口愈合、恶性肿瘤和血管生成。先前对颗粒胞分泌分子机制的研究已经确定了SNARE机制和膜融合所需的伴侣蛋白。本提案中描述的研究将评估这一前提，即该机制在将特定货物分类到不同的颗粒亚群中发挥作用，并在血小板激活时使颗粒内容物的差异释放。我们将研究VAMP-3、VAMP-5、VAMP-7和VAMP-8缺失小鼠的颗粒分离和分泌。在血小板扩散过程中发生的细胞骨架动力学可以影响SNARE相互作用并改变颗粒胞吐。我们将评估血小板扩散过程中的颗粒胞吐。这些研究还将评估胞吐作用在为粘附血小板周围生长的肌动蛋白结构提供膜以及在粘附蛋白微模式基质上定向扩散和运动方面的作用。使用高速活体显微镜将用于监测颗粒从单个血小板在体内血栓形成过程中的释放。这些方法将使我们能够评估黏附血小板中颗粒分泌机制与悬浮血小板相比发生改变的假设。我们还将评估动力蛋白在控制融合孔动力学中的作用，证明在snare介导的膜融合后发生的血小板胞吐的控制水平。这些研究将使用单细胞安培法来监测单个血小板中单个颗粒的释放。动态蛋白在体内血栓形成过程中控制血小板胞吐的作用将对血小板融合孔可调节以控制血栓形成的假设进行评估。这些实验将解决尚不清楚的血小板颗粒胞吐问题，并为血栓形成过程中选择性血小板膜融合事件的靶向研究提供基础。

DESCRIPTION (provided by applicant): Granule exocytosis is an essential platelet function, contributing not only to hemostasis and thrombosis, but also to inflammation, wound healing, malignancy, and angiogenesis. Previous efforts to understand the molecular mechanisms of granule exocytosis have identified the SNARE machinery and chaperone proteins required for membrane fusion. Studies described in this proposal will evaluate the premise that this machinery serves a role in sorting specific cargos to distinct subpopulations of ¿-granules and enabling differential release of granule contents upon platelet activation. We will study granule segregation and secretion in mice lacking VAMP-3, VAMP-5, VAMP-7, and VAMP-8. Cytoskeleton dynamics that occur during platelet spreading can influence SNARE interactions and modify granule exocytosis. We will evaluate granule exocytosis during platelet spreading. These studies will also assess the role of exocytosis in providing membrane for growing actin structures at the periphery of adherent platelets and for directed spreading and movement on micropatterned matrices of adhesion proteins. Use of high-speed intravital microscopy will be used to monitor release of granules from individual platelets during thrombus formation in vivo. These approaches will allow us to evaluate the hypothesis that the mechanism of granule secretion is altered in adherent platelets compared to suspension platelets. We will also evaluate the role of dynamins in controlling fusion pore dynamics, demonstrating a level of control of platelet exocytosis that occurs following SNARE-mediated membrane fusion. These studies will use single-cell amperometry to monitor the release of individual granules from single platelets. The role of dynamins in controlling platelet exocytosis during thrombus formation in vivo will evaluate the hypothesis that the platelet fusion pore can be regulated to control thrombus formation. These experiments will address poorly understood aspects of platelet granule exocytosis and provide a foundation for targeting select platelet membrane fusion events during thrombosis.