Platelet granule exocytosis and thrombus formation

描述(申请人提供)：颗粒胞吐是一种基本的血小板功能，不仅有助于止血和血栓形成，而且还有助于炎症、伤口愈合、恶性肿瘤和血管生成。先前为了了解颗粒胞吐的分子机制，已经确定了膜融合所需的陷阱机制和伴侣蛋白。这项建议中描述的研究将评估这样一个前提，即这种机制在将特定货物分选到不同的颗粒亚群以及使颗粒内容物在血小板激活时能够有区别地释放方面发挥作用。我们将研究缺乏VAMP-3、VAMP-5、VAMP-7和VAMP-8的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SNAR相互作用，并改变颗粒胞吐作用。我们将评估血小板扩散过程中的颗粒胞吐作用。这些研究还将评估胞吐作用在为黏附的血小板外围生长肌动蛋白结构提供膜以及在黏附蛋白的微图案基质上定向扩散和移动方面的作用。使用高速活体显微镜将被用来监测体内血栓形成过程中单个血小板释放颗粒的情况。这些方法将使我们能够评估这一假说，即与悬浮血小板相比，贴壁血小板的颗粒分泌机制发生了变化。我们还将评估动力素在控制融合孔动力学中的作用，展示在SNARE介导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颗粒从单个血小板中的释放。在体内血栓形成过程中，动力素在控制血小板胞吐中的作用将对血小板融合孔可以被调节以控制血栓形成的假说进行评估。这些实验将解决鲜为人知的血小板颗粒胞吐方面的问题，并为在血栓形成过程中靶向选择血小板膜融合事件提供基础。

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.