A systems approach to hemostasis and thrombosis

项目3 血小板活化对止血至关重要，也是血栓形成的促成因素。尽管最近的研究表明， 由于血小板在不同过程中的作用突出，大量血小板的快速积累仍然存在， 止血和动脉血栓形成的标志，是本项目的主要重点。我们最近的研究 小鼠微血管显示，对小损伤的止血反应产生相对简单的 一种结构，其中完全活化的血小板的核心被较少活化的血小板的壳覆盖。密集 堆芯中的填充物起到分子陷阱的作用，建立了扩散代替对流的环境。 这种结构允许凝血酶和其他激动剂形成重叠梯度，从而产生区域差异 血小板活化和纤维蛋白分布。认识到转运受血小板堆积密度的调节， 一种范式转变，表明血小板促凝活性源于形成一个受保护的环境， 而不仅仅是因为暴露在磷脂中。我们认为，这一概念是理解 抗血小板药物和动脉血栓形成事件。测试它需要扩大到更大的伤害， 血管，并将我们的分析从小鼠扩展到人类，从止血扩展到血栓形成，所有这些都具有 混合实验和计算的方法，集成并支持本PPG中的其他项目。 目标1将以高分辨率检查血小板活化的空间和时间分布，测量 在血小板之间的间隙中运输，并检查大动脉和静脉中的止血反应。的 最初的结果显示了更复杂的结构，具有更大和更小的血小板活化和堆积区域 密度，以及腔表面和腔外表面之间的较大差异。我们与布莱恩·斯托里在 阿肯色州大学将允许在亚微米级对较大的止血血栓进行三维重建 水平与项目4合作，我们将研究脓毒症和全身炎症对血小板的影响。 在体内的功能，并支持研究的影响PF 4定向抗体，KKO。µ-和m-的研究 钙蛋白酶缺陷小鼠将支持项目2的工作，但也是理解血块收缩作用的一部分。 限制通过较大止血结构的运输。目标#2将研究塑造 止血塞，检验止血结构需要严格调节血小板聚集程度的假设 活化和血小板货物在止血物质深处的递送。NBEAL 2-/-（灰色）的研究 血小板综合征）小鼠和项目1中开发的“空α-颗粒”小鼠将使我们能够检查 止血栓结构上的分泌物。目标3将确定有序止血结构是否 我们在小鼠中观察到的适用于人类，以及它在动脉血栓形成中与 止血。人体研究将由宾夕法尼亚创伤外科医生Carrie西姆斯在体内进行， 使用了一种新型的微流体设备，该设备是由宾夕法尼亚大学工程学院的Dan Huh开发的。人类研究 将与项目#2共同研究者John Weisel合作完成动脉血栓检查。

Project 3 Abstract Platelet activation is critical for hemostasis and a contributing factor in thrombosis. Although recent studies have highlighted roles for platelets in diverse processes, the rapid accumulation of large numbers of platelets remains the hallmark of hemostasis and arterial thrombosis, and is the major focus of this project. Our recent studies in the mouse microvasculature show that the hemostatic response to small injuries produces a relatively simple structure in which a core of fully-activated platelets is overlaid by a shell of less-activated platelets. Dense packing in the core acts as a molecular trap, establishing an environment in which diffusion replaces convection. This structure allows thrombin and other agonists to form overlapping gradients that produce regional differences in platelet activation and fibrin distribution. Recognizing that transport is regulated by platelet packing density is a paradigm shift, suggesting that platelet procoagulant activity arises from forming a sheltered environment and not just from phospholipid exposure. We believe that this concept is key to understanding the impact of antiplatelet agents and the events of arterial thrombosis. Testing it calls for scaling up to larger injuries in larger vessels, and for extending our analysis from mice to humans and from hemostasis to thrombosis, all with a hybrid experimental and computational approach that integrates with and supports the other projects in this PPG. Aim #1 will examine the spatial and temporal distribution of platelet activation at high resolution, measure transport in the gaps between platelets, and examine the hemostatic response in large arteries and veins. The initial results show a more complex architecture with regions of greater and lesser platelet activation and packing density, and large differences between the luminal and abluminal surfaces. Our subcontract with Brian Storrie at the University of Arkansas will allow 3-dimensional reconstruction of larger hemostatic thrombi at the sub-micron level. In collaboration with Project 4 we will examine the impact of sepsis and systemic inflammation on platelet function in vivo and support studies on the impact of the PF4-directed antibody, KKO. Studies with µ- and m- calpain deficient mice will support work in Project 2, but also be part of understanding the role of clot retraction in limiting transport through larger hemostatic structures. Aim #2 will examine the mechanisms that shape the hemostatic plug, testing the hypothesis that hemostatic structure requires tight regulation of the extent of platelet activation and the delivery of platelet cargoes deep within the hemostatic mass. Studies on NBEAL2-/- (gray platelet syndrome) mice and the “empty a-granule” mice developed in Project 1 will allow us to examine the role of secretion on hemostatic plug architecture. Aim #3 will determine whether the ordered hemostatic structure that we have observed in mice applies to humans, and how it differs in arterial thrombosis as compared to hemostasis. The human studies will be performed in vivo with Penn trauma surgeon, Carrie Sims, and ex vivo using a novel microfluidics device developed with Dan Huh in Penn’s School of Engineering. Studies of human arterial thrombi will be done in collaboration with Project #2 co-investigator John Weisel.