A systems approach to hemostasis and thrombosis

项目3摘要 血小板活化是止血的关键，也是血栓形成的一个促成因素。尽管最近的研究表明 强调了血小板在不同过程中的作用，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，也是这个项目的主要焦点。我们最近在中国的研究 小鼠的微血管系统显示，对轻微损伤的止血反应产生相对简单的 一种结构，在这种结构中，完全激活的血小板的核心被活性较低的血小板的外壳覆盖。稠密 堆芯中的堆积起到了分子陷阱的作用，建立了扩散取代对流的环境。 这种结构允许凝血酶和其他激动剂形成重叠的梯度，从而产生区域差异 在血小板活化和纤维蛋白分布方面。认识到转运是由血小板堆积密度调节的 一种范式的转变，表明血小板促凝血活性来自于形成一个庇护的环境和 不只是因为接触了磷脂。我们认为，这一概念是理解 抗血小板药物与动脉血栓形成事件。测试它需要扩大到更大规模的伤害 血管，并将我们的分析从小鼠扩展到人类，从止血到血栓，所有这些都是通过 混合实验和计算方法，与本PPG中的其他项目集成并提供支持。 目标1将在高分辨率下检查血小板激活的时空分布，测量 在血小板之间的缝隙中运输，并检查大动脉和静脉的止血反应。这个 初步结果显示一个更复杂的结构，有更多和更少的血小板活化和堆积区域 密度，以及管腔表面和管腔表面之间的巨大差异。我们与Brian Storrie的分包合同在 阿肯色大学将允许在亚微米范围内对更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 体内功能和支持研究PF4导向的抗体KKO的影响。使用µ-和m-的研究 钙蛋白缺乏的小鼠将支持项目2中的工作，但也是理解凝块回缩作用的一部分 限制通过较大止血结构的运输。目标#2将研究塑造 止血塞，测试止血结构需要严格调节血小板范围的假设 血小板的激活和输送深入止血块内部。NBEAL2-/-(灰色)的研究 血小板综合征)小鼠和在项目1中开发的“空a-颗粒”小鼠将允许我们检查其作用 止血塞结构上的分泌物。目标3将确定有序的止血结构 我们在老鼠身上观察到的结果也适用于人类，以及与人类相比，它在动脉血栓形成方面有何不同 止血。人体研究将与宾夕法尼亚大学创伤外科医生凯莉·西姆斯一起在体内进行，并在体外进行 使用一种与宾夕法尼亚大学工程学院的Dan Huh共同开发的新型微流体设备。关于人类的研究 动脉血栓将与项目2的合作研究员John Weisel合作完成。

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.