Resubmission: Structure/Function Determinants of Puncture Wound Thrombus Formation

项目摘要/摘要 科学前提和前期研究：刺伤，无论是意外、故意还是医疗原因造成的 实践，例如手术，可能是重大创伤事件的例行公事。此外，药物加重的出血是一个主要的 限制目前的抗血小板药物，这些药物构成了心血管患者治疗的基石和潜在的 对任何手术都负有重大责任。我们认为对以血小板为中心的反应的详细结构理解 对于填补先前研究中的空白并产生重要的 创伤性损伤的处理框架和常见的出血副作用。 知识上的差距(S)：以前血栓形成的方法大多集中在 暴露的血管基质保持完好，因此很少或根本没有出血发生和遭受 由于2P显微镜或扫描电子显微镜无法在单个血小板的水平上评估血小板的激活。 从这个受限的实验角度来看，血栓结构的“核-壳”模型，即 基质锚定的高活性血小板的水平核心，覆盖着一层弱激活的血小板外壳 核心内的哪些信号是凝血酶依赖的，而外壳内的哪些信号是ADP/血栓素依赖的 已经出现了。我们认为，该模型的应用可能仅限于实际出血， 伤口是一个开放的穿刺孔。 问题的解决方案：采用其他可视化方法来确定个体的激活状态 3D空间中的血小板，我们的初步研究已经产生了重新定义正常、止血的数据 如刺伤模型所示。我们的数据揭示了一种空间分散的血小板激活模式 强烈的迹象表明，血栓形成的最初步骤依赖于对血小板聚集物的捕获 这就从外面堵住了这个洞，而不是填满了这个洞。这导致我们制定了一个新的“上限和构建” 范例。如何将以血小板为中心的事件和凝血因子级联事件整合在 范式现在是一个重要的问题。为了通过实验理解这些多重输入，我们有 建议的目标首先集中在信号事件上，无论是自分泌(血小板分泌，特定目标1)还是 外分泌(从抗血小板药物或DOAC的效果推断，特定目标2)，以及 血小板黏附受体(特异性靶点3)。我们还建议测试上限和构建范型有多好 适用于较小、创伤较小的损伤。

PROJECT SUMMARY/ABSTRACT Scientific Premise and Prior Research: Puncture wounds, be they produced by accident, intent or medical practice, e.g., surgery, can be a routine to major traumatic event. Moreover, drug aggravated bleeding is a major limitation of current antiplatelet drugs that form a cornerstone of cardiovascular patient therapy and a potentially major liability to any surgery. We contend that detailed structure understanding of the platelet centric responses that occur within the actual puncture hole is essential to fill a void in prior research and yield an important framework for the management traumatic injuries and common bleeding side effects. Gap(s) in knowledge: Previous approaches to thrombus formation have focused mostly on examples in which the exposed vascular matrix remains intact and therefore little to no bleeding occurs and have suffered from the inability of 2P microscopy or SEM to assess platelet activation at the level of individual platelets. From this circumscribed experimental perspective, a “Core and Shell” model of thrombus structure, i.e., a horizontal core of matrix anchored, highly activated platelets covered by a shell of weakly activated platelets in which signaling within the Core is thrombin-dependent and within the Shell is ADP/thromboxane-dependent has emerged. We submit that this model may well be limited in its application to actual bleeding where the wound is an open puncture hole. Solution to problem: Taking alternate visualization approaches that determine the activation state of individual platelets in 3D space, our Preliminary Studies have already yielded data redefining normal, bleeding cessation as exemplified in a puncture wound model. Our data reveal a spatially dispersed, platelet activation pattern and strong indications that initial steps in thrombus formation are dependent on the capture of platelet aggregates that cap the hole from the outside rather than fill the hole. This has led us to formulate a new “Cap and Build” paradigm. How both platelet-centric and coagulation factor cascade events can be integrated within the paradigm is now an important question. To understand these multiple inputs through experimentation, we have proposed Aims that focus first on signaling events, be they autocrine (platelet secretion, Specific Aim 1) or exocrine (as inferred from the effects of anti-platelet drugs or DOACs, Specific Aim 2), and the importance of platelet adhesion receptors (Specific Aim 3). We also propose to test how well the Cap and Build paradigm apply to smaller, less traumatic injury.