PLATELET INTERACTIONS WITH ADHESIVE PROTEINS

要求提供资金，以继续研究支持血小板止血和血栓形成功能的机制。血小板对血管损伤的反应是通过与细胞外基质和内皮下基质的相互作用来启动和指导的，并且内皮下结构在这些过程中起关键作用，特别是在慢性动脉疾病期间。在具体目标1中，我们将确定介导血小板粘附和活化的亚内皮分子，并确定它们的差异表达如何影响血栓形成。 越来越多的实验证据表明，内衬器官血管的内皮细胞不仅在形态上而且在功能上具有明显的特异性特征。在目标2中测试的假设得到了初始实验结果的支持，即细胞外基质组成是区分不同来源的内皮细胞的结构和功能特征之一。因此，我们将从各种组织中分离小鼠内皮细胞，并通过使用共聚焦视频显微镜在真实的时间中测量流动血液中血小板粘附和聚集的诱导来测试基质促凝性。观察到的表型变异将导致识别来自不同组织的内皮细胞的遗传特征的差异，以及相应的细胞外基质的结构特性，这可能有助于解释血栓性血管疾病的发病机制。遗传诱导的小鼠血小板反应蛋白-2（TSP 2）缺陷导致出血时间延长。为了解释这种意想不到的表型，在具体目标3中，我们提出选择性地评价TSP2缺陷型内皮下基质的血栓形成特性以及TSP2缺陷型血小板粘附并聚集到暴露于流动血液的适当基质上的能力。这些研究将有助于确定TSP2在血管生物学中的作用。将血小板束缚到血栓形成表面的初始相互作用与快速活化相结合，这是进展到不可逆粘附和聚集所严格需要的。在具体目标4中，我们提出研究可能由不同底物引起的信号传导机制，这些机制可能直接促成流动血液中血栓形成的过程。此外，我们将使用遗传操作的小鼠血小板来定义整合素α IIb β 3的β 3亚基的胞质结构域中的靶向Tyr-> Phe突变的后果，这些突变似乎干扰信号传导过程。这些研究将有助于阐明与特定细胞外基质成分的相互作用和血小板活化与不同血流动力学条件之间的整合。在具体目标5中，我们将使用真实的三维测量来研究纤维蛋白原、纤连蛋白和玻连蛋白对暴露于以正常和病理动脉剪切速率流动的血液的不同基质上的vWF引发的血栓的发展和稳定性的影响。与拟议的研究所获得的结果将推进我们的理解血栓形成的机制，使用流动的血液中定义明确的定量分析，从而提供必要的新信息，以解释在体内的生物功能和病理疾病过程。

Funding is requested to continue studies on the mechanisms that support platelet function in hemostasis and thrombosis. Platelet response to vascular injury is initiated and directed by interactions with the extracellular matrix, and sub-endothelial matrix, and sub-endothelial structures play a key role in these processes particularly during chronic arterial disease. In specific aim 1 we will identify the sub-endothelial molecules that mediate platelet adhesion and activation, and define how their differential expression affects thrombus formation. Increasingly experimental evidence indicates that endothelial cells lining the vessels of organs have distinctly specific features, not only morphologic but also functional. The hypothesis tested in aim 2, supported by initiated experimental results, is that extracellular matrix composition is one of the structural and functional features distinguishing endothelial cells of different origin. Thus, we will isolate mouse endothelial cells from various tissues and test matrix thrombogenicity by measuring induction of platelet adhesion and aggregation in flowing blood using confocal videomicroscopy in real time. Observed phenotypic variations will lead to the identification of differences in the genetic characteristics of endothelial cells from different tissues as well as in the structural properties of the corresponding extracellular matrices that may help explain the pathogenesis of thrombotic vascular disease. Genetically induced deficiency of thrombospondin-2 (TSP2) in mice results in prolongation of the bleeding time. To explain this unexpected phenotype, in specific aim 3 we propose to evaluate selectively the thrombogenic properties of TSP2 deficient sub-endothelial matrix and the capacity of TSP2 deficient platelets to adhere to and aggregate onto appropriate substrates exposed to flowing blood. These studies will help define the role of TSP2 in vascular biology. Initial interactions tethering platelets to thrombogenic surfaces are coupled to rapid activation that is strictly required for the progression to irreversible adhesion and aggregation. In specific aim 4 we propose to investigate the signaling mechanisms that may be elicited by different substrates and may contributed directly to the process of thrombus formation in the flowing blood. Moreover, we will use genetically manipulated mouse platelets to define the consequences of targeted Tyr->Phe mutations in the cytoplasmic domain of the beta3 subunit of integrin alphaIIbbeta3 that appear to interfere with signaling processes. These studies will contribute to clarify the integration between interaction with specific extracellular matrix components and platelet activation in relation to varying hemodynamic conditions. In specific aim 5 we will use three-dimensional measurements in real time to study the effects of fibrinogen, fibronectin and vitronectin on the development and stability of vWF-initiated thrombi on different substrates exposed to blood flowing at normal and pathological arterial shear rates. The results obtained with the proposed studies will advance our understanding of the mechanisms of thrombus formation using well defined quantitative analysis in flowing blood, thus providing new information necessary to explain in vivo biological functions and pathological disease processes.