PLATELET INTERACTIONS WITH ADHESIVE PROTEINS

需要资金继续研究血小板在止血和血栓形成中的作用机制。血小板对血管损伤的反应是由与细胞外基质和亚内皮基质的相互作用引发和引导的，而亚内皮结构在这些过程中起着关键作用，特别是在慢性动脉疾病中。在具体目标1中，我们将识别介导血小板粘附和活化的内皮下分子，并定义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，器官血管内的内皮细胞不仅在形态上而且在功能上都具有明显的特异性。在aim 2中验证的假设得到初步实验结果的支持，即细胞外基质组成是区分不同来源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实时测量血流中血小板粘附和聚集的诱导来测试基质的血栓形成性。观察到的表型变异将导致鉴定来自不同组织的内皮细胞遗传特征的差异，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。基因诱导的小鼠血栓反应蛋白-2 （TSP2）缺乏导致出血时间延长。为了解释这种意想不到的表型，在特定的目的3中，我们建议有选择性地评估TSP2缺陷亚内皮基质的血栓形成特性，以及TSP2缺陷血小板粘附和聚集到暴露于流动血液的适当底物上的能力。这些研究将有助于明确TSP2在血管生物学中的作用。将血小板拴在血栓形成表面的初始相互作用与快速激活相结合，这是向不可逆粘附和聚集发展所严格要求的。在具体的目标4中，我们建议研究可能由不同底物引发的信号机制，并可能直接促进流动血液中血栓形成的过程。此外，我们将使用基因操作的小鼠血小板来确定整合素alphaIIbbeta3亚基的细胞质域靶向Tyr->Phe突变的后果，这些突变似乎干扰了信号传导过程。这些研究将有助于阐明与不同血流动力学条件相关的特定细胞外基质成分相互作用和血小板活化之间的整合。在具体目标5中，我们将使用实时三维测量来研究纤维蛋白原、纤维连接蛋白和玻璃体连接蛋白对不同底物上vwf引发的血栓的发展和稳定性的影响，这些血栓暴露于正常和病理动脉剪切速率下的血液流动。这些研究的结果将促进我们对血栓形成机制的理解，通过对流动血液进行定量分析，从而为解释体内生物学功能和病理疾病过程提供必要的新信息。

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.