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.
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