Thrombotic disease in both the arterial and venous circulation is a major cause of morbidity and mortality in the United States. In vivo studies in mouse models of thrombosis are beginning to challenge paradigms of initiation and extension of thrombi largely based on in vitro and ex vivo studies. Interaction of blood proteins and cells with the vessel wall is influenced by shear forces within the vessel and by other vascular bed specific factors including heterogeneity of endothelial cells. This heterogeneity of vascular beds leads to differences in the process of thrombus formation at various vascular sites. We have developed a unique brightfield and confocal intravital microscopy system to study thrombus formation in vivo. Using this instrument we have determined the kinetics of thrombus formation after laser induced endothelial cell injury in the arterioles of the mouse cremaster muscle, have begun to explore the incorporation of components of the thrombus and have shown a role for P-selectin and PSGL-1 in thrombus formation. Here we propose to explore differences in thrombus formation in additional vascular beds including venules of the cremaster muscle and the arterioles and venules of the pia mater with its unique endothelial cell population. We will study the incorporation of thrombus components and kinetics or thrombus formation in the arterioles and venules of the cremaster muscle and the pia materin three mouse models of thrombosis. We will determine incorporation of platelets, fibrin, tissue, factor, white cells, red cells and microparticles into the growing thrombus; the kinetics of thrombus formation; the stability of the thrombus and the exposure of sub-endothelial proteins in the different thrombosis models. We will extend our studies of the role of P-selectin in thrombus formation by determining the importance of endothelial cell and platelet P-selectin in three models of thrombus formation in different vascular beds. To distinguish the roles of P-selectin from platelets and endothelial cells we will use bone marrow transplant to generate mice that have P-selectin in only one cell type and repeat the studies of thrombus formation in these mice in the different vascular beds. In order to explore the importance of thrombin activation of platelets in thrombus formation we will repeat the studies of thrombus formation in the various vascular beds and in the different models of thrombosis in PAR 4 and PAR 3 null mice.
