This proposal characterizes the relationship between expression of selectins/selectin ligands and thrombus formation during acute vascular inflammation. We have established a link between selectins, inflammation and thrombosis: [1] leukocytes accumulate in the developing thrombus, and this accumulation and fibrin deposition are P-selectin-mediated; [2] P-selectin initiates the de novo synthesis of tissue factor in monocytes. Our goals are to explore this relationship employing in vivo mouse models, including mice genetically deficient in the selectins or the selectin ligands, using state-of- the-art real time intravital high speed confocal and wide-field microscopy to monitor laser-induced and endotoxin-induced vascular activation and thrombosis. The hypothesis to be tested is whether selectin-mediated leukocyte/leukocyte membrane fragment/microparticle accumulation in areas of acute inflammation is a physiologic pathway for delivering tissue factor for the initiation of blood coagulation in the injured vasculature. Our goal is to determine if this inflammatory process is required for thrombus generation using real time intravital high speed confocal and widefield microscopy and vascular activation by mild trauma, cytokines, endotoxin or laser-induced endothelial injury. The density of P- and E-selectin on the activated endothelium will be studied as a function of time after injury. The kinetics and magnitude of fibrin, vWf, leukocyte and platelet deposition in an experimental thrombus following vascular injury will be determined. Using antibodies to tissue factor, we will study by intravital microscopy the accumulation of tissue factor in the thrombus and surrounding vascular wall during thrombogensis. To detect tissue factor regardless of its biologic activity or cell surface exposure, a knockin mouse will be prepared that expresses tissue factor as a fusion protein with enhanced green fluorescent protein. The localization of tissue factor on leukocytes, platelets and microparticles derived from these cells will be monitored during thrombus formation. Furthermore, we will study the effect of PSGL-1 deficiency and selectin deficiency on tissue factor expression following endotoxin exposure in mice by intravital microscopy. Microparticles, tissue factor and fibrin in the thrombus will be quantitated in wild type and genetically modified mice. To prove the physiologic importance of this pathway, we will attempt to rescue homozygous protein C deficient mice from fatal thrombosis by PSGL-1 deficiency.
