DESCRIPTION (Applicant's list of Aims) Tissue factor (TF) is a cell surface glycoprotein that initiates coagulation. Recent studies have demonstrated that active TF circulates in the blood in microparticles and in a subset of leukocytes. This circulating TF may play an important role in initiating and propagating thrombosis. In addition to its well-characterized expression in monocyte/macrophages and endothelial cells, TF is also abundant in "activated" arterial smooth muscle cells (SMC) and in adult cardiomyocytes. We hypothesize that in response to arterial injury and myocardial ischemia, these cells release TF into the circulation, promoting a hypercoagulable state. We also hypothesize that SMC- and cardiomyocyte-derived TF are important in mediating the migratory/ proliferative response to vascular injury and the effects of myocardial ischemia. Aim 1- Examine the Role of TF Derived from Cardiomyocytes. Although abundant in adult cardiomyocytes, TF antigen is markedly reduced in areas of infarction. Recent studies have suggested that blood TF levels increase in patients with unstable angina and myocardial infarction (MI), and in patients undergoing coronary pulmonary bypass. The increase in blood TF may come from several sources including the coronary arterial wall, cadiomyocytes, and activated leukocytes. A number of recent reports have shown that TF inhibitors reduce ischemia/reperfusion injury. The mechanism underlying this reduction has not been determined. We will test the hypotheses that ischemia and infarction release TF from the heart into the circulation and that myocardial-derived TF plays a role in mediating ischemia/reperfusion injury. A) Initial studies will examine the expression of TF in a porcine model of MI. Changes in coronary sinus and peripheral blood TF activity will be correlated with levels of creatine kinase (CK) and infarct size and with the thrombogenicity of the blood assessed ex vivo using a flow chamber. We will also assess the ability of inhibitors of TF to reduce myocardial damage after infarction. B) We will generate mice lacking TF in their cardiomyocytes using the Cre/lox system. We will establish the contribution of cardiomyocytes to TF blood levels under basal conditions and after MI and will assess the potential role of myocardial TF in mediating infarct size. We will also determine whether myocardial TF plays a non-hemostatic role in adult hearts and isolated caridomyocytes. Aim 2 - Examine the role of TF Derived from SMC. TF is induced in the arterial media by injury and accumulates in the neointima. Thrombosis in animal models occurs predominantly when the neointima is injured (double injury) and not after injury to normal arteries (single injury), suggesting that exposure of potentially active TF, rather than de novo synthesis, is necessary. Studies using an ex vivo flow chamber suggest that active TF in the injured arterial wall may be released into the circulation. Experimentally induced and native thrombi stain intensely for TF; a substantial component of this TF is likely derived from circulating blood. These data raise the possibility that arterial thrombosis involves both exposure of active TF in the underlying artery and deposition of TF from the circulation. A number of studies have shown that inhibitors of TF reduce intimal hyperplasia after balloon arterial injury and may attenuate growth factor-induced SMC migration. A) We will confirm our preliminary observations that arterial injury in the rate releases TF into the blood. B) We will extend these studies to porcine single and double coronary arterial injury and will correlate changes in blood TF activity to the thrombogenicity of the blood. C) We will generate mice lacking TF in their SMC, using the Cre/lox system. We will determine the contribution of SMC to blood TF levels after arterial injury. We will establish the role of SMC-derived TF in the development of arterial thrombosis, intimal hyperplasia, and in mediating the effect of TF inhibitors on intimal hyperplasia and SMC migration.