Tissue factor in smooth and cardiac muscle

组织因子（TF）是一种启动凝血的细胞表面糖蛋白。最近的研究表明，活性TF在血液中以微粒和一部分白细胞的形式循环。这种循环的TF可能在血栓形成和传播中起重要作用。除了在单核细胞/巨噬细胞和内皮细胞中表达，TF也大量存在于“活化”的动脉平滑肌细胞（SMC）和成人心肌细胞中。我们推测，在动脉损伤和心肌缺血的反应中，这些细胞释放TF进入循环，促进高凝状态。我们还假设SMC和心肌细胞来源的TF在介导血管损伤和心肌缺血的迁移/增殖反应中起重要作用。目的1-研究心肌细胞来源的TF的作用。虽然在成人心肌细胞中大量存在，但在梗死区TF抗原明显减少。最近的研究表明，不稳定心绞痛和心肌梗死（MI）患者以及接受冠状动脉旁路手术的患者血TF水平升高。血TF的增加可能来自几个来源，包括冠状动脉壁、心肌细胞和活化的白细胞。最近的一些报道表明，TF抑制剂可以减轻缺血/再灌注损伤。这种减少背后的机制尚未确定。我们将验证缺血和梗死将TF从心脏释放到循环中，以及心肌来源的TF在介导缺血/再灌注损伤中发挥作用的假设。A)初步研究将检查猪心肌梗死模型中TF的表达。冠状动脉窦和外周血TF活性的变化将与肌酸激酶（CK）水平和梗死面积以及体外血液的血栓形成性评估相关。我们还将评估TF抑制剂减少梗死后心肌损伤的能力。B)我们将使用Cre/lox系统生成心肌细胞中缺乏TF的小鼠。我们将确定心肌细胞在基础条件下和心肌梗死后对TF血液水平的贡献，并评估心肌TF在调节梗死面积方面的潜在作用。我们还将确定心肌TF是否在成人心脏和分离心肌细胞中起非止血作用。目的2 -研究SMC衍生的TF的作用。TF在动脉介质中由损伤引起，并在新生内膜中积累。在动物模型中，血栓形成主要发生在新内膜损伤时（双损伤），而不是在正常动脉损伤后（单损伤），这表明暴露潜在活性TF而不是从头合成是必要的。使用离体血流室的研究表明，损伤动脉壁中的活性TF可能被释放到循环中。TF实验诱导和天然血栓染色；这种TF的很大一部分可能来自循环血液。这些数据提高了动脉血栓形成的可能性，即既涉及底层动脉中活性TF的暴露，也涉及循环中TF的沉积。许多研究表明，TF抑制剂可减少球囊动脉损伤后的内膜增生，并可能减弱生长因子诱导的SMC迁移。A)我们将证实我们的初步观察结果，即动脉损伤将TF释放到血液中。B)我们将把这些研究扩展到猪单冠状动脉和双冠状动脉损伤，并将血液TF活性的变化与血液的血栓形成性联系起来。C)我们将使用Cre/lox系统生成SMC中缺乏TF的小鼠。我们将确定动脉损伤后SMC对血TF水平的贡献。我们将建立SMC衍生的TF在动脉血栓形成、内膜增生的发展中的作用，并介导TF抑制剂对内膜增生和SMC迁移的影响。

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.