Tissue factor in smooth and cardiac muscle

组织因子（TF）是启动凝血的细胞表面糖蛋白。最近的研究表明，活性TF在血液中以微粒和白细胞亚群的形式循环。这种循环TF可能在引发和传播血栓形成中起重要作用。除了其在单核细胞/巨噬细胞和内皮细胞中的充分表征的表达之外，TF还在“活化的”动脉平滑肌细胞（SMC）和成体心肌细胞中丰富。我们推测，在动脉损伤和心肌缺血的反应，这些细胞释放TF进入循环，促进高凝状态。我们还假设SMC和心肌细胞衍生的TF在介导血管损伤的迁移/增殖反应和心肌缺血的影响中是重要的。目的1-检查心肌细胞来源的TF的作用。尽管TF抗原在成年心肌细胞中含量丰富，但在梗死区域明显减少。最近的研究表明，不稳定型心绞痛和心肌梗死（MI）患者以及接受冠状动脉肺动脉搭桥术的患者血液TF水平升高。血液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抑制剂减少缺血/再灌注损伤。这种减少的机制尚未确定。我们将测试缺血和梗死释放TF从心脏进入循环和心肌来源的TF在介导缺血/再灌注损伤中发挥作用的假设。A）初始研究将检查TF在猪MI模型中的表达。冠状窦和外周血TF活性的变化将与肌酸激酶（CK）水平和梗死面积相关，并与使用流动室离体评估的血液血栓形成相关。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系统产生心肌细胞中缺乏TF的小鼠。我们将建立在基础条件下和心肌梗死后心肌细胞对TF血液水平的贡献，并将评估心肌TF在介导梗死面积中的潜在作用。我们还将确定心肌TF是否在成人心脏和离体心肌细胞中起非止血作用。目的2 -检查来自SMC的TF的作用。TF由损伤在动脉中膜中诱导并在新生内膜中积聚。动物模型中血栓形成主要发生在新生内膜损伤（双重损伤）而非正常动脉损伤（单一损伤）后，表明暴露潜在活性TF而非从头合成是必要的。使用离体流动室的研究表明，损伤动脉壁中的活性TF可释放到循环中。实验诱导的和天然的血栓对TF有强烈的染色;这种TF的主要成分可能来自循环血液。这些数据提高了动脉血栓形成涉及暴露于下层动脉中的活性TF和来自循环的TF沉积的可能性。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，即动脉损伤的速率释放TF到血液中。B）我们将这些研究扩展到猪单、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相关联。C）我们将使用Cre/lox系统产生SMC中缺乏TF的小鼠。我们将确定动脉损伤后SMC对血液TF水平的贡献。我们将确定SMC衍生的TF在动脉血栓形成、内膜增生中的作用，以及TF抑制剂对内膜增生和SMC迁移的介导作用。

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.