A Novel Form of Tissue Factor and Cardiovascular Disease

描述（由申请人提供）：组织因子（TF）是一种调节止血的跨膜糖蛋白，被认为在介导动脉血栓形成中起关键作用。在动物模型中，TF还介导了动脉损伤的内膜反应和缺血再灌注损伤的损伤程度。TF被认为以单一形式存在，其活性取决于其在细胞膜中的插入。最近在组织和循环中的微粒中发现了TF。此外，还发现了一种选择性剪接形式的TF (asTF)，它不包括外显子5，并包含一个帧移，产生一个独特的没有跨膜结构域的c端。asTF的活性需要暴露于脂质，但不需要插入脂质双分子层。asTF已从血液中分离出来，是体外和体内血栓的主要成分。研究人员推测，asTF在调节动脉血栓形成、介导正常心脏止血、介导内膜增生和缺血再灌注反应等方面发挥着关键作用。本提案将研究asTF和全长TF （flTF）在细胞培养和基因工程小鼠中的调控和生物学作用。目的1将建立平滑肌细胞、巨噬细胞、内皮细胞和心肌细胞中asTF表达的时间过程，并确定选择性剪接是否具有激动剂、组织和发育特异性。它还将确定，作为一种可溶性分子，asTF是否与血管细胞特异性结合，并表现出与其启动凝血能力不同的激动剂特性。目的2将建立asTF在止血、血栓形成和tnf介导的损伤反应中的作用。小鼠将产生一种或两种形式的TF由内源性TF启动子调节。研究人员将验证以下假设：1)flTF对胚胎存活至关重要；2) asTF是动脉损伤部位形成明显血栓的关键；3) asTF在动脉损伤时调节内膜反应中起关键作用；4) flTF在介导正常止血中起主导作用，而asTF在心肌毛细血管水平介导止血中起重要作用；5) asTF和flTF在缺血-再灌注损伤中起互补作用；6) asTF介导正常细胞功能，包括生长、迁移和粘附。这些研究将为一种可能在心血管疾病中起关键作用的新型循环TF提供见解。

DESCRIPTION (provided by applicant): Tissue factor (TF) is a transmembrane glycoprotein that regulates hemostasis and is thought to play a critical role in mediating arterial thrombosis. In animal models, TF also mediates the intimal response to arterial injury and the extent of damage resulting from ischemia-reperfusion injury. TF was thought to exist as a single form whose activity was dependent upon its insertion in the cell membrane. TF has recently been found in microparticles that are present in tissues and in the circulation. In addition, an alternatively spliced form of TF (asTF) has been identified that excludes exon 5 and contains a frame shift that generates a unique C-terminus without a transmembrane domain. asTF activity requires exposure to lipids, but not insertion into a lipid bilayer. asTF has been isolated from blood and is a major component of ex vivo and in vivo thrombi. The investigators hypothesize that asTF plays critical roles in regulating arterial thrombosis, in mediating normal cardiac hemostasis, and in mediating intimal hyperplasia and the response to ischemia-reperfusion. This proposal will examine the regulation and biologic role(s) of asTF and full-length TF (flTF) in cell culture and in genetically engineered mice. Aim 1 will establish the time course of asTF expression in smooth muscle cells, macrophages, endothelial cells, and cardiocytes and determine whether alternative splicing exhibits agonist, tissue and developmental specificity. It will also determine whether, as a soluble molecule, asTF binds specifically to vascular cells and exhibits agonist properties distinct from its ability to initiate coagulation. Aim 2 will establish the role of asTF in hemostasis, thrombosis, and in TF-mediated responses to injury. Mice will be generated in which one or both forms of TF are regulated by the endogenous TF promoter. The investigators will test the hypotheses that: 1) flTF is critical for embryonic survival; 2) asTF is critical for generating significant thrombosis at sites of arterial injury; 3) asTF plays a pivotal role in regulating the intimal response to arterial injury; 4) flTF plays the dominant role in mediating normal hemostasis, but asTF is important in mediating hemostasis at the level of the myocardial capillaries; 5) asTF and flTF play complementary roles in mediating ischemia-reperfusion injury; and 6) asTF mediates normal cell functions, including growth, migration, and adhesion. These studies will provide insights into a novel form of circulating TF that may play a key role in cardiovascular disease.