Circulating Tissue Factor: Characterization and Interactions with Factor IX

说明(申请人提供)：组织因子(Tf)是一种完整的膜糖蛋白，在接触血液成分后启动凝血级联反应。在经典模型中，TF在上皮细胞和血管外膜细胞中结构性表达，从而在周围形成一个止血包膜，但与血液分离。然而，内皮细胞和造血细胞可以表达TF，尤其是在炎症刺激后，已经推测循环TF的激活对血栓的传播是重要的。我们已经使用Tie2-Cre转基因来切除一个Tf等位基因，以产生造血和内皮细胞Tf已经被去除的小鼠(‘Tf Hem/Endo Knokout’)。使用这些小鼠的数据表明，内皮和造血因子对于内毒素血症小鼠模型的弥散性血管内凝血是必要的。然而，内皮和造血因子在体内平衡和炎症状态中的正常作用尚不清楚。我们应该问：特定的目标：1.内皮和造血因子对止血和血栓形成重要吗？在TFHEM/Endo基因敲除小鼠中，我们将通过定量RT-PCR检测Tf mRNA来评估循环Tf的消融。止血和血栓形成将使用经过充分验证的方案在小鼠身上进行测试。具体目的2.炎症刺激诱导内皮细胞和造血细胞因子是否促进止血和血栓形成？我们将通过暴露内毒素来诱导TF mRNA的表达，然后检测出血时间和血栓形成情况。增强的止血和/或血栓形成将支持内皮和造血因子可能在炎症和血栓形成之间提供联系的假设。特定目的3.缺乏因子IX的小鼠能否提供一种致敏系统，使我们可以检测循环中的转铁蛋白的正常作用？我们将通过检查缺乏循环转铁蛋白和因子IX的小鼠的止血和血栓形成来检验这些系统部分多余的假设。我们的研究试图确定循环转铁蛋白的作用，循环转铁蛋白在重要的人类炎症疾病状态中增加，以及它与内在凝血途径的相互作用。这些研究可能为这些炎症状态的治疗提供有吸引力的治疗靶点。

DESCRIPTION (provided by applicant): Tissue factor (TF) is an integral membrane glycoprotein that initiates the coagulation cascade after exposure to blood components. In classical models, TF is constitutively expressed in epithelia and vascular adventitial cells so as to create a "hemostatic envelope" around but separated from blood. However, endothelial cells and hematopoietic cells can express TF especially after inflammatory stimuli, and it has been postulated that activation of circulating TF is important for clot propagation. We have used a Tie2-Cre transgene to excise a floxed Tf allele to generate mice in which hematopoietic and endothelial cell Tf has been ablated ('Tf hem/endo knockout"). Data using such mice suggest that endothelial and hematopoietic Tf is necessary for disseminated intravascular coagulation in mouse models of endotoxemia. However, the normal role of endothelial and hematopoietic TF in homeostasis and in inflammatory states is unknown. We shallask: Specific Aim 1. Is endothelial and hematopoietic TF important for hemostasis and thrombosis? In Tf hem/endo knockout mice, we will assess ablation of circulating TF by quantitative RT- PCR for Tf mRNA. Hemostasis and thrombosis will be tested in mice using well-validated protocols. Specific Aim 2. Does induction of endothelial and hematopoietic TF by inflammatory stimuli promote hemostasis and thrombosis? We will induce TF mRNA expression via exposure to endotoxin and then examine bleeding times and thrombosis. Enhanced hemostasis and/or thrombosis would support the hypothesis that endothelial and hematopoietic TF may provide a link between inflammation and thrombosis. Specific Aim 3. Might mice lacking factor IX provide a sensitized system in which we might detect normal roles for circulating TF? We will test the hypothesis that these systems are partially redundant by examining hemostasis and thrombosis in mice that lack both circulating TF and factor IX. Our study seeks to define the role of circulating TF, which is increased in important human inflammatory disease states, and its interaction with the intrinsic coagulation pathway. These studies may provide attractive therapeutic targets for treatment of these inflammatory states.