Association of tissue factor pathway inhibitor with endothelium

摘要 组织因子途径抑制物（TFPI）是一种内皮相关抗凝蛋白 直接抑制组织因子-因子VIIa（TF-fVIIa）催化复合物， 血管内血栓形成、弥散性血管内凝血、肿瘤转移和 与脉管系统内TF产生相关的其他病理。TF-fviia 通过因子IX和X的裂解激活血液凝固，并通过 蛋白酶激活受体（PAR）。我们的长期目标是发展 预防和治疗血管内TF活性的副作用的方法 基于通过TFPI对其抑制的研究对其病理生物学的理解。 TFPI以三种不同组织分布的选择性剪接同种型制备， 结构域结构和细胞表面缔合机制。我们假设 检查TFPI的结构多样形式，具有不同的细胞增殖机制， 表面缔合将揭示其抑制TF介导的细胞凋亡的能力的可变功效。 促凝血和/或促炎活性。通过描述 TFPI如何下调TF活性，我们将更好地了解 TF血管内表达介导的疾病。设计了三个具体目标 使用体外和体内模型来确定不同形式的TFPI的功能 系统. SA 1将比较不同形式的GPI锚定的TFPI的抗TF活性 在CHO细胞和原代小鼠内皮细胞的表面上。SA 2将检查如何 膜结合模式（GPI锚定与跨膜）TFPI活性对 CHO细胞、气溶素抗性EA.hy926细胞和原代小鼠内皮细胞。 还将进行研究，以检查小窝蛋白-1和GPI锚定的共- TFPI-α受体影响TFPI抑制活性。在SA 3中，我们将产生转基因的 使用可诱导的和可逆的方法特异性表达TFPI-α或TFPI-β的小鼠 四环素控制的反式激活因子系统。转基因小鼠将与 TFPI（+/-）小鼠，以确定是否存在胚胎致死的同种型特异性拯救。

Abstract Tissue Factor Pathway Inhibitor (TFPI) is an endothelial associated anticoagulant protein that directly inhibits the Tissue Factor-factor VIIa (TF-fVIIa) catalytic complex preventing intravascular thrombosis, disseminated intravascular coagulation, tumor metastasis and other pathologies associated with production of TF within the vasculature. TF-fVIIa activates blood clotting via cleavage of factors IX and X and initiates inflammation via activation of Protease Activated Receptors (PARs). Our long-term goal is to develop methods for prevention and treatment of the adverse effects of intravascular TF activity based upon understanding its pathobiology through studies of its inhibition by TFPI. TFPI is made in three alternatively spliced isoforms that differ in their tissue distribution, domain structure, and mechanism for cell surface association. We hypothesize that examination of structurally diverse forms of TFPI with distinct mechanisms for cell surface association will reveal variable efficacies in their ability to inhibit TF-mediated pro-coagulant and/or pro-inflammatory activity. By characterizing the unique features of how TFPI down-regulates TF activity we will better understand the pathogenesis of diseases mediated by intravascular expression of TF. Three Specific Aims are designed to define the functions of different forms of TFPI using both in vitro and in vivo model systems. SA1 will compare the anti-TF activity of different forms of GPI-anchored TFPI on the surface of CHO cells and primary mouse endothelial cells. SA2 will examine how the mode of membrane association (GPI-anchored vs. transmembrane) TFPI activity on CHO cells, aerolysin resistant EA.hy926 cells and primary mouse endothelial cells. Studies will also be performed to examine how caveolin-1 and the GPI-anchored co- receptor for TFPI-alpha impact TFPI inhibitory activity. In SA3 we will produce transgenic mice specifically expressing TFPI-alpha or TFPI-beta using an inducible and reversible tetracycline controlled transactivator system. The transgenic mice will be bred with TFPI(+/-) mice to determine if there is isoform specific rescue of embryonic lethality.