Association of tissue factor pathway inhibitor with endothelium

描述（由申请人提供）：组织因子途径抑制剂（TFPI）是一种内皮相关抗凝蛋白，可直接抑制组织因子-因子VIIa（TF-fVIIa）催化复合物，预防血管内血栓形成、弥散性血管内凝血、肿瘤转移和与血管系统内TF产生相关的其他病理。TF-fVIIa通过切割因子IX和X激活血液凝固，并通过激活蛋白酶激活受体（PAR）引发炎症。我们的长期目标是通过研究TFPI对TF活性的抑制作用，在了解其病理生物学的基础上，开发预防和治疗血管内TF活性不良反应的方法。TFPI以三种选择性剪接的同种型制备，其在组织分布、结构域结构和细胞表面缔合机制方面不同。我们假设，检查具有不同细胞表面缔合机制的TFPI的结构多样形式将揭示其抑制TF介导的促凝血和/或促炎活性的能力的可变功效。通过表征TFPI如何下调TF活性的独特特征，我们将更好地理解由TF的血管内表达介导的疾病的发病机制。设计了三个特定目的，以使用体外和体内模型系统来定义不同形式的TFPI的功能。SA 1将比较不同形式的GPI锚定的TFPI在CHO细胞和原代小鼠内皮细胞表面上的抗TF活性。SA 2将检查膜结合模式（GPI锚定与跨膜）TFPI活性如何作用于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TFPI-a的GPI锚定共受体如何影响TFPI抑制活性。在SA 3中，我们将使用可诱导和可逆的四环素控制的反式激活因子系统产生特异性表达TFPI-α或TFPI-β的转基因小鼠。转基因小鼠将与TFPI（）小鼠交配，以确定是否存在同种型特异性挽救胚胎致死性。公共卫生相关性：该提案与公共卫生相关，因为它研究了细胞对损伤和炎症的反应，平衡了预防严重出血所需的血凝块的发展，同时避免了可能导致心脏病发作或中风并增强肿瘤转移的血管内血凝块的发展。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This proposal is relevant to public health as it investigates cellular responses to injury and inflammation, balancing the development of blood clots necessary to prevent severe bleeding while at the same time avoiding the development of intravascular blood clots that can result in heart attack or stroke and enhance tumor metastasis.