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）是一种内皮相关的抗凝蛋白，可直接抑制组织因子-因子VIIa （TF- fviia）催化复合物，防止血管内血栓形成、弥散性血管内凝血、肿瘤转移和其他与血管内TF产生相关的病理。TF-fVIIa通过分裂因子IX和X激活血液凝固，并通过激活蛋白酶激活受体（PARs）引发炎症。我们的长期目标是通过研究TFPI对血管内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是由三种选择性剪接的异构体组成的，它们在组织分布、结构域结构和细胞表面结合机制上有所不同。我们假设，对具有不同细胞表面关联机制的不同结构形式的TFPI进行检查，将揭示其抑制tf介导的促凝血和/或促炎活性的能力的不同功效。通过表征TFPI如何下调TF活性的独特特征，我们将更好地了解TF在血管内表达介导的疾病发病机制。三个特定目标旨在定义不同形式的TFPI在体外和体内模型系统中的功能。SA1将比较不同形式gpi锚定的TFPI在CHO细胞和原代小鼠内皮细胞表面的抗tf活性。SA2将研究细胞膜结合模式（gpi锚定与跨膜）TFPI活性对CHO细胞、抗气溶素EA.hy926细胞和原代小鼠内皮细胞的影响。还将进行研究，以检查小窝蛋白-1和gpi锚定的TFPI- α共受体如何影响TFPI抑制活性。在SA3中，我们将使用可诱导和可逆的四环素控制的反激活剂系统产生特异性表达tfpi - α或tfpi - β的转基因小鼠。将转基因小鼠与TFPI（）小鼠杂交，以确定是否存在同种异构体特异性的胚胎致死性拯救。公共卫生相关性：该建议与公共卫生相关，因为它调查了细胞对损伤和炎症的反应，平衡了防止严重出血所需的血凝块的发展，同时避免了可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.