Association of tissue factor pathway inhibitor with endothelium

说明(申请人提供)：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VIIa(TF-FVIIa)催化复合体，防止血管内血栓形成、弥漫性血管内凝血、肿瘤转移和其他与血管内TF产生相关的病理改变。Tf-FVIIa通过裂解因子IX和X来激活凝血，并通过激活蛋白酶激活的受体(PARs)来启动炎症。我们的长期目标是通过研究TFPI对血管内转铁蛋白活性的抑制作用，在了解其病理生物学的基础上，开发预防和治疗血管内转铁蛋白活性不良影响的方法。TFPI由三种不同的剪接异构体组成，它们的组织分布、结构域结构和细胞表面结合机制不同。我们假设，对具有不同细胞表面结合机制的不同结构形式的TFPI的研究将揭示它们抑制TF介导的促凝血和/或促炎活性的能力的不同效果。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TF血管内表达介导的疾病的发病机制。使用体外和体内模型系统，设计了三个特定的目标来定义不同形式的TFPI的功能。SA1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的抗TF活性。SA2将研究膜结合模式(GPI锚定与跨膜)TFPI在CHO细胞、抗溶气素的EA.hy926细胞和原代小鼠内皮细胞上的活性。还将进行研究，以检查小窝蛋白-1和GPI锚定的TFPI-α的共同受体如何影响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反式激活系统来产生特异性表达TFPI-α或TFPI-β的转基因小鼠。转基因小鼠将与TFPI()小鼠杂交，以确定是否存在同型特异性的胚胎致死性挽救。公共卫生相关性：这项建议与公共健康相关，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栓的发展，同时避免了可能导致心脏病发作或中风和增加肿瘤转移的血管内血栓的发展。

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.