TFPI ALPHA AND TFPI BETA

描述（由申请方提供）：组织因子途径抑制剂（TFPI）对因子VIIa/组织因子（FVIIa/TF）诱导的凝血产生因子Xa（FXa）依赖性反馈抑制。在人类中，TFPI表达为两种同种型。TFPI 1含有三个串联的Kunitz型蛋白酶抑制剂结构域，Kunitz-1结合FVIIa/TF复合物中的FVIIa，第二个结合FXa。Kunitz-3不具有蛋白酶抑制剂活性。TFPI 2缺少Kunitz-3和TFPI 1的C-末端。后者是TFPI 1对FXa的最佳抑制及其在一期凝血试验中的抗凝活性所需的。TFPI 1与蛋白S（PS）相互作用，增强其抗FXa活性，并与因子V（FV）相互作用，可延长其从血浆中的清除。两种形式的TFPI以糖基磷脂酰肌醇（GPI）-锚依赖性方式与细胞表面缔合，但通过不同的机制。存在于血浆中并由活化的血小板分泌的TFPI 1似乎与另一种GPI锚定蛋白结合，而TFPI 2含有内在的GPI锚。我们计划确定TFPI 1内负责其与PS和FV结合的结构，并进一步探索其与细胞表面的相互作用。将鉴定与脂蛋白结合的TFPI循环形式，并研究TFPI潜在的其他亚型TFPI 4的相关性。TFPI基因破坏的小鼠（TFPI KO）死于子宫内广泛的血栓形成和消耗性凝血病，但被低水平的两种具有FVIIa/TF抑制活性的转基因中的任一种所拯救。一种是Tie 2-hTFPI 1，其在内皮细胞中指导表达，另一种是Tf-mXK 1-hAlb，其在血浆中指导表达。转基因挽救的TFPI KO小鼠和适当的骨髓移植研究将用于评估血小板相关TFPI 1的作用，并使用小鼠模型确定低水平内源性TFPI对血栓形成、炎症和动脉粥样硬化的影响。公共卫生相关性：TFPI是一种血浆和细胞相关蛋白，以两种亚型（TFPI 1和TFPI 2）存在，在调节凝血起始中起重要作用。该提案的目标是确定其与循环脂蛋白，细胞表面和其他两种凝血相关蛋白，蛋白S和因子V的相互作用的机制和相关性，并确定TFPI缺乏对血栓形成，炎症和动脉粥样硬化的影响，使用小鼠模型。

DESCRIPTION (provided by applicant): Tissue factor pathway inhibitor (TFPI) produces factor Xa (FXa)-dependent feedback inhibition of factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. In humans, TFPI is expressed as two isoforms. TFPI1 contains three tandem Kunitz-type protease inhibitor domains, Kunitz-1 binds FVIIa in the FVIIa/TF complex and the second binds FXa. Kunitz-3 does not possess protease inhibitor activity. TFPI2 lacks the Kunitz-3 and the C-terminus of TFPI1. The latter is required for TFPI1's optimal inhibition of FXa and its anticoagulant activity in one-stage coagulation assays. TFPI1 interacts with protein S (PS), which enhances its anti-FXa activity, and with factor V (FV), which may prolong its clearance from plasma. Both forms of TFPI associate with the surface of cells in a glycosylphosphatidylinositol (GPI)-anchor dependent fashion, but through different mechanisms. TFPI1, present in plasma and secreted by activated platelets, appears to bind to another GPI-anchored protein(s), whereas TFPI2 contains an intrinsic GPI-anchor. We plan to determine the structures within TFPI1 responsible for its binding to PS and FV and further explore its interactions with cell surfaces. The form(s) of TFPI, which circulate bound to lipoproteins, will be identified and the relevance of a potential additional isoform of TFPI, TFPI4, will be investigated. TFPI gene-disrupted mice (TFPI KO) die intrautero of widespread thrombosis and a consumptive coagulopathy, but are rescued by a low level of either of two transgenes with FVIIa/TF inhibitory activity. One, Tie2-hTFPI1, directs expression in endothelial cells, the other, Tf-mXK1-hAlb directs expression into plasma. Transgene-rescued TFPI KO mice and appropriate bone marrow transplantation studies will be used to assess the role of platelet- associated TFPI1 and to determine the effect of low levels of endogenous TFPI on thrombosis, inflammation, and atherosclerosis using mouse models. Public Health Relevance: TFPI is a plasma and cell-associated protein, which exists as two isoforms (TFPI1 and TFPI2) and plays an important role in regulating the initiation of coagulation. The goals of this proposal are to determine the mechanism and relevance of its interactions with circulation lipoproteins, cell surfaces, and two other coagulation-related proteins, Protein S and Factor V, and to determine the effects of TFPI deficiency on thrombosis, inflammation, and atherosclerosis using mouse models.