TFPI ALPHA AND TFPI BETA

描述(申请人提供)：组织因子途径抑制物(TFPI)产生因子Xa(FXA)依赖的反馈抑制因子VIIa/组织因子(FVIIa/Tf)诱导的凝血。在人类中，TFPI表达为两种异构体。TFP Iα含有三个串联的kunitz型蛋白酶抑制域，kunitz-1结合FVIIa/Tf复合体中的FVIIa，第二个结合FxA。库尼茨-3不具有蛋白酶抑制活性。TfpIβ缺乏kunitz-3和α的C末端。后者是αS在一期凝血试验中对FXA的最佳抑制和抗凝活性所必需的。α可与S蛋白(PS)和因子V(FV)相互作用，前者可增强其抗FXA活性，后者可延长其从血浆中的清除。这两种形式的TFPI都以糖基磷脂酰肌醇(GPI)锚点依赖的方式与细胞表面结合，但机制不同。TFP Iα存在于血浆中，由活化的血小板分泌，似乎与另一种GPI锚定蛋白(S)结合，而TFP Iβ含有固有的GPI锚定蛋白。我们计划确定TFP Iα中负责其与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。将确定结合脂蛋白循环的TFP I的形式(S)，并将研究TFP I的一个潜在的附加异构体TFP I4的相关性。TFPI基因破坏的小鼠(TFPI KO)死于广泛的血栓形成和消耗性凝血障碍，但通过两种具有FVIIa/Tf抑制活性的转基因之一的低水平挽救。一种是Tie2-hTFP Iα，它在内皮细胞中直接表达，另一种是Tf-mXK1-HALB，它直接在血浆中表达。转基因挽救的TFP I KO小鼠和适当的骨髓移植研究将用于评估血小板相关TFP Iα的作用，并通过小鼠模型确定低水平内源性TFP I对血栓形成、炎症和动脉粥样硬化的影响。

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. TFPIα 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. TFPIβ lacks the Kunitz-3 and the C-terminus of TFPIα. The latter is required for TFPIα's optimal inhibition of FXa and its anticoagulant activity in one-stage coagulation assays. TFPIα 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. TFPIα, present in plasma and secreted by activated platelets, appears to bind to another GPI-anchored protein(s), whereas TFPIβ contains an intrinsic GPI-anchor. We plan to determine the structures within TFPIα 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-hTFPIα, 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 TFPIα and to determine the effect of low levels of endogenous TFPI on thrombosis, inflammation, and atherosclerosis using mouse models.