Protease Activated Receptor and Thrombomodulin Signaling by coagulation proteases

凝血蛋白酶激活的蛋白酶受体和血栓调节蛋白信号传导

基本信息

批准号：
10657134
负责人：
ALIREZA R. REZAIE
金额：
$ 42.63万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10657134
关键词：
Actins Active Sites Adenosine Monophosphate Angiopoietin-2 Anti-Inflammatory Agents Anticoagulants Binding Biological Assay Blood Vessels Catalytic Domain Cell Proliferation Cells Coagulation Process Complex Cytoplasmic Tail Cytoprotection Cytoskeleton Data Disease Docking EGF-Like Domain Endothelial Cells Endowment Exclusion F-Actin FOXO1A gene FRAP1 gene Gene Expression Genes Genetic Human Immunofluorescence Immunologic Impairment Inflammatory Inflammatory Response Intercellular Junctions Knock-out Knockout Mice Lectin Lentivirus Ligands Link Maintenance Mediating Membrane Microfilaments Monitor Myocardial Infarction NF-kappa B Neoplasm Metastasis Nuclear PAR-1 Receptor PTEN gene Pathway interactions Peptide Hydrolases Permeability Phenotype Phosphorylation Phosphotransferases Polymers Protein C Proteinase-Activated Receptors Proteins Prothrombin Proto-Oncogene Proteins c-akt Public Health Recombinants Regulation Reproducibility Research Sampling Sepsis Series Signal Transduction Signaling Molecule Site Specificity Stroke Surface System Tertiary Protein Structure Thrombin Thrombomodulin Thrombosis Tissue Sample Tissues Transfection Tumor Suppressor Proteins Vascular Endothelial Cell Western Blotting activated Protein C activated protein C receptor cadherin 5 novel therapeutics overexpression polymerization protein activation receptor receptor binding response thrombotic von Willebrand Factor

项目摘要

Project Summary Thrombomodulin (TM) is an integral membrane receptor on endothelial cells which binds to exosite- 1 of thrombin to switch the specificity of thrombin from a procoagulant to an anticoagulant protease. The interaction with TM enables thrombin to activate protein C to activated protein C (APC). In addition to its anticoagulant function, APC binds to endothelial protein C receptor (EPCR) to cleave protease-activated receptor 1 (PAR1) at Arg46 site to elicit cytoprotective responses in endothelial cells. The exosite-1-dependent interaction of thrombin with TM on endothelial cells inhibits the thrombin recognition of PAR1 and procoagulant substrates. TM exerts direct anti-inflammatory functions through its lectin-like domain by unknown mechanisms. Recent results have indicated an association between loss of TM expression and uncontrolled cell proliferation and metastasis. Based on our preliminary data in this application, we hypothesize that the cytoplasmic domain of TM is linked to the actin cytoskeleton and involved in the regulation of PTEN/AKT/mTOR signaling axis. TM endows a quiescence phenotype to endothelial cells through modulation of this signaling axis under basal and stimulated conditions. We demonstrate that interaction of EGF-like domains of TM with exosite-1 of thrombin and proexosite-1 of prothrombin leads to activation (phosphorylation at Ser172) of AMPK, thereby TM differentially regulating mTORC1 (inhibition) and mTORC2 (activation) signaling. We hypothesize by this signaling mechanism, TM contributes to regulation of the vascular tone, maintenance, and stabilization of the barrier permeability function of the vasculature under steady-state and stimulated conditions. We further demonstrate TM changes the PAR1 cleavage specificity of thrombin from Arg41 site to Arg46 site, thereby switching the PAR1- dependent signaling specificity of thrombin from a proinflammatory response to a cytoprotective one. We have prepared a series of TM receptor constructs, recombinant APC and thrombin derivatives and TM-null and PAR1-null endothelial cells to investigate the following three Specific Aims: Aim 1 will investigate the mechanism by which TM maintains a quiescence phenotype in endothelial cells. Aim 2 will investigate the hypothesis that the cytoplasmic domain of TM is linked to the actin cytoskeleton and involved in regulation of mTOR signaling upon interaction with ligands thrombin and prothrombin. Aim 3 will investigate mechanisms through which EPCR and TM modulate the signaling specificity of PAR1 cleavage by coagulation proteases in endothelial cells.

项目概要血栓调节蛋白 (TM) 是内皮细胞上的一种整合膜受体，可与外部位点结合 1 凝血酶将凝血酶的特异性从促凝蛋白酶转变为抗凝蛋白酶。与 TM 的相互作用使凝血酶能够将蛋白 C 激活为活化蛋白 C (APC)。在除了抗凝功能外，APC 还与内皮蛋白 C 受体 (EPCR) 结合以裂解 Arg46 位点的蛋白酶激活受体 1 (PAR1) 可引发内皮细胞的细胞保护反应细胞。内皮细胞上凝血酶与 TM 的外位点 1 依赖性相互作用抑制凝血酶识别 PAR1 和促凝血底物。 TM具有直接抗炎作用通过其凝集素样结构域以未知的机制发挥作用。最近的结果表明 TM 表达缺失与不受控制的细胞增殖和转移之间的关联。基于根据我们在本申请中的初步数据，我们假设 TM 的细胞质结构域是与肌动蛋白细胞骨架相连，参与 PTEN/AKT/mTOR 信号轴的调节。 TM 通过调节该信号轴赋予内皮细胞静止表型在基础和刺激条件下。我们证明了 TM 的 EGF 样结构域的相互作用与凝血酶的 exosite-1 和凝血酶原的 proexosite-1 结合导致激活（磷酸化 AMPK 的 Ser172)，从而 TM 差异调节 mTORC1（抑制）和 mTORC2 （激活）信号。我们假设通过这种信号机制，TM 有助于调节血管张力、维持和稳定屏障通透性功能稳态和刺激条件下的脉管系统。我们进一步证明 TM 改变了凝血酶的 PAR1 裂解特异性从 Arg41 位点到 Arg46 位点，从而将 PAR1- 凝血酶从促炎反应到细胞保护反应的依赖性信号传导特异性。我们制备了一系列TM受体构建体、重组APC和凝血酶衍生物以及 TM-null 和 PAR1-null 内皮细胞，以研究以下三个具体目标：目标 1 将研究TM在内皮细胞中维持静止表型的机制。目标 2 将研究 TM 的细胞质结构域与肌动蛋白相关的假设细胞骨架并参与与配体凝血酶相互作用时 mTOR 信号传导的调节和凝血酶原。目标 3 将研究 EPCR 和 TM 调节的机制内皮细胞中凝血蛋白酶对 PAR1 裂解的信号传导特异性。