Protease Activated Receptor Signaling by Coagulation Proteases

凝血蛋白酶激活的蛋白酶受体信号传导

基本信息

批准号：
8054797
负责人：
ALIREZA R. REZAIE
金额：
$ 37.53万
依托单位：
SAINT LOUIS UNIVERSITY
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 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8054797
关键词：
Alkaline Phosphatase Anterior Descending Coronary Artery Anti-Inflammatory Agents Anticoagulants Binding Biological Assay Blood coagulation Cardiac Myocytes Caveolae Cell surface Cellular Assay Chimera organism Cleaved cell Coagulation Process Collaborations Complex Coupling Data Development Disease Dissociation Drug Controls Endothelial Cells Engineering Enzyme Precursors Event Extracellular Domain FDA approved G-Protein-Coupled Receptors GTP-Binding Proteins Generations Glutamine Heart In Vitro Incubated Inflammation Inflammatory Lead Left Ligands Ligation Lung Mediating Membrane Microdomains Methods Modeling Monitor Mus Myocardial Infarction Names Organ Organ failure PAR-1 Receptor PAR-2 Receptor Pathway interactions Peptide Hydrolases Permeability Physiological Processes Plasma Proteins Platelet Factor 4 Property Protein C Proteinase-Activated Receptors Proteins Receptor Activation Receptor Signaling Recruitment Activity Regulation Reperfusion Injury Reporter Role Sepsis Septic Shock Signal Pathway Signal Transduction Signaling Molecule Specificity Sphingosine-1-Phosphate Receptor Therapeutic Thrombin Variant Vascular Endothelial Cell Vitamin K Vitamins activated Protein C angiogenesis base caveolin 1 chymotrypsin cytokine disulfide bond improved in vivo Model mutant novel prevent protease-activated receptor 4 public health relevance receptor response rho GTP-Binding Proteins

项目摘要

DESCRIPTION (provided by applicant): Coagulation proteases, in addition to their role in the regulation of blood coagulation, can modulate intracellular signaling events by activating a subfamily of G-protein coupled receptors named protease-activated receptors (PARs) expressed on the cell surface of various organs. In vitro studies have indicated that while the anticoagulant protease, activated protein C (APC), in complex with endothelial protein C receptor (EPCR) elicits a barrier protective response via activation of PAR-1 in vascular endothelial cells, thrombin elicits a barrier disruptive response via the activation of the same receptor. APC has been approved by the FDA for treatment of severe sepsis. It has been hypothesized that the activation of PAR-1 by APC on endothelial cells is responsible, at least partially, for its protective properties in preventing organ failure (i.e., lung and heart) which is caused by septic shock in severe sepsis. Thus, it is highly important to understand how the activation of PAR-1 by either APC or thrombin elicits paradoxical protective and disruptive cellular responses, respectively. We recently discovered that the activation of PAR-1 by thrombin can also elicit barrier protective cellular responses if EPCR is occupied by the Gla-domain of protein C. We demonstrated that the unoccupied EPCR together with PAR-1 are both associated with caveolin-1 within lipid-rafts of endothelial cells. Under these conditions, PAR-1 appears to signal via Gq and/or G12/13 subfamily of G-proteins, and thus thrombin activation of PAR-1 leads to disruptive cellular responses. However, we discovered that the occupancy of EPCR by protein C results in dissociation of EPCR from caveolin-1, thereby recruiting PAR-1 to protective pathways by likely coupling it to Gi-protein. Based on our recent preliminary data, we hypothesize that the occupancy of EPCR by protein C can also change the signaling specificity of PAR-2. Thus, it appears that the cell surface occupancy of EPCR determines the signaling specificity of activated PAR-1 and PAR-2 not the proteases that are cleaving them. To understand the PAR-dependent signaling specificity of coagulation proteases, we have prepared several mutant proteases and receptors and set up appropriate in vitro and in vivo models to investigate the following four Specific Aims. In Aim 1, we will investigate the PAR-1- and PAR- 2-dependent signaling specificity of coagulation proteases under conditions in which endothelial cells have been pre-incubated with vitamin K-dependent coagulation zymogens so that to occupy EPCR or other potential receptors by their natural ligands. In Aim 2, we will investigate the contribution of the Gla-domain of vitamin Kdependent coagulation proteases to the recognition and cleavage specificity of PARs localized into the lipidrafts. In Aim 3, we will develop novel APC variants which have improved PAR-1-dependent protective signaling properties, but have reduced anticoagulant activities. In Aim 4, we will characterize the cardioproperties of the improved APC variants or variants possessing only signaling activity or only anticoagulant activity in a mouse ischemia/reperfusion injury model. PUBLIC HEALTH RELEVANCE: The studies of this application will focus on the mechanism by which coagulation proteases activate a subfamily of G-protein coupled receptors called protease-activated receptors to alter the properties of vascular endothelial cells. The activation of these receptors by coagulation proteases is known to be important for the regulation of a range of (patho) physiological processes including coagulation, inflammation and angiogenesis. Understanding this role of coagulation proteases can lead to development of a new generation of therapeutic drugs for controlling thrombotic and inflammatory disorders, including heart attack and severe sepsis.

描述（由申请人提供）：凝血蛋白酶除了在调节血液凝固中发挥作用外，还可以通过激活细胞表面表达的称为蛋白酶激活受体（PAR）的 G 蛋白偶联受体亚家族来调节细胞内信号传导事件的各种器官。体外研究表明，抗凝蛋白酶、活化蛋白 C (APC) 与内皮蛋白 C 受体 (EPCR) 复合可通过激活血管内皮细胞中的 PAR-1 引发屏障保护反应，而凝血酶则会引发屏障破坏反应通过激活相同的受体。 APC已获得FDA批准用于治疗严重败血症。据推测，APC 对内皮细胞的 PAR-1 激活至少部分地导致了其预防器官衰竭（即肺和心脏）的保护特性，器官衰竭是由严重脓毒症中的感染性休克引起的。因此，了解 APC 或凝血酶激活 PAR-1 如何分别引发矛盾的保护性和破坏性细胞反应非常重要。我们最近发现，如果 EPCR 被蛋白 C 的 Gla 结构域占据，凝血酶对 PAR-1 的激活也可以引发屏障保护性细胞反应。我们证明，未占据的 EPCR 与 PAR-1 都与 Caveolin-1 相关。内皮细胞的脂筏内。在这些条件下，PAR-1 似乎通过 Gq 和/或 G 蛋白的 G12/13 亚家族发出信号，因此 PAR-1 的凝血酶激活会导致破坏性的细胞反应。然而，我们发现蛋白 C 占据 EPCR 会导致 EPCR 从 Caveolin-1 解离，从而可能通过将 PAR-1 与 Gi 蛋白偶联而将 PAR-1 招募到保护性途径。根据我们最近的初步数据，我们假设蛋白 C 占据 EPCR 也可以改变 PAR-2 的信号传导特异性。因此，看来 EPCR 的细胞表面占据决定了激活的 PAR-1 和 PAR-2 的信号传导特异性，而不是切割它们的蛋白酶。为了了解凝血蛋白酶的 PAR 依赖性信号传导特异性，我们制备了几种突变蛋白酶和受体，并建立了适当的体外和体内模型来研究以下四个具体目标。在目标 1 中，我们将研究在内皮细胞与维生素 K 依赖性凝血酶原预孵育的条件下凝血蛋白酶的 PAR-1 和 PAR-2 依赖性信号传导特异性，从而占据 EPCR 或其他潜在受体通过它们的天然配体。在目标 2 中，我们将研究维生素 K 依赖性凝血蛋白酶的 Gla 结构域对位于脂筏中的 PAR 的识别和切割特异性的贡献。在目标 3 中，我们将开发新型 APC 变体，这些变体改善了 PAR-1 依赖性保护信号传导特性，但降低了抗凝活性。在目标 4 中，我们将在小鼠缺血/再灌注损伤模型中表征改进的 APC 变体或仅具有信号活性或仅具有抗凝活性的变体的心脏特性。公共健康相关性：本申请的研究将重点关注凝血蛋白酶激活 G 蛋白偶联受体亚家族（称为蛋白酶激活受体）以改变血管内皮细胞特性的机制。已知凝血蛋白酶对这些受体的激活对于调节一系列（病理）生理过程（包括凝血、炎症和血管生成）非常重要。了解凝血蛋白酶的这种作用可以促进新一代治疗药物的开发，用于控制血栓形成和炎症性疾病，包括心脏病发作和严重败血症。