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：
8434881
负责人：
ALIREZA R. REZAIE
金额：
$ 35.37万
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 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8434881
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.

描述（由申请人提供）：凝血蛋白酶除了在调节血液凝结中的作用外，还可以通过激活称为蛋白酶激活受体（PARS）的G蛋白偶联受体的亚家族来调节细胞内信号传导事件，该受体表达在各种器官的细胞表面上表达。体外研究表明，虽然与内皮蛋白C受体（EPCR）复杂的抗凝蛋白酶激活的蛋白C（APC）通过血管内皮细胞中的PAR-1激活引起屏障保护反应，但凝血酶通过同一受体激活引起了屏障的障碍反应。 APC已获得FDA的批准严重败血症的治疗。假设APC对内皮细胞的PAR-1激活至少部分原因是其防止器官衰竭（即肺和心脏）的保护特性，这是严重败血症中败血性休克引起的。因此，非常重要的是要了解APC或凝血酶对PAR-1的激活分别引起矛盾的保护性和破坏性细胞反应。我们最近发现，如果蛋白质的gla域占据EPCR，凝血酶对PAR-1的激活也可能引起屏障保护性细胞反应。我们证明，无置型EPCR与PAR-1一起均与caveolin-1有关，这两者都与内皮细胞的脂质生命值中的小窝蛋白-1相关。在这些条件下，PAR-1似乎通过GQ和/或G12/13的G12/13信号信号，因此PAR-1的凝血酶激活会导致破坏性的细胞反应。但是，我们发现蛋白质C对EPCR的占用导致EPCR与Caveolin-1的解离，从而将PAR-1募集到可能通过将其偶联与Gi-protein耦合来保护途径。基于我们最近的初步数据，我们假设蛋白质占EPCR的占用也可以改变PAR-2的信号传导特异性。因此，似乎EPCR的细胞表面占用率决定了激活的PAR-1和PAR-2的信号传导特异性，而不是裂解它们的蛋白酶。为了了解凝血蛋白酶的分子依赖性信号传导特异性，我们准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，以研究以下四个特定目标。在AIM 1中，我们将研究凝血蛋白酶的PAR-1和2依赖性信号传导特异性，在这种条件下，内皮细胞已与维生素K依赖性凝血的Zymogens预孵育，从而通过其自然配体占据EPCR或其他潜在受体。在AIM 2中，我们将研究维生素KD依赖性凝血蛋白酶对局部固定在Lipidrafts中的PAR的识别和裂解特异性的GLA结构域的贡献。在AIM 3中，我们将开发新型的APC变体，这些变体已改善了PAR-1依赖性的保护性信号传导特性，但降低了抗凝活性。在AIM 4中，我们将表征改进的APC变体或仅具有信号活性或仅具有抗凝性活性的APC变体的心op毛。