Activated protein C system in stroke models

中风模型中激活的蛋白 C 系统

• 批准号: 8831264
• 负责人: Berislav V Zlokovic
• 金额: $ 66.73万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8831264
• 关键词: APC2 gene; Agonist; Anti-Inflammatory Agents; Anti-inflammatory; Anticoagulants; Apoptotic; Arrestins; Blood - brain barrier anatomy; Blood Vessels; Brain; Cells; Chronic; Cleaved cell; Clinic; Clinical Trials; Collaborations; Confocal Microscopy; Data; Disease; Distal; Endothelium; Engineering; Extravasation; Factor Va; Feasibility Studies; Female; Figs - dietary; Funding; GTP-Binding Proteins; Gene Expression; Generations; Goals; In Vitro; Inbred SHR Rats; Inflammatory; Ischemic Stroke; Knowledge; Ligands; Magnetic Resonance Imaging; Mediating; Middle Cerebral Artery Occlusion; Modeling; Mus; Mutation; N-terminal; Nerve Degeneration; Neuraxis; Neurologic; Neuronal Injury; Neurons; PAR-1 Receptor; Pathway interactions; Peptides; Phase; Phase II Clinical Trials; Platelet Activation; Protein C; Proteolysis; Public Health; Rattus; Resolution; Rodent Model; Role; Signal Transduction; Stroke; System; Testing; Therapeutic; Thrombin; Toxic effect; Translating; Translations; Traumatic Brain Injury; Variant; activated Protein C; activated protein C receptor; analog; arrestin 2; base; behavior test; improved; in vitro Model; in vivo; insight; mimetics; mutant; nerve stem cell; nervous system disorder; neuroprotection; novel; prevent; public health relevance; receptor; success

DESCRIPTION (provided by applicant): For this competing renewal project (R01HL63290-16), we propose to continue collaborative studies between the Zlokovic and Griffin labs aimed at advancing basic knowledge on protein C/activated protein C (APC) pathways in the central nervous system (CNS) with an overall goal of advancing from basic knowledge to translation to the clinic. Previous results funded by this R01 project led to discovery of direct vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective and anti-inflammatory activities of APC and its cytoprotective-selective mutants using rodent models of stroke, brain trauma and chronic neuronal degeneration. This project identified APC receptors and downstream pathways in the CNS that mediate cytoprotective APC signaling in neurons, brain endothelium and at the BBB and showed that protease activated receptor-1 (PAR1) has a major role in protection. Our progress was translated in 2014 into clinical Phase II trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective APC. For extending this project, our goals include: 1) providing proof of concept for hypothesized mechanisms for neuroprotective activities of APC, 2) characterizing novel 3rd generation APC-mimetic agents, and 3) discovering improved biologics for ischemic stroke with potential for treating other neurologic diseases. Our exciting new findings and preliminary data show that activation of PAR1 by APC involves a novel cleavage of this receptor's N-terminal domain at Arg46 which reveals a novel tethered ligand ending at residue Asn47 that causes APC's biased, ?-arrestin-dependent cytoprotective signaling. In contrast, classical activation of PAR1 by thrombin involves cleavage at Arg41 resulting in G-protein signaling and cellular toxicity. APC can cleave PAR1 at both Arg41 and Arg46, and in vivo proof of concept is lacking for the attractive hypothesis that APC's neuroprotection is based on PAR1 cleavage at Arg46. We hypothesize that: i) APC cleavage of PAR1 at Arg46 protects CNS, BBB and neurons (AIM 1); ii) an APC-mimetic peptide with the sequence of PAR1 residues 47-66 (TR47) (i.e., the tethered agonist generated after Arg46 cleavage) elicits ?-arrestin 2-dependent cytoprotective signaling in brain endothelium and neurons in vitro and in vivo after ischemic stroke (AIM 2); and iii) APC mutants with enhanced ability to cleave PAR1 at Arg46 may provide 3rd generation neuroprotective biologics. PRELIMINARY DATA include: i) generation of new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 to prevent cleavages at either Arg41 or Arg46; ii) proof for feasibility of studying the neuroprotective actions of the TR47 peptide; and iii) success at engineering novel APC variants with mutations that enhance PAR1 cleavage at Arg46. Neuroprotection will be assessed using in vitro models of BBB and neuronal injury, stroke models in mice and rats, magnetic resonance imaging, neuropathological analysis, confocal microscopy and behavioral testing. We expect that new knowledge generated from this project will likely have significant, direct impact for translation to the clinic for ischemic stroke and, by extension, implications for therapies for othr neurological disorders.

描述（由申请人提供）：对于这个竞争性更新项目（R01HL63290-16），我们建议继续在Zlokovic和Griffin Labs之间进行合作研究，旨在在中枢神经系统（CNS）中促进蛋白质C/活化蛋白C（APC）途径的基础知识，并以基本知识为临床知识进行了整体知识。该R01项目资助的先前结果导致使用APC及其细胞保护性突变体的直接血管保护，血脑屏障（BBB）稳定，神经保护和抗炎活性，使用啮齿动物的脑部创伤，脑部创伤和慢性神经元变性。该项目鉴定了中枢神经系统中的APC受体和下游途径，这些途径介导神经元，脑内皮和BBB中的细胞保护APC信号传导，并表明蛋白酶活化的受体1（PAR1）在保护中具有重要作用。我们的进度在2014年转化为第二代细胞保护APC的3K3A-APC缺血性中风的临床II期试验。为了扩展该项目，我们的目标包括：1）为APC的神经保护活性提供概念验证，2）表征新型的第三代APC模拟剂，3）发现缺血性中风的改善生物学，具有治疗其他神经学疾病的潜力。我们令人兴奋的新发现和初步数据表明，APC激活PAR1涉及该受体N端域在Arg46上进行了新的裂解，该裂解揭示了在残基ASN47处的一种新型的束缚配体，导致APC偏见，？？ - 依赖于arrarstin依赖的细胞保护信号。相比之下，凝血酶对PAR1的经典激活涉及在ARG41处进行裂解，从而导致G蛋白信号传导和细胞毒性。 APC可以在ARG41和ARG46上切割PAR1，并且在体内概念证明缺乏有吸引力的假设：APC的神经保护基于ARG46的PAR1裂解。我们假设：i）ARG46处PAR1的APC切割保护CNS，BBB和神经元（AIM 1）； ii）一种具有PAR1残基序列47-66（即，在Arg46裂解后产生的链状激动剂）的APC模拟肽？-artestin 2依赖性的细胞触发信号在脑部内皮和神经元中的体外和Invivo（Invivo）; iii）在ARG46裂解PAR1的能力增强的APC突变体可能会提供第三代神经保护生物制剂。初步数据包括：i）携带R41Q-PAR1和R46Q-PAR1的新鼠标生成，以防止在ARG41或ARG46处裂解； ii）证明了研究TR47肽的神经保护作用的可行性； iii）在工程新型APC变体方面的成功，突变增强了ARG46的PAR1裂解。将使用BBB和神经元损伤的体外模型，小鼠和大鼠的中风模型，磁共振成像，神经病理学分析，共聚焦显微镜和行为测试来评估神经保护作用。我们预计，该项目产生的新知识可能会对诊所的缺血性中风产生重大的直接影响，并扩大对OTHR神经系统疾病的疗法的影响。