Activated protein C system in stroke models

描述（由申请人提供）：对于该竞争性更新项目（R 01 HL 63290 -16），我们建议继续Zlokovic和Griffin实验室之间的合作研究，旨在推进中枢神经系统（CNS）中蛋白C/活化蛋白C（APC）通路的基础知识，总体目标是从基础知识向临床转化。由该R 01项目资助的先前结果导致使用中风、脑创伤和慢性神经元变性的啮齿动物模型发现APC及其细胞保护选择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。该项目鉴定了CNS中介导神经元、脑内皮和BBB中细胞保护性APC信号传导的APC受体和下游通路，并表明蛋白酶激活受体-1（PAR 1）在保护中具有重要作用。我们的进展在2014年转化为第二代细胞保护性APC 3 K3 A-APC治疗缺血性卒中的临床II期试验。为了扩展该项目，我们的目标包括：1）为APC的神经保护活性的假设机制提供概念证明，2）表征新型第三代APC模拟剂，以及3）发现具有治疗其他神经系统疾病潜力的缺血性卒中的改进生物制剂。我们令人兴奋的新发现和初步数据表明，APC对PAR 1的激活涉及该受体的N-末端结构域在Arg 46处的新切割，这揭示了一种终止于残基Asn 47的新型拴系配体，其导致APC的偏向性β-抑制蛋白依赖性细胞保护信号传导。相比之下，凝血酶对PAR 1的经典活化涉及Arg 41处的裂解，导致G蛋白信号传导和细胞毒性。APC可以在Arg 41和Arg 46处切割PAR 1，并且缺乏体内概念证明来证明APC的神经保护基于PAR 1在Arg 46处切割的有吸引力的假设。我们假设：i）PAR 1在Arg 46处的APC切割保护CNS、BBB和神经元（AIM 1）; ii）具有PAR 1残基47-66序列的APC模拟肽（TR 47）（即，Arg 46切割后产生的系留激动剂）缺血性中风（AIM 2）后体外和体内脑内皮和神经元中的依曲普利-抑制蛋白2-依赖性细胞保护性信号传导;和iii）在Arg 46切割PAR 1能力增强的APC突变体可提供第三代神经保护性生物制剂。初步数据包括：i）产生携带R41 Q-PAR 1和R46 Q-PAR 1以防止在Arg 41或Arg 46处裂解的新小鼠系; ii）证明研究TR 47肽的神经保护作用的可行性;和iii）成功地工程化具有增强PAR 1在Arg 46处裂解的突变的新型APC变体。将使用BBB和神经元损伤的体外模型、小鼠和大鼠中风模型、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。我们预计，从这个项目中产生的新知识将可能对缺血性卒中的临床转化产生重大的直接影响，并延伸到其他神经系统疾病的治疗。

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, ß-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 ß-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.