Activated protein C system in stroke models

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）途径的基础知识，总体目标是将基础知识转化为临床应用。该R01项目资助的先前研究结果表明，APC及其细胞保护选择性突变体在中风、脑外伤和慢性神经元变性的啮齿动物模型中具有直接的血管保护、血脑屏障（BBB）稳定、神经保护和抗炎活性。本项目确定了中枢神经系统中APC受体及其下游通路介导神经元、脑内皮和血脑屏障中APC信号的细胞保护作用，并表明蛋白酶激活受体-1 （PAR1）在保护中起主要作用。我们的进展于2014年转化为第二代细胞保护性APC 3K3A-APC的缺血性卒中临床II期试验。为了进一步扩展这个项目，我们的目标包括：1)为APC神经保护作用的假设机制提供概念证明；2)表征新的第三代APC模拟药物；3)发现改进的缺血性卒中生物制剂，并具有治疗其他神经系统疾病的潜力。我们令人兴奋的新发现和初步数据表明，APC对PAR1的激活涉及到Arg46受体n端结构域的一种新的切割，这揭示了一种新的系带配体，该配体位于残基Asn47处，导致APC的偏倚，？-抑制蛋白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Arg41的裂解，导致g蛋白信号传导和细胞毒性。APC可以在Arg41和Arg46位点切割PAR1，但缺乏体内概念证据来证明APC的神经保护作用是基于PAR1在Arg46位点的切割。我们推测：i) PAR1在Arg46位点的APC切割对中枢神经系统、血脑屏障和神经元（AIM 1）有保护作用；ii)具有PAR1残基47-66 （TR47）序列的apc模拟肽（即Arg46切割后产生的栓系激动剂）引发？-缺血性卒中后体外和体内脑内皮和神经元中依赖于阻滞蛋白2的细胞保护信号（AIM 2）；iii) APC突变体在Arg46位点切割PAR1的能力增强，可能提供第三代神经保护生物制剂。初步数据包括：i)产生携带R41Q-PAR1和R46Q-PAR1的新小鼠系，以防止Arg41或Arg46的切割；ii)研究TR47肽的神经保护作用的可行性证明；iii)成功设计出具有增强PAR1在Arg46位点切割的突变的新型APC变体。神经保护将通过血脑屏障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、神经病理分析、共聚焦显微镜和行为测试来评估。我们期望从这个项目中产生的新知识可能会对转化为缺血性中风的临床有重大的、直接的影响，并进一步影响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, ?-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.