Thrombin Mediated Cytotoxicity during Cerebral Ischemia

描述(申请人提供)：尽管急性中风的溶栓疗法继续得到更广泛的接受和使用，但仍然迫切需要神经保护疗法和新的治疗方法来减少溶栓并发症。凝血酶是一种丝氨酸蛋白酶，在凝血级联反应中起关键作用。凝血酶在低剂量(凝血酶预适应)时起到保护作用，但在高剂量时起到神经毒素的作用，通过蛋白酶激活的受体(PARs)杀死细胞。我们现在建议检验这一假说，即凝血酶通过PAR-1受体信号通路部分调节卒中时的水肿和细胞死亡。使用罗杰·钱存训博士开发的一种可切割的细胞穿透探针，我们将直接确定凝血酶激活是否介导了浮肿和组织损伤。使用高度可重复性和可量化的缺血性水肿和组织损伤模型，我们将测试凝血或PAR-1及其假定的信号通路的各种药理操作。利用慢病毒介导的RNA干扰，我们将在标准MCAO前降低成年小鼠顶叶皮质局灶区PAR-1的基因表达，然后确定PAR-1基因敲除对组织损伤的影响。利用PAR-1、PAR-2、PAR-3或PAR-4缺陷基因敲除的小鼠接受MCAO，我们将确定每个或所有PAR受体是否影响MCAO后的血管破坏和组织损伤。最后，我们将扩展我们之前的研究，将大脑损伤的行为测量包括在内，并试图确定凝血酶是否会加剧MCAO后的行为缺陷，以及阿加曲班是否会改善行为缺陷。我们将使用动脉内突变凝血酶，旨在激活PAR-1受体，但不会在缺血期间影响凝血。我们还将测试神经保护性药物，如突变的APC或其他筛选出的对中风预后的影响。综上所述，这些研究将揭示凝血酶细胞毒性是否在卒中中发挥作用，以及激活PAR-1信号通路的药物是否代表有效的治疗方法。相关：寻找有效的神经保护剂受到许多因素的阻碍：我们仍然缺乏对缺血性细胞死亡和血管破裂的分子机制，以及缺血性水肿、脑梗塞和行为障碍的病理解剖机制的充分了解。对分子和病理解剖机制的基础探索和治疗学的翻译研究对于理解和治疗中风仍然具有非常重要的意义和关键，中风是世界上最常见的成人残疾原因。

DESCRIPTION (provided by applicant): Although thrombolytic therapy for acute stroke continues to gain wider acceptance and usage, there remains a compelling need for neuroprotective therapy and new treatment to reduce thrombolytic complications. Thrombin is a serine protease that plays a critical role in the coagulation cascade. Thrombin induces protection at low doses (thrombin preconditioning) but acts as a neurotoxin at high doses, killing cells via the protease activated receptors (PARs). We now propose to test the hypothesis that thrombin partially mediates edema and cell death during stroke via the PAR-1 receptor signaling pathway. Using a protease-cleavable cell-penetrating probe developed by Dr. Roger Tsien, we will determine directly whether thrombin activation mediates edema and tissue injury. Using a highly reproducible and quantifiable model of ischemic edema and tissue injury, we will test a variety of pharmacological manipulations of coagulation or PAR-1 and its presumed signaling pathways. Using lentiviral mediated RNA interference, we will reduce gene expression of PAR-1 in focal areas of the parietal cortex of adult mice prior to standard MCAo and then determine the effect of PAR-1 knock-down on tissue injury. Using knock-out mice deficient in PAR-1, PAR-2, PAR-3, or PAR-4 subjected to MCAo, we will determine whether each or all PAR receptors influence vascular disruption and tissue injury after MCAo. Finally, we will extend our previous studies to include behavioral measures of cerebral injury and seek to determine whether thrombin exacerbates-and whether argatroban ameliorates-behavioral deficits after MCAo. We will use intra-arterial mutant thrombin designed to activate PAR-1 receptor but not affect coagulation during ischemia. We will also test neuroprotective drugs such as mutant APC or others screened for effect on stroke outcome. Taken together, these studies will reveal whether thrombin cytotoxicity plays a role in stroke and whether agents active at PAR-1 signaling pathway represent an effective therapy. Relevance The search for effective neuroprotectants is hampered by many factors: we continue to lack a full understanding of the molecular mechanisms of ischemic cell death and vascular disruption, as well as the patho-anatomic mechanisms of ischemic edema, infarction, and behavioral impairment. Fundamental exploration of molecular and patho- anatomic mechanisms AND translational studies of therapeutics remain highly significant and critical to understanding and treating stroke, the most common cause of adult disability in the world.