Thrombin Mediated Cytotoxicity during Cerebral Ischemia

描述（由申请人提供）：尽管急性卒中的溶栓治疗继续获得更广泛的接受和使用，但仍然迫切需要神经保护治疗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。凝血酶在低剂量下诱导保护作用（凝血酶预处理），但在高剂量下作为神经毒素，通过蛋白酶激活受体（PAR）杀死细胞。我们现在提出的假设，凝血酶部分介导水肿和细胞死亡，在中风期间通过PAR-1受体信号通路进行测试。使用Roger Tsien博士开发的蛋白酶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水肿和组织损伤。使用缺血性水肿和组织损伤的高度可重复和可量化的模型，我们将测试凝血或PAR-1及其假定的信号通路的各种药理学操作。使用慢病毒介导的RNA干扰，我们将减少PAR-1的基因表达的成年小鼠顶叶皮质的焦点领域之前，标准MCAo，然后确定PAR-1敲低对组织损伤的影响。使用PAR-1、PAR-2、PAR-3或PAR-4缺陷的敲除小鼠进行MCAo，我们将确定每种或所有PAR受体是否影响MCAo后的血管破裂和组织损伤。最后，我们将扩展我们以前的研究，包括脑损伤的行为措施，并寻求确定凝血酶是否加重和阿加曲班是否改善MCAo后的行为缺陷。我们将使用动脉内突变凝血酶，其设计用于激活PAR-1受体，但不影响缺血期间的凝血。我们还将测试神经保护药物，如突变APC或其他筛选对中风结局的影响。综上所述，这些研究将揭示凝血酶细胞毒性是否在卒中中起作用，以及在PAR-1信号通路中具有活性的药物是否代表有效的治疗方法。相关性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坏的分子机制以及缺血性水肿、梗死和行为障碍的病理解剖机制的充分理解。分子和病理解剖机制的基础探索以及治疗学的转化研究对于理解和治疗中风仍然非常重要和关键，中风是世界上成人残疾的最常见原因。 公共卫生相关性：中风是世界上成人残疾的最常见原因，也是美国第三大死亡原因，但我们对大多数患者缺乏有效的治疗。该提案旨在为中风的最常见原因找到新的，强大的治疗方法，并通过研究常见且重要的血液蛋白凝血酶来阐明脑缺血的一些基本机制。该提案将使我们能够确认和扩展我们有希望的初步发现，并帮助我们了解凝血酶如何促进中风后的脑损伤。

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. PUBLIC HEALTH RELEVANCE: Stroke, the most common cause of adult disability in the world, is the third leading cause of death in America, yet we lack effective therapy for most patients. This proposal seeks to find new, powerful treatments for the most common cause of stroke, and to elucidate some of the fundamental mechanisms underlying brain ischemia by studying a common and important blood protein, thrombin. The proposal will enable us to confirm and extend our promising preliminary findings as well as help us understand how thrombin contributes to brain injury after stroke.