Proteolytic Pathways in Thrombus Resolution

描述（由申请人提供）： 背景/依据：深静脉血栓形成（DVT）及其并发症是美国人发病率和死亡率的重要来源，并且在过去十年中在退伍军人群体中有所增加。沿着肺栓塞的潜在致命并发症，DVT经常导致显著的长期并发症，对此我们没有特异性治疗，血栓形成后综合征，其在25-60%的DVT患者中引起使人衰弱的肿胀、疼痛和腿部溃疡。DVT的常见风险因素包括癌症、严重创伤、手术、瘫痪、长时间不动和老年。由于空中和地面运输时间长、脱水、吸烟以及因严重受伤住院期间长时间不能移动，部署的军事人员面临的风险增加。目前的治疗依赖于抗凝剂来治疗DVT，这不能解决现有的血栓，而只能防止进一步的血栓形成。血栓消退是血栓后综合征发病机制中的关键因素，因为血栓消退不完全可导致血流阻塞和静脉瓣膜功能丧失。临床研究表明，血栓消退较快的患者比血栓消退较慢的患者预后更好。目前，对静脉血栓消退的细胞和分子机制知之甚少。然而，清楚的是，有效的血栓溶解需要炎性细胞来塑造免疫应答并动员蛋白水解途径来溶解血栓。目的：使用DVT的临床相关实验模型，我们发现小鼠中派- 2的基因缺失加速静脉血栓消退。我们的数据表明，派-2是一个新的目标，可能会影响两个独立的途径来调节静脉血栓的解决，通过炎症和尿激酶（uPA）激活机制，其活动可能是由G蛋白偶联受体PAR 2调节。这项建议的目标是确定这些发现背后的分子和细胞机制。待检验的假设是，静脉血栓消退速率受涉及uPA、派-2、派-1和PAR 2的共同途径调节。该研究计划提出（1）确定派-2在静脉血栓消退过程中调节炎症细胞的机制，（2）确定派-2在静脉血栓消退过程中作为uPA抑制剂的作用，以及（3）定义PAR 2激活调节静脉血栓消退的机制。研究方法：研究将在DVT模型中使用遗传缺陷小鼠，这些模型准确模拟了在人类DVT中观察到的许多临床和病理生理学特征。将通过免疫组织化学、流式细胞术、mRNA和蛋白质分析对静脉血栓进行分析，以确定炎症和血栓消退的分子指标。将使用离体血栓溶解测定和细胞凝块溶解测定以及采用转染和shRNA方法的细胞培养模型来研究蛋白水解途径。生物力学测定将评估实验性DVT期间的静脉壁损伤。发现/结果：所提出的实验将定义uPA、派-2、派-1和PAR 2在实验性DVT期间调节炎症和凝块溶解的机制。现状：这是一个新的项目，产生于大量的支持性初步数据。影响：这些研究的结果将是确定一种新的分子途径，连接炎症，凝血和纤维蛋白溶解，以调节静脉血栓的解决。这一点很重要，因为这些分子的特异性拮抗作用可以提供辅助治疗，促进血栓快速消退，以减少DVT中的血栓形成后并发症。因此，这项基础研究提案的目标与影响战士、军人家庭和美国公众健康的严重疾病直接相关。

DESCRIPTION (provided by applicant): Background / Rationale: Deep vein thrombosis (DVT) and its complications are a significant source of morbidity and mortality among Americans, and have increased in the Veteran population over the last decade. Along with the potentially fatal complication of pulmonary embolism, DVT frequently leads to a significant long- term complication for which we have no specific therapy, post-thrombotic syndrome, which causes debilitating swelling, pain and leg ulceration in 25-60% of DVT patients. Common risk factors for DVT include cancer, major trauma, surgery, paralysis, prolonged periods of immobility, and older age. Deployed military personnel are at increased risk due to prolonged air and ground transport, dehydration, tobacco use, and extended immobility during hospitalizations for severe injuries. Current therapies rely on anticoagulants to treat DVT, which do not resolve existing blood clots, but only prevent further clot development. Thrombus resolution is a critical factor in the pathogenesis of post-thrombotic syndrome since incomplete thrombus resolution can result in obstruction of flow and loss of venous valve function. Clinical studies show that patients with more rapid thrombus resolution have a better prognosis than those patients whose thrombus resolves much slower. At present, the cellular and molecular mechanisms involved in venous thrombus resolution are poorly understood. What is clear however, is that effective thrombus resolution requires inflammatory cells to sculpt immune responses and to mobilize proteolytic pathways to resolve the thrombus. Objectives: Using clinically relevant experimental models of DVT, we discovered that genetic deletion of PAI- 2 in mice accelerates venous thrombus resolution. Our data suggest that PAI-2 is a novel target that may affect two independent pathways to regulate venous thrombus resolution, through inflammatory and urokinase (uPA) activation mechanisms, and its activities may be regulated by the G protein coupled receptor PAR2. The goal of this proposal is to determine molecular and cellular mechanisms underlying these findings. The hypothesis to be tested is that the rate of venous thrombus resolution is regulated by a common pathway involving uPA, PAI-2, PAI-1 and PAR2. The research plan proposes (1) to determine mechanisms by which PAI-2 modulates inflammatory cells during venous thrombus resolution, (2) to determine the role of PAI-2 as a uPA inhibitor during venous thrombus resolution, and (3) to define mechanisms by which PAR2 activation modulates venous thrombus resolution. Methods: Studies will utilize genetically deficient mice in models of DVT that accurately mimic many of the clinical and pathophysiological features observed in human DVT. Venous thrombi will be analyzed by immunohistochemistry, flow cytometry, mRNA and protein analyses for molecular indicators of inflammation and thrombus resolution. Proteolytic pathways will be investigated using ex vivo thrombolysis assays and cellular clot lysis assays, as well as cell culture models employing transfection and shRNA approaches. Biomechanical assays will assess vein wall injury during experimental DVT. Findings/Results: The proposed experiments will define mechanisms by which uPA, PAI-2, PAI-1 and PAR2 modulate inflammation and clot dissolution during experimental DVT. Status: This is a new project arising from substantial supportive preliminary data. Impact: The outcome of these studies will be to define a novel molecular pathway that links inflammation, coagulation and fibrinolysis to modulate venous thrombus resolution. This is important since specific antagonism of these molecules could provide a complementary therapy for facilitating rapid thrombus resolution to reduce post-thrombotic complications in DVT. The objectives of this basic research proposal are therefore of direct relevance to a serious condition that impacts the health of the warfighter, the military family, and the American public.