Proteolytic Pathways in Thrombus Resolution

描述(由申请人提供)： 背景/原理：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中有所增加。伴随着潜在的致命的肺栓塞并发症，深静脉血栓形成常常会导致一种我们没有特殊治疗方法的重要的长期并发症--血栓形成后综合征，它会导致25%-60%的深静脉血栓形成患者出现衰弱的肿胀、疼痛和腿部溃疡。DVT的常见危险因素包括癌症、重大创伤、手术、瘫痪、长时间不动和高龄。由于空中和地面运输时间长、脱水、使用烟草以及严重受伤住院期间长时间不能动，部署的军事人员面临更大的风险。目前的治疗方法依赖于抗凝剂来治疗DVT，DVT并不能溶解现有的血栓，而只是防止进一步的血栓形成。血栓溶解是血栓形成后综合征发病的关键因素，因为血栓溶解不完全会导致血流受阻和静脉瓣膜功能丧失。临床研究表明，血栓消退较快的患者预后好于血栓消退较慢的患者。目前，静脉血栓溶解的细胞和分子机制尚不清楚。然而，有一点是明确的，有效的血栓溶解需要炎症细胞建立免疫反应，并动员蛋白分解途径来溶解血栓。目的：利用临床相关的深静脉血栓形成的实验模型，我们发现小鼠体内PAI-2基因缺失可加速静脉血栓的溶解。我们的数据表明，PAI-2是一个新的靶点，它可能通过炎症和尿激酶(UPA)激活机制影响两条独立的途径来调节静脉血栓的溶解，其活性可能受G蛋白偶联受体PAR2的调节。这项提议的目标是确定这些发现背后的分子和细胞机制。需要检验的假设是，静脉血栓的消退率受涉及uPA、PAI-2、PAI-1和PAR2的共同途径调节。该研究计划建议(1)确定PAI-2在静脉血栓溶解过程中调节炎症细胞的机制，(2)确定PAI-2作为uPA抑制物在静脉血栓溶解过程中的作用，以及(3)确定PAR2激活调节静脉血栓溶解的机制。方法：研究将利用遗传缺陷小鼠建立DVT模型，准确模拟人类DVT中观察到的许多临床和病理生理特征。静脉血栓将通过免疫组织化学、流式细胞术、mRNA和蛋白质分析进行分析，以确定炎症和血栓消退的分子指标。蛋白质分解途径将使用体外溶栓试验和细胞凝块溶解试验，以及使用转基因和shRNA方法的细胞培养模型来研究。生物力学分析将评估实验性DVT中的静脉壁损伤。发现/结果：拟议的实验将确定uPA、PAI-2、PAI-1和PAR2在实验性DVT中调节炎症和血栓溶解的机制。现状：这是一个从大量支持性初步数据中产生的新项目。影响：这些研究的结果将是确定一种新的分子途径，将炎症、凝血和纤溶联系起来，调节静脉血栓的溶解。这一点很重要，因为这些分子的特异性拮抗可以提供一种补充疗法，促进血栓的快速溶解，以减少深静脉血栓形成后的并发症。因此，这项基础研究提案的目标与影响战士、军人家属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.