Proteolytic Pathways in Venous Thrombus Resolution

背景/原理：静脉血栓栓塞症(VTE)，包括深静脉血栓形成(DVT)，以及 它的并发症是美国人发病率和死亡率的重要来源；并在#年有所增加 过去十年的退伍军人人数。最近的一项研究对近50万例手术进行了调查 退伍军人事务部医学中心部发现，超过一半的患者在 手术后90天。伴随着肺栓塞的潜在致命并发症，DVT经常 导致一种重大的长期并发症，我们没有针对血栓形成后的特殊治疗方法 综合症；在25%-60%的DVT患者中会导致虚弱的肿胀、疼痛和腿部溃疡。普普通通 DVT的危险因素包括：癌症、重大创伤、手术、瘫痪、长时间不动和 年纪大了。由于空中和地面运输时间延长，部署的军事人员面临更大的风险， 严重受伤住院期间出现脱水、吸烟和长时间不动。当前 治疗依赖于抗凝剂来治疗DVT，DVT不能溶解现有的血液凝块；而只是预防 进一步的血栓形成。血栓溶解是血栓形成后发病的关键因素 血栓溶解不全可导致血流阻塞和静脉瓣膜丧失的综合征 功能。临床研究表明，血栓溶解越快的患者预后越好。 比血栓消退慢得多的患者。目前，细胞和分子 目前对深静脉血栓形成的机制知之甚少，目前尚无加速其发生的治疗方法。 进程。 目的：利用临床相关的深静脉血栓形成的实验模型，我们发现了一种关键的分子。 在静脉血栓溶解过程中作为炎症关键调节因子发挥作用的途径。一个 相互连接的细胞介导的分子过程的全面图景，这些过程协调了 煽动性计划开始出现。我们的研究计划建议通过(1)来定义这一途径 纤溶酶原激活物抑制物-2(PAI-2)缺乏对早期调节机制的研究 急性DVT加速静脉血栓溶解，以及(2)确定纤溶酶产生的纤维蛋白的作用 降解产物(FDPs)在静脉血栓溶解过程中对炎性巨噬细胞的调节作用。 方法：研究将利用基因缺陷小鼠在DVT的实验模型中准确地模仿 在人类深静脉血栓中观察到的许多临床和病理生理特征。静脉血栓将被分析 通过免疫组织化学、流式细胞术、mRNA和蛋白质分析检测炎症的分子指标 和血栓消退。蛋白分解途径将使用体外溶栓试验进行研究，细胞 凝块溶解试验和体外细胞培养。形态测量分析和生物力学分析将评估静脉 墙壁受伤。这些发现的翻译潜力将在人类细胞和临床前模型中进行测试。 发现/结果：静脉血栓中调节炎症的新分子机制 将确定解决方案，并在临床前测试基于这些机制的治疗潜力 模特们。 现状：这是一个新项目，源于之前退伍军人事务部的大量支持性初步数据 奖品。 影响：从这些研究中获得的知识具有很强的临床翻译潜力，因为 静脉血栓溶解抑制剂或促溶解激动剂的拮抗作用可能是安全的 加速这一过程的有效疗法，与抗凝剂相结合，提供了一种 改良治疗减少深静脉血栓形成所致静脉肺栓塞和血栓后并发症 我们的退伍军人。

Background / Rationale: Venous thrombus embolism (VTE), which includes 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. A recent study of almost 500,000 surgeries performed at the Department of Veterans Affair Medical Centers found that over half of the patients developed VTE within 90 days after surgery. 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 an 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 DVT are poorly understood, and there currently is no therapy to accelerate this process. Objectives: Using clinically relevant experimental models of DVT, we have uncovered a critical molecular pathway that functions as a key modulator of inflammation during venous thrombus resolution. A comprehensive picture of interconnected cell-mediated molecular processes that orchestrate a precise inflammatory program is starting to emerge. Our research plan proposes to define this pathway by (1) determining mechanisms by which plasminogen activator inhibitor type 2 (PAI-2) deficiency modulates early acute DVT to accelerate venous thrombus resolution, and (2) determining the role of plasmin-generated fibrin degradation products (FDPs) in regulating inflammatory macrophages during venous thrombus resolution. Methods: Studies will utilize genetically deficient mice in experimental 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, cellular clot lysis assays, and ex vivo cell culture. Morphometric analyses and biomechanical assays will assess vein wall injury. The translational potential of these findings will be tested in human cells and in preclinical models. Findings/Results: Novel molecular mechanisms that modulate inflammation during venous thrombus resolution will be identified and the potential for therapies based on these mechanisms tested in preclinical models. Status: This is a new project arising from substantial supportive preliminary data from a previous VA Merit Award. Impact: The knowledge gained from these studies has strong potential for clinical translation since specific antagonism of inhibitors of venous thrombus resolution or resolution-promoting agonists could present safe and effective therapies for accelerating this process, in combination with anticoagulants, provide an improved treatment for reducing venous pulmonary embolism and post-thrombotic complications from DVT in our Veteran population.