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.
Deep venous thrombosis (DVT) is a common and serious clinical problem with between 350,000 to 600,000 cases per year in the U.S. and 200,000 deaths from pulmonary embolism. Military personnel are at increased risk of DVT due to significant periods of travel, prolonged evacuation times, dehydration, and tobacco use. The incidence in Veterans is likely to increase due to increasing age and obesity. Current therapies for DVT rely on anticoagulants, which do not enhance resolution of existing clots, but only prevent further clot development. Delayed or incomplete clot resolution frequently leads to post-thrombotic syndrome, a long-term complication associated with debilitating limb swelling, pain and recurrent skin ulceration. What is needed is a better understanding of the molecular and cellular processes that regulate clot resolution, so that specific therapies to accelerate the resolution process can be devised. The purpose of this project is to address this by identifying new regulators that may be developed for reducing post-thrombotic complications in DVT.