Proteolytic Pathways in Venous Thrombus Resolution

Background / Rationale: VTE (Venous Thromboembolism) is a common and serious cardiovascular disease
with significant mortality and morbidity. Prompt anticoagulation of patients with deep venous thrombosis (DVT)
reduces fatal pulmonary embolism (PE) but does not prevent long-term morbidity of VTE. Post-thrombotic
syndrome occurs in 25-50% of patients with clots in the deep veins of the arms and legs that consists of pain,
swelling, and recurrent skin ulceration. Common risk factors for DVT include cancer, major trauma, surgery,
paralysis, prolonged periods of immobility, and older age. DVT and its complications have increased in the
Veteran population over the last decade. 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. 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. Using novel
3D serial measurements of thrombus volume; we demonstrated that DVT patients with similar initial DVTs have
widely varying rates of thrombus resolution over time despite adequate anticoagulation. Longitudinal studies
show that patients with more rapid thrombus resolution have a better prognosis than those patients whose
thrombus resolves much slower. Despite its clinical importance, 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, a comprehensive picture of interconnected
cell-mediated molecular processes that orchestrate a precise inflammatory program is starting to emerge and
forms the foundation for this proposal. Our objectives are to build on our previous VA Merit findings to: 1) define
mechanisms by which plasminogen activator inhibitor-2 (PAI-2) deficiency modulates inflammatory leukocytes
to accelerate venous thrombus resolution; 2) test the therapeutic efficacy of blocking the PAI-2 pathway to
accelerate venous thrombus resolution; and 3) determine specific gene signatures for the temporal inflammatory
vascular remodeling events that occur during venous thrombus resolution and evaluate the prognostic value of
candidate biomarkers in patients with evolving and maladaptive thrombus resolution after VTE.
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. We will define mechanisms by
which PAI-2 deficiency calibrates immune regulation to accelerate venous thrombus resolution using ex vivo
thrombolysis assays, transmigration assays, and neutrophil adoptive transfer experiments. The translational
potential of suppressing PAI-2 expression or activity to accelerate venous thrombus resolution will be tested in
human cells and in preclinical models. Finally, we will use our well-established clinically relevant mouse models
of DVT resolution to identify gene regulatory signatures/potential biomarkers and then evaluate changes in gene
networks over time in blood from patients with evolving thrombus resolution after VTE.
Findings/Results: Molecular mechanisms that modulate inflammation during venous thrombus resolution in
experimental models and in human patients will be identified and 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: New knowledge from these studies regarding the inflammatory signatures in DVT patients may form the
basis for novel therapies for accelerating this process, and in combination with anticoagulants, control excessive
fibrosis, and prevent this disease.

Grant Number: 5I01BX001921-12
NIH Institute/Center: VA
Principal Investigator: Toni Antalis