Background: Venous thrombosis (DVT) is a serious health concern, with growing incidence in an aging population. DVT is marked by sterile inflammation and neutrophil (PMN) activation. PMNs extrude their DNA to form extracellular traps (NETs) which potentiate thrombosis. To identify therapeutic targets at the nexus of inflammation and coagulation, we focused on the adenosinergic enzyme CD73 which is expressed on the surface of neutrophils and the endothelium. CD73 phosphohydrolyzes AMP to produce the anti-thromboinflammatory nucleoside adenosine. We hypothesized that CD73 is a critical enzyme in venous homeostasis, restraining unchecked thrombosis in DVT.
Methods: DVT was induced in WT and CD73-/- mice using a “flow-restriction” model of inferior vena cava (IVC) stenosis. Thrombus frequency, size were quantified following IVC stenosis. Plasma cell-free DNA (cfDNA) as a marker of NETs was quantified with a PicoGreen assay. NETs were degraded in vivo by DNase or vehicle (Veh) infusion.
Results: CD73-/- mice developed significantly larger thrombi (2.5-fold), with more pronounced clot extension (2.6-fold) compared with WT mice 48 h after IVC stenosis (thrombus size: 23.4 ± 2.6 mg vs 9.2 ± 2.4 mg, n=14-15. P<0.0005; clot length: 7.6 ± 0.9 mm vs 2.9 ± 0.7 mm, n=22-36. P<0.0005). Plasma cfDNA was elevated in CD73-/- mice vs WT 6 h after surgery (P<0.05). DNase administration reduced frequency of thrombus in CD73-/- mice by 60% (n=8 P<0.05) compared with Veh-treated CD73-/- mice. In addition, clot burden was reduced by 65% (n=8 P<0.05), and clot extension by 67% (n=8 P<0.01) compared with Veh-treated CD73-/- mice.
Conclusion: The adenosinergic ecto-enzyme, CD73 protects against venous thrombosis. CD73-/- mice have increased DVT burden at 48 h, preceded by elevated cfDNA early after DVT induction, likely implicating NETs in CD73-/- thrombogenesis. Remarkably, cleavage of NETs in vivo by DNase rescued the exaggerated thrombotic phenotype in CD73-/- mice to the level of WT controls. CD73 represents a novel link between venous inflammation and thrombosis. Experiments are underway to delineate relative contributions of neutrophils and the endothelium to thrombosis in CD73-/- mice, and the molecular mechanisms underlying adenosinergic-mediated venous protection.