Abstract 050: The Vascular Ecto-enzyme, CD39 Protects from Venous Thrombogenesis by inhibiting Innate Immune Activation

Background: Venous thrombosis (DVT) is a serious health concern, with growing incidence in an aging, comorbid population. DVT is marked by sterile inflammation and innate immune activation. Early participants in DVT include platelets, and neutrophils (PMN) which extrude their DNA to form thrombus-potentiating extracellular traps (NETs). To identify improved therapeutic targets at the nexus of inflammation and coagulation, we focused on the vascular ectonucleotidase CD39, found on leukocytes and the endothelium. CD39 dissipates extracellular ATP & ADP, thrombo-inflammatory “danger” signals. We hypothesized that CD39 is a critical enzyme in venous homeostasis, restraining unchecked inflammation and thrombosis in DVT. Methods: DVT was induced in Cd39 +/- and WT control mice using a “flow-restriction” model of inferior vena cava (IVC) stenosis. Thrombus frequency and size were assessed 48h after DVT induction. A novel flow cytometry method, immunoblot, immunostaining were used to examine thrombus cellular content, fibrin, NETs, and inflammasome activation. Results: Cd39 +/- mice had a significantly higher thrombus frequency (2.5-fold) and clot size (3-fold), with more fibrin content by immunoblot. Flow cytometry revealed exaggerated PMN recruitment to the growing thrombus in Cd39 +/- mice, with more NETs in vitro , and within DVT compared with WT mice. Cd39 +/- mice also had amplified DVT inflammasome activity in vivo , measured by increased NFkB phosphorylation and mature IL-1 beta compared with WT DVT. Flow cytometry of the thrombus revealed an increase in activated platelet-PMN heteroaggregates within Cd39 +/- DVTs, indicating enhanced innate-coagulation system crosstalk. Conclusion: CD39 is a critical vasculoprotective ecto-enzyme in venous thrombogenesis. Cd39 +/- mice have increased DVT burden, fibrin deposition, activated platelet-PMN interactions, and exaggerated inflammasome activation compared with WT mice. By enrolling NET formation and inflammasome activation, CD39 posits a previously unexplored link between venous inflammation and thrombosis. Studies are underway to modulate inflammasome activation in Cd39 +/- mice, and to delineate the relative contributions of leukocyte/endothelial CD39 in venous thrombosis.