Abstract 12521: TMEM16 Antagonists Inhibit Phosphatidylserine-Induced Procoagulant Activity in Endothelial Cells and Protect From Thrombosis in vivo

Abstract

Introduction: Constitutively an anticoagulant surface, endothelial cells (ECs) switch to support coagulation following pathogenic stimuli, contributing to cardiovascular disease. To promote thrombosis, coagulation proteins must assemble on a membrane surface containing the anionic phospholipid phosphatidylserine (PS). PS is asymmetrically distributed on the inner leaflet of the plasma membrane but is rapidly externalized to the outer leaflet by calcium-activated phospholipid “scramblases”, such as TMEM16F. How phospholipid externalization on ECs supports coagulation and thrombosis has not been investigated. Hypothesis: We proposed that pharmacologic inhibition of TMEM16 phospholipid scramblases inhibits EC procoagulant activity and protects against thrombosis. Methods and Results: siRNA silencing of TMEM16F, and its closest paralog, TMEM16E, inhibited factor Xa generation and thrombin generation on the EC surface (60% and 90%, respectively, p < 0.0001), equivalent to the PS-binding protein lactadherin (100 nM). Two unrelated compounds with TMEM16 inhibitory activity, benzbromarone and CaCCinh-A01 (30 μM), prevented calcium-induced PS externalization on ECs, as determined by flow cytometry and immunofluorescence microscopy. These drugs did not inhibit intracellular calcium flux, suggesting they block the PS externalization activity of TMEM16. Benzbromarone and CaCCinh-A01 inhibited factor Xa generation on the EC surface in a dose-dependent manner (IC 50 3.2 μM and 2.0 μM, respectively). Thrombosis was evaluated using a laser injury model in the mouse cremasteric arteriole coupled with intravital microscopy to measure accumulation of fluorescently-labeled platelets and fibrin. Benzbromarone (5 mg/kg) inhibited platelet accumulation ( p < 0.05) and fibrin formation ( p < 0.01). Benzbromarone did not increase bleeding time in a tail-clip hemostasis assay. Conclusions: Activated ECs contribute anionic phospholipid to support coagulation, a process regulated by TMEM16 phospholipid scramblases. TMEM16 inhibition with benzbromarone protects from experimental thrombosis without resulting in excess bleeding. Benzbromarone is used worldwide to treat gout and could be repurposed for anti-thrombotic properties.