TMEM16E Regulates Endothelial Cell Procoagulant Activity and Thrombosis

Endothelial cells (ECs) are constitutively an anticoagulant surface but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane surface containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases", such as TMEM16F, following sustained elevation in intracellular Ca²⁺. TMEM16F-dependent externalization of PS is well-characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not well understood.

To identify regulators of PS exposure on ECs, we employed a focused genetic screen to evaluate genes implicated in outer leaflet externalization of PS in biological membranes, including six members of the Ca²⁺-activated TMEM16 family, three members of the apoptosis-activated Xk-related family, and three members of the P4-ATPase family of phospholipid flippases. We determined that two TMEM16 family members, TMEM16F, and its closest paralog, TMEM16E, were required for maximal procoagulant activity on the EC surface. TMEM16E has not previously been implicated in blood coagulation. Silencing TMEM16E or TMEM16F in TNF-α stimulated primary HUVECs inhibited factor VIIa catalyzed factor Xa generation by over 50% (p<0.0001), thrombin generation by over 75% (p<0.0001), and PS externalization by over 75% (p<.0001). Silencing TMEM16E or TMEM16F also suppressed calcium ionophore A23187-induced PS externalization by 50-80% (p<.01-.0001) and augmentation of tissue factor (TF)-dependent factor Xa generation (p < 0.001). To uncouple TF expression from PS externalization, we used an Ea.hy926 cell line stably expressing TF, and found that TMEM16E and TMEM16F were each required for PS externalization and Ca²⁺ ionophore-induced augmentation of factor Xa generation (p<0.001). TMEM16E or TMEM16F were not required for expression of TF on the EC surface. Silencing of TMEM16E or TMEM16F had no effect on intracellular Ca²⁺ flux in ECs stimulated with thrombin as determined by the fluorescent Ca²⁺ indicator Calbryte-520.

TMEM16E is highly expressed in skeletal muscle, and we found TMEM16E is expressed in human ECs from various tissues, but is not expressed in platelets, as determined by qPCR. TMEM16E^-/- mice are overtly healthy yet demonstrate defective muscle repair, but have not been studied with regard to blood coagulation. We used intravital microscopy following laser injury of the cremasteric arteriole to measure accumulation of fluorescently-labeled platelets and fibrin. TMEM16E^-/- mice demonstrated a small but significant decrease in fibrin formation (15.9%, <0.05) but no difference in platelet accumulation compared to TMEM16E^+/+ littermate controls. To eliminate the contribution of platelet-mediated fibrin formation and to clarify the contribution of platelet versus vessel wall PS externalization, animals were treated with eptifibatide to prevent platelet accumulation. Under eptifibatide treatment, TMEM16E^-/- mice demonstrated a larger reduction in fibrin formation (43.5%, p<0.01) following laser injury compared to littermate controls. We observed no differences in baseline coagulation parameters (aPTT or PT) or platelet count between TMEM16E^-/- and TMEM16E^+/+ littermates. TMEM16E^-/- mice did not demonstrate excessive bleeding in a tail clip bleeding assay as determined by bleeding time and total hemoglobin loss.

This investigation establishes TMEM16 proteins as drivers of EC procoagulant activity. We specifically identify TMEM16F, known to contribute to procoagulant activity in platelets, and TMEM16E, which is novel. Fibrin generation is impaired in TMEM16E-deficient mice in a vessel-wall dependent manner, independent of platelets. These observations suggest new areas of investigation into the role of endothelial membrane phospholipid dynamics in coagulation and the potential of TMEM16 inhibition for treatment of pathologic blood clotting.

Flaumenhaft:Platelet Diagnostics: Consultancy, Current equity holder in private company, Other: Founder, Patents & Royalties; Function Therapeutics: Consultancy, Current holder of stock options in a privately-held company; Xap Therapeutics: Consultancy.