TMEM16E regulates endothelial cell procoagulant activity and thrombosis

Abstract

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 containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases", such as TMEM16F. TMEM16F-dependent PS externalization is well-characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified two TMEM16 family members, TMEM16F, and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

Two to tango: endothelial cell TMEM16 scramblases drive coagulation and thrombosis

“…An intriguing finding of Schmaier et al is the absence of additive suppression for factor Xa generation following dual silencing of TMEM16E and TMEM16F ( 15 ). Thus, TMEM16E and TMEM16F appear to be epistatic to one another or to function in a linear pathway.…”

“…In this issue of the JCI , Schmaier and coauthors identified five proteins as important regulators of factor VIIa–catalyzed activation of factor X in TNF-α–stimulated human umbilical vein endothelial cells (HUVECs), including TMEM16F, TMEM16E, the closest paralog of the canonical Ca 2+ -activated scramblase TMEM16F ( 6 ), and Xkr9, a member of the Xk-related family of caspase-activated phospholipid scramblases ( 15 ). Expression of TMEM16E and TMEM16F was confirmed in unstimulated HUVECs and primary endothelial cells from human coronary artery and microvascular tissue, whereas XKR9 expression was undetectable with quantitative PCR (qPCR) in these cell types.…”

“…Consistently, silencing of TMEM16E or TMEM16F markedly reduced PS externalization in HUVECs in response to TNF-α or A23187 without affecting intracellular Ca 2+ flux, TF cell-surface expression, or TF pathway inhibitor (TFPI) expression ( 16 ). Furthermore, pharmacological blockade of TMEM16 proteins with the unrelated small molecules benzbromarone and CaCCinh-A01 inhibited Ca 2+ -ionophore–stimulated PS externalization in parallel with activation of factor X, but without affecting intracellular Ca 2+ flux ( 15 ). These results indicate that TMEM16E and TMEM16F regulate endothelial procoagulant activity through PS externalization ( Figure 1A ).…”

“…Schmaier and coauthors used intravital microscopy to monitor PS externalization and platelet and fibrin accumulation in cremaster arterioles following laser injury to the vessel wall and observed PS externalization along the injured vessel wall, not in the growing platelet aggregate ( 15 ). PS externalization also spread to the opposite wall in about one-third of the injuries without platelet accumulation or fibrin formation, indicating that these processes require adhesion proteins or TF in addition to PS externalization.…”

OxLDL enhances procoagulant activity of endothelial cells by TMEM16F‐mediated phosphatidylserine exposure

“…Moreover, inactivating mutations of TMEM16F result in Scott's syndrome, a bleeding disorder in humans (Millington‐Burgess & Harper, 2020). Recently, TMEM16F had been identified that could regulate PCA of EC by mediating PS exposure (Schmaier et al, 2023). Therefore, further investigation of the role and the molecular mechanism of TMEM16F‐mediated PS exposure in hyperlipidemia is necessary.…”