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 Ca2+-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. Silencing of either TMEM16E or TMEM16F resulted in a similar (approximately 50%) reduction of factor Xa generation and factor VIIa–catalyzed factor X activation. The degree of inhibition was similar to that observed following treatment with lactadherin, which neutralizes externalized PS. HUVECs stimulated with TNF-α, the Ca2+ ionophore A23187, or lipopolysaccharide also required TMEM16E and TMEM16F to support thrombin generation in human plasma. Consistently, silencing of TMEM16E or TMEM16F markedly reduced PS externalization in HUVECs in response to TNF-α or A23187 without affecting intracellular Ca2+ 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 Ca2+-ionophore–stimulated PS externalization in parallel with activation of factor X, but without affecting intracellular Ca2+ flux (15). These results indicate that TMEM16E and TMEM16F regulate endothelial procoagulant activity through PS externalization (Figure 1A).

TMEM16E and TMEM16F regulate endothelial cell procoagulant activity and thrombosis. (A) Endothelial cells express the Ca2+-activated phospholipid scramblases TMEM16E and TMEM16F. Activation and/or injury triggers increases in intracellular Ca2+ and conformation changes in scramblases, leading to externalization of anionic phospholipids, most commonly PS. TMEM16E and TMEM16F might be epistatic to one another or function in a linear pathway. One possible mechanism involves the shuttling of PS to the plasma membrane by TMEM16E, where it can be directly externalized by TMEM16F. Alternatively, TMEM16E and TMEM16F could form a heterodimer in which TMEM16E functions as a regulator of TMEM16F. The precise localization of TMEM16E and TMEM16F, within the same or separate compartments, remains unclear. Externalized PS binds to and allosterically regulates coagulation factors Xa and Va, promoting activation of factor X by the TF–factor VIIa complex and the factor Xa–factor Va–prothrombinase complex, leading to thrombin formation and fibrin generation. (B) Laser injury to the vessel wall induces PS externalization on the endothelium, fibrin deposition, and platelet accumulation at the injured sites in mice. Mice with genetic depletion of either TMEM16E or TMEM16F showed reduced PS externalization and fibrin deposition, but platelet accumulation similar to that of WT littermates. Prevention of platelet accumulation with eptifibatide did not reduce PS externalization on the endothelium or diminish fibrin formation. Treatment with the TMEM16 inhibitor benzbromarone reduced platelet accumulation, endothelial PS externalization, and fibrin deposition. These results indicate that PS externalization on the vessel wall drives 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. A possible mechanism for independent activities could involve the step-wise shuttling of PS, first by TMEM16E to the plasma membrane, then by TMEM16F to externalize it from the plasma membrane. Alternatively, TMEM16E could function as a regulator of TMEM16F, perhaps by forming a heterodimer, as TMEM16 proteins are capable of assembling into homo- and heterodimers (7, 17). Whether TMEM16E and TMEM16F interact in so-to-speak tango or shuffle or whether they are located in the same or separate compartments remains to be investigated. Of note, a recent study implicates TMEM16E as having a role in coordinating membrane fusion between muscle precursor cells to produce multinucleated skeletal muscle fibers (18).