(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.