TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca²⁺ ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca²⁺-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.Phospholipids are asymmetrically distributed between the inner and outer leaflets of plasma membranes as a result of the activity of flippase(s), which specifically translocates phosphatidylserine (PtdSer) and phosphatidylethanolamine from the outer to the inner leaflet of plasma membranes (1). PtdSer is preferentially exposed on the cell surface during certain physiological processes. During apoptosis, cell-surface PtdSer functions as an “eat me” signal to induce engulfment by phagocytic cells, and, during platelet activation, it serves as a scaffold for the activation of clotting factors. Exposed PtdSer is also implicated in pathological processes and may promote the retention of Ca²⁺ oxalate in kidneys, leading to kidney stone formation (2).PtdSer exposure is accomplished by the inactivation of flippase(s), along with the activation of scramblases (3). We recently identified two protein families (TMEM16 and Xkr) that support phospholipid scrambling (4–6). The TMEM16 family consists of 10 members with 10 transmembrane regions, and TMEM16C, 16D, 16F, 16G, and 16J support the Ca²⁺-dependent scrambling of phospholipids. Scott syndrome is a mild bleeding disorder caused by a defect in platelet procoagulant activity (7, 8). Platelets, red blood cells, and EBV-transformed B cells from patients with Scott syndrome exhibit defective PtdSer exposure following platelet activation or treatment with Ca²⁺ ionophore (9–11). We and others reported that patients with Scott syndrome carry null mutations in the TMEM16F gene (6, 12).The fetal thymocyte cell lines established from TMEM16F^−/− mouse embryos exhibit defective PtdSer exposure upon treatment with Ca²⁺ ionophore (5), reminiscent of the EBV-transformed B-cell lines from patients with Scott syndrome. In contrast, Yang et al. (13) reported that TMEM16F-deficient mouse platelets exhibit only a mild defect in Ca²⁺ionophore–induced PtdSer exposure and tissue factor-induced thrombin generation. Furthermore, in contrast to a human patient with Scott syndrome (14) and dogs with a similar hereditary syndrome (15), neither of which exhibits apparent bleeding-time defects, TMEM16F^−/− mice exhibit a prolonged bleeding time—twice that of WT mice (13).To address the apparent discrepancies between TMEM16F^−/− mouse phenotypes and the clinical presentation of patients with Scott syndrome, and to examine the role of platelet-expressed TMEM16F in blood clotting, we generated a platelet-specific TMEM16F deletion in mice. Our in vitro and in vivo analyses of thrombus formation induced by TMEM16F-null platelets suggested a role for TMEM16F in activation-induced PtdSer exposure, and supported the model in which Ca²⁺-induced PtdSer exposure is involved in the generation of thrombin and fibrin, but not clot retraction (16, 17). Mice with the platelet-specific deletion of TMEM16F exhibited a phenotype similar to that of human patients with Scott syndrome, and may provide a useful model for this human disease.