Endothelial-specific knockout of the scramblase TMEM16F impairs in vivo clot formation.

Abstract

Objective: Loss of function of the phospholipid scramblase (PLS) TMEM16F results in Scott Syndrome, a hereditary bleeding disorder generally attributed to intrinsic platelet dysfunction. The role of TMEM16F in endothelial cells, however, is not well understood. We sought to test the hypothesis that endothelial TMEM16F contributes to hemostasis by measuring bleeding time and venous clotting in endothelial-specific knockout (ECKO) mice.

Materials and methods: We initially evaluated the extent to which TMEM16F contributes to endothelial calcium events produced by trauma factors in vitro, using a pharmacological approach. Cultured endothelial cells were exposed to histones in the presence or absence of the PLS inhibitor, niclosamide, for live-cell calcium imaging and flow cytometry with annexin V staining. We then applied a genetic approach to specifically ablate TMEM16F in vascular endothelial cells in vivo using a murine tamoxifen-inducible cre-lox system under control of a Cdh5 promoter. Hemostasis was evaluated by measuring tail bleeding time after a distal 5 mm tail resection. Venous thrombus formation was evaluated by creating a surgical stenosis of the inferior vena cava (IVC) and harvesting the resultant clot 24 hours post-procedure for measurement. Blood samples were obtained via IVC cannulation to assay plasma-based coagulation. Mesenteric arteries were isolated and cannulated for assessment of endothelial-dependent vasodilation by pressure myography.

Results: Pretreatment with the PLS inhibitor niclosamide prevented pathological calcium signals and mitigated PS translocation in cultured endothelial cells exposed to extracellular histones. TMEM16F ECKO mice exhibited prolonged bleeding compared to controls (time, 205.6 +/- 234.5 vs. 38.1 +/- 29.11 sec; p < 0.05). The ECKO mice also generated significantly smaller IVC thrombi (length, 0.9 +/- 1.4 vs. 4.7 +/- 3.3 mm; p < 0.05). TMEM16F ablation did not impact prothrombin time or endothelial-dependent vasodilatory function.

Conclusions: Endothelial TMEM16F function is essential for normal hemostasis. ECKO of TMEM16F is sufficient to produce a coagulopathic phenotype, as shown by the prolonged bleeding time after tail transection and decreased thrombus generation in response to IVC stenosis. Because endothelial calcium events are pathologically amplified in response to trauma factors, these results suggest that TMEM16F may play a role in trauma-induced coagulopathy.