TMEM16E regulates endothelial cell procoagulant activity and thrombosis
Alec A. Schmaier, Papa F. Anderson, Siyu M. Chen, Emale El-Darzi, Ivan Aivasovsky, Milan P. Kaushik, Kelsey D. Sack, H. Criss Hartzell, Samir M. Parikh, Robert Flaumenhaft, Sol Schulman
Alec A. Schmaier, Papa F. Anderson, Siyu M. Chen, Emale El-Darzi, Ivan Aivasovsky, Milan P. Kaushik, Kelsey D. Sack, H. Criss Hartzell, Samir M. Parikh, Robert Flaumenhaft, Sol Schulman
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Research Article Hematology Vascular biology

TMEM16E regulates endothelial cell procoagulant activity and thrombosis

Abstract

Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, 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 2 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.

Authors

Alec A. Schmaier, Papa F. Anderson, Siyu M. Chen, Emale El-Darzi, Ivan Aivasovsky, Milan P. Kaushik, Kelsey D. Sack, H. Criss Hartzell, Samir M. Parikh, Robert Flaumenhaft, Sol Schulman

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Figure 5

TMEM16 antagonists reduce EC procoagulant activity by inhibiting PS externalization.

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TMEM16 antagonists reduce EC procoagulant activity by inhibiting PS exte...
(A) HUVECs were treated with Ca2+ ionophore A23187 for 20 minutes, and PS externalization was measured by flow cytometry. Histograms were generated after gating on live (DAPI negative) cells only. (B) HUVECs were treated with TNF-α (10 ng/mL) for 3.5 hours and/or A23187 for 20 minutes at indicated concentrations and analyzed for factor VIIa–catalyzed activation of factor X. Anti-TF antibody (10 μg/mL) and lactadherin (100 nM) were used to block TF and PS, respectively. All cells not receiving anti-TF antibody were treated with IgG isotype control. Asterisks denoting statistical significance show comparison with cells treated with TNF-α, no ionophore, unless otherwise specified, with brackets to indicate pairwise comparison. (C) HUVECs were treated with A23187 (6 μM) for 20 minutes in the presence of TMEM16 inhibitors CaCCinh-A01 (A01) and BBR (both 10 μM) and analyzed for PS externalization as in A. (D) TMEM16 antagonists were assayed for their ability to inhibit factor VIIa–catalyzed activation of factor X on HUVECs stimulated with TNF-α (10 ng/mL, 3.5 hours) followed by A23187 (6 μM, 20 minutes). (E) HUVECs were transfected with indicated siRNAs, treated with A01 or BBR, stimulated with TNF-α (10 ng/mL, 3.5 hours), and assayed for their ability to support factor Xa generation. (F and G) HUVECs were treated with A01 or BBR or the phospholipase C inhibitor U73122, and intracellular Ca2+ transients were measured with Calbryte 520 AM following stimulation with thrombin (1 U/mL). Time course of Calbryte 520 fluorescence, normalized to background (F), and AUC values (G), normalized to vehicle-treated cells, are shown. n = 3–6 independent experiments. Error bars indicate mean ± SD (A–E and G) or mean ± SEM (F). ANOVA with Tukey’s post test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.