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 1

TMEM16E and TMEM16F regulate EC procoagulant activity.

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TMEM16E and TMEM16F regulate EC procoagulant activity. (A) Heatmap illus...
(A) Heatmap illustrating the relative positive or negative regulation of factor VIIa–catalyzed activation of factor X following silencing of the indicated genes in HUVECs. Indicated genes were silenced with a pool of 4 distinct siRNAs and tested in triplicate. Each box represents an independent experimental plate, with scale bar depicting percentage of factor Xa generated compared with cells transfected with untargeted control siRNA. Lighter color indicates lower percentage factor Xa generation compared with control. (B–G) HUVECs were transfected with individual siRNAs for 72 hours and assayed for their ability to support factor VIIa–catalyzed activation of factor X (B–D) or thrombin generation in plasma-treated ECs (E–G). Cells were stimulated with TNF-α (10 ng/mL) for 3.5 hours (B, C, E, and F), TNF-α for 3.5 hours plus Ca2+ ionophore A23187 (6 μM) for 20 minutes (D), or A23187 alone for 20 minutes (G). Representative experiments are depicted as mean absorbance for factor Xa generation (B) or the first derivative of arbitrary fluorescent units for thrombin generation (E) as a function of time. 16E, 16F, and TF denote siRNA targeting TMEM16E, TMEM16F, and TF, respectively, and #1 and #2 denote distinct siRNA sequences. When indicated, lactadherin (100 nM) was added to cells treated with control siRNA. n = 3–5 independent experiments. Error bars indicate mean ± SEM (B and E) or mean ± SD (C, D, F, and G). Asterisks denoting significance are in reference to control siRNA, unless otherwise specified with brackets to indicate pairwise comparison, ANOVA with Tukey’s post test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.