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 2

TMEM16E and TMEM16F are required for PS externalization on ECs.

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TMEM16E and TMEM16F are required for PS externalization on ECs. HUVECs w...
HUVECs were transfected with indicated siRNAs for 72 hours, stimulated with TNF-α (10 ng/mL) for 16 hours (A) or Ca2+ ionophore A23187 (6 μM) for 20 minutes (B), and stained with annexin V (green) to detect PS externalization and Zombie Red (red) to detect cell death. Total annexin V fluorescence was normalized to number of nuclei (blue) and dead cells. (C) PS externalization following treatment with ionophore A23187 was detected using annexin V by flow cytometry. Histograms were generated after gating on live (DAPI negative) cells only. (D) HUVECs were transfected with indicated siRNAs for 72 hours, stimulated with TNF-α (10 ng/mL) for 3.5 hours, and stained for TF (green). Mean fluorescent intensity (MFI) was normalized to background for each image. Representative images are shown. (E and F) Intracellular Ca2+ flux was measured with Calbryte 520 AM in siRNA-transfected HUVECs following stimulation with thrombin (1 U/mL). Silencing of the store-operated Ca2+ regulator STIM1 served as a positive control. Time course of Calbryte 520 fluorescence after thrombin stimulation normalized to background fluorescence (E) and AUC values (F) normalized to cells treated with control siRNA are shown. (G) TFPI protein was determined by SDS-PAGE and immunoblotting with anti-TFPI antibody in HUVECs transfected with indicated siRNAs. Numbers refer to fold-change normalized to GAPDH (± SD). Scale bars: 100 μm (A and B); 50 μm (D). n = 3–6 independent experiments. Error bars indicate mean ± SD (A–D and F) or mean ± SEM (E). Asterisks denoting significance are in reference to control siRNA, ANOVA with Tukey’s post test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.