TMEM16E regulates endothelial cell procoagulant activity and thrombosis

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.

each biologic sample using a QuantStudio 6 Flex real-time PCR system. Gene expression was compared to ACTB expression using the DDCt method.
Beginning after 48 hours, the viral supernatant was collected twice every 24 hours and replaced with fresh DMEM supplemented with 10% fetal bovine serum. Primary HUVECs (passage [1][2] were cultured in the presence of viral supernatant for 6 hours and then replaced with complete endothelial cell growth media (see Endothelial cell culture and siRNA transfection) for 48 hours before selecting for transduced cells with addition of blasticidin (10 µg/mL) to the cell culture media.

Cell Viability Assay
To assess cell viability of cells transfected with indicated siRNAs, an XTT Cell Proliferation Assay (ATCC) was performed according to manufacturer protocol. Absorbance at 630 nm and 450 nm were measured on an xMark Spectrophotometer (Bio-Rad).

Confocal intravital microscopy for Z-stack images
Wild-type C57BL/6J male mice were anesthetized and prepped as described in "Intravital microscopy and laser-induced vessel wall injury model" and the cremaster arteriole was injured using the Ablate! (3i) laser ablation system. Z-stack images were obtained on an CSU-W1 spinning disk confocal microscope with SoRa super resolution optical unit (Yokagawa) using a 6-line laser illumination system (3i), 63X high numerical aperture water corrected lens (Zeiss) and Orca-Fusion BT sCMOS digital video camera (Hamamatsu). Z-stack images were rendered into 3-dimensional images using using Slidebook version 6.0 (Intelligent Imaging Innovations). HUVECs stably expressing TMEM16E containing a C-terminal V5 tag were transfected with indicated siRNA targeting TMEM16E or untargeted control siRNA for 72 h. TMEM16E protein was determined by SDS-PAGE and immunoblotting with antibodies against TMEM16E, V5, and actin (loading control). TMEM16E is detected as a band running just above 100 kd. B. Primary HUVECs were transfected with siRNA targeting TMEM16E for 72 h before determining endogenous TMEM16E mRNA level by quantitative PCR. C. HUVECs were transfected with siRNA targeting TMEM16F for 72 h. TMEM16F protein was determined by SDS-PAGE and immunoblotting with anti-TMEM16F antibody. D. Primary HUVECs or HUVECs stably expressing TMEM16E-V5 were transfected with indicated siRNA for 72 h prior to determination of TMEM16E and TMEM16F protein by SDS-PAGE and immunoblotting with anti-TMEM16F and anti-V5 antibody. E. HUVECs were transfected with indicated siRNA for 72 h and cell viability was determined by XTT assay. #1 and #2 denote independent siRNA sequences. TF denotes siRNA targeting tissue factor. Figure 3. TMEM16E and TMEM16F regulate endothelial cell (EC) procoagulant activity. A. Primary HUVECs were stimulated with TNF-a (10 ng/mL), LPS complex (LPS [100 ng/mL], LBP [10 ng/mL], and sCD14 [100 ng/mL]) or vehicle control for 3.5 h and assayed for their ability to support factor VIIa-catalyzed activation of factor X. B. HUVECs were transfected with individual siRNAs for 72 h and assayed for their ability to support factor Xa generation following stimulation with LPS complex for 3.5 h. C. An Ea.hy926 cell line stably expressing TF was transfected with siRNAs targeting TMEM16E, TMEM16F, or TF for 72 h. Cells were treated with Ca 2+ ionophore A23187 (6 µM) for 20 min and assayed for their ability to support factor VIIa-catalyzed activation of factor X. 16E, 16F and TF denote siRNA targeting TMEM16E, TMEM16F, and tissue factor, respectively. #1 and #2 denote independent siRNA sequences. Error bars indicate mean ± SEM (A) or mean ± SD (B and C), ANOVA with Tukey's posttest, ****p<0.0001.

Supplemental Figure 4. TMEM16E and TMEM16F are required for PS externalization on
ECs. HUVECs were transfected with indicated siRNAs for 72 h, stimulated with TNF-a (10 ng/mL) for 16 h (A) or calcium ionophore A23187 (6 µM) for 20 min (B), and stained with annexin V to detect PS externalization and Zombie Red to detect cell death. Each dot represents the total fluorescent area of Zombie Red per image normalized to the number of nuclei present. Note no increase in cell death (Zombie Red positivity) in cells treated with TNF-a or A23187 compared to control. C. Representative flow cytometric analysis of PS exposure (annexin V, x-axis) and cell death (DAPI, y-axis). Numbers refer to the percentage of total cells in each quadrant. Q1 represents dead, PS-negative population, Q2 represents dead, PSpositive population, Q3 represents live, PS-positive population, Q4 represents live, PS-negative population. Figure 5. PS externalization during thrombus formation following laser injury. Thrombus formation was monitored for 180 seconds in wild-type mice following laser injury of the cremasteric arteriole (A) and additionally in the presence of the platelet aggregation inhibitor eptifibatide (B, 10 µg/g of body weight). Representative images at indicated time points of the PS probe annexin V (red, Alexa Fluor 647), platelets (anti-CD42b antibody, blue, Dylight 405), and fibrin (anti-fibrin antibody, green, Dylight 488). Note annexin V positivity on the vessel wall and in the absence of platelet aggregation. Arrowheads denote extent of vessel-wall injury and "X" indicates site of laser ablation. Arrows indicate extension of annexin V binding to the vessel wall opposite of laser ablation. To better visualize annexin V binding, platelet fluorescence is omitted from the bottom images in (A). Asterisk (*) indicates the platelet aggregate. C. 3-dimensional renderings of Z-stack images of annexin V binding following laser injury. Dotted yellow lines indicate the vessel wall boundaries. Scale bar is 25 µm unless otherwise indicated. and activated partial thromboplastin time (aPTT, E). Wild-type C57BL/6J mice were treated with intraperitoneal injection of benzbromarone (5 µg/g of body weight). After 1 h, plasma was assessed for prothrombin time (PT, F) and activated partial thromboplastin time (aPTT, G).

Supplemental
Error bars indicate mean ± SEM, n = 5-8 animals per genotype or treatment.

Supplemental Movie 1. PS externalization and thrombus formation following vascular
injury. The following probes were injected into the mouse vasculature: annexin V conjugated to Alexa Fluor 647 to detect PS externalization (red), anti-CD42b antibody conjugated to Dylight 405 to detect platelets (blue), anti-fibrin antibody conjugated to Dylight 488 to detect fibrin (green). Vessel wall injury was induced by laser ablation in a cremasteric arteriole and intravital video microscopy was performed for 180 seconds.