TMEM16E regulates endothelial cell procoagulant activity and thrombosis
Alec A. Schmaier, … , Robert Flaumenhaft, Sol Schulman
Alec A. Schmaier, … , Robert Flaumenhaft, Sol Schulman
Published March 23, 2023
Citation Information: J Clin Invest. 2023;133(11):e163808. https://doi.org/10.1172/JCI163808.
View: Text | PDF
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

×

Figure 3

PS externalization visualized via intravital microscopy occurs on the vessel wall and is unaffected by platelet inhibition.

Options: View larger image (or click on image) Download as PowerPoint
PS externalization visualized via intravital microscopy occurs on the ve...
Thrombus formation was monitored for 180 seconds in WT mice following laser injury of the cremasteric arteriole in the presence or absence of the platelet aggregation inhibitor eptifibatide (10 μg/g of body weight). (A) Representative images at indicated time points of the PS probe annexin V (red, Alexa Fluor 647), platelets (blue, anti-CD42b antibody, DyLight 405), and fibrin (green, anti-fibrin antibody, DyLight 488). Note annexin V positivity on the vessel wall and in the absence of platelet aggregation. Kinetics and magnitude of median integrated RFUs for platelet accumulation (B) and PS externalization (D) are shown following laser injury. AUC for fluorescence intensity was determined for platelets (C) and annexin V (E). Lines represent the median AUC for individual thrombi (vehicle n = 34, eptifibatide n = 35) analyzed by Mann-Whitney U test. ****P < 0.0001. A vessel-wall pattern for PS externalization is also observed using alternative PS probes pSIVA (F, red pseudocolor) and lactadherin-FITC (G, red pseudocolor), shown 180 seconds following laser injury. In both F and G, platelets are labeled blue, and representative images are shown from 10 individual thrombi. Arrowheads denote extent of vessel-wall injury and x indicates sites of laser ablation. Scale bars: 25 μm.