TMEM16E and TMEM16F Regulate the Generation and Procoagulant Activity of Extracellular Vesicles during Endothelial Cell Inflammation

Background: Inflammatory activation of endothelial cells (ECs) is strongly associated with cardiovascular disease, but mechanisms of EC-specific procoagulant activity (PCA) are poorly understood. Cell membrane externalization of phosphatidylserine (PS) by the Ca²⁺-activated phospholipid “scramblase” TMEM16F is a key step in thrombin formation, a process well-characterized in platelets. Our previous work has demonstrated that the EC membrane is a significant source of procoagulant PS and that TMEM16E, a scramblase not found in platelets, is a key regulator of EC PCA, alongside TMEM16F. In addition to procoagulant membrane in situ, inflammatory stimuli promote release of extracellular vesicles (EVs) from ECs. Platelets release procoagulant EVs in a process requiring TMEM16F. We sought to investigate whether the release and/or PCA is of inflammation-induced, EC-derived EVs (ECEVs) is regulated by TMEM16F and/or the newly identified TMEM16E.

Methods and results: To induce EV release, primary HUVECs were treated with TNF-α (10 ng/mL) for 16 h. This stimulation led to non-apoptotic PS exposure. EVs were isolated from HUVEC conditioned media by serial ultracentrifugation at 100,000g and analyzed by nanoparticle tracking analysis (NTA). TNF-⍺ led to a 50-fold increase in EVs released (p<0.001) and produced EVs of smaller size and more negative charge (p<0.01). ECEVs exhibited heterogeneous staining for exosome markers (CD63 and CD9) via immunogold transmission electron microscopy (TEM). Immunoblotting demonstrated exosome and ectosome markers (Alix, exosome; CD31, plasma membrane) and lack of non-EV protein calnexin, confirming the purity of the preparation. TNF-⍺-induced ECEVs had significant PCA as determined by extrinsic and intrinsic tenase, prothrombinase, and plasma thrombin generation. ECEV extrinsic tenase and thrombin generation were inhibited by anti-tissue factor antibody and all PCA was inhibited by the PS-binding protein lactadherin. TMEM16E or TMEM16F were silenced in primary HUVECs using one of two independent siRNAs for each gene for 60 h. In the absence of TMEM16E or 16F, EV release in response to TNF-⍺ was inhibited 10-fold (p<0.001 for NTA-detected EVs compared to control siRNA+TNF-⍺). EVs from TMEM16E- and 16F-deficient ECs had minimal PCA, even after normalizing for the decreased EV number (p<0.0001 for extrinsic and intrinsic tenase and prothrombinase; p<0.001 and p<0.0001 for peak thrombin generation, and p<0.001 and p<0.05 for thrombin lag time for 16E or 16F siRNA compared to control siRNA, respectively). Treatment of ECs with non-specific TMEM16 inhibitors also inhibited release of ECEVs and EV PCA (p<0.001 for CaCCinh-A01 and p<0.0001 for benzbromarone and niclosamide).

We used a mouse endotoxemia model to determine whether systemic inflammation upregulates procoagulant EVs in vivo. Mice were treated with LPS (10 µg/g i.p.) and EVs were analyzed in platelet-depleted plasma by bead-based multiplex flow cytometry (MACSplex, Miltenyi Biotec), analyzed with MPApass software that allows for stitched analysis and normalization across experiments. We observed a significant increase in EC- and leukocyte-derived PS-positive EVs, without an increase in platelet-derived EVs. We generated EC-specific TMEM16F KO mice (TMEM16F^fl/fl; VE-cadherin-Cre) and used global TMEM16E KO mice. 16F-ECKO mice had fewer total and EC-derived EVs and fewer PS+ EVs at baseline and during endotoxemia. TMEM16E-KO mice exhibited decreased CD31+/PS+ EVs at baseline and during endotoxemia.

To determine how TMEM16E and 16F regulate EV release, we performed TEM to visualize multivesicular bodies (MVBs), the intracellular precursors to secreted exosomes. Following TNF-⍺ stimulation, HUVECs silenced for TMEM16E had fewer MVBs, whereas cells silenced for TMEM16F had increased MVBs (p<0.01 vs. control siRNA) with no difference in intraluminal vesicles per MVB.

Conclusions: TMEM16E and 16F regulate both the secretion and intrinsic PCA of ECEVs. Mouse models suggest that TMEM16E and 16F are involved in the host-inflammatory response via release of procoagulant EVs from ECs. TMEM16E may regulate exosome formation at the endosomal level whereas 16F may regulate exosome release at the plasma membrane.