Abstract 121: TMEM16E And TMEM16F Are Required For The Formation Of Prothrombotic Extracellular Vesicles From Endothelial Cells

• Published in: Arteriosclerosis, thrombosis, and vascular biology Vol. 43; no. Suppl_1
• Main Authors: Anderson, Papa Freduah A, Schmaier, Alec A
• Format: Journal Article
• Language: English
• Published: 01.05.2023
• ISSN: 1079-5642; 1524-4636
• DOI: 10.1161/atvb.43.suppl_1.121

Abstract only Procoagulant extracellular vehicles (EVs) have been implicated in thrombotic cardiovascular disease. Externalization of phosphatidylserine (PS) supports the assembly of coagulation enzyme complexes and the formation of EVs. Prior work has demonstrated that the endothelial cell (EC) membrane is a significant source of procoagulant PS and that the transmembrane proteins TMEM16E and TMEM16F are required for procoagulant PS externalization. We sought to determine whether formation of procoagulant EC EVs is regulated by TMEM16E or TMEM16F. TMEM16E or TMEM16F were silenced in HUVECS using siRNA and cells were treated with TNF-α (16 h, 10 ng/mL) to stimulate tissue factor expression and PS externalization. EC EVs were isolated from conditioned media via ultracentrifugation. EV charge, particle number, and size were analyzed using zeta potential, nanoparticle tracking analysis, and transmission electron microscopy (TEM). Procoagulant activity was measured via factor VIIa-catalyzed factor Xa generation, prothrombinase complex assay, and by thrombin generation in plasma. Silencing of TMEM16E or TMEM16F reduced factor Xa generation on EC EVs by 90% (P < 0.0001, 2 independent siRNA). TMEM16E and TMEM16F were required for thrombin generation, a process also inhibited by lactadherin, which blocks the accessibility of PS to coagulation enzymes. TNF-α resulted in a ~400-fold increase in total EC EV generation compared to vehicle control (P < 0.0001). TNF-α-induced EC EVs were smaller than that induced by vehicle control (88 nM vs 110 nM, P < 0.02). Silencing of TMEM16E or TMEM16F reduced TNF-α-induced EV production by 100-fold (P < 0.0001) and EVs from TMEM16E or TMEM16F silenced cells trended larger in size. We confirmed the exosome identity of EC EVs via CD63 immunogold staining and TEM. Zeta potential showed that EC EVs were negatively charged and EC EVs from TNF-α-stimulated cells showed the most negative charge. Silencing of TMEM16F reduced the negative charge of EC EVs (-38.57mV vs -20.51mV, P < 0.004) suggesting less abundance of negatively charged phospholipids. This study identifies TMEM16E and TMEM16F as essential for the production of PS-positive, procoagulant EC EVs. TMEM16 proteins may therefore be therapeutic targets to protect against thrombosis.