Mechanism of FVIIa-induced biogenesis of EVs in endothelial cells. (A) HUVECs were transfected with scr RNA or β-arrestin 1 (β-Arr.1) or β-arrestin 2 (β-Arr.2) siRNA (200 nM each) for 48 hours. The extent of β-arrestin knockdown was examined by immunoblot analysis (top) and quantified by densitometry (bottom). (B) β-Arrestin 1– or β-arrestin 2–silenced endothelial cells were serum starved for 1 hour and then treated with a control vehicle (Con) or FVIIa (100 nM) for 24 hours. EVs were isolated from the conditioned medium and quantified by NTA. (C-D) Serum-starved HUVECs were treated for 1 hour with specific inhibitors of AKT, ERK1/2, or ROCK: LY294002 (LY, 25 µM), U0126 (U, 20 µM), or Y27632 (Y, 10 µM), respectively. The cells were treated with a control vehicle or FVIIa (100 nM) for 1 hour (C) or 20 minutes (D), to assess the activation of AKT or ERK1/2, respectively, by immunoblot analysis (top), and the band intensities were quantified by densitometry to determine the extent of activation (bottom). (E) HUVECs were treated with LY294002, U0126, or Y27632, alone or in combination, at the concentrations and for the times described for panels C and D and then exposed to the control vehicle or FVIIa (100 nM). After 24 hours, EVs were isolated from the conditioned medium and quantified by NTA. (F) The potential signaling mechanism involved in FVIIa-induced generation of EVs. *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, not significant. GAPDH, glyceraldehyde phosphate dehydrogenase.