FVIIa treatment induces the release of procoagulant EVs from the endothelium in vivo via EPCR and PAR1 activation–dependent mechanism. (A) WT mice were administered with FVIIa (0.25 mg/kg body weight) via the tail vein. At varying times following FVIIa administration, mice were killed, blood was collected, and EVs were isolated from the plasma and counted by NTA NanoSight. (B) WT, EPCR-KO, EPCR-OX, PAR1-R41Q, and PAR1-R46Q mice were injected with saline or FVIIa (0.25 mg/kg) via the tail vein. After 2 hours, blood was collected, and the number of EVs in the blood was quantified. (C) EVs isolated from the blood of WT, EPCR-KO, or EPCR-OX mice treated with saline or FVIIa (0.25 mg/kg) for 2 hours were subjected to immunoblot analysis to probe for VE-cadherin (endothelial cell marker), CD41 (platelet marker), CD14 (monocyte marker), TER119 (RBC marker), or VWF. Lysates of murine brain endothelial cells (E), platelets (P), peripheral blood mononuclear cells (M), and RBC lysate (R) were used as positive controls and to attest to the specificity of antibodies. (D-H) Band intensities of immunoblots were quantified by densitometric analysis. (I-J) The procoagulant activity of EVs generated in vivo. EVs, isolated from WT mice treated with saline or FVIIa as described in panel B, were assayed for their ability to support the activation of FX (I) or prothrombin (J). *P < .05; **P < .01; ***P < .001; ****P < .0001.