Endothelial activation by heme-laden MPs. Confluent HUVEC monolayers were treated for 2 hours with erythrocyte MPs or synthetic vesicles. Some MPs were preincubated for 1 hour with specific inhibitors of MPs PS (10 μg/mL annexin-a5) and heme (Hpx, 2 μM). HUVECs were incubated with MPs derived from SCD or control erythrocytes (50 MPs/μL; ie, ∼20 nM heme for control MPs and 65 nM heme for SCD MPs) (A), or synthetic heme-laden MLVs (50 MLVs/μL) (B), washed with PBS, and lysed. Heme incorporation was estimated by spectrophotometry (Abs₃₉₈) with respect to serial heme dilutions and normalized for protein contents; *P < .05 vs control; ^#P < .05 vs control MPs; ^$P < .05 vs MPs + none. (C) We analyzed ROS generation after incubation of HUVECs with SCD and control erythrocyte MPs (25 MPs/μL), with or without preincubation for 1 hour with annexin-a5 or Hpx. (D) HUVECs were preincubated for 1 hour with reduced NAD phosphate inhibitor DPI (10 μM). Here, fluorescent H₂-DFF-DA was added after 30 minutes with MPs, and ROS production measured after 90 minutes. *P < .05 vs control; ^#P < .05 vs MPs + none. (E) Alternatively, we assessed ROS production by erythrocyte MPs alone for 1 hour in H₂-DFF-DA, in absence of HUVECs. *P < .05 vs none; ^#P < .05 vs control MPs; ^$P < .05 vs SCD MPs without endothelium. (F) We assessed the effects of heme-laden MLVs on HUVEC ROS production, after incubation with annexin-a5 (10 μg/mL) or Hpx (2 μM). *P < .05 vs control; ^#P < .05 vs MLVs + none.