Endothelial apoptosis by heme-laden MPs. Confluent HUVEC monolayers were treated with purified erythrocyte MPs (25 MPs/μL), synthetic heme-laden MLVs, or heme alone (5 μM) for 16 hours. Some MPs were preincubated with annexin-a5 (10 μg/mL) or Hpx (2 μM) for 1 hour. HUVECs were then fixed. Total DNA contents were determined by FACS after coloration with propidium iodide. Cells undergoing apoptosis (sub-G1 phase) or proliferation (G2/M) were quantified. (A) Representative DNA content profiles. We compared the effects of serial dilutions of control and SCD erythrocyte MPs (B) vs high serum (10% serum) or proapoptotic etoposide (100 μM). Triangles, SCD MPs; squares, control MPs. *P < .05 vs none; ^#P < .05 vs control MPs. (C) In other experiments, HUVECs were fixed in situ and stained with 4,6-diamidino-2-phenylindole. Nuclei displaying fragmentation, pyknosis, or condensed chromatin by fluorescence microscopy were counted as apoptotic and expressed as percentage. (D) Representative images (×400). Arrows designate fragmented and condensed nuclei. (E) Quantification of degraded nuclei after incubation with erythrocyte MPs (25 MPs/μL), vs high serum (10% serum), or proapoptotic etoposide. Some HUVECs were preincubated for 30 minutes with NAC (5 mM), DPI (10 μM), or apocynin (100 μM). *P < .05 vs none; ^$P < .05 vs control MPs; ^#P < .05 vs SCD MPs. (F) Effects of synthetic heme-laden MLVs preincubated with annexin-a5 or Hpx. *P < .05 vs none; ^#P < .05 vs SCD MPs.