title: Enhanced Eryptosis Following Exposure to Lopinavir.
abstract: OBJECTIVE
  The protease inhibitor lopinavir, used for the treatment of HIV infections, triggers suicidal death or apoptosis of nucleated cells. Side effects of lopinavir include anemia, which could in theory result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and by phospholipid scrambling of the cell membrane leading to phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether lopinavir induces eryptosis.
  
  
  METHODS
  Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide abundance utilizing labelled specific antibodies. Hemolysis was estimated from haemoglobin concentration of the supernatant.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to lopinavir significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥15 µg/ml), significantly increased hemolysis (≥ 15 µg/ml), significantly increased Fluo3-fluorescence (20 µg/ml), and significantly increased DCFDA fluorescence (20 µg/ml) but did not significantly modify ceramide abundance. The effect of lopinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+.
  
  
  CONCLUSIONS
  Lopinavir treatment of erythrocytes from healthy volunteers is followed by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.
authors.forename: Rosi
authors.initials: R
authors.lastname: Bissinger
authors.forename: Sabrina
authors.initials: S
authors.lastname: Waibel
authors.forename: Ghada
authors.initials: G
authors.lastname: Bouguerra
authors.forename: Abdulla
authors.initials: A
authors.lastname: Al Mamun Bhuyan
authors.forename: Salem
authors.initials: S
authors.lastname: Abbès
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D005978:Glutathione / Q000097:blood; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D061466:Lopinavir / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017320:HIV Protease Inhibitors; D017382:Reactive Oxygen Species; D061466:Lopinavir; D005978:Glutathione