title: Saquinavir Induced Suicidal Death of Human Erythrocytes.
abstract: OBJECTIVE
  The antiretroviral protease inhibitor saquinavir is used for the treatment of HIV infections. Effects of saquinavir include induction of apoptosis, the suicidal death of nucleated cells. Saquinavir treatment may further lead to anemia. In theory, anemia could result from accelerated erythrocyte loss by enhanced suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress with increase of reactive oxygen species (ROS) and ceramide. The present study explored, whether and how saquinavir induces eryptosis.
  
  
  METHODS
  To this end, flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ROS abundance from DCFDA fluorescence and ceramide abundance utilizing specific antibodies.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to saquinavir significantly decreased forward scatter (≥ 5 µg/ml), significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly increased Fluo3-fluorescence (15 µg/ml), significantly increased DCFDA fluorescence (15 µg/ml), but did not significantly modify ceramide abundance. The effect of saquinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+.
  
  
  CONCLUSIONS
  Saquinavir triggers 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: Sabrina
authors.initials: S
authors.lastname: Waibel
authors.forename: Rosi
authors.initials: R
authors.lastname: Bissinger
authors.forename: Ghada
authors.initials: G
authors.lastname: Bouguerra
authors.forename: Salem
authors.initials: S
authors.lastname: Abbès
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D019258:Saquinavir / Q000633:toxicity*; D013050:Spectrometry, Fluorescence; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D002518:Ceramides; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D014966:Xanthenes; C059715:Fluo-3; D019258:Saquinavir; D002118:Calcium