title: Micafungin-Induced Suicidal Erythrocyte Death.
abstract: OBJECTIVE
  The antifungal drug Micafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. Side effects of Micafungin treatment include microangiopathic hemolytic anemia and thrombocytopenia with microvascular thrombosis. The development of thrombosis may be fostered by stimulation of eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. The present study explored, whether Micafungin induces eryptosis.
  
  
  METHODS
  Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to Micafungin (10 - 25 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Micafungin (25 µg/ml) did not significantly modify Fluo3-fluorescence, DCFDA fluorescence, or ceramide abundance. The effect of Micafungin on annexin-V-binding was not significantly modified by removal of extracellular Ca2+, by PKC inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM).
  
  
  CONCLUSIONS
  Micafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane.
authors.affiliation: Departments of Cardiology, Cardiovascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Peter
authors.forename: Rosi
authors.initials: R
authors.lastname: Bissinger
authors.forename: Elena
authors.initials: E
authors.lastname: Signoretto
authors.forename: Andreas F
authors.initials: AF
authors.lastname: Mack
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D047389:Casein Kinase I / Q000037:antagonists & inhibitors / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D054714:Echinocandins / Q000494:pharmacology*; D000072817:Eryptosis / Q000187:drug effects*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D055666:Lipopeptides / Q000494:pharmacology*; D000077551:Micafungin; D018613:Microscopy, Confocal; D010718:Phosphatidylserines / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C493177:4-(4-(2,3-dihydrobenzo(1,4)dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D000935:Antifungal Agents; D001549:Benzamides; D002518:Ceramides; D054714:Echinocandins; D007093:Imidazoles; D055666:Lipopeptides; D010718:Phosphatidylserines; D011725:Pyridines; D017382:Reactive Oxygen Species; D047389:Casein Kinase I; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases; D019311:Staurosporine; C093642:SB 203580; D000077551:Micafungin; D002118:Calcium