title: Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes.
abstract: Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.
authors.affiliation: Sanquin Research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
authors.forename: P M
authors.initials: PM
authors.lastname: Hilarius
authors.forename: I G
authors.initials: IG
authors.lastname: Ebbing
authors.forename: D W C
authors.initials: DW
authors.lastname: Dekkers
authors.forename: J W M
authors.initials: JW
authors.lastname: Lagerberg
authors.forename: D
authors.initials: D
authors.lastname: de Korte
authors.forename: A J
authors.initials: AJ
authors.lastname: Verhoeven
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000494:pharmacology; D017304:Annexin A5 / Q000097:blood; D001692:Biological Transport / Q000187:drug effects; D017301:Ca(2+) Mg(2+)-ATPase / Q000097:blood; D002118:Calcium / Q000037:antagonists & inhibitors / Q000737:chemistry; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008565:Membrane Proteins / Q000097:blood / Q000737:chemistry; D008751:Methylene Blue / Q000031:analogs & derivatives* / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000494:pharmacology; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000097:blood* / Q000737:chemistry*; D017319:Photosensitizing Agents / Q000494:pharmacology; D011485:Protein Binding; D026082:Singlet Oxygen / Q000097:blood* / Q000737:chemistry*
chemical_list: C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D017304:Annexin A5; D002352:Carrier Proteins; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D017319:Photosensitizing Agents; C435946:dimethylmethylene blue; D026082:Singlet Oxygen; D004533:Egtazic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D009327:4-Chloro-7-nitrobenzofurazan; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D005033:Ethylmaleimide; D002118:Calcium; D008751:Methylene Blue