title: Prolonged storage of red blood cells affects aminophospholipid translocase activity.
abstract: OBJECTIVE
  Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting phospholipid asymmetry: the ATP-dependent aminophospholipid translocase (or flippase, transporting PS from the outer to the inner leaflet) and phospholipid scrambling (which will move PS from the inner to the outer leaflet).
  
  
  METHODS
  Standard leukodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) at 4 degrees C for up to 7 weeks. PS exposure was determined by measurement of AnnexinV-FITC binding to the cells, flippase activity by measurement of the inward translocation of NBD-labelled PS. Scrambling activity was determined by following the inward translocation of fluorescent NBD-phosphatidylcholine. In parallel, intracellular ATP levels were determined.
  
  
  RESULTS
  PS exposure amounted to only 1.5 +/- 0.3% positive cells (n = 8) after 5 weeks of storage, which slightly increased to 3.5 +/- 0.7% (n = 8) after 7 weeks of storage. Flippase activity started to decrease after 21 days of storage and reached 81 +/- 5% of the control value after 5 weeks of storage (n = 6) and 59 +/- 6% (n = 6) after 7 weeks. Also in RBC obtained by apheresis, flippase activity decreased upon storage. Scrambling activity remained virtually absent during storage, explaining the low PS exposure despite the decrease in flippase activity. Rejuvenation of RBC after 7 weeks to increase ATP levels only partially restored flippase activity, but in combination with a correction of the intracellular pH to that of fresh cells, almost complete restoration was achieved. The decrease in flippase activity after prolonged storage did make the RBCs more prone to PS exposure after activation of phospholipid scrambling.
  
  
  CONCLUSIONS
  This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.
authors.affiliation: Sanquin Blood Supply Foundation, Division Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. a.verhoeven@sanquin.nl
authors.forename: A J
authors.initials: AJ
authors.lastname: Verhoeven
authors.forename: P M
authors.initials: PM
authors.lastname: Hilarius
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
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis; D001793:Blood Preservation / Q000009:adverse effects / Q000379:methods; D004910:Erythrocyte Membrane / Q000201:enzymology; D017707:Erythrocyte Transfusion / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D006461:Hemolysis; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000737:chemistry / Q000378:metabolism*; D012034:Refrigeration / Q000009:adverse effects; D013997:Time Factors
chemical_list: D008051:Lipid Bilayers; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D000255:Adenosine Triphosphate