Prolonged storage of red blood cells affects aminophospholipid translocase activity.

BACKGROUND AND OBJECTIVES: 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). MATERIALS AND 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. CONCLUSION: 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.

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes.

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.