Flippase Activity Decreases throughout Erythrocyte Life-Span in Normal and Sickle Mice.

Phosphatidylserine (PS) exposure contributes to recognition and removal of aged erythrocytes. PS exposure can only arise in cells in which the aminophospholipid translocase, or flippase, is defunct, or otherwise PS would be transported back into the inner cell monolayer. To provide evidence that flippase activity is reduced in old erythrocytes, we studied mouse erythrocytes that were biotinylated in vivo. This way we were able to distinguish the aging, biotinylated, RBC population from the newly formed, unbiotinylated RBC. Detection of the biotinylation with fluorescent streptavidin was combined with a flow cytometric assay for flippase activity using NBD-PS. We found that flippase activity decreased over time during the life-span of the RBC, as shown by a decrease in flippase activity of the biotinylated RBC compared to the newly formed RBC. A particularly high flippase activity was found in CD71-positive reticulocytes. Murine sickle cells show a much higher erythrocyte turnover than normal erythrocytes, with a 25–50% newly formed population each day and complete elimination of all biotinylated cells in 10 days. As in control mice, flippase activity decreased during the (short) life-span of sickle RBC and at all times, a subpopulation of sickle cells had a markedly reduced flippase activity. High flippase activity was only found in newly released erythrocytes. Our data indicate that the flippase activity decreases throughout the life-span of erythrocytes and can be markedly different in subpopulations of RBC in vivo. This decrease reduces the cell’s potential to maintain its phospholipid asymmetry, which results in PS exposure as a removal signal for the damaged cell. We conclude that the inability of sickle RBC to maintain flippase activity is related to the decreased survival time of those cells.