High Calcium Requirement for Phosphatidylserine Exposure in Sickle Cell Disease.

While the adverse effects of red blood cells (RBC) that expose phosphatidylserine (PS) in the sickle cell circulation are well recognized, the mechanism that underlies the formation of these cells is not well understood. Diminished flippase activity is found in a subset of sickle cells, and while this explains how PS persists in the outer RBC monolayer, it does not explain how the phospholipids are randomized across the membrane bilayer. Rapid scrambling of phospholipids, resulting in PS exposure, requires elevation of intracellular free Ca⁺⁺ levels by Ca⁺⁺ influx into the RBC. In this study we assessed how much Ca⁺⁺ is required for PS exposure, and how this relates to previous findings of elevated Ca⁺⁺ influx during deoxygenation of sickle cells. We loaded RBC for 30 minutes at 37°C with accurately established levels of intracellular Ca⁺⁺ by balancing CaCl₂ solutions with the chelator BAPTA, followed by Annexin V labeling under conditions that scrambling did not continue. In both normal and sickle RBC from either human or mouse, PS exposure was only found at or above 10 microM Ca⁺⁺. Flippase inhibition with vanadate did not change this, indicating that phospholipid scrambling had not been initiated under these conditions. Scramblase sulfhydryl modification with NEM reduced the Ca⁺⁺ requirement to lower than 1 microM, as did treatment with the scramblase stimulator oligomycin. Further experiments showed that Ca⁺⁺ needs to be present during the entire duration of the scrambling process, and removal of Ca⁺⁺ arrests the scrambling process. With 1 microM Ca⁺⁺ in oligomycin-treated (scramblase stimulated) RBC and in absence of flippase activity (using vanadate), it took 5 minutes at 37°C to obtain a significant (>2%) population of PS-exposing cells. These conditions are unlikely met in all sickle cells. In addition, these experiments showed that the flippase is not inhibited by 1 or 100 microM Ca⁺⁺, despite previous reports suggesting that Ca⁺⁺ loading inhibits the flippase. Taken together, our data indicate that PS scrambling requires more Ca⁺⁺ influx than has been reported for deoxygenated sickle cells (50–100 nM). We suggest that a small percentage of individual sickle cells contain Ca⁺⁺ levels of 1 microM or above, which would lead to PS exposure in those RBC that have sustained flippase inhibition and scramblase modifications similar to those achieved with NEM or oligomycin. We have previously reported evidence for such scramblase modifications, as well as reduced flippase activity in a subset of sickle cells. Our data indicate that the degree of Ca⁺⁺ influx may be the determining parameter for the extent of PS exposure in sickle cells.