Enhanced Phosphatidylserine Scrambling in Oxidatively Challenged Red Blood Cells.
Authors: DeJong Kitty , Kuypers Frans A. 
Published: April 2020

Abstract
Red blood cells (RBC) that abnormally expose phosphatidylserine (PS) contribute to the pathophysiology of several hemoglobinopathies. PS exposure requires inactivation of the flippase that transports PS from the outer to the inner membrane monolayer, and activation of a phospholipid scrambling process. To evaluate the role of increased oxidative stress in this process, we compared RBC from transgenic sickle mice (Berkeley type) with RBC from peroxiredoxin 2 (prdx) knock-out mice (prdx −/−). These mice lack one of the most prevalent cytosolic antioxidant molecules. This molecule, also known as calpromotin, was previously implicated in membrane abnormalities of the dense sickle RBC population. Mice that lack prdx are slightly anemic, have reduced RBC survival and exhibit a subpopulation of older highly oxidized RBC. The prdx −/− strain did not show a subpopulation of PS-exposing cells in freshly collected blood and the flippase activity, measured by transbilayer kinetics of the fluorescent probe NBD-PS, was normal. In contrast, blood collected from the sickle mice showed a large subpopulation with decreased flippase activity and exhibited a subpopulation of PS-exposing RBC that lack flippase activity. Flippase inhibition induced with vanadate or NEM did not increase the PS exposure of prdx −/− RBC incubated with high levels of Ca2+, indicating that there was no increased Ca2+ influx. In sickle cells, elevated intracellular Ca2+ was evident under similar loading conditions. Loading RBC with 0.1 mM Ca2+, but not lower concentrations, using Ca-ionophore resulted in bilayer scrambling and PS exposure in both strains as well as in normal control mice. The rate of PS scrambling was increased 1.5-fold in sickle mice compared to normal mouse RBC. While the scrambling rate was normal in the young, not oxidized prdx−/− RBC, it was increased 3-fold in the older highly oxidized prdx −/− RBC as compared to normal mouse RBC. The sulfhydryl modifiers NEM or PDA caused flippase inhibition, and altered the PS scrambling rate in normal mouse RBC as reported earlier. Both sickle cells and the older oxidized prdx −/− RBC showed a reduced susceptibility to NEM and PDA, while the younger prdx −/− RBC exhibited a normal sensitivity to these compounds. This suggests that both prdx −/− RBC and sickle cells have sustained similar sulfhydryl damage leading to enhanced scrambling. Exposure to three well-known oxidants (0.1–0.5 mM cumene hydroperoxide, tert-butyl hydroperoxide or hydrogen peroxide) did not increase the percentage of oxidized cells or PS exposure in prdx −/− RBC compared to normal RBC. This indicates that targeted sulfhydryl modification but not general short-term oxidative stress impacts the loss of phospholipid asymmetry. These data confirm that increased oxidative sulfhydryl damage results in a higher propensity for phospholipid scrambling. The presence of active prdx is important to maintain PS asymmetry as it prevents accelerated phospholipid scrambling. In those cells in which the flippase is also inactivated, PS exposure becomes apparent. The loss of flippase activity is much more prevalent in sickle RBC, indicating that prdx does not play an important role in protecting the flippase from inactivation. It can be expected that PS-exposing cells are rapidly removed from the circulation, as they resemble apoptotic cells, which may explain their absence in blood from prdx−/− mice. The presence of PS-exposing RBC in the circulation of sickle mice suggests that the formation of these cells overwhelms their removal.