Dysregulation of Phosphatidylserine Scrambling by Oxidative Stress.
Authors: DeJong Kitty , Kuypers Frans A. 
Published: October 2019

An abnormal population of red blood cells (RBC) that exposes phosphatidylserine (PS) has been found in patients with sickle cell anemia and thalassemia. Enhanced formation of PS-exposing RBC results in their premature demise as these cells are removed from the circulation, and contributes to disease complications in the case that these cells are insufficiently removed. We hypothesize that increased oxidative stress, a common indication in these RBC, contributes to increased PS exposure. To evaluate the underlying mechanism, we compared the PS scrambling rate in three different mouse strains that sustain oxidative stress to their RBC: sickle mice, thalassemic mice (C57/Hbb[th3]), and mice deficient in peroxiredoxin II (prdx −/−). Oxidative damage is prominent in sickle and thalassemic RBC because of the presence of damaged hemoglobin and free heme components, and in prdx −/− mice because they lack a key component of oxidative damage repair. We determined the PS scrambling rate resulting from loading RBC with 100 microM intracellular Ca2+, in presence of either NEM (sulfhydryl alkylation), PDA (sulfhydryl modification), vanadate (flippase/ATPase inhibitor), or oligomycin (scrambling stimulation). As previously reported, sulfhydryl modifications cause flippase inhibition, and also change the PS scrambling rate with NEM treatment stimulating scrambling but PDA treatment reducing it. Sickle cells showed a 10% reduced susceptibility to NEM modification and no susceptibility to PDA, indicating shielding of the scramblase sulfhydryl groups. Thalassemic and Prdx −/− RBC showed a 30% and 10% reduced response to NEM modification, respectively, but normal reaction with PDA. The prdx −/− RBC contain a subpopulation that is severely oxidized as detected by an increase in autofluorescence. This cell population had a 10–15% lower susceptibility to NEM and PDA compared to the remaining population. The maximum percentage of PS-exposing cells (with oligomycin) was 10–20% reduced in all transgenic strains compared to normal, and even further reduced in the highly oxidized prdx−/− cells, indicating that a subset of cells had lost its ability to expose PS altogether. Yet, the baseline scrambling rate with 100 microM Ca2+ was increased 3-fold in thalassemic RBC, 1.5-fold in sickle mice, and 3-fold in the highly oxidized prdx −/− cells compared to normal mouse RBC, confirming the hypothesis that increased oxidative damage results in a higher propensity to scramble. Vanadate treatment had a stimulating effect on mouse RBC scrambling resulting in similar PS exposure in all mouse strains. While flippase activity was reduced in sickle RBC, this was not the case in thalassemic and prdx −/− RBC. In all RBC, increased scrambling activity resembled alterations similar to those seen after NEM treatment. Our data show that a subpopulation of oxidatively damaged cells is more prone to PS exposure during Ca2+ influx due to sulfhydryl modification. In addition, some oxidatively damaged cells lose their ability for Ca-induced scrambling. Together, we conclude that scrambling is dysregulated in oxidatively challenged RBC.