Oxidative Modifications to Flippase and Scramblase Affect Phosphatidylserine Exposure in Mouse Sickle Cells.
Authors: DeJong Kitty , Kuypers Frans A. 
Published: October 2019

A subset of sickle red blood cells shows phosphatidylserine (PS) exposure on the red blood cell (RBC) surface. PS exposure has consequences for sickle cell pathophysiology, including an imbalance in hemostasis, enhanced cell recognition and adhesion to the endothelium. PS-exposing RBC are found among young reticulocytes as well as old dense sickle cells, but it is poorly understood how the PS-exposing cells are formed. PS exposure occurs when phospholipids are randomized across the membrane by the calcium-activated phospholipid scramblase. In addition, the flippase, which is responsible for transporting PS from the outer to the inner monolayer, needs to be inactivated. Indeed, we have previously found that all highly PS-exposing sickle cells have lost their flippase activity. We hypothesized then that transient calcium influx in RBC without flippase activity would result in PS exposure in sickle cells. However, brief calcium influx in normal red cells is not sufficient to induce PS exposure. Here we show that the scramblase activity is also altered by oxidative damage in sickle cells, which contributes to a higher susceptibility to calcium-induced PS exposure. We modified the sulfhydryl moieties of the flippase and the scramblase to see their respective effect on PS scrambling in sickle mouse (Berkeley type) and normal mouse RBC. We evaluated the scrambling rate by monitoring the appearance of PS-exposing cells over time after loading the cells with 0.1 mM calcium using ionophore. Sickle cells scrambled at twice the rate of normal cells when loaded with the same level of calcium. This could be accounted for by reduced flippase activity, since flippase inhibition by vanadate increased the formation of PS-exposing cells by 2.5-fold in normal mouse cells. However, a similar stimulation was also seen in sickle cells, indicating that not only flippase inhibition, but also enhanced scrambling contributes to PS exposure in sickle cells. The sulfhydryl alkylating reagent n-ethylmaleimide (NEM) inhibits the flippase and enhances scrambling in human RBC, probably by changing the scramblase into an active conformation. This effect was similar in mouse RBC, leading to a markedly enhanced scrambling rate with more than 70% of the cells exposing PS after 10 minutes incubation. While most sickle cells also scrambled rapidly after NEM treatment, about 20% of mouse sickle cells had lost their ability to scramble. Another sulfhydryl modifying reagent, pyridyldithioethylamine (PDA), suppresses the scrambling process in human RBC even though it inhibits the flippase, indicating a different type of sulfhydryl modification of the scramblase. Mouse RBC were slightly less susceptible to PDA than human cells, but the observed scrambling resulted in a lower degree of PS exposure per cell than normally found. Sickle cells, on the other hand, doubled their scrambling rate in presence of PDA, showing that the inhibiting effect on the scramblase was inactivated in these cells. In conclusion, mouse sickle cells show a higher rate of scrambling than normal RBC at the same level of intracellular calcium, although it is suppressed in a subpopulation. Sulfhydryl modification of the scramblase at the site that can be modified by PDA could prevent its downregulation and increase its susceptibility to PS exposure in response to transient calcium influx. Our data show that both flippase and scramblase sustain damage by oxidative stress in sickle cells.