Phosphatidylserine Scrambling Is Faster in Sickle Mouse Erythrocytes Because of Oxidative Damage.
Authors: DeJong Kitty , Kuypers Frans A. 
Published: April 2020

Abstract
A subset of sickle red blood cells shows phosphatidylserine (PS) exposure on the red blood cell (RBC) surface. These cells contribute to the morbidity of the disease by increasing the patient’s risk for stroke, altering the adhesive properties of the RBC, and affecting RBC survival. The cause and mechanism of production of these cells has not yet been resolved. PS asymmetry is normally maintained by activity of the flippase, which was shown to be inactivated in the PS-exposing sickle cells. Oxidative damage inside the sickle cells seems to contribute to this inactivation. PS exposure requires that phospholipids are randomized across the membrane by the Ca++-activated phospholipid scramblase. Thus far it has been unclear how small and transient increases (&lt;100 nM) in intracellular Ca++ attained during sickle cell deoxygenation could facilitate this process. We now show that subtle oxidative modifications to the sulfhydryl groups on the scramblase can regulate its activity and lower its requirement for Ca++ to levels that might be reached in sickle cells. We evaluated the phospholipid scrambling rate, monitored by the appearance of PS-exposing cells over time, in sickle mouse (Berkeley type) and normal mouse RBC after loading the cells with Ca++ using ionophore. The level of intracellular Ca++ needed for scrambling was found to be at least 10 μM in normal mouse and human RBC and reached a maximum at 100 μM. Sickle cells scrambled at 1.5-fold the rate of normal cells when loaded with the same level of Ca++. Flippase inhibition by vanadate increased the scrambling rate both in normal and sickle cells, but did not lead to immediate scrambling in all cells and did not reduce the requirement for Ca++, suggesting that scramblase activation rather than flippase inhibition is the rate-limiting step in PS exposure. 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 in presence of 100 μM Ca++. In addition, we found that NEM treatment largely reduces the requirement for Ca++ with scrambling already effective below 1 μM. On the other hand, a similar compound, pyridyldithioethylamine (PDA), suppressed the scrambling process in normal mouse and human RBC by 50–80% even though it inhibits the flippase, showing that the scramblase can also be down-regulated by a different type of sulfhydryl modification. Interestingly, mouse sickle cells were not susceptible to inhibition by PDA, indicating altered sulfhydryl moieties on their scramblase. While most mouse sickle cells scrambled rapidly after NEM treatment similar to normal mouse cells, about 10% of the sickle cells were not responsive to NEM, also indicating previous sulfhydryl modifications. In conclusion, we show that the scramblase in mouse sickle cells is altered by sulfhydryl modifications, that could set up the sickle cells to expose PS more rapidly at lower intracellular Ca++ levels than normal RBC, leading to enhanced formation of PS-exposing cells in sickle cell disease.