Reactive Oxygen Species and RBC Membrane Integrity in Peroxyredoxin II Deficient Mice.
Authors: Bhagat Amrita , Emerson Renee , DeJong Kitty , Kuypers Frans A. 
Published: April 2020

Abstract
Red blood cells (RBCs) contain a complex set of enzymes and non-enzymatic scavengers as defense against reactive oxygen species (ROS). Peroxiredoxin II (PrxII), a member of a family of small proteins with strong antioxidant properties, is highly abundant in RBCs. PrxII is likely to play an essential role in ROS protection, as RBCs generate high levels of ROS due to their role in oxygen transport and the presence of redox active hemoglobin. Phosphatidylserine (PS) asymmetry in RBCs is maintained by the active transport of PS from outer to inner monolayer by the oxidant sensitive aminophospholipid translocase or flipase. PS exposure is found in RBCs where phospholipid scrambling is activated and the flipase is inhibited. In PrxII−/− mice, PrxII is absent from the RBC. These mice are anemic (Hct 41% vs. 46% in wild type mice (WT)), with increased reticulocyte count (4.7% vs. 2.0 % in WT), and a morphologically diverse RBC population. RBC indices after isovolumetric sphering showed a similar MCH (14.3 vs. 14.2), slightly increased MCV (49.2 fl vs. 45.5 fl) and slightly decreased MCHC (30.1 vs. 32.1) in PrxII−/− mice as compared to WT. In flow cytometric analysis, two distinct populations of RBC are found with either slight or significantly increased autofluorescence in the fluorescein channel (excitation 488 nm, emission 515 nm), indicative of oxidant damage. These two populations of low (LF) and high (HF) fluorescent cells comprise 70–80% and 20–30% of the total RBC population respectively. RBC from PrxII−/− and WT mice were biotinylated using EZ-Link Sulfo-NHS-Biotin (Pierce) allowing turnover studies of the LF and HF population. At set time points, the number of biotinylated cells was determined in small blood samples by flow cytometry using fluorescently labeled streptavidin. The data were mathematically fitted to 100–100*[1−(1/T)*t]exp(−kt), where t is the time point, T is the extinction time, and k the exponential rate of RBC removal. The data in the WT showed a linear removal rate (k=0), and a T of 40 days (R2=0.99). In PrxII−/−, an overall faster disappearance of biotinylated cells was noted, and the number of surviving (biotinylated) cells in the population followed an exponential pattern, consistent with random removal (k=0.08, R2= 0.98). At 20 days, 50% of biotinylated RBC were present in WT, but only 18% were found in PrxII−/− mice. In the non-biotinylated RBC, HF cells started to appear at day 13, indicating that autofluorescence is acquired in time. Using the fluorescent ROS membrane probe C11-BODIPY, our data indicate a higher level of ROS in the HF population. The HF population exhibited a lower flipase activity and increased phospholipid scrambling, as measured by labeling with annexin V. Together, our data indicate the importance of PrxII in the maintenance of RBC membrane integrity and suggest that oxidant induced PS exposure is in part responsible for shortened RBC survival in these mice. These findings indicate a role for oxidation in the exposure of PS on the RBC surface, which may clarify mechanisms in oxidant induced membrane alterations in hemoglobinopathies.