Red blood cells contain enzymatically active GPx4 whose abundance anticorrelates with hemolysis during blood bank storage

Abstract

The antioxidant function of the phospholipid hydroperoxide glutathione peroxidase (GPx4) is vital for the homeostasis of many cell types, from neoplastic cells to normal erythroid precursors. However, some functional proteins in erythroid precursors are lost during the development of red blood cells (RBCs); whether GPx4 is maintained as an active enzyme in mature RBCs has remained unclear. Our meta-analyses of existing RBC proteomics and metabolomics studies revealed the abundance of GPx4 to be correlated with lipid-anchored proteins. In addition, GPx4 anti-correlated with lyso-phospholipids and complement system proteins, further supporting the presence of active GPx4 in mature RBCs. To test the potential biological relevance of GPx4 in mature RBCs, we correlated the rate of hemolysis of human RBCs during storage with the abundance of GPx4 and other heritable RBC proteins. Of the molecules that anti-correlated with the rate of hemolysis of RBCs, proteins that mediate the cellular response to hydroperoxides, including GPx4, have the greatest enrichment. Western blotting further confirmed the presence of GPx4 antigenic protein in RBCs. Using an assay optimized to measure the activity of GPx4 in RBCs, we found GPx4 to be an active enzyme in mature RBCs, suggesting that GPx4 protects RBCs from hemolysis during blood bank storage.

The Post-Storage Performance of RBCs from Beta-Thalassemia Trait Donors Is Related to Their Storability Profile

“…For instance, glutathione peroxidase 4 (GPX4) levels anticorrelate with hemolysis, and extracellular antioxidant capacity with ROS accumulation and concentration of procoagulant EVs. Both findings have been previously observed during storage [39,40], so it seems that these antioxidants continue to protect stored RBCs after "re-addition" of plasma. Finally, pyridoxamine, previously shown to (a) ameliorate oxidative and ion transport defects in RBCs [41], and (b) be more abundant in βThal + stored erythrocytes [1], showed many negative correlations with an array of parameters, a great part of which were favorable in the group of heterozygotes post-mixing (e.g., hemolysis and lipid peroxidation).…”

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring

“…GSH synthesis rates decrease under hypothermic storage, leading to insufficient levels of this non-enzymatic antioxidant, and thus, inferior antioxidant defense [ 65 ]. At the same time, GSH serves as a co-factor for glutathione peroxidases, antioxidant enzymes linked to beneficial RBC storability [ 66 ]. It has been proposed that addition of the amino acids necessary for its biosynthesis in the storage solution could improve this lesion [ 67 ], thus the boosted production of GSH observed in this study is important.…”

Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage

“…Of note, polymorphisms in the gene coding for glutathione peroxidase 4 (GPX4) have been shown to correlate with increased susceptibility of stored RBCs to hemolysis following oxidant insults ( Page et al, 2021 ). This is relevant in light of the role of GPX4, which is present and active in human RBCs in the glutathione-dependent detoxification of oxidized lipids ( Stolwijk et al, 2021 ). Alternatively, increased autoxidation may induce vesiculation, thereby eliminating damaged cytoskeletal lipids and proteins, resulting in the transition from discocyte to spherocyte due to the progressive decrease in the surface-area-to-volume ratio ( Cloos et al, 2020 ; Kaczmarska et al, 2020 ).…”