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

Redox imbalance and oxidative stress have emerged as generative causes of the structural and functional degradation of red blood cells (RBC) that happens during their hypothermic storage at blood banks. The aim of the present study was to examine whether the antioxidant enhancement of stored RBC units following uric (UA) and/or ascorbic acid (AA) supplementation can improve their storability as well as post-transfusion phenotypes and recovery by using in vitro and animal models, respectively. For this purpose, 34 leukoreduced CPD/SAGM RBC units were aseptically split in 4 satellite units each. UA, AA or their mixture were added in the three of them, while the fourth was used as control. Hemolysis as well as redox and metabolic parameters were studied in RBC units throughout storage. The addition of antioxidants maintained the quality parameters of stored RBCs, (e.g., hemolysis, calcium homeostasis) and furthermore, shielded them against oxidative defects by boosting extracellular and intracellular (e.g., reduced glutathione; GSH) antioxidant powers. Higher levels of GSH seemed to be obtained through distinct metabolic rewiring in the modified units: methionine-cysteine metabolism in UA samples and glutamine production in the other two groups. Oxidatively-induced hemolysis, reactive oxygen species accumulation and membrane lipid peroxidation were lower in all modifications compared to controls. Moreover, denatured/oxidized Hb binding to the membrane was minor, especially in the AA and mix treatments during middle storage. The treated RBC were able to cope against pro-oxidant triggers when found in a recipient mimicking environment in vitro, and retain control levels of 24h recovery in mice circulation. The currently presented study provides (a) a detailed picture of the effect of UA/AA administration upon stored RBCs and (b) insight into the differential metabolic rewiring when distinct antioxidant "enhancers" are used.

Innate Variability in Physiological and Omics Aspects of the Beta Thalassemia Trait-Specific Donor Variation Effects.

The broad spectrum of beta-thalassemia (ßThal) mutations may result in mild reduction (ß ++), severe reduction (ß +) or complete absence (ß 0) of beta-globin synthesis. ßThal heterozygotes eligible for blood donation are "good storers" in terms of red blood cell (RBC) fragility, proteostasis and redox parameters of storage lesion. However, it has not been examined if heterogeneity in genetic backgrounds among ßThal-trait donors affects their RBC storability profile. For this purpose, a paired analysis of physiological and omics parameters was performed in freshly drawn blood and CPD/SAGM-stored RBCs donated by eligible volunteers of ß ++ (N = 4), ß + (N = 9) and ß 0 (N = 2) mutation-based phenotypes. Compared to ß +, ß ++ RBCs were characterized by significantly lower RDW and HbA2 but higher hematocrit, MCV and NADPH levels in vivo. Moreover, they had lower levels of reactive oxygen species and markers of oxidative stress, already from baseline. Interestingly, their lower myosin and arginase membrane levels were accompanied by increased cellular fragility and arginine values. Proteostasis markers (proteasomal activity and/or chaperoning-protein membrane-binding) seem to be also diminished in ß ++ as opposed to the other two phenotypic groups. Overall, despite the low number of samples in the sub-cohorts, it seems that the second level of genetic variability among the group of ßThal-trait donors is reflected not only in the physiological features of RBCs in vivo, but almost equally in their storability profiles. Mutations that only slightly affect the globin chain equilibrium direct RBCs towards phenotypes closer to the average control, at least in terms of fragility indices and proteostatic dynamics.

Corpuscular Fragility and Metabolic Aspects of Freshly Drawn Beta-Thalassemia Minor RBCs Impact Their Physiology and Performance Post Transfusion: A Triangular Correlation Analysis In Vitro and In Vivo.

The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor-recipient matching optimization. We hereby propose a "triangular" strategy for studying the links comprising the transfusion chain-donor, blood product, recipient-as exemplified in two cohorts of control and beta-thalassemia minor (ßThal+) donors (n = 18 each). It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules (e.g., phosphocreatine) and purine metabolism factors (IMP, hypoxanthine) were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed "triangularity model" can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes.

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

Blood donors with beta-thalassemia traits (ßThal+) have proven to be good "storers", since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored ßThal+ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted ßThal+ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that ßThal+ RBCs present a marginal trend for higher recovery, regardless of the recipient's immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored ßThal+ RBCs. Overall, it seems that the intrinsic physiology of ßThal+ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions.

Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor's characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (ßThal+). For this purpose, RBC units from fourteen ßThal+ donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the ßThal+ membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the ßThal+ interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in ßThal+ membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that ßThal+ erythrocytes maintain a considerable "proteo-vigilance" during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile.