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The favorable storability of bThal+ RBC
tions may contribute to explain one of the molecular mechanisms through which bThal+ protects from malaria infection.22
Reports on the oxidative profile of bThal+ RBC and on the plasma antioxidant capacity in vivo are quite contra- dicting.12 Many studies, however, pointed to increased activity and/or upregulation of several antioxidant enzymes and proteins41 in bThal+ RBC as a possible adap- tation to mild sustained oxidative stress. Metabolism exhibits a similar adaptive response according to the cur- rently reported data showing lower baseline levels of oxi- dant stress-induced purine deamination, higher levels of sulfur-containing metabolites (methionine and cysteine), increase in glycolysis upstream of pyruvate kinase and an apparent increase in fluxes through the PPP in bThal+ RBC. Combined with increases in PPP-derived reducing equivalents (NADPH), in the absence of significant alter- ations of total glutathione pools and ratios of oxidized/reduced forms, these data are suggestive of an overall higher antioxidant armamentarium in bThal+ RBC. “Training” to mild oxidative stress in vivo may mod- ulate bThal+ RBC storability,41 as evidenced by ROS accu- mulation, oxidative hemolysis, and oxidative defects to membrane components. In the same context, the mem- brane profile of stress markers and the low intracellular allantoin42 are suggestive of the protective action of molecular chaperones and other antioxidant molecules, such as urate, present in excess in bThal+ RBC. Despite the fact that urate can act as a pro- or anti-oxidant factor, its minimal oxidation to allantoin at the same period of low ROS generation in bThal+ RBC points towards its antioxidant effect during storage. Moreover, it mostly accounted for the elevated antioxidant capacity of bThal+ plasma/supernatant. Network paired analysis of fresh ver- sus stored samples confirmed that the extracellular antioxidant capacity (like osmotic fragility) is an almost “heritable” feature of RBC units in both donor groups,43 and further “signs” bThal+ status in stored RBC (Online Supplementary Figure S1). Of note, the levels of TAC and UAdAC in bThal+ units exhibited inverse correlations with acyl-carnitine RBC metabolites, such as the redox related acetyl-carnitine. These metabolites have been suggested as markers of osmotic fragility in blood donors undergoing testosterone-replacement therapy,44 consis- tent with a role of this pathway in the Lands cycle-depen- dent remodeling of oxidized membrane lipids.
Genetic variation among donors may have a positive or negative impact on transfusion performance. G6PD-defi- cient RBC, for instance, are susceptible to hemolysis and oxidative stress post-transfusion,6 and exhibit lower 24- hour recovery in autologous transfusion recipients.7 Similarly, sickle cell trait RBC were shown to have reduced recovery in animal models.28 Quite to the con- trary, the unique storability of bThal+ RBC is suggestive of good post-transfusion recovery. Apart from the low intracellular levels of hypoxanthine, a metabolic marker of storage lesion exhibiting negative correlation with recovery in vivo,45 the low extracellular Hb/potassium and RBC removal signaling are in favor of therapeutic out- comes mainly through preservation of nitric oxide bioavailability and alleviation of hemolysis/iron-associat- ed toxicity, including inflammatory responses and bacte- rial infections.46 In addition, the advantageous redox phe- notype of stored bThal+ is expected to promote their functionality in vivo and to ameliorate oxidative stress-
associated adverse clinical outcomes in susceptible patients with G6PD-deficiency and sickle cell disease.47 Finally, the modified arginine/nitric oxide metabolism (involved in the induction of HbF48) in bThal+ RBC during storage may be relevant to their transfusion in susceptible recipients, including hemoglobinopathies, cancer, lung and cardiovascular disease patients. In the light of those results, we have initiated a study on post-transfusion per- formance of bThal+ RBC.
Heterozygotes for bThal+ constitute a non-negligible proportion of blood donors not only in the Mediterranean, but also in Africa, Asia and in under-rep- resented minorities of US.49 Their typical profile of low Hb concentration, and thus, of lower dose of Hb per unit of RBC, would predispose to reduced Hb increment and lower efficacy of the transfusion therapy compared to control. However, the currently presented data show that bThal+ RBC are unique in terms of susceptibility to hemolysis, redox homeostasis and nitrogen/hexosamine- related metabolism during storage. This favorable stora- bility and thereof improved quality of stored bThal+ RBC may counterpoise the negative effects of lower Hb dose on the recipient. Despite the fact that further studies are needed to clarify the post-transfusion performance of bThal+ RBC in the context of intra-group genetic hetero- geneity and recipient variation, the currently reported data may allow considering bThal+ individuals as a novel, high quality, “good storer” group. Indeed, bThal+ RBC may be safely stored for long time periods before transfu- sion, highlighting the notion of molecular versus storage age of blood.5 Moreover, bThal+ RBC are probably ideal candidates for alternative storage strategies, like cryop- reservation, which involves a deglycerolization step dur- ing which RBC are highly susceptible to hypo-osmotic lysis.50 These promising data and potentials highlight the emergence of bThal+ donors for a distinct and potentially valuable role in blood transfusions.
Disclosures
Though unrelated to the contents of this manuscript, ADA declares that he is a founder of Omix Technologies Inc and Altis Biosciencens LLC and a consultant for Hemanext Inc. All other authors declare no conflicts of interest.
Contributions
VLT and ATA are co-first authors; VLT and MHA designed the study; VLT, ATA, MHA and ADA wrote the manuscript; VLT, ATA and AGK performed the physiological and biochem- ical experiments; DS, FC, LB and FG performed the metabolomics analyses; MD performed the proteomics experi- ments; MHA and PR performed the flow cytometry analyses; VLT, ATA, MHA and ADA analyzed the data; AV, VS, KS, and AGK provided project support and organized participant recruitment; MD, OET, ISP, KS, AGK and ADA provided expert knowledge.
Acknowledgements
The authors would like to thank all blood donors that volun- tarily participated in this study; M.S. Jacovides Hellas S.A. for the kind offer of the LTRC blood bags; Dr Pantelis Constantoulakis and Dr Stavros Bournazos (Genotypos Science Labs) for their contribution in the mutation identification; the NKUA students Dimitrios G. Karadimas (MSc) and Christos Christogeorgos for their participation in a part of the physiolog- ical experiments performed; Dr Hara Georgatzakou, Mrs
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