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V.L. Tzounakas et al.
the groups. The osmotic fragility of stored bThal+ RBC exhibited strong correlations with several biochemical (e.g., transferrin, r=0.560, P<0.01), cellular (e.g., RBC mechanical hemolysis, r=0.771, P<0.01), protein and metabolic (e.g., L-arginine, r=0.697, sphingosine-1-phos- phate, r=-0.636, P<0.01) parameters of bThal+ RBC and plasma, in striking contrast to the controls (Figure 7A). Notably, in the case of bThal+ RBC, fragility variation dur- ing storage was less correlated to the in vivo levels of fatty acids but more correlated to those of carboxylic acids, compared to control. Despite the above-mentioned dif- ferences, osmotic hemolysis during storage was found interrelated with the in vivo levels of RBC aging markers and energy metabolism in both donor groups.
In similarity to the MCF network, the extracellular antioxidant capacity of the bThal+ units presented higher connectivity with baseline parameters compared to con- trol (twice the number of connections) including several metabolites of energy metabolism (e.g., D- glucose/UAdAC, r=-0.705, P<0.01), purine oxidation (e.g., TAC/5-hydroxyisourate, r=0.756, P<0.01), urea cycle (e.g., ornithine/UAdAC, r=-0.769, P<0.01) and car- nitines (Figure 7B). Those findings suggested that that the basic physiology of bThal+ is strongly and intrinsically related with the unique resistance to osmotic hemolysis and the high antioxidant capacity of the RBC units throughout storage.
Discussion
According to an increasing number of studies, genetic diversity among donors substantially affects RBC storage stability. Results of the REDS-III-Omics study showed that female sex and African American or Asian race-eth- nicity2 predispose to low susceptibility to storage and stress hemolysis. RBC from G6PD-deficient donors exhibit control levels of storage hemolysis, opposite to sickle cell trait RBC that are more susceptible to sponta- neous hemolysis during storage.28 Our study reports for the first time that bThal+ RBC are superior to those of the general population in terms of all hemolysis measures (including the less studied mechanical one), either throughout the storage period or for the more susceptible last 2 weeks of it. Superior secondary metrics of storage quality (including removal signaling and extracellular potassium) were also detected in bThal+. As expected, the unique storage capacity of bThal+ RBC was found mech- anistically linked to changes in specific metabolic flows and membrane protein expression profiles.
A tentative explanation for resistance to hemolysis lies in the appreciation of decreased HCT, MCH and Hb lev- els in bThal+. Similar findings in high frequency blood donors have been associated with decreases in ferritin and total iron pools and improved hemolytic parame- ters,29 in keeping with a negative role of iron levels and metabolism with RBC storage quality and post-transfu- sion efficacy.30 Moreover, bThal+ RBC are intrinsically dif- ferent from control, with reduced cellular volume and increased surface:volume ratio.31 Altered ion transport homeostasis leading to efflux of osmotically active com- ponents32 underlies to great extent this phenotype. In sup- port, serum levels of osmotically active ions in bThal+ exhibit significant correlation with RBC osmotic fragili- ty.33 Under normal membrane and skeletal networks
(manuscript in preparation) these features contribute to increased resistance to osmotic (and probably to mechan- ical) stress.
The biological networks suggested that the resistance of stored RBC to osmotic stress is a multifactorial pheno- type exhibiting numerous correlations with the baseline RBC physiology (such as the energy metabolism) espe- cially in the case of bThal+ RBC. Indeed, the levels of sev- eral biochemical and metabolic features of bThal+ RBC in vivo exhibited strong correlations with those of osmotic hemolysis throughout storage, including L-arginine34 and sphingosine-1-phosphate, which significantly differed between the two groups under examination at baseline. Of note, similar correlations were also detected in G6PD- defficient donors.35 This observation suggests a genetical- ly determined aspect of osmotic hemolysis phenotype in stored RBC that deserves further examination. Since osmotic fragility is strongly donor-specific (storage levels are proportional to baseline levels in vivo),17 the superior osmotic hemolysis (and consequently part of storage hemolysis) of bThal+ RBC was rather anticipated. Resistance to oxidative hemolysis at late storage seemed to follow the fluctuation in ROS levels at the same period, as well as the modulation of purine and arginine metabo- lism, previously reported in control donor groups.36 Osmotic stress and additional hemolysis triggers may be further associated with the specific protein composition of RBC membrane in bThal+, since our preliminary analy- sis revealed variations in proteins critically involved in cell volume regulation and transmembrane water/cation flows responsive to osmotic and mechanical stimuli, including aquaporin and piezo. Variation in some of these transporters has already been reported in thalassaemic or other anemic subjects.37 Our study (Online Supplementary Figure S1A) showed for the first time that resistance to both spontaneous and induced hemolysis is a distinctive feature of bThal+ RBC storability. However, diversity in bThal+ mutations worldwide should be also taken into consideration. Indeed, certain bThal+ donors exhibited control baseline levels of RBC osmotic fragility (Table 1). The effects of such genetic variation on bThal+ RBC stor- ability and performance deserve further evaluation by future studies in various ethnic groups.
Apart from hemolysis, PS exposure (a potent signal for RBC clearance in vivo) was also lower in bThal+ RBC at late storage, when ROS accumulation and membrane expression of phospholipid scramblase38 were also low. In contrast, Ca2+ concentration was higher in bThal+ RBC at baseline (and slightly higher during storage) compared to controls, consistent with the distorted divalent cation homeostasis reported in bThal+.39 This finding deserves further attention in the light of bThal+-specific variation in Ca2+-dependent membrane proteins that are involved in critical signaling events in RBC. Finally, alterations in the hexosamine pathway in bThal+ RBC are suggestive of either up-regulation of UDP-N-acetyl-glucosamine usage for O-GlcNAcylation or decreased synthesis through blockade of the late steps in this biosynthetic pathway. To the best of our knowledge, this is the first report of this pathway being affected in bThal+. Interestingly, O- GlcNAcylation is a common post-translational modifica- tion in Plasmodium falciparum proteins following malaria infection, to the extent that therapeutic blockade of this pathway has been proposed as an intervention to shorten the life cycle of the Plasmodium.40 As such, our observa-
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haematologica | 2022; 107(1)