Cryopreserved red blood cells maintain allosteric control of oxygen binding when utilizing trehalose as a cryoprotectant.

One of the most common life-saving medical procedures is a red blood cell (RBC) transfusion. Unfortunately, RBCs for transfusion have a limited shelf life after donation due to detrimental storage effects on their morphological and biochemical properties. Inspired by nature, a biomimetics approach was developed to preserve RBCs for long-term storage using compounds found in animals with a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent and added to the extracellular freezing solution of porcine RBCs. Slow cooling (-1 °C min-1) resulted in almost complete hemolysis (1 ± 1 % RBC recovery), and rapid cooling rates had to be used to achieve satisfactory cryopreservation outcomes. After rapid cooling, the highest percentage of RBC recovery was obtained by plunging in liquid nitrogen and thawing at 55 °C, using a cryopreservation solution containing 300 mM trehalose. Under these conditions, 88 ± 8 % of processed RBCs were recovered and retained hemoglobin (14 ± 2 % hemolysis). Hemoglobin's oxygen-binding properties of cryopreserved RBCs were not significantly different to unfrozen controls and was allosterically regulated by 2,3-bisphosphoglycerate. These data indicate the feasibility of using trehalose instead of glycerol as a cryoprotective compound for RBCs. In contrast to glycerol, trehalose-preserved RBCs can potentially be transfused without time-consuming washing steps, which significantly facilitates the usage of cryopreserved transfusible units in trauma situations when time is of the essence.

Erythrocyte Adenosine A2B Receptor-Mediated AMPK Activation: A Missing Component Counteracting CKD by Promoting Oxygen Delivery.

BACKGROUND: Oxygen deprivation or hypoxia in the kidney drives CKD and contributes to end organ damage. The erythrocyte's role in delivery of oxygen (O2) is regulated by hypoxia, but the effects of CKD are unknown. METHODS: We screened all of the metabolites in the whole blood of mice infused with angiotensin II (Ang II) at 140 ng/kg per minute up to 14 days to simulate CKD and compared their metabolites with those from untreated mice. Mice lacking a receptor on their erythrocytes called ADORA2B, which increases O2 delivery, and patients with CKD were studied to assess the role of ADORA2B-mediated O2 delivery in CKD. RESULTS: Untargeted metabolomics showed increased production of 2,3-biphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite promoting O2 delivery, in mice given Ang II to induce CKD. Genetic studies in mice revealed that erythrocyte ADORA2B signaling leads to AMPK-stimulated activation of BPG mutase, promoting 2,3-BPG production and O2 delivery to counteract kidney hypoxia, tissue damage, and disease progression in Ang II-induced CKD. Enhancing AMPK activation in mice offset kidney hypoxia by triggering 2,3-BPG production and O2 delivery. Patients with CKD had higher 2,3-BPG levels, AMPK activity, and O2 delivery in their erythrocytes compared with controls. Changes were proportional to disease severity, suggesting a protective effect. CONCLUSIONS: Mouse and human evidence reveals that ADORA2B-AMPK signaling cascade-induced 2,3-BPG production promotes O2 delivery by erythrocytes to counteract kidney hypoxia and progression of CKD. These findings pave a way to novel therapeutic avenues in CKD targeting this pathway.

Insights into the Progression of Labile Hb A1c to Stable Hb A1c via a Mechanistic Assessment of 2,3-Bisphosphoglycerate Facilitation of the Slow Nonenzymatic Glycation Process.

Nonenzymatic glycation (NEG) of human hemoglobin (Hb A) consists of initial non covalent, reversible steps involving glucose and amino acid residues, which may also involve effector reagent(s) in the formation of labile Hb A1c (the conjugate acid of the Schiff base). Labile Hb A1c can then undergo slow, largely irreversible, formation of stable Hb A1c (the Amadori product). Stable Hb A1c is measured to assess diabetic progression after labile Hb A1c removal. This study aimed to increase the understanding of the distinctions between labile and stable Hb A1c from a mechanistic perspective in the presence of 2,3-bisphosphoglycerate (2,3-BPG). 2,3-Bisphosphoglycerate is an effector reagent that reversibly binds in the Hb A1c pocket and modestly enhances overall NEG rate. The deprotonation of C2 on labile Hb A1c in the formation of the Amadori product was previously proposed to be rate-limiting. Computational chemistry was used here to identify the mechanism(s) by which 2,3-BPG facilitates the deprotonation of C2 on labile Hb A1c. 2,3-Bisphosphoglycerate is capable of abstracting protons on C2 and the α-nitrogen of labile Hb A1c and can also deprotonate water and/or amino acid residues, therefore preparing these secondary reagents to deprotonate labile Hb A1c. Parallel reactions not leading to an Amadori product were found that include formation of the neutral Schiff base, dissociation of glucose from the protein, and cyclic glycosylamine formation. These heretofore under appreciated parallel reactions may help explain both the selective removal of labile from stable Hb A1c and the slow rate of NEG.

Erythrocyte metabolism.

Our aim is to present an updated overview of the erythrocyte metabolism highlighting its richness and complexity. We have manually collected and connected the available biochemical pathways and integrated them into a functional metabolic map. The focus of this map is on the main biochemical pathways consisting of glycolysis, the pentose phosphate pathway, redox metabolism, oxygen metabolism, purine/nucleoside metabolism, and membrane transport. Other recently emerging pathways are also curated, like the methionine salvage pathway, the glyoxalase system, carnitine metabolism, and the lands cycle, as well as remnants of the carboxylic acid metabolism. An additional goal of this review is to present the dynamics of erythrocyte metabolism, providing key numbers used to perform basic quantitative analyses. By synthesizing experimental and computational data, we conclude that glycolysis, pentose phosphate pathway, and redox metabolism are the foundations of erythrocyte metabolism. Additionally, the erythrocyte can sense oxygen levels and oxidative stress adjusting its mechanics, metabolism, and function. In conclusion, fine-tuning of erythrocyte metabolism controls one of the most important biological processes, that is, oxygen loading, transport, and delivery.

Alteration in the number, morphology, function, and metabolism of erythrocytes in high-altitude polycythemia.

Introduction: High-altitude polycythemia (HAPC) is a common chronic high-altitude disease characterized by significantly increased erythrocyte, hemoglobin (Hb), and hematocrit values and decreased arterial oxygen saturation. The mechanisms underlying HAPC development are unclear; we aimed to investigate this in an HAPC rat model. Methods: Twelve Sprague-Dawley rats were divided into control and HAPC groups. The HAPC group was exposed to hypobaric hypoxia. This HAPC model was assessed using routine blood tests and blood gas analyses. Bone marrow, peripheral blood reticulocytes (RETs), and peripheral blood erythrocyte apoptosis were measured using flow cytometry. Erythrocyte osmotic fragility (EOF) tests were conducted. Abnormal erythrocytes were counted using electron microscopy. Plasma-free hemoglobin, 5'-nucleotidase (CD73), adenosine, erythrocyte cytosolic adenosine, sphingosine-1-phosphate (S1P), and 2,3-bisphosphoglycerate (BPG) levels were measured using enzyme-linked immunosorbent assays. Erythrocyte metabolic pathway-related protein [adenosine A2B receptor (ADORA2B), erythrocyte equilibrative nucleoside transporter 1 (eENT1), sphingosine kinase 1 (SPHK1), phospho-SPHK1, bisphosphoglycerate mutase (BPGM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] levels were assessed by Western blotting. Results: The HAPC rat model was successfully established (Hb > 210 g/L). Indices of bone marrow and peripheral blood RET proportions were significantly higher in the HAPC than the control group (p = 0.04 and p < 0.001, respectively). The proportion of peripheral blood erythrocytes in early apoptosis was significantly lower in the HAPC than the control group (p < 0.001). Vesicular erythrocyte and acanthocyte proportions were significantly higher in the HAPC than the control group (p < 0.001 and p = 0.019, respectively). The EOF tests revealed that 50% erythrocyte hemolysis occurred at 4.0-4.5 and 4.5-5.0 g/L NaCl in the control and HAPC groups, respectively. Plasma-free hemoglobin, CD73, adenosine, erythrocyte cytosolic adenosine, S1P, and 2,3-BPG levels and ADORA2B, eENT1, phospho-SPHK1, S1P, BPGM, and GAPDH erythrocyte expression levels (all p ≤ 0.02) were significantly higher in the HAPC than the control group. Conclusion: In model rats, an HAPC-related erythrocyte increase was associated with enhanced bone marrow hematopoietic function and reduced erythrocyte apoptosis, whereas numerous abnormal erythrocytes, increased EOF, and reduced hemolysis resistance were associated with erythrocyte metabolism. CD73/adenosine/S1P/2,3-BPG and eENT1/adenosine/BPGM/2,3-BPG metabolic pathways in erythrocytes were activated in HAPC rats, facilitating oxygen release. These findings further reveal the intrinsic HAPC mechanism and forms a basis for future development of preventive and therapeutic strategies for HAPC.

The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta.

Background: Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown. Methods: Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry. Results: We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation. Conclusions: This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR. Funding: This work was supported by the Weizmann Krenter Foundation and the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.

Synthetic Red Blood Cell-Specific Glycolytic Intermediate 2,3-Diphosphoglycerate (2,3-DPG) Inhibits Plasmodium falciparum Development In Vitro.

Mechanisms of malaria parasite interaction with its host red blood cell may provide potential targets for new antimalarial approaches. Pyruvate kinase deficiency has been associated with resistance to malaria in both experimental models and population studies. Two of the major pyruvate kinase deficient-cell disorders are the decrease in ATP and the increase in 2,3-biphosphoglycerate (2,3-BPG) concentration. High levels of this metabolite, only present in mammalian red blood cell, has an inhibitory effect on glycolysis and we hypothesized that its accumulation may also be harmful to the parasite and be involved in the mechanism of protection provided by that enzymopathy. We examined the effect of a synthetic form, 2,3-DPG, on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. Results showed an impairment of parasite growth with a direct effect on parasite maturation as significant lower progeny emerged from parasites that were submitted to 2,3-DPG. Further, adding the compound to the culture medium did not result in any effect on the host cell, but instead the metabolic profile of an infected cell became closer to that of a non-infected cell.