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Competing Interests: Competing interests statement:The authors declare no competing interest.

Amyloid beta peptides (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Soluble Aβ oligomers, rather than monomer or insoluble amyloid fibrils, show red blood cell (RBC) membrane-binding capacity and trigger several morphological and functional alterations in RBCs that can result in impaired oxygen transport and delivery. Since bioactive lipids have been recently proposed as potent protective agents against Aβ toxicity, we investigated the role of sphingosine-1-phosphate (S1P) in signaling pathways involved in the mechanism underlying ATP release in Ab-treated RBCs. In RBCs following different treatments, the ATP, 2,3 DPG and cAMP levels and caspase 3 activity were determined by spectrophotometric and immunoassay. S1P rescued the inhibition of ATP release from RBCs triggered by Ab, through a mechanism involving caspase-3 and restoring 2,3 DPG and cAMP levels within the cell. These findings reveal the molecular basis of S1P protection against Aβ in RBCs and suggest new therapeutic avenues in AD.

Red blood cells (RBCs) are the primary mediators of oxygen transport in the human body, and their function is mainly achieved through conformational changes of hemoglobin (Hb). Hb is a tetramer composed of four subunits, with HbA being the predominant Hb in healthy adults, existing in two forms: tense state (T state) and relaxed state (R state). Endogenous regulators of Hb conformation include 2,3-diphosphoglyceric acid, carbon dioxide, protons, and chloride ions, while exogenous regulators include inositol hexaphosphate, inositol tripyrophosphate, benzabate, urea derivative L35, and vanillin, each with different mechanisms of action. The application of Hb conformational regulators provides new insights into the study of hypoxia oxygen supply issues and the treatment of sickle cell disease.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Hemoglobin S (HbS) polymerization, red blood cell (RBC) sickling, chronic anemia, and vaso-occlusion are core to sickle cell disease (SCD) pathophysiology. Pyruvate kinase (PK) activators are a novel class of drugs that target RBC metabolism by reducing the buildup of the glycolytic intermediate 2,3-diphosphoglycerate (2,3-DPG) and increasing production of adenosine triphosphate (ATP). Lower 2,3-DPG level is associated with an increase in oxygen affinity and reduction in HbS polymerization, while increased RBC ATP may improve RBC membrane integrity and survival. There are currently 3 PK activators in clinical development for SCD: mitapivat (AG-348), etavopivat (FT-4202), and the second-generation molecule AG-946. Preclinical and clinical data from these 3 molecules demonstrate the ability of PK activators to lower 2,3-DPG levels and increase ATP levels in animal models and patients with SCD, as well as influence a number of potential pathways in SCD, including hemoglobin oxygen affinity, RBC sickling, RBC deformability, RBC hydration, inflammation, oxidative stress, hypercoagulability, and adhesion. Furthermore, early-phase clinical trials of mitapivat and etavopivat have demonstrated the safety and tolerability of PK activators in patients with SCD, and phase 2/3 trials for both drugs are ongoing. Additional considerations for this novel therapeutic approach include the balance between increasing hemoglobin oxygen affinity and tissue oxygen delivery, the cost and accessibility of these drugs, and the potential of multimodal therapy with existing and novel therapies targeting different disease mechanisms in SCD., (Copyright © 2023 by The American Society of Hematology.)

Innovative strategies to control malaria are urgently needed. Exploring the interplay between Plasmodium sp. parasites and host red blood cells (RBCs) offers opportunities for novel antimalarial interventions. Pyruvate kinase deficiency (PKD), characterized by heightened 2,3-diphosphoglycerate (2,3-DPG) concentration, has been associated with protection against malaria. Elevated levels of 2,3-DPG, a specific mammalian metabolite, may hinder glycolysis, prompting us to hypothesize its potential contribution to PKD-mediated protection. We investigated the impact of the extracellular supplementation of 2,3-DPG on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. The results showed an inhibition of parasite growth, resulting from significantly fewer progeny from 2,3-DPG-treated parasites. We analyzed differential gene expression and the transcriptomic profile of P. falciparum trophozoites, from in vitro cultures subjected or not subjected to the action of 2,3-DPG, using Nanopore Sequencing Technology. The presence of 2,3-DPG in the culture medium was associated with the significant differential expression of 71 genes, mostly associated with the GO terms nucleic acid binding, transcription or monoatomic anion channel. Further, several genes related to cell cycle control were downregulated in treated parasites. These findings suggest that the presence of this RBC-specific glycolytic metabolite impacts the expression of genes transcribed during the parasite trophozoite stage and the number of merozoites released from individual schizonts, which supports the potential role of 2,3-DPG in the mechanism of protection against malaria by PKD.

Malaria remains a major world public health problem, contributing to poverty and inequality. It is urgent to find new efficacious tools with few adverse effects. Malaria has selected red blood cell (RBC) alterations linked to resistance against infection, and understanding the protective mechanisms involved may be useful for developing host-directed tools to control Plasmodium infection. Pyruvate kinase deficiency has been associated with resistance to malaria. Pyruvate kinase-deficient RBCs display an increased concentration of 2,3-diphosphoglycerate (2,3-DPG). We recently showed that 2,3-DPG impacts in vitro intraerythrocytic parasite growth, induces a shift of the metabolic profile of infected cells (iRBCs), making it closer to that of noninfected ones (niRBCs), and decreases the number of parasite progenies that invade new RBCs. As an increase of 2,3-DPG content may also have an adverse effect on RBC membrane and, consequently, on the parasite invasion, in this study, we explored modifications of the RBC morphology, biomechanical properties, and RBC membrane on Plasmodium falciparum in vitro cultures treated with 2,3-DPG, using atomic force microscopy (AFM)-based force spectroscopy and other experimental approaches. The presence of infection by P. falciparum significantly increased the rigidity of parasitized cells and influenced the morphology of RBCs, as parasitized cells showed a decrease of the area-to-volume ratio. The extracellular addition of 2,3-DPG also slightly affected the stiffness of niRBCs, making it more similar to that of infected cells. It also changed the niRBC height, making the cells appear more elongated. Moreover, 2,3-DPG treatment influenced the cell surface charge, becoming more negative in treated RBCs than in untreated ones. The results indicate that treatment with 2,3-DPG has only a mild effect on RBCs in comparison with the effect of the presence of the parasite on the host cell. 2,3-DPG is an endogenous host metabolite, which may, in the future, originate a new antimalarial tool with few adverse effects on noninfected cells.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels., (Published by Elsevier Inc.)

Acute pain, the most prominent complication of sickle cell disease (SCD), results from vaso-occlusion triggered by sickling of deoxygenated red blood cells (RBCs). Concentration of 2,3-diphosphoglycerate (2,3-DPG) in RBCs promotes deoxygenation by preferentially binding to the low-affinity T conformation of HbS. 2,3-DPG is an intermediate substrate in the glycolytic pathway in which pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme; variants in PKLR may affect PKR activity, 2,3-DPG levels in RBCs, RBC sickling, and acute pain episodes (APEs). We performed a candidate gene association study using 2 cohorts: 242 adult SCD-HbSS patients and 977 children with SCD-HbSS or SCD-HbSβ0 thalassemia. Seven of 47 PKLR variants evaluated in the adult cohort were associated with hospitalization: intron 4, rs2071053; intron 2, rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964. All 7 variants showed consistent effect directions in both cohorts and remained significant in weighted Fisher's meta-analyses of the adult and pediatric cohorts using P < .0071 as threshold to correct for multiple testing. Allele-specific expression analyses in an independent cohort of 52 SCD adults showed that the intronic variants are likely to influence APE by affecting expression of PKLR, although the causal variant and mechanism are not defined., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The problem of analysis of clinical - diagnostic and biochemical criteria of postoperative cognitive dysfunction in abdominal oncosurgery, depending on the degree and structure of disorders, remains unresolved, which determines its relevance. The role of 2, 3-diphosphoglycerate is essential, because its increase in the concentration of red blood cells in hypoxic conditions is one of the adaptive mechanisms that improve oxygen transport to tissues. Purpose. The influence of the dynamics of 2,3 diphosphoglycerate content, as the main indicator of hypoxia, on the occurrence of cognitive dysfunction in the postoperative period in patients with neoplasms of the abdominal cavity. The study was conducted on the basis of departments for patients of the surgical profile of the municipal institution "Kharkiv City Clinical Hospital of Ambulance and Emergency Care named after Professor OI Meshchaninov ". To achieve this goal, we examined 80 patients with abdominal neoplasms who underwent surgery under general anesthesia using propofol and fentanyl. All patients were divided into 2 groups depending on the age of patients on the WHO scale, who underwent surgery using general anesthesia: Group 1 (n = 39) - middle-aged patients (50-59 years); Group 2 (n = 41) - elderly and senile patients (60-80 years). The control points of the examination were the day before the operation and the 1st, 7th, 30th day from the moment of the operation. The state of cognitive function in these patients was determined by conducting neuropsychological tests. To assess the state of cognitive function of patients, neuropsychological tests were used: MMSE scale (Mini-Mental State Examination, MMSE), the method of memorizing 10 words AR Luria, frontal dysfunction battery (FAB), Schulte technique. To assess the state of energy metabolism in patients, the level of erythrocytes and hemoglobin in the blood analysis was determined by well-known methods, the level of 2,3 diphosphoglycerate in erythrocytes and its ratio to hemoglobin. Anemia in the first week after surgery in patients of group 1 contributes to the development of a hypoxic state, in erythrocytes there is an increase in the content of 2,3 41 diphosphoglycerate, which promotes the transport of oxygen to tissues. During the week there is an increase in the intensity of the formation of 2,3 diphosphoglycerate, as evidenced by the ratio of 2,3 diphosphoglycerate to hemoglobin. In patients of group 2, the changes are more pronounced: anemia with a significant decrease in erythrocytes and hemoglobin in the blood, a decrease in 2.3 diphosphoglycerate in erythrocytes, reflects changes in erythrocyte metabolism, namely a decrease in biosynthesis of important organophosphorus compounds, in particular 2,3 diphosphogly by reducing the basic enzymes of glycolysis. Decreased energy metabolism in the elderly contributes to impaired cell function. With age, the content of adenosine triphosphate, 2,3 diphosphoglycerate decreases, thus increasing the affinity of hemoglobin for oxygen, impaired transport of oxygen to tissues, which leads to the development of hypoxia. According to the results of neuropsychological tests, we found postoperative cognitive dysfunction in patients with neoplasms of the abdominal cavity. Disruption of energy metabolism and changes in the activity of glycolysis enzymes in erythrocytes contributes to a decrease in the concentration of 2, 3 diphosphoglycerate, increase the affinity of hemoglobin for oxygen and the development of tissue hypoxia. The obtained results indicate the interdependence of these processes and allow continuing research in this direction with the development of appropriate clinical and diagnostic measures and areas of intensive care to improve the condition of patients with abdominal tumors and their quality of life after surgery.

Objective: To investigate the anti-oxidative effect of ethyl pyruvate (EP) and taurine (TAU) on the quality of red blood cells stored at 4±2 ℃, hemolysis, energy metabolism and lipid peroxidation of the red blood cells in the preservation solution were studied at different intervals., Methods: At 4±2 ℃, the deleukocyte red blood cells were stored in the citrate-phosphate-dextrosesaline-adenine-1 (CPDA-1) preservation (control group), preservation solution with EP (EP-AS), and TAU (TAU-AS) for long-term preservation. The enzyme-linked immunoassay and automatic blood cell analyzer were used to detect hemolysis and erythrocyte parameters. Adenine nucleoside triphosphate (ATP), glycerol 2,3-diphosphate (2,3-DPG) and malondialdehyde (MDA) kits were used to test the ATP, 2,3-DPG and MDA concentration., Results: During the preservation, the rate of red blood cell hemolysis in EP-AS and TAU-AS groups were significantly lower than that in CPDA-1 group (P<0.01). The MCV of EP-AS group was increased with the preservation time (r=0.71), while the MCV of the TAU-AS group was significantly lower than that in the other two groups (P<0.05). The concentration of ATP and MDA in EP-AS and TAU-AS groups were significantly higher than that in CPDA-1 group at the 14th day (P<0.01). The concentrations of 2,3-DPG in the EP-AS and TAU-AS groups were significantly higher than that in the CPDA-1 group from the 7th day (P<0.01)., Conclusion: EP and TAU can significantly reduce the red blood cell hemolysis rate, inhibit the lipid peroxidation level of red blood cells, and improve the energy metabolism of red blood cells during storage. The mechanism of EP and TAU may be related to their antioxidation and membrane protection effect, so as to improve the red blood cell quality and extend the preservation time.

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., Competing Interests: Author JAR was employed by company Clarify Analytical. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Morais, Medeiros, Carvalho, Morello, Teixeira, Maciel, Nhantumbo, Balau, Rosa, Nogueira, Rodrigues, Carvalho, Antunes and Arez.)

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia., (Copyright © 2022 The Author(s).)

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline., Competing Interests: The authors have declared that no competing interests exist.

Oxygen supply for ischemic brain tissue during stroke is critical to neuroprotection. Remote ischemic conditioning (RIC) treatment is effective for stroke. However, it is not known whether RIC can improve brain tissue oxygen supply. In current study, we employed a mouse model of stroke created by middle cerebral artery occlusion (MCAO) to investigate the effect of RIC on oxygen supply to the ischemic brain tissue using a hypoxyprobe system. Erythrocyte oxygen-carrying capacity and tissue oxygen exchange were assessed by measuring oxygenated hemoglobin and oxygen dissociation curve. We found that RIC significantly mitigated hypoxic signals and decreased neural cell death, thereby preserving neurological functions. The tissue oxygen exchange was markedly enhanced, along with the elevated hemoglobin P50 and right-shifted oxygen dissociation curve. Intriguingly, RIC markedly elevated 2,3-biphosphoglycerate (2,3-BPG) levels in erythrocyte, and the erythrocyte 2,3-BPG levels were highly negatively correlated with the hypoxia in the ischemic brain tissue. Further, adoptive transfusion of 2,3-BPG-rich erythrocytes prepared from RIC-treated mice significantly enhanced the oxygen supply to the ischemic tissue in MCAO mouse model. Collectively, RIC protects against ischemic stroke through improving oxygen supply to the ischemic brain tissue where the enhanced tissue oxygen delivery and exchange by RIC-induced 2,3-BPG-rich erythrocytes may play a role.

Hydrogen sulfide (H 2 S) is naturally synthesized in a wide range of mammalian tissues. Whether H 2 S is involved in the regulation of erythrocyte functions remains unknown. Using mice with a genetic deficiency in a H 2 S natural synthesis enzyme cystathionine- γ -lyase (CSE) and high-throughput metabolomic profiling, we found that levels of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), an erythroid-specific metabolite negatively regulating hemoglobin- (Hb-) oxygen (O 2 ) binding affinity, were increased in CSE knockout ( Cse -/- ) mice under normoxia. Consistently, the 50% oxygen saturation (P50) value was increased in erythrocytes of Cse -/- mice. These effects were reversed by treatment with H 2 S donor GYY4137. In the models of cultured mouse and human erythrocytes, we found that H 2 S directly acts on erythrocytes to decrease 2,3-BPG production, thereby enhancing Hb-O 2 binding affinity. Mouse genetic studies showed that H 2 S produced by peripheral tissues has a tonic inhibitory effect on 2,3-BPG production and consequently maintains Hb-O 2 binding affinity in erythrocytes. We further revealed that H 2 S promotes Hb release from the membrane to the cytosol and consequently enhances bisphosphoglycerate mutase (BPGM) anchoring to the membrane. These processes might be associated with S-sulfhydration of Hb. Moreover, hypoxia decreased the circulatory H 2 S level and increased the erythrocyte 2,3-BPG content in mice, which could be reversed by GYY4137 treatment. Altogether, our study revealed a novel signaling pathway that regulates oxygen-carrying capacity in erythrocytes and highlights a previously unrecognized role of H 2 S in erythrocyte 2,3-BPG production., Competing Interests: The authors declare no competing interests., (Copyright © 2021 Gang Wang et al.)

Murine serine racemase (SR), the enzyme responsible for the biosynthesis of the neuromodulator d-serine, was reported to form a complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in SR inhibition. In this work, we investigated the interaction between the two human orthologues. We were not able to observe neither the inhibition nor the formation of the SR-GAPDH complex. Rather, hSR is inhibited by the hGAPDH substrate glyceraldehyde 3-phosphate (G3P) in a time- and concentration-dependent fashion, likely through a covalent reaction of the aldehyde functional group. The inhibition was similar for the two G3P enantiomers but it was not observed for structurally similar aldehydes. We ruled out a mechanism of inhibition based on the competition with either pyridoxal phosphate (PLP) - described for other PLP-dependent enzymes when incubated with small aldehydes - or ATP. Nevertheless, the inhibition time course was affected by the presence of hSR allosteric and orthosteric ligands, suggesting a conformation-dependence of the reaction., (Copyright © 2020. Published by Elsevier B.V.)

Sphingosine-1-phosphate (S1P) is emerging as a hypoxia responsive bio-lipid; systemically raised levels of S1P are proposed to have potential hypoxia pre-conditioning effects. The study aims to evaluate the hypoxia pre-conditioning efficacy of exogenously administered S1P in rats exposed to acute (24-48 hs (h)) and sub-chronic (7 days) hypobaric hypoxia. Sprague-Dawley rats (200 ± 20 g) were preconditioned with 1 μg/kg body weight S1P intravenously for three consecutive days. On the third day, control and S1P preconditioned animals were exposed to hypobaric hypoxia equivalent to 7620 m for 24 h, 48 h and 7 days. Post exposure analysis included body weight quantitation, blood gas/chemistry analysis, vascular permeability assays, evaluation of oxidative stress/inflammation parameters, and estimation of hypoxia responsive molecules. S1P preconditioned rats exposed to acute HH display a significant reduction in body weight loss, as a culmination of improved oxygen carrying capacity, increased 2,3- diphosphoglycerate levels and recuperation from energy deficit. Pathological disturbances such as vascular leakage in the lungs and brain, oxidative stress, pro-inflammatory milieu and raised level of endothelin-1 were also reined. The adaptive and protective advantage conferred by S1P in the acute phase of hypobaric hypoxia exposure, is observed to precipitate into an improved sustenance even after sub-chronic (7d) hypobaric hypoxia exposure as indicated by decreased body weight loss, lower edema index and improvement in general pathology biomarkers. Conclusively, administration of 1 μg/kg body weight S1P, in the aforementioned schedule, confer hypoxia pre-conditioning benefits, sustained up to 7 days of hypobaric hypoxia exposure., Competing Interests: Declaration of competing interest The authors express no conflict of interest with any researcher, research group or organization., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ammonium, an end-product of catabolism, in low doses can promote adaptation of metabolic pathways in erythrocytes under conditions of extreme physical exercise. We compared the effects of two ammonium salts, ammonium chloride and ammonium carbonate, in two doses on biochemical parameters of rat erythrocytes 1 day after extreme physical exercise in a 4-week cycle of forced swimming. Of 16 analyzed parameters, the maximum number of significant shifts from the control was revealed in the groups of rats receiving ammonium chloride in doses of 20 and 10 mg/kg, and the minimal number of differences was found in groups treated with ammonium carbonate in the same doses. The comparison of the levels of reduced glutathione and 2.3-bisphosphoglicerate and activities of 5'-nucleotidase and Ca 2+ - and Na/K-ATPases attested to more rigorous control of the mechanism of oxygen delivery to tissues by erythrocytes after administration of ammonium chloride in a dose of 20 mg/kg.

Background: Rejuvenation of stored red blood cells (RBCs) increases levels of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) to those of fresh cells. This study aimed to optimize and validate the US-approved process to a UK setting for manufacture and issue of rejuvenated RBCs for a multicenter randomized controlled clinical trial in cardiac surgery., Study Design and Methods: Rejuvenation of leukoreduced RBC units involved adding a solution containing pyruvate, inosine, phosphate, and adenine (Rejuvesol, Zimmer Biomet), warming at 37°C for 60 minutes, then "manual" washing with saline adenine glucose mannitol solution. A laboratory study was conducted on six pools of ABO/D-matched units made the day after donation. On Days 7, 21, and 28 of 4 ± 2°C storage, one unit per pool was rejuvenated and measured over 96 hours for volume, hematocrit, hemolysis, ATP, 2,3-DPG, supernatant potassium, lactate, and purines added (inosine) or produced (hypoxanthine) by rejuvenation. Subsequently, an operational validation (two phases of 32 units each) was undertaken, with results from the first informing a trial component specification applied to the second. Rejuvenation effects were also tested on crossmatch reactivity and RBC antigen profiles., Results: Rejuvenation raised 2,3-DPG to, and ATP above, levels of fresh cells. The final component had potassium and hemolysis values below those of standard storage Days 7 and 21, respectively, containing 1.2% exogenous inosine and 500 to 1900 μmoles/unit of hypoxanthine. The second operational validation met compliance to the trial component specification. Rejuvenation did not adversely affect crossmatch reactivity or RBC antigen profiles., Conclusion: The validated rejuvenation process operates within defined quality limits, preserving RBC immunophenotypes, enabling manufacture for clinical trials., (© 2019 Crown copyright. Transfusion © 2019 AABB.)

Glycolysis is a central metabolic pathway that provides energy and products of primary metabolites. 2,3-Biphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is a key enzyme that catalyses the reversible interconversion of 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA) in glycolysis. Low temperature is a common abiotic stress in rice production. However, the mechanism for rice iPGAM genes is not fully understood at low temperature. In this study, the rice mutant tcm12, with chlorosis, malformed chloroplasts and impaired photosynthesis, was grown at a low temperature (<20 °C) to the three-leaf stage, while the normal phenotype at 32 °C was used. Chlorophyll fluorescence analysis and transmission electron microscopy were used to examine features of the tcm12 mutant. The inheritance behaviour and function of TCM12 were then analysed thorough map-based cloning, transgenic complementation and subcellular localisation. The thermo-sensitive chlorosis phenotype was caused by a single nucleotide mutation (T→C) on the fifth exon of TCM12 (LOC&#95;Os12g35040) encoding iPGAM, localised to both nucleus and membranes. In addition, TCM12 was constitutively expressed, and its disruption resulted in down-regulation of some genes associated with chlorophyll biosynthesis and photosynthesis at low temperatures (20 °C). This is the first report of the involvement of rice iPGAM gene in chlorophyll synthesis, photosynthesis and chloroplast development, providing new insights into the mechanisms underlying early growth of rice at low temperatures., (© 2019 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Fetal hemoglobin (HbF) induction therapy has become the most promising strategy for treating β-hemoglobinopathies, including sickle-cell diseases and β-thalassemia. However, subtle but critical structural difference exists between HbF and normal adult hemoglobin (HbA), which inevitably leads to reduced binding of the endogenous modulator 2,3-bisphosphoglycerate (2,3-BPG) to HbF and thus increased oxygen affinity and decreased oxygen transport efficiency of HbF. We combined the oxygen equilibrium experiments, resonance Raman (RR) spectroscopy, and molecular docking modeling, and we discuss 2 phthalides, z-butylidenephthalide and z-ligustilide, that can effectively lower the oxygen affinity of HbF. They adjust it to a level closer to that of HbA and make it a more satisfactory oxygen carrier for adults. From the oxygen equilibrium curve measurements, we show that the 2 phthalides are more effective than 2,3-BPG for modulating HbF. The RR spectra show that phthalides allosterically stabilize the oxygenated HbF in the low oxygen affinity conformation, and the molecular docking modeling reveals that the 2 chosen phthalides interact with HbF via the cleft around the γ 1 /γ 2 interface with a binding strength ∼1.6 times stronger than that of 2,3-BPG. We discuss the implications of z-butylidenephthalide and z-ligustilide in boosting the efficacy of HbF induction therapy to mitigate the clinical severities of β-hemoglobinopathies., (© 2019 by The American Society of Hematology.)

The antibiotic moenomycin A is a phosphoglycerate derivative with a C 25 -moenocinyl chain and a branched oligosaccharide. Formation of the C 25 -chain is catalyzed by the enzyme MoeN5 with geranyl pyrophosphate (GPP) and the sugar-linked 2-Z,E-farnesyl-3-phosphoglycerate (FPG) as its substrates. Previous complex crystal structures with GPP and long-chain alkyl glycosides suggested that GPP binds to the S1 site in a similar way as in most other α-helical prenyltransferases (PTs), and FPG is likely to assume a bent conformation in the S2 site. However, two FPG derivatives synthesized in the current study were found in the S1 site rather than S2 in their complex crystal structures with MoeN5. Apparently S1 is the preferred site for prenyl-containing ligand, and S2 binding may proceed only after S1 is occupied. Thus, like most trans-type PTs, MoeN5 may employ a sequential ionization-condensation-elimination mechanism that involves a carbocation intermediate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 A 1c (the conjugate acid of the Schiff base). Labile Hb A 1c can then undergo slow, largely irreversible, formation of stable Hb A 1c (the Amadori product). Stable Hb A 1c is measured to assess diabetic progression after labile Hb A 1c removal. This study aimed to increase the understanding of the distinctions between labile and stable Hb A 1c 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 A 1c pocket and modestly enhances overall NEG rate. The deprotonation of C2 on labile Hb A 1c 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 A 1c . 2,3-Bisphosphoglycerate is capable of abstracting protons on C2 and the α-nitrogen of labile Hb A 1c and can also deprotonate water and/or amino acid residues, therefore preparing these secondary reagents to deprotonate labile Hb A 1c . 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 A 1c and the slow rate of NEG.

Although excessive plasma adenosine is detrimental in sickle cell disease (SCD), the molecular mechanism underlying elevated circulating adenosine remains unclear. Here we report that the activity of soluble CD73, an ectonucleotidase producing extracellular adenosine, was significantly elevated in a murine model of SCD and correlated with increased plasma adenosine. Mouse genetic studies demonstrated that CD73 activity contributes to excessive induction of plasma adenosine and thereby promotes sickling, hemolysis, multiorgan damage, and disease progression. Mechanistically, we showed that erythrocyte adenosine 5'-monophosphate-activated protein kinase (AMPK) was activated both in SCD patients and in the murine model of SCD. AMPK functions downstream of adenosine receptor ADORA2B signaling and contributes to sickling by regulating the production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O 2 binding affinity. Preclinically, we reported that treatment of α,β-methylene adenosine 5'-diphosphate, a potent CD73 specific inhibitor, significantly decreased sickling, hemolysis, multiorgan damage, and disease progression in the murine model of SCD. Taken together, both human and mouse studies reveal a novel molecular mechanism contributing to the pathophysiology of SCD and identify potential therapeutic strategies to treat SCD., (© 2018 by The American Society of Hematology.)

Background: Refrigerated red blood cell (RBC) storage results in the progressive accumulation of biochemical and morphological alterations collectively referred to as the storage lesion. Storage-induced metabolic alterations can be in part reversed by rejuvenation practices. However, rejuvenation requires an incubation step of RBCs for 1 hour at 37°C, limiting the practicality of providing "on-demand," rejuvenated RBCs. We tested the hypothesis that the addition of rejuvenation solution early in storage as an adjunct additive solution would prevent-in a time window consistent with the average age of units transfused to sickle cell recipients at Duke (15 days)-many of the adverse biochemical changes that can be reversed via standard rejuvenation, while obviating the incubation step., Study Design and Methods: Metabolomics analyses were performed on cells and supernatants from AS-1 RBC units (n = 4), stored for 15 days. Units were split into pediatric bag aliquots and stored at 4°C. These were untreated controls, washed with or without rejuvenation, performed under either standard (37°C) or cold (4°C) conditions., Results: All three treatments removed most metabolic storage by-products from RBC supernatants. However, only standard and cold rejuvenation provided significant metabolic benefits as judged by the reactivation of glycolysis and regeneration of adenosine triphosphate and 2,3-diphosphoglycerate. Improvements in energy metabolism also translated into increased capacity to restore the total glutathione pool and regenerate oxidized vitamin C in its reduced (ascorbate) form., Conclusion: Cold and standard rejuvenation of 15-day-old RBCs primes energy and redox metabolism of stored RBCs, while providing a logistic advantage for routine blood bank processing workflows., (© 2018 AABB.)

Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression. Functional studies revealed that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure at 1.9 Å resolution deciphered that S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to α1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with α2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying S1P-mediated pathogenic metabolic reprogramming in SCD and novel therapeutic avenues.

Sepsis induces a wide range of effects on the red blood cell (RBC). Some of the effects including altered metabolism and decreased 2,3-bisphosphoglycerate are preventable with appropriate treatment, whereas others, including decreased erythrocyte deformability and redistribution of membrane phospholipids, appear to be permanent, and factors in RBC clearance. Here, we review the effects of sepsis on the erythrocyte, including changes in RBC volume, metabolism and hemoglobin's affinity for oxygen, morphology, RBC deformability (an early indicator of sepsis), antioxidant status, intracellular Ca 2+ homeostasis, membrane proteins, membrane phospholipid redistribution, clearance and RBC O₂-dependent adenosine triphosphate efflux (an RBC hypoxia signaling mechanism involved in microvascular autoregulation). We also consider the causes of these effects by host mediated oxidant stress and bacterial virulence factors. Additionally, we consider the altered erythrocyte microenvironment due to sepsis induced microvascular dysregulation and speculate on the possible effects of RBC autoxidation. In future, a better understanding of the mechanisms involved in sepsis induced erythrocyte pathophysiology and clearance may guide improved sepsis treatments. Evidence that small molecule antioxidants protect the erythrocyte from loss of deformability, and more importantly improve septic patient outcome suggest further research in this area is warranted. While not generally considered a critical factor in sepsis, erythrocytes (and especially a smaller subpopulation) appear to be highly susceptible to sepsis induced injury, provide an early warning signal of sepsis and are a factor in the microvascular dysfunction that has been associated with organ dysfunction., Competing Interests: The authors declare no conflict of interest.

As a prelude to separating tertiary from quaternary structure contributions to the Bohr effect, we employed the Wyman equation to analyze Bohr data for human hemoglobin to which 2,3-bisphosphoglycerate, 2,3-BPG, is bound. Changes in the pK a s of the histidine Bohr groups result in a net reduction of their contributions to the Bohr effect at pH 7.4 compared to their contributions in stripped hemoglobin. The non-histidine 2,3-BPG binding groups - the β-chain terminal amino group and Lys82β - make negative and positive contributions, respectively, to the Bohr effect. The final result is that the Bohr effect at physiological pH is higher for 2,3-BPG bound compared to stripped hemoglobin. Contributions linked to His2β, His77β and His143β enable us to separate tertiary from quaternary Bohr contributions in stripped and in 2,3-BPG bound hemoglobin. Both contributions serve to make the Bohr effect for 2,3-BPG bound hemoglobin higher than for stripped hemoglobin at physiological pH., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A new algorithm for comparison of protein dynamics is presented. Compared protein structures are superposed and their modes of motions are calculated using the anisotropic network model. The obtained modes are aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. Dynamical comparison of hemoglobin in the T and R2 states reveals that the dynamics of the allosteric effector 2,3-bisphosphoglycerate binding site is different in the two states. These differences can contribute to the selectivity of the effector to the T state. Similar comparison of the ionotropic glutamate receptor in the kainate+(R,R)-2b and ZK bound states reveals that the kainate+(R,R)-2b bound states slow modes describe upward motions of ligand binding domain and the transmembrane domain regions. Such motions may lead to the opening of the receptor. The upper lobes of the LBDs of the ZK bound state have a smaller interface with the amino terminal domains above them and have a better ability to move together. The present study exemplifies the use of dynamics comparison as a tool to study protein function. Proteins 2017; 85:1507-1517. © 2014 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (>82% of original values)., Study Design and Methods: At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at -80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters., Results: Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively., Conclusion: Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3., (© 2017 AABB.)

Phosphoglycerate mutase 1 (PGAM1) catalyzes the eighth step of glycolysis and is often found upregulated in cancer cells. To test the hypothesis that the phosphorylation of tyrosine 26 residue of PGAM1 greatly enhances its activity, we performed both conventional and steered molecular dynamics simulations on the binding and unbinding of PGAM1 to its substrates, with tyrosine 26 either phosphorylated or not. We analyzed the simulated data in terms of structural stability, hydrogen bond formation, binding free energy, etc. We found that tyrosine 26 phosphorylation enhances the binding of PGAM1 to its substrates through generating electrostatic environment and structural features that are advantageous to the binding. Our results may provide valuable insights into computer-aided design of drugs that specifically target cancer cells with PGAM1 tyrosine 26 phosphorylated.

Erythrocyte-based drug delivery systems are emerging as potential new solutions for the release of drugs into the bloodstream. The aim of the present work was to assess the performance of a fully automated process (EDS) for the ex-vivo encapsulation of the pro-drug dexamethasone sodium phosphate (DSP) into autologous erythrocytes in compliance with regulatory requirements. The loading method was based on reversible hypotonic hemolysis, which allows the opening of transient pores in the cell membrane to be crossed by DSP. The efficiency of encapsulation and the biochemical and physiological characteristics of the processed erythrocytes were investigated in blood samples from 34 healthy donors. It was found that the processed erythrocytes maintained their fundamental properties and the encapsulation process was reproducible. The EDS under study showed greater loading efficiency and reduced variability compared to previous EDS versions. Notably, these results were confirmed using blood samples from Ataxia Telangiectasia (AT) patients, 9.33±1.40 and 19.41±2.10mg of DSP (mean±SD, n=134) by using 62.5 and 125mg DSP loading quantities, respectively. These results support the use of the new EDS version 3.2.0 to investigate the effect of erythrocyte-delivered dexamethasone in regulatory trials in patients with AT., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Each unit of blood donated is processed and stored individually resulting in variability in the amount of red blood cells (RBCs) collected, RBC properties, and the 24-hour posttransfusion RBC survivability. As a result, each unit differs in its ability to deliver oxygen and potentially its effects on the recipient. The goal of this study was to investigate the storage of pooled RBCs from multiple donors in comparison to control standard RBC units., Study Design and Methods: Two units of irradiated, leukoreduced RBCs of same ABO, D, E, C, and K antigen phenotype were collected from each of five donors using apheresis. One unit from each donor was pooled in a 2-L bag and remaining units were used as controls. After being pooled, RBCs were separated in five bags and stored at 4°C along with the controls. Quality indexes were measured on Days 2, 14, and 28 for all the units., Results: Adenosine triphosphate assays for both pooled and controls showed a slight decrease from Day 2 to Day 28 (pooled/control from 5.22/5.24 to 4.35/4.33 µmol/g hemoglobin [Hb]). 2,3-Diphosphoglycerate was successfully rejuvenated for all RBC units on Day 28 (pooled 11.46 µmol/g Hb; control 11.86 µmol/g Hb). The results showed a nonsignificant difference between pooled and control units, with a general trend of lower standard deviation for pooled units when compared to controls., Conclusion: Pooled units have reduced unit-to-unit variability. Future exploration of their immunogenicity is required before using pooled units for transfusion., (© 2016 AABB.)

Long-term administration of simvastatin to rats, irrespective of the baseline cholesterol levels, induced biochemical changes in erythrocytes attesting to hypoxic damage (accumulation of lactate and 2,3-diphosphoglycerate), disturbances in ATP-dependent mechanisms of ion homeostasis regulation (decrease in total ATPase and Ca(2+)-ATPase activities), and antioxidant enzymes system imbalance. These changes can be considered as a sensitive indicator and molecular basis of cell damage during long-term administration of statins.

Sphingosine-1-phosphate (S1P) is a bioactive signalling lipid highly enriched in mature erythrocytes, with unknown functions pertaining to erythrocyte physiology. Here by employing nonbiased high-throughput metabolomic profiling, we show that erythrocyte S1P levels rapidly increase in 21 healthy lowland volunteers at 5,260 m altitude on day 1 and continue increasing to 16 days with concurrently elevated erythrocyte sphingonisne kinase 1 (Sphk1) activity and haemoglobin (Hb) oxygen (O2) release capacity. Mouse genetic studies show that elevated erythrocyte Sphk1-induced S1P protects against tissue hypoxia by inducing O2 release. Mechanistically, we show that intracellular S1P promotes deoxygenated Hb anchoring to the membrane, enhances the release of membrane-bound glycolytic enzymes to the cytosol, induces glycolysis and thus the production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific glycolytic intermediate, which facilitates O2 release. Altogether, we reveal S1P as an intracellular hypoxia-responsive biolipid promoting erythrocyte glycolysis, O2 delivery and thus new therapeutic opportunities to counteract tissue hypoxia.

Background: Anaerobic red blood cell (RBC) storage reduces oxidative damage, maintains adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) levels, and has superior 24-hour recovery at 6 weeks compared to standard storage. This study will determine if removal of CO2 during O2 depletion by gas exchange may affect RBCs during anaerobic storage., Study Design and Methods: This is a matched three-arm study (n = 14): control, O2 and CO2 depleted with Ar (AN), and O2 depleted with 95%Ar/5%CO2 (AN[CO2 ]). RBCs in additives AS-3 or OFAS-3 were evenly divided into three bags, and anaerobic conditions were established by gas exchange. Bags were stored at 1 to 6°C in closed chambers under anaerobic conditions or ambient air, sampled weekly for up to 9 weeks for a panel of in vitro tests. A full metabolomics screening was conducted for the first 4 weeks of storage., Results: Purging with Ar (AN) results in alkalization of the RBC and increased glucose consumption. The addition of 5% CO2 to the purging gas prevented CO2 loss with an equivalent starting and final pH and lactate to control bags (p > 0.5, Days 0-21). ATP levels are higher in AN[CO2 ] (p < 0.0001). DPG was maintained beyond 2 weeks in the AN arm (p < 0.0001). Surprisingly, DPG was lost at the same rate in both control and AN[CO2 ] arms (p = 0.6)., Conclusion: Maintenance of ATP in the AN[CO2 ] arm demonstrates that ATP production is not solely a function of the pH effect on glycolysis. CO2 in anaerobic storage prevented the maintenance of DPG, and DPG production appears to be pH dependent. CO2 as well as O2 depletion provides metabolic advantage for stored RBCs., (© 2015 AABB.)

Erythrocytes are the key target in 5'-AMP induced hypometabolism. To understand how regulation of endogenous erythrocyte AMP levels modulates systemic metabolism, we generated mice deficient in both CD73 and AMPD3, the key catabolic enzymes for extracellular and intra-erythrocyte AMP, respectively. Under physiological conditions, these mice displayed enhanced capacity for physical activity accompanied by significantly higher food and oxygen consumption, compared to wild type mice. Erythrocytes from Ampd3(-/-) mice exhibited higher half-saturation pressure of oxygen (p50) and about 3-fold higher levels of ATP and ADP, while they maintained normal 2,3-bisphosphoglycerate (2,3-BPG), methemoglobin levels and intracellular pH. The affinity of mammalian hemoglobin for oxygen is thought to be regulated primarily by 2,3-BPG levels and pH (the Bohr effect). However, our results show that increased endogenous levels of ATP and ADP, but not AMP, directly increase the p50 value of hemoglobin. Additionally, the rise in erythrocyte p50 directly correlates with an enhanced capability of systemic metabolism.

Genetically based modifications of hemoglobin (Hb) function that increase blood-O2 affinity are hallmarks of hypoxia adaptation in vertebrates. Among mammals, felid Hbs are unusual in that they have low intrinsic O2 affinities and reduced sensitivities to the allosteric cofactor 2,3-diphosphoglycerate (DPG). This combination of features compromises the acclimatization capacity of blood-O2 affinity and has led to the hypothesis that felids have a restricted physiological niche breadth relative to other mammals. In seeming defiance of this conjecture, the snow leopard (Panthera uncia) has an extraordinarily broad elevational distribution and occurs at elevations above 6000 m in the Himalayas. Here, we characterized structural and functional variation of big cat Hbs and investigated molecular mechanisms of Hb adaptation and allosteric regulation that may contribute to the extreme hypoxia tolerance of the snow leopard. Experiments revealed that purified Hbs from snow leopard and African lion exhibited equally low O2 affinities and DPG sensitivities. Both properties are primarily attributable to a single amino acid substitution, β2His→Phe, which occurred in the common ancestor of Felidae. Given the low O2 affinity and reduced regulatory capacity of feline Hbs, the extreme hypoxia tolerance of snow leopards must be attributable to compensatory modifications of other steps in the O2-transport pathway., (© 2015. Published by The Company of Biologists Ltd.)

Background: Over the past decade there has been a growth in the development of pathogen reduction technologies to protect the blood supply from emerging pathogens. This development has proven to be difficult for red blood cells (RBCs). However the S-303 system has been shown to effectively inactivate a broad spectrum of pathogens, while maintaining RBC quality., Study Design and Methods: A paired three-arm study was performed to compare the in vitro quality of S-303-treated RBCs with RBCs stored at room temperature (RT) for the duration of the treatment (18-20 hr) and control RBCs stored at 2 to 6°C. Products were sampled weekly over 42 days of storage (n = 10) and tested using an array of in vitro assays to measure quality, metabolism, and functional variables., Results: During S-303 treatment there was a slight loss of RBCs and hemoglobin (Hb < 5 g). Hemolysis, glucose consumption, and potassium release were similar in all groups during the 42 days of storage. S-303-treated RBCs had a significantly lower lactate concentration and pH compared to the paired controls. The S-303-treated RBCs had significantly higher adenosine triphosphate than the RT and control RBCs. There was a significant loss of 2,3-diphosphoglycerate in the S-303-treated products, which was also observed in the RT RBCs. Flow cytometry analysis demonstrated similar RBC size, morphology, expression of CD47, and glycophorin A in all groups., Conclusion: RBCs treated with S-303 for pathogen reduction had similar in vitro properties to the paired controls and were within transfusion guidelines., (© 2014 Australian Red Cross Blood Service. Transfusion © 2014 AABB.)

Blood banking underpins modern medical care, but blood storage, necessary for testing and inventory management, reduces the safety and efficacy of individual units of red blood cells (RBCs). Stored RBCs are damaged by the accumulation of their own waste products, by enzymatic and oxidative injury, and by metabolically programmed cell death. These chemical activities lead to a complex RBC storage lesion that includes haemolysis, reduced in vivo recovery, energy and membrane loss, altered oxygen release, reduced adenosine tri-phosphate and nitric oxide secretion, and shedding of toxic products. These toxic products include lysophospholipids that can cause transfusion-related acute lung injury, free iron that can potentiate infections and cause inflammation, and shed microvesicles that can scavenge nitric oxide and potentiate inflammation and thrombosis. However, most of the obvious negative outcomes of RBC storage are uncommon and appear to be related to exceptionally bad units. Generally, the quality of stored RBCs is highly related to the conditions of storage, so refrigerator temperature, intact bags, residual leucocyte counts and visible haemolysis remain excellent general measures. Specific biochemical measures, such as adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) concentrations, calcium and potassium content or lipid breakdown products, require specialized measures that are not widely available, involve destructive testing and generally reflect only a part of the storage lesion. This review describes a number of components of the storage lesion and their measurement and attempts to access the utility of the measures., (© 2014 International Society of Blood Transfusion.)

Bisphosphoglycerate mutase (BPGM) is a multi-activity enzyme. Its main function is to synthesize the 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. This enzyme can also catalyze the 2,3-bisphosphoglycerate to the 3-phosphoglycerate. In this study, the reaction mechanisms of both the phosphatase and the synthase activities of human bisphosphoglycerate mutase were theoretically calculated by using the quantum mechanics/molecular mechanics method based on the metadynamics and umbrella sampling simulations. The simulation results not only show the free energy curve of the phosphatase and the synthase reactions, but also reveal the important role of some residues in the active site. Additionally, the energy barriers of the two reactions indicate that the activity of the synthase in human bisphosphoglycerate mutase is much higher than that of the phosphatase. The estimated reaction barriers are consistent with the experimental data. Therefore, our work can give important information to understand the catalytic mechanism of the bisphosphoglycerate mutase family.

This study aimed to investigate the effect of mesenteric lymph drainage on the acute kidney injury induced by hemorrhagic shock without resuscitation. Eighteen male Wistar rats were randomly divided into sham, shock, and drainage groups. The hemorrhagic shock model (40 mmHg, 3 h) was established in shock and drainage groups; mesenteric lymph drainage was performed from 1 h to 3 h of hypotension in the drainage group. The results showed that renal tissue damage occurred; the levels of urea, creatinine, and trypsin in the plasma as well as intercellular adhesion molecule-1 (ICAM-1), receptor of advanced glycation end-products (RAGE), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), lactic acid (LA), and 2,3-DPG in the renal tissue were increased in the shock group after 3 h of hypotension. Mesenteric lymph drainage lessened the following: renal tissue damage; urea and trypsin concentrations in the plasma; ICAM-1, RAGE, TNF-α, MDA, and LA levels in the renal tissue. By contrast, mesenteric lymph drainage increased the 2,3-DPG level in the renal tissue. These findings indicated that mesenteric lymph drainage could relieve kidney injury caused by sustained hypotension, and its mechanisms involve the decrease in trypsin activity, suppression of inflammation, alleviation of free radical injury, and improvement of energy metabolism.

Background: Cryopreservaton of packed human red blood cells requires the use of cryoprotectants., Objective: The study assessed physiological parameters of 40 RBC units frozen with either 40% glycerol or 6.7% HES., Methods: After thawing, they were suspended in NaCl or in 6% HES. Tests of Hct, Hb, Na+ and K+ ions, ATP, 2,3-DPG, pH and erythrocyte stability were measured 30 minutes and 24 hours after thawing., Results: Hct was lower after thawing but did not differ significantly between two groups. Hb was lower after thawing, but was statistically significant higher in the HES group (43.8 g/unit vs 35.4 g/unit). K+ concentration increased after thawing and was significantly higher after 24 hours in the glycerol group (29.0 mEq/l vs 8.7 mEq/l). ATP concentration in the HES group was significantly lower (2.15 micromol/g) in comparison with the glycerol group (6.30 micromol/g) 24 hours after thawing. 2,3-DPG levels did not differ significantly between the methods. Stability of RBCs frozen in glycerol were better (94.58%) than RBCs frozen in HES (80.75%) measured 24 hours after thawing. ATP is better protected in erythrocytes frozen in glycerol than in HES., Conclusion: Erythrocytes frozen with HES preserved more hemoglobin than with glycerol. Membrane permeability for Na+ and K+ ions was preserved better with HES. HES compared to glycerol offered better protection for erythrocytes.

Background: Studies are currently under way examining whether the age of stored red blood cells (RBCs) affects clinical outcome in transfusion recipients. The effects of storage duration on the RBC storage lesion are well documented, while fewer studies are available regarding the effect of RBC production method. In this study, we compared in vitro RBC quality variables and markers of inflammatory response in apheresis and whole blood (WB)-derived RBCs, specifically those prepared after an overnight room temperature hold (RTH) of WB., Study Design and Methods: SAGM RBCs, prepared from WB after overnight RTH (n = 10), were compared to SAGM RBCs prepared using an apheresis device (Alyx, n = 10). As a control, SAGM RBCs were also prepared within 2 hours of WB collection (2-hr WB, n = 10). All RBCs were stored at 4°C for 42 days with weekly assay of in vitro variables, cytokines and/or chemokines, and neutrophil activation after incubation with RBC supernatant., Results: RTH WB RBCs exhibited decreased levels of 2,3-diphosphoglycerate acid (2.3 μmol/g hemoglobin [Hb] ± 2.1 vs. 13.7 ± 1.3 μmol/g Hb) and morphology (160 ± 10 vs. 192 ± 5) on Day 1 and increased hemolysis (0.45 ± 0.21% vs. 0.31 ± 0.09%) and microparticles (6.1 ± 2.8/10(3) RBCs vs. 3.9 ± 1.1/10(3) RBCs) on Day 42 compared to apheresis RBCs. Gro-α and ENA-78 cytokine levels were significantly higher in RTH WB than Alyx RBCs during storage. CD11b expression was highest in neutrophils exposed to supernatant from RTH WB RBCs (p < 0.05)., Conclusion: RBC preparation method has a meaningful effect on the RBC storage lesion, which should be taken into account in addition to length of storage., (© 2013 American Association of Blood Banks.)

Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J. Mol. Biol. 2013, 425, 647-661), now applicable to systems with multiple coupled binding sites, such as hemoglobin. First, we predict the relative stabilities of substates and microstates of hemoglobin, which are determined primarily by entropy within our model. Next, we predict the impact of 866 annotated mutations on hemoglobin's oxygen binding equilibrium. We then discuss a subset of 30 mutations that occur in the presence of the sickle cell mutation and whose effects on polymerization have been measured. Seven of these HbS mutations occur in three predicted druggable binding pockets that might be exploited to directly inhibit polymerization; one of these binding pockets is not apparent in the crystal structure, but only in structures generated by AllosMod. For the 30 mutations, we predict that mutation-induced conformational changes within a single tetramer tend not to significantly impact polymerization; instead, these mutations more likely impact polymerization by directly perturbing a polymerization interface. Finally, our analysis of allostery allows us to hypothesize why hemoglobin evolved to have multiple subunits and a persistent low frequency sickle cell mutation.

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb-NO). Many aspects of the formation and persistence of Hb-NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O2. Partially nitrosylated Hb samples had predominantly hexacoordinate NO-heme geometry and resisted oxidation when exposed to O2 in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO-heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO-heme geometry also affected O2 binding equilibria. O2-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O2 affinity T-state of the Hb by increasing percentages of NO-heme, much as occurs with increasing levels of CO-heme. Samples containing IHP showed small decreases in O2 affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O2-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO-heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O2 transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Analysis of metabolic changes in rat erythrocytes against the background of long-term simvastatin treatment revealed an increase in glycolysis metabolite concentrations and glucose-6-phosphate dehydrogenase activity and oppositely directed shifts in antioxidant defense enzyme activities. It was demonstrated that hypoxia and imbalance in the antioxidant enzyme system contribute to the development of side effects of statins.