Biopreservation and Reversal of Oxidative Injury During Blood Storage by a Novel Curcumin-based Gel Formulation.

Blood storage lesion induces cytosolic and membrane changes driven in part by hemoglobin (Hb) oxidation reactions within red blood cells (RBCs). A novel gel formulation containing the antioxidant curcuminoids in a biocompatible solvent system was used to deliver curcumin into RBCs. Incubation of peroxide treated RBCs stored in PBS with curcumin gel led to a reduction in prooxidant ferrylHb and recovery in ATP. Curcumin treatment prevented band 3 tyrosine (Y359 and Y21) phosphorylation. RBCs stored in AS-3 solutions for 28, 35, 42 and 49 days, following a single-dose of 100µM curcuminoids at each time points, caused reduction in protein carbonylation and considerable recovery in ATP levels. Proteomic analysis revealed minimal changes in the proteomic landscape in 35 days. However, a downregulation in fibrinogen was observed in the treated samples which may reduce RBC aggregation. Additionally, we used a guinea pig model where the circulation of infused aged RBCs can be extended (approximately 10%) when treated with curcumin gel at the start of storage. Our data therefore provide mechanistic insights and supportive animal data into benefits of treating stored RBCs with a novel curcuminoid formulation based on the biopreservation of RBC membrane integrity, redox balance, and increased longevity in circulation.

Changes in hemoglobin oxidation and band 3 during blood storage impact oxygen sensing and mitochondrial bioenergetic pathways in the human pulmonary arterial endothelial cell model.

Red blood cells (RBCs) undergo metabolic, oxidative, and physiological changes during storage, collectively described as the "storage lesion." The impact of storage on oxygen homeostasis, following transfusion, is not fully understood. We show that RBC storage induces changes in oxygen binding that were linked to changes in oxygen sensing (hypoxia-inducible factor, HIF-1α) mechanisms and mitochondrial respiration in human pulmonary arterial endothelial cells (HPAECs). A decrease in oxygen affinity (P50) to approximately 20 from 30 mmHg was seen at the first week but remained unchanged for up to 42 days. This led to the suppression of HIF-1α in the first 3 weeks due to limited oxygen supplies by RBCs. Furthermore, membrane oxidative damage, band 3 alterations, and subsequent microparticle (MP) formation were also noted. Mass spectrometric analysis revealed the upregulation of transitional endoplasmic reticulum ATPase, essential for clearing ROS-damaged membrane proteins and the protein DDI1 homolog, a proteasomal shuttle chaperone. Band 3 complex proteins and superoxide dismutase were among the downregulated proteins. Mitochondrial oxygen consumption rates measured in HPAECs incubated with RBC-derived MPs (14-day and 42-day) showed a rise in maximal respiration. Intervention strategies that target intracellular hemoglobin (Hb)'s redox transitions and membrane changes may lead to the reestablishment of oxygen homeostasis in old RBCs.

Antimicrobial 405 nm violet-blue light treatment of ex vivo human platelets leads to mitochondrial metabolic reprogramming and potential alteration of Phospho-proteome.

Continued efforts to reduce the risk of transfusion-transmitted infections (TTIs) through blood and blood components led to the development of ultraviolet (UV) light irradiation technologies known as pathogen reduction technologies (PRT) to enhance blood safety. While these PRTs demonstrate germicidal efficiency, it is generally accepted that these photoinactivation techniques have limitations as they employ treatment conditions shown to compromise the quality of the blood components. During ex vivo storage, platelets having mitochondria for energy production suffer most from the consequences of UV irradiation. Recently, application of visible violet-blue light in the 400-470 nm wavelength range has been identified as a relatively more compatible alternative to UV light. Hence, in this report, we evaluated 405 nm light-treated platelets to assess alterations in energy utilization by measuring different mitochondrial bioenergetic parameters, glycolytic flux, and reactive oxygen species (ROS). Furthermore, we employed untargeted data-independent acquisition mass spectrometry to characterize platelet proteomic differences in protein regulation after the light treatment. Overall, our analyses demonstrate that ex vivo treatment of human platelets with antimicrobial 405 nm violet-blue light leads to mitochondrial metabolic reprogramming to survive the treatment, and alters a fraction of platelet proteome.

Oxidation reactions of cellular and acellular hemoglobins: Implications for human health.

Oxygen reversibly binds to the redox active iron, a transition metal in human Hemoglobin (Hb), which subsequently undergoes oxidation in air. This process is akin to iron rusting in non-biological systems. This results in the formation of non-oxygen carrying methemoglobin (ferric) (Fe3+) and reactive oxygen species (ROS). In circulating red blood cells (RBCs), Hb remains largely in the ferrous functional form (HbF2+) throughout the RBC's lifespan due to the presence of effective enzymatic and non-enzymatic proteins that keep the levels of metHb to a minimum (1%-3%). In biological systems Hb is viewed as a Fenton reagent where oxidative toxicity is attributed to the formation of a highly reactive hydroxyl radical (OH•) generated by the reaction between Hb's iron (Fe2+) and hydrogen peroxide (H2O2). However, recent research on both cellular and acellular Hbs revealed that the protein engages in enzymatic-like activity when challenged with H2O2, resulting in the formation of a highly reactive ferryl heme (Fe4+) that can target other biological molecules before it self-destructs. Accumulating evidence from several in vitro and in vivo studies are summarized in this review to show that Hb's pseudoperoxidase activity is physiologically more dominant than the Fenton reaction and it plays a pivotal role in the pathophysiology of several blood disorders, storage lesions associated with old blood, and in the toxicity associated with the infusion of Hb-derived oxygen therapeutics.

Hemoglobin Oxidation Reactions in Stored Blood.

Hemoglobin (Hb) inside and outside the red blood cells (RBCs) undergoes constant transformation to an oxidized form in a process known as autoxidation. The ferrous heme iron (Fe2+) of the prosthetic group is spontaneously transformed into an oxidized ferric (Fe3+) form, but under oxidative stress conditions a higher oxidation ferryl heme (Fe4+) is also formed. Although Fe3+ is a non-functional form of Hb, the Fe4+ is also extremely reactive towards other biological molecules due to its high redox potential. The RBC contains an effective reductive machinery that maintains Hb in the functional form with little oxidation during its life span. The redox transformation of Hb occurs to a lesser extent in young RBCs; it may, however, have detrimental effects on the integrity of these cells during ex vivo storage or when RBCs are subjected to pathogen reduction processes. In this review, Hb oxidation reactions ("oxidative lesion") will be described, including details of how these reactions might impact the clinical use of stored or processed blood for therapeutic purposes.

Mitapivat increases ATP and decreases oxidative stress and erythrocyte mitochondria retention in a SCD mouse model.

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.

HIF-1α-Dependent Metabolic Reprogramming, Oxidative Stress, and Bioenergetic Dysfunction in SARS-CoV-2-Infected Hamsters.

The mechanistic interplay between SARS-CoV-2 infection, inflammation, and oxygen homeostasis is not well defined. Here, we show that the hypoxia-inducible factor (HIF-1α) transcriptional pathway is activated, perhaps due to a lack of oxygen or an accumulation of mitochondrial reactive oxygen species (ROS) in the lungs of adult Syrian hamsters infected with SARS-CoV-2. Prominent nuclear localization of HIF-1α and increased expression of HIF-1α target proteins, including glucose transporter 1 (Glut1), lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase-1 (PDK1), were observed in areas of lung consolidation filled with infiltrating monocytes/macrophages. Upregulation of these HIF-1α target proteins was accompanied by a rise in glycolysis as measured by extracellular acidification rate (ECAR) in lung homogenates. A concomitant reduction in mitochondrial respiration was also observed as indicated by a partial loss of oxygen consumption rates (OCR) in isolated mitochondrial fractions of SARS-CoV-2-infected hamster lungs. Proteomic analysis further revealed specific deficits in the mitochondrial ATP synthase (Atp5a1) within complex V and in the ATP/ADP translocase (Slc25a4). The activation of HIF-1α in inflammatory macrophages may also drive proinflammatory cytokine production and complement activation and oxidative stress in infected lungs. Together, these findings support a role for HIF-1α as a central mediator of the metabolic reprogramming, inflammation, and bioenergetic dysfunction associated with SARS-CoV-2 infection.

The Impact of COVID-19 Infection on Oxygen Homeostasis: A Molecular Perspective.

The novel coronavirus (2019-nCoV/SARS-CoV-2) causes respiratory symptoms including a substantial pulmonary dysfunction with worsening arterial hypoxemia (low blood oxygenation), eventually leading to acute respiratory distress syndrome (ARDS). The impact of the viral infection on blood oxygenation and other elements of oxygen homeostasis, such as oxygen sensing and respiratory mitochondrial mechanisms, are not well understood. As a step toward understanding these mechanisms in the context of COVID-19, recent experiments revealed contradictory data on the impact of COVID-19 infection on red blood cells (RBCs) oxygenation parameters. However, structural protein damage and membrane lipid remodeling in RBCs from COVID-19 patients that may impact RBC function have been reported. Moreover, COVID-19 infection could potentially disrupt one, if not all, of the other major pathways of homeostasis. Understanding the nature of the crosstalk among normal homeostatic pathways; oxygen carrying, oxygen sensing (i.e., hypoxia inducible factor, HIF) proteins, and the mitochondrial respiratory machinery may provide a target for therapeutic interventions.

Caffeic acid: an antioxidant with novel antisickling properties.

It is well documented that caffeic acid (3,4-dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H2 O2 ), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe4+ ). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H2 O2 (0.5-fold over heme), we observed a fivefold reduction in the amount of HbFe4+ accumulated in a mixture of ferric and H2 O2 solution. Higher levels of H2 O2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe4+ , respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5-molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to ßCys93 than to ßCys112 and αCys104 . The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD.

Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells.

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1ß, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.

ßCysteine 93 in human hemoglobin: a gateway to oxidative stability in health and disease.

ßcysteine 93 residue plays a key role in oxygen (O2)-linked conformational changes in the hemoglobin (Hb) molecule. This solvent accessible residue is also a target for binding of thiol reagents that can remotely alter O2 affinity, cooperativity, and Hb's sensitivity to changes in pH. In recent years, ßCys93 was assigned a new physiological role in the transport of nitric oxide (NO) through a process of S-nitrosylation as red blood cells (RBCs) travel from lungs to tissues. ßCys93 is readily and irreversibly oxidized in the presence of a mild oxidant to cysteic acid, which causes destabilization of Hb resulting in improper protein folding and the loss of heme. Under these oxidative conditions, ferryl heme (HbFe4+), a higher oxidation state of Hb is formed together with its protein radical (.HbFe4+). This radical migrates to ßCys93 and interacts with other "hotspot" amino acids that are highly susceptible to oxidative modifications. Oxidized ßCys93 may therefore be used as a biomarker of oxidative stress, reflecting the deterioration of Hb within RBCs intended for transfusion or RBCs from patients with hemoglobinopathies. Site specific mutation of a redox active amino acid(s) to reduce the ferryl heme or direct chemical modifications that can shield ßCys93 have been proposed to improve oxidative resistance of Hb and may offer a protective therapeutic strategy.

The Providence Mutation (ßK82D) in Human Hemoglobin Substantially Reduces ßCysteine 93 Oxidation and Oxidative Stress in Endothelial Cells.

The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe4+) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (ßK82D) mutation in sickle cell Hb (ßE6V/ßK82D) and in a recombinant crosslinked Hb (rHb0.1/ßK82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of ßCys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the ßK82D mutation showed considerably less cysteic acid formation, a byproduct of cysteine irreversible oxidation. Next, we performed a novel study aimed at exploring the impact of introducing ßK82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying ßK82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing ßK82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of ßK82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that ßK82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics.

Effects of α subunit substitutions on the oxidation of ßCys93 and the stability of sickle cell hemoglobin.

The ß subunit substitutions, F41Y and K82D, in sickle cell hemoglobin (Hb) (ßE6 V) provides significant resistance to oxidative stress by shielding ßCys93 from the oxidizing ferryl heme. We evaluated the oxidative resistance of ßCys93 to hydrogen peroxide (H2O2) in α subunit mutations in ßE6 V (at both the putative and lateral contact regions) that included (1) αH20Q/ßE6 V; (2) αH50Q/ßE6 V; (3) αH20Q/H50Q/ßE6 V; (4) αH20R/ßE6 V; and (5) αH20R/H50Q/ßE6 V. Estimation by mass spectrometry of irreversible oxidation of ßCys93 to cysteic acid (CA) was unchanged or moderately increased in the single mutants harboring a H20Q or H50Q substitution when compared to control (ßE6 V). The introduction of Arg (R) singularly or in combination with Q enhanced the pseudoperoxidative cycle by slightly decreasing the ferryl in favor of ferrous and ferric species after treatment with H2O2. Higher rates for heme loss from the ferric forms of the Q species to the receptor high affinity recombinant apomyglobin were observed in contrast to the R mutants and control. Because of their improved solubility, a combination of Q and R substitutions together with mutations carrying redox active variants (F41Y/K82D) may provide dual antioxidant and antisickling targets in the design of gene therapy-based candidates.

Post-translational modification as a response to cellular stress induced by hemoglobin oxidation in sickle cell disease.

Intracellular oxidative stress and oxidative modification of sickle hemoglobin (HbS) play a role in sickle cell disease (SCD) pathogenesis. Recently, we reported that Hb-dependent oxidative stress induced post-translational modifications (PTMs) of Hb and red blood cell (RBC) membrane proteins of transgenic SCD mice. To identify the mechanistic basis of these protein modifications, we followed in vitro oxidative changes occurring in intracellular Hb obtained from RBCs and RBC-derived microparticles (MPs) from the blood of 23 SCD patients (HbSS) of which 11 were on, and 12, off hydroxyurea (HU) treatment, and 5 ethnic matched controls. We used mass spectrometry-based proteomics to characterize these oxidative PTMs on a cross-sectional group of these patients (n = 4) and a separate subgroup of patients (n = 2) studied prior to initiation and during HU treatment. Collectively, these data indicated that band-3 and its interaction network involved in MPs formation exhibited more protein phosphorylation and ubiquitination in SCD patients than in controls. HU treatment reversed these oxidative PTMs back to level observed in controls. These PTMs were also confirmed using orthogonal immunoprecipitation experiments. Moreover, we observed specific markers reflective of oxidative stress, including irreversible oxidation of ßCys93 and ubiquitination of Hb ßLys145 (and ßLys96). Overall, these studies strongly suggest that extensive erythrocyte membrane protein phosphorylation and ubiquitination are involved in SCD pathogenesis and provide further insight into the multifaceted effects of HU treatment.

Antisickling Drugs Targeting ßCys93 Reduce Iron Oxidation and Oxidative Changes in Sickle Cell Hemoglobin.

Sickle cell disease is a genetic blood disorder caused by a single point mutation in the ß globin gene where glutamic acid is replaced by valine at the sixth position of the ß chain of hemoglobin (Hb). At low oxygen tension, the polymerization of deoxyHbS into fibers occurs in red blood cells (RBCs) leading to an impaired blood vessel transit. Sickle cell hemoglobin (HbS), when oxidized with hydrogen peroxide (H2O2), stays longer in a highly oxidizing ferryl (Fe4+) form causing irreversible oxidation of ßCys93 to a destabilizing cysteic acid. We have previously reported that an antisickling drug can be designed to bind specifically to ßCys93 and effectively protect against its irreversible oxidation by H2O2. Here, we report oxygen dissociation, oxidation, and polymerization kinetic reactions for four antisickling drugs (under different preclinical/clinical developmental stages) that either site-specifically target ßCys93 or other sites on the HbS molecule. Molecules that specifically bind to or modify ßCys93, such as 4,4'-di(1,2,3-triazolyl) disulfide (TD-3) and hydroxyurea (HU) were contrasted with molecules that target other sites on Hb including 5-hydroxymethyl-2-furfural (5-HMF) and L-glutamine. All reagents induced a left shift in the oxygen dissociation curve (ODC) except L-glutamine. In the presence of H2O2 (2.5:1, H2O2:heme), both TD-3 and HU reduced the ferryl heme by 22 and 37%, respectively, which corresponded to a 3- to 2-fold reduction in the levels of ßCys93 oxidation as verified by mass spectrometry. Increases in the delay times prior to polymerization of HbS under hypoxia were in the following order: TD-3 > HU > 5-HMF = L-glutamine. Designing antisickling agents that can specifically target ßCys93 may provide a dual antioxidant and antisickling therapeutic benefits in treating this disease.

Redox states of hemoglobin determine left ventricle pressure recovery and activity of mitochondrial complex IV in hypoxic rat hearts.

Cardiovascular effects were reported to occur in humans and in animal models during transfusion with hemoglobin (Hb)-based oxygen therapeutics. The effects of Hb's iron redox states on cardiac parameters during hypoxia/reoxygenation are however poorly defined. We hypothesize that acute exposures to ferric Hb during hypoxia leads to cardiomyocyte injury and an impaired left ventricular response accompanied by cardiac mitochondrial bioenergetic dysfunction. Recovery of left ventricular functions in an isolated rat heart Langendorff perfusion system was observed following perfusion with ferrous but not with ferric Hb. Ferric Hb induced the development of heart lesions, and impairment of the respiratory chain complex activity. Under normoxia, a sharp decline in cardiac parameters was observed following co-perfusion of low (20 µM) and high (100 µM) ascorbic acid (Asc) with ferrous Hb. This trend continued with ferric Hb co-perfusion, but only at the higher concentration of Asc. These observations suggest that perfusion of the hypoxic heart with ferric Hb increases oxidative stress thereby resulting in cardiac dysfunction. Intervention with Asc to reduce ferric Hb may offer a strategy to control Hb toxicity; however, timing of administration, and dosage of Asc may require individual optimization to target specific redox forms of Hb.

Interactions of an Anti-Sickling Drug with Hemoglobin in Red Blood Cells from a Patient with Sickle Cell Anemia.

The pathophysiology associated with sickle cell disease (SCD) includes hemolytic anemia, vaso-occlusive events, and ultimately end organ damage set off by the polymerization of deoxygenated hemoglobin S (HbS) into long fibers and sickling of red blood cells (RBCs). One approach toward mitigating HbS polymerization is to pharmacologically stabilize the oxygenated (R) conformation of HbS and thereby reduce sickling frequency and SCD pathology. GBT440 is an α-subunit-specific modifying agent that has recently been reported to increase HbS oxygen binding affinity and consequently delay in vitro polymerization. In addition, animal model studies have demonstrated the potential for GBT440 to be a suitable therapeutic for daily oral dosing in humans. Here, we report an optimized method for detecting GBT440 intermediates in human patient hemolysate using a combination of HPLC and mass spectrometry analysis. First, oxygen dissociation curves (ODCs) analyzed from patient blood showed that oxygen affinity increased in a dose dependent manner. Second, HPLC and integrated mass spectrometric analysis collectively confirmed that GBT440 labeling was specific to the α N-terminus thereby ruling out other potential ligand binding sites. Finally, the results from this optimized analytical approach allowed us to detect a stable α-specific GBT440 adduct in the patient's hemolysate in a dose dependent manner. The results and methods presented in this report could therefore potentially help therapeutic monitoring of GBT440 induced oxygen affinity and reveal critical insight into the biophysical properties of GBT440 Hb complexes.