High O2 Affinity Research Articles

Convergent High O2 Affinity but Distinct ATP-Mediated Allosteric Regulation of Hemoglobins in Oviparous and Viviparous Eremias Lizards from the Qinghai-Tibet Plateau.

The functional adaptation and underlying molecular mechanisms of hemoglobins (Hbs) have primarily concentrated on mammals and birds, with few reports on reptiles. This study aimed to investigate the convergent and species-specific high-altitude adaptation mechanisms of Hbs in two Eremias lizards from the Qinghai-Tibet Plateau. The Hbs of high-altitude E. argus and E. multiocellata were characterized by significantly high overall and intrinsic Hb-O2 affinity compared to their low-altitude populations. Despite the similarly low Cl- sensitivities, the Hbs of high-altitude E. argus exhibited higher ATP sensitivity and ATP-dependent Bohr effects than that of E. multiocellata, which could facilitate O2 unloading in respiring tissues. Eremias lizards Hbs exhibited similarly low temperature sensitivities and relatively high Bohr effects at lower temperatures, which could help to stably deliver and release O2 to cold extremities at low temperatures. The oxygenation properties of Hbs in high-altitude populations might be attributed to varying ratios of β2/β1 globin and substitutions on the β2-type globin. Notably, the Asn12Ala in lowland E. argus could cause localized destabilization of the E-helix in the tetrameric Hb by elimination of hydrogen bonds, thereby resulting in its lowest O2 affinity. This study provides a valuable reference for the high-altitude adaptation mechanisms of hemoglobins in reptiles.

Effect of autologous umbilical cord blood transfusion in the development of retinopathy of prematurity: randomized clinical trial - study protocol.

Currently, the treatment of anemia in preterm infants is based on packed red blood cell (RBC) transfusions from adult donors. Oxygen (O2) is mainly transported to the tissues bound to hemoglobin (Hb). In extremely low gestational age neonates (ELGANs), fetal hemoglobin (HbF), which has a higher affinity for O2, represents up to 95% of circulating hemoglobin. During the first month of life, the majority of ELGANs will require an adult-donor RBC transfusion causing HbF levels to rapidly drop. HbA releases 50% more oxygen in peripheral tissues than HbF. Increased release of O2 in the retina is one of the main factors related to the development of retinopathy of prematurity (ROP). Collecting umbilical cord blood and using autologous umbilical cord whole blood (UCB) transfusions would contribute to maintaining physiological HbF concentrations in newborns and avoid oxygen-in-excess derived damage. This is a randomized, double-blinded, multicenter clinical trial. ELGANs ≤28 weeks of gestational age will be randomized 1:1 to receive an autologous umbilical cord blood transfusion (intervention arm) or standard transfusion of packed RBC from an adult donor (control arm) to assess ROP development. Assuming a 50% reduction in ROP incidence, 134 patients (67 per group) will be recruited. When blood transfusion is indicated, the Blook Bank will supply UCB or RCB according to the patient's group. The primary endpoint is the incidence of any ROP. Secondary endpoints are assessessment of treatment safety, results of biomarkers related to ROP and its chronology, and urine oxidative stress markers. In addition, the cellular composition of umbilical cord blood and its relationship with prematurity-related pathologies will be analyzed. All patients will be followed-up to 24 months of corrected age to evaluate their neurodevelopment. ROP is a major cause of irreversible blindness in preterm newborns. Transfusions with adult donor blood can lead to complications, including ROP. UCB transfusions offer advantages by maintaining physiological HbF levels and potentially optimizing postnatal development. Moreover, autologous UCB transfusion could reduce risks associated with heterologous blood products, although volume collection remains challenging. UCB contains growth factors and progenitor cells that may impact ROP.

Metalloporphycene is an Attractive Cofactor for Hemoproteins

Abstract Porphycene, a constitutional isomer of porphyrin, is an attractive ligand, and its metal complexes have been investigated as alternative metal cofactors for hemoproteins such as myoglobin. Iron, cobalt and manganese complexes of porphycene are smoothly inserted into apomyoglobin after removal of native heme b, resulting in stable reconstituted proteins. Myoglobin reconstituted with iron porphycene exhibits extremely high O2 affinity compared to native myoglobin (nMb). In addition, the reconstituted protein also shows catalytic activity toward one-electron oxidation of phenol derivatives and sulfoxidation of thioanisole, although the natural function of nMb is O2 storage. Furthermore, myoglobin reconstituted with manganese porphycene can promote H2O2-dependent hydroxylation of inert alkane species as seen with cytochrome P450s. Myoglobin reconstituted with iron porphycene can act as a catalyst for abiological reactions such as cyclopropanation with ethyl diazoacetate. These results clearly indicate that replacement of heme with metalloporphycenes can dramatically alter the function of hemoproteins.

Structural and dynamic characterization of the hexa-coordinated globin from Spisula solidissima

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where ‘nerve hemoglobins’ (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb ‘classic’ globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.-

Quantitative assessment of the in-vitro binding kinetics of antisickling aromatic aldehydes with hemoglobin A: A universal HPLC-UV/Vis method to quantitate Schiff-base adduct formation

Aromatic aldehydes act as allosteric effectors of hemoglobin (AEH), forming Schiff-base adducts with the protein to increase its oxygen (O2) affinity; a desirable property in sickle cell disease (SCD) treatment, as the high-O2 affinity hemoglobin (Hb) does not polymerize and subsequently prevents erythrocytes sickling. This study reports the development, validation, and application of a weak cation-exchange HPLC assay – quantifying the appearance of Hb-AEH adduct – as a “universal” method, allowing for the prioritization of AEH candidates through an understanding of their Hb binding affinity and kinetics. Concentration- and time-dependent Hb binding profiles of ten AEHs were determined with HPLC, followed by the appropriate non-linear modeling to characterize their steady-state binding affinity (KDss), and binding kinetics second-order association (kon) and first-order dissociation (koff) rate constants. Vanillin-derived AEHs exhibited enhanced binding affinity to Hb, primarily due to their faster kon. Across AEH, kon and koff values are strongly correlated (r = 0.993, n = 7), suggesting that modifications of the AEH scaffold enhanced their interactions with Hb as intended, but inadvertently increased their Hb-AEH adduct dissociation. To our knowledge, the present study is the first to provide valuable insight into Hb binding kinetics of antisickling aromatic aldehydes, and the assay will be a useful platform in screening/prioritizing drug candidates for SCD treatment.

Three Haemoglobin Variants for Congenital Erythrocytosis and Sickle Cell Disease HbSC

The inherited haemoglobin disorders and the clinical laboratory is essential in the diagnosis. Phlebotomy is the therapeutic procedure indicated to reduce blood viscosity and decreasing the risk of thrombotic events in the haemoglobin variants with high affinity for O2 or vaso-occlusive crises in Sickle Cell Disease (SCD) with double heterozygosity HbSC. We describe how therapeutic bleeding remains the treatment in two variant haemoglobinopathies, Haemoglobin Regina and the Haemoglobin Trollhattan, reported for the first time in the Italian population and a double heterozygosity HbSC. In patients with high haematocrit level when other secondary conditions of polyglobulia are excluded, a High-Performance Liquid Chromatography (HPLC) analysis for haemoglobin study should be suggested. Also in children with erythrocytosis frequently, such as the finding of Haemoglobin Okazaky.

On the Origin of Hemoglobin Cooperativity under Non-equilibrium Conditions.

Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve, which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the oxygen dissociation curve of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of red blood cells through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium oxygen dissociation curve and show that under these conditions the true nature of hemoglobin cooperativity is revealed as emerging solely from the consecutive binding of oxygen to each one of the four subunits of hemoglobin until the entire tetramer is saturated. We call this form of cooperativity the sequential cooperativity of hemoglobin and define the simplest model that includes it as the minimalist model of hemoglobin. A single instantiation of this model accounts for ~70% of hemoglobin cooperativity under non-equilibrium conditions. The total cooperativity of hemoglobin can be viewed more correctly as the summation of two instantiations of the minimalist model (each one corresponding to a tetramer of low and high affinity for O2, respectively) in equilibrium with each other, as in the Monod-Wyman-Changeux model of hemoglobin. In addition to offering new insights on the nature of hemoglobin reaction with oxygen, the methodology described here for the determination of hemoglobin non-equilibrium oxygen dissociation curve provides a simple, fast, low-cost alternative to complex spectrophotometric methods, which is expected to be particularly valuable in regions where hemoglobinopathies are a significant public health problem, but where highly specialized laboratories capable of determining a traditional oxygen dissociation curve are not easily accessible.

Safety profile and efficacy of low and high oxygen affinity polymerized human hemoglobin in trauma model

Hemoglobin (Hb)‐based O2 carriers (HBOC) are being developed as alternatives to blood. Large molecular weight polymerized human Hb (PolyhHb) represents a new class of HBOC with promising results. It has been shown that PolyhHb can be locked in the T‐state (low O2 affinity) or R‐state (high O2 affinity) to control and target O2 delivery and avoid damage. The present study hypothesized that PolyhHb’s quaternary state could differently affect safety and hemodynamics restoration, by controlling O2 distribution. Thus, we aimed to compare PolyhHb T‐state and R‐state to restore hemodynamics after trauma, and toxicity 24 hrs after resuscitation. To test the efficacy to restore hemodynamics, Wistar rats were instrumented with catheters in the femoral artery and vein, and subjected to TBI through a pneumatic controlled cortical impact. Hemorrhagic Shock (HS) was induced by blood withdrawal to achieve a mean arterial pressure (MAP) between 35‐40mmHg, and the hypotensive state was maintained for 90 min. Animals were separated into 3 groups based on the resuscitation solution: Blood, PolyhHb T‐state, or R‐state and, followed for 2 hrs after resuscitation. For toxicity evaluation guinea pigs (GP) (a more clinically relevant model due to lack of ascorbic acid production) were anesthetized and the carotid artery and jugular vein were catheterized, HS was induced by 40 % blood volume (BV) withdrawal, and the hypovolemic state was maintained for 50 min. GP was divided into 2 groups, and 25% of their BV was reinfused with PolyhHb T‐state or R‐state. Rats resuscitated with Blood and T‐state had a higher increase in MAP 30 min after resuscitation when compared to R‐state. Showing higher efficacy of T‐state to restore hemodynamics compared to R‐state. The T‐state group showed lower O2 saturation 30 min after reperfusion compared to R‐state and Blood groups. However, 2 hrs after resuscitation both the Blood and T‐state groups presented lower O2 saturation compared to R‐state. Resuscitation with R‐state in the GP increased AST, ALT, and liver chemokine ligand‐1 (CXCL1) (markers of liver damage and inflammation), suggesting that R‐state caused greater liver damage compared to T‐state. It was also observed an increase in the BUN, urine NGAL, and serum creatinine in the R‐state group compared to the T‐state group, showing an increased kidney injury in the animals resuscitated with R‐State PolyhHb. Also was observed higher levels of serum Il‐6, CXCL‐1, and Il‐10, in the R‐state group compared to the T‐State group, showing elevated inflammation in this group. Finally, was observed by increased levels of cardiac IL‐6, tumor necrosis factor‐alpha, monocyte chemoattractant protein‐1, troponin, and c‐reactive protein, higher cardiac damage in the R‐sate group when compared to the T‐state group. Our results showed that T‐state has higher efficacy on restoring hemodynamics in a model of TBI followed by HS, and also presented reduced vital organs toxicity in GP, when compared to R‐state. These results are promising and encourage the use of this new generation of PolyhHb T‐state as an alternative when blood is not available.

Cardiac baroreflex sensitivity and heart rate variability are lower in humans with high affinity hemoglobin compared to typical affinity hemoglobin

BackgroundHumans with high O2 affinity hemoglobin (HAH) have a markedly lower partial pressure of O2at which 50% of hemoglobin is saturated (P50) and a leftward shift of the oxy‐hemoglobin dissociation curve. Humans with HAH have attenuated heart rate responses during both acute and chronic hypoxic exposure relative to humans with typical P50. Despite the attenuated heart rate responses to hypoxia, there are typically no blood pressure differences found in humans with HAH relative to humans with typical P50. Due to the blunted heart rate and normal blood pressure responses to hypoxia in humans with HAH, this study aimed to investigate the impact of HAH on cardiac autonomic function during normoxia and hypoxia. We hypothesized that humans with HAH would have lower cardiac baroreflex sensitivity (cBRS) and heart rate variability (HRV) during normoxia and hypoxia relative to individuals with typical P50.MethodsNine humans with a HAH mutation (6 female, Age: 40±12yrs, P50: 16±1 mmHg) and 12 controls with typical affinity hemoglobin (6 female, Age: 40±10yrs, P50: 26±1 mmHg) laid supine for 10‐minutes prior to 20‐minutes of isocapnic hypoxia, titrated to reduce arterial O2 pressure to ~50 mmHg (Avg. FiO2 = 11.9±0.0%). Beat‐to‐beat arterial pressures (arterial catheter) and heart rate (electrocardiogram) were continuously recorded. Data were averaged in five‐minute segments starting with the last five minutes of supine normoxic rest. Spontaneous cBRS was quantified using the sequence method. HRV was measured in time and frequency domains. Standard deviation of the RR interval (SDRR) was calculated as the standard deviation of all RR intervals in a data segment. Low frequency (LF) HRV was calculated using Fast Fourier Transformation and assessed as absolute spectral intensity in the LF band (0.04‐0.15Hz).ResultsArterial oxygen (PaO2) and carbon dioxide (PaCO2) pressures were not different between humans with HAH and controls during normoxia or isocapnic hypoxia [PaO2: (HAH vs. Control): 48±3 vs. 47±2 mmHg, p=0.612; PaCO2: (HAH vs. Control): 40±3 vs. 39±1 mmHg, p=0.328] exposure. cBRS was lower in humans with HAH compared to controls in normoxia and throughout isocapnic hypoxia [normoxia (HAH vs. Control): 7±4 vs. 16±10 ms/mmHg; hypoxia minutes 15‐20 (HAH vs. Control): 4±3 vs. 14±11 ms/mmHg; Hb type, p=0.024]. Several metrics of HRV were lower in humans with HAH relative to control (SDRR: Hb type, p≤0.001; LF: Hb type, p=0.030) and HRV was blunted to a similar extent in both groups with hypoxia (SDRR: time effect, p=0.009).ConclusionThe findings of this study are twofold. Compared to controls, humans with HAH have: 1) attenuated cardiac baroreflex sensitivity and 2) lower heart rate variability in both normoxia and isocapnic hypoxia.

Bimetallic ZIF-derived cobalt nanoparticles anchored on N- and S-codoped porous carbon nanofibers as cathode catalyst for Li-O2 batteries

Metal-organic frameworks (MOFs) have shown great application potential for improving Lithium-oxygen (Li-O2) batteries performance with high O2 affinity and accessible active centers. Herein, a novel type of cobalt nanoparticles embedded in porous nitrogen (N)- and sulfur (S)-codoped carbon nanofiber (Co@N/S-CNF) structure is developed by employing electrospinning technology and bimetallic zeolitic imidazole framework (ZIF-8/ZIF-67, a kind of MOF) precursor with the following annealing and hydrothermal treatment route. When utilized as cathode catalysts for Li-O2 batteries, the Co@N/S-CNF sample exhibits higher discharge capacity (9290.7 mAh g−1 at 50 mA g−1) and better cycling stability (42 cycles at 100 mA g−1 under a curtailing capacity of 500 mAh g−1) compared to Co@N-CNF electrode. The enhanced performance can be attributed to the 1D porous morphology and N/S doping effect, which can effectively improve mass transport, increase exposed active sites, and induce structural defect, thereby accelerating the formation and decomposition progress of discharge products. This simple synthetic strategy may provide a new insight for designing and developing MOFs-based multifunctional catalysts in the field of energy and electrocatalysis.

Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, Alligator mississippiensis.

In the developing embryos of egg-laying vertebrates, O2 flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O2 flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O2 transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O2). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O2 affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O2 affinity and high CO2 sensitivity. These results indicate that whole blood O2 affinity is mainly regulated by ATP in the early embryo and by CO2 and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

Thromboembolic and Vascular Complications in Patients with Secondary Erythrocytosis Caused By High Oxygen Affinity Variant Hb Coimbra

There are 99 hemoglobin (Hb) variants with increased oxygen affinity inducing compensatory erythrocytosis (Globin Gene Server Web Site: http://globin.cse.psu.edu). One of these mutations, named Hb Coimbra, was described in 1991 in Portugal by Tamagnini et al. (HBB c.300T>A, p.Asp100Glu). Limited amount of published clinical and laboratory data of this condition and the absence of specific guidelines for its management suggest it would be a very benign condition. Here we report clinical and laboratorial findings of carriers of Hb Coimbra with some unexpectedly more severe clinical presentations.We collected peripheral blood venous samples from 10 patients (6 female), aged 31-72 years, upon signed informed consent. All of them were diagnosed with erythrocytosis, with hematocrits in the 46.9-78.1% range at the time of diagnosis, and were heterozygous for the Hb Coimbra mutation as confirmed by direct sequencing of the β-globin gene. The β-globin cluster haplotypes were determined by TaqMan SNP Genotyping Assay (rs7482144, rs968857, rs16911905) and other polymorphisms in the β-globin gene by direct sequencing (rs713040, rs10768683, rs7480526, rs7946748). We were able to identify a common haplotype for the Hb Coimbra allele, which strongly suggests a common origin despite the patients belonging to 5 different unrelated families.Three patients had complications possibly associated with the disease. Patient 1, male, 64 years old, presented with pulmonary embolism in July 2012. Patient 2, male, 60 years old, suffered a left lower limb amputation due to complicated venous thromboembolism in 2010 before he was diagnosed with Hb Coimbra and, in January of 2015, he also developed right pulmonary artery thromboembolism. Patient 3, male, 72 years old, presented with left hemiparesis in 2016 due to intraparenchymatous hemorrhage in the right basal ganglia.Differently from the other patients, elevated levels of serum erythropoietin were observed only in patients 1, 2, and 3 (81.1 mUI/mL, 423.5 mUI/mL, and 35.4 mUI/mL, respectively, reference range 2.6-18.5 mUI/mL). We speculate this abnormality could be associated with the therapeutic phlebotomy treatment performed in these cases.Oxygen affinity was evaluated by determining the p50 in whole blood samples without Hb isolation. O2 dissociation and saturation curves demonstrated that samples containing Hb Coimbra had considerably high affinity for oxygen in comparison to wild type (p50deoxi and p50oxi for Hb Coimbra were, 8.22 [SD 0.64] and 7.94 [SD 0.98], respectively, vs. wild type p50deoxi = 15.56 [SD 0.50] and p50oxi = 15.56 [SD 0.77]). Heme-heme cooperativity was also significantly reduced, tending to a non-cooperative event. Hill coefficients were ndeoxi - Hb Coimbra= 1.51 (SD 0.19) and noxi - Hb Coimbra = 1.48 (SD 0.32), ndeoxi - wild type = 2.52 (SD 0.22) and noxi - wild type = 2.41 (SD 0.32). In spite of the very high O2 affinity of Hb Coimbra, no significant difference was observed between samples from patients with clinical complications and from the remaining Hb Coimbra carriers.Alterations in the α1β2 interface, such as the one in Hb Coimbra, promote conformational changes in the structure of the hemoglobin, and tend to promote O2 binding, reducing oxygen supply to peripheral tissues and resulting in compensatory erythrocytosis (Wajcman et al, 2005). Mutations that generate hemoglobin variants are very rare, but should be considered in the context of familial erythrocytosis or in cases in which more frequent hematological diseases such as polycythemia vera have been excluded.In general, hemoglobin variants with high oxygen affinity manifest exclusively with erythrocytosis and a benign clinical course; however, our data suggest that this might not be the case in some patients, particularly in carriers of Hb Coimbra. Our experience supports that some patients may benefit from the prophylactic use of aspirin or therapeutic phlebotomy as attempts to reduce the incidence of thromboembolic or other vascular complications.Taken together, our data underscore that larger and multicentric studies of the clinical complications secondary to high oxygen affinity hemoglobins are necessary to define guidelines for the optimal management of this type of erythrocytosis. DisclosuresFertrin:Alexion Pharmaceuticals: Consultancy.

MetAP2 inhibition modifies hemoglobin S to delay polymerization and improves blood flow in sickle cell disease

Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and βS-globin. Kinetic studies with MetAP2 show that βS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood–derived erythroid progenitor 2 cells shows more extensive modification of α-globin than β-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on βS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor–treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2–affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.

A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

Safety profile of high molecular weight polymerized hemoglobins

Hemoglobin (Hb)-based oxygen (O2 ) carriers (HBOCs) are being developed as alternatives to red blood cells and blood when these products are unavailable. Clinical trials of previous HBOC generations revealed side effects, including hypertension and vasoconstriction, that were not observed in preclinical studies. Large molecular weight (MW) polymerized bovine Hb (PolybHb) represents a new class of HBOC with promising results. We evaluated the safety profile of PolybHb after an exchange transfusion (ET) in guinea pigs (GPs). This study compares changes in indices of cardiac, inflammatory, and organ function after ET with high (R-state) and low (T-state) O2 affinity PolybHb with high MW. Guinea pigs underwent a 20% ET with PolybHb. To assess the implication of PolybHb ET on the microcirculation, hamsters instrumented with a dorsal window chamber were subjected to a similar volume ET. T and R-state PolybHb did not induce significant alterations in cardiac function. T-state PolybHb induced mild vasoconstriction shortly after transfusion, while R-state did not have acute effects on microvascular tone. Large MW PolybHbs were found to be safe and efficacious in increasing O2 carrying capacity and the O2 affinity of the PolybHb did not affect O2 delivery or extraction by tissues in relevant preclinical models. In conclusion, these results suggest that both T-state and R-state PolybHb are safe and do not impair O2 delivery. The results are encouraging and support further evaluation of high MW PolybHbs and their future feasibility compared to allogenic blood in a trauma model.

Polymerized human hemoglobin increases the effectiveness of cisplatin-based chemotherapy in non-small cell lung cancer.

Cisplatin is a promising therapeutic for the treatment of non-small cell lung cancer (NSCLC). Unfortunately, a significant portion of NSCLC patients relapse due to cisplatin chemoresistance. This chemoresistance is thought to be primarily associated with hypoxia in the tumor microenvironment. Administration of hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) is a promising strategy to alleviate hypoxia in the tumor, which may make cisplatin more effective. In this study, we administered a high O2 affinity, relaxed state (R-state) polymerized hemoglobin (PolyHb) to three different NSCLC cell lines cultured in vitro and implanted in vivo into healthy mice. The R-state PolyHb administered in this study is unable to deliver O2 unless under severe hypoxia which significantly limits its oxygenation potential. In vitro sensitivity studies indicate that the administration of PolyHb increases the effectiveness of cisplatin under hypoxic conditions. Additional animal studies revealed that co-administration of PolyHb with cisplatin attenuated tumor growth without alleviating hypoxia. Analysis of reactive O2 species production in the presence of hypoxic culture indicates that exogenous ROS production by oxidized PolyHb may the mechanism of chemosensitization. This ROS mechanism, coupled with oxygenation, may be a potential chemosensitizing strategy for use in NSCLC treatment.

Immediate effects of systemic administration of normal and high O2-affinity haemoglobin vesicles as a transfusion alternative in a rat pneumonectomy model

BackgroundHaemoglobin vesicles (HbVs) are red blood cell (RBC) substitutes with a phospholipid bilayer membrane and a polyethylene modified surface (diameter=250 nm; P50=28 Torr). They can be preserved for years and...

Polymerized Hemoglobin Maintains Cardiac Function After Extreme Anemia

BackgroundBlood transfusion as a treatment for anemia is vital in ensuring oxygen (O2) carrying capacity. However, fears of blood shortages and adverse reactions to stored blood transfusion have driven the development of oxygen carrying alternatives to blood. One class of alternatives to blood for transfusion medicine are hemoglobin (Hb) based oxygen carriers (HBOCs), which have been in development for decades. A recent advancement in the development of HBOCs is the discovery that their O2 affinity can be tightly regulated by locking Hb in the T state (low O2 affinity), or R state (high O2 affinity). By regulating O2 affinity, O2 extraction by tissues can be controlled, and targeted O2 delivery to hypoxic tissues is made possible. As the heart is one of the most sensitive organs to changes in oxygenation due to its high metabolic rate, the objective of this study was to test the efficacy of high and low O2 affinity HBOCs in maintaining cardiac function and systemic O2 delivery during severe anemia.MethodsPolymerized hemoglobin (PolyHb), the most scalable method of producing HBOCs, was synthesized in the high and low O2 affinity state (R and T, respectively) with glutaraldehyde and then subjected to 8–9 cycles of diafiltration as previously described. This resulted in a PolyHb solution containing only polymerized Hb molecules > 500kDa suspended in Lactated Ringers solution. Rats were instrumented with a pressure‐volume catheter to monitor cardiac function. Severe anemia was induced via a 50% isovolumic hemodilution with 5% human serum albumin. Rats were then resuscitated by a 40% isovolumic infusion of a 10 g/dL solution of R PolyHb, T PolyHb, or fresh blood (drawn from the animal during the first exchange).ResultsSevere anemia resulted in impaired cardiac function and O2 delivery. Transfusion of fresh blood, T PolyHb, and R PolyHb restored vascular resistance, blood pressure, and cardiac contractility. T PolyHb also promoted increased O2 extraction and myocardial energy utilization. Severe anemia did not cause significant changes in blood electrolytes relative to baseline. Severe anemia resulted in blood acidification that only recovered for animals resuscitated with T PolyHb. Furthermore, resuscitation with T PolyHb resulted in higher arterial pO2 and lower pCO2 than other groups. R PolyHb increased lactate and glucose, suggesting that the high O2 affinity prevented proper O2 release to some tissues, despite maintaining cardiac function.ConclusionsBoth T and R PolyHb maintain myocardial O2 extraction compared to fresh blood. T PolyHb appears to be the optimal molecule for increasing O2 delivery during anemic conditions. Although R PolyHb apparently decreased systemic O2 extraction during anemia, it can be potentially useful to increase O2 uptake in the lungs during hypoxic‐hypoxia. This study indicates that PolyHb is as efficacious as fresh blood in restoring cardiac function and oxygenation in rats during severe anemia.Support or Funding InformationThis work was supported by the NIH Heart Lung and Blood Institute under Grants T32‐HL105373, R01‐HL126945, and R01‐HL138116.

Structural and functional properties of Antarctic fish cytoglobins-1: Cold-reactivity in multi-ligand reactions

While the functions of the recently discovered cytoglobin, ubiquitously expressed in vertebrate tissues, remain uncertain, Antarctic fish provide unparalleled models to study novel protein traits that may arise from cold adaptation. We report here the spectral, ligand-binding and enzymatic properties (peroxynitrite isomerization, nitrite-reductase activity) of cytoglobin-1 from two Antarctic fish, Chaenocephalus aceratus and Dissostichus mawsoni, and present the crystal structure of D. mawsoni cytoglobin-1. The Antarctic cytoglobins-1 display high O2 affinity, scarcely compatible with an O2-supply role, a slow rate constant for nitrite-reductase activity, and do not catalyze peroxynitrite isomerization. Compared with mesophilic orthologues, the cold-adapted cytoglobins favor binding of exogenous ligands to the hexa-coordinated bis-histidyl species, a trait related to their higher rate constant for distal-His/heme-Fe dissociation relative to human cytoglobin. At the light of a remarkable 3D-structure conservation, the observed differences in ligand-binding kinetics may reflect Antarctic fish cytoglobin-1 specific features in the dynamics of the heme distal region and of protein matrix cavities, suggesting adaptation to functional requirements posed by the cold environment. Taken together, the biochemical and biophysical data presented suggest that in Antarctic fish, as in humans, cytoglobin-1 unlikely plays a role in O2 transport, rather it may be involved in processes such as NO detoxification.

Artificial oxygen carrier improves fatigue resistance in slow muscle but not in fast muscle in a rat in situ model.

The effects of liposome-encapsulated hemoglobin with high O2 affinity (h-LEH), an artificial O2 carrier in skeletal muscle, were studied by in situ fatigue resistance test in fast-type plantaris (PLT) and slow-type soleus (SOL) muscles with or without ischemia in the rat. The distal tendons of PLT and SOL muscles were isolated in situ and individually attached to the force transducers to record the developed tension in response to stimuli (80Hz tetanus train, 1.5minutes) to the ipsilateral sciatic nerve. The fatigue resistance test (five sets separated by 2-minute rests) was evaluated in terms of tension attenuation (fatigue) from the initial to the last tension (A) during each set, attenuation of the initial (B) or last tension (C) in each set, as compared to the first set in the presence or absence of ischemia or h-LEH (10mL/kg). While ischemia significantly enhanced fatigue only in PLT, h-LEH showed no effect regardless of the perfusion pattern (normal/ischemia) or muscle-type (PLT/SOL) during each set (A). In parameter (B), set-by-set fatigue development was observed in PLT, whereas h-LEH-SOL showed a trend of advanced fatigue resistance. Such trends became clear in the parameter C (last tension), because h-LEH-SOL exerted, rather than decreased, the tension enhancement regardless of the presence or absence of ischemia, whereas there were no h-LEH effects in PLT. In addition, faster recovery of the nicotinamide adenine dinucleotide content in the muscle after 10minutes of all fatigue tests was observed in h-LEH-SOL, while saline-SOL still showed a significantly higher value than that of control. These results suggested that additional O2 supply by h-LEH may accelerate the tricarboxylic acid cycle/electron transport chain in slow-type aerobic SOL muscle containing abundant mitochondria and contribute to the faster removal of muscle fatigue substances such as lactate.