Hb-O2 Affinity Research Articles

Effector-Linked High-Frequency Thermal Fluctuations of Globin (Changes in Protein Dynamics) Regulate the Oxyge-Affinity and Cooperativity of Hemoglobin

Diatomic ligands such as O2, CO, and NO, which are dissociating from their binding sites (hemes) of myoglobin (Mb) and hemoglobin (Hb), are tentatively trapped at physical barriers of globin matrix (the “Caged” states) [1-5], before either [A] undergoing bimolecular dissociation to (deoxy state + free ligand) or [B] reverting back to re-bind to the hemes (geminate-recombination) [5]. Majorities of the un-bonded ligands take Route [B], while only small percentages of the un-bonded ligands proceed through Route [A] [5]. This causes substantial (>103 to 104-fold) increases in the apparent ligand-affinity for Mb and Hb, compared with that of free protoheme-N-base complexes [5]. It is well known that binding of heterotropic effectors reduces the O2-affinity of Hb (increases in P50), whereas binding of O2 does not change the P50 value of Hb. Effector-linked enhancements of high-frequency thermal fluctuations of globin [6] increase the transparency of globin matrix toward migrating diatomic ligands to enhance Route [A] and to diminish Route [B], resulted in substantial (up to >103-fold) decrease in the apparent O2-affinity (KT and KR) or (up to 100-fold increases in P50) without detectable changes in static crystallographic structures of Hb as well as the coordination/electronic structures of the hemes in T(deoxy)- and R(oxy)-Hb [5,7,8].

Bohr effect and temperature sensitivity of hemoglobins from highland and lowland deer mice

An important means of physiological adaptation to environmental hypoxia is an increased oxygen (O2) affinity of the hemoglobin (Hb) that can help secure high O2 saturation of arterial blood. However, the trade-off associated with a high Hb–O2 affinity is that it can compromise O2 unloading in the systemic capillaries. High-altitude deer mice (Peromyscus maniculatus) have evolved an increased Hb–O2 affinity relative to lowland conspecifics, but it is not known whether they have also evolved compensatory mechanisms to facilitate O2 unloading to respiring tissues. Here we investigate the effects of pH (Bohr effect) and temperature on the O2-affinity of high- and low-altitude deer mouse Hb variants, as these properties can potentially facilitate O2 unloading to metabolizing tissues.Our experiments revealed that Bohr factors for the high- and low-altitude Hb variants are very similar in spite of the differences in O2-affinity. The Bohr factors of deer mouse Hbs are also comparable to those of other mammalian Hbs. In contrast, the high- and low-altitude variants of deer mouse Hb exhibited similarly low temperature sensitivities that were independent of red blood cell anionic cofactors, suggesting an appreciable endothermic allosteric transition upon oxygenation. In conclusion, high-altitude deer mice have evolved an adaptive increase in Hb–O2 affinity, but this is not associated with compensatory changes in sensitivity to changes in pH or temperature. Instead, it appears that the elevated Hb–O2 affinity in high-altitude deer mice is compensated by an associated increase in the tissue diffusion capacity of O2 (via increased muscle capillarization), which promotes O2 unloading.

Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level.

A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.

GBT440 Demonstrates High Specificity for Red Blood Cells in Nonclinical Species

Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes upon deoxygenation with subsequent formation of sickled red blood cells (RBCs). GBT440 modulates O2 affinity of hemoglobin (Hb) by binding to the N-terminal α chain of Hb via a reversible Schiff base. We previously demonstrated that GBT440 preventedsickling of RBCs from sickle cell patients, in vitro. Also, in a murine model of sickle cell disease (Townes SS mice), GBT440 prevented ex vivo sickling of RBCs and prolonged RBC half-life.Pharmacokinetic (PK) studies of GBT440 were conducted in mouse, rat, dog and monkey following IV and oral administration. Both blood and plasma samples were collected and assayed for GBT440 concentration using LCMS. Following IV and oral administrations, GBT440 quickly partitions into the RBC with a high blood/plasma ratio of ~70:1 which corresponded to a RBC/plasma ratio of ~150:1. Volume of distribution (Vss) was small in whole blood (0.041-0.171 L/kg) but much larger in plasma (1.44-8.45 L/kg) indicating that RBCs are a reservoir of GBT440. Systemic clearance (CLs) was low in both blood (0.016-0.113 mL/min/kg) and plasma (0.943-3.16 mL/min/kg) indicating that GBT440 was mostly bound to hemoglobin and only a small fraction of unbound GBT440 re-distributed into the plasma and was available for clearance. Terminal elimination half-life (t1/2) was similar between whole blood and plasma for each species and was long, ranging from 6.4 hours in mouse plasma to 93.5 hours in dog plasma. GBT440 was well absorbed and absolute oral bioavailability ranged from 33% to 70% in four species.A quantitative whole body autoradiography study to determine tissue distribution of GBT440 was conducted in male rats following an oral dose of 14C-GBT440 (10 mg/kg; 150 µCi/kg PO). The data showed that GBT440 is co-located in hematopoietic tissues as expected for a molecule whose target is hemoglobin, including blood, spleen, liver and bone marrow.A mass balance study of 14C-GBT440 (10 mg/kg; 150 µCi/kg PO) was conducted in rats to determine route of elimination of GBT440. The 14C-GBT440-derived radioactivity was well absorbed and rapidly excreted after oral administration. By 240 hours postdose, mean values of 79.0 ± 3.86 and 9.74 ± 3.02% of the administered radioactivity were excreted in feces and urine, respectively. The mean overall recovery of radioactivity was 92.4 ± 0.875%. Metabolism via both Phase I and Phase II pathways was the major route of elimination of GBT440. These data indicate that despite its high affinity binding toward Hb, GBT440 could be released from the hemoglobin complex and completely eliminated from the body.To further correlate PK to pharmacological activity (hemoglobin modification based on changes in the oxygen equilibrium curve), mice were given an oral dose of 30, 50 and 100 mg/kg and blood were collected at 4 and 6 hr postdose for hemoximetry analysis. Data showed good correlation between blood concentrations and changes in p50. Blood concentrations following 30, 50 and 100 mg/kg at 4 hr were 243, 446, and 806 µM, which resulted in changes in p50 of 11%, 25% and 55%, respectively, indicating that GBT440 elicits an ex vivo dose dependent increase in Hb-O2 affinity following increasing dosage to mice.Based on PK data from 4 animal species, PK profile of GBT440 in human was predicted using a simple allometric scaling technique. The predicted PK profile following an oral administration was highly concordant with actual data from healthy subjects for Cmax, AUC and T½, which suggested that the disposition kinetics of GBT440 in humans were consistent to that in animals.In summary, nonclinical PK studies support previous findings that GBT440 partitions to RBCs, binds specifically to Hb with a slow off rate, modulates Hb-O2 affinity, and is completely eliminated from the body. GBT440 is in clinical trials for the treatment of SCD. DisclosuresHutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Silva:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Consultancy, Equity Ownership.

Abstract 10826: GBT1118, a Potent Allosteric Modifier of Hemoglobin Oxygen Affinity Increases Tolerance to Hypoxia in Mice

Introduction: Adaptation to hypoxia involves several mechanisms that influence oxygen (O2) transport and utilization, including a reduction in hemoglobin (Hb) O2 affinity to favor the release of O2 to tissues. Paradoxically, increased Hb O2 affinity has been observed to have beneficial effects during severe hypoxia. Hypothesis: This study was designed to test the hypothesis that pharmacologically increasing Hb O2 affinity with GBT1118 can augment O2 transport during severe hypoxia (10 and 5% O2). Method: Mice were orally dosed with GBT1118 (70 or 140 mg/kg) or only vehicle (Control), and Hb O2 affinity was determined by hemoximetry. During hypoxia, changes in systemic and microvascular hemodynamics, tissue partial pressure of O2 (PO2) and tissue hypoxia were evaluated. At each timepoint, mice with adequate systolic blood pressure (BP) were counted as survived and mice with severe hypotension (BP ≤60 mmHg) were counted as non-survived and were euthanized. Results: Similar trends were observed at 10 and 5% O2 hypoxia. GBT1118 at 70 and 140 mg/kg decreased the P50 (PO2 at which blood is 50% saturated with O2) by 29 and 35 mmHg, respectively. During 5% O2 hypoxia, GBT1118 at 70 and 140 mg/kg increased SaO2 from 40% (Control) to 57% (70mg/kg) and 80% (140mg/kg), respectively. Moreover, mean BP and heart rate (HR) decreased to 50 mmHg and 375 bpm in control mice, whereas GBT1118-dosed mice sustained higher mean BP and HR in a dose-dependent fashion and improved survival (Figure 1). In addition, GBT1118 preserved microvascular blood flow and tissue oxygenation; consequently, GBT1118 reduced heart and brain hypoxic areas compared with control groups during hypoxia. Conclusion: Under severe hypoxia, GBT1118-mediated increase in Hb O2 affinity increases SaO2. At the tissue level, local milieu allows for efficient O2 extraction leading to improved oxygenation as measured by decreased lactic acidosis, improved cardiovascular function and ultimately, survival.

Contribution of a mutational hot spot to hemoglobin adaptation in high-altitude Andean house wrens

A key question in evolutionary genetics is why certain mutations or certain types of mutation make disproportionate contributions to adaptive phenotypic evolution. In principle, the preferential fixation of particular mutations could stem directly from variation in the underlying rate of mutation to function-altering alleles. However, the influence of mutation bias on the genetic architecture of phenotypic evolution is difficult to evaluate because data on rates of mutation to function-altering alleles are seldom available. Here, we report the discovery that a single point mutation at a highly mutable site in the β(A)-globin gene has contributed to an evolutionary change in hemoglobin (Hb) function in high-altitude Andean house wrens (Troglodytes aedon). Results of experiments on native Hb variants and engineered, recombinant Hb mutants demonstrate that a nonsynonymous mutation at a CpG dinucleotide in the β(A)-globin gene is responsible for an evolved difference in Hb-O2 affinity between high- and low-altitude house wren populations. Moreover, patterns of genomic differentiation between high- and low-altitude populations suggest that altitudinal differentiation in allele frequencies at the causal amino acid polymorphism reflects a history of spatially varying selection. The experimental results highlight the influence of mutation rate on the genetic basis of phenotypic evolution by demonstrating that a large-effect allele at a highly mutable CpG site has promoted physiological differentiation in blood O2 transport capacity between house wren populations that are native to different elevations.

Rapid regulation of blood parameters under acute hypoxia in the Amazonian fish Prochilodus nigricans

Prochilodus nigricans, locally known as curimatã, is an Amazonian commercial fish that endures adverse environmental conditions, in particular low dissolved oxygen, during its migration. Poorer environmental conditions are expected in the near future. Prochilodus nigricans overcomes current seasonal and diurnal changes in dissolved oxygen by adjusting erythrocytic levels of ATP and GTP, modulators of Hb-O2 affinity. Will this fish species be endangered under more extreme environmental conditions as hypoxia and acidification tend to occur in a shorter period of time? As P. nigricans does not exhibit any apparent morphological alterations to exploit the air–water interface, it must rely on fast adjustments of blood properties. To investigate this aspect, basic hematology indices, pHe, pHi, plasma lactate, erythrocytic levels of ATP and GTP and functional properties of the hemolysate of P. nigricans were analyzed over a period of 6h in hypoxia and subsequent recovery in normoxia. The levels of erythrocytic GTP were four times higher than ATP and were reduced to ¼ of the original level after 3h under hypoxia. Erythrocytic levels of ATP were unaffected over the experimental period. All other analyzed blood parameters exhibited a time-course change in animals under hypoxia and returned to normoxic levels. Considering the hemolysate functional properties and the ability to regulate the above mentioned blood characteristics, P. nigricans is able to endure short-term changes in dissolved oxygen.

Intraspecific Polymorphism, Interspecific Divergence, and the Origins of Function-Altering Mutations in Deer Mouse Hemoglobin

Major challenges for illuminating the genetic basis of phenotypic evolution are to identify causative mutations, to quantify their functional effects, to trace their origins as new or preexisting variants, and to assess the manner in which segregating variation is transduced into species differences. Here, we report an experimental analysis of genetic variation in hemoglobin (Hb) function within and among species of Peromyscus mice that are native to different elevations. A multilocus survey of sequence variation in the duplicated HBA and HBB genes in Peromyscus maniculatus revealed that function-altering amino acid variants are widely shared among geographically disparate populations from different elevations, and numerous amino acid polymorphisms are also shared with closely related species. Variation in Hb-O2 affinity within and among populations of P. maniculatus is attributable to numerous amino acid mutations that have individually small effects. One especially surprising feature of the Hb polymorphism in P. maniculatus is that an appreciable fraction of functional standing variation in the two transcriptionally active HBA paralogs is attributable to recurrent gene conversion from a tandemly linked HBA pseudogene. Moreover, transpecific polymorphism in the duplicated HBA genes is not solely attributable to incomplete lineage sorting or introgressive hybridization; instead, it is mainly attributable to recurrent interparalog gene conversion that has occurred independently in different species. Partly as a result of concerted evolution between tandemly duplicated globin genes, the same amino acid changes that contribute to variation in Hb function within P. maniculatus also contribute to divergence in Hb function among different species of Peromyscus. In the case of function-altering Hb mutations in Peromyscus, there is no qualitative or quantitative distinction between segregating variants within species and fixed differences between species.

GTx011, a Potent Allosteric Modifier of Hemoglobin Oxygen Affinity, Prevents RBC Sickling in Whole Blood and Prolongs RBC Half-Life in Vivo in a Murine Model of Sickle Cell Disease

Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life.We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry.In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of <40 mm Hg) evaluated. Moreover, at an O2 tension mimicking typical hypoxic conditions in tissue capillaries (40 mm Hg), 300 µM of GTx011 was sufficient to prevent sickling of human SSRBCs in whole blood (20% Hct). Similarly, ex vivo sickling analysis indicated that, relative to blood from vehicle-treated SS mice, blood from GTx011-treated SS mice showed a pronounced reduction in the number of sickled RBCs under hypoxic conditions with a concurrent reduction in p50. For example, at a pO2 of 10 mm Hg, 19% of SSRBCs in blood from GTx011-treated mice sickled ex vivo compared with 56% in blood from vehicle-treated SS mice.In SS mice chronically dosed with GTx011, a prolongation of the RBC half-life from 2.4 days to 3.8 days was achieved together with a marked decrease in reticulocyte count. This increase in RBC half-life and accompanying reduction in reticulocyte count was observed in mice with GTx011 concentrations in blood that corresponded to >30% calculated Hb target occupancy.Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD.TableSS miceRBC half lifeReticulocytesSickled RBCsHemoximetryChronic treatment, PO, BID, 10-12 days(Days)(%)(% at 10 mm Hg)p20 (mm Hg)p50 (mm Hg)Vehicle-treated2.453561832GBT440-treated (100mg/kg)3.832194.521 DisclosuresDufu:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Zhou:Global Blood Therapeutics: Research Funding. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Archer:Global Blood Therapeutics: Consultancy, Research Funding.

Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Prevents Hypoxia-Induced Tissue Injury in High Altitude By Inducing 2,3-Bisphosphoglycerate

To function effectively in O2 uptake, delivery and release, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)-O2 affinity by allosteric modulators, especially 2,3-bisphophosphoglycerate (2,3-BPG). Earlier studies had shown that elevated erythrocyte 2,3-BPG level is correlated to increased availability of oxygen to tissues in high altitudes. However, nothing is known how 2,3-BPG is induced in high altitude. Recent study revealed that plasma adenosine is increased in patient and mice with sickle cell disease and elevated adenosine signaling via A2B receptor (ADORA2B) induces 2,3-BPG and thereby promotes deoxy-sickle hemoglobin and subsequent sickling. However, whether adenosine is induced and its effect in 2,3-BPG induction in high altitude are unknown. To address this question, we recruited 24 low-land volunteers and placed them in high altitude for different time points. Here we report that: 1) plasma adenosine, a molecule well known to be induced under hypoxia, is increased and its elevation is correlated to elevated erythrocyte 2,3-BPG levels and decreased Hb-O2 affinity; and 2) elevated plasma adenosine is correlated to increased circulating ecto-5’-nucleotidase (CD73), a key enzyme responsible for generation of extracellular adenosine. Similar to humans, increased circulating CD73 activity, plasma adenosine levels and erythrocyte 2,3-BPG and decreased Hb-O2 binding affinity were observed in wild type (WT) mice under hypoxia condition mimicking our human high altitude studies. Moreover, we found that hypoxia-induced increased plasma adenosine, erythrocyte 2,3-BPG and decreased Hb-O2 binding affinity were significantly attenuated in CD73-deficient mice (CD73-/-). As such, hypoxia-induced tissue injury and cell apoptosis were significantly elevated in CD73-/- mice compared to WT mice. Mechanistically, we provide both in vitro and in vivo genetic evidence that erythrocyte adenosine A2B receptor (ADORA2B) is essential for 2,3-BPG induction and subsequent O2 release. This finding led us to further discover that AMP-Activated Protein Kinase (AMPK) functions downstream of ADORA2B underlying adenosine-induced 2,3-BPG induction and O2 release by direct phosphorylation of 2,3-BPG mutase, a key enzyme for 2,3-BPG production. Finally, we demonstrated that treatment of metformin, a FDA approved potent AMPK agonist, induced erythrocyte 2,3-BPG levels and triggered O2 release and thereby prevented hypoxia-induced tissue injury in both CD73-/- mice and erythrocyte specific ADORA2B-deficient mice. Overall, our studies have revealed that erythrocyte ADORA2B-mediated AMPK activation is a novel mechanism underlying hypoxia-induced 2,3-BPG levels and thereby highlight its beneficial role to prevent hypoxia-induced tissue injury. DisclosuresNo relevant conflicts of interest to declare.

Epistasis constrains mutational pathways of hemoglobin adaptation in high-altitude pikas.

A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. This question can be addressed most directly by using site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here, we evaluate how pleiotropic trade-offs and epistatic interactions influence the accessibility of alternative mutational pathways during the adaptive evolution of hemoglobin (Hb) function in high-altitude pikas (Mammalia: Lagomorpha). By combining ancestral protein resurrection with a combinatorial protein-engineering approach, we examined the functional effects of sequential mutational steps in all possible pathways that produced an increased Hb–O2 affinity. These experiments revealed that the effects of mutations on Hb–O2 affinity are highly dependent on the temporal order in which they occur: Each of three β-chain substitutions produced a significant increase in Hb–O2 affinity on the ancestral genetic background, but two of these substitutions produced opposite effects when they occurred as later steps in the pathway. The experiments revealed pervasive epistasis for Hb–O2 affinity, but affinity-altering mutations produced no significant pleiotropic trade-offs. These results provide insights into the properties of adaptive substitutions in naturally evolved proteins and suggest that the accessibility of alternative mutational pathways may be more strongly constrained by sign epistasis for positively selected biochemical phenotypes than by antagonistic pleiotropy.

Blood oxygen affinity increases during digestion in the South American rattlesnake, Crotalus durissus terrificus

Digesting snakes experience massive increases in metabolism that can last for many days and are accompanied by adjustments in the oxygen transport cascade. Accordingly, we examined the oxygen-binding properties of the blood in the South American rattlesnake (Crotalus durissus terrificus) during fasting and 24 and 48h after the snakes have ingested a rodent meal corresponding to 15% (±2%) of its own body mass. In general, oxygen–hemoglobin (Hb–O2) affinity was significantly increased 24h post-feeding, and then returned toward fasting values within 48h post-feeding. Content of organic phosphates ([NTP] and [NTP]/[Hb]), hemoglobin cooperativity (Hill's n), and Bohr Effect (ΔlogP50/ΔpH) were not affected by feeding. The postprandial increase in Hb–O2 affinity in the South American rattlesnake can be almost entirely ascribed by the moderate alkaline tide that follows meal ingestion. In general, digesting snakes were able to regulate blood metabolites at quite constant levels (e.g., plasma osmolality, lactate, glucose, and total protein levels). The level of circulating lipids, however, was considerably increased, which may be related to their mobilization, since lipids are known to be incorporated by the enterocytes after snakes have fed. In conclusion, our results indicate that the exceptional metabolic increment exhibited by C. d. terrificus during meal digestion is entirely supported by the aerobic pathways and that among the attending cardiorespiratory adjustments, pulmonary Hb–O2 loading is likely improved due to the increment in blood O2 affinity.

Osmoregulatory bicarbonate secretion exploits H(+)-sensitive haemoglobins to autoregulate intestinal O2 delivery in euryhaline teleosts.

Marine teleost fish secrete bicarbonate (HCO3 −) into the intestine to aid osmoregulation and limit Ca2+ uptake by carbonate precipitation. Intestinal HCO3 − secretion is associated with an equimolar transport of protons (H+) into the blood, both being proportional to environmental salinity. We hypothesized that the H+-sensitive haemoglobin (Hb) system of seawater teleosts could be exploited via the Bohr and/or Root effects (reduced Hb-O2 affinity and/or capacity with decreasing pH) to improve O2 delivery to intestinal cells during high metabolic demand associated with osmoregulation. To test this, we characterized H+ equilibria and gas exchange properties of European flounder (Platichthys flesus) haemoglobin and constructed a model incorporating these values, intestinal blood flow rates and arterial–venous acidification at three different environmental salinities (33, 60 and 90). The model suggested red blood cell pH (pHi) during passage through intestinal capillaries could be reduced by 0.14–0.33 units (depending on external salinity) which is sufficient to activate the Bohr effect (Bohr coefficient of −0.63), and perhaps even the Root effect, and enhance tissue O2 delivery by up to 42 % without changing blood flow. In vivo measurements of intestinal venous blood pH were not possible in flounder but were in seawater-acclimated rainbow trout which confirmed a blood acidification of no less than 0.2 units (equivalent to −0.12 for pHi). When using trout-specific values for the model variables, predicted values were consistent with measured in vivo values, further supporting the model. Thus this system is an elegant example of autoregulation: as the need for costly osmoregulatory processes (including HCO3 − secretion) increases at higher environmental salinity, so does the enhancement of O2 delivery to the intestine via a localized acidosis and the Bohr (and possibly Root) effect.

High blood oxygen affinity in the air-breathing swamp eel Monopterus albus

The Asian swamp eel (Monopterus albus, Zuiew 1793) is a facultative air-breathing fish with reduced gills. Previous studies have shown that gas exchange seems to occur across the epithelium of the buccopharyngeal cavity, the esophagus and the integument, resulting in substantial diffusion limitations that must be compensated by adaptations in others steps of the O2 transport system to secure adequate O2 delivery to the respiring tissues. We therefore investigated O2 binding properties of whole blood, stripped hemoglobin (Hb), two major isoHb components and the myoglobin (Mb) from M. albus. Whole blood was sampled using indwelling catheters for blood gas analysis and determination of O2 equilibrium curves. Hb was purified to assess the effects of endogenous allosteric effectors, and Mb was isolated from heart and skeletal muscle to determine its O2 binding properties. The blood of M. albus has a high O2 carrying capacity [hematocrit (Hct) of 42.4±4.5%] and binds O2 with an unusually high affinity (P50=2.8±0.4mmHg at 27°C and pH7.7), correlating with insensitivity of the Hb to the anionic allosteric effectors that normally decrease Hb-O2 affinity. In addition, Mb is present at high concentrations in both heart and muscle (5.16±0.99 and 1.08±0.19mg ∙ g wet tissue-1, respectively). We suggest that the high Hct and high blood O2 affinity serve to overcome the low diffusion capacity in the relatively inefficient respiratory surfaces, while high Hct and Mb concentration aid in increasing the O2 flux from the blood to the muscles.

Enthalpic partitioning of the reduced temperature sensitivity of O2 binding in bovine hemoglobin

The oxygenation enthalpy of the heme groups of hemoglobin (Hb) is inherently exothermic, resulting in decreased Hb-O2 affinity with rising temperature. However, oxygenation is coupled with endothermic dissociation of allosteric effectors (e.g. protons, chloride ions and organic phosphates) from the protein, which reduces the overall oxygenation enthalpy. The evolution of Hbs with reduced temperature sensitivity ostensibly safeguards O2 unloading in cold extremities of regionally-heterothermic vertebrates permitting energy-saving reductions in heat loss. Ungulate (e.g. bovine) Hbs have long served as a model system in this regard in that they exhibit numerically low oxygenation enthalpies that are thought to correlate with the presence of an additional Cl− binding site (compared to human Hb) comprised of three cationic residues at positions 8, 76 and 77 of the β-chains of Hb. However, ungulate Hbs also exhibit distinctive amino acid exchanges at the N-termini of the β-chains that stabilize the low-affinity deoxystructure of the Hb, mimicking the action of organic phosphates. In order to assess the relative contributions from these two effects, we measured the temperature sensitivity of Hb-O2 affinity in bovine and human Hbs in the absence and presence of Cl− ions under strictly controlled pH conditions. The data indicate that Cl−-binding accounts for a minority (~30%) of the total reduction in the oxygenation enthalpy manifested in bovine compared to human Hb, whereas the majority of this reduction is ascribable to structural differences, including increased β-chain hydrophobicity that would increase the heat of oxygenation-linked conformational change in bovine Hb.

Localized increase of tissue oxygen tension by magnetic targeted drug delivery

Hypoxia is the major hindrance to successful radiation therapy of tumors. Attempts to increase the oxygen (O2) tension (PO2) of tissue by delivering more O2 have been clinically disappointing, largely due to the way O2 is transported and released by the hemoglobin (Hb) within the red blood cells (RBCs). Systemic manipulation of O2 transport increases vascular resistance due to metabolic autoregulation of blood flow to prevent over oxygenation. This study investigates a new technology to increase O2 delivery to a target tissue by decreasing the Hb-O2 affinity of the blood circulating within the targeted tissue. As the Hb-O2 affinity decreases, the tissue PO2 to satisfy tissue O2 metabolic needs increases without increasing O2 delivery or extraction. Paramagnetic nanoparticles (PMNPs), synthetized using gadolinium oxide, were coated with the cell permeable Hb allosteric effector L35 (3,5-trichlorophenylureido-phenoxy-methylpropionic acid). L35 decreases Hb affinity for O2 and favors the release of O2. The L35-coated PMNPs (L35-PMNPs) were intravenously infused (10 mg kg−1) to hamsters instrumented with the dorsal window chamber model. A magnetic field of 3 mT was applied to localize the effects of the L35-PMNPs to the window chamber. Systemic O2 transport characteristics and microvascular tissue oxygenation were measured after administration of L35-PMNPs with and without magnetic field. The tissue PO2 in untreated control animals was 25.2 mmHg. L35-PMNPs without magnetic field decreased tissue PO2 to 23.4 mmHg, increased blood pressure, and reduced blood flow, largely due to systemic modification of Hb-O2 affinity. L35-PMNPs with magnetic field increased tissue PO2 to 27.9 mmHg, without systemic or microhemodynamic changes. These results indicate that localized modification of Hb-O2 affinity can increase PO2 of target tissue without affecting systemic O2 delivery or triggering O2 autoregulation mechanisms. This technology can be used to treat local hypoxia and to increase O2 in tumors, enhancing the efficacy of radiation therapies.

A unique mode of tissue oxygenation and the adaptive radiation of teleost fishes

Teleost fishes constitute 95% of extant aquatic vertebrates, and we suggest that this is related in part to their unique mode of tissue oxygenation. We propose the following sequence of events in the evolution of their oxygen delivery system. First, loss of plasma-accessible carbonic anhydrase (CA) in the gill and venous circulations slowed the Jacobs-Stewart cycle and the transfer of acid between the plasma and the red blood cells (RBCs). This ameliorated the effects of a generalised acidosis (associated with an increased capacity for burst swimming) on haemoglobin (Hb)-O2 binding. Because RBC pH was uncoupled from plasma pH, the importance of Hb as a buffer was reduced. The decrease in buffering was mediated by a reduction in the number of histidine residues on the Hb molecule and resulted in enhanced coupling of O2 and CO2 transfer through the RBCs. In the absence of plasma CA, nearly all plasma bicarbonate ultimately dehydrated to CO2 occurred via the RBCs, and chloride/bicarbonate exchange was the rate-limiting step in CO2 excretion. This pattern of CO2 excretion across the gills resulted in disequilibrium states for CO2 hydration/dehydration reactions and thus elevated arterial and venous plasma bicarbonate levels. Plasma-accessible CA embedded in arterial endothelia was retained, which eliminated the localized bicarbonate disequilibrium forming CO2 that then moved into the RBCs. Consequently, RBC pH decreased which, in conjunction with pH-sensitive Bohr/Root Hbs, elevated arterial oxygen tensions and thus enhanced tissue oxygenation. Counter-current arrangement of capillaries (retia) at the eye and later the swim bladder evolved along with the gas gland at the swim bladder. Both arrangements enhanced and magnified CO2 and acid production and, therefore, oxygen secretion to those specialised tissues. The evolution of β-adrenergically stimulated RBC Na(+)/H(+) exchange protected gill O2 uptake during stress and further augmented plasma disequilibrium states for CO2 hydration/dehydration. Finally, RBC organophosphates (e.g. NTP) could be reduced during hypoxia to further increase Hb-O2 affinity without compromising tissue O2 delivery because high-affinity Hbs could still adequately deliver O2 to the tissues via Bohr/Root shifts. We suggest that the evolution of this unique mode of tissue O2 transfer evolved in the Triassic/Jurassic Period, when O2 levels were low, ultimately giving rise to the most extensive adaptive radiation of extant vertebrates, the teleost fishes.

Oxygen-Affinity of Hemoglobin is Regulated by Effector-Linked Dynamic Modulations of High-Frequency Thermal Fluctuations

Heterotropic effectors reduce the O2-affinity of hemoglobin (Hb) as much as >10\3-folds: P50 = 0.4 → 200 mmHg, Klow = 0.3 → 0.004 mmHg-1, and Khigh = 10 → 0.004 mmHg-1. However, there are no detectable changes in either static T/R-quaternary and associated tertiary structures or the stereochemical and electronic structures of the heme coordination that are attributable to such changes in the O2-affinity of Hb, upon binding of the heterotropic effectors to either T(deoxy)- or R(oxy)-Hb1,2, indicating that the O2-affinity of Hb is not regulated by static T/R-quaternary and associated tertiary structures of Hb and that the reactivity (or the O2-affinity) of the heme Fe itself is not altered1, in sharp contrast to the mainstream idea of the heme-centric regulation of the O2-affinity in Hb3. Our 6ns-molecular dynamics simulations4,5 indicated that the amplitudes of very high-frequency (>GHz) thermal fluctuations of the globin matrix are entropically enhanced by the dissociation of O2 from oxy-Hb and/or binding of heterotropic effectors to deoxy- and oxy-Hb, in parallel to the reduction of the O2-affinity. The rate of dissociation of O2 from oxy-Hb is enhanced by the effector-linked enhanced thermal fluctuations, which simultaneously reduce the rate of geminate-recombination of O2. This results in the reduction of the apparent O2-affinity of Hb by heterotropic effectors.

Adenosine Is A Common Factor Regulating Erythrocyte 2,3-Bisphosphate Induction In Normal Individuals At High Altitude and In Patients With Sickle Cell Disease

To function effectively in O2 uptake, transport and delivery, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)-O2 affinity by endogenous allosteric modulators. One of the best know allosteric modulators is 2,3-bisphophosphoglycerate (2,3-BPG), a metabolic byproduct of glycolysis synthesized primarily in erythrocytes for the purpose of regulating Hb-O2 affinity. Earlier studies demonstrated that erythrocyte 2,3-BPG levels are elevated at a high altitude and in patients with sickle cell disease (SCD). We have recently shown that adenosine, a molecule well known to be induced under hypoxic conditions, is significantly elevated in SCD and contributes to increased 2,3-BPG induction in SCD erythrocytes. However, whether adenosine contributes to increased erythrocyte 2,3-BPG production at high altitude is unknown. To address this question we recruited 24 individuals who normally lived at sea level, and placed them at high altitude for different time points. Similarly, blood was collected from 45 SCD patients and 50 controls (at sea level). Here we report that 1) Plasma adenosine and erythrocyte 2,3-BPG levels were significantly elevated in normal individuals at high altitude after 24 hours compared to sea level; 2) The elevations of both molecules are further enhanced at high altitude after 16 days; 3) Elevated circulating adenosine levels significantly correlated with increased erythrocyte 2,3-DPG levels in normal individuals at high altitude and in SCD patients. These results suggest that adenosine is a common factor responsible for elevated 2,3-BPG as a function in normal individuals and in SCD patients. To test this hypothesis we took pharmacologic and genetic approaches. We found that NECA, a stable adenosine analog, significantly increased 2,3-BPG production in cultured erythrocytes from normal and SCD mice and humans. Additional genetic and pharmacologic experiment showed that adenosine-induced 2,3-BPG production was mediated by ADORA2B receptor activation and downstream signaling by protein kinase A. To examine the in vivo physiological relevance of our findings we used adenosine deaminase (ADA)-deficient mice that accumulate high levels of circulating adenosine as a result of their enzyme deficiency. We found that elevated adenosine correlated to elevated erythrocyte 2,3-BPG and decreased Hb-O2 affinity. Experimental strategies to lower plasma adenosine or inhibit ADORA2B signaling in ADA-deficient mice resulted in reduced 2,3-BPG production and increased Hb-O2 affinity in erythrocytes. These studies provide in vivo evidence that elevated adenosine signaling via ADORA2B contributes to elevated of 2,3-BPG production and thereby triggers O2 release from erythrocytes. Taken together, our studies show that adenosine is a common factor regulating erythrocyte 2,3-BPG induction as a function of altitude and in patients with sickle cell disease. Our studies reveal numerous approaches to enhance oxygen release from oxyHb to physiologically or environmentally induced conditions of hypoxia. Disclosures:No relevant conflicts of interest to declare.

GTx011, a Novel Agent That Improves Rheological Properties Of Sickle Cell Blood By Increasing Oxygen Affinity For Hemoglobin

Sickle cell disorder (SCD) is characterized by the presence of non-deformable red blood cells. Literature reports indicate that the T-state structure of hemoglobin (Hb) (highly correlated with the deoxy form) is responsible for the formation of HbS polymers that lead to rigid cells. We hypothesized that the likelihood of polymer formation will be reduced if sufficient HbS remains in the R-state (oxy) conformation. We have designed and synthesized a novel series of compounds that increase the O2 affinity of HbS and improve the rheological properties of SCD blood. In a novel 96-well format oxygen dissociation assay (ODA), compounds including GTx006, GTx007 and GTx011 were all more potent than 5-hydroxy furfural (5HMF), an agent being tested in clinical trials in SCD patients. After two hours of passive deoxygenation, GTx011, at an equimolar concentration to Hb, increases the O2 affinity by six-fold and drastically delays polymerization of HbS. Even at substoichiometric concentrations (GTx011:Hb= 1:3) GTx011 elicits a two-fold improvement in O2 affinity for Hb that translated to 16% more oxy-Hb relative to control (Table 1). We then analyzed the agents in a TCS Hemox analyzer using purified Hb at 25µM. At a GTx011:Hb ratio of 1:3 the oxygen affinity was improved by 15%, while at stoichiometric concentrations, the oxygen affinity was increased by 70% compared to Hb control. These biochemical assays indicated that the GTx agents were altering Hb O2 affinity and should therefore assist in maintaining the oxy conformation of Hb and prevent the formation of polymers.Table 1AssayHb in ODAWashed RBC OECWhole Blood OECViscosityunit(Δoxy state)(%Δp50)(%Δp50)(ΔcP)[Hb]3 µM1 mM1 mM1.5 mM[cmp]1 µM3 µM1 mM3 mM1 mM3 mM1.6 mM8 mM5-HMF<1<1306510470.042.4GTx006210597849711.7>2.5GTx007623697863>802.1>2.5GTx0111656768380>802.5>2.5To test the agents in a more physiological system, oxygen equilibrium curves (OECs) were measured in washed red blood cells (RBCs) and in whole blood at 20% hematocrit (∼1 mM Hb). In washed RBCs, 5HMF, GTx006, GTx007 and GTx011 at a concentration of 1mM produced partial O2 pressures (p50) of 20, 12, 9 and 7 mm Hg, respectively (control RBCs = 30 mm Hg). To determine the effects of plasma proteins, OECs were measured in whole blood from SCD patients, giving p50s of 27, 18, 11 and 6 mm Hg compared to the control blood p50 of 30 mm Hg (agent concentration of 1 mM). Table 1 shows that 5HMF and GTx006 activities were affected by the presence of plasma proteins but GTx007 and GTx011 activities were not altered.SCD patients develop anemia due to hemolysis, which partially compensates for an increase in blood viscosity caused by the non-deformable RBCs (ssRBCs). We monitored the effect of our agents on SCD patient blood rheology, ex vivo, to determine if they were capable of decreasing the viscosity of SS blood under hypoxic conditions. We incubated whole blood from SCD patients (30% hematocrit, ∼1.5 mM Hb) with 5HMF, GTx006, GTx007 or GTx011 for 30 mins and then subjected the blood to 2 hours of hypoxia (2.4% O2). Blood viscosity was then measured in a cone-plate viscometer at shear rates ranging from 60 s-1 to 415 s-1. Of the four compounds, GTx011 showed the most pronounced improvement in rheologic measures (see Table 1), changing the viscosity from 6.46 cP (no GTx011) to 4.00 cP (equimolar GTx011). Normoxic SCD blood had a viscosity of 4.28 cP. A similar improvement in blood viscosity under physiologic conditions may be predicted to decrease the residence time of ssRBCs in hypoxic tissue, and allow for a lower level of polymerization in individual red blood cells. In addition, GTx011 has also been shown to delay polymerization and delay sickling. Thus, GTx011 has the potential to elicit a decrease in HbS polymer concentration, reducing the likelihood of forming the rigid cells that cause vaso-occlusion in SCD patients. Disclosures:Patel:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Silva:Global Blood Therapeutics: Employment, Equity Ownership. Betz:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Employment, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.