Hb-O2 Affinity Research Articles

FT-4202, a selective pyruvate kinase R activator for sickle cell disease.

Anemia in patients with sickle cell disease (SCD) increases 2,3-diphosphoglycerate (2,3-DPG), decreasing hemoglobin-oxygen (Hb-O2) affinity to improve oxygen offloading and promote hemoglobin polymerization (sickling) of red blood cells (RBCs). We report the discovery of FT-4202, an investigational, selective pyruvate kinase type-R (PKR) activator with a multimodal mechanism of action and potential to increase ATP and decrease 2,3-DPG, resulting in increased Hb-O2 affinity, decreased Hb polymerization, and improved RBC health. FT-4202 was identified via structure-enabled lead optimization medicinal chemistry using X-ray crystallography, molecular modeling, and thermal shift assays. FT-4202, an allosteric PKR activator, stabilizes the tetrameric enzyme and increases PKR activity in human and mouse RBCs in vitro. Seven-day oral administration of FT-4202 in Berkeley SCD mice reduced 2,3-DPG, increased Hb-O2 affinity, and reduced RBC sickling versus control. There were no adverse in vitro safety findings. FT-4202 offers a therapeutic opportunity to modify the course of SCD.

Common tenrecs (Tenrec ecaudatus) reduce oxygen consumption in hypoxia and in hypercapnia without concordant changes to body temperature or heart rate.

Common tenrecs (Tenrec ecaudatus) are fossorial mammals that use burrows during both active and hibernating seasons in Madagascar and its neighboring islands. Prevailing thought was that tenrecs hibernate for 8-9months individually, but 13 tenrecs were removed from the same sealed burrow 1m deep from the surface. Such group hibernation in sealed burrows presumably creates a hypoxic and/or hypercapnic environment and suggests that this placental mammal may have an increased tolerance to hypoxia and hypercapnia. Higher tolerances to hypoxia and hypercapnia have been documented for other mammals capable of hibernation and to determine if this is the case for tenrecs, we exposed them to acute hypoxia (4h of 16 or 7% O2), progressive hypoxia (2h of 16, 10 and 4% O2), or progressive hypercapnia (2h of 2, 5 and 10% CO2) at cold (16°C) or warm (28°C) ambient temperatures (Ta). Oxygen equilibrium curves were also constructed on the whole blood of tenrecs at 10, 25, and 37°C to determine if hemoglobin (Hb)-O2 affinity contributes to hypoxia tolerance. In animals held at 16°C, normoxic and normocapnic levels of oxygen consumption rate ( ), body temperature (Tb), and heart rate (HR) were highly variable between individuals. This inter-individual variation was greatly reduced in animals held at 28°C for oxygen consumption rate and body temperature. Both hypoxia (acute and progressive) and progressive hypercapnia led to decreases in as well as the variation in between animals held at 16°C. The fall in oxygen consumption rate in 7% O2 independent of changes in body temperature in tenrecs held at 16°C is unique and not consistent with the typical hypoxic metabolic response seen in other hibernating species that depends on concomitant falls in Tb. In animals held at 28°C, exposure to O2 levels as low as 4% and CO2 levels as high as 10% had no significant effect on , HR, or Tb, indicative of high tolerance to both hypoxia and hypercapnia. High variation in heart rate remained between individuals in all gas compositions and at all temperatures. Tenrec Hb-O2 affinity was similar to other homeothermic placental mammals and likely does not contribute to the increased hypoxia tolerance. Ultimately, our results suggest changes in Ta dictate physiological responses to hypoxia or hypercapnia in tenrecs, responses more characteristic of reptiles than of most placental mammals. Given that numerous anatomical and physiological characteristics of tenrecs suggest that they may be representative of an ancestral placental mammal, our findings suggest the typical hypoxic metabolic response evolved later in mammalian evolution.

Soluble adenylyl cyclase is an acid-base sensor in rainbow trout red blood cells that regulates intracellular pH and haemoglobin-oxygen binding.

To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O2) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O2 transport. Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid-base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3 --dependent sAC signaling. Inhibition of sAC decreased Hb-O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During invitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%. sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

Nitric Oxide Ameliorates the Effects of Hypoxia in Mice by Regulating Oxygen Transport by Hemoglobin.

Xiaoying Zhou, Wenting Su, Quanwei Bao, Yu Cui, Xiaoxu Li, Yidong Yang, Chengzhong Yang, Chengyuan Wang, Li Jiao, Dewei Chen, and Jian Huang. Nitric oxide ameliorates the effects of hypoxia in mice by regulating oxygen transport by hemoglobin. High Alt Med Biol. 25:174-185, 2024.-Hypoxia is a common pathological and physiological phenomenon in ischemia, cancer, and strenuous exercise. Nitric oxide (NO) acts as an endothelium-derived relaxing factor in hypoxic vasodilation and serves as an allosteric regulator of hemoglobin (Hb). However, the ultimate effects of NO on the hematological system invivo remain unknown, especially in extreme environmental hypoxia. Whether NO regulation of the structure of Hb improves oxygen transport remains unclear. Hence, we examined whether NO altered the oxygen affinity of Hb (Hb-O2 affinity) to protect extremely hypoxic mice. Mice were exposed to severe hypoxia with various concentrations of NO, and the survival time, exercise capacity, and other physical indexes were recorded. The survival time was prolonged in the 5 ppm NO (6.09 ± 1.29 minutes) and 10 ppm NO (6.39 ± 1.58 minutes) groups compared with the 0 ppm group (4.98 ± 1.23 minutes). Hypoxia of the brain was relieved, and the exercise exhaustion time was prolonged when mice inhaled 20 ppm NO (24.70 ± 6.87 minutes vs. 20.23 ± 6.51 minutes). In addition, the differences in arterial oxygen saturation (SO2%) (49.64 ± 7.29% vs. 42.90 ± 4.30%) and arteriovenous SO2% difference (25.14 ± 8.95% vs. 18.10 ± 6.90%) obviously increased. In ex vivo experiments, the oxygen equilibrium curve (OEC) left shifted as P50 decreased from 43.77 ± 2.49 mmHg (0 ppm NO) to 40.97 ± 1.40 mmHg (100 ppm NO) and 38.36 ± 2.78 mmHg (200 ppm NO). Furthermore, the Bohr effect of Hb was enhanced by the introduction of 200 ppm NO (-0.72 ± 0.062 vs.-0.65 ± 0.051), possibly allowing Hb to more easily offload oxygen in tissue at lower pH. The crystal structure reveals a greater distance between Asp94β-His146β in nitrosyl -Hb(NO-Hb), NO-HbβCSO93, and S-NitrosoHb(SNO-Hb) compared to tense Hb(T-Hb, 3.7 Å, 4.3 Å, and 5.8 Å respectively, versus 3.5 Å for T-Hb). Moreover, hydrogen bonds were less likely to form, representing a key limitation of relaxed Hb (R-Hb). Upon NO interaction with Hb, hydrogen bonds and salt bridges were less favored, facilitating relaxation. We speculated that NO ameliorated the effects of hypoxia in mice by promoting erythrocyte oxygen loading in the lung and offloading in tissues.

The impact of cannabinoids on methemoglobin formation and hemoglobin oxygen affinity: An ex-vivo study

The affinity of hemoglobin (Hb) to oxygen (O2) influences processes of oxygen delivery and extraction at the tissue level. Despite cannabinoids being utilized or ingested in various ways, their possible impact on Hb-O2 affinity has barely been studied. This is an experimental ex-vivo trial. Venous blood samples were drawn from 5 male and 6 female healthy volunteers and subsequently exposed to different cannabinoid types: (delta-9-tetrahydrocannabinol [Δ9-THC], delta-8-tetrahydrocannabinol [Δ8-THC], cannabidiol [CBD]) at different concentrations. Oxygen dissociation curves (ODC) were measured and blood gas analyses were performed for methemoglobin (MetHb) determination. The results revealed no MetHb formation. Besides two statistically significant changes (+1.4 mmHg and -0.9 mmHg) in the female cohort, following Δ9-THC and Δ8-THC exposure, no further P50 changes could be observed. The study demonstrated an in-vitro effect of selected cannabinoids and dosages on P50 values in female participants, with variations not observed at other dosages, leaving the underlying mechanisms open for debate. MetHb formation, as potential mechanism, was not detected in this study. The precise reasons why changes only occurred at specific dosages remain unclear, indicating a need for further in-vivo research to understand the interaction between cannabinoids and Hb-O2 affinity completely.

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.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Rapid Assessment of Hemoglobin-Oxygen Dissociation

Introduction: Oxygen (O 2) delivery to tissues depends on the hemoglobin (Hb)-O 2 affinity and dissociation of O 2 from Hb in red blood cells (RBCs). Mutations that impact α and β-globin subunits of Hb often alter the Hb-O2 affinity of Hb and Hb-O 2 dissociation. A point mutation in the β-globin gene yields sickle hemoglobin (HbSS) which results in sickle cell disease (SCD). Recently, there has been an upsurge in improving SCD pathophysiology through modifying HbSS O 2 affinity. The ability to alter cellular phosphate such as 2,3-DPG that influences Hb-O 2 affinity is also under investigation. In this context, measuring Hb-O 2 affinity has gained additional significance as a clinical and laboratory parameter. Hb-O 2 affinity is determined by measuring Hb-O 2 saturation (SO 2, %) vs. partial pressure of oxygen (pO 2, mmHg). The Hb-O 2 affinity is commonly represented by the p50 value, which is the pO 2 value at 50% SO 2. p50 measurement is underutilized in clinical practice mainly due to cost, complexity, and limited throughput of existing instruments. Herein, we present a rapid assay that can detect Hb-O 2 affinity disorders, determine cellular oxygen availability, and examine the effectiveness of existing and novel therapies for SCD patients. Methods: In this assay, we optically measure the Hb-O 2 dissociation by induction of physiologic acidic pH (6.9). We exploited the acidic pH environment to expedite chemically induced partial deoxygenation of Hb (deoxy-Hb) during which the conformational changes in the tertiary structure of Hb are manifested as significant peak shifts in the optical absorption spectra. Hb absorption spectra in the 350-700 nm wavelength range primarily reflect the conformation of heme-heme interactions, which are sensitive indicators of changes in the Hb-O 2 affinity. We validated the assay by comparing it with the reference standard Hemox analyzer using blood from healthy individuals (HbAA) and from patients with SCD (HbSS) i.e., low Hb-O 2 affinity. Venous blood samples were collected in EDTA-coated tubes from subjects with HbSS (n=44), HbAS (n=14), and HbAA (n=17) following an IRB-approved study protocol. 75 µl of whole blood diluted 100-fold with pH 6.9 phosphate buffer was incubated for 2 minutes at room air to attain full oxygenation. The mixture of blood and the buffer was then deoxygenated with 0.053M of sodium metabisulphite (Na 2S 2O 5) for 3 minutes to achieve partial deoxy-Hb. We then obtained 350-450 nm spectra of deoxy-Hb using a spectroscopy microplate reader with a 2 nm resolution. We recorded each sample's Soret band shift in 4 minutes using the 414 nm Hb fully oxygenated absorption peak as the reference. Data were reported as SEM. Results: Hb exhibits three characteristic light absorption peaks. The Soret peak is located at 414 nm under full oxygenation at pO 2 of 162 mmHg, and upon full deoxy-Hb at pO 2 of 5 mmHg, the peak shifts to 430 nm ( Figure 1A). We initially tested different concentrations of Na 2S 2O 5 and the time of deoxygenation to achieve partial deoxy-Hb. At 75 mmHg we analyzed the Soret peak which was used as an optical marker of oxygen affinity, an equivalent to the p50 value. The peak shift significantly correlated to the p50 value measured by the Hemox device (PCC=0.81, p=0.004, n=28) ( Figure 1A). We also observed significant differences between HbAA (n=17), HbAS(n=14), SCD samples on various treatments (transfusion (Tx) (n=8), and Hydroxyurea (n=15) and SCD treatment-naïve patients' samples (n=20). HbAA vs. HbAS; p=0.01, HbAS vs. HbSS+Tx; p=0.02, HbSS+Tx vs. HbSS+Hu; p=0.04, HbAA vs. HbSS treatment naïve; p=0.000, HbSS treatment Naive vs. HbAS; p=0.001 ( Figure 1B). Additionally, the peak shift for HbSS correlated with the percentage of HbSS (PCC = 0.87, p = 0.000, n=20, data not shown). Discussion: We demonstrate fast, effective differentiation of abnormal Hb-O 2 affinity and characterize the changes in the HbSS-O 2 affinity in response to treatments by using chemically induced partial deoxygenation of Hb at a physiologic acidic pH.The ubiquity of optical absorption spectrophotometers positions this assay as an accessible, equitable, and accurate Hb-O 2 dissociation measurement resource for research and clinical use. The assay will facilitate the diagnosis and prognosis of red blood cells and Hb disorders requiring minimal technical expertise and laboratory infrastructure and assist with testing the effect of new targeted and genome-editing-based therapies for SCD.

Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller's sea cows.

The extinct Steller's sea cow (Hydrodamalis gigas; †1768) was a whale-sized marine mammal that manifested profound morphological specializations to exploit the harsh coastal climate of the North Pacific. Yet despite first-hand accounts of their biology, little is known regarding the physiological adjustments underlying their evolution to this environment. Here, the adult-expressed hemoglobin (Hb; a2β/δ2) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position (β/δ82Lys→Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrate that the Hb-O2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas β/δ82Asn→Lys mutant Hb further reveal this exchange renders Steller's sea cow Hb unresponsive to the potent intraerythrocytic allosteric effector 2,3-diphosphoglycerate, a radical modification that is the first documented example of this phenotype among mammals. Notably, β/δ82Lys→Asn moreover underlies the secondary evolution of a reduced blood-O2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O2 delivery. This conclusion is bolstered by analyses of two Steller's sea cow prenatal Hb proteins (Hb Gower I; z2e2 and HbF; a2g2) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF (g38Thr→Ile and g101Glu→Asp) that increased Hb-O2 affinity relative to dugong HbF. Finally, the β/δ82Lys→Asn replacement of the adult/fetal protein is shown to increase protein solubility, which may have elevated red blood cell Hb content within both the adult and fetal circulations and contributed to meeting the elevated metabolic (thermoregulatory) requirements and fetal growth rates associated with this species cold adaptation.

Effects of GBT1118, a voxelotor analog, on intestinal pathophysiology in sickle cell disease.

Voxelotor is an allosteric haemoglobin (Hb) modulator that binds covalently and reversibly to Hb alpha chain to facilitate improved Hb-O2 affinity and arterial oxygen. It, therefore, reduces the susceptibility of erythrocytes carrying Haemoglobin S to sickle. In this study, we have used GBT1118, an analog of voxelotor, to treat male Townes sickle cell disease (SCD) mice to investigate whether the Hb modulator could attenuate the intestinal pathophysiologic changes associated with SCD. Compared with mice fed with control chow, GBT1118-treated mice showed improvement in the intestinal pathophysiology. These mice exhibited improved small intestinal barrier functions, reduced intestinal microbial density, reduced enterocyte injury, lower serum lipopolysaccharides and smaller spleens. These improvements were observed after only 3 weeks of GBT1118 treatment. Benefits were also observed after experimentally-induced vaso-occlusive crisis (VOC). Recovery from the VOC-induced changes was faster in mice that were treated with GBT1118. The improved small intestinal barrier function was associated with higher expression of genes encoding enterocyte E-cadherin, JAM-A, ZO-1, MUC-2 and occludin while the lower intestinal microbial density associated with higher expression of genes encoding the antimicrobial peptides defensin-α 1 and defensin-α 4. Our findings provide the evidence to support the beneficial effects of GBT1118 in SCD-related intestinal pathophysiology.

Erythrocytes Functionality in SARS-CoV-2 Infection: Potential Link with Alzheimer's Disease.

Coronavirus disease 2019 (COVID-19) is a rapidly spreading acute respiratory infection caused by SARS-CoV-2. The pathogenesis of the disease remains unclear. Recently, several hypotheses have emerged to explain the mechanism of interaction between SARS-CoV-2 and erythrocytes, and its negative effect on the oxygen-transport function that depends on erythrocyte metabolism, which is responsible for hemoglobin-oxygen affinity (Hb-O2 affinity). In clinical settings, the modulators of the Hb-O2 affinity are not currently measured to assess tissue oxygenation, thereby providing inadequate evaluation of erythrocyte dysfunction in the integrated oxygen-transport system. To discover more about hypoxemia/hypoxia in COVID-19 patients, this review highlights the need for further investigation of the relationship between biochemical aberrations in erythrocytes and oxygen-transport efficiency. Furthermore, patients with severe COVID-19 experience symptoms similar to Alzheimer's, suggesting that their brains have been altered in ways that increase the likelihood of Alzheimer's. Mindful of the partly assessed role of structural, metabolic abnormalities that underlie erythrocyte dysfunction in the pathophysiology of Alzheimer's disease (AD), we further summarize the available data showing that COVID-19 neurocognitive impairments most probably share similar patterns with known mechanisms of brain dysfunctions in AD. Identification of parameters responsible for erythrocyte function that vary under SARS-CoV-2 may contribute to the search for additional components of progressive and irreversible failure in the integrated oxygen-transport system leading to tissue hypoperfusion. This is particularly relevant for the older generation who experience age-related disorders of erythrocyte metabolism and are prone to AD, and provide an opportunity for new personalized therapies to control this deadly infection.

A novel mechanism for high-altitude adaptation in hemoglobin of black-spotted frog (Pelophylax nigromaculatus)

Understanding how animals living in highland adapt to extreme conditions is critical to evolutionary biology. In contrast to birds and mammals, little information was available on the adaptation mechanisms for O2 transport in high-altitude ectothermic vertebrates. Here we report for the first time on hematological parameters, amino acid sequences of α and β chains of hemoglobin (Hb), O2 affinity of purified hemoglobins (Hbs) and their sensitivities to anion allosteric effector (H+, Cl−, ATP) and temperature in the high-altitude (2,292 m) black-spotted frogs (Pelophylax nigromaculatus) from the Qinghai-Tibet plateau (QTP) compared with the low-altitude (135 m) population. Our results showed that high-altitude black-spotted frogs exhibit significantly increased relative lung mass, hematocrit, and hemoglobin concentration, but significantly decreased body mass and erythrocyte volume, which could improve the blood O2 carrying capability. Compared with the low-altitude population, the purified Hbs of high-altitude black-spotted frogs possessed significantly higher intrinsic Hb-O2 affinity, similar low anion allosteric effector sensitivities, Bohr effects and temperature sensitivities. The elevated Hb-O2 affinity of highland frogs could maximize the O2 extraction from the lungs. Molecular dynamics simulations showed that the Gln123Glu substitution on α2 chain in highland frogs could form a hydrogen bond with 127Lys on α2 chain, resulting in the elimination of a hydrogen bond between 127Lys on α2 chain and 141Arg on α1 chain. This could weaken the interaction between two semirigid dimers (α1β1 and α2β2) and then lead to the high intrinsic Hb-O2 affinity in high-altitude black-spotted frogs.

The effect of temperature on haemoglobin-oxygen binding affinity in regionally endothermic and ectothermic sharks.

Haemoglobin (Hb)-O2 binding affinity typically decreases with increasing temperature, but several species of ectothermic and regionally endothermic fishes exhibit reduced Hb thermal-sensitivity. Regionally endothermic sharks, including the common thresher shark (Alopias vulpinus) and lamnid sharks such as the shortfin mako shark (Isurus oxyrinchus), can maintain select tissues and organs warmer than ambient temperature by retaining metabolic heat with vascular heat exchangers. In the ectothermic bigeye thresher shark (Alopias superciliosus), diurnal movements above and below the thermocline subject the tissues, including the blood, to a wide range of operating temperatures. Therefore, blood-O2 transport must occur across internal temperature gradients in regionally endothermic species, and over the range of environmental temperatures encountered by the ectothermic bigeye thresher shark. While previous studies have shown temperature-independent Hb-O2 affinity in lamnid sharks, including shortfin mako, the Hb-O2 affinity of the common and bigeye thresher sharks is unknown. Therefore, we examined the effect of temperature on whole blood Hb-O2 affinity in common thresher shark and bigeye thresher shark. For comparison, analyses were also conducted on the shortfin mako shark and two ectothermic species, blue shark (Prionace glauca) and spiny dogfish (Squalus acanthias). Blood-O2 binding affinity was temperature-independent for common thresher shark and shortfin mako shark, which should prevent internal temperature gradients from negatively affecting blood-O2 transport. Blue shark and spiny dogfish blood-O2 affinity decreased with increasing temperature, as expected, but bigeye thresher shark blood exhibited both a reduced temperature-dependence and a high Hb-O2 affinity, which likely prevents large changes in environment temperature and low environmental oxygen from affecting O2 uptake.

Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin

The extraordinary breath-hold diving capacity of crocodilians has been ascribed to a unique mode of allosterically regulating hemoglobin (Hb)-oxygenation in circulating red blood cells. We investigated the origin and mechanistic basis of this novel biochemical phenomenon by performing directed mutagenesis experiments on resurrected ancestral Hbs. Comparisons of Hb function between the common ancestor of archosaurs (the group that includes crocodilians and birds) and the last common ancestor of modern crocodilians revealed that regulation of Hb-O2 affinity via allosteric binding of bicarbonate ions represents a croc-specific innovation that evolved in combination with the loss of allosteric regulation by ATP binding. Mutagenesis experiments revealed that evolution of the novel allosteric function in crocodilians and the concomitant loss of ancestral function were not mechanistically coupled and were caused by different sets of substitutions. The gain of bicarbonate sensitivity in crocodilian Hb involved the direct effect of few amino acid substitutions at key sites in combination with indirect effects of numerous other substitutions at structurally disparate sites. Such indirect interaction effects suggest that evolution of the novel protein function was conditional on neutral mutations that produced no adaptive benefit when they first arose but that contributed to a permissive background for subsequent function-altering mutations at other sites. Due to the context dependence of causative substitutions, the unique allosteric properties of crocodilian Hb cannot be easily transplanted into divergent homologs of other species.

Compound Nanoparticle 5HMF-Prodrug Formulations for Optimized Anti-Sickling Therapy

BACKROUND: Sickle cell disease (SCD) pathology stems from deoxygenation-induced hemoglobin (HbS) polymerization; allosteric Hb adducts that increase oxyHbS target this mechanistic trigger. 5-hydroxymethylfurfural (5HMF) binds to Hb αVal1, reducing O2 affinity & sickling and protecting hypoxic SS mice (survival, organ injury). However, 5HMF did not advance beyond PhI/II trials, due to pharmacokinetic/dynamic (PK/PD) limitations. A related molecule (Voxelotor) was approved based on surrogate endpoints (Hb level and hemolytic markers), but without improved vaso-occlusive morbidities. We reformulated 5HMF, a potentially superior agent, to address translation limitations arising from disfavorable PK/PD. 5HMF PK/PD: RBCs from SCD patients were incubated with 5HMF (20% Hct, 37C, HMF: 0-5mM, 0-3 hrs). 5HMF-induced Hb modification was quantified by HPLC (Waters e2695XE, Swift WCX-PEEK); O2 dissociation curves (ODC) were measured in intact RBCs (HEMOX); pO2's at the point of sickling (POS) and point of unsickling (POU) were determined during controlled Hb O2 unloading/loading, respectively (LORRCA Oxygenscan). We observed a dose dependent increase in 5HMF modified Hb, that was maximal (~75%) at 1 hr and 5mM [5HMF], and that resulted in: (1) 35% increase in HbO2 affinity (ODC p50 pH 7.4; 31.1 ± 3.1 Torr vs 20.3 ± 1.5 Torr; 0 vs 5mM 5HMF), (2) 30% POS reduction (56.9 ± 10.1 vs 39.8 ± 11.6 Torr; 0 vs 5mM 5HMF) and (3) 45% POR reduction (38.3 ± 9.1 vs 21.1 ± 5.7 Torr; 0 vs 5mM 5HMF) (p < 0.05 for all, RMANOVA). PRODRUG NPs: We designed two complementary 5HMF (phospholipid and graphitic) prodrugs (Lipid Prodrug; LP and carbon dot (CD)) that were evaluated individually and as an integrated self-assembled compound nanoparticle (CompNP). Phospholipid LP 'pro-HMF' synthesis: 5HMF was treated with NaH and THF (RT, N2), then mixed with 1-Lysophosphatidylcholine (16:0 lysoPC) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-succinimide (NHS) (anhydrous chloroform, RT, ON). Graphitic CD 'pro-HMF' synthesis: sucrose in water (0.2 μM, 18 MΩ cm) was heated (270C, 30m), followed by sonication (Q700, 20m: Amp, 1, on: 2s, off: 1s), filtering (0.2 μm) and dialysis (10 kD MWCO, 2d). Simple LNPs (comprised only of LP pro-5HMF) were prepared by combining 5HMF LP (5 mM) with L-α-Phosphatidylcholine (1:1) in chloroform/methanol, then drying (thin film) that was resuspended (PBS), vortexed and sonicated (amp 1, on 2s, off 1s, 10m) and dialyzed (2d, PBS). Compound Nanoparticle (CompNP) self-assembly: CDs in PBS were mixed with the above lipid film, followed by sonication and dialysis (as above) resulting 152±12nm (hydrodynamic size) particles with -34±6mV surface charge. All formulations were extensively characterized (physicochemical features and biodegradation mapping). NP PK/PD: We found that the prodrugs release 5HMF in RBCs through complimentary mechanisms (acutely from the LP shell by phospholipase A2 (PLA2) activity and slowly/sustained from the CD core by enzyme-triggered and peroxide-based degradation). Then, using human SS RBCs, we quantified time- and concentration-dependent in vitro PK/PD parameters (partially presented here): 3h data for the composite NP: Hb-5HMF modification (HPLC, 76.7±8.9% 5HMF modificatied HbS), HbO2 affinity (p50 15.4±3.8Torr) and antisickling potency (LORRCA deformability/elongation index rose by 0.28±3.8.07AU & point of sickling fell by 21.4±8.2Torr); imaging: ~75% reduced hypoxia-induced sickling (p<0.05, ANOVA, all). Notably, PK (5HMF modified SHb) for LNP & CompNP was sustained well beyond that for free 5HMF and was progressive beyond 24h (likely due to increased PLA2 and oxidizing milieu in SRBCs). PD (anti-sickling) for the CompNP formulation exhibited the greatest efficacy for (POS) (eg hypoxia tolerance) and POR (simulating O2 loading during pulmonary circulatory transit); as for our PK data, effect upon POS/POR progressed from 1h to 3h. NB: No produg/NP formulation resulted in hemolysis > 1-2%. SUMMARY: We developed first in class RBC-targeted, protected, biocompatible, self-assembled 5HMF prodrug nanoparticles for SCD therapy that are designed for eventual transdermal delivery. In vitro proof of concept data demonstrates: (1) improved potency, (2) targeted payload delivery and subsequent extended (>24h) prodrug release within RBCs, and (3) with prolonged pharmacodynamic (antisickling) effect.

Increased Hemoglobin Affinity for Oxygen with GBT021601 Reduces Pulmonary Hypertension and Lung Vascular Inflammation in a Murine Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin gene coding sequence that results in the production of hemoglobin S (HbS). Under conditions of red blood cell (RBC) deoxygenation, HbS polymerizes and leads to RBC sickling, resulting in hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Despite improvements in treatment options, SCD remains a debilitating condition, and most patients experience progressive end-organ damage with associated complications and repeated hospitalizations. Pulmonary hypertension (PH), which is associated with increased mortality, is a common complication in patients with SCD, with nearly 10% of the SCD population reporting PH. PH in patients with SCD is multifactorial and may be caused by hemolysis, nitric oxide scavenging, and/or tissue hypoxia. Pharmacological increase of the concentration of oxygenated HbS (oxyHbS) in RBCs by voxelotor, a first-generation HbS polymerization inhibitor, reduces RBC sickling and preserves oxygen (O2) delivery by reducing anemia and improving blood rheology. Limited data are currently available on whether inhibiting HbS polymerization by increasing oxyHbS concentration may improve end-organ damage and associated complications, including PH. Objective: To examine the effects of GBT021601, a small-molecule, investigational, second-generation HbS polymerization inhibitor, on hemoglobin (Hb)-O2 affinity, PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Methods: Townes HbSS mice treated with GBT021601 (75 mg/kg orally once daily for 14 days) were exposed to acute hypoxia (8% O2) for 1 hour and reoxygenated for 72 hours. Untreated HbAA Townes mice were used as controls. Lung inflammation was assessed in terms of accumulation of inflammatory cells, activation of nuclear factor kappa B subunit 1, and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. To evaluate the impact of GBT021601 treatment on Hb-O2 affinity and anemia, p50 (partial O2 pressure at which Hb is 50% saturated with O2) and hematocrit levels were measured in SCD mice. In vivo pulmonary hemodynamic measurements included pulmonary artery pressure and right atrial pressure, whereas right ventricular hypertrophy was assessed using the Fulton index, which is the ratio of right ventricular weight to the weight of the left ventricle plus septum. Results: The total number of inflammatory cells and the specific numbers of lymphocytes, macrophages, and neutrophils were lower in untreated HbAA Townes mice and in HbSS Townes mice treated with GBT021601 compared with vehicle-dosed HbSS mice (Figure, panel A). Chronic treatment with GBT021601 increased Hb-O2 affinity, reducing the p50 in SCD mice from 32 mm Hg to 14 mm Hg. The treatment significantly reduced anemia, increasing hematocrit from 27.2% in the vehicle-dosed HbSS mice to 38.5% in the mice treated with GBT021601. Chronic GBT021601 treatment in HbSS mice also reduced pulmonary artery and right atrial pressure compared with those in untreated HbSS mice. Additionally, GBT021601 reduced right ventricular hypertrophy in HbSS mice, as evidenced by a reduction in the Fulton ratio (Figure, panel B). Conclusions: GBT021601 reduced PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Our findings suggest that reducing RBC sickling, anemia, and hemolysis by increasing HbS affinity for O2 plays a critical role in modulating the immune system in PH and lung vasculature. These findings highlight the potential therapeutic efficacy of GBT021601 based on its ability to increase HbS affinity for O2 and prevent HbS polymerization and RBC sickling to reduce PH in SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GBT021601 Prevents Microvascular Dysfunction and Improves Hypoxia-Reoxygenation-Induced Vaso-Occlusion in a Murine Model of Sickle Cell Disease

Background: In sickle cell disease (SCD), a single nucleotide change in the β-globin gene coding sequence results in the production of hemoglobin S (HbS), which polymerizes when deoxygenated and leads to red blood cell (RBC) sickling, hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Pharmacological increase of the oxygenated HbS concentration in RBCs has been shown to be a well-tolerated and effective approach to inhibiting HbS polymerization, reducing RBC sickling, and improving oxygen (O2) delivery by reducing anemia and improving blood rheology. GBT021601, a potent second-generation small-molecule HbS polymerization inhibitor, is currently under investigation and has previously been shown to increase hemoglobin (Hb), improve hemolysis, and improve RBC health in a murine model. However, little is known regarding its impact on vaso-occlusion and microvascular dysfunction. Objective: To examine the effects of GBT021601 on SCD-induced microvascular dysfunction, capillary perfusion and density, Hb-O2 oxygen affinity, and Hb and hematocrit levels in a murine model of SCD. Methods: Capillary perfusion was measured by functional capillary density (FCD, a marker of capillary perfusion defined as the number of capillaries with RBCs in transit) in a murine model of SCD with an implanted dorsal window chamber (Figure). HbSS Townes mice were treated with GBT021601 (75 mg/kg) by oral gavage daily for 14 days and were compared with untreated HbSS and HbAA Townes mice. Results: Chronic treatment of HbSS mice with GBT021601 led to increased Hb-O2 affinity, evidenced by a reduction in blood p50 (partial O2 pressure at which Hb is 50% saturated with O2) from 32 mm Hg to 14 mm Hg. GBT021601 significantly reduced anemia by increasing total Hb and hematocrit from 8.3 g/dL and 26% in untreated HbSS mice to 11.2 g/dL and 37% in GBT021601-treated HbSS mice, respectively (P<0.01). Moreover, GBT021601 treatment prevented microvascular dysfunction (reduced RBC velocities and blood flow) in the arterioles and venules of HbSS mice. Increased capillary density is indicative of greater oxygen transport. Challenging HbSS mice with hypoxia (8% O2 for 1 hour) followed by reoxygenation (21% O2) induced vaso-occlusion, causing reduced microvascular blood flow and FCD in untreated mice. However, in GBT021601-treated HbSS mice, microvascular blood flow and FCD were significantly higher than those in untreated HbSS mice. Unlike in untreated HbSS mice, microvascular blood flow and FCD returned to baseline values after 72 hours of reoxygenation in treated HbSS mice. These results suggest that by increasing HbS-O2 affinity and thereby inhibiting HbS polymerization, GBT021601 may attenuate hypoxia-induced vaso-occlusion and impaired capillary perfusion. Furthermore, GBT021601 treatment in HbSS mice reduced both circulating adhesion markers and leukocyte-endothelium interactions in venules after hypoxia and reoxygenation. Conclusions: GBT021601 increased microvascular perfusion, reduced vaso-occlusion, and improved the hypoxia-induced reduction in capillary perfusion in HbSS mice, suggesting that chronic GBT021601 treatment prevents SCD-induced microvascular dysfunction and mitigates the rebound vaso-occlusion induced by hypoxia and reoxygenation in a murine model of SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Temperature independence of haemoglobin-oxygen affinity in smalleye Pacific opah (Lampris incognitus) and swordfish (Xiphias gladius).

Smalleye Pacific opah and swordfish can conserve metabolic heat and maintain specific body regions warmer than ambient water temperature (i.e. regional heterothermy). Consequently, blood O2 uptake at the gills occurs at the environmental temperature at which the individual is found, but O2 offloading will occur at different temperatures in different tissues. While several regionally heterothermic fishes (e.g. billfishes, tunas and sharks) show a reduced temperature effect on haemoglobin (Hb)-O2 affinity, the temperature dependence of Hb-O2 affinity in opah and swordfish is unknown. We hypothesized that the Hb of opah and swordfish would also show a reduced temperature dependence. Opah whole-blood-O2 affinity exhibited a reverse temperature dependence above 50% Hb-O2 saturation (10-20°C, pH7.2-8.0), while the temperature dependence of swordfish blood-O2 affinity (10-25°C) was saturation and pH dependent, becoming temperature independent below 50% Hb-O2 saturation and pH7.4. Experiments on stripped haemolysates showed that adding ATP ([ATP]/[Hb]=30) decreased the temperature sensitivity of Hb-O2 affinity, changing the overall oxygenation enthalpy (ΔH') values of opah (10-20°C) and swordfish (10-25°C) Hbs at pH7.4 from -15 and -42 kJ mol-1 O2, respectively, to +84 and -9 kJ mol-1 O2. Swordfish blood-O2 affinity was high compared with that of other large, pelagic, marine teleosts, which may be the result of unusually low ATP/Hb levels, but might also enable swordfish to forage in the potentially low-oxygenated water of the upper reaches of the oxygen minimum layer. The existence of Hbs with reduced temperature sensitivity in regionally heterothermic fishes may prevent marked changes in Hb-O2 affinity between the cold and warm tissues.

The effect of desflurane, isoflurane and sevoflurane on the hemoglobin oxygen dissociation curve in human blood samples

Desflurane, isoflurane and sevoflurane, three halogenated ethers, are commonly used inhaled anesthetics, both in the operating room and in the intensive care unit (ICU). The potency and dosage of these drugs is expressed by the MAC value (minimum alveolar concentration). Their interaction with hemoglobin and its affinity for oxygen, best described by the oxygen dissociation curve (ODC), has already been investigated, with conflicting results. Altered by many factors, the ODC can be shifted to the left or to the right, therefore increasing or decreasing hemoglobin oxygen (Hb-O2) affinity. In venous blood samples of 22 healthy participants (11 female, 11 male) ODC were measured with a high-throughput method in vitro. Blood samples were either exposed to control or to three different concentrations of desflurane, isoflurane or sevoflurane prior to and during measurements (low, medium and high corresponding to MAC 0.5, MAC 1.0 and MAC 2.0). With increasing concentrations from control to medium, desflurane and isoflurane significantly decreased Hb-O2 affinity by shifting the ODC to the right (p = 0.016 and p < 0.001) but sevoflurane showed no effects. When further increasing concentrations from medium to high, all three inhaled anesthetics shifted the ODC back to the left (p < 0.001). Comparing only controls to high concentrations, a significant increase in Hb-O2 affinity for desflurane (p = 0.005) and sevoflurane (p < 0.001) was detected. Our study shows a varying effect at different doses of inhaled anesthetics on Hb-O2 affinity. While the underlying mechanisms remain unclear, these results show an effect which needs to be further investigated to determine if patients undergoing anesthesia may potentially benefit or get disadvantage from this slightly increased (e.g. impaired pulmonary oxygen uptake), or decreased Hb-O2 affinity (e.g. arterial vascular disease).Trial registration: This study is registered with clinicaltrials.gov (NCT04612270).

The impact of nebulized epoprostenol and iloprost on hemoglobin oxygen affinity: an ex vivo experiment.

Inhalational prostacyclins act as strong vasodilators, potentially improving oxygenation by reducing shunt fraction and ventilation-perfusion mismatch. As prostacyclin receptors are known to be present on human erythrocytes, possible direct effects on hemoglobin oxygen transport were further explored by examining the sole in vitro influence of prostacyclins on hemoglobin oxygen (Hb-O2) affinity. Venous blood samples from 20 healthy volunteers were exposed in vitro to supramaximal doses of epoprostenol, iloprost, and compared with control. By high-throughput measurements, hemoglobin oxygen dissociation curves (ODCs) were derived. Hb-O2 affinity, expressed by P50 and Hill coefficient, was determined and analyzed for three subgroups: males (n = 10), females not taking oral contraceptives (n = 4), and females taking oral contraceptives (n = 6). Epoprostenol significantly decreased P50 in all (males, females without contraceptives, and females taking oral contraceptives) [27.5 (26.4–28.6) mmHg (control) vs. 24.2 (22.7–25.3) mmHg; P < 0.001. median (interquartile range, IQR)] thereby increasing Hb-O2 affinity. Inversely, iloprost only showed significant effects in females taking oral contraceptives where P50 was markedly increased and therefore Hb-O2 affinity decreased [28.4 (27.9–28.9) mmHg (control) vs. 34.4 (32.2–36.0) mmHg; P < 0.001]. Prostacyclin-receptor stimulation and subsequent cAMP-mediated ATP release from erythrocytes are discussed as a possible underlying mechanism for the effect of epoprostenol on Hb-O2 affinity. The reason for the sex hormone-modified iloprost effect remains unclear. Being aware of potentially differing effects on Hb-O2 affinity might help select the right prostacyclin (epoprostenol vs. iloprost) depending on the patient and the underlying disease (e.g., acute respiratory distress syndrome vs. peripheral arterial disease).