Oxygen Affinity Of Hb Research Articles

A Rare Case of Homozygous Hemoglobin G-Philadelphia & Alpha Thalassemia

Abstract Introduction/Objective Hemoglobin (Hb) G-Philadelphia (GPh) is an alpha chain gene variant caused by a missense mutation resulting in a change from Glu to Asp at position 68. This change leads to alterations in the Hb structure and oxygen affinity. The mutation is inherited in an autosomal codominant pattern allowing for an asymptomatic carrier state. While the most common alpha chain variant, GPh heterozygosity is still infrequent, estimated at 1:5000 in African Americans. In African Americans, GPh is frequently linked to an alpha thalassemia deletion. On its own it is considered a benign variant that does not result in significant health issues. Homozygous HbGPh frequency is estimated at 1 in 25 million. Methods/Case Report A postpartum woman in her 4th decade of life was found to have a Hb of 10.8 and a mean corpuscular volume of 74. Initial work up included Hb electrophoresis that quantified HbA at 0%, Hb F at 0%, HbA2 at 0%, and 2 Hb variants representing 96.1% and 3.8% of the total globin. The major Hb variant migrated in the D or G position by alkaline and acid gel Hb electrophoresis. The minor variant was consistent with HbG2. Due to this abnormal Hb electrophoresis, a sample was sent out for a Hb evaluation reflexive cascade (HERC). HERC analysis by high-performance liquid chromatography and capillary gel electrophoresis found 99.9% of Hb consistent with the alpha chain variant HbG. Analysis of the alpha globin gene region revealed 2 copies of an alpha globin gene deletion that was interpreted as alpha-thal trait (-a/-a). This result is consistent with homozygous alpha-thal-2 and homozygous HbGPh (alpha^G/thal + alpha^G/thal). Results (if a Case Study enter NA) NA Conclusion Alpha^G/thal + alpha^G/thal presents a unique and complex challenge for diagnosis due to its rarity and because HbG2 is not always visualized by alkaline gel electrophoresis. The presence of a Hb G or D variant, a HbG2 variant, and the lack of both HbA and HbA2 are key electrophoretic findings. Clinically, alpha^G/thal + alpha^G/thal behaves essentially like a homozygous alpha-thal-2 trait with persistent microcytosis and erythrocytosis. Thus, co- inheritance of the alpha-thal trait will be suggested by a thalassemic complete blood count pattern.

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.

Mechanisms Underlying the Effects of Chloroquine on Red Blood Cells Metabolism.

Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug's ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 μM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

Molecular Insights of an Avian Species with Low Oxygen Affinity, the Crystal Structure of Duck T-State Methemoglobin.

Hemoglobin (Hb) is the key metalloprotein within red blood cells involved in oxygen transportation from lungs to body cells. The heme-iron atom inherent within Hb effectuates the mechanism of oxygen transportation and carbon dioxide removal. Structural investigations on avian Hb are limited when compared with the enormous work has been carried out on mammalian Hb. Here, the crystal structure of T-state methemoglobin (T-metHb) from domestic duck (Anas platyrhynchos), a low oxygen affinity avian species, determined to 2.1Å resolution is presented. Duck T-metHb crystallized in the orthorhombic space group C2221 with unit cell parameters a = 59.89, b = 109.42 and c = 92.07Å. The final refined model with R-factor: 19.5% and Rfree: 25.2% was obtained. The structural analysis reveals that duck T-metHb adopts a unique quaternary structure that is distinct from any of the avian liganded Hb structures. Moreover, it closely resembles the deoxy Hb of bar-headed goose, a high oxygen-affinity species. Besides the amino acid αPro119 located in the α1β1 interface, a unique quaternary structure with a constrained heme environment is attributed for the intrinsic low oxygen-affinity of duck Hb. This study reports the first protein crystal structure of low oxygen-affinity avian T-metHb from Anas platyrhynchos.

The Preliminary Study on Preparation Technology of PolyHb-SOD-CAT-CA - The Effects of Different Extractants.

To explore the alternative to extractants for the preparation technology of polyHb-SOD-CAT-CA derived both from bovine blood and human cord blood, compared to the toluene currently used in the existing studies, several organic solvents, including n-hexane and ethyl ether, were investigated during the polyHb-SOC-CAT-CA preparation process. During the technological process, the effects of investigated organic extractants on the properties and stability of hemoglobin and enzymes, including SOD, CAT and CA, were investigated by monitoring the property indexes of macromolecules, including Hb concentrations, MetHb contents, molecular weight distribution, oxygen affinity of Hb, enzymes activities, and so on. Among the investigated experimental groups, the results, including the Hb recoveries, MetHb contents, oxygen affinity, the molecular weight distribution of obtained complex, and enzyme activity, presented to be the best in n-hexane groups, followed by those in toluene groups, and these results in ether groups showed to be the worst. Meanwhile, during the preparation process, both for bovine and human cord derivatives, the changing of properties indexes of Hb and enzymes investigated demonstrated similar declining trends on the whole, and the oxygen-carrying properties and enzyme activities showed to be in the effective range. Among the investigated organic extractants, both for preparation of bovine and human cord-derived polyHb-SOD-CAT-CA complex, n-hexane indicated to have much less negative influence on the properties and stability of Hb and enzyme molecules, including SOD, CAT, and CA. Moreover, the obtained human cord blood polyHb-SOD-CAT-CA also showed effective oxygen-carrying properties and enzyme activities, suggesting the promising application prospects of polyHb-SOD-CAT-CA and the new generation HBOC products in the future.

Safety and Efficacy of Mitapivat in Adult Patients with Erythrocyte Membranopathies (SATISFY)

Background: Membranopathies encompass hemolytic disorders arising from genetic defects in erythrocyte membrane proteins and include hereditary spherocytosis and stomatocytosis. Congenital dyserythropoietic anemia type II (CDA II) is associated with SEC23B gene variants and its phenotype may be similar to hereditary spherocytosis. Anemia with or without the need for chronic transfusion, jaundice, splenomegaly, iron overload are complications common in both hereditary membranopathies and CDA II. Current treatment options for membranopathies and CDA II 1, apart from splenectomy, are primarily supportive. Mitapivat, an investigational pyruvate kinase-R (PKR) activator, has demonstrated efficacy in improving anemia and reducing hemolysis in adult patients with PK deficiency, thalassemia, sickle cell disease, and a mouse model of hereditary spherocytosis. Study Design and Methods: This study (NCT05935202) is an investigator initiated, prospective, multicenter, single-arm phase 2 trial. The study will be conducted in the European Union in Denmark and The Netherlands. It is the first clinical study in the context of the European Reference Network EuroBloodNet and will be sponsored by its closely associated non-for-profit EuroBloodNet Association. A sibling study in Toronto, Canada will be registered separately, and data will be pooled in a prespecified statistical analysis plan. The study will include approximately twenty-five adult patients (aged 18 years and older) diagnosed with a membranopathy or CDA II. Diagnosis must be confirmed genetically. Average hemoglobin concentration (Hb) must be less than 13.0 g/dL for males and 11.0 g/dL for females. Patients with average Hb >10.0 g/dL for males and females at screening must meet at least one of the following additional criteria: Splenomegaly, fatigue attributed to hemolysis, or paraclinical hemolysis. Adequate organ function is required. Patients cannot be included if they have a diagnosis of PK deficiency or if they have received red blood cell (RBC) transfusions (≥5 units the last 12 months or any within the last 3 months). During the 8-week Dose Escalation Period, subjects will receive an initial dose of 50 mg mitapivat twice daily (BID). Based on safety and changes in Hb levels, dosing may be increased to 100 mg BID at week 4. Patients who tolerate mitapivat may be eligible to continue in two consecutive 24-week Fixed Dose Periods, with an interim analysis in between the two fixed dose periods. The primary objective of this study is to evaluate the safety of mitapivat, assessed through the occurrence of treatment-emergent adverse events. Secondary objectives include assessing the effects of mitapivat on Hb concentration (≥1.0 g/dL increase sustained at two scheduled visits), hemolysis, erythropoiesis, patient-reported outcome measures, and spleen size. Exploratory endpoints include enzyme activity and stability of PK, Hb oxygen affinity, proteomics and metabolomics. To assess how activation of PK will impact RBC function we will measure RBC deformability (osmotic gradient ektacytometry and cell membrane stability) in whole blood and in different RBC subpopulations. Acknowledgement: This project is carried out within the framework of European Reference Network on Rare Haematological Diseases (ERN-EuroBloodNet)-Project ID No 101085717. ERN-EuroBloodNet is partly co-funded by the European Union within the framework of the Fourth EU Health Programme. The study is funded by a grant from Agios Pharmaceuticals.

One-Year Safety and Efficacy of Mitapivat in Sickle Cell Disease: Follow-up Results of a Phase 2, Open-Label Study

Background Targeting the primary pathogenic event of sickle cell disease (SCD), polymerization of deoxygenated sickle hemoglobin (HbS), may prevent downstream clinical events. Mitapivat, an oral allosteric activator of pyruvate kinase (PK), has shown therapeutic potential by increasing adenosine triphosphate (ATP) and decreasing 2,3-diphosphoglycerate (2,3-DPG), a glycolytic red blood cell (RBC) intermediate. In addition to improving anemia and hemolysis, mitapivat may reduce HbS polymerization and inhibit sickling by decreasing 2,3-DPG levels in patients with SCD. In the previously reported 8-week dose-finding period (DFP) of this phase 2 study, mitapivat was well tolerated and showed efficacy in SCD ( Van Dijk et al. Am J Hematol. 2022). Here, the safety and efficacy of treatment with mitapivat in patients with SCD from the 1-year fixed-dose extension period (FDEP) are reported (EudraCT 2019-003438-18, ESTIMATE study). Methods The ESTIMATE study is a phase 2, investigator initiated, open-label study in which subjects ≥16 years with SCD (HbSS, HbS/β 0, HbS/β +) with a baseline hemoglobin (Hb) >4.0 g/dL and ≤11.1 g/dL, no chronic transfusion and adequate organ function were eligible. After the 8-week DFP in which patients were dosed mitapivat 20 mg, 50 mg or 100 mg twice daily depending on safety, patients could continue in the 1-year FDEP. The primary endpoints were safety, evaluated by frequency and severity of adverse events (AEs), and efficacy of mitapivat including the evaluation of a hematological response (improvement in mean Hb level of ≥1 g/dL compared to baseline during the FDEP). RBC sickling was evaluated by oxygen gradient ektacytometry (point of sickling, Lorrca). Secondary endpoints included changes in hematological parameters including Hb, absolute reticulocyte count (ARC), total bilirubin, lactate dehydrogenase (LDH), levels of ATP and 2,3-DPG and Hb-oxygen affinity, as reflected by the oxygen tension at which Hb is 50% saturated (p50, Hemox Analyzer). Exploratory endpoints included changes in annualized rates of vaso-occlusive events (VOEs) and annualized SCD-related hospital admission days. Intention-to-treat analysis was performed using IBM SPSS Statistics (version 27.0.0.0) and Graphpad Prism (version 9.3.0) with the paired sample t-test or Wilcoxon signed-rank test used when appropriate. Results In total, ten patients were included in this study. Safety analysis showed mostly mild treatment-emergent adverse events (most commonly [n>2 patients]: transaminase increase [all grade 1] and headache [grade 1-2]). Apart from the non-treatment-related serious adverse event (SAE) of a urinary tract infection in the DFP in a patient who was lost to follow-up afterwards, one non-treatment-related SAE occurred in the FDEP in a patient who died of massive pulmonary embolism due to COVID-19. Importantly, sustained improvements in Hb level (mean increase: 1.1 ± 0.7 g/dL; p=0.0014) were seen in the nine patients who continued treatment with mitapivat 50 mg twice daily (n=2) or 100 mg twice daily (n=7) in the FDEP (Table 1). These improvements were accompanied by decreases in markers of hemolysis (ARC, total bilirubin and LDH) (Table 1). On a cellular level, the ATP/2,3-DPG ratio and Hb-oxygen affinity increased significantly, and RBC sickling was reduced, though non-significantly (Table 1). In addition, the annualized rate of vaso-occlusive events reduced significantly from 1.33 ± 1.32 to 0.64 ± 0.87 (p=0.0489) when combining the DFP and FDEP (Table 1 and Figure 1). Conclusion Overall, this study demonstrated long-term safety and efficacy of treatment with mitapivat in SCD, warranting further evaluation in the ongoing phase 2/3 RISE UP study (ClinicalTrials.gov NCT05031780).

Red Blood Cell Deformability and Morphological Dynamic Sickling As a Metric to Monitor Pharmacodynamic Response to Voxelotor

The recent introduction of new therapies, such as hemoglobin (Hb) oxygen affinity modifiers, fetal Hb (HbF) inducers, and gene therapy (replacement of HbS with non-sickling Hb), that affect the Hb polymerization rate for sickle cell disease (SCD), highlight the need for new metrics to assess changes in sickling characteristics as therapeutic response. Primary outcomes in clinical trials often focus on indirect clinical improvement i.e. decrease in vaso-occlusive events (VOC), Hb increase, fatigue and pain score. There is currently a lack of direct biomarkers to assess the pharmacodynamic impact of these therapies. Two methods have been developed to assess downstream Hb polymerization, LoRRca (oxygenscan), a commercial oxygen gradient ektacytometry that measures deformability as a function of oxygen tension (pO 2), and dynamic sickling assay (DSA), an enzymatic oxygen scavenging system that directly assesses morphological sickling. In the present study, we investigate the relationship between red blood cell (RBC) deformability and kinetics of sickling morphology after a treatment with different doses of voxelotor (GBT440, Global Blood Therapeutics), the only FDA approved Hb oxygen modifier. Blood samples from eight SCD patients were collected following the IRB #041718MP2E & IRB #Pro00066220 protocols. RBCs were incubated with voxelotor at five different doses based on sample hematocrit (Hct): 25% (0.32 ± 0.06 mM), 50% (0.65 ± 0.11 mM), 75% (0.97 ± 0.17 mM), 100% (1.29 ± 0.23 mM) and 125% (1.62 ± 0.28 mM), or with an equal volume of DMSO-containing buffer (vehicle). Oxygenscan is performed (LoRRca, RR Mechatronics, The Netherlands) as per manufacturer's instructions. The following parameters are calculated: i) elongation index (EImin), measured at minimum RBC deformability, ii) point of sickling (PoS), the pO 2 at which RBC deformability decreases below 5% of EImax during deoxygenation and iii) ΔEI, the difference between EImin and EImax. DSA uses an enzymatic reaction that allows for tight control of the rate and depth of hypoxia, resulting in RBC morphological changes observed through time-lapse photography. Sickling profiles were constructed to represent: i) morphological points of sickling at 50% of induced fraction (mPoS i@50%), ii) rate of sickling at 50% of induced fraction (rate i@50%) and iii) area under the curve at 15 minutes (AUC 15). Paired t-test analysis of the LoRRca elongation curves show a significant dose response in Elmin (Vehicle: 0.16 ± 0.16; 25%: 0.20 ± 0.16, p=0.0033; 50%: 0.23 ± 0.15, p=0.0001; 75%: 0.27 ± 0.15, p=0.0004; 100%: 0.30 ± 0.14, p=0.0009; 125%: 0.35 ± 0.13, p=0.0015), ΔEl (Vehicle: 0.26 ± 0.11; 25%: 0.22 ± 0.11, p=0.0016; 50%: 0.18 ± 0.11, p=0.0003; 75%: 0.15 ± 0.08, p=0.0002; 100%: 0.11 ± 0.06, p=0.0004; 125%: 0.09 ± 0.07, p=0.0011) and PoS (Figure 1; Vehicle: 35 ± 11 mmHg; 25%: 32 ± 6, p=0.0092; 50%: 28 ± 7, p=0.0038; 75%: 25 ± 6, p<0.0001; 100%: 17 ± 5, p=0.0007; 125%: 14 ± 4, p=0.0015). These findings indicate improvements in RBC deformability under hypoxic conditions when the drug is present. Furthermore, paired t-test of DSA data showed a significant dose response with AUC 15 (Vehicle: 958 ± 104; 25%: 909 ± 105, p=0.0061; 50%: 845 ± 146, p=0.0004; 75%: 768 ± 194, p=0.0014; 100%: 672 ± 179, p<0.0001; 125%: 623 ± 192, p=0.0002), rate i@50% (Vehicle: 0.87 ± 0.26; 25%: 0.77 ± 0.30, p=0.1236; 50%: 0.61 ± 0.29, p=0.0003; 75%: 0.33 ± 0.13, p<0.0001; 100%: 0.28 ± 0.10, p<0.0001; 125%: 0.25 ± 0.09, p=0.0004) and mPoS i@50% (Figure 2; Vehicle: 4.4 ± 0.7 minutes; 25%: 4.7 ± 0.9, p=0.0260; 50%: 5.0 ± 1.2, p=0.0407; 75%: 5.9 ± 1.7, p=0.0082; 100%: 7.0 ± 1.6, p=0.0002; 125%: 7.8 ± 1.8, p=0.0004). These results indicate a delay in morphological sickling due to drug treatment. Based on the results of our study there is a dose-dependent decrease in RBC deformability and in the kinetics of morphologic sickling based of DSA following voxelotor treatment. These two methods have demonstrated a comparable ability to assess downstream therapeutic effects on delaying/inhibiting HbS polymerization. The combination of these two unique properties, RBC deformability and morphology change, can possibly provide a comprehensive approach to describe the cellular level of phenotypes, which may predict SCD severity. With further development these complementary assays have the potential to become important tools for drug development and monitoring response to SCD therapies.

Fetal Hemoglobin Decrease during Voxelotor Treatment

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to the formation of hemoglobin S (HbS). The polymerization of deoxygenated HbS is the first event in the molecular pathogenesis of SCD resulting in sickling of red blood cells (RBC), and hemolytic anemia which has been shown to be associated with organ damage and early death. Hemolytic anemia is defined by an accelerated rate of destruction RBC resulting in a lowered Hb level and a reduced gas transport capacity. Because Hb S in its oxy (R) conformation do not polymerize, drugs modifying hemoglobin (Hb) to increase the proportion of oxy-HbS have been propose to inhibit HbS polymerization. Voxelotor is such a molecule, which allosterically modifies Hb-oxygen affinity improving anemia and reducing hemolysis. However, the impact of Voxelotor on HbF levels is unknown. We describe here variations of %HbF and Mean corpuscular HbF (MCHbF) in a cohort of homozygous SCD patients treated with Voxelotor in our Sickle Cell Referral center. We conducted a prospective register study to analyze clinical and biological evolution of SCD patients treated with Voxelotor at 1500mg taken orally once daily. From December 2021 through December 2022, 25 SCD adult patients were followed during a median of 7 months [1-12]. Among them, HbF values were analyzable for 15 patients (SS gentype n=14; women in 73%; median age of 50 years [24-61]) who completed 6 months of Voxelotor with no transfusion during the same period; and with a stable dose of hydroxyurea (HU), for at least 3 months before inclusion and during the study in case of patients treated by HU (n=10). The dose of Voxelotor was stable for 13/15 patients and was decreased in 2 patients due to skin allergy (n=1) and moderate digestive intolerance. One patient died after 6 months of treatment in a context of end-stage renal failure and severe pulmonary hypertension not related to Voxelotor treatment. Mean corpuscular HbF (MCHbF) was calculated (MCHbF (pg) = MCH x %HbF). HbF percentage was measured by high performance liquid chromatography (Biorad, Variant II, beta-thal dual program). All participants provided written informed consent (CPP Erythropedie). From baseline to 6 months of follow up mean Hb (g/dL) concentration increased from 7.4 [5.4-8.5] to 8 [5-11.9] (p=0,0003; Figure A); absolute reticulocyte count (ARC ; Giga/L) decreased from 158 to 145 (p=0,005), %HbF decreased by 63% in all patients from 7,1% to 3,3% (p=0,008; Figure B) and MCHbF (pg) decreased from 3,36 to 1,03 (p=0,001; Figure D). A decrease in % HbF was observed from the first 15 days of treatment in both groups of patients treated or not with HU (Figure C) ; no changes in mean corpuscular volume (p=0.15) was observed in patients treated with HU during follow up. The HbF decrease in SCD patients treated with Voxelotor could be explained by different mechanisms: - Reduction of stress erythropoiesis which is due to an increase in erythroid output in response to anemic stress associated with a reduced expression of BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. This hypothesis is supported by the reduction in reticulocytes in patients treated with Voxelotor - The loss of an HbF-related survival advantage for SCD RBCs, while the normal RBC lifespan is approximatively 120 days, in SCD RBCs it is only a sixth of it. HbF is a major modulator of SCD and prolonge SCD RBCs survival. Its protective effect relies on its quantity and distribution among the RBC population, which is often heterogeneous in SCD patients. Protection against polymerisation of Voxelotor is probably more homogeneous among RBCs. As a result, RBCs containing low levels of HbF, which would normally disappear, have a prolonged lifespan. Similarly, it was presumed that sickling of erythroblasts could lead to ineffective terminal erythroid differentiation with an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. However we found no significant difference for index of ineffective erythropoiesis (soluble transferrin receptor / absolute reticulocyte count) before and after 6 months of treatment. The question raised is therefore that of the risk of a rebound effect upon discontinuation of Voxelotor, which has a short half-life, during the time it takes for HbF to increase. To better study this hypothesis, distribution of HbF/RBC will be studied in the HEMOPROVE trial (NCT05199766) currently in progress.

One-year safety and efficacy of mitapivat in sickle cell disease: follow-up results of a phase 2, open-label study

AbstractTargeting the primary pathogenic event of sickle cell disease (SCD), the polymerization of sickle hemoglobin (HbS), may prevent downstream clinical events. Mitapivat, an oral pyruvate kinase (PK) activator, has therapeutic potential by increasing adenosine triphosphate (ATP) and decreasing 2,3-diphosphoglycerate (2,3-DPG), a glycolytic red blood cell (RBC) intermediate. In the previously reported 8-week dose-finding period of this phase 2, investigator-initiated, open-label study, mitapivat was well tolerated and showed efficacy in SCD. Here, the 1-year fixed-dose extension period is reported in which 9 of 10 included patients (90%) aged ≥16 years with SCD (HbSS, HbS/β0, or HbS/β+) continued with mitapivat. Mostly mild treatment-emergent adverse events (AEs) (most commonly, transaminase increase and headache) were still reported. Apart from the reported nontreatment-related serious AE (SAE) of a urinary tract infection in the dose-finding period, 1 nontreatment-related SAE occurred in the fixed-dose extension period in a patient who died of massive pulmonary embolism due to COVID-19. Importantly, sustained improvement in Hb level (mean increase, 1.1 ± 0.7 g/dL; P = .0014) was seen, which was accompanied by decreases in markers of hemolysis. In addition, the annualized rate of vaso-occlusive events reduced significantly from a historic baseline of 1.33 ± 1.32 to 0.64 ± 0.87 (P = .0489) when combining the dose-finding period and fixed-dose extension period. Cellularly, the ATP:2,3-DPG ratio and Hb-oxygen affinity significantly increased and RBC sickling (point of sickling) nonsignificantly reduced. Overall, this study demonstrated 1-year safety and efficacy of treatment with mitapivat in SCD, supporting further evaluation in ongoing phase 2/3 study (RISE UP, NCT05031780). This trial was registered at https://www.clinicaltrialsregister.eu/ as NL8517 and EudraCT 2019-003438-18.

A Phase 2/3, Double-Blind, Randomized, Placebo-Controlled, Multicenter Study of Mitapivat in Patients with Sickle Cell Disease: RISE UP Phase 2 Results

Background: Sickle cell disease (SCD) is characterized by the presence of hemoglobin S (HbS), a structural variant caused by a mutation in the HBB gene. Deoxygenated HbS molecules polymerize into “fibers”, causing red blood cells (RBCs) to sickle and hemolyze. Sickled RBCs have a shortened lifespan and impede blood flow to tissues, causing painful vaso-occlusive crises. Recurrent microvascular damage and chronic hemolytic anemia result in progressive multi-organ damage. Mitapivat is an oral, small-molecule allosteric activator of pyruvate kinase, a key enzyme in RBC metabolism, with a dual mechanism of action under investigation in SCD: it increases adenosine triphosphate (ATP) in RBCs, improving survival, and decreases 2,3−diphosphoglycerate, increasing Hb oxygen affinity and thus diminishing HbS polymerization and RBC sickling. RISE UP is a global Phase (Ph) 2/3 double-blind, randomized, placebo (PBO)-controlled trial evaluating the efficacy and safety of mitapivat in patients (pts) with SCD (NCT05031780). Methods: In the Ph 2 double-blind period (DBP), 2 dose levels of mitapivat were evaluated against PBO for 12 weeks (wks). Eligible pts were ≥16 years with ≥5.5 to ≤10.5 g/dL Hb and ≥2 to ≤10 sickle cell pain crises (SCPCs, including pain, priapism, acute chest syndrome, hepatic sequestration, and splenic sequestration) in year prior to entry. Pts were randomized 1:1:1 to mitapivat 50 mg twice daily (BID), mitapivat 100 mg BID, or matched PBO. Pts completing the DBP were eligible to receive mitapivat in a 216-wk open-label extension. Primary endpoints were Hb response (≥1.0 g/dL increase in average Hb concentrations from Wks 10 through 12 compared with baseline [BL], tested with Fisher's exact test at 2-sided 0.05 significance level) and adverse events (AEs), including the type, severity, and relationship to study drug. Prespecified secondary endpoints included average change from BL from Wks 10 through 12 in Hb, markers of hemolysis, markers of erythropoiesis, and pt-reported fatigue score, as well as annualized rate of SCPCs. Results: In the Ph 2 DBP, 79 pts were randomized and treated with mitapivat 50 mg BID (n=26), mitapivat 100 mg BID (n=26), or PBO (n=27). In the 50 mg BID, 100 mg BID, and PBO arms, mean (SD) age was: 29.9 (7.79), 30.2 (10.52), and 28.5 (10.30) years; female: 57.7%, 61.5%, and 74.1%; mean (SD) BL Hb: 8.76 (1.295), 8.82 (0.898), and 8.49 (1.141) g/dL; mean (SD) number of SCPCs during previous year: 3.1 (1.83), 3.2 (1.65), and 3.4 (1.91), respectively. The primary endpoint was achieved; 46.2% (12/26) of pts in the 50 mg BID arm and 50.0% (13/26) in the 100 mg BID arm demonstrated a significant increase in Hb response compared with 3.7% (1/27) of pts in the PBO arm (2-sided p=0.0003 and p=0.0001, respectively). Mitapivat was generally well tolerated; observed safety profile was consistent with previously reported data of mitapivat in SCD and other hemolytic anemias. No serious treatment-emergent AEs were related to study drug; no AEs led to study drug discontinuation or death. Least-squares mean (LSM [95% CI]) for average change from BL in Hb levels from Wks 10 through 12 was 1.11 (0.77, 1.45) g/dL, 1.13 (0.79, 1.47) g/dL, and 0.05 (−0.28, 0.39) g/dL for 50 mg BID, 100 mg BID, and PBO arms, respectively ( Figure). Improvements were observed in markers of hemolysis and erythropoiesis. SCPCs annualized rates (95% CI) were 0.83 (0.34, 1.99) and 0.51 (0.16, 1.59) for 50 mg BID and 100 mg BID arms, respectively, compared to 1.71 (0.95, 3.08) for PBO. LSM (95% CI) for average changes from BL in pt-reported fatigue score from Wks 10 through 12 was −3.80 (−7.16, −0.45), −0.10 (−3.27, 3.08), and −0.17 (−3.40, 3.07) for 50 mg BID, 100 mg BID, and PBO arms, respectively ( Table). Conclusion: During the Ph 2 DBP of RISE UP, treatment with mitapivat demonstrated statistically significant and clinically meaningful improvement in Hb response at both dose levels (50 mg BID and 100 mg BID) compared to PBO. Improvements in markers of hemolysis/erythropoiesis and annualized rate of SCPCs were observed for both doses as compared with PBO; the magnitude of improvement in markers of hemolysis and annualized rate of SCPCs was larger in the mitapivat 100 mg BID arm. Mitapivat was well tolerated with a safety profile consistent with previous studies. These data suggest that mitapivat, through its dual mechanism of action, may provide clinical benefit to pts with SCD and support continued development in the Ph 3 portion of the RISE UP trial.

Design, Synthesis, and Antisickling Investigation of a Thiazolidine Prodrug of TD-7 That Prolongs the Duration of Action of Antisickling Aromatic Aldehyde.

The synthetic allosteric effector of hemoglobin, TD-7 has been investigated as a potential therapeutic agent for the treatment of sickle cell disease. The pharmacologic activity of TD-7 is due to formation of a Schiff-base interaction between its aldehyde group and the two N-terminal αVal1 amines of hemoglobin, effectively inhibiting sickling of red blood cells. However, TD-7 faces a challenge in terms of poor oral bioavailability due to rapid in-vivo oxidative metabolism of its aldehyde functional group. To address this shortcoming, researches have explored the use of a L-cysteine ethyl ester group to cap the aldehyde group to form a thiazolidine aromatic aldehyde prodrug complex, resulting in the improvement of the metabolic stability of this class of compounds. This report details the synthesis of a thiazolidine prodrug of TD-7, referred to as Pro-7, along with a comprehensive investigation of Pro-7 functional and biological properties. In an in-vitro Hb modification and Hb oxygen affinity studies using normal whole blood, as well as erythrocyte sickling inhibition using sickle whole blood, Pro-7 exhibited a gradual onset but progressive increase in all activities. Additionally, in-vivo pharmacokinetic studies conducted with Sprague Dawley rats demonstrated that Pro-7 can undergo hydrolysis to release TD-7. However, the blood concentration of TD-7 did not reach the desired therapeutic level. These findings suggest that the incorporation of the L-cysteine ethyl ester group to TD-7 represents a promising strategy to enhance the metabolic stability of aromatic aldehydes that could lead to the development of a more effective drug for the treatment of sickle cell disease.

Phylogeny and evolution of erythrocytes in mammals.

In the past century, several authors have investigated the allometry of haematological parameters in mammals. As haematocrit and haemoglobin (Hb) concentration are almost constant within the Mammalia (although with notable exceptions), differences in other haematological parameters are mainly reducible to red blood cell size (mean corpuscular volume, MCV). Past studies testing for correlation between MCV and body mass have given contradictory results. Using phylogenetically informed regressions, here I demonstrate that the correlation between MCV and body mass is indirect, and is in reality due to the correlation between MCV and basal metabolic rate. This could be explained by the fact that small erythrocytes allow a fast release of oxygen in tissues with high metabolic demand. Nonetheless, hypoxia-adapted species show MCV greater than that predicted by their metabolic rate, while Ruminantia show the inverse. Interestingly, these species show the highest and lowest, respectively, Hb affinity for oxygen. In the present paper, I suggest that Hb-oxygen affinity, acting as a biological constraint for oxygen exchange, determines the size of red blood cells. Hb intrinsic affinity for oxygen shows little variation during evolution and modifying the levels of allosteric factors can be viewed as an adaption to adjust Hb-oxygen affinity to metabolic demands (the same also happens during ontogeny). Nonetheless, in some lineages, mutations raising Hb-oxygen affinity allowed some species to colonize hypoxic environments; in Ruminantia, instead, there was a drastic decrease, which cannot be adaptively explained.

5589320 FOLLOW-UP RESULTS OF A PHASE 2 STUDY ASSESSING THE SAFETY AND EFFICACY OF MITAPIVAT TREATMENT, AN ORAL PYRUVATE KINASE ACTIVATOR, FOR UP TO 60 WEEKS IN SUBJECTS WITH SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is a devastating red blood cell (RBC) disorder. The root cause of SCD is polymerization of sickle hemoglobin (HbS) upon deoxygenation, resulting in poorly deformable sickled RBCs with shortened lifespan. 2,3-diphosphyglycerate (2,3-DPG), a glycolytic intermediate in RBCs, promotes deoxygenation by lowering hemoglobin (Hb)-oxygen affinity. Mitapivat (AG-348) is an oral allosteric activator of pyruvate kinase (PK), a key enzyme in RBC glycolysis generating adenosine triphosphate (ATP) and reducing 2,3-DPG levels. Aims: To report follow-up data of the safety and efficacy of mitapivat treatment in subjects with SCD (ESTIMATE study; www.trialregister.nl NL8517; EudraCT 2019-003438-18). Methods: The ESTIMATE study is a phase 2, investigator initiated, open-label study. Subjects ≥16 years with SCD (HbSS, HbS/β0 or HbS/β+), 1-10 vaso-occlusive crises (VOCs) in the prior year and/or prior SCD-related complications, a Hb level >6.1 g/dL and ≤11.1 g/dL, and no chronic transfusions are eligible and treated with mitapivat after obtaining informed content. In the 8-week dose finding period, initial dosing is 20 mg twice daily, escalated to max. 100 mg twice daily, and followed by an up to 52-week fixed dose extension period (FDEP). Primary endpoints are safety, based on adverse events (AEs), and efficacy of mitapivat treatment on RBC sickling (Point of Sickling (PoS), oxygen gradient ektacytometry). Key secondary endpoints are changes in Hb level, markers of hemolysis, ATP/2,3-DPG levels, and Hb-oxygen affinity (p50, Hemox Analyzer). Results: Until Jan. 2022, 9 subjects were treated with mitapivat. Baseline characteristics were: median age 22 years (range 16-59 years), 6/9 (67%) female, and 6/9 (67%) used hydroxyurea. 7/9 (78%) had HbSS, 1/9 (11%) had HbS/β0, and 1/9 (11%) had HbS/β+. One subject with a non-treatment related serious AE (SAE) of a urinary tract infection (grade 4), was lost to follow-up shortly after first dosing. No other SAEs or treatment-emergent AEs (TEAEs) grade ≥3 occurred. The remaining 8 subjects had a median treatment duration of 38 weeks (range 11-60 weeks). 6/8 (75%) took 100 mg mitapivat twice daily in the FDEP, and 2/8 (25%) took 50 mg mitapivat twice daily after a single dose reduction because of progressive transaminase increase. The most common TEAEs were (n>2 subjects): ALT or AST increase (both 5/9, 56%; all grade 1), and headache (4/9, 44%; grade 1-2). Three VOCs occurred: one related to excessive alcohol consumption with no need for hospitalization, and two in subjects with documented noncompliance the week before hospitalization. Mean annual VOC rate and SCD-related hospital admission days were, respectively, 1.5±1.3 and 5.9±7.1 days at baseline, and reduced to 0.5±0.7 and 1.6±3.1 days when weighting cases by follow-up duration (p=0.021 and p=0.134, respectively). Table 1 summarizes mean changes in parameters of primary and key secondary endpoints of scheduled visits in the FDEP compared to baseline. Hb level significantly increased, accompanied by a decrease in markers of hemolysis (absolute reticulocyte count, total bilirubin, lactate dehydrogenase). Mean PoS decreased, and p50, 2,3-DPG level and ATP/2,3-DPG ratio significantly improved. Conclusion: Treatment with mitapivat for up to 60 weeks in subjects with SCD showed no treatment related TEAEs grade ≥3. Improvements in anemia, markers of hemolysis, oxygen affinity, 2,3-DPG level and ATP/2,3-DPG ratio were seen.

Slow dynamics measured by phosphorescence lifetime reveals global conformational changes in human adult hemoglobin induced by allosteric effectors.

The mechanism underlying allostery in hemoglobin (Hb) is still not completely understood. Various models describing the action of allosteric effectors on Hb function have been published in the literature. It has also been reported that some allosteric effectors-such as chloride ions, inositol hexaphosphate, 2,3-diphospho-glycerate and bezafibrate-considerably lower the oxygen affinity of Hb. In this context, an important question is the extent to which these changes influence the conformational dynamics of the protein. Earlier, we elaborated a challenging method based on phosphorescence quenching, which makes characterizing protein-internal dynamics possible in the ms time range. The experimental technique involves phosphorescence lifetime measurements in thermal equilibrium at varied temperatures from 10 K up to 273 K, based on the signal of Zn-protoporphyrin substituted for the heme in the β-subunits of Hb. The thermal activation of protein dynamics was observed by the enhancement of phosphorescence quenching attributed to O2 diffusion. It was shown that the thermal activation of protein matrix dynamics was clearly distinguishable from the dynamic activation of the aqueous solvent, and was therefore highly specific for the protein. In the present work, the same method was used to study the changes in the parameters of the dynamic activation of human HbA induced by binding allosteric effectors. We interpreted the phenomenon as phase transition between two states. The fitting of this model to lifetime data yielded the change of energy and entropy in the activation process and the quenching rate in the dynamically activated state. The fitted parameters were particularly sensitive to the presence of allosteric effectors and could be interpreted in line with results from earlier experimental studies. The results suggest that allosteric effectors are tightly coupled to the dynamics of the whole protein, and thus underline the importance of global dynamics in the regulation of Hb function.

Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown.This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

GBT021601 Increases Hemoglobin and Improves Red Blood Cell Deformability without Rebound Hyperviscosity upon Drug Clearance in a Sickle Mouse Model

Background: Whole-blood viscosity and red cell deformability are markedly abnormal in sickle cell disease (SCD) due to sickling of red blood cells (RBCs). Increased whole-blood viscosity is a risk factor for vaso-occlusive crisis (VOC), avascular necrosis, and proliferative retinopathy. Blood viscosity is strongly impacted by hemoglobin (Hb) levels and RBC deformability. GBT021601 is a potent next-generation sickle hemoglobin (HbS) polymerization inhibitor, with anti-sickling properties, that increases the oxygen affinity of Hb. In preclinical studies, treatment with GBT021601 achieved higher levels of Hb than the US Food and Drug Administration-approved first-generation compound voxelotor, raising concern that this rise in Hb would substantially increase whole-blood viscosity and impose a VOC risk after treatment cessation. Objective: To investigate a potential rebound hyperviscosity effect from GBT021601 cessation in a murine model of SCD by determining the effect on whole-blood viscosity and on RBC deformability under a range of oxygen concentrations using oxygen gradient ektacytometry. Methods: Nine-week-old Townes HbSS mice were fed chows containing 0.2% GBT021601 for 5 weeks, then transitioned to control chow. Whole blood was collected in an EDTA tube. All tests were conducted within 4 hours of collection. Using an Element HT5 hematology analyzer (Heska), complete blood count was obtained from mice while on GBT021601 (day 0) and then on days 1, 3, and 5 after transition to control chow. Blood viscosity was measured with a cone/plate viscometer (AMETEK Brookfield) at 37 °C at shear rates of 45 s-1 and 225 s-1. Hematocrit to viscosity ratio (HVR) was calculated to determine RBC oxygen-carrying capacity. RBC deformability was measured by oxygen gradient ektacytometry (Lorrca, RR Mechatronics). Results: Treatment with GBT021601 significantly increased Hb from 8.0 to 12.9 g/dL (P<0.0001) without increasing HVR. Compared with previous studies with the voxelotor analog GBT1118, Hb dropped off more slowly with GBT021601 treatment; this allowed for the ideal assessment of whole-blood viscosity and HVR when the drug had cleared, and HbS was no longer modified, despite elevated Hb levels. HVR at both 45 s-1 and 225 s-1 showed no significant change with drug clearance. GBT021601 treatment also improved RBC deformability, as indicated by a higher elongation index (EI) under both normoxic (EImax, P<0.001) and hypoxic (EImin, P<0.0001) conditions. Point of sickling (PoS) was also reduced (P<0.0001), indicating that treated RBCs tolerated lower oxygen concentrations before sickling. When the drug was withdrawn, PoS and EImin returned to baseline levels, whereas EImax remained elevated until day 5 off treatment. Hb also remained elevated until day 5 off treatment, despite the return to baseline levels of the EImin, PoS, and p50 (partial oxygen pressure at which Hb is 50% saturated with oxygen), indicating drug clearance. Conclusions: In response to GBT021601, sickle cell mice showed a robust increase in Hb, comparable to that of a HbAS mouse, as well as a marked improvement in PoS and RBC deformability. When normalized to HVR, there was no significant difference in blood viscosity during treatment and after cessation of treatment. This finding suggests that oxygen-carrying capacity is not impaired and provides no indication for rebound hyperviscosity with drug clearance. Moreover, the observation that EImax remained elevated until 5 days after drug cessation suggests that some residual rheological improvement occurred in the RBC population that did not dissipate when the drug was cleared. In vitro addition of GBT021601 (and other related compounds) to blood rapidly increases the EImin and reduces the PoS of RBCs but does not alter the EImax. In contrast, an increase in the EImax of RBCs has been consistently observed with GBT021601 treatment in vivo, including in early clinical studies in patients with SCD. The precise mechanism by which this benefit is obtained requires further study. Funding: Global Blood Therapeutics.

Dynamic Sickling Assay: Using Enzymatically Induced Hypoxia to Assess Real-Time Sickling Scalable from Large Erythrocyte Populations to Individual Cells

Background: There is a pressing need for assays to assess both patient condition and to monitor the progress of Sickle Cell Disease (SCD) therapies. It would be beneficial for an assay to allow assessment of oxygen dissociation, hemoglobin polymerization and Red Blood Cell (RBc) sickling under both equilibrium (mimicking longer-term effects) and non-equilibrium (simulating in-vivo process) conditions. Also desirable is the CLIA-waived system suitable for use in clinical trials and ultimately for clinical management of patients on SCD therapies including those those targeting Hb oxygen affinity. Our proposed work introduces an RBC hypoxia-induced sickling assay as a clinical tool to assess SCD patient condition and to assist in development, monitoring, and optimization of SCD therapies. Methods: The system uses an enzymatic oxygen scrubbing system allowing for a wide range of deoxygenation rates and final hypoxia severity. Diluted blood samples were supplemented with Protocatechuic acid (PCA) and then with Protocatechuate 3,4-Dioxygenase (PCD) enzyme inducing hypoxia at a predetermined rate. The sample was injected into an anaerobic microfluidic chamber with the resulting Red Blood Cell (RBC) morphological changes observed through time-lapsed photography. Image analysis allows for identification and quantification of sickled RBC linked to different polymerization scenarios/mechanisms. An oxygen probe was used to track the rate of medium deoxygenation. Sickling profiles were constructed to represent a percent of sickled RBC in a total cell population measured every two seconds after the enzymatic reaction is initiated. Profiles were assessed though i) percent sickling at normoxia, ii) morphological Point of Sickling (mPoS) and iii) time delay of sickling both measured at 5% increase in sickling over the normoxia sickling baseline, iv rate of sickling, v) size of the sickling fraction and vi) Area under the Curve (AUC). The last four parameters are measured for each sickling fraction if more than one such is present. Results: In control experiments PCA/PCD system was shown to allow reliable and reproducible sample deoxygenation currently enabled in a range from seconds to hours. Comprehensive multidimensional profiles were constructed presenting fraction of sickled RBC as a function of a) time from enzymatic reaction initiation, and b) one or more of rate of oxygen consumption, end of reaction hypoxia level, or oxygen tension in the medium. Such profiling allows for fast optimization of assay parameters to target assay conditions most suitable for detection of desired effects.Significant changes in sickling profiles, with marked delay in sickling, were observed upon treatment of RBC with hemoglobin-modifying drugs like voxelotor.Sickling profiles of Sickle Trait (SCT) patients showed significant delay in sickling compared to SS-type SCD patients. The method shows the potential to differentiate SCT patients based on the risk of adverse conditions to occur due to the patient's trait status.By stopping the flow, the system allows tracking of morphological status of individual cells over the full course of medium deoxygenation. Individual cells show pronounced differences in their propensity to sickle, potentially correlated to individual cell Hb F content. Change in morphology of sickling cells from biconcave disk to crescent and then to holly leaf and granular forms had been observed upon progressively decreasing medium oxygen tension.RBC microparticle formation can be observed and tracked over time as an additional metric of erythrocyte response to developing hypoxia Conclusions: By using enzymatically induced hypoxia in a microfluidic environment, presented system allows comprehensive analysis of RBC sickling dynamics in a wide range of deoxygenation rates and conditions. It would find utility in SCD therapy development by offering a robust and high throughput method for assessing critical RBC response to therapies. With no need for cumbersome gas cylinders and low training requirements, a stand-alone system with single use disposable cartridges would also have utility in clinical trials both in the US and abroad, due to simplicity of use suitable for developing countries with limited infrastructure. When validated, the system can be adapted for Point of Care use either as complementary to other blood tests or in an independent monitoring mode.

P-023: 2,3-DIPHOSPHOGLYCERATE DETECTION VIA DIRECT INFUSION HIGH RESOLUTION MASS SPECTROMETRY CORRELATES WITH QUANTITATIVE DETECTION IN BLOOD OF PATIENTS WITH SICKLE CELL DISEASE

Purpose: Sickle cell disease (SCD) is a hereditary and chronic life-threatening disorder, characterized by haemolytic anaemia. Increased 2,3-diphosphoglycerate (2,3-DPG) concentrations, along with decreased oxygen affinity of hemoglobin, may be related to the variability of clinical outcomes in SCD. Furthermore, genomic health data holds promise to improve the prediction of disease severity in SCD. Based on the integration of genomics, metabolomics and clinical data from 1000 SCD patients, to be included in 2022, GenoMED4all aims to develop Artificial Intelligence (AI) based deep learning algorithms to improve the prediction of disease severity and phenotype in SCD. This study aims to correlate non-quantitative metabolomics data obtained from dried blood spots (DBS), one of the inputs for GenoMED4all, to quantitative measurement of 2,3-DPG. Aiming to improve the potential of non-quantitative metabolomics from DBS to asses 2,3-DPG. Materials and methods: In snap frozen blood samples from 37 SCD patients and 29 healthy controls, 2,3-DPG was quantified by liquid chromatography mass spectrometry. 2,3-DPG was also detected in DBS from the same subjects by direct infusion high resolution mass spectrometry (DIHRMS). The oxygen tension at 50% Hb saturation (p50) was determined using a Hemox Analyzer (TCS). Statistical analysis were performed by Spearman’s correlation coefficients (SPSS v26.0.0.1) and Mann Whitney testing (GraphPad Prism v9.3.0). Results: After correcting for Hb, 2,3-DPG concentrations were higher in SCD patients than in controls (p<0.001) and Z-scores for 2,3-DPG, as assessed by DIHRMS, were similar in patients and controls. The Z-scores positively correlated with 2,3-DPG concentrations (Fig1A, 0.353, p=0.004). Because of the anaemia in SCD, red blood cells (RBCs) and plasma make up lower and higher volumes in the blood, respectively, compared to healthy controls. This affects the ratio of RBC and plasma metabolites on the DBS. DIHRMS detects a wide range of RBC and plasma metabolites, whereas the quantitative measurement is restricted to measuring 2,3-DPG of RBCs. To correct for those differences between methods, we applied a correction factor to the DIHRMS data using (1/Ht)*(1-Ht/1), correcting for the RBC volume (1/Ht) and the plasma volume (1-Ht/1). This resulted in a trend towards higher Z-scores in patients than controls (p=0.0597). Moreover, the positive correlation with 2,3-DPG concentrations increased to 0.526 (Fig1B, p<0.001). As 2,3-DPG affects the oxygen affinity of Hb, all measurements were correlated to p50. Expectedly, 2,3-DPG concentrations positively correlated with p50 (0.842, p<0.001). After applying the correction factor to the DIHRMS data, p50 correlations increased from 0.361 (p=0.003) to 0.529 (p<0.001). Conclusion: Strongest correlation between quantitative and non-quantitative methods for 2,3-DPG detection were observed after correcting for both RBC and plasma volumes in non-quantitative metabolomics. After correction, DIHRMS can be used to assess 2,3-DPG concentrations in DBS. This translation of non-quantitative to quantitative metabolomics for 2,3-DPG and potentially other RBC metabolites adds significant value to the use of DIHRMS, as DIHRMS is far more efficient in obtaining extensive information about the total metabolome than quantitative methods. The large population size and the AI based deep learning algorithms of GenoMED4all will enable to evaluate the potential of non-quantitative metabolomics in SCD. Funding: Horizon2020, GenoMED4all(https://genomed4all.eu/), Agios Pharmaceuticals Inc., ERN-EuroBloodNetFig 1. correlations between non-quantitative (Z-score) and quantitative 2,3-DPG detection A) non-quantitative and quantitative 2,3-DPG detection B) non-quantitative detection corrected for RBC and plasma volume and quantitative 2,3-DPG detection The authors do not declare any conflict of interest

P1065: CONGENITAL ERYTHROCYTOSIS DUE TO HETEROZYGOUS VARIANTS IN THE BISPHOSPHOGLYCERATE MUTASE GENE

Background: Congenital erythrocytosis is an inherited condition characterized by increased red blood cell mass, reflected by persistently elevated hemoglobin (Hb) and hematocrit levels. Erythrocytosis can be primary due to an intrinsic defect of erythropoiesis, or secondary, including germline variants in genes involved in the oxygen-sensing pathway and Hb variants with altered oxygen affinity. A very rare cause involves variants in bisphosphoglyerate mutase (BPGM), which regulates the red blood cell concentration of 2,3-bisphosphoglycerate (2,3-BPG), an important modifier of Hb-oxygen affinity. Aims: We report three European families with erythrocytosis and partial BPGM deficiency due to heterozygosity for variants in the BPGM gene, including one novel variant. Methods: The study was performed on adults of three families with JAK2-negative erythrocytosis. DNA analysis concerned sequencing of relevant coding exons, including flanking splice-site consensus sequences, of the eight major genes involved in secondary congenital erythrocytosis (EPOR, HBB, HBA1, HBA2, VHL, EPAS1, ELGN1, and BPGM). Identified variants were evaluated using Ensembl, gnomAD, PolyPhen-2, and SIFT. BPGM enzyme activity was determined as described by Beutler. Quantitative analysis of 2,3-BPG was performed using liquid chromatography tandem mass spectrometry. Hb-oxygen affinity, reflected by p50 levels (the oxygen pressure when Hb is 50% saturated with oxygen), was analyzed using the Hemox Analyzer (TCS Scientific). Results: In the first family (Table 1), a novel missense variant (c.535C>T p.(Arg179Cys) was identified in BPGM. Both the 65-year-old mother with complaints of headaches, and her 39-year-old son with a medical history of retinal vein occlusion were heterozygous for this variant. Allele frequency in gnomAD was 3.98-5. The predicted effect ranged from either deleterious (SIFT) to benign (HumVar model of Polyphen-2). Subsequent functional analysis showed low-normal BPGM activity, whereas 2,3-BPG and p50 levels were decreased (Table 1). In the second and third families, three male individuals were heterozygous for the c.269G>A p.(Arg90His) missense variant in BPGM. This variant was only once previously reported, and has an allele frequency of 1.06-5. All affected subjects had mild erythrocytosis without any additional clinical features, and showed decreased BPGM activity, decreased 2,3-BPG levels and decreased p50 (Table 1). Image:Summary/Conclusion: We describe five individuals from three families in which heterozygosity for missense variants in BPGM is associated with congenital erythrocytosis. Our findings further strengthen the hypothesis of an autosomal dominant inheritance pattern of BPGM variants. Our data also demonstrate the added value of functional analyses to validate gene panel and in silico analysis results for their clinical relevance. Future cases may help to interpret the impact of BPGM variants on hematological and clinical phenotype.