Hemopexin (Hpx) is an acute phase plasma protein that is responsible for sequestration and removal of cell-free heme with very high affinity (Kd < 1 pM). Hpx expression in liver cells is induced following an inflammatory event such as severe hemolysis. Therefore, plasma Hpx has potential clinical relevance due to its' ability to bind free heme, thus reducing lipid peroxidation and activation of inflammatory pathways in genetic anemias, such as sickle cell disease (SCD). This provides the rationale for purifying Hpx at high purity and at large scale. Starting from human Cohn fraction IV, we purified a protein cocktail using tangential flow filtration for use in this study as the starting material for purification of Hpx. Hpx was purified from the protein cocktail using immobilized metal ion affinity chromatography. SDS-PAGE and densitometric analysis showed that Hpx was purified to homogeneity and was approximately 99% pure. To gain further insight into Hpx activity and its' ligand binding properties, we employed comparative biophysical techniques such as UV-visible, circular dichroism, electron paramagnetic resonance and stopped flow spectroscopy. Finally we evaluated Hpx in a mouse model of SCD to test the ability of the novel material to attenuate tissue iron accumulation.

Haptoglobin (Hp) is a polymorphic acute phase α-2 glycoprotein found in plasma that plays a critical role in binding, neutralizing, and removing cell-free hemoglobin (Hb) from the circulation. Under clinical conditions characterized by high levels of hemolysis, such as in patients with sickle cell disease (SCD), large quantities of cell-free Hb are released from lysed red blood cells (RBCs) into the circulation and bind to Hp. This interaction reduces the plasma Hp concentration below basal levels and diminishes its Hb-binding capacity. Therefore, plasma-derived Hp has the potential to be used therapeutically to scavenge, neutralize, and remove excess cell-free Hb from the blood, thus preventing Hb-mediated toxicity. This provides strong motivation to purify Hp at high purity using sustainable sources, such as waste plasma fractions from the Cohn plasma fractionation process used to produce human serum albumin from pooled plasma. Starting from human Cohn fraction IV, we first enriched an Hp-rich fraction using tangential flow filtration (TFF), which was then used as the starting material for purification. Hp was subsequently purified from this fraction using hydrophobic interaction chromatography (HIC) to homogeneity. The final Hp purity reached 98% by SDS-PAGE densitometry and 87% by trypsin digest LC-MS/MS analysis. The purified Hp was further characterized to determine its molecular weight, secondary structure, Hb-binding capacity, and binding kinetics using biophysical techniques including MALDI-TOF, circular dichroism, size exclusion HPLC, and UV-visible stopped-flow spectroscopy. More importantly, the simplicity and efficiency of the TFF-HIC workflow demonstrated strong potential for scalable Hp production.

Sickle cell disease-associated pulmonary hypertension (SCD-PH) affects approximately 10% of adults with SCD and markedly increases mortality, yet mechanistic and haemodynamic heterogeneity complicates classification, trial design, and treatment selection. We propose an integrated framework linking five interacting axes-anaemia/high-output, haemolysis/haem/iron toxicity, hypoxia, inflammation, and thrombosis-to clinically defined phenotypes (post-capillary, pre-capillary, combined, chronic thromboembolic PH [CTEPH], and acute cor pulmonale). Chronic anaemia drives high-output physiology, left ventricular diastolic dysfunction, and post-capillary PH. Intravascular and erythrophagocytic haemolysis cause convergent inside-out and outside-in pulmonary vascular injury via nitric oxide depletion and oxidative damage, promoting pre-capillary PH; hypoxia, inflammation, and thrombosis amplify remodelling, helping explain why combined phenotypes predominate. Management prioritises hydroxyurea and transfusion, while PDE5 inhibition (sildenafil) has shown harm. Emerging avenues include soluble guanylate cyclase stimulation, L-arginine, haemoglobin/haem scavenging (haptoglobin, hemopexin), anti-inflammatory strategies, and iron-targeted interventions. This mechanism-to-phenotype map supports phenotype-stratified, mechanism-guided trials in SCD-PH.

Pulmonary hypertension (PH) is a severe vascular complication of sickle cell disease (SCD); yet, not all patients with SCD develop PH, and PH also arises independently. This duality underscores the need to understand their intersecting biology. We integrated metabolomic, proteomic, and elemental analyses of human peripheral blood mononuclear cells (PBMCs) from individuals with SCD, PH, combined SCD-PH, and healthy controls to define shared and distinct mechanisms. PBMCs from SCD patients, regardless of PH status, displayed significantly elevated intracellular iron, consistent with chronic hemolysis and erythrophagocytosis. Multi-omic profiling revealed condition-specific immune-metabolic signatures: SCD PBMCs showed mitochondrial suppression and reduced oxidative phosphorylation; PH PBMCs showed dysregulated arginine and creatine metabolism, implicating nitric oxide and polyamine pathways; and SCD-PH PBMCs displayed amplified hemoglobin/iron handling, oxidative stress, and immune activation. Unsupervised clustering confirmed discrete phenotypes, with greatest overlap between SCD and SCD-PH, reflecting the additive impact of hemolysis-driven iron loading and PH-driven metabolic remodeling. Histological validation of SCD-PH lung tissue demonstrated iron accumulation in perivascular macrophages, supporting a mechanistic link between systemic PBMC remodeling and pulmonary vascular pathology. Together, these findings establish PBMCs as a readily accessible compartment that mirrors disease-specific metabolic and immune alterations. By capturing iron, arginine, and redox pathways across SCD, PH, and SCD-PH, our study positions PBMC profiling as a novel tool for mechanistic insight, patient stratification, and biomarker discovery and novel interventions.

Chronic intravascular hemolysis releases cell-free hemoglobin (Hb) and heme, promoting oxidative stress, inflammation, and tissue injury that drive cardiovascular and renal complications in hemolytic disorders. Haptoglobin (Hp) and hemopexin (Hpx) mitigate Hb and heme toxicity. However, their levels during chronic hemolysis decrease, allowing for Hb and heme tissue partitioning and multi-organ injury. To address this, we evaluated the apohemoglobin–haptoglobin (ApoHb–Hp) complex, a dual-function scavenger that neutralizes acellular Hb and free heme, in a chronic Hb-infusion mouse model. Male and female mice received daily Hb or Hb + ApoHb–Hp injections for six weeks. Cardiac and renal function were assessed by echocardiography and transdermal glomerular filtration rate (GFR), along with inflammatory and injury biomarkers. Chronic Hb impaired cardiac and renal function and elevated renal and cardiac-associated injury biomarkers, such as troponin and KIM-1. ApoHb–Hp treatment preserved cardiac and renal function, lowered inflammatory and injury biomarkers, while also reduced circulating heme and urinary iron. Although females showed partial resilience to Hb-induced dysfunction, both sexes benefited from ApoHb–Hp therapy. These results demonstrate that ApoHb–Hp effectively mitigates Hb- and heme-driven organ injury, supporting its potential as a targeted therapeutic for chronic hemolytic pathologies.

Sickle cell disease (SCD) is a genetic inherited hemoglobinopathy arising from homozygosity or compound heterozygosity for a single base pair mutation in hemoglobin β-globin gene (HBB) and the severity is affected by allelic combinations, haplotypes and gene products. Numerous SCD mouse models exist to study mechanism and therapeutic intervention, and each display some phenotypic features of human disease. Berkeley SCD mice demonstrate clinically relevant pulmonary hypertension (mean pulmonary artery pressure = 25-35 mmHg) when housed under sub-chronic (3-months) exposure to a moderately decreased oxygen level, approximately 15%. This model accelerates red blood cell sickling and hemolysis, which perpetuates precapillary pulmonary vascular disease and right ventricular dysfunction. Iron restriction in SCD is reported to attenuate the frequency and severity of vaso-occlusive crisis through reducing HbS in RBCs. Vamifeport is an oral clinical stage ferroportin inhibitor shown to improve microcirculatory blood flow in Townes SS mice. We hypothesized that vamifeport treatment may attenuate right ventricular dysfunction and pulmonary vascular remodeling in Berkeley SCD mice that express a pulmonary hypertension phenotype. Further, we hypothesized that lung and right ventricle metabolism and protein expressions would show an antioxidant and iron regulatory response that favored the attenuation of cardiopulmonary dysfunction. Indeed, attenuation of red cell sickling, less extra- and intravascular hemolysis and normalization of cardiopulmonary dysfunction was observed after vamifeport treatment. We suggest that induction of mild iron deficiency anemia may attenuate deadly sequelae of SCD, including cardiopulmonary dysfunction.

This study investigated the pathophysiological effects of cell-free hemoglobin (Hb) generated by mechanical hemolysis during venovenous extracorporeal circulation (VVECC). We hypothesized that Hb scavenger protein constructs that bind Hb, heme and iron, could attenuate end-organ injury caused by intravascular hemolysis during VVECC. Scavenger constructs consisted of an apohemoglobin-haptoglobin (apoHb-Hp) complex designed to bind Hb and heme, as well as a separate preparation of haptoglobin, albumin, hemopexin, transferrin, termed the protein cocktail. To test the hypothesis, Golden Syrian hamsters were instrumented with dorsal window chambers and catheters, and VVECC was maintained for a total of 2 h, with a maximum flow rate equivalent to 50% of the animal’s cardiac output. VVECC circuits were primed with either two binding materials, the apoHb-Hp and the protein cocktail, or a control solution of 5% human serum albumin (HSA). Microvascular Hb oxygen saturation in arterioles (saO2) and venules (svO2) were studied. All groups displayed a significant decrease in saO2 and svO2 at maximum VVECC when compared to baseline, while statistically significant changes between treatment groups showed no consistent trend. The protein cocktail bound 24% of cell-free Hb, while the apoHb-Hp bound 66% of cell-free Hb. Additionally, markers of renal damage and inflammation such as plasma creatinine, urinary NGAL, 4-HNE, and KIM-1 were significantly reduced in both Hb scavenger groups as compared to the HSA control. Results from this study suggest that Hb, heme and iron scavenging solutions used to prime VVECC circuits are indicated to support organ function.

Red blood cells (RBCs) are transcriptionally silent yet dynamically remodel metabolism in response to oxygen tension. Using ultra-pure human RBCs, we generated the deepest contamination-free proteome to date (3,775 proteins) and mapped the oxygen-dependent interactome. These datasets reveal an oxygen-responsive metabolon centered on the Band 3 (SLC4A1) N-terminus. We identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that dissociates under hypoxia, coincident with increased Band 3-deoxyhemoglobin contacts. This reversible assembly functions as an oxygen-sensitive switch coordinating redox and glycolytic remodeling. Humanized mice lacking Band 3 N-terminal segments exhibit impaired oxygen-dependent regulation of BLVRB binding to band 3, impaired hypoxic activation of glycolysis, reduced 2,3-bisphosphoglycerate synthesis, and diminished exercise tolerance, demonstrating physiological relevance. Population-scale cis-pQTLs for SLC4A1 and BLVRB suggest functions beyond canonical heme catabolism. Mechanistically, biochemical analyses in vitro suggest that hemoglobin β (HBB), Band 3, and BLVRB can undergo S-nitrosation and may participate in trans-nitrosation reactions with the glycolytic enzyme GAPDH, whose modification at C152 inhibits enzymatic activity in vitro. Collectively, these findings define a Band 3-BLVRB axis that integrates oxygen-dependent protein interactions with thiol-based redox chemistry, providing a framework for understanding how an anucleate cell achieves metabolic adaptability through reversible protein-protein interactions and post-translational modification. These findings suggest that perturbation of the Band 3-BLVRB axis may influence oxygen delivery and metabolic flexibility during hypoxic stress, with potential relevance to high-altitude adaptation, exercise physiology, and cardiopulmonary disease.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymatic disorder, affects over 500 million people worldwide and is often linked to exercise intolerance due to oxidative stress, but its true impact on physical performance remains unclear. This study aimed to evaluate the physiological and metabolic effects of G6PD deficiency on endurance capacity. Using humanized mice carrying the African G6PD variant [V68M; N126D] (hG6PDA-), we show that despite reduced pentose phosphate pathway activity, these mice exhibit a 10.8% increase in treadmill critical speed (CS)-suggesting enhanced endurance capacity. Multi-omics profiling across red blood cells, plasma, skeletal muscle, spleen, kidney, and liver reveals metabolic adaptations, including elevated glycolysis, fatty acid oxidation, and increased mitochondrial activity, alongside heightened oxidative phosphorylation in muscle and accelerated red blood cell turnover in the spleen and liver. These findings indicate that systemic metabolic reprogramming may offset antioxidant deficiencies, potentially conferring a performance advantage. Given that G6PD deficiency affects up to 13% of African Americans and is associated with cardiovascular health disparities, our results challenge conventional exercise restrictions and highlight the need for personalized exercise guidelines for affected individuals.

Red blood cells (RBCs) have long been regarded as passive oxygen carriers, yet growing evidence reveals a complex, dynamic proteome independent of de novo gene expression. Here, we define the erythrocyte as an oxygen-responsive system organized around a Band 3 (SLC4A1)-centered metabolon. Using deep proteomics of ultra-pure RBCs and cross-linking interactomics, we identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that binds the N-terminal cytosolic domain under normoxia and dissociates under hypoxia, when band 3-deoxyhemoglobin interactions increase threefold. This reversible interaction forms an oxygen-sensitive switch coupling structural, redox, and metabolic remodeling. In humanized mice, truncation of the Band 3 N-terminus disrupted glycolytic activation, reduced 2,3-bisphosphoglycerate synthesis, and impaired exercise tolerance despite preserved cardiopulmonary function, establishing the physiological relevance of this module. Population-scale proteome quantitative trait locus (pQTL) analyses revealed coordinated variation of SLC4A1 and BLVRB abundance but minimal association of biliverdin levels with BLVRB genotype, suggesting alternative functions beyond heme catabolism. Mechanistically, BLVRB Cys109 acts as a nitric oxide (NO) relay, trans-nitrosating glycolytic enzymes such as GAPDH at active site Cys152, transiently inhibiting glycolysis. This S-nitrosation-mediated feedback mirrors conserved mechanisms in plants, where GAPDH-SNO redirects carbon flow toward the Calvin-Benson cycle under nitrosative stress, revealing an evolutionary convergence in gas-responsive metabolic control. Collectively, our findings define a Band 3-BLVRB-hemoglobin axis that links oxygen sensing, NO signaling, and redox homeostasis, providing a unifying model for how an anucleate cell achieves environmental adaptability through reversible protein-protein interactions and post-translational chemistry. Graphic abstract:Issaian et al. define the most comprehensive proteome of ultra-pure human red blood cells (3,775 proteins) and map the O₂-dependent interactome, revealing a Band 3-BLVRB-hemoglobin module that links oxygen sensing to metabolic remodeling via reversible inhibitory S-nitrosation of GAPDH C152. In plants this redirects carbon toward photosynthesis, illustrating a conserved NO-dependent metabolic reprogramming mechanism across oxygen-regulated systems. Highlights:Deep proteomics defines a complete, contamination-free RBC proteome (3,775 proteins)Cross-linking proteomics maps an oxygen-sensitive Band 3-centered interactomeO2-dependent BLVRB-Band 3 binding regulates metabolism via S-nitrosation of GAPDHBand 3 N-terminus is required for hypoxic remodeling and exercise tolerance in vivo.

Hemolysis and the downstream consequences of cell-free hemoglobin (Hb) and heme contribute to the development of sickle cell disease pulmonary hypertension (SCD-PH). The plasma concentrations of Hb and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hpx) in sickle cell patients are observed to be significantly lower than healthy donors. The unchecked exposure to Hb and heme contribute to vasculopathy and aberrant cardiac function. This is consistent with vascular remodeling co-localized within iron rich macrophages. Based on these observations in patients, we hypothesize that a joint Hb and heme scavenger approach, combining Hp + Hpx as a therapeutic will attenuate hemolysis driven SCD-PH progression in a SCD mouse model. To test the hypothesis, we utilized our validated Berk-SS mouse model of SCD-PH driven by a 10-week moderate hypoxia exposure and weekly subcutaneous administration of Hp + Hpx. At study termination, we analyzed changes in cardiopulmonary iron deposition, right ventricular and pulmonary functional parameters, and multi-omic indices associated with SCD-PH. Our data demonstrates that Hp+Hpx improves pulmonary vascular resistance and right ventricular function including stiffness, afterload, cardiac output, ventricular to vascular coupling ratio, pulmonary vascular resistance and medial hypertrophy. Histological evaluation of lung and right ventricular tissue demonstrates attenuation of cardiopulmonary pathology. Finally, a multi-omic analysis of whole lung and heart tissue demonstrates a rebalancing of proteins related to PH, iron, inflammation, and oxidative stress. This data provides strong pre-clinical evidence for the clinical study of combined Hb and heme scavenger proteins in the treatment of PH-associated SCD.

Plasma transferrin (Tf) is the transport protein central to the process of iron recycling and metabolism. Holo-Tf serves as the body's pool of ferric iron, facilitating transport from tissues such as the intestine, liver, spleen, and finally bone marrow, where iron is incorporated into erythropoiesis. In sickle cell disease (SCD), iron overload is primarily caused by chronic blood transfusions in patients at risk of stroke or frequent acute pain crisis. However, we have identified that pulmonary vascular iron accumulation, independent of transfusion, is a driver of pulmonary hypertension in SCD patients and murine models. Therefore, we hypothesize that intra-pulmonary administration of apo-Tf localizes the protein to sites of iron accumulation within the lung, where reactive iron-driven pathology develops. This approach to therapeutic development focuses on optimizing administration using aerosol drug delivery, which can increase clinical compliance compared to subcutaneous or intravenous administration. The goal of this study was to purify apo-Tf using a novel process, perform biochemical characterization on the material, and test the proof of concept that apo-Tf protein can be delivered to lung regions where iron accumulation occurs in SCD pulmonary hypertension. We conclude that apo-Tf can be isolated from plasma Cohn fraction IV paste using a simple process and that characterization of the material identified a high-purity apo-Tf product with functional iron binding properties. Further, this material was administered to SCD mice to target pulmonary anatomical regions where pathology occurs. This data suggests an intriguing approach to iron chelation applicable to a relevant clinical population.

Sickle cell disease and β-Thalassemia are two of the most prevalent hemoglobinopathies worldwide. Both occur due to genetic mutations within the HBB gene and are characterized by red blood cell dysfunction, anemia, and end-organ injury. The spleen and liver are the primary organs where erythrophagocytosis, engulfing the red blood cells, occurs in these diseases. Understanding metabolism and protein composition within these tissues can therefore inform the extent of hemolysis and disease progression. We utilized a multiomics approach to highlight metabolomic and proteomic differences in the spleen and liver. The Berkley sickle cell disease (Berk-SS), heterozygous B1/B2 globin gene deletion (HbbTh3/+) a known β-Thalassemia model, and wildtype (WT, C57/Bl6) murine models were evaluated in this report. This analysis showed Berk-SS and HbbTh3/+ shared distinct antioxidant and immunosuppressive splenic phenotypes compared to WT mice with divergence in purine metabolism, gluconeogenesis, and glycolysis. In contrast, Berk-SS mice have a distinct liver pro-inflammatory phenotype not shared by HbbTh3/+ or WT mice. Together, these data emphasize that metabolic and proteomic reprogramming of the spleen and livers in Berk-SS and HbbTh3/+mice may be relevant to the individual disease processes.

Inflammation drives the initiation and progression of pulmonary hypertension (PH). Platelets, increasingly recognized as immune cells, are activated and increased in the lungs of patients with PH. Platelet activation leads to the release of α-granule chemokines, many of which are implicated in PH. We hypothesized that hypoxia-induced secretion of platelet α-granule stored proteins and PH would be prevented in Nbeal2 -/-, α-granule deficient mice. WT and Nbeal2 -/- mice were maintained in normoxia or exposed to 10% hypobaric hypoxia for 3, 14, 21, or 35 days. We observed macrothrombocytopenia, increased circulating neutrophils and monocytes, and increased lung interstitial macrophages in Nbeal2 -/- mice at baseline. Hypoxia-induced platelet activation was attenuated, and hypoxia-induced increase in lung PF4 and platelets was delayed in Nbeal2 -/- mice compared to WT mice. Finally, although pulmonary vascular remodeling (PVR) and PH were attenuated at day 21, Nbeal2 -/- mice were not protected against hypoxia-induced PVR and PH at day 35. While this mutation also impacted circulating monocytes, neutrophils, and lung IMs, all of which are critical in the development of experimental PH, we gained further support for the role of platelets and α-granule proteins, such as PF4, in PH progression and pathogenesis and made several observations that expand our understanding of α-granule deficient mice in chronic hypoxia.

Schistosomiasis and sickle cell disease (SCD) both cause pulmonary hypertension (PH). We identified a subject with sickle cell trait and hepatosplenic schistosomiasis, who on right heart catheterization had PH, but due to high cardiac output. In a pre-clinical model, we found SCD mice were protected from developing schistosomiasis-induced PH.

ABSTRACT:Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects 500 million people globally, affecting red blood cell (RBC) antioxidant pathways and increasing susceptibility to hemolysis under oxidative stress. Despite the systemic generation of reactive oxygen species during exercise, the effects of exercise on individuals with G6PD deficiency remain poorly understood This study used humanized mouse models expressing the G6PD Mediterranean variant (S188F, with 10% enzymatic activity) to investigate exercise performance and molecular outcomes. Surprisingly, despite decreased enzyme activity, G6PD-deficient mice have faster critical speed than mice expressing human canonical G6PD. After exercise, deficient mice did not exhibit differences in RBC morphology or hemolysis, but had improved cardiac function, including cardiac output, stroke volume, sarcomere length, and mitochondrial content. Proteomics analyses of cardiac and skeletal muscles (gastrocnemius, soleus) from G6PD-deficient compared with sufficient mice revealed improvements in mitochondrial function and increased protein turnover via ubiquitination, especially for mitochondrial and structural myofibrillar proteins. Mass spectrometry-based metabolomics revealed alterations in energy metabolism and fatty acid oxidation. These findings challenge the traditional assumptions regarding hemolytic risk during exercise in G6PD deficiency, suggesting a potential metabolic advantage in exercise performance for individuals carrying noncanonical G6PD variants.

Pulmonary hypertension (PH) significantly impairs exercise capacity and the quality of life in patients, which is influenced by dysfunctions in multiple organ systems, including the right ventricle, lungs, and skeletal muscles. Recent research has identified metabolic reprogramming and mitochondrial dysfunction as contributing factors to reduced exercise tolerance in PH patients. In this study, we investigated the therapeutic potential of enhancing mitochondrial function through the activation of the mitochondrial deacetylase SIRT3, using SIRT3 activator Honokiol combined with the SIRT3 co-factor nicotinamide adenine dinucleotide (NAD), in a Sugen/Hypoxia-induced PH rat model. Our results show that Sugen/Hypoxia-induced PH significantly impairs RV, lung, and skeletal muscle function, leading to reduced exercise capacity. Treatment with Honokiol and NAD notably improved exercise endurance, primarily by restoring SIRT3 levels in skeletal muscles, reducing proteolysis and atrophy in the gastrocnemius, and enhancing mitochondrial complex I levels in the soleus. These effects were independent of changes in cardiopulmonary hemodynamics. We concluded that targeting skeletal muscle dysfunction may be a promising approach to improving exercise capacity and overall quality of life in PH patients.

A comprehensive national study investigated education outside the classroom (EOTC) in Aotearoa New Zealand and revealed the negative impact of safety legislation on EOTC. To understand the work the safety legislation does, we analysed data from a survey and interviews, and developed a framework of spheres of responsibilities: student safety; legislative requirements; staff competence; and paperwork. Findings show that health and safety legislation has reduced the amount of EOTC in many schools. There was strong evidence that educators cared for the learning and safety of students, and this generated anxiety for staff. This anxiety was further heightened by threats of personal legal liability. Instead of threats, some respondents felt that teachers' commitment to student learning through EOTC should be celebrated. Other respondents strongly continued to support EOTC. The contribution of school culture, EOTC champions, effective systems and teacher education are seen as pivotal to reducing anxiety and sustaining EOTC.

Sickle cell disease (SCD) is characterized by central (cardiac) and peripheral vascular dysfunctions, significantly diminishing exercise capacity and quality of life. Although central cardiopulmonary abnormalities in SCD are known to reduce exercise capacity and quality of life; the impact of hemolysis and subsequent cell-free hemoglobin (Hb)-mediated peripheral vascular abnormalities on those outcomes are not fully understood. Despite the recognized benefits of exercise training for cardiovascular health and clinical management in chronic diseases like heart failure, there remains substantial debate on the advisability of regular physical activity for patients with SCD. This is primarily due to concerns that prolonged and/or high-intensity exercise might trigger metabolic shifts leading to vaso-occlusive crises. As a result, exercise recommendations for patients with SCD are often vague or nonexistent, reflecting a gap in knowledge about the mechanisms of exercise intolerance and the impact of exercise training on SCD-related health issues. This mini-review sheds light on recent developments in understanding how SCD affects exercise tolerance, with a special focus on the roles of hemolysis and the release of cell-free hemoglobin in altering cardiovascular and skeletal muscle function. Also highlighted here is the emerging research on the therapeutic effects and safety of exercise training in patients with SCD. In addition, the review identifies future research opportunities to fill existing gaps in our understanding of exercise (in)tolerance in SCD.