Executive Overview
Red blood cells (RBCs) rely entirely on glycolysis to sustain their structural integrity, membrane flexibility, and essential physiological functions. In sickle cell disease (SCD), this energy balance is frequently disrupted, leading to chronic hemolytic anemia, painful vaso-occlusive crises (VOCs), and progressive end-organ damage. A promising therapeutic approach involves targeting red blood cell pyruvate kinase (PKR) with small-molecule activators such as mitapivat (AG-348). These pharmacological agents are designed to enhance intracellular adenosine triphosphate (ATP) production while simultaneously suppressing upstream 2,3-diphosphoglycerate (2,3-DPG) levels, thereby inhibiting sickling and improving erythrocyte survival.
Despite the growing clinical interest in PKR activators, their impact on the export of ATP from red blood cells—a critical physiological mechanism that regulates vascular tone, prevents excess capillary permeability, and limits intercellular adhesion—has remained largely unexplored. To bridge this knowledge gap, researchers at Duke Health conducted a comprehensive ex vivo investigation to evaluate the effects of mitapivat on intracellular and exported ATP levels, as well as on cellular adhesivity, in blood samples obtained from healthy controls and pediatric SCD patients.
The study’s findings reveal a striking dichotomy: while ex vivo treatment with mitapivat significantly increases both intracellular ATP content and ATP export in healthy control RBCs under normoxic and hypoxic conditions, it fails to produce similar intracellular or extracellular ATP elevations in SCD red blood cells. However, microfluidic analyses demonstrated that whole-blood exposure to the pyruvate kinase activator (PKRA) effectively mitigates sickle red blood cell adhesion to vascular basement membrane components like laminin under physiologically relevant shear stresses. These insights shed critical light on the complex metabolic wiring of sickle erythrocytes and provide vital context for optimizing emerging metabolic therapies.
Detailed Chronology and Experimental Design
To evaluate the translational potential and cellular consequences of pharmacological PKR activation, the research team structured a rigorous, multi-tiered laboratory investigation using human whole-blood samples collected under institutional review board (IRB)-approved protocols.
Phase I: Sample Acquisition and Patient Cohorts
Whole-blood samples were gathered from healthy adult volunteers and pediatric SCD outpatients. To maintain clinical consistency, the SCD cohort was carefully filtered: patients who had experienced blood transfusions or emergency department admissions within the preceding 30 days, those exhibiting fetal hemoglobin (HbF) levels exceeding 20%, and individuals actively prescribed Voxelotor or L-glutamine were excluded from the study. Ultimately, samples from 15 pediatric SCD patients—all with confirmed HbSS genotypes and the vast majority undergoing concurrent hydroxyurea (HU) therapy—alongside samples from 13 healthy control subjects, were utilized for the primary evaluations.
Phase II: Ex Vivo Drug Exposure and Tonometry
Whole blood from both cohorts was incubated with either the non-selective pyruvate kinase activator mitapivat (AG-348 at a concentration of 10 µM) or a control vehicle (DMSO). Initial optimization experiments involving healthy control blood established that an incubation period of 18 to 24 hours at 4 °C successfully enhanced intra-RBC ATP and ATP export. Consequently, this protocol was applied uniformly across the SCD patient samples.
Following incubation, isolated RBCs were washed and subjected to controlled gas-mixture tonometry at 37 °C using a final hematocrit of 1% in Krebs buffer. The cell suspensions were equilibrated for 8 minutes under strict normoxic (21% $O_2$, 5% $CO_2$) or hypoxic (1% $O_2$, 5% $CO_2$) conditions. Following centrifugation, supernatant ATP and intracellular ATP concentrations were quantified using a sensitive luciferase assay, alongside spectroscopic hemoglobin quantification to monitor and calculate post-assay hemolysis rates.
Phase III: Microfluidic Adhesion Assays
To determine whether PKR activation influences the adhesive properties of sickle erythrocytes, parallel aliquots of AG-348-treated and vehicle-exposed SCD whole-blood and washed RBC samples were perfused through microfluidic channels. These channels were pre-coated with recombinant human laminin and blocked with bovine serum albumin (BSA) to prevent non-specific binding. Using a precision syringe pump, the channels were subjected to escalating physiological shear stresses ranging from 0.3 to 10 dynes/$cm^2$. Automated digital microscopy captured images at baseline and following each shear increment, allowing researchers to calculate cellular adhesivity as the percentage of cells remaining bound relative to baseline.
Supporting Context & Metrics
The experimental outcomes revealed distinct physiological responses between healthy and sickle cell erythrocytes, challenging several assumptions regarding metabolic modulation in hemoglobinopathies.
Intracellular ATP Dynamics
In healthy control samples, ex vivo exposure to mitapivat significantly elevated intracellular ATP levels under both normoxic (increasing from 57.84 µM with vehicle to 77.59 µM with AG-348) and hypoxic conditions. However, in the SCD patient cohort, treatment with AG-348 failed to elicit a statistically significant increase in intra-RBC ATP under normoxia (71.48 µM vs. 75.17 µM) or hypoxia. Furthermore, basal intracellular ATP levels in the pediatric SCD cohort (nearly all treated with hydroxyurea) did not differ significantly from those of healthy controls, aligning with previous observations that hydroxyurea therapy can partially restore baseline energy reserves in sickle erythrocytes.
ATP Export and Hemolysis Rates
In untreated healthy control samples, extracellular ATP export increased markedly following exposure to hypoxia (94.28 nM vs. 79.70 nM in normoxia). Treatment with mitapivat further amplified ATP export in healthy RBCs under both normoxic (80.31 nM to 109.0 nM) and hypoxic conditions.
For sickle cell disease erythrocytes, while hypoxia similarly triggered a modest rise in ATP export, ex vivo treatment with AG-348 did not significantly alter exported ATP levels in either normoxia or hypoxia. Crucially, post-assay hemolysis remained minimal across all experimental arms, registering between 1.0% and 1.5% with no significant variation attributable to drug treatment, oxygen tension, or disease state, confirming that the measured extracellular ATP was actively exported rather than passively released via cellular lysis.
| Experimental Parameter | Healthy Controls (Vehicle) | Healthy Controls (AG-348) | SCD Patients (Vehicle) | SCD Patients (AG-348) |
|---|---|---|---|---|
| Normoxic Intra-RBC ATP ($mu$M) | 57.84 $pm$ 5.64 | 77.59 $pm$ 10.12 | 71.48 $pm$ 11.96 | 75.17 $pm$ 19.41 |
| Hypoxic Intra-RBC ATP ($mu$M) | 57.67 $pm$ 5.69 | 81.03 $pm$ 9.98 | Not significantly altered | Not significantly altered |
| Normoxic Exported ATP (nM) | 80.31 $pm$ 22.27 | 109.0 $pm$ 30.23 | 79.70 $pm$ 20.58 | 77.12 $pm$ 19.91 |
| Hypoxic Exported ATP (nM) | 94.28 $pm$ 24.34 | Significantly Increased | Modestly Increased | No significant change |
| Post-Assay Lysis (%) | ~1.0% – 1.4% | ~1.0% – 1.4% | ~1.0% – 1.5% | ~1.0% – 1.5% |
Adhesion Characteristics
Microfluidic evaluation of sickle erythrocyte interactions with laminin yielded notable mechanistic insights. While direct treatment of washed SCD red blood cells with AG-348 did not significantly alter cellular adhesivity, whole-blood exposure to the pyruvate kinase activator resulted in a significant interaction with shear stress, showing reduced cell adhesion at physiologically relevant low shear rates (0.3, 1, and 3 dynes/$cm^2$). This discrepancy strongly implies that plasma components, leukocytes, or platelets act in concert with red blood cell metabolic shifts to modulate vascular adhesion.
Official Statements & Investigator Perspectives
Reflecting on the implications of these findings, lead author Dr. Apoorva Jagadish and senior investigator Dr. Tim J. McMahon emphasized the importance of dissecting the precise downstream consequences of metabolic therapeutics.
"Our data demonstrate for the first time that ATP export from healthy red blood cells increases reliably following treatment with a pyruvate kinase activator," stated Dr. Jagadish. "However, the failure of a single ex vivo dose of mitapivat to substantially expand internal or exported ATP pools in pediatric sickle cell disease patients—many of whom are on hydroxyurea therapy—highlights the complex metabolic baseline of these cells. It underscores that drug responses cannot be universally extrapolated from healthy models to diseased states."
Dr. McMahon added context regarding the observed anti-adhesive properties of the compound: "The reduction in sickle erythrocyte adhesion when whole blood, rather than isolated red blood cells, is exposed to AG-348 suggests an intricate cross-talk within the vascular compartment. While direct ATP export from SCD cells did not spike in our ex vivo setup, the functional benefit on cell-matrix interactions points to broader vasoprotective pathways that warrant deep clinical investigation as these therapeutic classes move through advanced trials."
Future Outlook & Clinical Implications
The development of red blood cell pyruvate kinase activators represents a paradigm shift in the management of hemolytic anemias and hemoglobinopathies. By simultaneously enhancing intracellular ATP synthesis and driving down elevated 2,3-DPG levels—thereby increasing hemoglobin-oxygen affinity and inhibiting HbS polymerization—agents like mitapivat, etavopivat, and tebapivat offer multimodal disease-modifying potential.
However, several critical questions remain open for future research. Investigators note that subsequent clinical studies must explore whether continuous, long-term in vivo administration of PKR activators overcomes the limitations observed during acute ex vivo exposure. Furthermore, researchers plan to evaluate whether baseline differences exist in patients not currently maintained on hydroxyurea therapy, and to fully characterize the cellular determinants governing purine nucleotide export in inflamed vascular microenvironments. As clinical trials advance, mapping these metabolic and hemorheological pathways will be essential to maximizing therapeutic efficacy and ensuring long-term vascular safety for patients living with sickle cell disease.