Basal and stimulated intra-RBC and exported ATP and RBC adhesivity ex vivo in children with sickle cell disease Free

Sickle cell disease (SCD) represents a significant global health burden, and is characterized by hemolytic anemia, vaso-occlusive crises, and end-organ damage.

RBCs depend on glycolysis due to virtual absence of mitochondria. RBC glycolysis generates adenosine triphosphate (ATP) and regulates levels of 2,3-diphosphoglycerate (2,3-DPG). Intracellular ATP is essential for numerous RBC functions, eg maintaining ion homeostasis, cell membrane integrity, redox protection, and RBC deformability. Sickle (SS) RBCs have reduced intracellular ATP compared to normal RBCs.

RBCs also export ATP, which can modulate blood flow in response to hypoxia, inhibit intercellular adhesion, and prevent excess capillary permeability. We sought primarily to investigate the effects of exported ATP in SS vs. healthy RBCs.

Pyruvate kinase (PK) catalyzes the final step of glycolysis, and generating intra-RBC ATP. RBC-specific PK (PKR) activators (PKRAs), such as etavopivat and mitapivat, are now approved in PK deficiency, and are being studied in SCD patients. They act by increasing intracellular ATP while decreasing upstream 2,3-DPG. Our secondary aim was to determine the effects of a PKRA on ATP export by RBCs in SCD vs. healthy (HC) individuals.

Leftover whole blood (WB) samples were collected from SCD patients, and fresh blood samples from healthy adult volunteers (IRB-approved). WB was incubated with AG-348 (mitapivat, 10 µM) or vehicle (DMSO) at 4°C for 18-24 h. After incubation, RBCs were separated from WB by centrifugation and washed twice with PBS. Washed RBCs then underwent RBC tonometry (gentle, controlled gas exposure) at a final hematocrit of 1% RBCs in Krebs buffer containing 0.5% bovine serum albumin (BSA). RBC suspensions were equilibrated in normoxia (74% N2, 21% O2, 5% CO2) or hypoxia (94% N2, 1% O2, 5% CO2). After centrifugation, supernatant and intracellular ATP were assayed (luciferase method). Hemoglobin (Hb) was assayed to quantify intra-assay hemolysis.

In parallel, aliquots of treated or vehicle-exposed SCD WB were diluted with PBS for adhesion assays. Ibidi microfluidic channels were coated with laminin, incubated at 25°C for 1 h, coated with 1% BSA, and incubated at 37°C for 1 h. After incubation, slides were washed with PBS and loaded with either WB or RBCs alone. Using a syringe pump, the channels were exposed to increasing shear stress levels (0.3, 1, 3, 10 dynes/cm²) on a microscope platform using DPBS (containing calcium and magnesium). Photomicrographs of the channels were obtained basally and after each shear stress increment, and were processed (Image J) to count cells and calculate adhesivity (the % cells remaining at each shear).

We obtained WB from 15 SCD patients (8 females, ages 3 to 19 y). Exported ATP was increased after exposure to hypoxia (94.3 ± 24.3 nM (mean ± SEM) vs. 79.7 ± 20.6 nM in normoxia). Exported ATP did not change significantly in SCD samples treated with the PKRA AG-348 (79.7 ± 20.6 nM vs. 77.1 ± 19.9 nM). ATP export from HCs increased after treatment with the PKRA AG-348 (80.3 ± 22.3 nM vs. 109.0 ± 30.2 nM).

PKRA treatment ex vivo did not increase intra-RBC ATP significantly in SCD blood, while PKRA raised intra-RBC ATP in HC blood, consistent with prior reports.

Post-tonometry lysis levels were 1% or lower under all conditions. These low levels of lysis in ATP export assays did not differ as a function of PKRA treatment or between HC and SCD RBCs.

When correlating the experimental findings with clinical data, we found near-significance (p = 0.056) for an association of fetal Hb and changes in exported ATP in SCD samples treated with PKRA. Neither age nor sex had associations with ATP export findings.

As compared to the effects of vehicle alone, RBC adhesion to laminin after WB exposure to AG-348 was decreased at the physiologically relevant shear stresses of 0.3, 1, and 3 dyne/cm².

To our knowledge, our data show for the first time that ATP export from HC RBCs increases after treatment with a PKRA (which also increased intra-RBC ATP, confirming prior reports). In contrast, similar exposure to PKRA did not significantly increase intra-RBC ATP in SCD, and ATP export from SS RBCs rose in hypoxia but not after ex vivo PKRA treatment. Ongoing work addresses the determinants of cellular and exported ATP in HC and SCD RBCs (basal and treated) and the basis of the antiadhesive effect of this PKRA on SS RBCs.