Red Blood Cell ATP Release Correlates with Red Blood Cell Hemolysis
Authors: Ferguson Brian S. , Neidert Leslie E. , Rogatzki Matthew J. , Lohse Keith R. , Gladden L. Bruce , Kluess Heidi A. 
Published: November 2023

The elegant and precise matching of blood flow to skeletal muscle from the onset of exercise through the steady state remains an important area of investigation. Release of adenosine triphosphate (ATP) from the red blood cell (RBC) has been postulated as a mediator of peripheral vascular tone in response to shear stress, hypoxia, and mechanical deformation. While these stimuli coexist during exercise, questions persist concerning the regulated release of ATP. Alternatively, intravascular hemolysis may contribute significantly to the appearance of ATP in circulation. We aimed to test the following hypotheses: 1) RBCs within different density ranges contain different quantities of ATP, 2) hypoxia serves as a stimulus for ATP release from RBCs, 3) ATP release from RBCs due to hypoxia is dependent on RBC lysis.Human blood was drawn from recreationally active male and female volunteers (n=11) by venipuncture of a prominent antecubital vein. Hematocrit was determined, then RBCs were washed and isolated. A Percoll gradient was used to separate RBCs based on cellular density (low density (LD)<1.068 g/ml; medium density (MD)=1.068–1.076 g/ml; high density (HD)>1.076 g/ml. Each density group was then re‐suspended to 4% hematocrit and exposed to eight minutes of hypoxia or normoxia in a Tonometer at 37°C. Equilibrated samples were drawn and centrifuged to obtain supernatant samples for analysis. ATP was quantified by luminescence via a luciferase‐catalyzed reaction and hemolysis was determined with the Harboe spectrophotometric absorbance assay.Normoxic conditions were: PO2 = 106±8 mmHg, PCO2 = 33±2 mmHg, pH = 7.32±0.03. Hypoxic conditions were: PO2 = 11±2 mmHg, PCO2 = 38±1 mmHg, pH = 7.28±0.03 (p≤0.05, different from normoxia). RBCs separated by density showed no difference in diameter (LD 80% of cells; MD 86% of cells; HD 85% of cells at 6–7μm; p≥0.05). Total ATP release was not different following matched exposure time for LD, MD, or HD cell groups in normoxia (1.03±0.43, 0.82±0.38, and 1.22±0.74 nM ATP) or hypoxia (1.52±1.09, 0.53±0.23, and 1.07±0.72 nM ATP; p≥0.05) respectively. Using ATP curves generated by lysing a known number of RBCs in each density group, we calculated the number of lysed RBCs necessary for our measured ATP and found no difference between normoxia (39±18, 34±17, and 42±16 RBCs) and hypoxia (65±59, 20±7, and 35±12 RBCs; p≥0.05) respectively. Correlations were examined between ATP release and hemolysis for raw measures (r=0.5769, p≤0.05) as well as for release calculated per lysed RBC (r=0.5081, p≤0.05) indicating all measured ATP could be explained by hemolysis.With concomitant probing for free hemoglobin and ATP in the supernatant of each sample, we conclude that hemolysis is the primary cause for the appearance of ATP from RBCs. With equal durations of exposure in a Tonometer we found no increases in the appearance of ATP resulting from a hypoxic stimulus indicating that hypoxia per se does not stimulate release of ATP from RBCs.