American Journal of Physiology – Scientific Paper – Hypoxia and Red Blood Cells Studies

University: Duke Cancer Institute of Duke University Medical Center
Authors: Brett S. Kirby, Gabi Hanna, Hansford C. Hendargo, and Timothy J. McMahon
Journal: American Journal of Physiology

TITLE: Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion.

ABSTRACT: Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested.

We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ∼42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., “PIPA”) restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ∼120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo. red blood cell (rbc) transfusion is a common and sometimes lifesaving medical practice, but accumulating evidence questions both the criteria indicating a transfusion requirement and the relative benefits of transfusing RBCs later in their storage period (42-day maximum duration in the United States) (2, 5, 16–18, 20). Banking of RBCs elicits a “storage lesion” characterized by time-dependent metabolic and biochemical alterations including diminished intracellular ATP and 2,3-DPG, and a loss of bioactive nitric oxide (NO) derivatives (2, 30, 31, 33).

Functionally, longer-banked RBCs are less deformable, promote hypoxic vasodilation poorly, and adhere excessively to blood vessel walls (2, 30, 31, 43). Such effects from RBC banking could contribute to the disappointing, adverse clinical outcomes (e.g., increased hospital days and duration of mechanical ventilation, multiple organ failure, transfusion-related lung injury, mortality) often associated with transfusion of longer-stored RBCs (20, 31, 32). Remarkably, RBCs rely solely on glycolysis for intracellular ATP production, and hemoglobin (Hb) deoxygenation is a primary and potent activator of glycolytic flux within the RBC (4, 25, 39). Glycolytic enzymes normally bound to the RBC cytoplasmic domain of the membrane protein band 3 (cdb3; band 3 is also called anion exchanger-1) during R-state Hb conformation are liberated upon T-state Hb transition, thus shifting RBC metabolism from the pentose phosphate pathway (PPP; supporting generation of NADPH) toward the Embden Myerhof pathway (glycolytic production of NADH and ATP) (4, 23, 25). In this regard, coupling of RBC energy metabolism and O2-sensing is directly governed by O2-dependent regulation of binding of Hb by cdb3 (23, 25). 

Increasing intracellular ATP production, particularly in hypoxia, is essential for ion transport processes, phosphorylation reactions, and the production of reducing equivalents. These ATP-dependent events in turn regulate osmotic balance, cellular integrity, and redox reactions (9, 35, 39). Over a decade of investigation now indicates that ATP is more than a mere intracellular energy molecule, but also undergoes regulated export to assist in controlling extracellular processes (3, 7). In RBCs, functional glycolysis and cdb3 activity are two key factors regulating the hypoxia-sensitive controlled export of ATP (3, 13). 

We recently demonstrated that stored RBCs (sRBCs) exhibit a marked impairment in deoxygenation-induced ATP export (43). Furthermore, we observed that extracellular ATP acts as an anti-adhesive molecule and that RBCs deficient in ATP export are excessively adherent to the vascular endothelium (43). Given the cumulative evidence demonstrating that glycolytic inhibition significantly blunts ATP export from fresh RBCs (fRBCs) (13) and that RBC band 3 content is lowered with RBC storage (22), we questioned whether banked RBCs are functionally defective in glycolysis-mediated ATP production in response to deoxygenation and whether restoration of ATP production and export might attenuate RBC adhesion after transfusion. 

We performed a series of experiments to investigate 1) the capacity for sRBCs to increase glycolytic activity and ATP generation in hypoxia, 2) the impact of raising intracellular ATP content of banked RBCs on the controlled release of ATP from RBCs, and 3) how these interventions influence adhesion of transfused RBCs to the microvasculature in vivo. 

Accordingly, we first tested the hypothesis that banked RBCs are defective in the control of low O2-stimulated glycolysis (evidenced via intracellular ATP measures) and that RBC supplementation with an FDA-approved additive solution containing glycolytic intermediate/purine/phosphate precursors (i.e., pyruvate, inosine, inorganic phosphate, and adenine; here called “PIPA”) reverses this deficiency. 

Second, we tested the hypothesis that augmentation of hypoxia-mediated increases in intracellular ATP of banked RBCs is associated with an improvement in controlled, hypoxia-triggered ATP export. 

Third, we tested the hypothesis that banked human RBCs are markedly adhesive in vivo following transfusion and that pretransfusion treatment of RBCs with glycolytic intermediate / purine / phosphate precursors known to increase both intracellular ATP and the inducible extracellular release of ATP attenuates storage-mediated adhesion of transfused RBCs. To do so, we used an FDA-approved additive solution, PIPA, rich in RBC metabolic precursors and used a murine model of RBC transfusion to examine human RBC function in vivo.

FULL AND ORIGINAL SCIENTIFIC PAPER: American Journal of Physiology

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