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.

Rejuvenation of allogenic red cells: benefits and risks

“…Messana and colleagues demonstrated that rejuvenation did not restore this metabolic modulation after 21 days of storage, and in fact increased glucose conveyance to the PPP despite restoring 2,3‐DPG levels . Conversely, Kirby and colleagues demonstrated that rejuvenation restored ATP synthesis and export in red cells stored in AS‐3 for >35 days, and reduced cell adhesion to murine vascular endothelium in vivo .…”

“…Red cell rejuvenation refers to a process whereby red cells, prior to transfusion, are incubated with a solution containing pyruvate, inosine, phosphate and adenine (PIPA) at 37°C for 1 h, and then washed. Rejuvenation acts by restoring glycolytic flux via three processes . The first is the conversion of exogenous inosine towards ribose‐5‐phosphate, which can then be converted to the key glycolytic intermediates, fructose‐6‐phosphate and glyceraldehyde‐3‐phosphate.…”

Predicting storage‐dependent damage to red blood cells using nitrite oxidation kinetics, peroxiredoxin‐2 oxidation, and hemoglobin and free heme measurements

“…Several retrospective studies, together with experimental studies in different animal models, have shown that older RBCs provide a second hit that augments microcirculatory dysfunction, inflammatory tissue injury, and infection. These consequences underscore the associations between the age and number of RBCs transfused and morbidity and mortality in transfusion recipients . Specific proposed mechanisms of injury include increased oxidative stress and inflammation, inhibition of nitric oxide (NO) signaling and perturbation of iron homeostasis.…”

Liberation of ATP secondary to hemolysis is not mutually exclusive of regulated export

“…[2][3][4][6][7][8]10,11,13 Even the earliest investigations, from Bergfeld and Forrester, 9 parsed authentic ATP release from that due to cell damage, and numerous publications document regulated ATP export from RBCs while controlling for hemolysis. If increases in [ATP] ec are solely from hemolysis, then [ATP] ec should be unaffected by antagonizing ATP-export signaling.…”