Synopsis Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to the RBC. It has been suggested that these changes have a potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion related toxicities. However, mechanisms linking RBC storage and toxicity remain unclear. In this study we tested the hypothesis that storage of leukodepleted RBC result in cells that inhibit nitric oxide (NO)-signaling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from hemolysis or microparticles, our data indicate that NO-consumption rates increased ~40-fold and NO-dependent vasodilation was inhibited 2-4 fold with 42d old vs. 0d RBC. These results are likely due to the formation of smaller RBC with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBC to affect NO-formation via deoxygenated RBC mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBC did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBC stored for >25d, but not with younger RBC. Collectively, these data suggest that increased rates of reactions between intact RBC and NO and nitrite may contribute to mechanisms that lead to storage lesion-related transfusion risk

Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory and nitric oxide (NO) scavenging properties of stored RBC are thought to underlie this association. In this study we determined the effects of RBC washing, nitrite and anti-heme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57bl/6) model of trauma-hemorrhage and resuscitation with 1 or 3 units of RBC stored for 0–10d was used. Tested variables included whether washing RBC to remove lower MWt components that scavenge NO, NO-repletion therapy using nitrite or mitigation of free heme-toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5d RBC increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5d RBC prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10d RBC elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5d RBC. Both washing and nitrite therapy significantly protected against 10d RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBC. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBC. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5d and 10d mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy and / or anti-heme and TLR4 strategies may prevent stored RBC toxicities.

These data highlight the potential for slower Prx-2 recycling and β93Cys oxidation in modulating storage-dependent damage of RBCs and in mediating post-transfusion toxicity.

The blood storage lesion involves morphological and biochemical changes of red blood cells (RBCs) that occur during storage. These include conversion of the biconcave disc morphology to a spherical one, decreased mean corpuscular hemoglobin concentration, varied mean corpuscular volume, reduced integrity of the erythrocyte membrane with formation of microparticles, and increased cell-free hemoglobin. We studied the extent that older stored red blood cells scavenge nitric oxide (NO) faster than fresher stored red blood cells. Using electron paramagnetic resonance spectroscopy and stopped-flow absorption spectroscopy to measure the rate of NO uptake and reaction with hemoglobin in red cells, we found that older stored red blood cells scavenge NO about 1.8 times faster than fresher ones. Based on these experimental data, we simulated NO scavenging by fresher or older stored red blood cells with a biconcave or spherical geometry, respectively, in order to explore the mechanism of NO scavenging related to changes that occur during blood storage. We found that red blood cells with a spherical geometry scavenges NO about 2 times slower than ones with a biconcave geometry, and a smaller RBC hemoglobin concentration or volume increases NO scavenging by red blood cells. Our simulations demonstrate that even the most extreme possible changes in mean corpuscular hemoglobin concentration and mean corpuscular volume that favor increased NO scavenging are insufficient to account for what is observed experimentally. Therefore, RBC membrane permeability must increase during storage and we find that the permeability is likely to increase between 5 and 70 fold. Simulations using a two-dimensional blood vessel show that even a 5-fold increase in membrane permeability to NO can reduce NO bioavailability at the smooth muscle.BackgroundTransfusion of older stored blood may be harmful.ResultsOlder stored red blood cells scavenge nitric oxide more than fresher cells.ConclusionAs stored red blood cells age, structural and biochemical changes occur that lead to faster scavenging.SignificanceIncreased nitric oxide scavenging by red blood cells as a function of storage age contributes to deleterious effects upon transfusion.

Nitric oxide (NO) is a key modulator of vascular homeostasis controlling critical functions related to blood flow, respiration, cell death and proliferation, and protecting the vasculature from pro‐inflammatory and coagulative stresses. Inhibition of NO formation, and/or diversion of NO away from its physiological signalling targets lead to dysregulated NO bioavailability, a hallmark of numerous vascular and pulmonary diseases. Current concepts suggest that the balance between NO formation and NO scavenging is critical in disease development, with the corollary being that redressing the balance offers a target for therapeutic intervention. Evidence presented over the last two decades has seen red blood cells (RBCs) and haemoglobin specifically emerge as prominent effectors in this paradigm. In this symposium review article, we discuss recent insights into the mechanisms by which RBCs may modulate the balance between NO‐formation and inhibition. We discuss how these mechanisms may become dysfunctional to cause disease, highlight key questions that remain, and discuss the potential impact of these insights on therapeutic opportunities.