Red blood cells stored for increasing periods produce progressive impairments in nitric oxide-mediated vasodilation

Abstract

Background: Clinical outcomes in transfused patients may be affected by the duration of blood storage, possibly due to red blood cell (RBC)-mediated disruption of nitric oxide (NO) signaling, a key regulator of vascular tone and blood flow.

Study design and methods: AS-1 RBC units stored up to 42 days were sampled at selected storage times. Samples were added to aortic rings ex vivo, a system where NO-mediated vasodilation could be experimentally controlled.

Results: RBC units showed storage-dependent changes in plasma hemoglobin (Hb), RBC 2,3-diphosphoglycerate acid, and RBC adenosine triphosphate conforming to expected profiles. When freshly collected (Day 0) blood was added to rat aortic rings, methacholine (MCh) stimulated substantial NO-mediated vasodilation. In contrast, MCh produced no vasodilation in the presence of blood stored for 42 days. Surprisingly, the vasoinhibitory effects of stored RBCs were almost totally mediated by RBCs themselves: removal of the supernatant did not attenuate the inhibitory effects, while addition of supernatant alone to the aortic rings only minimally inhibited MCh-stimulated relaxation. Stored RBCs did not inhibit vasodilation by a direct NO donor, demonstrating that the RBC-mediated vasoinhibitory mechanism did not work by NO scavenging.

Conclusions: These studies have revealed a previously unrecognized vasoinhibitory activity of stored RBCs, which is more potent than the described effects of free Hb and works through a different mechanism that does not involve NO scavenging but may function by reducing endothelial NO production. Through this novel mechanism, transfusion of small volumes of stored blood may be able to disrupt physiologic vasodilatory responses and thereby possibly cause adverse clinical outcomes.

Fig. 1

Alterations in plasma free Hb, 2,3-DPG, and ATP during RBC storage. Serial aliquots, removed from saRBC units every 7 days over 42 days of storage, were assayed as described under Materials and Methods (n = 8-15 samples per data point). (A) Compared to Day 0, hemolysis was significantly greater on Days 35 and 42 of storage (*p < 0.05). However, mean plasma Hb on Day 42 (0.42 g/dL) represents less than 1% hemolysis (approx. 0.45 g/dL). (B) 2,3-DPG levels declined progressively with storage, as previously documented (*p < 0.05 vs. Day 0 sample). (C) ATP levels seen in stored blood were comparable to those previously described in the literature; compared to Day 28, the decrease seen on Day 42 was significant (*p < 0.05).

Fig. 2

Quantitation of plasma nitrite and nitrate during RBC storage. Plasma samples obtained during the storage period were tested by ion chromatography to quantify nitrite (A) and nitrate (B) concentrations (n = 9-15 samples per data point). No significant storage-related changes were observed at any of the time points (p > 0.05).

Fig. 3

Blood storage time correlates with decreased endothelium-dependent vasorelaxation. Vasorelaxation of rat aortic segments was quantified in response to increasing concentrations of the endothelium-dependent vasorelaxant MCh. (A) MCh stimulated marked vasorelaxation in the presence of Day 0 saRBCs (, 1% final concentration), but not Day 42 saRBCs (, *p < 0.001; n = 14-19 experiments per data point). (B) Time course analysis shows that blood stored 28 days or longer significantly inhibits vasodilation compared to Day 0 saRBCs (* p < 0.01; n = 62 [no blood], n = 10-19 [data points with added blood]). (C) Day 42 saRBCs () were more effective than Day 0 samples () at stimulating vasoconstriction when added to prerelaxed vessels (*p < 0.01 for two curves by two-way analysis of variance; n = 6 samples per data point).

Fig. 4

The vasoinhibitory effects of saRBCs are primarily mediated by RBCs, not the supernatant, and do not act by scavenging NO. Day 0 (A) and Day 42 (B) saRBC samples were washed to replace the supernatant with 0.9% NaCl (), or left unmanipulated (), and then added to aortic rings before MCh. Removal of supernatant by washing did not significantly affect the vasoinhibition (p > 0.05; n = 5-11 samples per data point). (C) Compared to the no supernatant added control (○), addition of Day 0 saRBC supernatant (■, n = 6) did not inhibit MCh-stimulated vasodilation (p > 0.05; NS). However, Day 42 supernatant (▲) did cause a small but significant inhibition of vasodilation (*p < 0.01) at the highest four MCh concentrations when compared to no supernatant, but not when compared to Day 0 supernatant (p > 0.05; NS). These results suggest that saRBC supernatant accounts for only a small fraction of the vasoinhibitory activity of unmanipulated saRBC samples. (D) In contrast to the inhibitory activity of saRBCs on endothelium-dependent MCh-stimulated vasodilation, saRBCs had no effect on vasodilation induced by the endothelium-independent NO-donor SNP (p > 0.05; n = 4). () Day 0; () Day 42.