Impaired adenosine-5'-triphosphate release from red blood cells promotes their adhesion to endothelial cells: a mechanism of hypoxemia after transfusion

Abstract

Objective: Transfusion of red blood cells has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that red blood cell storage impairs the ability of red blood cells to release adenosine-5'-triphosphate and that impaired adenosine-5'-triphosphate release was injurious in vivo, in part through increased red blood cell adhesion.

Design: Prospective, controlled, mechanistic study.

Setting: University research laboratory.

Subjects: Human and mouse blood donors; nude mouse transfusion recipients.

Interventions: Manipulation of adenosine-5'-triphosphate release, supplemental adenosine-5'-triphosphate, and antibodies to red blood cell and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released adenosine-5'-triphosphate and adhesion in responses to (transfused) red blood cells.

Measurements and main results: The ability of stored red blood cells to release adenosine-5'-triphosphate declined markedly within 14 days after collection despite relatively stable levels of adenosine-5'-triphosphate within the red blood cells. Inhibiting adenosine-5'-triphosphate release promoted the adhesion of stored red blood cells to endothelial cells in vitro and red blood cell sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human red blood cells, stored red blood cell transfusion in mice decreased blood oxygenation and increased extravasation of red blood cells into the lung's alveolar air spaces. Similar findings were seen with transfusion of fresh red blood cells treated with the adenosine-5'-triphosphate release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either coinfusion of an adenosine-5'-triphosphate analog or pretransfusion incubation of the red blood cells with an antibody against the erythrocyte adhesion receptor Landsteiner-Wiener (intercellular adhesion molecule-4).

Conclusions: The normal flow of red blood cells in pulmonary microvessels depends in part on the release of antiadhesive adenosine-5'-triphosphate from red blood cells, and storage-induced deficiency in adenosine-5'-triphosphate release from transfused red blood cells may promote or exacerbate microvascular pathophysiology in the lung, in part through increased red blood cell adhesion.

Figure 1. Basal and stimulated release of ATP from RBCs declines during conventional storage

(A) RBCs sampled at varying times after collection and storage were added to chambers filled with Krebs buffer and bubbled with normoxic (21% O₂) or hypoxic (~1% O₂) gas. Extracellular ATP was measured by the luciferase assay in the supernatant of centrifuged samples. Normoxic ATP release was not assayed at 21 days. (B, C) Influence of glibenclamide (Glib.) or carbenoxolone (CBX) on ATP release from fresh RBCs in (B) normoxia or (C) hypoxia. * = p<0.05 compared to control; ANOVA with Tukey’s was used. n=3–6 for each timepoint and condition.

Figure 2. Influence of storage and ATP release on the adhesion of RBCs to cultured endothelial cells

HUVECs adherent to gelatin-coated glass slides were exposed to either stored (35–42 days) or fresh human RBCs treated or not with 10 µm glibenclamide. Adhesion was measured at varying shear stresses as described. (A) shows results from a typical experiment, and (B) shows the mean % adhesion (+ SEM) at a shear stress of 2 dyne/cm²; (n=3). (C) shows the influence of apyrase (5 U/ml) on adhesion of fresh human RBCs to HUVECs at 2 dyne/cm² (mean ± SEM; n=3). * = p<0.05 the pairwise comparison. ANOVA with Tukey’s was used in (B) and paired t-test in (C).

Figure 3. Influence of storage and ATP release in a mouse model of the pulmonary sequelae of RBC transfusion

(A) Typical recordings of arterial Hb O₂ saturation in mice transfused with either stored (35–42 days) human RBCs, untreated (Con) fresh human RBCs, or fresh human RBCs treated with 10 µm glibenclamide (Glib.). RBC transfusions (over 10 seconds) began where indicated by arrow. (B) Mean (+ SEM) peak changes in oxygenation in nude mice after transfusion of stored or fresh, human RBCs treated or not (Con) with Glib. n=6–13. (C) Mean (+ SEM) RBC counts in the bronchoalveolar lavage fluid (BALF) of nude mice after the indicated transfusions (n=5–10). (D,E) Typical fluorescence micrographs, and (F) mean (+ SEM, n=8) fluorescence intensity in the lungs of nude mice transfused with fresh, Dil-labeled RBCs pretreated (E) or not (D) with the ATP-release inhibitor GLIB. Lungs were counterstained with DAPI (blue). * =p<0.05; ANOVA with Tukey’s was used.

Figure 3. Influence of storage and ATP release in a mouse model of the pulmonary sequelae of RBC transfusion

(A) Typical recordings of arterial Hb O₂ saturation in mice transfused with either stored (35–42 days) human RBCs, untreated (Con) fresh human RBCs, or fresh human RBCs treated with 10 µm glibenclamide (Glib.). RBC transfusions (over 10 seconds) began where indicated by arrow. (B) Mean (+ SEM) peak changes in oxygenation in nude mice after transfusion of stored or fresh, human RBCs treated or not (Con) with Glib. n=6–13. (C) Mean (+ SEM) RBC counts in the bronchoalveolar lavage fluid (BALF) of nude mice after the indicated transfusions (n=5–10). (D,E) Typical fluorescence micrographs, and (F) mean (+ SEM, n=8) fluorescence intensity in the lungs of nude mice transfused with fresh, Dil-labeled RBCs pretreated (E) or not (D) with the ATP-release inhibitor GLIB. Lungs were counterstained with DAPI (blue). * =p<0.05; ANOVA with Tukey’s was used.

Figure 4. (A) Extracellular ATP prevents glibenclamide (GLIB)-induced RBC adhesion to endothelial cells

Adhesion of GLIB-treated fresh RBCs to HUVECs was measured in the absence or presence of ATP applied extracellularly (final conc. 1 µM). Fresh RBCs incubated with 10 µm GLIB were washed in PBS. Just before adhesion assays, ATP was applied either to the RBC suspension (RBCs + ATP) or to the HUVECs. Results are mean % adhesion (+ SEM) at a shear stress of 2 dyne/cm² from 3 experiments. * = p<0.05 (differs significantly from adhesion to HUVECs of RBCs treated with GLIB alone). (B–C), Influence of co-infusion of an ATP analog on (B) changes in oxygenation and (C) BALF RBC counts in nude mice transfused with glibenclamide (GLIB)-treated fresh human RBCs. p=0.08 for Con vs. Glib in (A); * indicates p<0.05 for comparison (ANOVA with Tukey’s) with GLIB treatment alone. n=6.

Figure 5. (A) ATP-sensitive RBC adhesion to cultured endothelial cells is mediated by LW (ICAM-4)

Adhesion of glibenclamide (GLIB)-treated fresh RBCs to HUVECs was measured in the absence or presence of antibodies (Abs) to the RBC adhesion receptors LW, CD44, or CD47. RBCs were first incubated with one antibody (20–25 µg/ml) for 45 min followed by washing twice in PBS. *, differs significantly from adhesion of GLIB-treated RBCs to HUVECs in the absence of the mAbs (p<0.05). (B), Influence of exposure of HUVECs to a mAb vs. either CD44 or α_vβ₃ integrin on endothelial adhesion of GLIB-treated RBCs at a shear stress of 2 dyne/cm²; n=3. * p<0.05 by paired t-test. (C, D) Role of the RBC adhesion receptor LW in changes in oxygenation and BALF RBC counts. Nude mice were transfused with GLIB-treated, fresh human RBCs exposed or not to Abs against the adhesion receptors LW, CD44, or CD47; or with the nonreactive immunoglobulin P3. (B) Changes in SaO₂ in nude mice after transfusion of GLIB-treated fresh RBCs. (C) RBC counts in the BALF of nude mice after the indicated transfusions. Mean + SEM; n = 3–7. * indicates p<0.05 for comparison vs. GLIB treatment alone. ANOVA with Tukey’s was used.

Figure 6. Influence of ATP-release and LW on the pulmonary sequelae of transfusion of mouse RBCs

(A) Mean (+ SEM) changes in SaO₂ in nude mice after transfusion of glibenclamide (Glib.)-treated or control (Con) fresh RBCs from C57BL6 mice. * = p<0.05 for Con. vs. Glib. (B) Mean (+ SEM) RBC counts in the BALF of nude mice after the indicated transfusions. * p<0.05 (paired t-tests). n = 5–7.