Red blood cell endothelial nitric oxide synthase does not modulate red blood cell storage hemolysis

Abstract

Background: The red blood cell (RBC) endothelial nitric oxide synthase (eNOS) has been shown to regulate intrinsic RBC rheologic properties, such as membrane deformability, suggesting that a functional eNOS could be important in RBC viability and function during storage. This study examines the correlation between RBC eNOS deficiency and the propensity of RBCs to hemolyze under selected stress conditions including prolonged hypothermic storage.

Study design and methods: Fresh or stored RBCs from normal and eNOS knockout (KO) mice or from healthy human volunteers were subjected to selected hemolytic stress conditions including mechanical stress hemolysis, osmotic stress hemolysis, and oxidation stress hemolysis and evaluated during standard storage in CPDA-1 solutions.

Results: Fresh RBCs from normal and eNOS KO mice demonstrated comparable susceptibility to hemolysis triggered by mechanical stress (mechanical fragility index 6.5 ± 0.5 in eNOS KO vs. 6.4 ± 0.4 for controls; n = 8-9), osmotic stress, and oxidative stress. Additionally, RBCs from both mouse groups exhibited similar hemolytic profile at the end of 14-day hypothermic storage, analogous to 42 days of human RBC storage. Storage of human RBCs (28 days in CPDA-1) in the presence of NOS cofactors (L-arginine and tetrahydro-L-biopterin) or inhibitor (N(5) -[imino(methylamino)methyl]-L-ornithine monoacetate) did not affect cell recovery or hemolytic response to the selected stressors.

Conclusion: These studies suggest that RBC eNOS does not modulate susceptibility to hemolysis in response to selected stress conditions or prolonged hypothermic storage. Other strategies to increase nitric oxide (NO) bioactivity after prolonged storage utilizing NOS-independent pathways such as the nitrate-nitrite-NO pathway may prove a more promising approach.

Figure 1

Hemolytic response of eNOS KO RBCs versus C57BL/6J RBCs (normal controls): A. RBC mechanical fragility index (MFI) following 1.5 h rocking in the presence or absence of 3/32” stainless steel bead; B. RBC osmotic hemolysis (%) following 3 h or 24 h incubation in Pink Test buffer at 20 °C; C. Formation of fluorescent heme degradation products following 48 min incubation with 0.5 mmol/L H₂O₂; D. RBC oxidative hemolysis (%) following 3 h incubation at 37 °C in the presence or absence of 50 mmol/L 2,2′-azobis(2- methylpropionamidine) dihydrochloride (AAPH); Mean±SD, n=8–9 per strain.

Figure 1

Hemolytic response of eNOS KO RBCs versus C57BL/6J RBCs (normal controls): A. RBC mechanical fragility index (MFI) following 1.5 h rocking in the presence or absence of 3/32” stainless steel bead; B. RBC osmotic hemolysis (%) following 3 h or 24 h incubation in Pink Test buffer at 20 °C; C. Formation of fluorescent heme degradation products following 48 min incubation with 0.5 mmol/L H₂O₂; D. RBC oxidative hemolysis (%) following 3 h incubation at 37 °C in the presence or absence of 50 mmol/L 2,2′-azobis(2- methylpropionamidine) dihydrochloride (AAPH); Mean±SD, n=8–9 per strain.

Figure 2

Hemolytic response of mouse RBCs stored with CPDA-1 for 14 days: RBCs were collected from eNOS KO strain or from its background control (C57BL/6J). A. RBC storage hemolysis (%); B. RBC mechanical fragility index (MFI) following 1.5 h rocking in the presence or absence of 3/32″ stainless steel bead; C. RBC osmotic hemolysis (%) in fresh versus stored RBCs following 3 h incubation in Pink Test buffer at 20 °C; D. Formation of fluorescent heme degradation products in fresh versus stored RBCs following 48 min incubation with 0.5 mmol/L H₂O₂. Mean±SD, n=8–9 per strain.

Figure 2

Hemolytic response of mouse RBCs stored with CPDA-1 for 14 days: RBCs were collected from eNOS KO strain or from its background control (C57BL/6J). A. RBC storage hemolysis (%); B. RBC mechanical fragility index (MFI) following 1.5 h rocking in the presence or absence of 3/32″ stainless steel bead; C. RBC osmotic hemolysis (%) in fresh versus stored RBCs following 3 h incubation in Pink Test buffer at 20 °C; D. Formation of fluorescent heme degradation products in fresh versus stored RBCs following 48 min incubation with 0.5 mmol/L H₂O₂. Mean±SD, n=8–9 per strain.

Figure 3

Evaluation of RBC mechanical and osmotic fragility in L-arginine-treated RBCs: Human RBCs were subjected to mechanical or osmotic fragility test in the presence of L-arginine at 0, 2.5, 10, 100 and 1000 μmol/L. A. RBC mechanical fragility index (MFI) following 1.5 h rocking in the presence or absence of 3/32” stainless steel bead; B. RBC osmotic hemolysis (%). Mean±SD, n=8.

Figure 4

Hemolytic response of human RBCs stored with CPDA-1 for 28 days in the presence or absence of eNOS cofactors or inhibitor: RBCs were supplemented with the cofactors L-arginine (10 μmol/L) and tetrahydrobiopterin (BH4, 100 μmol/L) or with the eNOS inhibitor N⁵-[imino(methylamino)methyl]-L-ornithine, monoacetate (L-NMMA, 1 mmol/L). A. RBC storage hemolysis (%); B. RBC mechanical fragility index (MFI) following 1.5 h rocking in the presence or absence of 3/32” stainless steel bead; C. RBC osmotic hemolysis (%) following 24 h incubation in Pink Test buffer at 20 °C. Mean±SD, n=3.

Figure 4

Hemolytic response of human RBCs stored with CPDA-1 for 28 days in the presence or absence of eNOS cofactors or inhibitor: RBCs were supplemented with the cofactors L-arginine (10 μmol/L) and tetrahydrobiopterin (BH4, 100 μmol/L) or with the eNOS inhibitor N⁵-[imino(methylamino)methyl]-L-ornithine, monoacetate (L-NMMA, 1 mmol/L). A. RBC storage hemolysis (%); B. RBC mechanical fragility index (MFI) following 1.5 h rocking in the presence or absence of 3/32” stainless steel bead; C. RBC osmotic hemolysis (%) following 24 h incubation in Pink Test buffer at 20 °C. Mean±SD, n=3.

Figure 5

Oxidative response of human RBCs stored with CPDA-1 for 28 days in the presence or absence of eNOS cofactors or inhibitor: RBCs were supplemented with the cofactors L-arginine (10 μmol/L) and tetrahydrobiopterin (BH4, 100 μmol/L) or with the eNOS inhibitor N5-[imino(methylamino)methyl]-L-ornithine, monoacetate (L-NMMA, 1 mmol/L). A. Formation of fluorescent heme degradation products following 48 min incubation with 0.5 mmol/L H₂O₂; B. RBC oxidative hemolysis (%) following 24 h incubation at 37 °C in the presence or absence of 50 mmol/L 2,2′-azobis(2- methylpropionamidine) dihydrochloride (AAPH). Mean±SD, n=3.

Figure 5

Oxidative response of human RBCs stored with CPDA-1 for 28 days in the presence or absence of eNOS cofactors or inhibitor: RBCs were supplemented with the cofactors L-arginine (10 μmol/L) and tetrahydrobiopterin (BH4, 100 μmol/L) or with the eNOS inhibitor N5-[imino(methylamino)methyl]-L-ornithine, monoacetate (L-NMMA, 1 mmol/L). A. Formation of fluorescent heme degradation products following 48 min incubation with 0.5 mmol/L H₂O₂; B. RBC oxidative hemolysis (%) following 24 h incubation at 37 °C in the presence or absence of 50 mmol/L 2,2′-azobis(2- methylpropionamidine) dihydrochloride (AAPH). Mean±SD, n=3.