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


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.


CONCLUSIONS
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.
Vascular Medicine Institute and the Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Tamir
T
Kanias
Ling
L
Wang
Ashley
A
Lippert
Daniel B
DB
Kim-Shapiro
Mark T
MT
Gladwin
D000818:Animals; D001793:Blood Preservation / Q000379:methods; D015925:Cryopreservation / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology* / Q000473:pathology; D006461:Hemolysis / Q000502:physiology*; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency / Q000378:metabolism*; D013312:Stress, Physiological; D013997:Time Factors
C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse