New Strategy for the Preparation of NO-Treated Red Blood Cells as a Blood Substitute

Summary

Our previous studies on the α-nitrosyl derivative of human adult hemoglobin (HbA), tetrameric Hb in which only the two α-subunits are ligated with nitric oxide (NO) [Yonetani et al., (1998) J Biol Chem 273: 20323], have shown that the α-nitrosyl HbA is a cooperative, low-affinity oxygen carrier. We have developed a simple method to convert all the intra-human red blood cells (RBCs) to a 50% saturation of hemes with NO exclusively bound to the α-subunits. Oxygen equilibrium measurements showed that the α-NO RBCs also exhibit reduced oxygen affinity (increased P₅₀) and diminished Bohr effect (i.e., the pH dependence of P₅₀). Despite the fact that its oxygen-carrying capacity is reduced by 50%, since two α-subunits are already ligated with NO, and only two β-subunits are capable of oxygen binding, α-NO RBCs can efficiently deliver oxygen to tissues under normal physiological conditions, making this material an excellent candidate for blood transfusion. Crucial steps in our protocol for the preparation of α-NO Hb-containing RBCs were revised and technical adjustments were made in order to improve the quality and integrity of RBCs during and after treatment, while avoiding the use of potentially noxious chemicals that could entail harmful effects. We were also able to reduce the preparation time. All of which will make viable an increased production volume of quality NO-treated RBCs for practical use.