Adenosine-5'-triphosphate-adenosine-glutathione cross-linked hemoglobin as erythropoiesis-stimulating agent

An effective hemoglobin (Hb)-based blood substitute that acts as a physiological oxygen carrier and volume expander ought to stimulate erythropoiesis. A speedy replacement of blood loss with endogenous red blood cells should be an essential feature of any blood substitute product because of its relatively short circulatory retention time and high autoxidation rate. Erythropoiesis is a complex process controlled by oxygen and redox-regulated transcription factors and their target genes that can be affected by Hb physicochemical properties. Using an in vitro cellular model, we investigated the molecular mechanisms of erythropoietic action of unmodified tetrameric Hb (UHb) and Hb cross-linked with adenosine-5'-triphosphate (ATP), adenosine, and reduced glutathione (GSH). These effects were studied under normoxic and hypoxic conditions. Results indicate that these Hb solutions have different effects on stabilization and nuclear translocation of hypoxia-inducible factor (HIF)-1 alpha, induction of the erythropoietin (EPO) gene, activation of nuclear factor (NF)-kappa B, and expression of the anti-erythropoietic agents-tumor necrosis factor-alpha and transforming growth factor-beta 1. UHb suppresses erythropoiesis by increasing the cytoplasmic degradation of HIF-1 alpha and decreasing binding to the EPO gene while inducing NF-kappa B-dependent anti-erythropoietic genes. Cross-linked Hb accelerates erythropoiesis by downregulating NF-kappa B, stabilizing and facilitating HIF-1 alpha binding to the EPO gene, under both oxygen conditions. ATP and adenosine contribute to normoxic stabilization of HIF-1 and, with GSH, inhibit the NF-kappa B pathway that is involved in the suppression of erythroid-specific genes. Proper chemical/pharmacological modification is required to consider acellular Hb as an erythropoiesis-stimulating agent.