Vaso-occlusion is a hallmark of sickle cell disease (SCD). Congestion of blood vessels by sickle red blood cells (RBC) is caused by hemoglobin polymerization (sickling), increased adhesion, and vasoconstriction due to a disregulated nitric oxide (NO) metabolism. NO generated from L-arginine by NO synthases plays a key role in vasodilation. Under hypoxia hemoglobin has been shown to be a potent alternative source of NO by reducing nitrite (NO2−). In SCD, modification of hemoglobin may reduce sickling and increase its ability to generate NO, and as such improve blood flow. In this study, we examined hemoglobin modification and consequent change in nitrite reductase capacity by 1-bromoacetyl-3,3-dinitroazetidine (ABDNAZ). This is a compound that has recently been described to bind to hemoglobin (Scicinski et al. Drug Met Disp. 2012) and has promising anticancer activity by altering blood flow in hypoxic tumors (Ning et al. Cancer Res. 2012).
Blood samples from SCD patients and control blood from healthy volunteers was collected with IRB approval. Red blood cells were isolated and resuspended to physiological hemactocrit in HEPES buffered saline (HBS) containing 2% bovine serum albumin (BSA). Samples were incubated with ABDNAZ, hydroxyurea (HU) or iodoacetamide at a final concentration of 3 mM for 30 minutes at room temperature. Intact RBC, hemolysates or purified hemoglobin samples were exposed to a constant nitrogen (N2) flow (200 mL/minute) at 37°C in a tonometer. At time zero, sodium nitrite was added to a final concentration of 5 mM. The influx of NO2− into the RBC was measured using Griess reagent on supernatant samples. The gas outflow from the tonometer was collected in Mylar balloons at various time points, and NO released in the gas phase was measured using a Nitric Oxide Analyzer.
Upon NO2− addition under deoxygenated conditions, influx in RBC was rapid. An equilibrium between supernatant and RBC was reached within 10 minutes and indicated active recruitment of RBC rather than a simple diffusion equilibrium. Release of NO from SCD patient RBC was increased compared to normal control (HbAA) RBC. Under these conditions, hemolysates with the identical hemoglobin concentration as compared to intact RBC showed a pronounced increase in NO release. Using purified hemoglobin NO release was reduced to 50% as compared to hemolysates. Blood samples from both normal control and sickle cell patients treated with ABDNAZ, showed a significant increase in NO release as compared to untreated samples. Iodoacetamide and HU treatment, described to increase NO (Gladwin et al. Br. J. Hematol. 2002), also showed increased NO release but at a markedly lower level as compared to ABDNAZ treatment.
Episodes of vaso-occlusion result in pain and organ damage in SCD. Local generation of NO will improve vasodilation, and is potentially beneficial. The novel compound ABDNAZ, currently in human clinical trials, greatly increases nitrite reductase capacity of hemoglobin, in both normal and SCD blood samples. Our results indicate that this agent improves NO release from RBC and suggests that modification of sickle patient blood by ABDNAZ in the presence of plasma nitrite should be considered as a potential therapeutic approach.
Scicinski:RadioRx, Inc: Employment. Oronsky:RadioRx, Inc: Employment.
Asterisk with author names denotes non-ASH members.