Nitric Oxide Attenuates Adherence of Normal and Sickle Red Blood Cells and May Contribute to the Beneficial Effects of Hydroxyurea

Abstract 892 Hematology-Oncology II Poster Symposium, Sunday, 5/2

Increased endothelial adherence of sickle red blood cells (RBC) is implicated as an initiating event in vaso-occlusion. Nitric oxide (NO) inhibits adhesion of white blood cells and platelets to endothelium and decreases expression of VCAM-1, an endothelial receptor involved in adherence of sickle RBS (Khan et al, Proc Natl Acad Sci, 93;1996:9114). We hypothesized that NO also inhibits adherence of normal and sickle RBC to endothelium We investigated this question using a parallel plate flow chamber. Bovine pulmonary artery endothelial monolayers were exposed to RBC isolated from normal (Hb AA) or sickle cell (Hb SS) donors in the chamber with a combination of 10 minutes of RBC flow and 5 minutes of static incubation. Endothelial monolayers exposed to 100µM nitro-L-arginine (NLA), an inhibitor of NO production, for 30 minutes had an 87% increase in normal RBC adherence over baseline values (7.8 ± 0.82 adherent RBC/mm2 vs. 14.6 ± 2.08, n=19, p=0.002). Because adherence of normal RBC was low at baseline, the effect of an NO donor was tested after activating endothelium with TNF-α (5 units/ml for 1 hour) which increased adherence by 130% (17.9 ± 2.33, n=9, p<0.001). Addition of 2mM DETA-NO, an NO donor, produced a 42% decrease in the TNF-α-induced enhancement of normal RBC to endothelium (10.3 ± 1.84, n=5, p=0.03). DETA-NO also decreased adherence of sickle RBC to unactivated endothelium by 54% (387.4 ± 110.81 vs. 177.2 ± 63.79, n=6, p=0.04). These data indicate that NO inhibits adherence of normal and sickle RBC to endothelium. Hydoxyurea (HU) decreases the frequency of vaso-occlusive crises in sickle cell disease. Mechanisms responsible for this effect are uncertain. In vitro studies have shown that oxidation of HU in the presence of heme generates NO (Pacelli et al, Lancet, 1996;347:90). Rats injected with HU generated increasing levels of nitrosyl hemoglobin, indicative of NO production (Jiang et al, Mol Pharmacol, 1197;52;1081). In a second series of experiments we investigated whether HU triggers generation of NO by measuring stable NO metabolites, Nox (nitrate and nitrite), with chemiluminescence using vanadium reduction. RBC isolated from normal individuals (Hb AA) were resuspended at a HCT of 35% in buffered saline and incubated with 10mM HU or vehicle control. Samples were analyzed hourly for Nox levels. Control samples were unchanged from baseline over the 5 hour study period (3.8µM ± 1.0 to 3.5µM ± 0.7, n=4). Exposure to HU resulted in a steady increase in Nox, with a tripling of levels at 5 hours (4.8µM ± 0.4 to 18.7 ± 8.8, n=4, p=0.03). Thus, HU stimulates generation of NO by normal RBC. It is possible that some of the beneficial effect of HU in ameliorating the vaso-occlusive symptoms of sickle cell disease is due to increased generation of NO resulting in decreased endothelial adherence of sickle RBC. Other strategies which enhance NO production, such as nitrovasodilators, inhaled NO, or supplemental L-arginine, may also have a therapeutic role in sickle cell disease. (Figure)

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