S-nitrosothiols are stored by platelets and released during platelet-neutrophil interactions

Interaction between platelets and neutrophils is important in vascular injury. We have investigated the storage and release of nitric oxide (NO) by platelets interacting with neutrophils. Shear-activated platelets were added to neutrophils in suspension and both superoxide and peroxynitrite formations monitored by lucigenin- and luminol-enhanced chemiluminescence. In addition, intraplatelet S-nitrosothiols were measured by dichlorofluorescein fluorescence following displacement of NO by mercuric chloride. Addition of activated platelets to neutrophils caused free radical production and platelet-neutrophil rosette formation. Pretreatment of platelets with 20 microM S-nitrosoglutathione changed the balance between luminol and lucigenin chemiluminescence in favor of luminol, whereas S-nitrosoglutathione in platelet-free plasma did not produce these changes. This pattern was also observed both following inhibition of neutrophil NO synthase and in a neutrophil-free superoxide-generating system. Inhibition of platelet NO synthase decreased luminol and increased lucigenin chemiluminescence. These effects were reversed by L-arginine. Platelet activation increased intraplatelet S-nitrosothiols from 1.93+/-0.19 (mean +/- SD) to 4.9+/-1.10 x 10(-18) mol/platelet (P < 0.01); this increase halved following NO synthase inhibition, but was enhanced by approximately 220% following incubation with S-nitrosoglutathione. These results show that during shear stress platelets store S-nitrosothiols, which can be derived either endogenously from NO synthesis or exogenously by sequestration of S-nitrosoglutathione. Release of stored NO during platelet-neutrophil interaction alters the interaction of NO with superoxide and could modulate vascular inflammation.