Figure 4

Coelenterazine luminescence triggered by exosomes suggests the presence of reactive oxygen and nitrogen generation. The graph represents exosome coelenterazine (5 μM) luminescence above background. Exosomes were incubated with NADPH and L-arginine. Exosomes (10 μg protein content) obtained from platelets exposed to the nitric oxide donor diethylamine NONOate (NONOate; 0.5 μM) or lipopolysaccharide generate reactive oxygen species in a similar fashion to exosomes obtained from septic patients, whereas particles obtained from platelets exposed to saline (control) or thrombin have very low activity. Luminescent signals were consistently inhibited by the addition of the superoxide dismutase mimetic Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (SOD, 10 μM) and by the NO synthase inhibitors L-NMA (N^G-methyl-L-arginine acetate; 5 mM), or N^ω-Nitro-L-arginine methyl ester (L-NAME; 1 mM), suggesting the generation of reactive oxygen species and reactive nitrogen species by the exosomes. Results are means ± SD of seven experiments. *P < 0.05 versus control, ^†P < 0.05 versus untreated. RLU, relative luminescence units.