Abstract 921: Platelet Exosomes Induce Endothelial Cell Apoptosis through Peroxynitrite Generation: Evidence for a Septic Vascular Dysfunction Pathway
Platelet-derived microparticles (PMP) affect vascular cell signaling. Previously, we showed PMP from septic patients induce endothelial apoptosis through NADPH oxidase-dependent super-oxide release. Here, we further characterized the septic microparticle-dependent vascular injury pathway. In septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed to the NO donor diethylamine-NONOate (0.5μM), LPS (100 ng/ml), TNF-α 40 ng/ml, or thrombin (5 IU/ml) for 1h. PMP were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or western blot. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and 4,5-diaminofluorescein (10mM) and 2′,7′-dichlorofluorescein (10mM) fluorescence. Endothelial cell apoptosis was detected by annexinV and by ELISA caspase-3 activity measurement. Size, morphology, high exposure of the tetraspanin CD9, CD63, CD81 together with low phosphatidylserine, demonstrated that platelets exposed to NONO and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, with specific inhibitors use, revealed concomitant superoxide and NO generation. Western blots showed presence of NO synthase II (but not I and III) and of the NADPH oxidase subunits p22phox, PDI and Nox. Endothelial cells exposed to exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by superoxide dismutase mimetic, and were totally blocked by urate (1mM), suggesting a role for peroxynitrite. None of these redox properties and pro-apoptotic effects could be demonstrated for PMP after thrombin or TNF-α. Therefore, in sepsis NO and bacterial elements are responsible for specific platelet-derived exosome generation. Those exosomes play active role in vascular signaling as redox active particles, inducing endothelial cell caspase-3 activation and apoptosis by superoxide, NO and peroxynitrite generation. Thus, exosomes must be considered for further developments in comprehension and treatment of vascular dysfunction.