RT Journal Article SR Electronic T1 Pneumolysin mediates platelet activation in vitro JF European Respiratory Journal JO Eur Respir J FD European Respiratory Society SP PA2593 DO 10.1183/13993003.congress-2016.PA2593 VO 48 IS suppl 60 A1 Nel, Jan-Gert A1 Durnadt, Chrisna A1 Mitchell, Timothy A1 Feldman, Charles A1 Anderson, Ronald A1 Tintinger, Gregory YR 2016 UL http://erj.ersjournals.com/content/48/suppl_60/PA2593.abstract AB INTRODUCTION: Platelets are key players in inflammatory responses, with platelet activation contributing to the pathogenesis of acute lung injury (ALI) and acute coronary events (ACE) in community-acquired pneumonia.AIM: To explore mechanisms underpinning platelet activation in severe pneumococcal disease, by investigating the effects of exposure of human platelets to the pneumococcal cytotoxin, pneumolysin (Ply) in vitro.METHODS: Recombinant Ply was used at pathologically relevant concentrations (10–80 nanograms/mL), while platelet activation was measured flow cytometrically according to the level of expression of P-selectin (CD62P). The influx of extracellular calcium, an event which precedes and is a prerequisite for platelet activation, including upregulation of CD62P, was measured using a Fura-2AM-6-based spectrofluorimetric procedure.RESULTS: Exposure of platelets to Ply resulted in a dose-related increase in the expression of CD62P which was significant at concentrations of >20 ng/ml (p<0.008–p<0.0001), reaching levels comparable to those following receptor-mediated activation with platelet-activating factor (PAF, 400nM). Ply-mediated upregulation of expression of CD62P on platelets was associated with increases in the concentrations of cytosolic calcium and attenuated by depletion of calcium from the cell-suspending medium, as well as by exposure of the cells to a pneumolysoid devoid of pore-forming activity. These observations are consistent with Ply-mediated platelet activation involving sub-lytic pore formation, calcium influx, and discharge of CD62P-expressing α-granules.CONCLUSION: If operative in the setting of severe pneumococcal disease, Ply-mediated platelet activation may contribute to the pathogenesis of ALI and ACE.