PT - JOURNAL ARTICLE AU - Nel, Jan-Gert AU - Durnadt, Chrisna AU - Mitchell, Timothy AU - Feldman, Charles AU - Anderson, Ronald AU - Tintinger, Gregory TI - Pneumolysin mediates platelet activation <em>in vitro</em> AID - 10.1183/13993003.congress-2016.PA2593 DP - 2016 Sep 01 TA - European Respiratory Journal PG - PA2593 VI - 48 IP - suppl 60 4099 - http://erj.ersjournals.com/content/48/suppl_60/PA2593.short 4100 - http://erj.ersjournals.com/content/48/suppl_60/PA2593.full SO - Eur Respir J2016 Sep 01; 48 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 &gt;20 ng/ml (p&lt;0.008–p&lt;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.