Abstract Rationale Platelets are implicated in the pathogenesis of acute lung injury (ALI), though the factors regulating platelet activation are incompletely understood. Platelet activation is intimately linked to reactive oxygen species (ROS). We recently demonstrated that treatment with an antioxidant, superoxide dismutase (SOD) mimetic MnTE-2-PyP protected against platelet activation, pulmonary thrombophilia, and ALI in Staphylococcus aureus pneumonia. We now investigate the redox regulation of platelet activation in S. aureus pneumonia by examining potential redox-regulated platelet proteins in the spleen tyrosine kinase (Syk)-dependent signaling pathway. We hypothesized that S. aureus pneumonia changes platelet proteome and MnTE-2-PyP attenuates platelet activation via Syk-dependent signaling pathway. Methods Adult male and female C57BL/CJ wild-type (WT) mice were treated with one dose of subcutaneous (s.q.) MnTE-2-PyP (7.5mg/kg) or PBS 6hrs prior to receiving i.t. S. aureus (108 cfu) or PBS for control. 24hrs post infection, anticoagulated whole blood with DiOC6-labeled platelets was tested for platelet aggregation and accumulation with microfluidic flow assay. Remaining washed platelets were tested for activation of Syk-dependent signaling with immunoblotting for phosphorylated and total Syk, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2), membrane-localized adapter protein LAT, and phospholipase C-y2 (PLCy2). For proteomics, platelet pellets from 5 mice per experimental group (S. aureus vs PBS) were pooled to yield 600uG of protein. General protein quantification was obtained with mass spectrometry and analyzed with Ingenuity Pathway Analysis. Results MnTE-2-PyP SOD mimetic attenuated S. aureus-induced activation of the Syk-dependent signaling as evidenced by reduced phosphorylation of SHP2, Syk, and LAT in the mimetic-treated S. aureus-infected mice compared to controls. S. aureus induced platelet accumulation and aggregation that was attenuated by SOD mimetic. Lastly, we detected over 2,200 proteins by mass spectrometry in the platelets of WT S. aureus-infected mice and PBS controls. We observed that 394 proteins increased by at least 2-fold and 126 proteins decreased by at least 50% in platelets from the S. aureus-infected mice compared to PBS. Ingenuity pathway analysis identified most significant changes in the complement and metabolic pathways. Conclusions Overall, these findings demonstrate that SOD mimetic attenuates S. aureus-induced platelet activation and that Syk-dependent signaling pathway may be subject to redox regulation. Furthermore, we have identified pneumonia-induced changes in platelet proteome and in specific biological, cellular, and molecular pathways which will inform further investigations. Our findings offer novel insight into platelet-driven pathogenesis of ALI and have demonstrated that MnTE-2-PyP may be therapeutic in ALI. This abstract is funded by: NIH, NHLBI, NICHD, The Bobrow-Tanabe Family Pilot Award, Children’s Hospital Colorado Foundation
Osier et al. (Fri,) studied this question.