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Signalling pathways involved in p47 phox ‐dependent reactive oxygen species in platelets of endotoxemic rats
Author(s) -
Lopes Pires Maria E.,
Antunes Naime Ana C.,
Oliveira Jessica G. F.,
Anhe Gabriel F.,
Garraud Oliver,
Cognasse Fabrice,
Antunes Edson,
Marcondes Sisi
Publication year - 2019
Publication title -
basic and clinical pharmacology and toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.805
H-Index - 90
eISSN - 1742-7843
pISSN - 1742-7835
DOI - 10.1111/bcpt.13148
Subject(s) - wortmannin , reactive oxygen species , nadph oxidase , protein kinase c , platelet , kinase , phosphorylation , platelet activation , lipopolysaccharide , protein kinase b , chemistry , signal transduction , pharmacology , microbiology and biotechnology , medicine , biology , endocrinology , biochemistry , immunology
Thrombocytopenia during sepsis is associated with a less favourable clinical outcome. Overproduction of reactive oxygen species ( ROS ) by different cell types contributes to sepsis. Platelets generate ROS , but the upstream pathways of NADPH oxidase activation are not completely understood. Here, we designed experiments in washed platelets from lipopolysaccharide ( LPS )‐treated rats to investigate the p47 phox activation and ROS generation, and its modulation by c‐Src family kinase (c‐Src), phosphoinositide 3‐kinase ( PI 3K), protein kinase C ( PKC ) and protein kinase G ( PKG ). Rats were injected intraperitoneally with LPS (1 mg/kg), and at 48 hours thereafter, arterial blood was collected and washed platelets were obtained. Washed platelets were pre‐incubated with different inhibitors and subsequently activated or not with ADP . Flow cytometry, Western blotting and ELISA were performed. We found that LPS significantly increased the p47 phox phosphorylation and ROS generation compared with the control group ( P < 0.05). The enhanced ROS production in the LPS group was unaffected by the non‐selective SFK s inhibitor PP 2, the PI 3K inhibitor wortmannin or the Akt inhibitor PPI ‐1. The cyclic GMP levels were 115% higher in activated platelets of LPS compared with the saline group ( P < 0.05). Moreover, in the LPS group, the sGC inhibitor ODQ , the PKG inhibitor Rp‐8‐Br and the PKC inhibitor GF 109203X abrogated the increased p47 phox phosphorylation and reduced the ROS levels. In conclusion, selective inhibitors of cGMP ‐ PKG and PKC ‐p47 phox pathways that regulate ROS generation by LPS in platelets may help control the redox balance in sepsis improving the survival of patients.