Signalling pathways involved in p47^phox‐dependent reactive oxygen species in platelets of endotoxemic rats

Abstract

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 (PI3K), 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 SFKs inhibitor PP2, the PI3K 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 GF109203X 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.

Protein Disulphide Isomerase and NADPH Oxidase 1 Cooperate to Control Platelet Function and Are Associated with Cardiometabolic Disease Risk Factors

“…Interestingly, both Akt [ 45 ] and ERK1/2 [ 43 ] were shown to be involved in PKC signalling in platelets. Indeed, PKC is a signalling hub downstream of several platelet receptors that can also induce p47phox phosphorylation, and thus activate Nox-1 [ 46 ]. Therefore, we investigated the effect of dual inhibition of PDI and Nox-1 on the phosphorylation of PKC substrates, p38 MAPK and p47phox, in addition to Akt and ERK1/2.…”

Platelets in Sepsis: An Update on Experimental Models and Clinical Data

“…Rats are favorable models for in vitro analyses of platelets as their larger body weight and thus blood volume allows various concomitant measurements. Indeed, rat models have unveiled a number of crucial intracellular responses of platelets to sepsis, including activation of the NLRP3 inflammasome (187), upstream regulation of NADPH subunit p47 phox -dependent ROS production (188) and the contribution of protein kinase C for platelet-mediated leukocyte infiltration and organ injury (189). Furthermore, rats are more commonly used when investigating effects of therapeutic intervention strategies on clinical parameters e.g., hemodynamics.…”

Clotting Dysfunction in Sepsis: A Role for ROS and Potential for Therapeutic Intervention

“…However, any role of NOXs in platelet signalling remains somewhat contradictory [ 128 , 129 , 130 , 131 ]. Some studies have reported a fundamental role in experimental platelet ROS production, while others have shown that endotoxemia in rats increased TNF-α levels and promoted platelet-NADPH oxidase activity, via cGMP-PKG and PKC-p47phox signalling pathways [ 22 , 124 , 132 ]. NADPH oxidase (NOX2) activity within neutrophils and specifically ROS production has been suggested to stimulate NETosis, as seen in both human and experimental sepsis [ 104 , 133 ].…”