Mitochondria-derived ATP participates in the formation of neutrophil extracellular traps induced by platelet-activating factor through purinergic signaling in cows.

Neutrophil extracellular trap (NET) formation eliminates/prevents the spread of infectious agents. Platelet activating factor (PAF) is involved in infectious diseases of cattle because it recruits and activates neutrophils. However, its ability to induce NET release and the role of metabolism in this process is not known. We investigated if inhibition of glycolysis, mitochondrial-derived adenosine triphosphate (ATP) synthesis and purinergic signaling though P2X purinoceptors interfered with NET formation induced by PAF. We inhibited bovine neutrophils with 2-deoxy-d-glucose, rotenone, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and NF449 to evaluate PAF-mediated NET extrusion. PAF induced mitochondrial hyperpolarization and triggered extracellular ATP release via pannexin-1. Inhibition of mitochondrial metabolism prevented extracellular ATP release. Inhibition of glycolysis, complex-I activity and oxidative phosphorylation prevented NET formation induced by PAF. Inhibition of P2X purinergic receptors inhibited mitochondrial hyperpolarization and NET formation. We concluded that PAF-induced NET release is dependent upon glycolysis, mitochondrial ATP synthesis and purinergic signaling.