Pirfenidone induced inhibition of p38 downregulates procoagulant microparticle generation by human alveolar epithelia cells

Abstract

Background: Pirfenidone is a drug approved for idiopathic pulmonary fibrosis but its mechanisms of action are partially unknown. We have shown that the airways of patients with idiopathic pulmonary fibrosis contain procoagulant microparticles (MP) that activate coagulation factor X to its active form, Xa, a protease that signals fibroblast growth and differentiation. We also reported that in vitro exposure of human alveolar cells (A549) to H 2 O 2 caused MP generation [Novelli et. all.,PLoS One 2014,e95013]. Aim: To investigated the hypothesis that H 2 O 2 -induced generation of MP by A549 is dependent on p38 phosphorylation and is inhibited by pirfenidone. Methods: p-38 phosphorylation was analyzed by western blot analysis. MP generation was assessed by a functional assay that measures phosphatidylserine concentration (PS) and by cytofluorimetric analysis. MP-associated tissue factor (TF) activity was assessed by a one-stage clotting assay. Results: H 2 O 2 (100μM) stimulation of A549 caused p38 phosphorylation that was inhibited by pirfenidone (0.5mM ) (Fig. 1). The drug also inhibited H 2 O 2 induced MP generation as assessed by functional assay and flow cytometry (Fig. 2a,b). The shedding of MP-bound tissue factor activity was also inhibited by pirfenidone (Fig. 2c) Conclusion: Inhibition of p38-mediated generation of procoagulant MP is a previously unrecognized mechanism of action of pirfenidone.