PB1435 - Canonical activation of protease-activated receptor-1 drives experimental liver fibrosis

Background: Clinical and experimental evidence supports a pathologic role of the blood coagulation cascade in chronic liver injury. Coagulation proteases drive cellular signaling via activation of protease-activated receptors (PARs), a family of G-protein-coupled receptors which are activated upon cleavage of the N-terminus to generate a tethered agonist ligand. PAR-1 expressed by hepatic stellate cells (HSCs) drives HSC activation and collagen deposition in experimental liver fibrosis. The PAR-1 N-terminus can be cleaved at different sites which activate distinct downstream signaling pathways. The cleavage site responsible for the pro-fibrotic effects of PAR-1 activation is unknown.

Aims: Using novel mice expressing normal levels of PAR-1 which is mutated at either the Arg-41 cleavage site (R41Q mice) or Arg-46 cleavage site (R46Q mice), we tested the hypothesis that canonical PAR-1 cleavage at the Arg-41 cleavage site drives experimental hepatic fibrosis.

Methods: Male wildtype, R41Q, and R46Q mice on a C57Bl6/J background were challenged twice weekly with carbon tetrachloride (CCl4, 1 mL/kg i.p.) for 6 weeks.

Results: Chronic CCl4 challenge caused activation of HSCs to a myofibroblast phenotype and hepatic collagen deposition, indicated by labeling for the myofibroblast marker α-smooth muscle actin (αSMA) and collagen type I, respectively, in liver tissues of wildtype, R41Q, and R46Q mice. HSC activation and collagen deposition were similar in CCl4-challenged R46Q mice and wild-type mice. In contrast, αSMA labeling and collagen deposition were significantly reduced in R41Q mutant mice. Interestingly, Col1a1 gene expression was not reduced in R41Q mice, implying reduced collagen protein is not fully explained by reduced collagen gene induction.

Conclusion(s): The results indicate that the PAR-1 Arg-41 cleavage site mediates pro-fibrotic effects of PAR-1 activation. Notably, the Arg-46 cleavage site was dispensable for hepatic fibrosis. Identification of the thrombin-cleavage site at Arg-41 for PAR-1 driven hepatic fibrosis provides a novel mechanistic insight into the links between coagulation activation and liver fibrosis.