Protease‐activated receptor‐1 cleaved at R46 mediates cytoprotective effects

Abstract

Background Activated protein C (aPC) mediates powerful cytoprotective effects through protease activated receptor (PAR)-1 that translate into reduced harm in mouse injury models. However, it remains elusive how aPC-activated PAR1 can mediate cytoprotective effects while thrombin activation does the opposite. Objectives We hypothesized that aPC and thrombin might induce distinct active conformations in PAR1 causing opposing effects. Methods We analyzed antibody binding to, and cleavage and signalling of PAR1 in either endogenously expressing endothelial or overexpressing 293T cells. Results In thrombin-cleaved PAR1 neither the tethered ligand nor the hirudin like domain were available for anti-PAR1 ATAP2 and WEDE15 binding unless the tethered ligand was quenched. In contrast, aPC irreversibly prevented ATAP2 binding while not affecting WEDE15 binding. Reporter constructs with selective glutamine substitutions confirmed R41 as the only thrombin cleavage site in PAR1, whereas aPC preferentially cleaved at R46. Similarly, we report distinct cleavage sites on PAR3, K38 for thrombin and R41 for aPC. A soluble peptide corresponding to R46-cleaved PAR1 enhanced the endothelial barrier function and reduced staurosporine toxicity in endothelial as well as in 293T cells if PAR1 was expressed. Overexpression of PAR1 variants demonstrated that cleavage at R46 but not R41 is required for cytoprotective aPC signaling. Conclusions We provide a novel concept on how aPC and thrombin mediate distinct effects. We propose that the enzyme specific cleavage sites induce specific conformations which mediate divergent downstream effects. This unexpected model of PAR1 signaling might lead to novel therapeutic options for treatment of inflammatory diseases.

Biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46

“…35 That recent report and our studies coincide in advancing the paradigm that APC's PAR1-dependent protective actions are based on Arg46 cleavage. This present paper provides a number of novel aspects for For personal use only.…”

Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

“…to reveals a the tethered ligand 42 KVDGTS, which, like the trypsin-exposed AP SLIGRL, stimulates Ca 2ϩ signaling in HeLa cells, albeit with reduced potency (58). However, we found no evidence that Cat-S induced mobilization of intracellular Ca 2ϩ in KNRK or HEK cells expressing PAR 2 , but instead observed that Cat-S stimulates a TRPV4-dependent influx of extracellular Ca 2ϩ ions in nociceptive neurons.…”

“…Elastase cleavage of PAR 2 at S 68 2V 69 induces PAR 2 -dependent activation of extracellular signal regulated kinases 1/2 (ERK1/2) by a Rho-kinase dependent pathway (34) that is distinct from trypsin-induced MAPK activation that is mediated by ␤-arrestins (36). Potential biased agonists of PAR 1 include elastase (37), matrix metalloprotease-1 (MMP-1) (38 -40), and activated protein C (APC) (41,42). However, although biased agonism is emerging as potential mechanism of PAR activation, the patho-physiological relevance of biased agonism is not fully understood, and nothing is known about the contribution of biased agonism for proteaseinduced inflammation and pain.…”

Factor VII activating protease (FSAP) regulates the expression of inflammatory genes in vascular smooth muscle and endothelial cells

“…APC has a cytoprotective effect in various pathologies and tissues, and this cytoprotective effect is mediated through cleavage of PAR1 at position R 46 97,141 . Indeed it was shown that APC protects the heart from ischemia reperfusion (I/R) injury through PAR1 signaling.…”