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


Schuepbach, R A; Madon, J; Ender, M; Galli, P; Riewald, M (2012). Protease-activated receptor-1 cleaved at R46 mediates cytoprotective effects. Journal of Thrombosis and Haemostasis (JTH), 10(8):1675-1684.

Abstract

UNLABELLED: BACKGROUND: Activated protein C (aPC) mediates powerful cytoprotective effects through the protease-activated receptor-1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC-activated PAR1 can mediate cytoprotective effects whereas 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 the treatment of inflammatory diseases.

Abstract

UNLABELLED: BACKGROUND: Activated protein C (aPC) mediates powerful cytoprotective effects through the protease-activated receptor-1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC-activated PAR1 can mediate cytoprotective effects whereas 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 the treatment of inflammatory diseases.

Citations

28 citations in Web of Science®
29 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Division of Surgical Research
04 Faculty of Medicine > University Hospital Zurich > Division of Surgical Intensive Care Medicine
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2012
Deposited On:11 Dec 2012 17:23
Last Modified:05 Apr 2016 16:09
Publisher:Wiley-Blackwell
ISSN:1538-7836
Publisher DOI:https://doi.org/10.1111/j.1538-7836.2012.04825.x
PubMed ID:22712885

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