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


OBJECTIVE
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.
Surgical Intensive Care Medicine, University Hospital Zurich, University of Zurich, Zurich, Switzerland. reto.schuepbach@usz.ch
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Schuepbach
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Madon
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Ender
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Galli
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Riewald
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D001120:Arginine; D001666:Binding Sites, Antibody; D019610:Cytoprotection; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D057809:HEK293 Cells; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011486:Protein C / Q000378:metabolism; D011487:Protein Conformation; D034622:RNA Interference; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000633:toxicity; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism; D014162:Transfection; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors
D000906:Antibodies; D007191:Indazoles; D010446:Peptide Fragments; D011486:Protein C; C424723:RWJ 58259; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C105295:protease-activated receptor 3; D014508:Urea; D001120:Arginine; D013917:Thrombin; D019311:Staurosporine; D010975:Platelet Aggregation Inhibitors