Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin
BACKGROUND: Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin.
METHODS: We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags.
RESULTS: Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a β-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both β-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by β-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin.
CONCLUSIONS: These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.
| Errataetall: |
CommentIn: Arterioscler Thromb Vasc Biol. 2024 Mar;44(3):617-619. - PMID 38269587 |
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:44 |
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| Enthalten in: |
Arteriosclerosis, thrombosis, and vascular biology - 44(2024), 3 vom: 03. März, Seite 603-616 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Biswas, Indranil [VerfasserIn] |
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| Links: |
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| Themen: |
Beta-Arrestins |
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| Anmerkungen: |
Date Completed 23.02.2024 Date Revised 09.03.2024 published: Print-Electronic CommentIn: Arterioscler Thromb Vasc Biol. 2024 Mar;44(3):617-619. - PMID 38269587 Citation Status MEDLINE |
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| doi: |
10.1161/ATVBAHA.123.320185 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 500 | |a CommentIn: Arterioscler Thromb Vasc Biol. 2024 Mar;44(3):617-619. - PMID 38269587 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a BACKGROUND: Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin | ||
| 520 | |a METHODS: We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags | ||
| 520 | |a RESULTS: Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a β-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both β-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by β-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin | ||
| 520 | |a CONCLUSIONS: These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively | ||
| 650 | 4 | |a Journal Article | |
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