Proteomics

Dataset Information

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Paradigm of biased PAR1 activation and inhibition in endothelial cells dissected by phosphoproteomics


ABSTRACT: Objective: Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of protease-activated receptors (PARs). The predominant thrombin receptor is PAR1 and in endothelial cells (ECs) thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear if thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signaling is lacking. In addition, signaling networks of biased PAR1 activation after differential cleavage of the PAR1 N-terminus have remained an unresolved issue.Approach and Results: Here, we used a quantitative phosphoproteomics approach to show that ‘classical’ and ‘peptide’ activation of PAR1 induce highly similar signaling, that low thrombin concentrations initiate only limited phosphoregulation, and that the PAR1 inhibitors vorapaxar and parmodulin-2 demonstrate distinct antagonistic properties. Subsequent analysis of the thrombin-regulated phosphosites in presence of PAR1 inhibitors revealed that biased activation of PAR1 is not solely linked to a specific G-protein downstream of PAR1. In addition, we showed that only the canonical thrombin PAR1 tethered ligand induces extensive early phosphoregulation in ECs.Conclusions: Our study provides detailed insight in the signaling mechanisms downstream of PAR1. Our data demonstrates that thrombin-induced EC phosphoregulation is mediated exclusively through PAR1, that thrombin and thrombin-TL peptide induce similar phosphoregulation and that only canonical PAR1 cleavage by thrombin generates a tethered ligand that potently induces early signaling. Furthermore, platelet PAR1 inhibitors directly affect EC signaling, indicating it will be a challenge to design a PAR1 antagonist that will target only those pathways responsible for tissue pathology.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell, Blood

SUBMITTER: Bart van den Eshof  

LAB HEAD: Maartje van den Biggelaar

PROVIDER: PXD004264 | Pride | 2017-08-22

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
Bart_TVPM_N1.raw Raw
Bart_TVPM_N2.raw Raw
Bart_TVPM_N3.raw Raw
LT_N1_TiO1.raw Raw
LT_N1_TiO2.raw Raw
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Publications

Paradigm of Biased PAR1 (Protease-Activated Receptor-1) Activation and Inhibition in Endothelial Cells Dissected by Phosphoproteomics.

van den Eshof Bart L BL   Hoogendijk Arie J AJ   Simpson Pelle J PJ   van Alphen Floris P J FPJ   Zanivan Sara S   Mertens Koen K   Meijer Alexander B AB   van den Biggelaar Maartje M  

Arteriosclerosis, thrombosis, and vascular biology 20170817 10


<h4>Objective</h4>Thrombin is the key serine protease of the coagulation cascade and mediates cellular responses by activation of PARs (protease-activated receptors). The predominant thrombin receptor is PAR1, and in endothelial cells (ECs), thrombin dynamically regulates a plethora of phosphorylation events. However, it has remained unclear whether thrombin signaling is exclusively mediated through PAR1. Furthermore, mechanistic insight into activation and inhibition of PAR1-mediated EC signali  ...[more]

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