PXD004264

PXD004264 is an original dataset announced via ProteomeXchange.

Title	Paradigm of biased PAR1 activation and inhibition in endothelial cells dissected by phosphoproteomics
Description	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.
HostingRepository	PRIDE
AnnounceDate	2017-08-22
AnnouncementXML	Submission_2017-08-22_06:37:29.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Bart van den Eshof
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; monohydroxylated residue; acetylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion ETD

Revision	Datetime	Status	ChangeLog Entry
0	2016-06-02 01:34:40	ID requested
⏵ 1	2017-08-22 06:37:30	announced

van den Eshof BL, Hoogendijk AJ, Simpson PJ, van Alphen FPJ, Zanivan S, Mertens K, Meijer AB, van den Biggelaar M, Paradigm of Biased PAR1 (Protease-Activated Receptor-1) Activation and Inhibition in Endothelial Cells Dissected by Phosphoproteomics. Arterioscler Thromb Vasc Biol, 37(10):1891-1902(2017) [pubmed]

curator keyword: Biological

submitter keyword: Thrombin, PAR1, PAR1-inhibitors, endothelial cells, phosphoproteomics, SILAC

Maartje van den Biggelaar
contact affiliation	Dept. Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands
contact email	m.vandenbiggelaar@sanquin.nl
lab head
Bart van den Eshof
contact affiliation	Sanquin Research
contact email	b.vandeneshof@sanquin.nl
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD004264
     1. Label: PRIDE project
     2. Name: Paradigm of biased PAR1 activation and inhibition in endothelial cells dissected by phosphoproteomics