Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells

van den Biggelaar, M., Hernández-Fernaud, J. R., van den Eshof, B. L., Neilson, L. J., Meijer, A. B., Mertens, K. and Zanivan, S. (2014) Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells. Blood, 123(12), e22-e36. (doi:10.1182/blood-2013-12-546036)

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Publisher's URL: http://dx.doi.org/10.1182/blood-2013-12-546036

Abstract

Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor (PAR)1-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Due to its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phospho-peptide enrichment and high resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which has not been previously related to thrombin. Those sites were localized on proteins which are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites which may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Zanivan, Professor Sara Rossana and Hernandez, Dr Juan and Neilson, Ms Lisa
Authors: van den Biggelaar, M., Hernández-Fernaud, J. R., van den Eshof, B. L., Neilson, L. J., Meijer, A. B., Mertens, K., and Zanivan, S.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Blood
Publisher:American Society of Hematology
ISSN:0006-4971
ISSN (Online):1528-0020

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