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

Abstract

Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1hasbecomeanappealingtargetforanticoagulanttherapies. However,theinhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of 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 phosphopeptide enrichment, and high-resolution mass spectrometry and performed a timeresolvedanalysisofthethrombin-inducedsignalinginhumanprimaryendothelialcells.We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not beenpreviouslyrelatedtothrombin.Thosesiteswerelocalizedonproteinsthatarenovelto thrombinsignaling,butalsoonwell-knownplayerssuchasPAR1,Rho-associatedkinase2, phospholipaseC,andproteinsrelatedtoactincytoskeleton,cell-celljunctions,andWeibelPalade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that 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.