PARadigms of vascular signaling

Abstract

The inside of all blood vessels is lined with a tight network of specialized endothelial cells that form a semipermeable barrier between blood and underlying tissues. By virtue of their strategic position at the interface between blood and tissue, endothelial cells are constantly exposed to blood cells, proteins and other blood components. To sense their environment, endothelial cells are equipped with a wide range of transmembrane receptors that convert information from the extracellular environment into intracellular signaling pathways. To better understand the swift adaptive endothelial response it is crucial to map these communication networks. To cover the rapid and reversible post-translational modifications a system-wide analysis of cellular signaling networks is required. Therefore in the present thesis, we investigated endothelial signaling pathways, with particular emphasis on coagulation, using an unbiased quantitative mass spectrometry approach. Thrombin is the key serine protease of the coagulation cascade and its predominant receptor is protease-activated receptor 1 (PAR1). Our data demonstrated that thrombin dynamically regulates a plethora of phosphorylation events, including actin cytoskeleton and cell junctions signaling. Next, we showed that thrombin-induced phosphoregulation is mediated exclusively through PAR1. In addition, we showed that PAR1 inhibitors vorapaxar and parmodulin-2 demonstrated distinct antagonistic properties and that only canonical PAR1 cleavage by thrombin, and not by other proteases, generates a tethered ligand that potently induces early signaling. These data provided mechanistic inside into the activation and inhibition of PAR1-mediated signaling in endothelial cells. To dissect diverging and converging signaling pathways, we analyzed the communication networks initiated by histamine and other members of the PAR family (PAR1,2,3,4). Our data showed that PAR1 and PAR2 induced similar phosphoregulation of proteins involved in Gαq- and the MAPK pathway. In contrast, they differentially regulated proteins related to clathrin-mediated endocytosis. Thrombin and histamine induced highly similar phosphoregulation, while they differentially phosphorylated proteins related to the Gα12/13 pathway. Furthermore, we assessed phosphoproteomic regulation in endothelial cells induced by platelet proteins and compared it to vascular endothelial growth factor (VEGF)-mediated phosphoregulation. These data showed that platelet releasate and lysate induced highly similar signal transduction and that their effect was not primarily driven by VEGF. In parallel, we used a proteomics approach to compare cord blood platelets with adult peripheral blood platelets and showed that their protein abundance was highly similar, while limited differences were related to proteins involved in mitochondrial energy metabolism. Finally, we performed an integrated analysis of all phosphoproteomic analyses to reveal unique signaling signatures and core activation pathways. Taken together, this thesis provides a unique overview of communication networks in endothelial cells, which may contribute to a better understanding of the interplay of coagulation factors, platelets and the vessel wall and may prompt future investigations to identify key targets for therapeutic intervention.