Proteomics and biomolecular mass spectometry of hemostatic processes
Thrombin is the key enzyme of the coagulation cascade. Because it acts as pleiotropic factor, it has additional roles in a variety of pathological conditions, including arterial and venous thrombosis, sepsis, disseminated intravascular coagulation, angiogenesis, inflammation and wound healing. Thrombin signaling has, so far, mostly been investigated at the level of single protein or single pathway, Maartje van den Biggelaar reasoned that an unbiased and comprehensive phosphoproteomic approach would be powerful to complement the current knowledge and contribute to unravel the complexity of the thrombin signaling in endothelial cells. Therefore, thrombin-mediated PAR signaling in endothelial cells has been studied employing functional proteomics in close collaboration with Dr. Sara Zanivan at the Beatson Institute for Cancer Research, in Glasgow, UK. A global phosphoproteomic approach including quantitative mass spectrometry-based phosphoproteomics based on Stable Isotope Labelling using Amino acids in Cell culture (SILAC), was used to investigate the dynamics of thrombin-induced protein phosphorylation. This study provided the first unbiased, in-depth and time-resolved systems-wide view of thrombin signaling in primary human endothelial cells. The major finding of this study is that thrombin signaling involves a large number of intracellular signaling molecules and thousands of phosphorylation sites, which are regulated in a highly dynamic fashion. We identified 2224 dynamically regulated phosphorylation sites, including phosphorylation sites on well-known players in thrombin signaling, and more interestingly a plethora of novel proteins which have not been previously related to thrombin, including proteins that contain multiple phosphorylation sites and as such may be key hubs for signaling pathways, proteins that are immediately phosphorylated upon thrombin activation and several protein kinases.
Figure: Time-dependent changes in phorphorylation of proteins involved in Weibel-Palade body release by thrombin-stimulated endothelial cells.
van den Biggelaar M, Hernández-Fernaud JR, van den Eshof BL, Neilson LJ, Meijer AB, Mertens K, Zanivan S. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells. Blood 2014;123(12):e22-36.