PA-BJ is a serine proteinase isolated from Bothrops jararaca venom that induces platelet aggregation in platelet-rich plasma and washed platelets suspensions. This effect of PA-BJ on platelets aggregation is mediated by thrombin receptors PAR1 and PAR4 (Proteinase-Activated Receptors) that are G-protein coupled signaling receptors that require the cleavage of their extracellular N-terminal domain by proteinases such as thrombin, trypsin, and tryptase, generating a new N-terminal. PA-BJ cleaves in vitro the recombinant exodomain of PAR1 at Arg41-Ser42 and Arg46-Asn47 peptides bonds, resulting in the inactivation of the tethered ligand. PA-BJ also promotes calcium mobilization in fibroblasts transfected with PAR4 and desensitizes these cells to the thrombin action. PA-BJ is composed of 232 amino acid residues and contains one N- and one O-glycosidically linked carbohydrate moiety at residues Asn20 and Ser23 respectively. In order to better understand the signaling events caused by cleavage of the PAR1 receptor and following platelet activation we will employ a combination of proteomics and phosphoproteomics profiling and computational analyses to analyze the proteome of platelets activated by PA-BJ and thrombin. The purpose of this study is to provide insights into the mechanism of interaction of serine proteinases with their substrates and to describe new signaling pathways in platelets.
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