Novel regulators identified by quantitative phosphoproteomics analysis during ticagrelor-induced inhibition of platelet

Abstract

Purpose: To study the antiplatelet mechanisms of ticagrelor. Experimental design: Platelets underwent activation with 20 μM ADP for 30 seconds followed by inhibition with 2 nM ticagrelor for another 30 seconds. Mass-spectrometry-based phosphoproteomic technique was applied to obtain phosphorylation spectra in platelets. Results: We successfully quantified 2285 phosphopeptides with high confidence in 1189 phosphoproteins. Compared with intact platelets, ADP-activated platelets showed significant upregulation of PDE5ASer102 and downregulation of 178 phosphopeptides in 154 proteins. Gene Ontology analysis showed that downregulated phosphoproteins were enriched in molecular functions and pathways associated with RNA processing and surveillance. After ticagrelor treatment, we identified 53 significantly regulated phosphopeptides, including 17 upregulated and 36 downregulated, in 45 phosphoproteins. Eight phosphopeptides in STIM1, DENND4C, TNIK, BCL9L, DBN1, DOCK10, FRMD4B, and PRKAR2B were significantly downregulated after ADP stimulation and significantly upregulated after adding ticagrelor. They were mainly implicated in regulation of Ca2+ flow, Wnt/β-catenin signaling, and cytoskeleton remodeling, suggesting their potential role as mediators in ticagrelor-related signaling pathways. Conclusions and clinical relevance: By sequential activation and inhibition of platelets using mutual competitive inhibitors, ADP and ticagrelor, we demonstrated alternations in phosphorylation status of phosphoproteins, which could help to interpret the mechanism of bleeding complications associated with ticagrelor.