Time-resolved characterization of cAMP/PKA-dependant signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways

One of the most important physiological platelet inhibitors is endothelium-derived prostacyclin which stimulates the platelet cAMP/PKA signaling cascade and inhibits virtually all platelet activating key mechanisms. Using quantitative mass spectrometry, we analyzed time resolved phosphorylation patterns in human platelets after treatment with Iloprost, a stable prostacyclin analogue, for 0, 10, 30 and 60 seconds to characterize key mediators of platelet inhibition and activation in three independent biological replicates. We quantified over 2,700 different phosphorylated peptides of which 360 were significantly regulated upon stimulation. This comprehensive and time-resolved analysis indicates that platelet inhibition is a multi-pronged process involving different kinases and phosphatases as well as many previously unanticipated proteins and pathways.