Temporal phosphoproteomics reveals key regulators of procoagulant COAT platelet generation

Procoagulant platelets play a major role for stabilizing the primary hemostatic plug formed by aggregated platelets. The intracellular signaling pathways driving the shift from an aggregatory to a procoagulant (COAT) platelet phenotype upon collagen-plus-thrombin stimulation are only partially elucidated. Aiming at identifying differentially phosphorylated proteins and potential regulators of the procoagulant response, we mapped time-dependent changes in the phosphoproteome of aggregatory versus procoagulant COAT platelets using a liquid chromatography-mass spectrometry-based temporal phosphoproteomics approach. Upon stimulation with convulxin (an agonist of the collagen receptor glycoprotein VI) and thrombin, we observed that all platelets rapidly increased their overall phosphorylation during the first minute. Afterwards, aggregatory platelets maintained or further increased their phosphorylation levels, whereas procoagulant COAT platelets exhibited a progressive and marked decrease in phosphorylation. Of note, during the first minute post-activation, prior to the phenotypic development of the procoagulant response, several ion channels (calcium, sodium, hydrogen, chloride), receptor negative feedback loops, cyclic nucleotide-dependent regulatory proteins, GTPases, and key kinases were already differentially phosphorylated in procoagulant COAT versus aggregatory experimental conditions. At the onset of the procoagulant response (1-3 min post-activation), some calcium channels, phosphatidylinositol-transfer proteins, α2-adrenergic receptors, and intracellular and extracellular mechanisms involved in the regulation of integrin αIIbβ3 functionality were also differentially phosphorylated. This study highlights the usefulness of assessing phosphorylation dynamics to identify very early phosphorylation events and to understand the rapid phenotypic switch from aggregatory to procoagulant COAT platelets. This approach identified several candidate proteins and mechanisms likely to initiate and modulate the procoagulant response.