Temporal phosphoproteomics reveals key regulators of procoagulant COAT platelet generation

Procoagulant platelets play a major role in 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 vs 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. Afterward, aggregatory platelets maintained or further increased their phosphorylation levels, whereas procoagulant COAT platelets exhibited a progressive and marked decrease in phosphorylation. Notably, during the first minute after activation, before the phenotypic development of the procoagulant response, several ion channels (calcium, sodium, hydrogen, and chloride), proteins involved in receptor negative feedback loops, cyclic nucleotide-dependent regulatory proteins, GTPases, and key kinases were already differentially phosphorylated in procoagulant COAT vs aggregatory experimental conditions. At the onset of the procoagulant response (1-3 minutes after activation), some calcium channels, phosphatidylinositol transfer proteins, α2-adrenergic receptors, and proteins involved in intracellular and extracellular mechanisms regulating 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.