Temporal phosphoproteomics reveals key regulators of procoagulant COAT platelet generation

ABSTRACT: The intracellular signaling generating and sustaining the procoagulant response of platelets from a common aggregatory phenotype upon simultaneous collagen and thrombin (COAT) stimulation is only partially elucidated. Here we described the phosphoproteome of aggregatory and procoagulant COAT platelets using a temporal phosphoproteomic approach (tandem-mass-tag labelling and liquid-chromatography mass-spectrometry) to reveal differentially regulated proteins and pathways and possibly regulators of the procoagulant response. Upon stimulation, all platelets acquire an overall increased phosphorylation status up to one minute. Then, as procoagulant COAT platelets are generated from aggregatory ones, the global phosphorylation status is maintained in aggregatory platelets whereas it is gradually and strongly decreased in procoagulant COAT platelets over time. Prior the procoagulant response (0-1min post-activation), calcium/sodium/hydrogen/chloride channels, glycoprotein VI-dependent negative feedback loops, cyclic nucleotide-dependent inhibitory pathways, and cytoskeleton interactors were already differentially phosphorylated in COAT versus AGG conditions. At the onset of the procoagulant response (1-3min post-activation), calcium channels, phosphatidylinositol-transfer proteins, thromboxane and α2-adrenergic receptors were differentially phosphorylated. When both platelet phenotypes were stable (8min), numerous cytoskeleton proteins were decreased and signaling proteins were importantly dephosphorylated. Moreover, adhesive proteins, small GTPases, cytoskeleton-related proteins, and calcium-regulating proteins were differentially phosphorylated. This study therefore proposes several relevant targets for modulating the procoagulant response and highlights the relevance of studying time-resolved phosphorylation events in the frame of the rapid phenotypic switch from aggregatory to procoagulant platelet.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Platelet

SUBMITTER: Roman Mylonas  

LAB HEAD: Lorenzo Alberio

PROVIDER: PXD062271 | Pride | 2026-09-14

REPOSITORIES: Pride