Temporal phosphoproteomics reveals key regulators of procoagulant COAT platelet generation

Introduction Abnormal procoagulant platelet generation leads to bleeding or thrombotic events. Procoagulant (COAT) platelets develop from aggregating (AGG) ones upon stimulation with collagen-plus-thrombin. The mechanisms driving this dichotomous activation are only partially described. We employed temporal phosphoproteomics to identify proteins regulating the procoagulant differentiation. Method Washed platelets from healthy donors (n=5) were activated by convulxin-plus-thrombin with (COAT-generating condition) or without calcium (AGG-generating condition). Platelets were sampled at different timepoints up to 8min post-activation. Phosphoproteomes of AGG and COAT platelets were analysed by Liquid-Chromatography-Mass-Spectrometry (LC-MS). Phosphosites significantly differentially regulated among AGG versus COAT conditions were identified by Mann-Whitney test. Results Temporal LC-MS analysis identified 1404 consistently quantified phosphosites. During the first minute post-activation, prior to the development of the procoagulant response, some phosphosites were already differentially phosphorylated in COAT versus AGG conditions (five hyper- and 38 hypo-phosphorylated). These were ion channels (calcium, sodium, hydrogen, chloride), negative feedback loops, cyclic nucleotide-dependent inhibitory pathways, and cytoskeleton interactors. During the phenotypic development of the procoagulant response (1-3 min post-activation), additional calcium channels, phosphatidylinositol-transfer proteins and regulators of fibrinogen receptor activation were differentially phosphorylated (out of 18 hyper- and 457 hypo-phosphorylated). Conclusion Temporal phosphoproteomics identified several proteins differentially phosphorylated early upon platelet activation by convulxin-plus-thrombin. In particular, the 43 phosphorylation events preceding the development of procoagulant platelets from aggregating ones, most likely orchestrate this dichotomous activation pathway. For instance, they may regulate the reverse sodium-calcium exchanger (NCX) functional mode, which is required for the procoagulant response (Thromb Haemost 2021;121:309-321). This work highlights the utility of temporal phosphoproteome analysis to capture timely-regulated processes driving the procoagulant response. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany