Decreased or enhanced procoagulant platelet generation may lead to bleeding or thrombotic events, respectively. The intracellular program underlying the dichotomous generation of aggregating (AGG) and procoagulant (COAT) platelets upon combined activation by Collagen-And-Thrombin is only partially described. In this study, we investigated the utility of time-lapse phosphoproteomics to find potential regulators of the procoagulant response. Subsequently, we validated the phosphorylation pattern of Vasodilator-Stimulated Phosphoprotein at Serine 239 (VASP S239) with a flow cytometry based intracellular staining technique (phosphoflow).

Platelets were activated at RT with convulxin plus thrombin in presence or absence of calcium, which generated procoagulant or aggregating phenotypes, respectively. Platelets were sampled at baseline and at different timepoints up to 8 min after activation. The phosphoproteomes of resting, AGG and COAT platelets were analysed by isobaric Tandem-Mass-Tag based Mass Spectrometry strategy. Protein-specific phosphorylation sites (phosphosites) of interest were monitored at different time points in both AGG and COAT phenotypes by phosphoflow.

Upon stimulation, we identified 4223 differently regulated phosphosites corresponding to 1643 unique proteins showing significant regulation (Fig. 1A). Overall, proteins gradually dephosphorylate in procoagulant platelets and hyper-phosphorylate in aggregating platelets (Fig 1B). Phosphosites with similar and significant time-dependent phosphorylation changes were clustered in 4 groups (Fig 1C). Interestingly, a dichotomous phosphorylation status was observed for VASP S239 with a significant downregulation in procoagulant platelets (Fig 1D). This observation was also confirmed by phosphoflow (Fig. 1E) which validated the results from the phosphoproteomic analysis. Lower VASP S239 phosphorylation is involved in greater cytoskeleton remodelling, which is reported in the development of the procoagulant response, and adhesive events.

The present study highlights the utility of phosphoproteome analysis to detect time-dependent changes of key molecular regulators of the dichotomous response leading to the generation of procoagulant besides aggregating platelets. We propose that VASP could be an interesting target to modulate the procoagulant response.