Identification of key regulators of procoagulant COAT platelet generation by quantitative phosphoproteomic analysis and phosphoflow

Introduction At the site of vascular injury, by combined activation of COllagen And Thrombin (COAT), a fraction of platelets lose their aggregating properties and become procoagulant enhancing local thrombin generation and fibrin deposition. Decreased or enhanced procoagulant platelet generation lead to bleeding or thrombotic events, respectively. The intracellular signalling underlying this dichotomous activation is only partially described. Here, we investigated whether time-lapse phosphoproteomic analysis could identify key regulators of the procoagulant response. Moreover, a flow cytometry based intracellular staining technique (phosphoflow) was developed to confirm and expand on the phosphoproteomic data.

Method Platelets from healthy donors were activated at RT with convulxin and thrombin in presence or absence of calcium, which generated procoagulant or aggregating phenotypes, respectively. Platelets were sampled at baseline and different time points up to 8 min after activation. The phosphoproteomes of unstimulated, aggregating, and procoagulant COAT platelets were analysed by Tandem Mass Tag and quantitative Mass Spectrometry. For the phosphoflow, both phenotypes were generated in the same tube by COAT stimulation in presence of calcium at RT. The aggregant, procoagulant and phosphorylation status were sequentially monitored over 8 min.

Results We identified over 7200 different phosphorylation sites (phosphosites) corresponding to 1886 unique proteins, out of which 1643 (87%) showed significant regulation upon stimulation. Our data indicate that during the procoagulant response proteins are gradually dephosphorylated (and hyper-phosphorylated during aggregation) compared to baseline ([Fig. 1]). We identified 65 antithetically regulated phosphosites in the two activation end-points at 8 min: 29 phosphosites were down-regulated in procoagulant and 36 in aggregating platelets. Among these, we observed an antithetical phosphorylation status of sodium-calcium-exchanger (NCX) at Serine381 and Serine382 in procoagulant versus aggregating platelets. This observation is in line with our previous data showing a critical functional role of NCX for the dichotomous activation leading to procoagulant platelets (Thromb Haemost 2021;121:309). Additionally, a differential phosphorylation status was also observed at Serine 239 of the vasodilator stimulated phosphoprotein (VASP). We selected this phosphosite for the development and validation of the phosphoflow. The downregulation of this phosphosite in procoagulant platelets by phosphoflow confirmed the results from the phosphoproteomic analysis ([Fig. 2]).

Fig. 1  Phosphorylation status of aggregant and procoagulant platelets by phosphoproteomics; Median phosphorylation intensity (2409 phosphosites) during aggregant (AGG) and procoagulant (COAT) platelets generation compared to baseline. Results are shown in arbitrary unit (AU) of intensity as mean ± SD of 3 healthy donors.

Fig. 2  Phosphorylation status of pVASP Ser239 during COAT stimulation by phosphoflow; Timelapse of procoagulant convulxin and thrombin (COAT) platelet generation (top panels) and phosphorylation status (bottom panels) of vasodilator-stimulated phosphoprotein (VASP) at Serine 239 in aggregant (AGG, black) versus procoagulant (COAT, red) platelets. (Top panels) the fractions of each subpopulation are indicated next to the gates. (Bottom panels) the median fluorescence of the phosphorylation signal of pVASP Ser239 is indicated for each subpopulation (bottom) in arbitrary unit (AU).

Conclusion The present study highlights and confirms the utility of both phosphoproteomic and phosphoflow analysis to detect and observe time-dependent changes of critical molecular regulators of the dichotomous response leading to the generation of procoagulant platelets besides aggregating ones at the site of vascular injury.

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Artikel online veröffentlicht:
20. Februar 2023