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

Introduction 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 timelapse phosphoproteomics to identify potential early regulators of the procoagulant response and further describe the procoagulant phenotype.

Method Human platelets from 3 to 5 healthy controls were activated at RT simultaneously with convulxin (agonist of the collagen receptor GPVI) 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. Phosphosites significantly changing compared to baseline and differentially regulated among AGG versus COAT platelets were identified by non-parametric ANOVA test.

Results We identified 4223 differently regulated phosphosites corresponding to 1643 unique proteins showing significant changes at 8 min after stimulation ([Fig. 1a]). Starting at 1 min after activation, proteins gradually dephosphorylated in COAT platelets and hyper-phosphorylated in AGG platelets ([Fig. 1b]). Phosphosites were clustered in 5 groups based on their common patterns of phosphorylation changes over 8 min ([Fig. 1c]). All clusters showed decreased phosphorylation in COAT platelets at late timepoints.Potential regulators of the dichotomous program may be proteins showing early (during the first 45 sec after activation) differential phosphorylation, such as actors of cytoskeleton remodelling (DOCK families and small GTPase regulators), lipid transferases (DAPP1 and C2CD2L), ion exchangers (SLC9A1, TRPC6) and mitochondrial fission (DNM1L, MFF).

Fig. 1 Snapshot and timelapse phosphoproteome analysis of aggregant and procoagulant platelets; (a) Volcano plot of all 7219 phosphosites mean intensity values. Procoagulant (COAT) platelet proteins demonstrated downregulated phosphorylation status compared to aggregant (AGG) ones at 8 min (n=3). (b) Time-dependent changes of the overall phosphorylation status during convulxin-plus-thrombin stimulation in AGG (blue circle) compared to COAT (red square) platelets. (n=5, 1865 phosphosites). (c) Heatmap for each phosphosite up to 8 min of activation, in which we could identify 5 clustering groups (n=3-5, all 8186 phosphosites). P-values, *< 0.05, **< 0.01, ****< 0.0001

Conclusion The present study highlighted the utility of phosphoproteome analysis to detect time-dependent changes of key molecular regulators of the dichotomous response leading to the generation of COAT besides AGG platelets. We showed a global dephosphorylation in COAT platelets, possibly initiated by profound membrane remodelling and translocation of phosphatidylserine. We hypothesize that this decrease is due to the disassembly in the inner leaflet of platelet membrane of several protein kinase (PK) complexes requiring phosphatidylserine for binding, such as Fyn, PKC, and phospholipase A2 [1] [2] [3]. Moreover, the global dephosphorylation may also be explained by the differential phosphorylation of several actin phosphatase regulators and by the activation of a proteasome pathway. Finally, we identified several potential early regulators of the dichotomous platelet response upon combined convulxin and thrombin activation.

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