Phosphoproteomic delineation of platelet activation states

Background:

Primary hemostasis is a tightly orchestrated process initiated by the adhesion, activation and aggregation of blood platelets to stop bleeding. This multi-step process depends on receptor interactions, including VWF-Glycoprotein Ib collagen fibers -Glycoprotein VI/α2β1 integrin, epinephrin to A2α and released of platelet granule interactions of thromboxane A2, (TxA2 [TP]) with TP and ADP with P2Y1 and P2Y12.These interaction in platelet functions are regulated by kinases and phosphatases. In platelet function disorders, each of the aforementioned receptors can be impaired in response to stimuli, resulting in increased bleeding risk. The extent of diverging and converging signaling pathways between the intra-platelet signaling is unresolved. Further resolving these is called for to better understand biological mechanisms in the platelet function disorders that these receptors are involved in. To this end, we aimed to delineate receptor-specific platelet activation states for key receptors on platelets.

Method: Activation of platelets was induced with a panel of agonists, including arachidonic acid (AA), TxA2 mimic U46619, ristocetin, collagen, epinephrin, PAR-1 AP and PAR-4 AP, by stimulation for 30sec, 1min, 2min, 5min and 10min. After lysis, denaturation, reduction and alkylation, proteins were digested and phosphopeptides were enriched from the peptides with Fe3+-IMAC and analyzed with data independent acquisition on a TimsTof HT.

Results: A large coverage of phosphosites across the range of platelet activation states was obtained with a total of 25680 quantified phosphorylation sites. As previously reported, we observed MAPK1 phosphorylation (T185-MAPK1 and Y187-MAPK1) as well as dephosphorylation of the PAR-1 receptor (S412-F2R, S413-F2R, S418-F2R and Y420-F2R) upon activation with PAR-1 AP. Longitudinal statistical analysis showed limited (de)phosphorylation upon stimulation with epinephrine and ristocetin, whereas strong agonists (PAR-1 AP, PAR-4 AP and U46619) demonstrated a large impact on the platelet phosphoproteome. For each of the agonists we observed unique differentially expressed phosphorylation sites including dephosphorylation of the receptor for the agonists, including S314-A2α by epinephrine, S418-F2R by PAR-1 AP, S331-TP by U46619 and S366-F2RL3 by PAR-4 AP. Furthermore, the strong agonists shared over 2000 differentially regulated phosphorylation sites. These might be involved in TxA2 synthesis and platelet granule secretion, which are specific to strong agonists. Notably, focusing on the TxA2 synthesis pathway through the thromboxane A2 precursor AA, revealed that the AA phosphorylation signature was greater than the other strong agonists including thromboxane A2 itself.

Conclusion: This study dissected the longitudinal changes upon stimulation of receptors that are important in primary hemostasis. It highlights the overlap and unique signatures between the platelet activation states.