Abstract 108: Quantitative Phosphoproteomics And Causal Analysis Reveal Distinct And Combinatorial Signaling Mechanisms In Protease-Activated Receptor PAR1 And PAR4 Platelet Activation Programs

Abstract

Intracellular signaling pathways downstream of platelet protease-activated receptors (PARs) mediate hemostasis - and, also contribute to thrombosis in vascular diseases - through mechanisms that remain unspecified. Here, we assess the hypothesis that platelet PAR1 and PAR4 each activate specific, as well as overlapping signaling systems to drive platelet responses underlying hemostasis and thrombosis. Our systems biology approach incorporates state-of-the-art mass spectrometry, computational and cell physiological tools to measure and map phosphorylation events in platelet PAR responses. Following isolation from n=4 healthy human donors, washed platelets were treated with PAR1 agonist (TRAP6), PAR4 agonist (AYPGFK), thrombin, or vehicle, prior to lysis, digestion, phosphopeptide enrichment and 16plex tandem mass tag (TMT) labeling. Relative to resting platelets, we measured >1,000 significant phosphorylation events in response to PAR agonists (fold-change >1.5; false discovery rate <0.01), including >600 phosphorylation events common to TRAP6, AYPGFK and thrombin stimulation. These included phosphorylation of well-established mediators (GSK3α, PAK2) and more novel and emerging effectors in platelet activation pathways (BIN2, NKX3-2). Specific PAR1 and PAR4 agonist responses of mechanistic and translational interest were also noted, including phosphorylation of PAR1 T410 or PAR4 S369; thrombin uniquely activated tyrosine kinase Fer Y714 phosphorylation, in a manner that may integrate PAR1 and PAR4 signaling. CausalPath analysis identified >100 signaling relations among site-specific phosphorylation changes downstream of PARs, around MAPK, PI3K/Akt, mTOR/S6K and other pathways. Finally, physiological assays of platelet adhesion, secretion and aggregation, as well as biochemical assays of platelet signaling validated roles for several effectors and pathways in platelet PAR responses. In conclusion, we provide a quantitative omics study and causal analysis of platelet PAR signaling, including specific PAR1 and PAR4 agonist responses. Ultimately, this work will help to specify essential effectors, as well as biomarkers and therapeutic targets in platelet dysregulation, hyperactivity and thrombotic diseases.