Abstract 367: Quantitative Phosphoproteomic Profiling and Causal Pathway Analysis Reveal Novel Mediators and Mechanisms Supporting Glycoprotein VI (GPVI) Signaling and Platelet Function

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Intracellular signaling systems associated with the platelet glycoprotein VI receptor (GPVI) are essential to hemostasis and also initiate atherothrombotic events in cardiovascular disease contexts. While many separable molecular entities linked to GPVI-mediated platelet activation have been characterized, a systemic understanding of GPVI signaling over the context of the entire platelet proteome remains far from complete. To test the hypothesis that specific, intact signaling systems downstream of GPVI regulate platelet function, we used a combination of tandem mass tag (TMT) labeling and high-resolution, multidimensional mass spectrometry and informatics tools to quantify and model protein phosphorylation events reproducibly measurable in platelets from healthy human subjects (n=5) in response to the GPVI specific agonist collagen-related peptide (CRP). Following CRP treatment, platelets rapidly regulated the phosphorylation state of 1004 serine, threonine and tyrosine residues on 542 proteins (fold change > 1.5; false discovery rate < 0.05). In addition to regulatory sites on kinases, phospholipases and other well-known targets, the majority of phosphorylation events following CRP stimulation occurred on proteins with uncharacterized roles in platelet function. CausalPath analysis of this data mapped 46 high confidence pathway relations, suggesting that Syk, PKCs and MAPKs ultimately drive the phosphorylation of several novel effectors specifically downstream of GPVI in platelet regulation, including CRMP-2, RAD23B and caldesmon. In addition to comprehensively mapping out known signaling relations while illuminating previously unrecognized regulatory mechanisms of platelet function around GPVI, our work provides a platform to generate novel, mechanistic and testable hypotheses from omics data sets for subsequent discovery-driven efforts to identify platelet-based therapeutic and biomarker targets relevant to cardiovascular disease risk, progression and mortality.