Thrombin is known to induce the activation of platelet aggregation by binding to and cleaving the extracellular N-terminal domains of protease-activated receptors 1 and 4 (PAR1 and PAR4). To date, many direct thrombin inhibitors (DTIs) have proved to be potent inhibitors of thrombin-activated platelet aggregation. Such DTI can be used as pharmacological agents for the management of Acute Coronary Syndrome (ACS). Recent significant advances in platelet proteomics have led to the development of new nanoLC/ESI-MS/MS platforms for enhanced research of platelet pathophysiology. Herein we present the development of a pharmacoproteomics platform that highlights changes occurring in the protein expression profiles of healthy human platelets treated with selected DTI using the nanoLC-ESI MS/MS on a Q-Exactive quadrupole orbitrap mass spectrometer, and the label free quantification (LFQ) method. Ingenuity Pathways Analysis (IPA) highlighted that many proteins involved in the actin, ILK, rhoA, rhoGDI and integrin signaling pathways are down-regulated in the lead DTI-treated platelets. Specifically, actin related protein 2/3 complex, ADP-ribosylation factor 5, subunit 5-like, integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor), ras homolog family member A, Rho-associated, coiled-coil containing protein kinases 1 and 2, phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2 gamma, protein phosphatase 1, regulatory (inhibitor) subunit 14B, protein phosphatase 2, catalytic subunit, beta isozyme were the major proteins deficient in the thrombin activated-platelets that were treated with the novel DTIs. Our results advocate for the LFQ-based platelets proteomics profiling as a reliable assay for monitoring the efficacy of selected drug treatment during Acute Coronary Syndrome (ACS) management.

Label free proteomics profiling unveils downregulation of thrombin mediated signaling and aggregation of human platelets upon treatment with thrombin inhibitors

253rd National Meeting (2017)

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