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 (DTI) 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. This research presents the structure-based drug design (SBDD), synthesis and evaluation of novel tetrapeptide DTI inhibitors of thrombin-activated platelet aggregation, all done by means of proteomics. Analogs of the lead DTI, [D-Phe(P3)-Pro(P2)-D-Arg(P1)-D-Thr-(P1’)-CONH2], have been designed to improve the hydrophobic driven interactions with the S3 pocket of thrombin by replacing the D-Phe (in P3) with D-3,3-diphenylalanine (DIP). The newly DTI lead compounds completely inhibited threshold a-thrombin-induced platelet aggregation at concentrations of 25-8000 nM. The global changes in the protein expression profiles in the human platelets treated with the lead DTI in presence of thrombin were further analyzed by nanoLC-MS/MS using a Q-Exactive-Orbitrap mass spectrometer. 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, sustaining the new designed DTI as an efficacious inhibitor of thrombin-activated platelet aggregation.

Synthesis and proteomic evaluation of novel peptidic inhibitors for the thrombin-induced activation of platelet aggregation

251st National Meeting (2016)

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