title: In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist.
abstract: Vorapaxar is a novel protease-activated receptor-1 (PAR1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. The present study provides a comprehensive in vitro pharmacological characterization of vorapaxar interaction with the PAR1 receptor on human platelets. Similar studies were performed with a metabolite of vorapaxar (M20). Vorapaxar and M20 were competitive PAR1 antagonists that demonstrated concentration-dependent, saturable, specific, and slowly reversible binding to the receptor present on intact human platelets. The affinities of vorapaxar and M20 for the PAR1 receptor were in the low nanomolar range, as determined by saturation-, kinetic- and competitive binding studies. The calculated Kd and Ki values for vorapaxar increased in the presence of plasma, indicating a decrease in the free fraction available for binding to the PAR1 receptor on human platelets. Vorapaxar was also evaluated in functional assays using thrombin or a PAR1 agonist peptide (SFLLRN). Vorapaxar and M20 completely blocked thrombin-stimulated PAR1/β-arrestin association in recombinant cells and abolished thrombin-stimulated calcium influx in washed human platelets and vascular smooth muscle cells. Moreover, vorapaxar and M20 inhibited PAR1 agonist peptide-mediated platelet aggregation in human platelet rich plasma with a steep concentration response relationship. Vorapaxar exhibited high selectivity for inhibition of PAR1 over other platelet GPCRs. In conclusion, vorapaxar is a potent PAR1 antagonist exhibiting saturable, reversible, selective binding with slow off-rate kinetics and effectively inhibits thrombin's PAR1-mediated actions on human platelets.
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, USA.
authors.forename: Brian E
authors.initials: BE
authors.lastname: Hawes
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhai
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, USA.
authors.forename: David
authors.initials: D
authors.lastname: Hesk
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Wirth
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, USA.
authors.forename: Huijun
authors.initials: H
authors.lastname: Wei
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, USA.
authors.forename: Madhu
authors.initials: M
authors.lastname: Chintala
authors.affiliation: Merck & Co., Inc., Kenilworth, NJ, USA. Electronic address: dietmar.seiffert@merck.com.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Seiffert
mesh_terms: D019390:Arrestins / Q000378:metabolism; D001667:Binding, Competitive; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007783:Lactones / Q000378:metabolism / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology*; D044463:Receptor, PAR-1 / Q000378:metabolism; D013379:Substrate Specificity; D000071557:beta-Arrestins; D010975:Platelet Aggregation Inhibitors
chemical_list: D019390:Arrestins; D001798:Blood Proteins; D007783:Lactones; D011725:Pyridines; D044463:Receptor, PAR-1; D000071557:beta-Arrestins; D002118:Calcium; C530299:vorapaxar; D010975:Platelet Aggregation Inhibitors
keywords: In vitro pharmacology; Myocardial infarction; PAR1 antagonist; Peripheral arterial disease; Platelets; Vorapaxar