Elsevier

European Journal of Pharmacology

Volume 762, 5 September 2015, Pages 221-228
European Journal of Pharmacology

Molecular and cellular pharmacology
In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist

https://doi.org/10.1016/j.ejphar.2015.05.046Get rights and content

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.

Introduction

Thrombin is a serine protease that plays a key role in hemostasis and thrombosis. Thrombin enzymatically cleaves fibrinogen to fibrin, which then polymerizes, creating a mesh scaffold essential for clot formation. Moreover, acting through protease-activated receptors (PARs), thrombin activates platelets and affects a wide range of cell types including endothelial cells, vascular smooth muscle cells, monocytes, T lymphocytes, and fibroblasts (Martorell et al., 2008; Hirano 2007; Macfarlane et al., 2001; Coughlin 2005; Derian et al., 2003). Three thrombin-activated PAR receptors (PAR1, PAR3 and PAR4) have been identified (Vu et al., 1991, Ishihara et al., 1997, Xu et al., 1998), and human platelets have been shown to express at least two of these, PAR1 and PAR4 (Kahn et al., 1998, Kahn et al., 1999). Thrombin activates platelet PAR1 and PAR4 receptors by cleaving the receptors to expose a new amino terminus that acts as a tethered agonist ligand (Coughlin, 2005, Vu et al., 1991, Kahn et al., 1998, Kahn et al., 1999). Although thrombin-mediated cleavage of both PAR1 and PAR4 triggers G-protein-coupled intracellular signaling and subsequent platelet activation, lower concentrations of thrombin are required for PAR1- compared to PAR4-mediated platelet activation (Kahn et al., 1998).

Vorapaxar is a novel PAR1 antagonist recently approved in the U.S. for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease (brand name Zontivity®) (vorapaxar label, 2014). Clinical studies assessing efficacy and safety of vorapaxar in randomized, placebo-controlled Phase III trials include the Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events (TRA 2°P TIMI 50) trial (Morrow et al., 2012) and the Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRACER) trial (Tricoci et al., 2012). Vorapaxar was identified utilizing a binding assay against the PAR1 selective agonist peptide, SFLLRN, on washed human platelet membranes. Functional evidence of PAR1 inhibition with vorapaxar was provided by platelet aggregation assays using platelet rich plasma (PRP)(Chackalamannil et al., 2008).

Vorapaxar effectively blocks platelet aggregation stimulated by thrombin or SFLLRN. Vorapaxar is extensively metabolized after oral administration and one active circulating metabolite, M20 was identified (Ghosal et al., 2011). The relative contribution of M20 to the overall pharmacological activity has not been precisely quantified. Despite the extensive investigation of vorapaxar efficacy in clinical studies, a thorough characterization of the binding kinetics of vorapaxar to PAR1 on human platelets has not been reported. In the present studies, the in vitro binding of vorapaxar and M20 to intact washed human platelets was assessed, including saturation binding as well as competition and kinetic binding assays. In addition, vorapaxar and M20 were profiled using in vitro functional assays, including a calcium flux assay (FLIPR) in washed human platelets, a β-arrestin assay in a recombinant DiscoverX cell line, and a platelet aggregation assay using human PRP. The data demonstrate that vorapaxar and M20 bind to PAR1 in a selective, competitive, and reversible manner with a slow off-rate from the PAR1 receptor. In addition, vorapaxar Ki and Kd values are right-shifted in the presence of human plasma likely due to the high plasma protein binding of the molecule.

Section snippets

Synthesis of radiolabeled vorapaxar and the M20 metabolite

Vorapaxar, its M20 metabolite (Ghosal et al., 2011), and the respective radiolabeled compounds, were synthesized at Merck Research Labs (Fig. 1) (Chackalamannil et al., 2008). [3H]-vorapaxar was synthesized by palladium-mediated sodium borotritide reduction of the bromo-olefin precursor. [3H]-M20 was prepared by sodium borotritide reduction of the aldhehyde precursor. Radiochemical purity was determined on an Ascentis Express C18 column. The specific activity of [3H]-vorapaxar and [3H]-M20 were

Results

Saturation binding data of [3H]-vorapaxar to washed human platelets are shown in Fig. 2. Based on the specific binding determined by subtracting non-specific binding in the presence of excess (10 μM) unlabeled PAR1 antagonist (SCH590709) from total binding, the calculated Kd value for [3H]-vorapaxar was 1.2±0.3 nM.

To assess if vorapaxar binding to PAR1 is reversible, dissociation binding studies were performed (Fig. 3). Following preincubation with [3H]-vorapaxar, washed human platelets were

Discussion

Vorapaxar is a novel, first-in-class protease-activated receptor-1 (PAR1) antagonist that has been approved in the United States of America for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease (vorapaxar label. 2014). The present studies provide a comprehensive in vitro pharmacological characterization of the interaction of vorapaxar and M20 with the PAR1 receptor present on intact human platelets. These in

Conflict of interest

Brian Hawes, Ying Zhai, David Hesk, Mark Wirth, and Dietmar Seiffert are employees of and hold stock in Merck & Co., Inc. Huijun Wei and Madhu Chintala are former employees of Merck & Co., Inc.

Acknowledgments

This study was funded by Merck & Co., Inc., Kenilworth, NJ, USA The authors wish to thank Steve Fried and Stan Kurowski (Merck & Co., Inc.), Qiu Li, and Deborra Mullins (former employees of Merck & Co., Inc.) for their contributions to this study. The authors also wish to thank Alan Meehan (Merck & Co., Inc.) for editorial assistance and Jennifer Rotonda (Merck & Co., Inc.) for assistance in preparing this paper for publication.

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BH, YZ, DH, MW, HW, MC and DS are responsible for the work described in this paper. All authors were involved in at least one of the following: Conception, design, acquisition, analysis, statistical analysis, and interpretation of data in addition to drafting the manuscript and/or revising/reviewing the manuscript for important intellectual content. All authors provided final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

2

Madhu Chintala and Huijun Wei are former employees of Merck & Co., Inc.

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