PAR4 (Protease-Activated Receptor 4) Antagonism With BMS-986120 Inhibits Human Ex Vivo Thrombus Formation

Abstract

Objective: BMS-986120 is a novel first-in-class oral PAR4 (protease-activated receptor 4) antagonist with potent and selective antiplatelet effects. We sought to determine for the first time, the effect of BMS-986120 on human ex vivo thrombus formation.

Approach and results: Forty healthy volunteers completed a phase 1 parallel-group PROBE trial (Prospective Randomized Open-Label Blinded End Point). Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured at 0, 2, and 24 hours after (1) oral BMS-986120 (60 mg) or (2) oral aspirin (600 mg) followed at 18 hours with oral aspirin (600 mg) and oral clopidogrel (600 mg). BMS-986120 demonstrated highly selective and reversible inhibition of PAR4 agonist peptide (100 μM)-stimulated P-selectin expression, platelet-monocyte aggregates, and platelet aggregation (P<0.001 for all). Compared with pretreatment, total thrombus area (μm²/mm) at high shear was reduced by 29.2% (95% confidence interval, 18.3%-38.7%; P<0.001) at 2 hours and by 21.4% (9.3%-32.0%; P=0.002) at 24 hours. Reductions in thrombus formation were driven by a decrease in platelet-rich thrombus deposition: 34.8% (19.3%-47.3%; P<0.001) at 2 hours and 23.3% (5.1%-38.0%; P=0.016) at 24 hours. In contrast to aspirin alone, or in combination with clopidogrel, BMS-986120 had no effect on thrombus formation at low shear (P=nonsignificant). BMS-986120 administration was not associated with an increase in coagulation times or serious adverse events.

Conclusions: BMS-986120 is a highly selective and reversible oral PAR4 antagonist that substantially reduces platelet-rich thrombus formation under conditions of high shear stress. Our results suggest PAR4 antagonism has major potential as a therapeutic antiplatelet strategy.

Clinical trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02439190.

Keywords: antiplatelet; human; novel; protease-activated receptor 4; thrombosis.

Figure 1.

Pharmacokinetics of BMS-986120. BMS-986120 was rapidly absorbed with a half-life of 4 h. Data shown are mean plasma concentrations of BMS-986120 (±95% confidence intervals) after administration of a single oral 60-mg dose.

Figure 2.

BMS-986120 demonstrated highly selective, potent, and reversible inhibition of PAR (protease-activated receptor) 4-stimulated platelet activation and aggregation. Box plots of platelet activation and aggregation in response to (A–C) PAR4 Agonist peptide (AP; 100 μM), (D and E) PAR1 AP (100 μM), (F) PAR1 AP (25 μM), (G) ADP (10 μM), and (H) arachidonic acid (AA; 5 mmol/L), in volunteers randomized to BMS-986120. Data shown are the adjusted mean (+) normalized to unstimulated values. The line within the box represents the median, upper and lower edges of the box represent the 75th and 25th percentiles, and upper and lower whiskers represent the 95th and 5th percentiles. Statistical comparisons (least significance difference test) vs 0 h are represented above each plot. ns indicates nonsignificant. *P<0.05, **P<0.01, ***P<0.001.

Figure 3.

BMS-986120 reduced thrombus formation at high shear but not at low shear. A, Representative image of porcine aortic media exposed to human blood at high shear stained to quantify total thrombus area. Sections were stained with polyclonal goat antihuman fibrin(ogen) antibody and CD61 monoclonal mouse antihuman antibody before treatment with 3,3′-diaminobenzidine substrate chromogen. Sections were then counterstained with a modified Masson trichrome (hematoxylin and sirius red, 0.1%). Effect of (B) BMS-986120 and (C) aspirin (ASA)±clopidogrel (Clop.) on total thrombus area at high and low shear. Statistical comparisons (least significance difference test) vs 0 h are represented above each plot. ns indicates nonsignificant. *P<0.05, **P<0.01, ***P<0.001.

Figure 4.

Reductions in thrombus formation were driven by a decrease in platelet-rich thrombus formation. Representative image of thrombus formed at high shear stained to allow quantification of (A) platelet-rich and (B) fibrin-rich thrombus area. Sections were stained with polyclonal goat antihuman fibrin(ogen) antibody and CD61 monoclonal mouse antihuman antibody before counterstaining with tyramide Cy3 (cyanine 3) and FITC (fluorescein isothiocyanate). Effect of (C) BMS-986120 and (D) aspirin (ASA)±clopidogrel (Clop.) on platelet and fibrin deposition at low and high shear. Data shown are adjusted means±95% confidence intervals. Statistical comparisons (least significance difference test) vs 0 h are represented above each plot. ns indicates nonsignificant. *P<0.05, **P<0.01, ***P<0.001.