PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies

BACKGROUND: BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. METHODS: Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. RESULTS: BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)–induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (−21%; P=0.001) and in patients receiving ticagrelor alone (−28%; P=0.001), aspirin alone (−23%; P=0.018), or both in combination (−24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP–induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). CONCLUSIONS: PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

do not eradicate all atherothrombotic events, and current proven benefits come with an increased hazard of bleeding. 11,14There remains a need for improved therapy to enhance antithrombotic efficacy and to reduce any bleeding liability.
PAR (protease-activated receptor) type 4 antagonism is a key target of interest in the development of novel antiplatelet therapies.6][17] Unlike most other antiplatelet therapies, PAR4 antagonists target the later stages of platelet activation and deposition in response to high concentrations of thrombin, resulting in a slow sustained signaling response.][20][21][22] We have previously demonstrated in healthy volunteers that PAR4 antagonism reduces thrombus formation in an ex vivo model of acute arterial injury 18 as well as demonstrated that PAR4 antagonism provides additive antithrombotic effects when used in combination with factor Xa inhibition. 23However, the antithrombotic efficacy of PAR4 antagonism in patients with coronary artery disease and its additive benefit to contemporary antiplatelet therapy has not been established.Using the same ex vivo model of acute arterial injury, we sought to investigate the antithrombotic efficacy of BMS-986141, a novel highly selective, potent, and orally active PAR4 antagonist, when used in combination with conventional antiplatelet therapies in patients with stable coronary artery disease.

MATERIALS AND METHODS
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Study Design and Study Population
This was a phase 2a single-dose open-label single-center 4-arm study.The study population was composed of men and women aged between 18 and 75 years, with a body mass index between 18 and 35 kg/m 2 and included 45 patients with coronary artery disease and 10 healthy volunteers.Patients with coronary artery disease were recruited from local cardiology clinics, and healthy volunteers were recruited through local advertisement.Participants fasted for 8 hours and abstained from alcohol and caffeine for 72 hours before study attendance.Subjects were excluded if they had evidence of acute illness, clinically significant abnormalities in screening assessments (including clinical findings, ECG changes, or clinical laboratory findings), were pregnant, or could not tolerate repeated venipuncture.Patients were excluded if they were taking any prohibited medication known to interact with platelet aggregation (including opiates).All healthy volunteers had no clinically significant medical history and were not receiving any medication (except oral contraceptives).Patients with stable coronary artery disease had a history of at least 1 of the following: (1) prior angiographically proven coronary artery disease (>50% stenosis of a proximal coronary artery), (2) prior coronary revascularization (percutaneous coronary intervention or coronary artery bypass grafting), or (3) prior myocardial infarction.Patients were excluded if there was a history of acute coronary syndrome or coronary revascularization within 3 months of study attendance.The study was approved by the Scotland Research Ethics Committee A and conducted in accordance with the principles of the Declaration of Helsinki.Written informed consent was obtained from all participants.Clinical Trial Authorization was provided by the Medicines and Healthcare products Regulatory Agency of the United Kingdom.

Study Protocol
Patients with coronary artery disease were assigned to 1 of 3 treatment arms (n=15 per group) at the discretion of the investigators.In view of the potential risk of increased bleeding in patients taking multiple antiplatelet therapies, only patients who were already taking dual antiplatelet therapy (aspirin and a P2Y12 receptor antagonist) were eligible for allocation to combination ticagrelor and aspirin therapy.Where appropriate, patients' usual antiplatelet regimen was amended for 7 days before BMS-986141 administration in accordance with treatment arm allocation: (1) ticagrelor 90 mg BID, (2) aspirin 75 mg QD, or (3) combined ticagrelor 90 mg BID and aspirin 75 mg QD.Patients allocated to the ticagrelor or ticagrelor and aspirin treatment arm who were not already taking ticagrelor were provided with ticagrelor tablets to replace their usual P2Y12 receptor antagonist for 7 days before the first study visit.Healthy volunteers did not receive any background antiplatelet therapy.

Highlights
• PAR (protease-activated receptor) type 4 antagonism with BMS-986141 reduces thrombus formation in patients with coronary artery disease taking ticagrelor alone, aspirin alone, or the combination of ticagrelor and aspirin.• PAR4 antagonism provides specific inhibition of PAR4-AP (agonist peptide)-driven platelet aggregation and activation, irrespective of background antiplatelet therapy.• PAR4 antagonism used in combination with contemporary antiplatelet therapies could provide a novel therapeutic strategy in patients at high risk of secondary cardiovascular events.
Participants attended on 2 consecutive days following the 7-day run-in period as appropriate.All participants received a single 4-mg dose of BMS-986141 on day 1, ≈4 hours after any background antiplatelet therapy.Ex vivo platelet aggregation, activation, and thrombus formation were measured 2 hours before and 2 and 24 hours following administration of BMS-986141 (Figure S1).Participants were followed up for 7 days after the PAR4 antagonist dose.

Blood Sampling and Agonists
Blood samples for platelet function and pharmacokinetic assessments were obtained directly from the antecubital vein immediately before each chamber perfusion run.

Platelet Aggregation
Platelet aggregation stimulated by PAR4-activating peptide (A-Phe[4-F]-PGWLVKNG; GenScript Biotech, the Netherlands) and PAR1-activating peptide (SFLLRN; Merck, NJ) was assessed at each time point.Blood (18 mL) was transferred into tubes containing 2 mL of 3.8% sodium citrate and centrifuged at 300g for 8 minutes at room temperature to obtain plateletrich plasma.Plasma (2 mL) was further centrifuged at 4000g for 3 minutes to obtain platelet-poor plasma.Aggregation of platelet-rich plasma was evaluated by optical aggregometry (PAP-8E; Bio/Data Corp, Horsham, PA).For each patient, the aggregometer channels were blanked with an equivalent volume of platelet-poor plasma.Platelet-rich plasma was added to prewarmed (37 °C) siliconized glass cuvettes with a disposable magnetic stirrer, and the agonist was added to the appropriate platelet-rich plasma and the aggregometry curve was traced for 8 minutes.The final agonist concentrations were 12.5, 25, 50, and 100 μM of PAR4-activating peptide and 25 μM of PAR1activating peptide.

Pharmacokinetic Analysis
For BMS-986141 pharmacokinetic sampling, blood was drawn into 3 mL dipotassium ethylenediaminetetraacetic-coated tubes, gently inverted, and stored at room temperature for 15 minutes.Samples were then centrifuged at 1900g for 10 to 15 minutes at room temperature, and the plasma was decanted and stored at −80 °C before analysis.Plasma BMS-986141 concentrations were determined using a validated liquid chromatography-tandem mass spectrometry method with a lower limit of quantification of 0.050 ng/mL, with an accuracy percentage deviation of <5% and precision (intra-assay and interassay) coefficients of variation <10% during the analysis of the plasma study samples.

Ex Vivo Perfusion Chamber
4][25][26] Participants were cannulated with a 17-G peripheral venous catheter in the antecubital fossa at the beginning of each visit.A motorized pump set at a constant rate of 10 mL/min was used to draw blood from the antecubital vein through 3 perfusion chambers maintained at 37 °C in a water bath.The base of each chamber was lined by a strip of porcine aorta, which had the intima and thin layer of media removed, allowing blood to be exposed to tissue constituents and conditions common to deep arterial injury as encountered following atherosclerotic plaque rupture. 27Rheological conditions in the first chamber were set at low shear stress (≈212 s −1 ) simulating patent coronary arteries, and the second and third chambers were set at high shear stress (≈1690 s −1 ) simulating stenosed coronary arteries. 27,28Each chamber run lasted for 5 minutes and was conducted using the same chambers by the same operator.

Histomorphometric Analysis
Following each perfusion chamber run, porcine strips were fixed using 1% paraformaldehyde solution for 24 hours.Each 1-mm edge of the exposed strip was discarded, and the remaining tissue was cut into 8 segments.Tissue was embedded in paraffin wax and sectioned (4 μm) before histomorphometric analysis (please see the Major Resources Table in the Supplemental Material).
To examine total thrombus area, sections were deparaffinized using xylene and rehydrated through a series of graded alcohol solutions to water and then loaded onto a Leica BOND-MAX or BOND RX automated immunostainer.Antigen retrieval was performed using proteinase K (Dako, Glostrup, Denmark) for 5 minutes, and endogenous hydrogen peroxide activity was blocked using 3% hydrogen peroxide solution (Leica Microsystems GmbH, Wetzlar, Germany).Sections were then incubated for 1 hour at room temperature with polyclonal rabbit anti-human fibrinogen antibody (1:5000; Dako, Glostrup, Denmark) and monoclonal mouse anti-human CD61 antibody (1:50; Dako).Antigen visualization was performed using a bond polymer refine detection kit (Leica Microsystems GmbH) and 3,3ʹ-diaminobenzidine chromogenic substrate, counterstained with a modified direct red protocol.
A semiautomated slide scanner (Axioscan Z1; Zeiss, Jena, Germany) was used at ×20 magnification to acquire images of stained sections.Profiles were created for bright-field imaging of total thrombus area stained using 3,3ʹ-diaminobenzidine chromogenic immunohistochemistry or for 3-channel fluorescence using a halide light source with matched excitation and narrow band pass filters for platelet-rich (CD61) and fibrinrich (fibrinogen) thrombus areas.Images were exported and cropped to include the blood exposed region at 50% resize.Image cropping to the thrombus region of interest was consistent across all specimens examined.Images were batch analyzed by a blinded operator using a custom script with ImageJ (FIJI, University of Wisconsin).Regions of stained tissue were detected using thresholding and filtering (Figure 1).Thresholding was determined using positive and negative controls and were consistent across all images.

Statistical Analysis
On completion of the clinical study activities, data were cleaned, and the database was locked.Statistical analyses were performed independent of the study sponsor.Baseline demographics of participants are expressed as mean±SD for continuous variables and number (%) for categorical variables.Thrombus area in high shear stress conditions was taken as the mean of the 2 high shear chambers.The effect of BMS-986141 was examined by mean difference from baseline (pre-dose) at 2 and 24 hours using a 1-way ANOVA with post hoc pair-wise comparisons using the Fisher least significant difference test.Associations between plasma concentrations of BMS-986141 and pharmacodynamic measures were determined using the Spearman rank-order correlations.Statistical analyses were performed using GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA) and R (version 4.2.2;R Foundation for Statistical Computing, Vienna, Austria).Two-sided P<0.05 was considered to be statistically significant.

RESULTS
Of the 87 subjects who were screened, 56 participants were enrolled and 55 completed the study.One healthy volunteer was withdrawn on visit 2 due to problems with venous access.Patient demographics and baseline characteristics were similar across treatment arms (Table 1), although healthy volunteers were substantially younger with a lower body mass index than the patient groups (Table S1).

Effect of BMS-986141 on Platelet Aggregation and Platelet Activation
Across all treatment arms, BMS-986141 had detectable plasma concentrations 2 hours after administration reducing by 6-fold to 11-fold at 24 hours (Table 2).
BMS-986141 demonstrated strong, selective, and reversible inhibition of PAR4-AP (agonist peptide)-stimulated platelet aggregation, which was unaffected by background antiplatelet therapy (Figure 2; Figure S2).P-selectin expression and platelet-monocyte aggregates stimulated by PAR4-AP were selectively inhibited in all treatment arms at all time points following administration of BMS-986141 (P≤0.001 for all; Figure 2).At 24 hours, there was a persistent and demonstrable dosedependent inhibition of PAR4-AP-stimulated platelet aggregation and activation (Figure 2; Figure S2).Plasma concentrations of BMS-986141 strongly correlated with PAR4-AP-stimulated platelet aggregation, plateletmonocyte aggregates, and P-selectin expression in all treatment arms (P≤0.001 for all; Table 3).With one exception (aspirin arm at 2 hours), there were no demonstrable effects of BMS-986141 on the control PAR1-AP-stimulated platelet aggregation at any time point.

Healthy Volunteers
In high shear conditions, BMS-986141 reduced total thrombus area at 2 and 24 hours (Figure 3; Table S2).This was predominantly driven by reductions in platelet-rich thrombus.Plasma BMS-986141 concentrations correlated with total thrombus area under conditions of high shear stress (P=0.01;Table 3).BMS-986141 had no effect on total thrombus area, platelet-rich thrombus, or fibrin-rich thrombus in low shear conditions (Figure S3; Table S2).

Patients With Coronary Heart Disease
PAR4 antagonism reduced thrombus formation by 28% in those taking ticagrelor, 23% in those taking aspirin, and 24% in those taking combination therapy 2 hours after administration in high shear environments (Figure 3; Table S2), and this returned to baseline at 24 hours.Plasma concentrations of BMS-986141 correlated with total thrombus areas in each treatment arm (Table 3).In patients assigned to ticagrelor with or without aspirin, BMS-986141 provided further thrombus reduction in total thrombus and fibrin-rich thrombus areas (Figure S4).
In those taking aspirin alone or in combination with ticagrelor, PAR4 antagonism reduced platelet-rich, fibrinrich, and total thrombus area after 2 hours (Figure S4).A similar pattern was detected in low shear conditions (Figure S3; Table S3).

Safety and Tolerability
One enrolled subject experienced a serious adverse event, which was unrelated to study activities and was withdrawn before treatment arm allocation.Four participants experienced minor bruising during the run-in phase after receiving ticagrelor, which self-resolved and did not worsen following administration of BMS-986141.BMS-986141 was generally well tolerated by all participants, with no clinically significant effects on clinical safety assessments (Tables S3 and S4).

STUDIES -T
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DISCUSSION
This phase 2a study assessed the additive antithrombotic effect of PAR4 antagonism in patients with coronary artery disease receiving contemporary antiplatelet therapy.We have demonstrated that BMS-986141 strongly and selectively inhibits PAR4-induced platelet aggregation and activation, and these effects are demonstrable in patients with coronary artery disease and are unaffected by concomitant antiplatelet therapy.Importantly, we have established that PAR4 antagonism reduces thrombus formation in patients with stable coronary artery disease despite a range of intensities of background antiplatelet therapies.Our results establish that PAR4 antagonism has additive antithrombotic effects and holds major promise as a potential antiplatelet strategy in the prevention and treatment of atherothrombotic cardiovascular events.
We have characterized platelet aggregation and activation in response to PAR4 antagonism over a 24-hour period.In each subject group, a single dose of the PAR4 antagonist resulted in near complete inhibition of PAR4-AP-induced platelet aggregation.There was persistent suppression of aggregation at 24 hours in all patient groups using 25 μM of PAR4-AP.Similarly, PAR4-APstimulated platelet activation was strongly inhibited by PAR4 antagonism, with sustained effect at 24 hours.BMS-986141 had no effect on PAR1-AP-stimulated aggregation in all participants and at all time points except a minimal reduction in platelet aggregation at 2 hours in participants assigned to aspirin, which likely represents a type 2 error.Considering PAR1-AP as a gold  standard control agonist, this indicates BMS-986141 is a highly selective PAR4 antagonist, which carries persistent inhibition of platelet aggregation and activation in the presence of concomitant antiplatelet therapies.Consistent with our previous studies, 18,23 thrombus formation in healthy volunteers was reduced following PAR4 antagonism due to loss of platelet-rich thrombus area, predominately seen in conditions of high shear stress.At baseline, patient groups had proportionately lower platelet-rich thrombus formation under conditions of high shear stress than that observed in healthy volunteers, consistent with a background antithrombotic effect of concomitant antiplatelet therapies.This was particularly apparent in patients receiving ticagrelor alone or in combination with aspirin.Despite this background antiplatelet effect, BMS-986141 continued to have a demonstrable additive antithrombotic effect.Although most prominent under conditions of high shear stress (equivalent to a stenosed coronary artery), this effect was also apparent under conditions of low shear stress (equivalent to a patent coronary artery).
In the present study, we found that PAR4 antagonism had no effect specifically on platelet-rich thrombus area in participants taking aspirin or combination ticagrelor and aspirin therapy; yet there was an overall reduction in total thrombus area.PAR4 has been reported to provide a specific role in mediating platelet activation events that are dependent on initial thrombin-induced intracellular calcium signaling in platelets and subsequent phosphatidylserine exposure, which is reliant on sustained intracellular calcium concentrations. 29Activation of PAR4 promotes sustained platelet responses leading to the generation and stabilization of thrombus.Our results indicate that specific inhibition of PAR4-induced platelet activity may provide a synergistic effect in the presence of other antiplatelet therapies with alternative mechanisms of action where there is an already established reduction in platelet-rich thrombus area.
We have previously demonstrated a modest reduction in fibrin-rich thrombus formation in healthy volunteers administered a PAR4 antagonist. 18Ex vivo studies of human blood have indicated that selective inhibition of PAR4 substantially impairs thrombin activity and formation of fibrin. 29Moreover, in a microfluidic model, inhibition of PAR1 and PAR4 resulted in substantial reductions in platelet adhesion, thrombus growth, and fibrin formation. 30With prolonged PAR inhibition, fibrin formation remained moderately reduced, which corresponded with a reduction in thrombus size.Our results in a population of patients with coronary artery disease are also consistent with these findings.They suggest that PAR4 antagonism combined with other antiplatelet therapies may reduce platelet-induced fibrin formation, and this may be a further mechanism for PAR4 antagonism-induced reductions in total thrombus area.
In a previous study using this ex vivo model of thrombus formation, we demonstrated that high-dose aspirin alone or combined with high-dose clopidogrel (another P2Y12 inhibitor) markedly reduced thrombus area in both high and low shear conditions, 18 although the combination with PAR4 antagonism was not explored.In ex vivo studies using a microfluidic model of occlusive thrombosis, PAR4 and PAR1 antagonism prevented thrombus formation in conditions where dual antiplatelet therapy was insufficient. 31Our results add to these studies, suggesting a further antithrombotic benefit when PAR4 inhibition is combined with contemporary single or dual antiplatelet therapy and could indicate a novel antiplatelet strategy in patients at particularly high risk of atherothrombotic events.
Our study has some limitations.Although primary end point assessors were blinded to study allocation, the study was open label and treatment arm allocation in subjects with coronary artery disease was not randomized.Only patients who were already taking a P2Y12 antagonist were eligible for allocation to the dual antiplatelet therapy arm.Despite this, patient groups were well balanced in terms of baseline characteristics.Healthy volunteers were not age or sex matched to patient groups, prohibiting direct comparisons to be drawn.Most patients recruited were male, which limits the generalizability of our findings to a female population.Given the higher rates of mortality observed in women with coronary artery disease than men, 32,33 this is an important issue that must be addressed in future studies.We should also acknowledge that we assessed the effect of a single oral administration of BMS-986141, and the effect of prolonged maintenance PAR4 antagonism was not assessed.This will be important for its application as a preventative therapy for coronary artery disease and stroke.Moreover, we did not explore the dose response of BMS-986141 in this study.However, we did explore pharmacodynamic responses at 24 hours when plasma concentrations of BMS-986141 were <20% of the concentration at 2 hours.We were able to demonstrate continued pharmacodynamic effects that were consistent with, and proportional to, the residual plasma concentrations, suggesting a dose-dependent effect of BMS-986141.Finally, BMS-986141 was generally well tolerated with no serious adverse reactions or concerning safety signals in any of the participants.However, further study to characterize the safety profile of PAR4 antagonism is warranted in larger studies.This is of particular importance where dual or triple antiplatelet therapy with PAR4 antagonism is considered in patients at high risk of atherothrombotic events, where the risk of hazardous bleeding should be evaluated.
In conclusion, we have demonstrated that PAR4 antagonism reduces human thrombus formation when added to ticagrelor, aspirin, or their combination in patients with stable coronary artery disease.Our results suggest that PAR4 antagonism holds major potential in providing additive antithrombotic efficacy on a background of

Figure 1 .
Figure 1.Representative images of porcine aorta following perfusion chamber under conditions of high shear stress.Tissue is stained with polyclonal goat anti-human fibrin antibody and CD61 monoclonal mouse anti-human antibody to allow quantification.A, Total thrombus area in porcine aorta exposed to human blood.B through D, Platelet-rich thrombus and fibrin-rich thrombus in porcine aorta exposed to human blood.Platelet-rich thrombus is counterstained with fluorescein isothiocyanate (green; C), and fibrinrich thrombus is countered stained with cyanine 3 (red; D).

Figure 2 .
Figure 2. Platelet aggregation and activation in response to PAR4-AP.Platelet aggregation in response to (A) PAR (protease-activated receptor) type 4-AP (agonist peptide; 25 µmol/L) and (B) PAR1-AP (25 µmol/L).Platelet activation: (C) P-selectin expression and (D) platelet-monocyte aggregates (PMAs) in response to PAR4-AP (25 µmol/L) in each treatment arm.Box plots represent pre-dose and 2 and 24 hours following BMS-986141 in each treatment arm.The line within the box represents the median; lower and upper edges of the box represent the 25th and 75th centiles; and lower and upper whiskers represent the 5th and 95th centiles.Statistical comparisons of means (+) are presented using the Fisher least significant difference test compared with predose.GMFI indicates geometric mean fluorescent intensity.***P<0.001.

Figure 3 .
Figure 3. Mean total thrombus area, platelet-rich thrombus area, and fibrin-rich thrombus area under conditions of high shear stress in each allocated treatment arm.The upper edge of the column represents the mean thrombus area, and error bars represent 95% CIs.Statistical comparisons of means (Fisher least significant difference test) are presented at 2 hours (+2 h) and 24 hours (+24 h) compared with pre-dose.*P<0.05,**P<0.01,***P<0.001.