http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–7 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2014.902924
ORIGINAL ARTICLE
PAR1 antagonists inhibit thrombin-induced platelet activation whilst leaving the PAR4-mediated response intact Heather M. Judge1, Lisa K. Jennings2,3, David J. Moliterno4, Edward Hord3, Rosemary Ecob1, Pierluigi Tricoci5, Tyrus Rorick5, Jayaprakash Kotha3, & Robert F. Storey1 Department of Cardiovascular Science, University of Sheffield, UK, 2Vascular Biology Center, University of Tennessee Health Science Center, Memphis, TN, USA, 3CirQuest Labs, LLC, Memphis, TN, USA, 4Lexington VA Medical Center and Gill Heart Institute, University of Kentucky, Lexington, KY, USA, and 5Cardiovascular Research Unit, Duke Clinical Research Institute, Duke University Medical Center, Durham, NC, USA
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Abstract
Keywords
Thrombin-induced platelet activation is initiated by PAR1 and PAR4 receptors. Vorapaxar, a PAR1 antagonist, has been assessed in patients with acute coronary syndromes (ACS) and stable atherosclerotic disease in addition to standard-of-care treatment. In clinical trials, vorapaxar has been observed to reduce the frequency of ischaemic events in some subgroups though in others has increased the frequency of bleeding events. Among patients undergoing CABG surgery, which is associated with excess thrombin generation, bleeding was not increased. The aim of these studies was to investigate the effects of selective PAR1 antagonism on thrombin-induced platelet activation in patients receiving vorapaxar or placebo in the TRACER trial and to explore the roles of PAR1 and PAR4 in thrombin-induced platelet activation in healthy volunteers. ACS patients receiving vorapaxar or placebo in the TRACER trial were studied at baseline and 4 hours, 1 and 4 months during drug administration. Thrombin-induced calcium mobilisation in platelet-rich plasma was assessed by flow cytometry. In vitro studies were performed in healthy volunteers using the PAR1 antagonist SCH79797 or PAR4 receptor desensitisation. Vorapaxar treatment significantly inhibited thrombin-induced calcium mobilisation, leaving a residual, delayed response. These findings were consistent with calcium mobilisation mediated via the PAR4 receptor and were reproduced in vitro using SCH79797. PAR4 receptor desensitization, in combination with SCH79797, completely inhibited thrombininduced calcium mobilisation confirming that the residual calcium mobilisation was mediated via PAR4. In conclusion vorapaxar selectively antagonises the PAR1-mediated component of thrombin-induced platelet activation, leaving the PAR4-mediated response intact, which may explain why vorapaxar is well tolerated in patients undergoing CABG surgery since higher thrombin levels in this setting may override the effects of PAR1 antagonism through PAR4 activation, thus preserving haemostasis. Further assessment may be warranted.
Acute coronary syndromes, platelet aggregation inhibitors, protease-activated receptors, thrombin, vorapaxar
Introduction Platelets play a key role in haemostasis and thrombosis where they adhere to exposed areas of extracellular matrix, leading to platelet activation and recruitment and the formation of a platelet plug or thrombus. Platelet activation is mediated via subendothelial components such as collagen and several soluble mediators including ADP, thromboxane A2 (TxA2) and thrombin. Currently, standard antiplatelet therapy following acute coronary syndromes includes the dual administration of aspirin and a P2Y12 receptor antagonist. Aspirin irreversibly acetylates a serine residue in cyclooxygenase-1, preventing arachidonic acid binding, prostaglandin synthesis and subsequent activation of platelets. ADP is released from platelets as a consequence of activation by many different stimuli. Inhibition of the P2Y12 receptor by receptor antagonists prevents ADP-induced amplification of platelet activation that occurs via this receptor. Dual antiplatelet therapy
Correspondence: Robert F. Storey, MD, DM, FESC, Department of Cardiovascular Science, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, United Kingdom. Tel: 44 114 2261124. Fax: 44 114 2711863. E-mail: [email protected]
History Received 20 January 2014 Revised 21 February 2014 Accepted 6 March 2014 Published online 18 April 2014
with aspirin and a P2Y12 antagonist has been shown to reduce ischaemic events in patients with acute coronary syndromes and in patients undergoing percutaneous coronary interventions [1–4]. However, it has been noted that these patients still have recurrent thrombotic events. Indeed, platelets of patients on effective P2Y12 antagonist therapy are still able to respond to other agonists including thrombin [5]. Thrombin is a potent mediator of platelet activation via two subtypes of protease-activated receptor (PAR) on human platelets, PAR1 and PAR4 [6]. Thrombin binding to PARs on the platelet surface results in cleavage of the receptor and exposure of a tethered ligand, which binds and activates the receptor. PAR1 and PAR4 are coupled to Ga12/13 and Gaq and activation of these intracellular pathways leads to shape change, calcium mobilisation, platelet aggregation, TXA2 production, granule secretion and an increase in platelet procoagulant activity. PAR1 is a high affinity receptor that mediates the effect of thrombin at low concentrations whereas PAR4 has a lower affinity for thrombin, requiring higher concentrations of thrombin for its activation. Elevated markers of thrombin generation have been observed in patients who have previously had a myocardial infarction [10] and high levels may persist for up to five years post event [11].
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Increased thrombin levels have also been demonstrated to be a key mediator of ischaemia-reperfusion myocardial damage during coronary artery bypass graft (CABG) surgery [12]. In addition to activating platelets, thrombin may also induce endothelial cell and smooth muscle cell migration, proliferation, adhesion molecule expression and chemokine secretion [13]. There have been several studies of the oral PAR1 receptor antagonist vorapaxar (formerly SCH530348) in patients with acute coronary syndromes or stable atherosclerotic disease. PAR1 receptor antagonists block thrombin-induced platelet activation but do not affect the ability of thrombin to cleave fibrinogen, thereby maintaining coagulation pathways that contribute to haemostasis [14]. Recently published data from the TRACER study demonstrated significant inhibition of PAR1-mediated platelet aggregation in ACS patients receiving vorapaxar in addition to aspirin and a P2Y12 antagonist [15]. A study of vorapaxar (TRA2P-TIMI50) demonstrated a reduction in cardiovascular death in patients with a history of myocardial infarction, ischaemic stroke or peripheral arterial disease (10.5%, reduced to 9.3%, p50.001) and a reduced incidence of ischaemic events (12.4, reduced to 11.2%, p50.001) in patients who received vorapaxar compared to placebo. However, this was associated with an increased risk of bleeding in the vorapaxar group (4.2%) compared to the placebo group (2.5%, p50.001) [16]. In a study of vorapaxar in patients with acute coronary syndromes (TRACER), vorapaxar did not reduce the incidence of the primary composite endpoint of death from cardiovascular causes, myocardial Figure 1. Thrombin-induced calcium mobilization in fluo-4AM-loaded platelets from patients enrolled in the TRACER sub-study. Figure 1(A–C) show the results for patients receiving placebo (n ¼ 17, (n ¼ 12 at 4 months)) and Figure 1(D and E) for patients receiving vorapaxar (40 mg loading dose, 2.5 mg daily; n ¼ 13 (n ¼ 6 at 4 months)) showing the results for (A and D) 0.1 U/ml thrombin, (B and E) 0.3 U/ml thrombin, and (C and F) 1.0 U/ml thrombin. Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication. The graphs show the increase in fluorescence above background levels.
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infarction, stroke, recurrent ischaemia or urgent coronary revascularization (p ¼ 0.07) but was associated with a lower rate of the secondary composite endpoint of cardiovascular death, myocardial infarction or stroke. There was, as in previous studies, an increased incidence of bleeding although the rates of CABG-related bleeding were similar in the vorapaxar and placebo groups (7.3% placebo, 9.7% vorapaxar, p ¼ 0.13) [17]. A recent subgroup analysis from the TRACER trial demonstrated a reduction in ischaemic events in ACS patients undergoing CABG without an increase in major bleeding (0% versus 0.3%) [18]. Interestingly, Duvernay et al. have demonstrated that PAR4 stimulation leads to more efficient and potent procoagulant activity of platelets than PAR1 stimulation [19], suggesting a key role for PAR4 in maintaining haemostasis in the presence of a PAR1 antagonist. We propose that selective antagonism of the PAR1 receptor, whilst leaving thrombin activation via the PAR4 receptor intact, may result in effective inhibition of platelet activation but maintain haemostasis under conditions of high thrombin levels. The effects of selective PAR1 antagonism on thrombininduced platelet activation in patients receiving vorapaxar or placebo in the TRACER trial and the roles of PAR1 and PAR4 in thrombin-induced platelet activation in healthy volunteers were explored. Flow cytometry was utilised to study thrombin-induced calcium mobilisation in platelets from patients receiving vorapaxar (as part of the TRACER trial) and also in platelets
DOI: 10.3109/09537104.2014.902924
from healthy volunteers where PAR receptors had been antagonised either with a PAR1 receptor antagonist, SCH79797, or by inducing receptor desensitisation by prior exposure to PAR agonist peptides.
PAR1 antagonists inhibit PAR1 but not PAR4
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Blood tubes were 0.105 mol/l (3.2%) citrate Vacutainer tubes from Becton Dickinson (Oxford, UK). Fluo-4AM, probenecid (water soluble) and CaCl2 were from Fisher Scientific (Loughborough, UK). Thrombin was from SigmaAldrich (Gillingham, UK). HEPES Tyrodes buffer was 129 mmol/l NaCl, 8.9 mmol/l NaHCO3, 2.8 mmol/l KCl, 0.8 mmol/l KH2PO4, 5.6 mmol/l dextrose and 10 mmol/l HEPES. SCH79797 was from Tocris Chemicals (Bristol, UK).
PAR1 agonist peptide (SFLLRNPNDKYEPF) was synthesised by Almac Group Ltd (Craigavon, UK). PAR4 agonist peptide (AYPGKF) and scrambled peptide (YAPGKF) were synthesised by Peptide 2.0 Inc (Chantilly, VA). The TRACER pharmacodynamic substudy recruited 249 patients with non-ST elevation acute coronary syndromes from 24 locations (13 European and 11 North American) [15]. Ninty-eight percent patients were on aspirin, a thienopyridine or a combination of these prior to randomisation. Blood was taken from 30 ACS patients enrolled in the TRACER pharmacodynamic sub-study at the Sheffield site during 2010.[15]. Blood was also obtained from healthy volunteers (n ¼ 6–8) who had not taken anti-platelet medication in the previous 10 days. All studies had local ethics committee approval. TRACER patients received either a loading dose of 40 mg vorapaxar followed by 2.5 mg daily
Figure 2. Thrombin-induced peak median fluorescence in fluo-4AMloaded platelets from patients enrolled in the TRACER pharmacodynamics sub-study and treated with either placebo (n ¼ 17 (n ¼ 12 at 4 months)) or vorapaxar (n ¼ 13 (n ¼ 6 at 4 months)), showing the results for (A) 0.1 U/ml thrombin, (B) 0.3 U/ml thrombin, and (C) 1.0 U/ml thrombin. Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication. *p50.05, **p50.01 and ***p50.001 (ANOVA).
Figure 3. Time to reach peak median fluorescence in response to (A) 0.1 U/ml thrombin, (B) 0.3 U/ml thrombin, and (C) 1.0 U/ml thrombin in fluo-4AM-loaded platelets from patients enrolled on the TRACER pharmacodynamics sub-study and receiving either placebo (n ¼ 17 (n ¼ 12 at 4 months) or vorapaxar (n ¼ 13 (n ¼ 6 at 4 months)). Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication *p50.05 and ***p50.001 (ANOVA).
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Materials and methods
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maintenance oral dose or matching placebo loading and maintenance doses. Samples were taken before drug administration (baseline), 4 hours following administration of study medication and 1 month and 4 month whilst on study medication. A total of 30 patients were studied; 17 received placebo and 13 received vorapaxar which reduced to 12 patients receiving placebo and 6 patients receiving vorapaxar at the 4 month time point only as a result of study discontinuation. Blood samples were taken into vacutainer tubes containing tri-sodium citrate 0.105 mol/l. Samples were centrifuged at 140 g for 10 minutes to produce platelet-rich plasma (PRP) and diluted to 250 109/l with platelet-poor plasma, which was prepared by centrifuging the remaining blood at 2500 g for 15 minutes. PRP was labelled with fluo-4 AM (3 mmol/l) in the presence of probenecid (2.5 mmol/l). To block PAR-1 receptors, platelets were pre-incubated with 2 mmol/L SCH79797 or DMSO control (final concentration 0.02% v/v DMSO) for 30 minutes and SCH79797 or DMSO control was included in all subsequent incubation buffers. To induce receptor desensitisation, PRP was pre-incubated for 30 minutes with either control, 2 mmol/l PAR4 peptide, 2 mmol/l PAR4 scrambled peptide or 25 mmol/l PAR1 peptide [20]. For the calcium mobilisation studies, aliquots of fluo-4 labelled PRP were diluted 1:50 in Hepes–Tyrodes buffer containing 1 mmol/l CaCl2 which was maintained at 37 C. Samples were stimulated with 2 mmol/l PAR4 agonist peptide, 25 mmol/l PAR1 agonist peptide or 0.1, 0.3, 0.5 or 1.0 U/ml thrombin and the fluorescence measured at baseline (pre-stimulation) and 5, 15, 30, 60 and 120 seconds following stimulation using a LSR II flow cytometer (Becton Dickinson, Oxford, UK). Median fluorescence values were obtained and baseline values were subtracted. Data was compared by ANOVA using GraphPad Prism software version 5.0.
Results Ex vivo studies Prior to exposure to study medication, platelets that were stimulated with thrombin underwent a rapid calcium mobilisation that decayed to baseline levels over 2 minutes (Figure 1, baseline (BL)). In the patients that received placebo (n ¼ 17) there was no change in this response over the 4 month time period of the study (Figure 1A–C). Vorapaxar administration (n ¼ 13) changed the thrombin-induced calcium mobilisation profile to a reduced and delayed response and this effect occurred within 4 hours of drug administration and persisted throughout the study period (Figure 1D and E). The apparent difference between the results obtained at 1 month and 4 months in the patients receiving vorapaxar (Figure 1D–F) was found to be non-significant (ANOVA) when matched data comparisons were made (n ¼ 6 paired data sets). The inhibition of calcium mobilisation by vorapaxar was demonstrated as a significant reduction in the peak calcium mobilisation in response to all concentrations of thrombin studied (0.1–1.0 U/ml) (Figure 2A–C). Further analysis of this data demonstrated that vorapaxar significantly increased the time taken to reach a peak calcium mobilisation (Figure 3A–C), however there was a residual, delayed calcium mobilisation that was not prevented by vorapaxar administration. This was quantitated by performing area-under-the-curve calculations which showed only one significant difference between placebo and vorapaxar administration at a low concentration of thrombin (0.1 U/ml) (Figure 4A–C). In vitro studies Additional experiments were performed on blood obtained from healthy volunteers to further investigate the individual
Figure 4. The area under the curve for calcium mobilisation in response to (A) 0.1 U/ml thrombin, (B) 0.3 U/ml thrombin, and (C) 1.0 U/ml thrombin in fluo-4 AM-loaded platelets from patients enrolled on the TRACER pharmacodynamics sub-study and treated with either placebo (n ¼ 17 (n ¼ 12 at 4 months)) or vorapaxar (n ¼ 13 (n ¼ 6 at 4 months)). Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication. *p50.05 (ANOVA).
roles of PAR1 and PAR4 receptors in the calcium mobilisation induced by thrombin. PAR1 antagonist SCH79797 (2 mmol/L) significantly inhibited thrombin-induced calcium mobilisation (Figure 5A); however, there was a delayed calcium mobilisation which concurred with that seen during vorapaxar administration in the patient group (Figure 1D and E). SCH79797 at this concentration significantly inhibited PAR1 agonist peptide-induced calcium mobilisation (Figure 5B). SCH79797 has recently been described as having off-target effects that lead to platelet shape change and phosphatidlyserine exposure on the outer membrane [21]. These were only evident when the concentration of SCH79797 reached 10 mmol/L. The experiments carried out here used 2 mmol/L SCH79797 and demonstrated no inhibitory effect on PAR4 agonist peptide- or ADP-induced calcium mobilisation (results not shown).
PAR1 antagonists inhibit PAR1 but not PAR4
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DOI: 10.3109/09537104.2014.902924
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Figure 5. Calcium mobilisation in fluo-4 AM-loaded platelets from healthy volunteers showing (A) the effect of SCH79797 (2 mmol/l) on the response induced by thrombin 0.5 U/ml (n ¼ 7); (B) the effect of SCH79797 and pre-incubation with AYPGKF (2 mmol/l) to desensitise the PAR4 receptor on the response induced by PAR1 agonist peptide (25 mmol/l) (n ¼ 4), (C) the effects of pre-incubating with AYPGKF (2 mmol/l) or control peptide, YAPGKF (2 mmol/l) on the response to the PAR4 agonist peptide AYPGKF 2 mmol/l (n ¼ 7), (D) the effect of pre-incubation with PAR4 agonist peptide (2 mmol/l AYPGKF) or control peptide (2 mmol/l YAPGKF) in combination with SCH79797 (2 mmol/l) compared to SCH79797 alone on the response to thrombin 0.5 U/ml (n ¼ 7), and (E) the effect of pre-incubation with AYPGKF or YAPGKF on the response to thrombin 0.5 U/ml (n ¼ 7). Results show the increase in median fluorescence units above background. *p50.05, **p50.01 and ***p50.001 (ANOVA).
PAR4 desensitisation was induced by pre-incubating platelets with 2 mmol/L AYPGKF for 30 minutes. Subsequent stimulation with additional AYPGKF (2 mmol/L) showed complete inhibition of calcium mobilisation (Figure 5C), but no effect on PAR1 agonist peptide-induced calcium mobilisation (Figure 5B). Pre-incubating platelets with the scrambled peptide, YAPGKF, had no effect on AYPGKF-induced calcium mobilisation (Figure 5C). The delayed calcium mobilisation in response to thrombin seen in the presence of the PAR1 receptor antagonist, SCH79797, and in patients receiving vorapaxar in the pharmacodynamics sub-study was not seen in platelets that had been preincubated with AYPGKF to desensitize the PAR4 receptor (Figure 5D). This would suggest that the residual calcium mobilisation seen in the presence of a PAR1 antagonist is mediated via the PAR4 receptor. The control peptide, YAPGKF,
plus SCH79797 demonstrated similar results to those obtained with SCH79797 alone (Figure 5D). Interestingly, PAR4 desensitisation resulted in strong inhibition of thrombin-induced calcium mobilisation (Figure 5E). The control peptide (YAPGKF) did not have any effect on thrombin responses.
Discussion Inhibition of thrombin-induced, PAR1-mediated calcium mobilisation by vorapaxar or SCH79797 inhibited the magnitude of the response to thrombin but revealed a residual delayed calcium mobilisation. This finding agrees with studies with SCH79797 by Harper et al. [22]. Studies that utilised PAR desensitization revealed that this was mediated via the PAR4 receptor which is consistent with observations from other
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groups; PAR1 mediates a rapid, transient response and PAR4 mediates a slower, sustained calcium mobilisation in response to thrombin [7]. The maintenance of elevated calcium levels via PAR4 stimulation has been documented to support the stabilisation of platelet aggregates and production of TxA2, an important mediator of platelet activation [8, 9]. There is evidence in the literature that PAR1 and PAR4 co-operate to effect thrombininduced responses. Falker et al. [23] described a role for PAR4 in restoring PAR1 receptor responses following PAR1 desensitisation. Previous studies have also described a synergistic effect of both PAR1 and PAR4 antagonists on thrombin-induced P-selectin expression [24]. Leger et al. [25] suggested that PAR1 and PAR4 form a stable heterodimer and that PAR1 is able to promote the cleavage of PAR4. Interestingly, PAR4 desensitisation in our studies resulted in strong inhibition of thrombininduced calcium mobilisation but PAR1 agonist peptide responses were unaffected. This confirms that co-stimulation of both PAR1 and PAR4 are required for effective thrombin-induced platelet activation (calcium mobilisation). This finding supports the work of Arachiche et al. [26] who recently described the formation of PAR1 and PAR4 heterodimers in response to thrombin stimulation but not in response to combined PAR1 and PAR4 agonist peptide stimulation. Strong inhibition of thrombininduced responses was also observed in other studies of PAR4 receptor desensitisation [20]. Although vorapaxar did not achieve a benefit in terms of the primary end point in the TRACER study of patients with ACS, we postulate that there may be a role for PAR1 antagonists in conditions that are associated with excessive thrombin generation. During myocardial infarction (MI), thrombin levels are elevated and levels can remain high for many years after the event [11]. Increased thrombin levels post MI are still evident in patients on dual anti-platelet therapy [10] with aspirin and a P2Y12 antagonist. Vorapaxar was associated with reduced incidence of myocardial infarction in a sub-group analysis from the TRACER study [27]. Scirica et al. [28] reported findings from the TRA 2oP-TIMI 50 trial that show that vorapaxar administration reduces the incidence of cardiovascular death or ischaemic events in patients who have had a previous myocardial infarction. The authors suggest that vorapaxar administration may be of benefit in myocardial infarction patients who have no history of stroke, have a body weight of greater than 60 kg and are less than 75 years old. Thrombin has also been demonstrated to be an important mediator of myocardial injury following ischaemia-reperfusion injury during CABG surgery [29]. Studies with the PAR1 antagonist SCH79797 in a rat model of ischaemia-reperfusion injury resulted in improved left ventricular systolic function [30]. El Eter et al. [31] showed that this effect may be a consequence of reduced proinflammatory cytokine production by SCH79797. Wheelan et al. [18] demonstrated a 37% reduction in the incidence of primary endpoint (CV death, MI, stroke, recurrent rehospitalisation or urgent coronary revascularization) in patients who were administered vorapaxar and who underwent CABG surgery and this was not associated with any significant increase in CABG-related major bleeding [18]. The studies described here could have been further consolidated if we had utilized SCH530348 (vorapaxar) in place of SCH79797 for the in vitro studies. However, this compound wasn’t commercially available at the time we performed these studies. It would also have been interesting to have studied the role of the PAR4 receptor in the samples obtained from the patients in the TRACER substudy but the PAR4 antagonist, TcYN2 that was commercially available at the time the studies were carried out, was found to have partial agonist effects.
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In conclusion, PAR1 antagonism with vorapaxar inhibits the PAR1-mediated component of thrombin-induced platelet calcium mobilisation but does not inhibit the PAR4-mediated delayed increase in intracellular calcium. Vorapaxar has demonstrated clinical benefits in conditions where elevated thrombin levels are evident. We suggest that, under conditions of particularly high thrombin levels, PAR1 antagonists may result in clinical benefits and that the remaining PAR4 response would help maintain haemostasis.
Declaration of interest The ex vivo studies were funded by Merck & Co. Inc. NJ, USA and this funding was administered by CirQuest Labs. In vitro studies were funded by the University of Sheffield. RF Storey reports receiving research grants from AstraZeneca, Eli Lilly/Daiichi Sankyo, and Merck; research support from Accumetrics; honoraria from AstraZeneca, Eli Lilly/Daiichi Sankyo, Merck, Novartis, Iroko, Sanofi-Aventis, BMS, Accumetrics, and Medscape; consultancy fees from AstraZeneca, Daiichi Sankyo, Merck, Novartis, Accumetrics, Regeneron and Roche. Dr. Moliterno declares past consultancy and research funding from Merck-Schering-Plough. Dr. Kotha declares institutional contracts from Merck, Portola and Johnson and Johnson. LK Jennings reports receiving research grants from AstraZeneca and Merck; consultancy fees from Portola. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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23. Falker K, Haglund L, Gunnarsson P, Nylander M, Lindahl TL, Grenegard M. Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets. Biochem J 2011;436:469–480. 24. Wu CC, Teng CM. Comparison of the effects of PAR1 antagonists, PAR4 antagonists, and their combinations on thrombin-induced human platelet activation. Eur J Pharmacol 2006;546:142–147. 25. Leger AJ, Jacques SL, Badar J, Kaneider NC, Derian CK, AndradeGordon P, Covic L, Kuliopulos A. Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis. Circulation 2006;113:1244–1254. 26. Arachiche A, Mumaw MM, de la Fuente M, Nieman MT. Protease activated receptor 1 (PAR1) and PAR4 heterodimers are required for PAR1 enhanced cleavage of PAR4 by alpha-thrombin. J Biol Chem;288:32553–32562. 27. Leonardi S, Tricoci P, White HD, Armstrong PW, Huang Z, Wallentin L, Aylward PE, Moliterno DJ, Van de Werf F, Chen E, et al. Effect of vorapaxar on myocardial infarction in the thrombin receptor antagonist for clinical event reduction in acute coronary syndrome (TRA{middle dot}CER) trial. Eur Heart J 2013;34: 1723–1731. 28. Scirica BM, Bonaca MP, Braunwald E, De Ferrari GM, Isaza D, Lewis BS, Mehrhof F, Merlini PA, Murphy SA, Sabatine MS, et al. Vorapaxar for secondary prevention of thrombotic events for patients with previous myocardial infarction: A prespecified subgroup analysis of the TRA 2 degrees P-TIMI 50 trial. Lancet 2012;380: 1317–1324. 29. Raivio PM, Lassila R, Kuitunen AH, Eriksson H, Suojaranta-Ylinen RT, Petaja J. Increased preoperative thrombin generation and low protein S level associated with unfavorable postoperative hemodynamics after coronary artery bypass grafting. Perfusion 2011;26: 99–106. 30. Sonin DL, Wakatsuki T, Routhu KV, Harmann LM, Petersen M, Meyer J, Strande JL. Protease-activated receptor 1 inhibition by SCH79797 attenuates left ventricular remodeling and profibrotic activities of cardiac fibroblasts. J Cardiovasc Pharmacol Ther 2013; 18:460–475. 31. El Eter E, Aldrees A. Inhibition of proinflammatory cytokines by SCH79797, a selective protease-activated receptor 1 antagonist, protects rat kidney against ischemia-reperfusion injury. Shock 2012; 37:639–644.
ORIGINAL ARTICLE
PAR1 antagonists inhibit thrombin-induced platelet activation whilst leaving the PAR4-mediated response intact Heather M. Judge1, Lisa K. Jennings2,3, David J. Moliterno4, Edward Hord3, Rosemary Ecob1, Pierluigi Tricoci5, Tyrus Rorick5, Jayaprakash Kotha3, & Robert F. Storey1 Department of Cardiovascular Science, University of Sheffield, UK, 2Vascular Biology Center, University of Tennessee Health Science Center, Memphis, TN, USA, 3CirQuest Labs, LLC, Memphis, TN, USA, 4Lexington VA Medical Center and Gill Heart Institute, University of Kentucky, Lexington, KY, USA, and 5Cardiovascular Research Unit, Duke Clinical Research Institute, Duke University Medical Center, Durham, NC, USA
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Abstract
Keywords
Thrombin-induced platelet activation is initiated by PAR1 and PAR4 receptors. Vorapaxar, a PAR1 antagonist, has been assessed in patients with acute coronary syndromes (ACS) and stable atherosclerotic disease in addition to standard-of-care treatment. In clinical trials, vorapaxar has been observed to reduce the frequency of ischaemic events in some subgroups though in others has increased the frequency of bleeding events. Among patients undergoing CABG surgery, which is associated with excess thrombin generation, bleeding was not increased. The aim of these studies was to investigate the effects of selective PAR1 antagonism on thrombin-induced platelet activation in patients receiving vorapaxar or placebo in the TRACER trial and to explore the roles of PAR1 and PAR4 in thrombin-induced platelet activation in healthy volunteers. ACS patients receiving vorapaxar or placebo in the TRACER trial were studied at baseline and 4 hours, 1 and 4 months during drug administration. Thrombin-induced calcium mobilisation in platelet-rich plasma was assessed by flow cytometry. In vitro studies were performed in healthy volunteers using the PAR1 antagonist SCH79797 or PAR4 receptor desensitisation. Vorapaxar treatment significantly inhibited thrombin-induced calcium mobilisation, leaving a residual, delayed response. These findings were consistent with calcium mobilisation mediated via the PAR4 receptor and were reproduced in vitro using SCH79797. PAR4 receptor desensitization, in combination with SCH79797, completely inhibited thrombininduced calcium mobilisation confirming that the residual calcium mobilisation was mediated via PAR4. In conclusion vorapaxar selectively antagonises the PAR1-mediated component of thrombin-induced platelet activation, leaving the PAR4-mediated response intact, which may explain why vorapaxar is well tolerated in patients undergoing CABG surgery since higher thrombin levels in this setting may override the effects of PAR1 antagonism through PAR4 activation, thus preserving haemostasis. Further assessment may be warranted.
Acute coronary syndromes, platelet aggregation inhibitors, protease-activated receptors, thrombin, vorapaxar
Introduction Platelets play a key role in haemostasis and thrombosis where they adhere to exposed areas of extracellular matrix, leading to platelet activation and recruitment and the formation of a platelet plug or thrombus. Platelet activation is mediated via subendothelial components such as collagen and several soluble mediators including ADP, thromboxane A2 (TxA2) and thrombin. Currently, standard antiplatelet therapy following acute coronary syndromes includes the dual administration of aspirin and a P2Y12 receptor antagonist. Aspirin irreversibly acetylates a serine residue in cyclooxygenase-1, preventing arachidonic acid binding, prostaglandin synthesis and subsequent activation of platelets. ADP is released from platelets as a consequence of activation by many different stimuli. Inhibition of the P2Y12 receptor by receptor antagonists prevents ADP-induced amplification of platelet activation that occurs via this receptor. Dual antiplatelet therapy
Correspondence: Robert F. Storey, MD, DM, FESC, Department of Cardiovascular Science, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, United Kingdom. Tel: 44 114 2261124. Fax: 44 114 2711863. E-mail: [email protected]
History Received 20 January 2014 Revised 21 February 2014 Accepted 6 March 2014 Published online 18 April 2014
with aspirin and a P2Y12 antagonist has been shown to reduce ischaemic events in patients with acute coronary syndromes and in patients undergoing percutaneous coronary interventions [1–4]. However, it has been noted that these patients still have recurrent thrombotic events. Indeed, platelets of patients on effective P2Y12 antagonist therapy are still able to respond to other agonists including thrombin [5]. Thrombin is a potent mediator of platelet activation via two subtypes of protease-activated receptor (PAR) on human platelets, PAR1 and PAR4 [6]. Thrombin binding to PARs on the platelet surface results in cleavage of the receptor and exposure of a tethered ligand, which binds and activates the receptor. PAR1 and PAR4 are coupled to Ga12/13 and Gaq and activation of these intracellular pathways leads to shape change, calcium mobilisation, platelet aggregation, TXA2 production, granule secretion and an increase in platelet procoagulant activity. PAR1 is a high affinity receptor that mediates the effect of thrombin at low concentrations whereas PAR4 has a lower affinity for thrombin, requiring higher concentrations of thrombin for its activation. Elevated markers of thrombin generation have been observed in patients who have previously had a myocardial infarction [10] and high levels may persist for up to five years post event [11].
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Increased thrombin levels have also been demonstrated to be a key mediator of ischaemia-reperfusion myocardial damage during coronary artery bypass graft (CABG) surgery [12]. In addition to activating platelets, thrombin may also induce endothelial cell and smooth muscle cell migration, proliferation, adhesion molecule expression and chemokine secretion [13]. There have been several studies of the oral PAR1 receptor antagonist vorapaxar (formerly SCH530348) in patients with acute coronary syndromes or stable atherosclerotic disease. PAR1 receptor antagonists block thrombin-induced platelet activation but do not affect the ability of thrombin to cleave fibrinogen, thereby maintaining coagulation pathways that contribute to haemostasis [14]. Recently published data from the TRACER study demonstrated significant inhibition of PAR1-mediated platelet aggregation in ACS patients receiving vorapaxar in addition to aspirin and a P2Y12 antagonist [15]. A study of vorapaxar (TRA2P-TIMI50) demonstrated a reduction in cardiovascular death in patients with a history of myocardial infarction, ischaemic stroke or peripheral arterial disease (10.5%, reduced to 9.3%, p50.001) and a reduced incidence of ischaemic events (12.4, reduced to 11.2%, p50.001) in patients who received vorapaxar compared to placebo. However, this was associated with an increased risk of bleeding in the vorapaxar group (4.2%) compared to the placebo group (2.5%, p50.001) [16]. In a study of vorapaxar in patients with acute coronary syndromes (TRACER), vorapaxar did not reduce the incidence of the primary composite endpoint of death from cardiovascular causes, myocardial Figure 1. Thrombin-induced calcium mobilization in fluo-4AM-loaded platelets from patients enrolled in the TRACER sub-study. Figure 1(A–C) show the results for patients receiving placebo (n ¼ 17, (n ¼ 12 at 4 months)) and Figure 1(D and E) for patients receiving vorapaxar (40 mg loading dose, 2.5 mg daily; n ¼ 13 (n ¼ 6 at 4 months)) showing the results for (A and D) 0.1 U/ml thrombin, (B and E) 0.3 U/ml thrombin, and (C and F) 1.0 U/ml thrombin. Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication. The graphs show the increase in fluorescence above background levels.
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infarction, stroke, recurrent ischaemia or urgent coronary revascularization (p ¼ 0.07) but was associated with a lower rate of the secondary composite endpoint of cardiovascular death, myocardial infarction or stroke. There was, as in previous studies, an increased incidence of bleeding although the rates of CABG-related bleeding were similar in the vorapaxar and placebo groups (7.3% placebo, 9.7% vorapaxar, p ¼ 0.13) [17]. A recent subgroup analysis from the TRACER trial demonstrated a reduction in ischaemic events in ACS patients undergoing CABG without an increase in major bleeding (0% versus 0.3%) [18]. Interestingly, Duvernay et al. have demonstrated that PAR4 stimulation leads to more efficient and potent procoagulant activity of platelets than PAR1 stimulation [19], suggesting a key role for PAR4 in maintaining haemostasis in the presence of a PAR1 antagonist. We propose that selective antagonism of the PAR1 receptor, whilst leaving thrombin activation via the PAR4 receptor intact, may result in effective inhibition of platelet activation but maintain haemostasis under conditions of high thrombin levels. The effects of selective PAR1 antagonism on thrombininduced platelet activation in patients receiving vorapaxar or placebo in the TRACER trial and the roles of PAR1 and PAR4 in thrombin-induced platelet activation in healthy volunteers were explored. Flow cytometry was utilised to study thrombin-induced calcium mobilisation in platelets from patients receiving vorapaxar (as part of the TRACER trial) and also in platelets
DOI: 10.3109/09537104.2014.902924
from healthy volunteers where PAR receptors had been antagonised either with a PAR1 receptor antagonist, SCH79797, or by inducing receptor desensitisation by prior exposure to PAR agonist peptides.
PAR1 antagonists inhibit PAR1 but not PAR4
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Blood tubes were 0.105 mol/l (3.2%) citrate Vacutainer tubes from Becton Dickinson (Oxford, UK). Fluo-4AM, probenecid (water soluble) and CaCl2 were from Fisher Scientific (Loughborough, UK). Thrombin was from SigmaAldrich (Gillingham, UK). HEPES Tyrodes buffer was 129 mmol/l NaCl, 8.9 mmol/l NaHCO3, 2.8 mmol/l KCl, 0.8 mmol/l KH2PO4, 5.6 mmol/l dextrose and 10 mmol/l HEPES. SCH79797 was from Tocris Chemicals (Bristol, UK).
PAR1 agonist peptide (SFLLRNPNDKYEPF) was synthesised by Almac Group Ltd (Craigavon, UK). PAR4 agonist peptide (AYPGKF) and scrambled peptide (YAPGKF) were synthesised by Peptide 2.0 Inc (Chantilly, VA). The TRACER pharmacodynamic substudy recruited 249 patients with non-ST elevation acute coronary syndromes from 24 locations (13 European and 11 North American) [15]. Ninty-eight percent patients were on aspirin, a thienopyridine or a combination of these prior to randomisation. Blood was taken from 30 ACS patients enrolled in the TRACER pharmacodynamic sub-study at the Sheffield site during 2010.[15]. Blood was also obtained from healthy volunteers (n ¼ 6–8) who had not taken anti-platelet medication in the previous 10 days. All studies had local ethics committee approval. TRACER patients received either a loading dose of 40 mg vorapaxar followed by 2.5 mg daily
Figure 2. Thrombin-induced peak median fluorescence in fluo-4AMloaded platelets from patients enrolled in the TRACER pharmacodynamics sub-study and treated with either placebo (n ¼ 17 (n ¼ 12 at 4 months)) or vorapaxar (n ¼ 13 (n ¼ 6 at 4 months)), showing the results for (A) 0.1 U/ml thrombin, (B) 0.3 U/ml thrombin, and (C) 1.0 U/ml thrombin. Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication. *p50.05, **p50.01 and ***p50.001 (ANOVA).
Figure 3. Time to reach peak median fluorescence in response to (A) 0.1 U/ml thrombin, (B) 0.3 U/ml thrombin, and (C) 1.0 U/ml thrombin in fluo-4AM-loaded platelets from patients enrolled on the TRACER pharmacodynamics sub-study and receiving either placebo (n ¼ 17 (n ¼ 12 at 4 months) or vorapaxar (n ¼ 13 (n ¼ 6 at 4 months)). Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication *p50.05 and ***p50.001 (ANOVA).
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Materials and methods
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maintenance oral dose or matching placebo loading and maintenance doses. Samples were taken before drug administration (baseline), 4 hours following administration of study medication and 1 month and 4 month whilst on study medication. A total of 30 patients were studied; 17 received placebo and 13 received vorapaxar which reduced to 12 patients receiving placebo and 6 patients receiving vorapaxar at the 4 month time point only as a result of study discontinuation. Blood samples were taken into vacutainer tubes containing tri-sodium citrate 0.105 mol/l. Samples were centrifuged at 140 g for 10 minutes to produce platelet-rich plasma (PRP) and diluted to 250 109/l with platelet-poor plasma, which was prepared by centrifuging the remaining blood at 2500 g for 15 minutes. PRP was labelled with fluo-4 AM (3 mmol/l) in the presence of probenecid (2.5 mmol/l). To block PAR-1 receptors, platelets were pre-incubated with 2 mmol/L SCH79797 or DMSO control (final concentration 0.02% v/v DMSO) for 30 minutes and SCH79797 or DMSO control was included in all subsequent incubation buffers. To induce receptor desensitisation, PRP was pre-incubated for 30 minutes with either control, 2 mmol/l PAR4 peptide, 2 mmol/l PAR4 scrambled peptide or 25 mmol/l PAR1 peptide [20]. For the calcium mobilisation studies, aliquots of fluo-4 labelled PRP were diluted 1:50 in Hepes–Tyrodes buffer containing 1 mmol/l CaCl2 which was maintained at 37 C. Samples were stimulated with 2 mmol/l PAR4 agonist peptide, 25 mmol/l PAR1 agonist peptide or 0.1, 0.3, 0.5 or 1.0 U/ml thrombin and the fluorescence measured at baseline (pre-stimulation) and 5, 15, 30, 60 and 120 seconds following stimulation using a LSR II flow cytometer (Becton Dickinson, Oxford, UK). Median fluorescence values were obtained and baseline values were subtracted. Data was compared by ANOVA using GraphPad Prism software version 5.0.
Results Ex vivo studies Prior to exposure to study medication, platelets that were stimulated with thrombin underwent a rapid calcium mobilisation that decayed to baseline levels over 2 minutes (Figure 1, baseline (BL)). In the patients that received placebo (n ¼ 17) there was no change in this response over the 4 month time period of the study (Figure 1A–C). Vorapaxar administration (n ¼ 13) changed the thrombin-induced calcium mobilisation profile to a reduced and delayed response and this effect occurred within 4 hours of drug administration and persisted throughout the study period (Figure 1D and E). The apparent difference between the results obtained at 1 month and 4 months in the patients receiving vorapaxar (Figure 1D–F) was found to be non-significant (ANOVA) when matched data comparisons were made (n ¼ 6 paired data sets). The inhibition of calcium mobilisation by vorapaxar was demonstrated as a significant reduction in the peak calcium mobilisation in response to all concentrations of thrombin studied (0.1–1.0 U/ml) (Figure 2A–C). Further analysis of this data demonstrated that vorapaxar significantly increased the time taken to reach a peak calcium mobilisation (Figure 3A–C), however there was a residual, delayed calcium mobilisation that was not prevented by vorapaxar administration. This was quantitated by performing area-under-the-curve calculations which showed only one significant difference between placebo and vorapaxar administration at a low concentration of thrombin (0.1 U/ml) (Figure 4A–C). In vitro studies Additional experiments were performed on blood obtained from healthy volunteers to further investigate the individual
Figure 4. The area under the curve for calcium mobilisation in response to (A) 0.1 U/ml thrombin, (B) 0.3 U/ml thrombin, and (C) 1.0 U/ml thrombin in fluo-4 AM-loaded platelets from patients enrolled on the TRACER pharmacodynamics sub-study and treated with either placebo (n ¼ 17 (n ¼ 12 at 4 months)) or vorapaxar (n ¼ 13 (n ¼ 6 at 4 months)). Samples were obtained at baseline (BL), 4 hours (4 h) after loading dose and after 1 month (1 m) and 4 months (4 m) on study medication. *p50.05 (ANOVA).
roles of PAR1 and PAR4 receptors in the calcium mobilisation induced by thrombin. PAR1 antagonist SCH79797 (2 mmol/L) significantly inhibited thrombin-induced calcium mobilisation (Figure 5A); however, there was a delayed calcium mobilisation which concurred with that seen during vorapaxar administration in the patient group (Figure 1D and E). SCH79797 at this concentration significantly inhibited PAR1 agonist peptide-induced calcium mobilisation (Figure 5B). SCH79797 has recently been described as having off-target effects that lead to platelet shape change and phosphatidlyserine exposure on the outer membrane [21]. These were only evident when the concentration of SCH79797 reached 10 mmol/L. The experiments carried out here used 2 mmol/L SCH79797 and demonstrated no inhibitory effect on PAR4 agonist peptide- or ADP-induced calcium mobilisation (results not shown).
PAR1 antagonists inhibit PAR1 but not PAR4
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Figure 5. Calcium mobilisation in fluo-4 AM-loaded platelets from healthy volunteers showing (A) the effect of SCH79797 (2 mmol/l) on the response induced by thrombin 0.5 U/ml (n ¼ 7); (B) the effect of SCH79797 and pre-incubation with AYPGKF (2 mmol/l) to desensitise the PAR4 receptor on the response induced by PAR1 agonist peptide (25 mmol/l) (n ¼ 4), (C) the effects of pre-incubating with AYPGKF (2 mmol/l) or control peptide, YAPGKF (2 mmol/l) on the response to the PAR4 agonist peptide AYPGKF 2 mmol/l (n ¼ 7), (D) the effect of pre-incubation with PAR4 agonist peptide (2 mmol/l AYPGKF) or control peptide (2 mmol/l YAPGKF) in combination with SCH79797 (2 mmol/l) compared to SCH79797 alone on the response to thrombin 0.5 U/ml (n ¼ 7), and (E) the effect of pre-incubation with AYPGKF or YAPGKF on the response to thrombin 0.5 U/ml (n ¼ 7). Results show the increase in median fluorescence units above background. *p50.05, **p50.01 and ***p50.001 (ANOVA).
PAR4 desensitisation was induced by pre-incubating platelets with 2 mmol/L AYPGKF for 30 minutes. Subsequent stimulation with additional AYPGKF (2 mmol/L) showed complete inhibition of calcium mobilisation (Figure 5C), but no effect on PAR1 agonist peptide-induced calcium mobilisation (Figure 5B). Pre-incubating platelets with the scrambled peptide, YAPGKF, had no effect on AYPGKF-induced calcium mobilisation (Figure 5C). The delayed calcium mobilisation in response to thrombin seen in the presence of the PAR1 receptor antagonist, SCH79797, and in patients receiving vorapaxar in the pharmacodynamics sub-study was not seen in platelets that had been preincubated with AYPGKF to desensitize the PAR4 receptor (Figure 5D). This would suggest that the residual calcium mobilisation seen in the presence of a PAR1 antagonist is mediated via the PAR4 receptor. The control peptide, YAPGKF,
plus SCH79797 demonstrated similar results to those obtained with SCH79797 alone (Figure 5D). Interestingly, PAR4 desensitisation resulted in strong inhibition of thrombin-induced calcium mobilisation (Figure 5E). The control peptide (YAPGKF) did not have any effect on thrombin responses.
Discussion Inhibition of thrombin-induced, PAR1-mediated calcium mobilisation by vorapaxar or SCH79797 inhibited the magnitude of the response to thrombin but revealed a residual delayed calcium mobilisation. This finding agrees with studies with SCH79797 by Harper et al. [22]. Studies that utilised PAR desensitization revealed that this was mediated via the PAR4 receptor which is consistent with observations from other
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groups; PAR1 mediates a rapid, transient response and PAR4 mediates a slower, sustained calcium mobilisation in response to thrombin [7]. The maintenance of elevated calcium levels via PAR4 stimulation has been documented to support the stabilisation of platelet aggregates and production of TxA2, an important mediator of platelet activation [8, 9]. There is evidence in the literature that PAR1 and PAR4 co-operate to effect thrombininduced responses. Falker et al. [23] described a role for PAR4 in restoring PAR1 receptor responses following PAR1 desensitisation. Previous studies have also described a synergistic effect of both PAR1 and PAR4 antagonists on thrombin-induced P-selectin expression [24]. Leger et al. [25] suggested that PAR1 and PAR4 form a stable heterodimer and that PAR1 is able to promote the cleavage of PAR4. Interestingly, PAR4 desensitisation in our studies resulted in strong inhibition of thrombininduced calcium mobilisation but PAR1 agonist peptide responses were unaffected. This confirms that co-stimulation of both PAR1 and PAR4 are required for effective thrombin-induced platelet activation (calcium mobilisation). This finding supports the work of Arachiche et al. [26] who recently described the formation of PAR1 and PAR4 heterodimers in response to thrombin stimulation but not in response to combined PAR1 and PAR4 agonist peptide stimulation. Strong inhibition of thrombininduced responses was also observed in other studies of PAR4 receptor desensitisation [20]. Although vorapaxar did not achieve a benefit in terms of the primary end point in the TRACER study of patients with ACS, we postulate that there may be a role for PAR1 antagonists in conditions that are associated with excessive thrombin generation. During myocardial infarction (MI), thrombin levels are elevated and levels can remain high for many years after the event [11]. Increased thrombin levels post MI are still evident in patients on dual anti-platelet therapy [10] with aspirin and a P2Y12 antagonist. Vorapaxar was associated with reduced incidence of myocardial infarction in a sub-group analysis from the TRACER study [27]. Scirica et al. [28] reported findings from the TRA 2oP-TIMI 50 trial that show that vorapaxar administration reduces the incidence of cardiovascular death or ischaemic events in patients who have had a previous myocardial infarction. The authors suggest that vorapaxar administration may be of benefit in myocardial infarction patients who have no history of stroke, have a body weight of greater than 60 kg and are less than 75 years old. Thrombin has also been demonstrated to be an important mediator of myocardial injury following ischaemia-reperfusion injury during CABG surgery [29]. Studies with the PAR1 antagonist SCH79797 in a rat model of ischaemia-reperfusion injury resulted in improved left ventricular systolic function [30]. El Eter et al. [31] showed that this effect may be a consequence of reduced proinflammatory cytokine production by SCH79797. Wheelan et al. [18] demonstrated a 37% reduction in the incidence of primary endpoint (CV death, MI, stroke, recurrent rehospitalisation or urgent coronary revascularization) in patients who were administered vorapaxar and who underwent CABG surgery and this was not associated with any significant increase in CABG-related major bleeding [18]. The studies described here could have been further consolidated if we had utilized SCH530348 (vorapaxar) in place of SCH79797 for the in vitro studies. However, this compound wasn’t commercially available at the time we performed these studies. It would also have been interesting to have studied the role of the PAR4 receptor in the samples obtained from the patients in the TRACER substudy but the PAR4 antagonist, TcYN2 that was commercially available at the time the studies were carried out, was found to have partial agonist effects.
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In conclusion, PAR1 antagonism with vorapaxar inhibits the PAR1-mediated component of thrombin-induced platelet calcium mobilisation but does not inhibit the PAR4-mediated delayed increase in intracellular calcium. Vorapaxar has demonstrated clinical benefits in conditions where elevated thrombin levels are evident. We suggest that, under conditions of particularly high thrombin levels, PAR1 antagonists may result in clinical benefits and that the remaining PAR4 response would help maintain haemostasis.
Declaration of interest The ex vivo studies were funded by Merck & Co. Inc. NJ, USA and this funding was administered by CirQuest Labs. In vitro studies were funded by the University of Sheffield. RF Storey reports receiving research grants from AstraZeneca, Eli Lilly/Daiichi Sankyo, and Merck; research support from Accumetrics; honoraria from AstraZeneca, Eli Lilly/Daiichi Sankyo, Merck, Novartis, Iroko, Sanofi-Aventis, BMS, Accumetrics, and Medscape; consultancy fees from AstraZeneca, Daiichi Sankyo, Merck, Novartis, Accumetrics, Regeneron and Roche. Dr. Moliterno declares past consultancy and research funding from Merck-Schering-Plough. Dr. Kotha declares institutional contracts from Merck, Portola and Johnson and Johnson. LK Jennings reports receiving research grants from AstraZeneca and Merck; consultancy fees from Portola. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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DOI: 10.3109/09537104.2014.902924
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PAR1 antagonists inhibit PAR1 but not PAR4
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