physiology, pharmacology, and therapeutic potential of protease-activated receptors in vascular...

Pharmacology & Therapeutics 134 (2012) 246–259

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Pharmacology & Therapeutics

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Associate Editor: G.J. Dusting

Physiology, pharmacology, and therapeutic potential of protease-activated receptorsin vascular disease

Hannah Lee, Justin R. Hamilton ⁎Australian Centre for Blood Diseases, Monash University, Melbourne, Australia

Abbreviations: PARs, Protease-activated receptorsreceptor activating peptides; TBD, Thrombin-binding doreceptor; ADP, Adenosine di-phosphate; TxA2, ThromboxFactor.⁎ Corresponding author at: Australian Centre for Blood

89 Commercial Road, Melbourne, VIC 3004, Australia. Tel.9903 0228.

E-mail address: [email protected] (J.R. H

0163-7258/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.pharmthera.2012.01.007

a b s t r a c t
a r t i c l e i n f o
Keywords:
Protease-activated receptorThrombinPlateletsThrombosis
It has been 20 years since the discovery of the prototypical protease-activated receptor (PAR). In the timesince this landmark work, significant advances have been made in our understanding of this family of fourG protein-coupled receptors. Initially discovered and characterized in an attempt to determine the mecha-nism by which thrombin activates platelets, PARs have since been found to be widely expressed throughoutthe body, respond to multiple proteases, and to be involved in a vast array of physiological processes. Yet de-spite their wide-ranging expression, the function of PARs has been most extensively studied in the vascularsystem. In particular, the importance of PAR1 for platelet activation during arterial thrombosis has been thor-oughly investigated and has led to the development of a host of PAR1 antagonists—two of which are currentlyin Phase 3 trials as antiplatelet agents. Given the impending clinical use of the first PAR antagonists, it is time-ly to review the physiological roles of PARs in cells of the blood and blood vessels, the development and ex-perimental use of PAR antagonists in these systems, and the potential clinical application of these agents astherapeutics for vascular diseases.

© 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2462. Physiology of PARs in the vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2473. Pharmacology of PARs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2474. Therapeutic potential of PARs in vascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2485. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

246247249253255

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249255

1. Introduction

Serine proteases had long been observed to elicit intracellular signal-ling events and induce cellular responses in a variety of cells before themechanism by which they achieved these effects was elucidated. In1991 the landmark study from Shaun Coughlin's group used an expres-sion cloning screen to identify the first human thrombin receptor, nowknown as protease-activated receptor 1 (PAR1) (Vu et al., 1991). This

; PAR-AP, Protease-activatedmain; GPCR, G protein-coupledane A2; VWF, Von Willebrand

Diseases,Monash University, L6,: +61 3 9903 0125; fax: +61 3

amilton).

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receptor class numbers four in mammals, assigned PARs 1–4 (Coughlin,2000). PARs are G protein-coupled receptors found on the surface ofcells from a wide variety of tissues. In the vascular system, PARs areexpressed on platelets, most (if not all) leukocytes, vascular endothelialand smoothmuscle cells, cardiomyocytes, cardiac fibroblasts, and nervesinnervating the heart and blood vessels (for detailed PAR expression seeFig. 1 and also (Vu et al., 1991; A. J. Connolly et al., 1996; Bono et al., 1997;Ishihara et al., 1997; D'Andrea et al., 1998; Dery et al., 1998;Molino et al.,1998; Coughlin, 2000; Barnes et al., 2004).

The structure, activation mechanism, and signalling of PARs hasbeen reviewed extensively elsewhere (Dery et al., 1998; Coughlin,2000, 2005; Hamilton, 2009; Soh et al., 2010). Briefly, genes encodinghuman PARs 1–3 are located on chromosome 5(q13), and the gene forhuman PAR4 is on chromosome 19(p12). In the mouse, PARs 1–3 areon chromosome 13(p2), with PAR4 on chromosome 8(B3). Despitethe distinct location of PAR4, all four genes display a high degree of

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Fig. 1. PAR expression in the human vascular system. PARs are expressed on various cell types in the blood and blood vessels and facilitate a wide range of cellular responses. Ac-tivation of PAR1 and PAR4 on platelets causes platelet activation, secretion, and aggregation. Activation of endothelial cell PAR1 (and in inflammatory conditions, PAR2 and PAR4)causes vasodilation and consequent hypotension, as well as inducing endothelial cell retraction and increasing vascular permeability. Activation of vascular smooth muscle cellPAR1 leads to proliferation and hypertrophy, and is generally pro-inflammatory. *PARs are expressed on virtually all leukocytes.

247H. Lee, J.R. Hamilton / Pharmacology & Therapeutics 134 (2012) 246–259

structural similarity, an indication that the overall structure and func-tion of these receptors is conserved (Bohm et al., 1996; Kahn et al.,1998a; Xu et al., 1998). In both mice and humans, all four PARs havetwo exons: the first encoding a signal peptide with the second exonencoding the entire functional receptor protein (Kahn et al., 1998a).The prototypical PAR, human PAR1, is a 425 amino acid GPCR with aparticularly long N-terminal extracellular domain containing severalfeatures important for the normal functioning of the receptor (Vu etal., 1991; Grand et al., 1996). Firstly, a thrombin cleavage site is presentbetween amino acid residues 41 and 42 [LDPR41 / 42SFLLRN] (Vu et al.,1991). Secondly, a hirudin-like thrombin-binding domain lies betweenresidues 53 and 64 [DKYEPF], and is the primary binding site betweenPAR1 and the anion-binding exosite I of thrombin (Liu et al., 1991). Al-terations to the thrombin-binding site of PAR1 substantially reduce theefficacy of thrombin cleavage of the receptor (Liu et al., 1991). Cleavageof PAR1 reveals a neo-N-terminus, starting nowwith the amino acid se-quence 42SFLLRN47. This region of the receptor is known as the “teth-ered ligand”, as it activates the receptor by binding to its own secondextracellular loop (Vu et al., 1991). Critical interactions required for re-ceptor activation include R46 of the tethered ligand with L260 of the sec-ond extracellular loop of the receptor. This “self-activation” then leadsto conformational change of the receptor and the subsequent interac-tion with G proteins that these conformational changes allow(Nanevicz et al., 1995), as occurs with other GPCRs (Kristiansen,2004). Synthetic peptides that correspond to the sequence of the teth-ered ligand are capable of activating the receptor independently of N-terminal proteolysis, confirming the self-activation model and provid-ing a useful experimental tool for the specific activation of PARs(Faruqi et al., 2000).

Each of the four PARs is activated via this mechanism, with someminor modifications in each case. PAR2 is the most functionally distinctreceptor in the PAR family as it is the only PAR not cleaved by thrombin.PAR2 is most effectively cleaved by trypsin (Nystedt et al., 1994), tryp-tase (Molino et al., 1997b), coagulation factors VIIa and Xa (Camerer etal., 2000), and the membrane-bound serine proteases MTSP1(Takeuchi et al., 2000) and TMPRSS2 (Wilson et al., 2005). HumanPAR3 is activated in a very similar fashion to human PAR1, inasmuch asit is a thrombin cleaved receptor with a hirudin-like thrombin-bindingdomain downstreamof thrombin cleavage site (Ishihara et al., 1997). Re-markably, mouse PAR3 does not signal upon thrombin cleavage, but

functions instead via a unique co-factoringmechanism to support the ac-tivation of PAR4. Like human PAR3, mouse PAR3 binds thrombin via anN-terminal thrombin-binding domain and is cleaved by the protease atits consensus cleavage site. However, in contrast to human PAR3,mouse PAR3 is not activated in response to such cleavage. Indeed,while the expression of human PAR3 cDNA in either COS cells or Xenopusoocytes confers thrombin responsiveness in these cells, similar expres-sion of mouse PAR3 cDNA fails to permit thrombin signalling. Rather,coexpression of mouse PAR3 and PAR4 enhances thrombin sensitivityin such expression systems when compared with the expression onmouse PAR4 alone (Nakanishi-Matsui et al., 2000). Strikingly, when theN-terminal exodomain of mouse PAR3 was inserted into the plasmamembrane of these cells it was sufficient to reproduce this ‘PAR4 sensi-tising’ response, and mutation of the thrombin-binding domain in thissequence abolished the effect (Nakanishi-Matsui et al., 2000). On thebasis of this work, and in contrast to the human receptor, mouse PAR3appears unique among GPCRs in that it is incapable of mediating trans-membrane signalling but functions as a co-factor for the cleavage andactivation of mouse PAR4. In contrast to PAR1 and PAR3, PAR4 is athrombin-sensitive receptor which lacks a thrombin-binding domain,such that PAR4 cleavage by thrombin is significantly less efficient thanobserved for the other receptors (Kahn et al., 1998b). Activation ofPARs results in a multitude of cellular signalling events. Of the fourmajor Gα protein subclasses, both PAR1 and PAR2 have been shown tosignal through Gq, Gi, and G12/13, PAR3 through Gq, and PAR4 throughGq and G12/13 (Soh et al., 2010)—although the coupling of PAR1 andPAR4 to Gi remains controversial (Kim et al., 2002; Lova et al., 2004;Kim et al., 2006).

2. Physiology of PARs in the vasculature

2.1. Platelet PARs

Platelets are the main cellular component of arterial thrombi. Theplatelet surface expresses a number of adhesion receptors essentialfor the initial localization of platelets to sites of vascular damage,most notably receptors for the blood and vessel wall proteins vWF(glycoprotein (GP) complex Ib-V-IX), collagen (GPVI and the integrinα2β1), and fibrinogen (integrin αIIbβ3). Localized platelets are thenactivated by a number of mediators released or formed locally, most

248 H. Lee, J.R. Hamilton / Pharmacology & Therapeutics 134 (2012) 246–259

notably ADP (released from storage granules within platelets),thromboxane A2 (TxA2; synthesized by platelets in response to ago-nist stimulation), as well as thrombin (the end product of the coagu-lation cascade). Thrombin activates human platelets via two PARs,PAR1 and PAR4. PAR1 is a high affinity thrombin receptor, whilePAR4 is a lower affinity receptor. Important structural differences be-tween PAR1 and PAR4 underlie this dual affinity thrombin receptorsystem on platelets. The thrombin-binding domain of PAR1, whichis absent in PAR4, facilitates efficient (sub-nanomolar) thrombincleavage of PAR1 on platelets, such that PAR1 is considered the pri-mary platelet thrombin receptor, responsible for platelet activationby low concentrations of thrombin. Antibodies which block thethrombin-binding domain of PAR1 (Cook et al., 1995; Kahn et al.,1999) or mutation of the thrombin-binding domain (Liu et al.,1991) significantly reduce the sensitivity of PAR1 to thrombin. Theabsence of a thrombin-binding domain on PAR4 accounts for thehigh thrombin concentrations required for PAR4 to initiate plateletactivation in response to thrombin, emphasising the importance ofthe “docking” interaction between the protease and receptor forhigh sensitivity receptor activation. Despite the lower affinity ofPAR4 for thrombin, both PAR1 and PAR4 contribute to platelet activa-tion in a number of settings. One view is that thrombin-inducedplatelet activation by low thrombin concentrations (≤1 nM) is medi-ated predominantly by PAR1 alone and that responses to high(≥30 nM) thrombin concentrations are mediated by a combinationof both PAR1 and PAR4. Whether or not the two platelet thrombin re-ceptors have distinct functions or whether PAR4 performs purely aredundant function remain unknown. However, temporal differencesin the intracellular signalling events induced by PAR1 and PAR4(Shapiro et al., 2000; Kataoka et al., 2003) and the observation thatdistinct subsets of platelet granules are released in response toPAR1 and PAR4 activation on human platelets (Italiano et al., 2008)suggest non-redundant functions.

As in many cases, investigating the function of PARs on platelets hasrelied heavily on the use of animalmodels. However in the case of plate-let PARs these studies must be interpreted with caution as the expres-sion profile and potential activation mechanism of PARs in the mostcommonly used animal models is different to that in humans. Withregards to the platelet PAR expression profile, in contrast to humanplatelets, mouse, rat, and rabbit platelets do not express PAR1, but rath-er express PAR3 and PAR4 (Connolly et al., 1994, 1996; Nakanishi-Matsui et al., 2000; Khan et al., 2005) and guinea pigs platelets expressPARs 1–3 (Andrade-Gordon et al., 2001). The platelets of non-humanprimates, like humans, express PAR1 and PAR4 (Derian et al., 2003b)and therefore much of the pre-clinical work on PARs has beenperformed in non-human primate models. In addition to these expres-sion differences, distinct mechanisms of thrombin-induced platelet ac-tivation may also occur between some species. Most notably,thrombin activation of human platelets appears to be, at least in part,dependent on a distinct (non-PAR) thrombin-binding receptor on theplatelet surface—GPIb. As yet there is no evidence for a similar functionof GPIb in mouse platelets. Specifically, platelets from PAR4-deficientmice are completely unresponsive to thrombin, suggesting that mouseplatelets rely exclusively on PAR4 to mediate thrombin signalling. Incontrast to this situation in the mouse, binding of thrombin to GPIbhas been shown to accelerate the cleavage of PAR1 by thrombin onthe surface of human platelets (De Candia et al., 2001) and patients ge-netically deficient in GPIb (Bernard–Soulier syndrome; (Bernard &Soulier, 1948)) have impaired thrombin responses in their platelets(McNicol et al., 1996). In addition, there is some evidence for a directsignalling role for GPIb in human platelets (Yap et al., 2000; David etal., 2006).

Despite these expression and activation differences, much of theinformation regarding the functions and overall importance of plate-let PARs has been gleaned from the use of PAR-deficient mice. As pre-dicted, PAR1 knockout mice exhibit normal platelet functions due to

the lack of expression of PAR1 on mouse platelets (Connolly et al.,1996). In contrast, platelets from PAR3-deficient mice exhibit a subtledefect in thrombin stimulation (Kahn et al., 1998b; Weiss et al.,2002). Specifically, platelets from PAR3-deficient mice have mildlyimpaired platelet activation in response to low thrombin concentra-tions (Kahn et al., 1998b). Despite this modest effect on thrombin-induced activation in PAR3-deficient platelets, PAR3 appears to playan important role in thrombosis in mouse models as PAR3-deficientmice are protected against thrombosis in two experimental thrombo-sis models: thromboplastin-triggered pulmonary embolism and ferricchloride-induced injury of mesenteric arterioles (Weiss et al., 2002).Furthermore, PAR3-deficient mice have impaired haemostasis as evi-denced by a prolonged tail bleeding time—the gold standard mea-surement of haemostasis in the mouse. These in vivo findingssuggest that, although unable to signal directly, the co-factoring func-tion of PAR3 plays an important role in the activation of mouse plate-lets by thrombin (Nakanishi-Matsui et al., 2000). Platelets from PAR4-deficient mice do not respond to thrombin and have provided a toolfor studying the effects of thrombin-induced platelet activation inthrombus formation (Sambrano et al., 2001). In this regard, PAR4-deficient mice have markedly impaired haemostasis and are pro-tected against a variety of thrombotic challenges using distinctmodels of vascular injury, and to a greater extent than observed inPAR3-deficient mice (Sambrano et al., 2001; Weiss et al., 2002;Hamilton et al., 2004), emphasizing the importance of thrombin-induced platelet activation in haemostasis and thrombosis in mousemodels.

Whilst valuable for proof-of-concept, these studies of PARs inmouse models do not necessarily translate to humans due to the var-iation in the platelet PAR expression profile between the two species.However, non-human primates have the same platelet PARs ashumans and studies in African green (Cook et al., 1995) and cynomol-gus (Derian et al., 2003a; Chintala et al., 2010) monkeys have shownthat pharmacological blockade of PAR1 reduces thrombus formationand vascular occlusion in a similar manner to that observed in PAR-deficient mice. These studies provided further evidence for the poten-tial of PAR1 to become an effective drug target to combat thrombosisin humans and have driven the development of effective PAR antago-nists for this use (see Section 4.1, below).

2.2. Vascular PARs

All four PARs are expressed in vascular smoothmuscle and endothe-lial cells of several species. In humans, PAR1 has been detected in theendothelium and smoothmuscle layers of both artery and vein samples(Nelken et al., 1992; Yang et al., 1997), and appears to have severalfunctions. In cultured vascular smooth muscle cells, thrombin stimu-lates proliferation (McNamara et al., 1993, 1996), contraction (Yang etal., 1997; Chieng-Yane et al., 2011), hypertrophy (Chieng-Yane et al.,2011), extracellular matrix production (Dabbagh et al., 1998; Ivey &Little, 2008), and the release of cytokines (Kranzhofer et al., 1996) andmitogenic growth factors (Rauch et al., 2007) via activation of PAR1.In cultured endothelial cells, PAR1 activation stimulates the productionof prostacyclin and nitric oxide and causes cell retraction (Ngaiza &Jaffe, 1991; Malik & Fenton, 1992; Nagao & Vanhoutte, 1992; Garcia etal., 1993; Hollenberg et al., 1993; Minnear et al., 1993; Ku & Dai, 1997;Sen et al., 2011).When assessed in situ using intact artery preparations,these responses translate to modulation of vascular contractility: acti-vation of PAR1 causes endothelium-dependent, NO-mediated relaxa-tion of artery preparations from the rat (Muramatsu et al., 1992; Yanget al., 1992; Hollenberg et al., 1993), rabbit (Komuro et al., 1997), guineapig (Muramatsu et al., 1992), pig (Tesfamariam et al., 1993; Hwa et al.,1996; Hamilton & Cocks, 2000), dog (Tesfamariam, 1994b), monkey,and human (Hamilton et al., 1998, 2001b). Endothelial removal resultsin a PAR 1-mediated direct smooth muscle contraction of arteries fromrat (Antonaccio et al., 1993; Hollenberg et al., 1993), guinea pig


(Muramatsu et al., 1992), and dog (Tesfamariam, 1994a). Whilst PAR1activation does not contract human coronary arteries free of atheroma,thrombin contracts endothelium-denuded arteries displaying evidenceof atherosclerosis (Ku&Dai, 1997).Matching this observation, PAR1 ex-pression was observed to be limited to the endothelium of arteries freeof atheroma but was detected in both the endothelium and smoothmuscle in arteries which displayed a significant level of atheroma(Nelken et al., 1992). More recent studies have shown that vasodilatoryprostaglandins can downregulate the expression of PAR1 on humanvascular smooth muscle cells in culture (Pape et al., 2008; Rosenkranzet al., 2009), suggesting a further level of control of themitogenic effectsof thrombin via PAR1. Together, these findings suggest that PAR1 acti-vation in the vasculature normally causes endothelium-dependent,nitric oxide-mediated vasodilatation, but that upregulation of smoothmuscle PAR1 in atherosclerosis (and possibly other ‘pro-inflammatory’conditions) results in a PAR1-mediated contraction and may also con-tribute to restenotic events. This hypothesis is supported by the demon-stration that PAR1 activation causes acute hypotension whenadministered intravenously to mice (Cheung et al., 1998; Damiano etal., 1999), rats (Hwa et al., 1996), and humans (Gudmundsdottir et al.,2006, 2008). Furthermore, acute PAR1 activation has also been shownto cause increases in vascular permeability and edema formation in an-imal models (Vergnolle et al., 1999a, 1999b; de Garavilla et al., 2001),presumably due to direct endothelial contraction responses. Additionalpro-inflammatory effects of endothelial PAR1 activation include releaseof pro-inflammatory cytokines (Johnson et al., 1998; Chung et al., 2010)and increased surface expression of leukocyte adhesion molecules (Fuet al., 2005) and point to a role for PAR1 in mediating acute vascularinflammation.

In addition to these effects of PAR1 activation in the unaltered vas-culature, balloon-catheter injury to rat and baboon arteries rapidlyand markedly increased PAR1 expression in medial smooth musclecells and PAR1 is expressed in neointimal cells within the resulting le-sion, implicating the receptor in lesion formation and progression inresponse to injury (Wilcox et al., 1994). Furthermore, PAR1 mRNAlevels have been reported to increase approximately 10-fold in aorticsmooth muscle from rats with angiotensin II-induced hypertension(Capers IV et al., 1997). This increased PAR1 expression resulted inan increase in thrombin-induced contraction of endothelium-denuded rat aortic ring preparations in vitro (Capers IV et al., 1997)and suggests that changes in vascular smooth muscle PAR1 expres-sion are responsible, at least in part, for mediating the hypertensiveeffects of angiotensin II. Finally, constitutive endothelial PAR1 activa-tion, at least in mice, also plays important roles in vasculogenesis: halfof PAR1-deficient mouse embryos fail to develop through mid-gestation due to a defect in blood vessel formation (Connolly et al.,1996) which can be rescued by replacing PAR1 expression in endo-thelial cells alone (Griffin et al., 2001).

Cultured human vascular smoothmuscle (Bono et al., 1997) and en-dothelial cells (Molino et al., 1997c) also express PAR2. In human vascu-lature in situ, PAR2mRNA and protein has been detected in the smoothmuscle and endothelium of both arteries and veins (D'Andrea et al.,1998; Molino et al., 1998). Functionally, activation of PAR2 causes pro-liferation of cultured human aortic smooth muscle cells (Bono et al.,1997) and HUVECs (Mirza et al., 1996). Apart from these mitogenic ef-fects, PAR2 activation in HUVEC causes release of vonWillebrand factorand induces expression of tissue factor (Langer et al., 1999)—two vitalhaemostatic elements. Similar to PAR1 agonists, activators of PAR2cause endothelium-dependent vascular relaxation in several species,including rat (Hollenberg et al., 1996; Magazine et al., 1996;Saifeddine et al., 1996; Sobey et al., 1999), mouse (Moffatt & Cocks,1998) and pig (Hwa et al., 1996; Hamilton et al., 1999; Hamilton &Cocks, 2000; Hamilton et al., 2002). Although activation of PAR2 hasbeen reported to cause direct contraction of arterial smooth muscle inendothelium-denuded arteries of mouse (Moffatt & Cocks, 1998) andrabbit (Komuro et al., 1997), this is not the case in rat (Magazine et

al., 1996); (Sobey et al., 1999), pig (Hwa et al., 1996), or human(Hamilton et al., 2001a, 2001b). In vivo, selective PAR2 activation in-duces an acute hypotensive response in rats (Hwa et al., 1996;Emilsson et al., 1997) and mice (Cheung et al., 1998; Damiano et al.,1999). In contrast to these animal models, PAR2 activation causesendothelium-dependent NO-mediated relaxation of human arterypreparations only after an inflammatory insult (Hamilton et al.,2001a). In line with these findings, mRNA and protein levels for PAR2,but not PAR1, are upregulated in culturedHUVECs by inflammatoryme-diators such as interleukin 1α and tumour necrosis factorα (Nystedt etal., 1996) and PAR2 agonists mobilise Ca2+ in “inflamed” HUVECs(Mirza et al., 1996;Molino et al., 1997c, 1998; Langer et al., 1999). In ad-dition, in vivo treatment of rats with lipopolysaccharide similarly in-creased vascular PAR2 expression, which manifested as an increasedvasodilator response to PAR2 agonists in vitro and in vivo (Cicala etal., 1999). Again, this upregulation in response to an inflammatory stim-ulus was selective for PAR2 over PAR1. Finally, ex vivo treatment ofhuman arterial preparations with similar inflammatory mediators un-veiled an endothelium-dependent, NO-mediated vascular relaxationin response to selective PAR2 agonists (Hamilton et al., 2001a). Further-more, a recent study has shown that PAR2 is also upregulated in rataorta in response to oxidative stress (Aman et al., 2010). Perhaps sur-prisingly then, given these observations, PAR2-mediated vasodilatationhas been reported in humans in vivo (Robin et al., 2003; Roviezzo et al.,2005)—although at least in one study in the setting of diabetes, a pa-thology known to include vasculopathies. Regardless, these findingsraise the possibility that vascular endothelial cell PAR2 plays a role inendothelial cell responses, possibly in response to coagulation prote-ases, during inflammation and, possibly, angiogenesis (Riewald & Ruf,2005). While an explanation for the discrepancy in PAR2-mediated en-dothelial cell responses between humans and other species is wanting,it is tempting to speculate that it is related to an evolutionary divergentrole for PAR2 in the vasculature. Of note, as measured by endothelium-dependent arterial smooth muscle relaxation at least, the sensitivity toPAR2 agonists in comparison with PAR1 agonists across species ishumanbmonkeybpigbguinea pigbrat=mouse.

In comparisonwith PAR1 and PAR2, little is known of the function ofPAR3 and PAR4 in the vasculature, although both PAR3 (Bretschneideret al., 2003) and PAR4 (Bretschneider et al., 2001) are present and ap-pear functional in cultured human vascular smooth muscle cells. In ad-dition, PAR3 has been detected in HUVECs (Cupit et al., 1999) and incultured rat brain capillary endothelial cells (Bartha et al., 2000),while PAR4 is expressed on mouse endothelial cells and contributes toresponses to thrombin in these cells, in vitro and in vivo (Kataoka etal., 2003). PAR4 activation causes endothelium-dependent relaxationof rat aorta in a similar manner to that observed with PAR1 and PAR2activation (Hollenberg et al., 1999). However, as with PAR2, such re-sponses are only elicited in human artery preparations treated with in-flammatory mediators (Hamilton et al., 2001a). Clues to the function ofPAR3 and PAR4 in the vasculature come from recent studies showingthat activation of either PAR3 or PAR4 on human aortic smooth musclecells in culture supports ongoing thrombin production (Vidwan et al.,2010). In addition, the exposure of human vascular smooth musclecells to high glucose (22 mM as compared with the usual 5.5 mM) in-creased the expression and function of PAR4 on these cells (Dangwalet al., 2011), suggestive of a potential role for PAR4 in contributing tothe vasculopathy observed in diabetes.

3. Pharmacology of PARs

3.1. PAR agonists

3.1.1. PAR1For PAR1, five residues of the native peptide sequence of the re-

ceptor (SFLLR) are sufficient to provide the most potent PAR1-AP(Hollenberg et al., 1993; Ceruso et al., 1999). Amidation at the C-


terminus increases potency ~10 fold (Coller et al., 1992; Hollenberg etal., 1993; Lan et al., 2002), and SFLLR-NH2 has an EC50 of~0.5 μM. Al-though a comparatively high potency PAR1-AP and still widely used,SFLLR-NH2 activates both human and mouse PAR2 (Blackhart et al.,1996). In contrast, substitution of N-terminal Ser for Thr produces aPAR1-AP of high specificity, such that the best available PAR1-AP isTFLLR-NH2 (Hollenberg et al., 1997). This sequence appears to behighly specific for PAR1 and has an EC50 of~1 μM (Scarborough etal., 1992).

3.1.2. PAR2Amidated peptides corresponding to the native sequence of the neo-

N-terminus of mouse and human PAR2, SLIGRL-NH2 and SLIGKV-NH2

respectively, are PAR2-specific agonists with approximate equipotencyat PAR2 across several species and are generally used interchangeably.The EC50 of these PAR2-APs is 5–10 μM (Maryanoff et al., 2001). Substi-tution of the N-terminal Serwith a furoyl moiety improves potency, es-pecially in vivo, while maintaining selectivity for PAR2 (Ferrell et al.,2003; Kawabata et al., 2004).

3.1.3. PAR3In contrast to other PARs and their respective activating peptides,

synthetic peptides corresponding to human and mouse PAR3 havenot been useful experimentally. Activation of mouse PAR3 fails toelicit a signal regardless of the mode of activation, and the native se-quence of the human PAR3 tethered ligand (TFRGAP) also activatesPAR1 and PAR2 (Hansen et al., 2004; Kaufmann et al., 2005).

3.1.4. PAR4An amidated peptide corresponding to the native tethered ligand of

human PAR4 (GYPGQV-NH2) selectively activates PAR4butwith almostrestrictive potency, with concentrations of ~500 μM required (Faruqi etal., 2000). A peptide corresponding to the mouse PAR4 tethered ligandsequence (GYPGKF) is slightly more potent in activating both humanand mouse PAR4 (Kahn et al., 1998b). However, substitution of the N-terminal Gly with Ala in the murine sequence is the most potentPAR4-specific agonist to date: AYPGKF-NH2 has an EC50 of ~25 μM(Faruqi et al., 2000; Hollenberg et al., 2004) and has become the goldstandard PAR4-specific agonist.

3.2. PAR antagonists

To fully evaluate the physiological role of each of the PARs and toassess their therapeutic potential, specific antagonists against eachreceptor are required. The mechanism by which PARs are activatedis strikingly unique, and poses many questions and challenges regard-ing receptor pharmacology—in particular for the development of ro-bust antagonists. For example, receptor activation by proteolysis is ahighly efficient system in which many receptors can be activated bya single enzyme molecule. Furthermore, receptor cleavage is irrevers-ible. The self-activation model also likely hinders antagonist efficacy,since any antagonist must compete with an agonist with significantstearic advantage. Such issues—in some cases specific to PARs—havehindered the development of potent antagonists against this receptorclass, many of which are only just now being overcome.

3.2.1. PAR1 antagonists

3.2.1.1. Peptidomimetics. The potential use of PAR1 antagonists asantithrombotics has driven the development of large numbers ofthese agents, and antagonists against PAR1 are significantly more de-veloped than for PARs 2-4. The first PAR antagonists were peptide-based entities, in large part due to the relative ease of synthesis andthe specificity of the tethered ligand sequence to each receptor. Theearliest PAR1 antagonists contained modifications to the tethered li-gand such that the modified peptide retained binding capacity but

without the ability to activate the receptor. Specifically, modificationswere made to positions R1, R2, R3, and R4 side chains of the native se-quence (SFLLRN) which disrupted critical interactions between thepeptide sequence and the second extracellular loop of PAR1 andgave rise to a series of relatively high affinity peptide antagonists, ofwhich BMS-200261, RWJ-56110, and RWJ-58259 have been themost widely used experimentally (Seiler & Bernatowicz, 2003).

BMS-200261 is based on the most potent PAR1-AP, SFLLR-NH2, butwith the Ser replaced by a 3-mercaptopropionyl (Mpa) group, the addi-tion of a carbonyl group and aryl ring at the C-terminus, and a trans-cinnamoyl group at the N-terminus (Scarborough et al., 1992;Ogletree et al., 1993; Bernatowicz et al., 1996; McComsey et al., 1999).BMS-200261 inhibits PAR1-AP-induced platelet aggregation with anIC50 of 0.02 μM, and exhibits high selectivity for PAR1 relative to PAR2(O'Brien et al., 2000) and PAR4 (Kahn et al., 1999; Quinton et al.,2004). Due to the competitive nature of such antagonists and thestrong stearic advantage possessed by the receptor's tethered ligand,BMS 200261 has weaker effects on thrombin-induced cellularresponses.

Using the structure-activity information gained from peptide ago-nist studies, Hoekstra et al. (1998) designed libraries of conformation-ally constrained pentapeptides based on the PAR1-AP, SFLLR-NH2. Aseries of azole-based peptides with a p-F-Phe substituted at position 2of SFLLR inhibited platelet aggregation induced by PAR1-AP and throm-bin, albeit with an IC50 of approximately 20 μM. In attempts to increasethe potency of these compounds a “three-point model” was used tomap the spatial distribution of the ammonium, phenyl, and guanidiumgroups in the PAR1-AP (Andrade-Gordon et al., 1999; Zhang et al., 2001;Maryanoff et al., 2003; Zhang et al., 2003), which was then used in con-junction with different rigid molecular scaffolds for the design of pepti-domimetics. A 6-aminoindole template was selected and a diverserange of compounds screened. This approach yielded RWJ-56110,with an IC50 of 0.34 μM and 0.16 μM to thrombin and PAR1-AP-induced platelet aggregation, respectively (Andrade-Gordon et al.,1999; Derian et al., 2003b). In fact, using transfected murine myofibro-blasts, RWJ-56110was shown to be highly selective for PAR1, fully inhi-biting thrombin-induced calcium responses in the hPAR1 but nothPAR2 or hPAR4 transfected cells (Andrade-Gordon et al., 1999;Maryanoff et al., 2003).

Further optimization of this peptidomimetic class led to the devel-opment of RWJ-58259, in which the indole ring of RWJ-56110 isreplaced by indazole. RWJ-58259 retained the high selectivity of RWJ-56110 against PAR1 and increased potency in a number of assays. Invivo administration of RWJ-58259 to guinea pigs (0.3 mg/kg, i.v.) inhib-ited thrombin-induced aggregation of subsequently isolated platelets(Zhang et al., 2001; Derian et al., 2003a, 2003b, 2003c). When adminis-tered to the adventitial surface of the rat carotid artery in a rat model ofballoon angioplasty, RWJ-58259 reduced the extent of restenotic le-sions (Andrade-Gordon et al., 2001). The antithrombotic effects ofRWJ-58259 were also evaluated using an electrolytic injury model ofthrombosis in the carotid artery of cynomolgus monkeys in which theadministration of RWJ-58259 (as a 3 mg/kg i.v. loading dose followedby a maintenance dose of 0.123 mg/kg/min) significantly inhibitedthrombus formation at sites of vascular injury (Derian et al., 2003b).

3.2.1.2. Blocking antibodies. Antibodies that block the cleavage and/oractivation of PARs were among the earliest antagonist strategies.The first PAR blocking antibodies were used to study the individualroles of PAR1 and PAR4 on human platelets (Kahn et al., 1999).These rabbit polycolonal antibodies were generated using an antigenthat corresponded to the region of PAR1 spanning the thrombincleavage site, with the aim of competing for and preventing receptorcleavage (Fig. 2). Anti-PAR1 and anti-PAR4 antibodies were shown toinhibit thrombin-induced platelet activation by the respective recep-tors, although high concentrations were required: at 1 mg/ml, ananti-PAR1 antibody fully inhibited platelet aggregation induced by

A B

Fig. 2. Mechanisms of PAR antagonist function. A) PARs are G protein-coupled receptors that are activated upon cleavage of a consensus sequence in the extracellular N-terminus of thereceptor. The prototypical PAR, PAR1 (pictured) contains a thrombin-binding domain (TBD; white circle) within the N-terminus, which allows efficient cleavage of the receptors bythrombin. Once cleaved, the new N-terminus sequence activates the receptor by binding to its own second extracellular loop (pink). B) Existing PAR antagonists function by either:1) Blocking proteolytic cleavage of theN-terminus either by competing antibodies raised against the TBD (e.g. IGg9600) or against the thrombin cleavage site (yellow diamond); 2) block-ing the interaction of the new N-terminus with the second extracellular loop, e.g. SCH530348 and E55555 for PAR1, and YD3 for PAR4; and 3) blocking signal transduction by G proteinsafter receptor activation, e.g. pepducins.


1 nM thrombin, but required the addition of an anti-PAR4 antibody toabolish aggregation induced by 20 nM thrombin (Kahn et al., 1999).The thrombin-binding domain on PAR1 facilitates sub-nanomolar re-sponses to thrombin, and anti-PAR1 blocking antibodies have alsotargeted this region of the receptor. Specifically, IgG 9600 was pro-duced in rabbits immunized with a 15-residue peptide spanning thethrombin-binding domain of PAR1. It was shown to inhibitthrombin-induced aggregation and secretion in human plateletswith an IC50 of 20 μg/ml and 8 μg/ml, respectively, but had no effecton platelet activation induced by ADP, collagen, or a PAR1-AP (Cooket al., 1995). This approach provided the first in vivo evidence thatdisruption of the thrombin/PAR1 interaction is sufficient to inhibitthrombosis. Perhaps remarkably given the low potency of blockingantibodies and the presence of a distinct platelet thrombin receptor(PAR4) on non-human primate platelets, IgG 9600 (10 mg/kg, i.v.)exhibited significant antithrombotic activity in a Folts-typethrombosis model in African Green monkeys (Cook et al., 1995).

3.2.1.3. Small molecule antagonists. The peptidomimetic antagonistsand blocking antibodies provided major input into our understandingof PAR biology. However, as predicted, both types of antagonists expe-rience significant pharmacokinetic (and pharmacodynamic) limitationsin vivo, resulting in the subsequent development of small molecule an-tagonists. Two approaches were taken in the quest to develop selectiveand potent non-peptide PAR1 antagonists: rational design bymodifica-tion of PAR activating peptides and screening of chemical libraries.

FR17113 (methyl 2-(3-(4-chlorophenyl)-2-((2,4-dichlorobenzoyl)imino)-4-oxo-1,3-thiazolidin-5-ylidene)acetate)was one of the earliestsmall molecule PAR1 antagonists (Kato et al., 1999). FR17113 inhibitsPAR1-AP and thrombin-induced aggregation of human platelets withIC50 values of 0.15 μM and 0.29 μM respectively. However, FR17113was also shown to inhibit the proteolytic activity of thrombin directly(Kato et al., 1999). When used in vivo, administration of FR17113 toguinea pigs (0.1–3.2 mg/kg, s.c.) resulted in a dose-dependent inhibi-tion of ex vivo platelet aggregation and in vivo arterial thrombosis with-out impacting coagulation (Kato et al., 2003), suggesting limited impactof the drug's effects on thrombin activity contribute to its antithrombo-tic effects.

Vorapaxar (SCH-530348) is a small (MWof 591) inorganicmoleculederived from the structure of himbacine (Becker et al., 2009). Vorapaxar

inhibits PAR1 activation in a competitive manner by disrupting the in-teraction of the tethered ligand with the second intracellular loop(Ahn et al., 1999) and is currently in Phase 3 clinical trials as an antipla-telet agent for the prevention of arterial thrombosis (Barry et al., 2006).Precursors to vorapaxar that have been used experimentally includeSCH-79797 and SCH-203099, both of which are potent PAR1 antago-nists with marked selectivity (Ahn et al., 1999, 2000; Chackalamannilet al., 2001). SCH-79797 and SCH-203099 inhibit PAR1-AP-induced ag-gregation of human platelets with an IC50 of 70 and 45 nM respectively,and inhibit thrombin-induced platelet aggregation with an IC50 of 3 μMand 0.7 μM respectively. Despite a slightly inferior IC50, SCH-79797 hasbeen favoured for experimental use due to superior specificity (Ahn etal., 2000), and has become one of themost commonly used experimen-tal PAR1 antagonists. Importantly, SCH-79797 can be used in vivo in ro-dent models (Strande et al., 2007).

Further development of this series of PAR1 antagonists producedSCH-602539. Recently, a Folts-like model of thrombosis was used incynomolgus monkeys to show protection against thrombosis by SCH-602539 (0.1–1 mg/kg, i.v.) (Chintala et al., 2010). Synergywith cangrelor,a P2Y12 receptor antagonist currently in wide clinical use as an antiplate-let agent, was also observed (Chintala et al., 2010), suggesting that PAR1antagonists may be useful in the setting of combination antiplatelettherapies.

Atopaxar (E5555) is a small (MW of 609) inorganic molecule devel-oped as a competitive PAR1 antagonist for the treatment of thrombosis.Atopaxar is a bicyclic amidine derivative which appears to function bybinding at or near the tethered ligand binding site on the second extra-cellular loop of PAR1 (Matsuoka et al., 2004). Atopaxar inhibitsthrombin-induced aggregation of human platelets and other markersof platelet activationwith an IC50 of 64 nM(Kogushi et al., 2003) and in-hibits Ca2+ signalling in response to thrombin in cultured smoothmus-cle cells (Kai et al., 2007). Atopaxar has also been used in a number of invivo studies. Kai et al. (2007) reported that atopaxar (2 μg/kg, i.v.) pre-vented the thrombin-dependent cerebral vasospasm of the basilar ar-tery that occurs after haemorrhage in a rabbit model. Atopaxar hasrecently completed a phase 2 clinical trial as an antiplatelet agent andis seen as a therapeutic agent of high potential (see Section 4.1.2,below).

Recently, two additional classes of PAR1 antagonists have been de-scribed, although they have been characterised far less than those


discussed above. Firstly, a series of piperazine derivatives have beendeveloped (e.g. F16618; 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile and F16357; 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone) (Bocquet et al., 2009;Perez et al., 2009; Chieng-Yane et al., 2011; Letienne et al., 2010).These molecules inhibit PAR1-induced aggregation of human plate-lets and were shown to be active in the prevention of thrombosis inan arteriovensous shunt model of rat thrombosis when administeredeither orally or intravenously (Perez et al., 2009), although the ab-sence of PAR1 on rat platelets make these results difficult to interpret.Secondly, a number of triazolopyridazine derivatives have beenshown to exhibit IC50 values ranging from 0.098 to 24 μM againstPAR1 across a number of assays (Heinelt et al., 2009).

3.2.1.4. Pepducins. Classical inhibitors of GPCRs act by binding to theligand binding region of the extracellular surface of their target recep-tors. However, Covic et al. (2002a) developed a class of GPCR inhibi-tors, termed pepducins, which disrupt the intracellular interactionsof the receptor with its associated G protein, and used PARs as theirinitial receptor targets as a proof of principle. G proteins interactwith the third intracellular loop (i3) of GPCRs (Gilman, 1987;Okamoto et al., 1991; Luttrell et al., 1993). Covic et al. exploited thisby synthesising palmitolyated peptides with a sequence matchingthe i3 of PAR1, PAR2, PAR4, and the melanocortin-4 receptor. Thesepeptides insert into the cell membrane andmimic the G protein inter-acting regions of the GPCRs, thereby competing for free G proteinswithin the cell (Fig. 2). Pepducins targeting PARs, P1pal-12 (PAR1)and P4pal-10 (PAR4), are peptides designed to mimic the respectivei3 region of each receptor and are conjugated to an N-terminal palmi-tate (Kuliopulos & Covic, 2003). The hydrophobic nature of the palmi-tate anchors the peptide into the lipid membrane. Once inserted intothe lipid membrane, these peptides are proposed to bind G proteinsin place of receptors, thereby functioning as a G protein scavengerand preventing downstream signalling. P1pal-12 was shown to inhib-it PAR1-AP-induced aggregation of human platelets, but notaggregation induced by other agonists including PAR4-AP, U46619,ADP, collagen, or ristocetin (Covic et al., 2002b).

3.2.2. PAR2 antagonistsIn marked contrast to the significant number of PAR1 antagonists

developed, very little exists in the way of pharmacological inhibitorsof PARs 2–4. For PAR2, initial attempts at developing peptide-based an-tagonists used a similar approach to that used for early PAR1 antago-nists, but yielded potent PAR2 agonists instead of antagonists(Vergnolle et al., 1998). Yet two peptides, FSLLRY-NH2 and LSIGRL-NH2, have been reported to block activation of PAR2 by trypsin withIC50 values of 50 and 200 μM, respectively. However, neither compoundblocks PAR2-AP-induced PAR2 activation, even at concentrations ashigh as 400 μM (Al-Ani et al., 2002), suggesting that these peptidesmay not be functioning as direct receptor antagonists.

The most effective PAR2 antagonists were described by Kelso et al.(2006), where both a blocking antibody and a small molecule wereused to inhibit PAR2 for the treatment of joint inflammation in arthri-tis. The blocking antibody was a rabbit polyclonal antibody raisedagainst a 16-residue peptide sequence corresponding to the regionspanning the trypsin cleavage site of the rat PAR2. Administration ofthe blocking antibody inhibited the cleavage of PAR2 by trypsin andattenuated inflammation in a mouse model of rheumatoid arthritisinitiated by intra-articular injection of a mixture of the irritants carra-geenan and kaolin (Kelso et al., 2006). Similar results were obtainedwhen the small molecule PAR2 antagonist, ENMD-1068 (N1-3-methylbutyryl- N4-6-aminohexanoyl-piperazine) was used in thesame model. ENMD-1068 is a disubstituted piperazine derivativewhich has been shown to be an effective PAR2 antagonist that inhib-ited PAR2-AP-induced calcium signalling in a concentration-dependent manner in Lewis lung carcinoma cells, albeit at very high

concentrations (5 mM). Despite the high concentration required,the compound did not inhibit the proteolytic activity of trypsin, anddemonstrated selectivity for PAR2 (Kelso et al., 2006).

3.2.3. PAR3 antagonistsIn mice, PAR3 does not transduce a signal by itself and is considered

a co-factor for PAR4 activation. In humans, physiological functions forPAR3 have remained largely undetermined. As a consequence, noPAR3 antagonists have been developed to date. This may change asfunctions for PAR3 become known. Recent observations indicate thatthrombin enhances its interaction with PAR3 following stimulation byinflammatory cytokines such as TNF-α or IL-1β (Beri, 2006). Althoughthe consequences of this interaction remain undetermined, it hasbeen shown to induce GM-CSF production by lung fibroblasts to a sim-ilar level to that observed in asthma. These findings may stimulate thedevelopment of PAR3 antagonists for the treatment of asthma andother inflammatory conditions (Beri, 2006).

3.2.4. PAR4 antagonists

3.2.4.1. Peptidomimetics. As with the development of early PAR1 an-tagonists, a peptidomimetic approach was also taken to identify po-tential antagonists of PAR4. In order to study PAR4 functions in vivo,Hollenberg and Saifeddine (2001) generated a number of peptide an-alogues based on the human, mouse, and rat tethered ligand se-quences of PAR4. Similar to the BMS series of PAR1 antagonists, twopeptide sequences, mimicking the murine tethered ligand sequenceof PAR4 were modified by the addition of a trans-cinnamoyl (tc)group (tc-YPGKF-NH2 and tc-AYPGKF-NH2). Both tc-YPGKF-NH2 andtc-AYPGKF-NH2 inhibited PAR4 in rodents, with IC50 values of 64and 380 μM respectively as assessed by PAR4-AP-induced relaxationof isolated aorta (Hollenberg et al., 2004). tc-YPGKF-NH2 (400 μM)abolished PAR4-AP-induced aggregation of rat platelets and signifi-cantly reduced platelet aggregation induced by thrombin, but didnot affect aggregation induced by ADP (Hollenberg & Saifeddine,2001). tcYPGKF-NH2 has also been reported to inhibit thrombin-induced aggregation of human platelets as well thrombin-induced re-lease of VGEF and endostatin from human platelets (Ma et al., 2005),although whether or not tcYPGKF-NH2 is effective as an inhibitor ofhuman PAR4 remains controversial.

3.2.4.2. Small molecule antagonists. In order to identify small moleculePAR4 antagonists, various heterocyclic structures were screened fortheir ability to inhibit platelet aggregation, resulting in the identifica-tion of the indazole derivative YD-3 [1-benzyl-3(ethoxycarbonylphe-nyl)-indazole] (Wu et al., 2000). YD-3 was reported to selectivelyinhibit thrombin-induced aggregation of rabbit platelets with anIC50 of 28.3 μM. In human platelets, YD-3 partially inhibited plateletaggregation induced by low concentrations of thrombin (≤0.5 nM),but not higher concentrations (≥1 nM), and had little or no effecton platelet aggregation induced by platelet-activating factor, collagen,arachidonic acid or the thromboxane A2 mimetic U46619 (Wu et al.,2002, 2003). YD-3 was also shown to inhibit thrombin-induced Ras/ERK signalling in vascular smooth muscle cells in vitro leading to im-paired cell proliferation. Further, when orally administered to rats,YD-3 attenuated neointimal thickening and restenosis in balloon an-gioplasty injury model (Peng et al., 2004), suggesting YD-3 has suffi-cient bioavailability for use in in vivo studies. More recent studieshave focussed on increasing the efficacy of YD-3, with the aim ofusing these compounds to establish PAR4 as a therapeutic target foranti-angiogenic agents (Huang et al., 2006; Chen et al., 2008a,2008b). Several derivatives with superior potency to YD-3 havebeen tested in assays of neo-vascular formation and have beenshown to exhibit marked effects (Huang et al., 2006).


3.2.4.3. Pepducins. Analogous to the anti-PAR1 pepducin (see above),an anti-PAR4 pepducin was identified, named P4pal-10 and consist-ing of the sequence pal-SGRRYGHALR-NH2 (Covic et al., 2002a,2002b). P4pal-10 has been shown to inhibit PAR4-AP-induced aggre-gation of human platelets with an IC50 of ~1 μM. Similar effects wereobserved in mouse platelets (Covic et al., 2002a). P4pal-10 displayedsome level of cross-reactivity against PAR1, inhibiting platelet aggre-gation induced by PAR1-AP, although it did not affect aggregation inresponse to other agonists including ADP, U46619, collagen, or risto-cetin (Covic et al., 2002b). P4pal-10 also appears to function in vivo,as the treatment of mice with P4pal-10 increased tail bleeding time(Covic et al., 2002b). However, questions remain regarding the spec-ificity of P4pal-10, with reports of significant inhibitory effects byP4pal-10 on both collagen- and thromboxane A2-induced platelet ac-tivation (Stampfuss et al., 2003). In order to overcome the lack ofspecificity of P4pal-10, another pepducin, P4pal-i1, was synthesisedbased on the first intracellular loop of the receptor and was shownto inhibit PAR4-AP-induced platelet aggregation without effectingPAR1-AP-induced aggregation (Leger et al., 2006).

4. Therapeutic potential of PARs in vascular disease

4.1. Platelet PARs as targets for antithrombotics

Antiplatelet drugs are the primary pharmacological strategy forthe prevention of arterial thrombosis. However none of the currentlyused antiplatelet therapies affords a sufficiently potent, safe, and oral-ly active strategy for the prevention of arterial thrombosis, with eachof the most commonly used existing therapies—aspirin, clopidogrel,and glycoprotein (GP) IIb–IIIa antagonists—suffering from one ormore important limitations, including poor efficacy, significant riskof bleeding, drug resistance, or the need for parenteral administra-tion. The potential therapeutic benefits of inhibiting platelet PARsover these existing strategies are highlighted by the followingconsiderations:

1) Thrombin is the most potent known activator of platelets;2) In the setting of arterial thrombosis, the activation of platelets by

thrombin is important for thrombus growth. Of note, the existingantiplatelet drugs, aspirin and clopidogrel, inhibit distinct, but lesspowerful, mechanisms contributing to thrombus growth.

3) Drugs which inhibit either the generation or activity of thrombin(i.e. anticoagulants) are effective at inhibiting thrombus formation.

On this last point, although existing anticoagulants are capable of ef-fectively blocking thrombin function, one potential advantage of PARantagonism over thrombin inhibition is that other effects of thrombinmight be spared. With respect to haemostasis, these include fibrin for-mation as well as activation of coagulation factors V, VIII, XIII, IX, XIand TAFI. Therefore selective inhibition of thrombin-induced plateletactivation using PAR antagonists is predicted to provide a safer pharma-ceutical option (less bleeding) than existing anticoagulants, by sparingsome of the haemostatic functions of thrombin. To address this, recentstudies examined platelet activation and fibrin formation concurrentlyin a thrombosis model in PAR4-deficient mice. Despite marked reduc-tions in platelet accumulation and activation in thrombi of PAR4-deficient mice when compared with thrombi formed in control mice,no differences in the kinetics or quantity of fibrin accumulation wereobserved (Vandendries et al., 2007), suggesting that interfering withPAR-mediated platelet activation does not affect thrombin-dependentcoagulation at least in the mouse. These findings are likely of relevanceto the clinical setting because current therapies for the prevention of ar-terial thrombosis are either antiplatelet agents, which prevent plateletactivation, or anticoagulants, which impair fibrin formation by inhibit-ing the production or activity of thrombin. However, arterial thrombiare frequently insensitive to anticoagulant therapies, such that combi-nation anticoagulant/antiplatelet therapy impairs haemostasis without

providing additional antithrombotic effects. For example, numerousclinical studies (Becker, 2002; Fiore et al., 2002; Hurlen et al., 2002;Andreotti et al., 2006) have shown that concurrent administration ofthe most commonly used antiplatelet and anticoagulant, aspirin andwarfarin respectively, does not significantly reduce rates of myocardialinfarction yet causes a significant increase in major haemorrhage whencompared with administration of aspirin alone. As a consequence, thecurrent treatment recommendation for prevention of primary or sec-ondary cardiovascular events in patients with acute coronary syndromeis antiplatelet therapy alone (Becker et al., 2008). However the mostcommonly used antiplatelet agents, aspirin and clopidogrel, preventonly ~15 and 17% of lethal cardiovascular events respectively and areonly marginally more effective in combination than when either isused alone (CAPRIE Steering Committee, 1996). Therefore, novel anti-platelet approaches are required to meet the significant clinical needfor the safe and effective prevention and treatment of cardiovasculardisease, and PAR antagonists represent one group of the most likelycandidates for this purpose.

The potential therapeutic benefits of inhibiting the major plateletPAR, PAR1, have been extensively evaluated in pre-clinical studieswhere a number of PAR1 antagonists have displayed an ability to pro-vide protection against arterial thrombosis without causing significantbleeding liabilities. To date, two distinct PAR1 antagonists, vorapaxar(SCH-530348) and atopaxar (E-5555), have undergone significant de-velopmentwith the recent completion of phase 2 trials and progressionto phase 3.

4.1.1. Vorapaxar (SCH-530348)Vorapaxar is a high affinity, orally active, low molecular-weight,

non-peptide, competitive PAR1 antagonist (see Section 3.2.1.3, above).Following experiments conducted in non-human primates, whichshowed that the drugwas highly efficacious in the prevention of throm-bosis without significant bleeding side effects (Chintala et al., 2010), itwas fast tracked into clinical trials for the secondary prevention ofacute ischemic events. In human, vorapaxar has a Ki of 2.7 nM, andafter oral administration is rapidly absorbed, exhibits high bioavailabil-ity (86%), and has a terminal half-life of 126–269 h (Becker et al., 2009).Initial clinical trials in healthy volunteers showed that the drugwaswelltolerated and had long-lasting PAR1-inhibitory effects, with a singleloading dose ranging from 5 to 40 mg inhibiting PAR1-AP-inducedplatelet aggregation by greater than 90% for a period longer than 72 h.The drug is absorbed rapidly: a single dose of either 20 or 40 mg caused>90% inhibition of platelet aggregation after 1 hour (Kosoglou & Cutler,2005). In addition, administration of vorapaxar at 1, 3, or 5 mg per dayfor 28 days resulted in inhibition of platelet aggregation induced byPAR1-AP of greater than 90% in all volunteers by day 7 (Kosoglou etal., 2005). Pre-clinical and early clinical studies also reported no signif-icant increases in bleeding or any impact on coagulation (Kosoglou &Cutler, 2005; Kosoglou et al., 2005; Chintala et al., 2010).

Phase 2 studies of vorapaxar were performed in patients undergo-ing non-urgent percutaneous coronary intervention who received asingle loading dose of vorapaxar (10, 20, or 40 mg) followed by main-tenance daily doses (0.5, 1, or 2.5 mg) for 60 days. Vorapaxar waswell tolerated and caused no increase in major bleeding events,even when administered in combination with the existing antiplate-let agents aspirin and/or clopidogrel. Each of the loading doses of vor-apaxar inhibited PAR1-AP-induced platelet aggregation andmaintenance dosing prolonged this inhibition (≥80%) in most pa-tients at 30 and 60 days. These results led to the design of a multi-centre, randomised, double-blind, placebo controlled phase 3 trial(Morrow et al., 2009; TRACER Steering Committees, 2009).

The TRA•CER (thrombin receptor antagonist for clinical event re-duction in acute coronary syndrome) trial is designed to test theantithrombotic effects of vorapaxar when administered in combina-tion with the current standard of care antiplatelet agents (aspirinand/or clopidogrel). The incidence of the composite of cardiovascular


death, myocardial infarction, stroke, recurrent ischaemia with rehos-pitalisation, and urgent coronary revascularisation are being exam-ined, with safety assessed by monitoring bleeding events. Asubsection of the TRA•CER trial, named TRA 2°P-TIMI 50, is evaluatingthe efficacy and safety of vorapaxar for long-term treatment of pa-tients with established atherosclerotic diseases (Morrow et al.,2009). However, the TRA•CER trial was recently stopped because in-creased intracranial bleeding was detected in patients receiving vora-paxar who had a prior history of stroke. (The TRA 2°P-TIMI 50 trial iscontinuing but has been modified to exclude patients with priorstroke.) The specific cause of bleeding in these trials remains un-known. However, it should be noted that vorapaxar was added tostandard of care antiplatelet therapy in these studies that mostoften included both clopidogrel and aspirin. It is not yet knownwhether bleeding clustered in patients receiving triple antiplatelettherapy, or whether vorapaxar provided any incremental protectionagainst thrombosis when added to one or more existing antiplateletdrugs. Thus, there remains much to learn regarding how to best ma-nipulate thrombin signalling in platelets for the safe and effective pre-vention of arterial thrombosis. Furthermore, little is known about thepotential interactions of PAR antagonists with existing antiplateletagents.

4.1.2. Atopaxar (E5555)Atopaxar is another PAR1 antagonist being trialled for the treatment

and prevention of arterial thrombosis. Atopaxar, is a low molecular-weight (MW 609), competitive antagonist of PAR1 that has beenshown to inhibit PAR1-dependent aggregations of human plateletswhen used either in vitro and in vivo (see Section 3.2.1.3, above). Invivo, it does not significantly increase bleeding (Serebruany et al.,2009). The safety of atopaxar was evaluated in a small phase 2 trial(J-LANCELOT; Japanese-Lesson from Antagonising the Cellular Effectsof Thrombin) (Goto et al., 2010). J-LANCELOT was a multi-centre,randomised, double-blind, placebo controlled study that assessed thetolerability and safety of atopaxar in addition to standard antiplatelettherapy in Japanese patients with acute coronary syndrome or high-risk coronary artery disease. Patients were treated with atopaxar (50,100, or 200 mg) once daily for 12 or 24 weeks. Greater than 90% inhibi-tion of PAR1-AP-induced platelet aggregation was observed in patientstreatedwith the higher doses (100 or 200 mg),while 20–60% inhibitionwas observed in patients treated with 50 mg atopaxar. Atopaxar wasshown to be generally well tolerated, with no increase in bleedingreported other than a small increase in minor bleeding events not re-quiring medical intervention (e.g. bleeding from the site of catheterisa-tion) noted at the higher doses as well as some abnormalities in liverfunction. Although the study was not powered to demonstrate efficacy,a 50% numerical reduction in major adverse cardiac events was ob-served in patients receiving atopaxar compared with the placebogroup (Goto et al., 2010). These results showed that atopaxar has thepotential to be an effective treatment of thrombosis, and led to alarge-scalemulti-centre phase 3 trial for the evaluation of the therapeu-tic efficacy of atopaxar in the treatment and/or prevention of cardiovas-cular diseases. Whether or not atopaxar will impact haemostasis whenused in combination with other antiplatelet drugs, as observed withvorapaxar, is of great interest and remains to be seen.

In sum, the use of PAR1 antagonists appears an attractive strategyfor the primary and/or secondary prevention of arterial thrombosis inat-risk patients. However, as is always the case with the use of suchantiplatelet approaches, the potential impact on haemostasis is of para-mount importance and a key clinical limitation. To this end, safety andefficacy information from the various patient and treatment subgroupsin the ongoing Phase 3 trials will provide valuable clues on how to bestuse PAR1 antagonists in these clinical settings. For example, the com-bined use of aspirin and clopidogrel provides only a marginal increasein antithrombotic efficacy over the use of either antiplatelet drugalone (CAPRIE Steering Committee, 1996; Bhatt et al., 2006). Will the

use of a PAR1 antagonist in combination with one of either aspirin orclopidogrel provide a more potent antithrombotic setting than this cur-rent standard of care? Will PAR1 antagonists be useful as antiplateletdrugs in the estimated 5 to 20% of patients believed to be clopidogrel‘non-responders’ (Gurbel et al., 2003; Muller et al., 2003)? In additionto these issues, the prevalence of any side effects of chronic PAR1 antag-onism remains unknown. Although limited, if any, off target effects ofeither vorapaxar or atopaxar have been reported in the clinical studiesto date, the fact that PAR1 is widely expressed and performs a numberof important functions in the body suggests the potential for such ef-fects do exist. Notwithstanding the differences in PAR expression andfunction between rodents and humans, clues toward potential side ef-fects of chronic PAR1 antagonist administrationmay come from studiesof PAR1−/−mice, which have been reported to exhibit a range of phe-notypes, including impaired post-ischaemic neovasularisation(Thiyagarajan et al., 2008), defective leukocyte recruitment to sites ofinjury (Chen et al., 2008a, 2008b), and even impaired learning andmemory function (Almonte et al., 2007).

4.2. Vascular PARs as targets for anti-inflammatories?

The therapeutic use of PAR antagonists aimed at preventing acti-vation of vascular PARS is considerably more speculative than theproposed use of these agents as antithrombotics. However, giventhe role of thrombin in inflammatory processes associated withthrombosis and atherosclerosis (Griendling & Alexander, 1996;McNamara et al., 1996; Libby, 1998), it is not surprising that activa-tion of endothelial and smooth muscle cell PAR1 has pro-inflammatory effects (Cirino et al., 1996). For example, thrombin ac-cumulates at sites of vascular injury (Hatton et al., 1989; Gallo et al.,1998; Gandossi et al., 2000; Stoop et al., 2000) causing edema via en-dothelial cell retraction (Malik & Fenton, 1992) and promoting in-flammatory cell adhesion and infiltration (Rahman et al., 1999).These effects are mediated in large part by endothelial PAR1. Disrup-tion of the endothelial barrier and subsequent infiltration of inflam-matory cells are important processes in the initiation of vascularinflammatory responses. In addition to these early inflammatory pro-cesses, PAR1 expression is observed in neointimal smooth musclecells and is markedly increased in medial smooth muscle in athero-sclerosis (Stoop et al., 2000), hypertension (Capers IV et al., 1997)and after balloon-catheter injury (Wilcox et al., 1994). This increasedexpression of smooth muscle cell PAR1 upon disruption of the endo-thelium manifests as an increased mitogenic and contractile effect ofthe accumulating thrombin (Capers IV et al., 1997). In support ofthese studies, inhibition of PAR1 reduces neointimal formation afterballoon-catheter injury of porcine coronary artery (Gallo et al.,1998). Vascular injury-induced neointimal formation is also impairedin PAR1 knockout mice (Cheung et al., 1999). In contrast with PAR1,physiological roles and therefore the therapeutic potential of theother vascular PARs are virtually unknown. Several findings reportingcommon functions of PAR1 and PAR2 and that the generally regardedpro-inflammatory enzyme, mast cell tryptase, can activate PAR2(Corvera et al., 1997; Fox et al., 1997; Mirza et al., 1997; Molino etal., 1997a; Corvera et al., 1999; Steinhoff et al., 2000) have led tosuggestions that, like PAR1, vascular endothelial cell PAR2 areinvolved in pro-inflammatory responses (Bohm et al., 1998; Dery etal., 1998). For example, PAR2 activation stimulates mitogenic re-sponses (Mirza et al., 1996) and induces tissue factor expressionand von Willebrand factor release (Storck et al., 1996; Langer et al.,1999) in cultured HUVEC. Also, injection of PAR2-activating peptidesinto rats increases vascular permeability and leads to edema in thehindpaw (Kawabata et al., 1998; Vergnolle et al., 1999a, 1999b) andinduces leukocyte adhesion and recruitment (Vergnolle et al.,1999a, 1999b). Furthermore, as observed for PAR1, the expressionof PAR2 in medial and neointimal smooth muscle cells in rat carotidarteries is markedly increased following balloon injury (Damiano et


al., 1999). Finally, endothelial cell PAR2 upregulation in response toinflammatory stimuli (Nystedt et al., 1996; Cicala et al., 1999) hasbeen taken to indicate that PAR2 are involved in vascular inflammato-ry diseases (Dery et al., 1998). However, as argued by Cocks andMoffatt (2000), the opposite could apply: upregulation of endothelialcell PAR2 may in fact increase the protective barrier function of theendothelial cell layer and act to limit, for example, thrombosis in in-flammatory conditions such as sepsis, hypertension and atherosclero-sis. On this point, it is interesting to note that in hypertension, whilemost other endothelium-dependent vasodilator function is dimin-ished (Vanhoutte & Boulanger, 1995) and there is an increased poten-tial for thrombosis (Tomiyama et al., 1998), PAR2-mediatedvasodilatation of the rat basilar artery in vivo is preserved (Sobey etal., 1999). Therefore, whether PAR1 or PAR2 antagonists are likelyto be useful as anti-inflammatory agents requires further attention.

5. Concluding remarks

In the time since the initial discovery of PARs 20 years ago, rapidprogress has been made regarding the function of these unique GPCRsin physiology and pathologies. The importance of PARs in mediatingthrombin-induced platelet activation in the setting of arterial thrombo-sis led to major efforts in the development of PAR antagonists, most no-tably for PAR1. That two of these PAR1 antagonists arewell into Phase 3clinical trials as antiplatelet agents for the prevention of arterial throm-bosis shows the rapid progression in PAR biology over the last two de-cades. Despite this, many questions remain regarding PAR biology. Forexample, given the well documented and extensive effects that PAR ac-tivation is able to induce across a number of systems in the body, thelong-term effects of PAR1 antagonism may be significant and wideranging and there is virtually no information available on this. Further-more, comparatively little is known of the functions of PARs 2–4 in thevascular system (and beyond). What is the function of the so-called“second thrombin receptor”, PAR4, on human platelets? Does PAR4simply perform an overlapping, and potentially redundant, “backup”function for thrombin-induced platelet activation, or does it performdistinct functions? Andwhat impact will this have on the use and likelysuccess of PAR1 antagonists? Although studies in PAR-deficient micehave proven valuable in this regard, it is clear that more well character-ized and accessible antagonists for these receptors are required in orderto address questions such as these, particularly on human tissues.

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physiology, pharmacology, and therapeutic potential of protease-activated receptors in vascular...

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