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Protease-Activated Receptors Xu Han, Emma G. Bouck, Elizabeth R. Zunica, Amal Arachiche and Marvin T. Nieman Department of Pharmacology, Case Western Reserve University, Cleveland, OH, United States

DISCOVERY OF PROTEASE-ACTIVATED RECEPTORS (PARs) 243 GENERAL FACTS ABOUT PARs 243 Genomic Organization and Evolution of PARs 243 Protease-Activated Receptor-1 (PAR1) 243 Protease-Activated Receptor-3 (PAR3) 244 Protease-Activated Receptor-4 (PAR4) 244 EXPRESSION OF PARs ACROSS SPECIES 244 ACTIVATION OF PARs BY THROMBIN 244 Thrombin Recognition of PAR1 244 Thrombin Recognition of PAR4 246 PHYSICAL INTERACTIONS BETWEEN PARs 247 THROMBIN SIGNALING IN HUMAN PLATELETS 248 PAR1- AND PAR4-SPECIFIC SIGNALING 248 TERMINATION OF PAR SIGNALING 250 ACTIVATION OF PARs BY PROTEASES OTHER THAN THROMBIN 250 PHARMACOLOGICALLY BLOCKING PAR SIGNALING 250 Protease-Activated Receptor 1 Antagonists 250 Protease-Activated Receptor 4 Antagonists 251 STRUCTURAL STUDIES ON PARs 252 POLYMORPHISMS AND SEQUENCE VARIANTS 252 FUTURE DIRECTIONS 253 REFERENCES 253

DISCOVERY OF PROTEASE-ACTIVATED RECEPTORS (PARs) Thrombin is the most potent platelet agonist and consequently has important roles in hemostasis and thrombosis. In the late 1980s, several laboratories focused their work on solving the enigma of how thrombin, a serine protease, activates platelets. Originally, there were two proposed receptors for thrombin on platelets: glycoprotein Ib (GPIb) and glycoprotein V (GPV). However, neither GPV nor GPIb fully explain how platelets respond to thrombin. First, platelets from patients with Bernard-Soulier syndrome (BSS), deficient in both GPIb and GPV, still respond to thrombin, although to a lesser degree.1 Second, there is no correlation between the cleavage of GPV by thrombin and the kinetics of platelet activation.2 The search continued until Coughlin and colleagues used an expression cloning approach to identify the thrombin receptor, now known as protease-activated receptor 1 (PAR1).3 The cloning of PAR1 led to the identification of the three other family members (PARs 2–4).4–7 PARs are G-protein coupled receptors (GPCRs) that have a unique activation mechanism in which the N-terminus is cleaved by the activating protease to generate a tethered ligand Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00013-8 Copyright © 2019 Elsevier Inc. All rights reserved.

(Fig. 13.1A). The newly exposed ligand binds intramolecularly to the receptor, inducing a conformational change that initiates G-protein mediated signal transduction.8 Experimentally, PARs can be selectively activated independent of proteolysis with synthetic peptides, TRAPs (thrombin receptor activating peptides), which correspond to the tethered ligand sequence (Fig. 13.1B). PARs can couple to multiple G-proteins, leading to diverse intracellular signaling networks depending on the cellular context.9–11 In this chapter, we describe the expression and activation mechanism of PAR1, PAR3, and PAR4 by thrombin and their roles in platelet signaling. PAR2 is not expressed on platelets and is therefore not discussed.

GENERAL FACTS ABOUT PARs Genomic Organization and Evolution of PARs The genomic organization and function of PARs has been conserved in vertebrates from teleost to mammals.12,13 Phylogenetic tree analysis of 169 functional PAR genes shows that the four members of the PAR family have evolved from four separate ancestors.12 In most species, the genes encoding PAR1–3 (F2R, F2RL1, and F2RL2, respectively) are clustered on the same chromosome, whereas as the gene for PAR4 (F2RL3) is at a separate locus.12,14–16 The human F2R gene is located on chromosome 5 (5q13) and contains two exons separated by an intron of approximately 15 kb.14 Human F2RL2 colocalizes with F2R on chromosome 5 and has a similar genomic organization; one small exon and one large exon. In contrast to F2R, the two exons of F2RL2 are separated by a small intron of approximately 4.5 kb.14 Human F2RL3 maps to chromosome 19p12. The F2RL3 gene is also organized into two exons, but the intron separating the two exons is small (
0.25 kb) compared to other PARs.14

Protease-Activated Receptor-1 (PAR1) Protease-activated receptor 1 (PAR1) was the first family member to be identified and was cloned in 1991.3 PAR1 is widely expressed in a variety of tissues, but its expression on platelets is limited to primates and several other species (see section “Expression of PARs Across Species”). Human platelets have 1500–2000 copies of PAR1 on the surface.17 PAR1 is comprised of 425 amino acids, with a 99-amino acid N-terminal exodomain and a 51 amino acid C-terminal tail (Fig. 13.2).3 The Nterminus has two thrombin recognition sites, the cleavage site at arginine 41 (…LDPR41/SFLLRN…), and the hirudin-like sequence (K51YEPF).18,19 The synthetic peptide, SFLLRN, mimics the first six amino acids of the exposed amino terminal tethered ligand, and can activate PAR1 independent of cleavage (Fig. 13.1B).20 SFLLRN will also activate PAR2, so the peptide TFLLRN is used to achieve specific activation in cells coexpressing both PAR1 and PAR2, such as endothelial cells.21–23 PAR1 is also cleaved by proteases other than thrombin at alternative sites to generate a panel of tethered ligands (see section “Activation of PARs by Proteases Other Than Thrombin”).11

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cloned in 1998.7,31 PAR4 is expressed in platelets and a number of other human tissues such as the lungs, pancreas, thyroid, testes, and small intestine, but not in the brain, kidneys, spinal cord, and peripheral blood leukocytes.7,32 PAR4 contains 385 amino acids and has 27% and 30% sequence identity with PAR1 and PAR3, respectively.7 Thrombin activates PAR4 by the cleavage of the N-terminal of PAR4 at arginine 47 (…LPAPR47/GYPGQV…) (Fig. 13.2). In contrast to PAR1 and PAR3, PAR4 does not have a hirudin-like site and interacts differently with thrombin, which is described in detail below.7 The synthetic peptide (GYPGQV) corresponding to the unmasked amino terminus of PAR4 also activates the receptor, but with low potency.33,34 Subsequent peptide library screens demonstrated that the peptide AYPGKF is more potent and is commonly used experimentally.33,34

EXPRESSION OF PARs ACROSS SPECIES

Fig. 13.1 General activation mechanism of protease-activated receptors. (A) Physiologically, PARs are activated following cleavage by the activating protease. The newly exposed N-terminus serves as a tethered ligand, interacts with the receptor, and triggers a global structural rearrangement to induce PAR-mediated downstream signaling. (B) PARs can also be activated by synthetic peptides that mimic the tethered ligand sequences.

Protease-Activated Receptor-3 (PAR3) Protease-activated receptor-3 (PAR3) was cloned in 1997, becoming the second thrombin receptor and third member of the PAR family.4 PAR3 is expressed in a range of tissues and its platelet expression varies across species. Human platelets express very low levels (
150–200 copies) of PAR3, whereas it is highly expressed on mouse platelets.15 PAR3 consists of 373 amino acids and shares 27% amino acid sequence identity with PAR1 (Fig. 13.2). Similar to PAR1, the N-terminal exodomain of PAR3 has two thrombin recognition sites; the cleavage site at lysine 38 (…LPIK38/TFRGAP…) and a hirudin-like sequence (F48EEFP) which binds thrombin’s exosite I (Fig. 13.2).4,24,25 Notably, the C-terminal tail of PAR3 is significantly shorter than that of PAR1 (13 vs. 51 amino acids).4 The shorter C-terminal tail likely affects the coupling of PAR3 to intracellular signaling machinery. Studies with thrombin and the PAR3 activating peptide, TFRGAP, have not identified a direct signaling function for PAR3 in human platelets, mouse platelets, or COS7 cells transfected with human PAR3.4,26,27 The emerging role of PAR3 is to modulate signaling from other PARs.9,28,29 In mouse platelets, for example, PAR3 regulates the sensitivity and response of PAR4 at both low and high thrombin concentrations.27,30 Similar to PAR1, PAR3 is also cleaved at alternative sites by other proteases.29

Protease-Activated Receptor-4 (PAR4) Protease-activated receptor 4 (PAR4) was the fourth member of PAR family and the third thrombin receptor identified and

Animal models are essential to examining platelet function in vivo and have revealed important differences in how platelets across species respond to thrombin.35,36 The major contributor to species-specific responses to thrombin is the PAR expression profile on platelets (Table 13.1). Since mice express PAR3 and PAR4 on their platelets, but not PAR1 (Table 13.1), mice have limited utility as a preclinical model for developing PAR antagonists.37 This prompted the effort to develop a mouse model with “humanized” PAR platelet expression; however, to date, these efforts have been unsuccessful.38,39 Guinea pigs express PAR1 and PAR4 and have been used as an alternative to mice.40 However, they also express PAR3, which complicates the interpretation of thrombin signaling studies.41 Imposing an additional limitation on rodent models, PAR antagonists that are effective for human PARs often do not cross-react to rodent PARs, despite high sequence homology between species. For example, platelets from guinea pigs respond 10-fold less to the human TRAP (SFLLRN) in aggregation experiments, and the PAR1 inhibitor RWJ-58259 showed no significant antithrombotic effect in guinea pig thrombosis models in vivo.41,42 This has proven to be a common theme. The FDA-approved PAR1 antagonist, vorapaxar, does not effectively inhibit mouse PAR1.43 Further, the PAR4 antagonist BMS-986120 does not efficiently antagonize mouse, rat, or guinea pig PAR4.44 Although more costly and inconvenient, recent trials with a PAR4 antagonist have successfully circumvented this issue by using cynomolgus monkeys, which express PAR1 and PAR4.44

ACTIVATION OF PARs BY THROMBIN In addition to the active site, thrombin has two exosites that assist substrate recognition and proteolytic efficiency, which are allosterically linked to the active site.45,46 For example, when a substrate engages exosite I, the active site is held in the open “protease” conformation, thereby aiding proteolysis of the substrate in the active site.46 Like most thrombin substrates, PARs use a combination of interactions at exosite I and the active site. The extent to which PARs engage thrombin’s exosite I, the active site, or both dictates the rate of proteolysis of the N-terminus to expose the tethered ligand and initiating signaling.47

Thrombin Recognition of PAR1 PAR1 is an excellent thrombin substrate. Initial studies to determine the kinetic parameters of thrombin cleavage of PAR1 used peptides or purified recombinant exodomains, determining a Km of 10–28 μM and a kcat of 58–340 s1.19,48,49 These parameters are defined by the interactions between a hirudin-like sequence (D50KYPEK) on PAR1 that binds

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Fig. 13.2 Features of protease-activated receptors. PAR1, PAR3, and PAR4 sequences are shown as snake diagrams. The enzyme cleavage sites, polymorphisms, motifs, and residues that are critical for their physiological functions are highlighted.

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TABLE 13.1 PAR Expression in Platelets From Different Species PAR Expression Species

PAR1

Human Mouse Rat Rabbit Guinea pig Baboon Monkey

X

X X

PAR3 X X X X

PAR4 X X X X X X X

thrombin’s exosite I (Fig. 13.3).18,19,48 Mutations in the hirudin-like sequence increase the Km fourfold and decrease the kcat fourfold.48 We will use the nomenclature for enzyme substrates of Schechter where P1 is the cleavage site and P2, P3, etc. refer to each subsequent amino acid moving towards the N-terminus.50 Mutations in thrombin’s exosite I have the same impact on the kinetic parameters as mutating the hirudin-like site.24,51 In contrast, mutations around the thrombin cleavage site do not dramatically affect the Km of PAR1 thrombin cleavage, since the exosite I interaction is preserved. Instead, the kcat is reduced sevenfold when Leu38 at P4 is mutated to alanine and twofold when Pro40 at P2 is mutated (Fig. 13.3).49 The first structural studies examining the interaction between PAR1 exodomain peptides and thrombin revealed contacts of thrombin’s active site with P4-P1 of PAR1 (L38DPR) and thrombin’s exosite I with the hirudin-like sequence of PAR1 (K51YEPF).52 These results were corroborated with models generated from the data sets in Mathews et al. (PDB ID codes 1NRS and 1NRN) and subsequent structural studies that show a single PAR1 peptide interacting simultaneously with active site and exosite I (Fig. 13.3).53,54 The original structural studies by Mathews et al. provided the first detailed map of molecular interactions at the thrombin active site with the L38DPR sequence from PAR1 where Leu38 occupies the aryl-binding pocket formed by Ile174 and Trp215, as predicted by Bode et al.52,55 The exosite I interactions are

important for inducing conformational changes in the Ala190-Gly197 region of thrombin, prompting the Glu192 side chain to become disordered and accommodate the negatively charged Asp38 at P3 for PAR1.52,56 Although the charged residue is not ideal for thrombin substrates, it may facilitate efficient turnover of PAR1.49

Thrombin Recognition of PAR4 PAR4 has been characterized as a redundant, low-affinity backup thrombin receptor.57 This is a misnomer as the affinity (Km) for thrombin cleaving PAR4 is 56–61 μM and only around twofold greater than PAR1.49,58 The major difference is in the kcat, which is 17–18 s1 and four- to 20-fold lower than PAR1, depending on the study.49,58 The larger difference in kcat reflects the lack of a hirudin-like sequence in PAR4 and the resultant inability to allosterically modulate thrombin’s active site (Fig. 13.3).7 PAR4 primarily interacts at thrombin’s active site via amino acids Leu43 at P5, Pro44 at P4, and Pro46 at P2.49,58,59 These functional data are supported by structural and modeling studies. Ayala et al. showed via molecular modeling using peptides that Leu43 is important for interacting with thrombin residues Leu99, Ile174, and Trp215.24 However, NMR studies with PAR4 peptides by Cleary et al. demonstrate that this leucine is flexible and can interact with thrombin as well as Pro44 or Pro46 of PAR4 to stabilize secondary structure at the thrombin cleavage site.59 Even though PAR4 primarily interacts with thrombin at the cleavage site, individual point mutations at Leu43, Pro44, or Pro46 do not influence thrombin binding (i.e., do not affect the Km).49 In contrast, the kcat is reduced, indicating that PAR4 also has extended contacts with thrombin which minimize the influence of the individual amino acids at the cleavage site.49 These additional contacts are known to be mediated by the anionic cluster in the PAR4 exodomain (Asp57, Asp59, Glu62, Asp65) since mutating these residues decreases the Km of thrombin cleavage fourfold from 56 to 208 μM, and decreases the rate of PAR4 cleavage on cells.58,60 Structural studies show that PAR4’s anionic region

Fig. 13.3 Thrombin-PAR1 interactions. Thrombin interacts with PAR1 (left) and PAR4 (right) by different mechanisms as determined by co-crystallization studies with thrombin and PAR peptides. PAR1 contains a hirudin-like sequence that interacts with exosite I of the thrombin and locks the enzyme in its protease conformation. As a result, PAR1 is cleaved by thrombin at sub-nanomolar concentrations. PAR4 does not have a hirudin-like sequence and is a less efficient thrombin substrate that requires >10-fold more thrombin than PAR1. The anionic region of PAR4 interacts with the autolysis loop of thrombin. The protein data bank (PDB) references for the structures are 3LU9 (left), 2PV9 (right).

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Fig. 13.4 PAR1 serves as a cofactor for thrombin-mediated PAR4 activation when co-expressed on the same cell, such as human platelets. The enhanced rate of PAR4 activation by thrombin is facilitated by the hirudin-like sequence on PAR1. In mouse platelets, PAR3 serves as the co-factor for PAR4 activation.

Fig. 13.5 The physical interactions between PARs. PARs can form homo- and hetero-oligomers between PAR family members and with other platelet GPCRs. These physical interactions influence both the rate of the activation and receptor signaling. The heterodimeric assembly of PAR1 and PAR4 is required for PAR1 to serve as a cofactor to enhance the cleavage rate of PAR4 by thrombin.

interacts with thrombin’s gamma (autolysis) loop, confirming that PAR4 does not use thrombin’s exosite I (Fig. 13.3).25 PAR4 is not an efficient thrombin substrate when expressed on cells by itself.58,60 However, when PAR4 is co-expressed with PAR1 in heterologous systems as it is on human platelets, the rate of PAR4 cleavage is enhanced six- to 10-fold.40,58,60,61 The enhanced rate of PAR4 cleavage in the presence of PAR1 follows a similar mechanism to that first described for the functional interaction between PAR3 and PAR4 on mouse platelets.27 In this model, thrombin remains bound to PAR1 or PAR3 via the hirudin-like sequence following proteolysis at the cleavage site, which enhances the activation of an adjacent PAR4 (Fig. 13.4). A simple interpretation is that the higher affinity binding site on PAR1 or PAR3 (the hirudin-like sequence) increases the local concentration of thrombin near the platelet surface to facilitate the activation of PAR4. However, based on biochemical and structural data with other tight exosite I binders, there may be a more intricate cooperation between PARs. For example, the hirudin-like sequence of PAR1 or PAR3 likely induces thrombin into the protease conformation with an open active site to facilitate PAR4 cleavage.46,62 The latter model is also supported by structural models of murine thrombin bound to peptides from PAR3 and PAR4.25

PHYSICAL INTERACTIONS BETWEEN PARs Homo- and heterodimers of GPCRs may influence signaling and can be thought of as allosteric modulators of one another.63 However, the molecular organization of GPCRs within the plasma membrane is still controversial.64 There is

strong evidence of PARs forming physiologically relevant homodimers and heterodimers that influence the rate of activation, downstream signaling, and trafficking in several cell types.10,28,29,65–67 In platelets, the focus has been on PAR1 and PAR4 (Fig. 13.5). The major difficulty in ascribing a specific function to dimers is a reliable readout that distinguishes between monomers and oligomers in a primary cell or in vivo. As described above, the rate of PAR4 cleavage is enhanced
10-fold when it is co-expressed with PAR1.40,60,61 This cooperation is dependent upon dimerization. Leger et al. first reported PAR1PAR4 heterodimers using Forester Resonance Energy Transfer (FRET) and co-immunoprecipitation studies.40 This was later confirmed using Bioluminescence Resonance Energy Transfer (BRET) and the heterodimer interface was mapped to I2314.54 and W2274.50 in transmembrane helix 4 (TM4) of PAR1 and L1924.41, L1944.43, M1984.47, and L2024.51 of PAR4.61 The nomenclature is that of Ballesteros and Weinstein, where the most conserved residue in the TM helix is the designated as “50” and residues moving towards the N-terminus decrease in value while residues towards the C-terminus increase.68 Importantly, the ability of PAR1 to enhance the rate of PAR4 cleavage is abrogated when the dimer interface of PAR1 or PAR4 is mutated.61 This provides direct physiological consequences of PAR1–4 heterodimers. Determining a function for homodimers is more difficult. Using BRET assays, the PAR4 homodimer interface was mapped to the potential leucine zipper in TM4 (L1924.41, L1944.43, M1984.47, and L2024.51).69 Mutations that disrupt homodimer formation also disrupt calcium mobilization suggesting that dimers are the functional signaling unit for PAR4.69 Edelstein and colleagues recently described sequence variants

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that have different reactivities (see below for details on PAR polymorphisms and sequence variants).70 Platelets from individuals that are heterozygous for Val at amino acid 296 have a significantly decreased response to PAR4 agonists even in the presence of a hyperreactive allele.70 These data suggest that the alleles are forming homodimers and that the 296 V allele suppresses the PAR4 response via a dominant negative effect.70 PAR3 and PAR4 also form heterodimers and, in addition to enhancing PAR4 activation, PAR3 also influences PAR4 signaling.30 As described above, PAR3 does not signal on its own.27 However, PAR3 deficient mice have a 1.6-fold increase in the maximum Ca2+ mobilization and an increase in PKC activation compared to wild-type mice, suggesting that PAR3 is a negative regulator of PAR4 signaling.30 This may be analogous to the dominant negative effect between PAR4 sequence variants.10,70 PAR4, but not PAR1, also forms agonist dependent heterodimers with another platelet GPCR, the ADP receptor P2Y12.71–73 The interaction between P2Y12 and PAR4 enhances the recruitment of arrestin-2 to PAR4 and mediates sustained signaling through Akt in human platelets required to stabilize platelet plug formation. The interaction interface of the PAR4-P2Y12 heterodimer has also been mapped to transmembrane helix 4 of PAR4.71 A mutation in PAR4 at the heterodimer interface disrupts the interaction with P2Y12 and prevents arrestin-2 recruitment and Akt activation.71 Further studies are required to determine how these platelet GPCRs coordinate their interactions to regulate platelet responses to the variety of agonists in vivo (Fig. 13.5).

THROMBIN SIGNALING IN HUMAN PLATELETS Thrombin initiates multiple signaling cascades (e.g., calcium mobilization, Rap1B, and Rho) through PAR1 and PAR4 in human platelets. The activation of these pathways and their role in platelet adhesion, granule secretion, and aggregation is discussed in detail in Chapter 18. In this chapter, we will focus on the initial steps of signal transduction mediated by PAR1 and PAR4. The importance of G-protein signaling in platelets is well characterized (Fig. 13.6).74 Gαq stimulates the formation of inositol triphosphate (IP3) and diacylglycerol (DAG), leading to intracellular calcium mobilization and protein kinase C (PKC) activation.75,76 Gα12/13 mediates Rho guanine nucleotide exchange factors and RhoA activation, which controls platelet shape change, spreading, and thrombus stability.77–79 Gαi signaling in platelets inhibits adenylate cyclase activity and induces platelet shape change, granule secretion, and calcium mobilization. PAR1 and PAR4 directly couple to both Gαq and Gα12/13. Although PAR1 directly binds to Gαi in fibroblast and when expressed heterologously in COS7 cells,75,80 there are conflicting data in platelets. Consistent with other cell types, Voss and colleagues showed that PAR1 signaled through Gαi in human platelets using a panel of pharmacological tools.81 In other studies, signaling through the Gαi pathway via PAR1 and PAR4 was indirect and mediated by secondary release of ADP, which acts on the Gαi-coupled ADP receptor, P2Y12.82–84 PAR1 and PAR4 activate the same signaling pathways and both receptors can mediate full platelet responses in vitro. Stimulation of platelets with either the PAR1 or PAR4 agonist peptide leads to granule secretion, integrin activation, and ultimate aggregation.3,32,33 More specifically, RhoA activation downstream G12/13 and Gq, induced the same level of dense granule release in response to PAR1 or PAR4 activation.85 Similarly, a mass spectrometry (MS)-based quantitative proteomic analysis and enzyme-linked immunosorbent assay (ELISA) showed that α-granule release is also the same following PAR1 or PAR4 activation.86 Despite earlier reports that PAR1 and PAR4 lead to

release of differential α-granule cargo, more recent studies show any differences in release are due to kinetics.87

PAR1- AND PAR4-SPECIFIC SIGNALING The evolutionary purpose for the emergence of a dual thrombin receptor system on platelets in which both receptors couple to the same signaling machinery remains unclear. However, recent developments have begun to shed light on differential roles for PAR1 and PAR4. Due to the differences in PAR expression across species (mice do not express PAR1 on their platelets; see Table 13.1), delimitation of signaling pathways can be achieved in ex vivo experiments with human platelets, but do not allow in vivo studies. These studies are dependent on the availability and quality of agonists and antagonists to each receptor. Recent advances in PAR agonist development have given insight on the function of each receptor.44,88 With growing use of these compounds in humans, we will gain novel views into the individual physiological roles of PAR1 and PAR4. The first described and most dramatic difference between PAR1 and PAR4 signaling in platelets are the kinetics and duration of intracellular Ca2+ signaling (Fig. 13.7).89,90 Prior to the identification of PAR4, it was recognized that the PAR1 agonist peptide is a partial agonist for human platelets.91 The agonist peptide recapitulates the initial burst in intracellular Ca2+ observed with thrombin, but the signal quickly dissipates.91 The overall magnitude and duration of the Ca2+ signal was greater in thrombin-stimulated platelets than for the agonist peptide.92 The identification of PAR4 reconciled these differences when it was demonstrated that there is a distinct wave of Ca2+ signaling from PAR4.89,93 The prolonged Ca2+ stimulus associated with thrombin activation of PAR4 appears to be required for stable clot formation and full spreading on fibrinogen in a p38 and ERK1/2 dependent manner.94,95 The sustained calcium signaling from PAR4 also impacts microparticle release, procoagulant platelets, and thrombin generation.96–98 French and colleagues have mechanistically linked phosphatidyl serine (PS) exposure and thrombin generation to PAR4 activation in human platelets using flow chambers and PAR4 specific antibodies.97 They went on to show that initial platelet responses mediated by acute increases in calcium, such as αIIbβ3 integrin activation and granule secretion are independent of PAR4 signaling. The increase in PS exposure and the generation of procoagulant platelets were linked to larger thrombi and increased fibrin formation. These results are consistent with a recent study with a new PAR4 antagonist (BMS 986120) that showed a reduction in the fibrin component of an ex vivo thrombus.99 Sustained PAR4 signaling also impacts the Gα12/13 pathways. For example, PAR4 stimulation with the agonist peptide, AYPGKF, enhances the surface expression of Factor V (1.6-fold) and P-selectin (0.8-fold) compared to PAR1 activation with SFLLRN.96 PAR4 activation also induced a threefold greater production of platelet microparticles compared with PAR1 activation. The RhoA pathway inhibitor Y-27632, reduced Factor V translocation and microparticle release downstream PAR4 stimulation to levels observed with PAR1 stimulation. These combined data show that PAR4 is mediating these events through the G12/13-RhoA signaling axis. The sustained activation of PAR4 also impacts lipid signaling in platelets. For example, thrombin stimulates the generation of thromboxane A2 (TXA2), which, in turn, activates platelets via the thromboxane receptor (TP) and amplifies platelet activation to causes irreversible aggregation. PAR4 stimulation results in significantly greater TXA2 generation compared to PAR1.100 In addition, a specific inhibitor of cytosolic phospholipase A2α (cPLA2α), giripladib, selectively inhibited PAR4, but not PAR1-mediated P-selectin expression

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Fig. 13.6 Signaling cascades mediated by PAR activation. Upon thrombin activation, PAR1 and PAR4 recruit the G-proteins, Gq and G12/13. The initiation of these G-protein-mediated signals both contribute to platelet shape change and granule secretion. The ADP secreted by dense granules increases the local ADP concentration and activates P2Y12 further for Gi-mediated signaling. The combination of these signaling cascades leads to irreversible platelet aggregation and formation of a stable thrombus.

Fig. 13.7 PAR1 and PAR4 have overlapping and distinct downstream signaling events. PAR1 and PAR4 both signal through Gq and G12/13. PAR1 results in a rapid burst of intracellular calcium release that is quickly dissipated in the absence of PAR4 activation. In contrast, PAR4 has a slower onset and a sustained signal that facilitates growth and stability of the thrombus.

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in human platelets. However, specific inhibition of intracellular membrane-associated calcium-independent phospholipase A2γ (iPLA2γ) with bromoenol lactone (BEL) significantly reduced P-selectin expression after PAR1, but not PAR4 activation in human platelets.101 As these data have emerged, the concept that PAR1 and PAR4 may reflect different roles platelets have in the blood has been solidified. The efficient response of PAR1 to low thrombin concentration allows the platelets to monitor and respond to damage or breeches in the vasculature. However, if a robust response is not needed, the transient signal does not induce an unwanted thrombus. In contrast, PAR4 activation occurs when a high level of thrombin is generated following a more severe insult to the endothelium. Once PAR4 is activated, a sustained signal leads to robust platelet activation and stable thrombus formation.

TERMINATION OF PAR SIGNALING The activation of PARs is irreversible due to their unique activation mechanism. After enzymatic cleavage, the exposed ligand remains tethered to the receptor and cannot simply diffuse away to terminate PAR signaling. Thus, internalization and degradation are the primary mechanisms through which PAR signaling is discontinued.102–104 The regulation of PAR trafficking and desensitization has primarily been studied in endothelial cells. On the surface of human platelets, thrombin efficiently cleaves the majority of the PARs, which leads to the receptor internalization or shedding into the platelet microparticles during platelet aggregation.105,106 Due to their nature, platelets are only able to respond to thrombin once in the process of aggregating to form a thrombus. Thus, it is reasonable that platelets have evolutionarily favored a short half-life and a functionally irreversible terminal role in hemostasis, which makes the termination of PAR signaling less critical.

ACTIVATION OF PARs BY PROTEASES OTHER THAN THROMBIN In addition to thrombin, several other serine proteases are capable of activating PARs such as Factor Xa, plasmin, matrix metalloproteases 1, 2, and 13, elastase, activated protein C (APC), proteinase-3, granzyme, cathepsin G, and calpain.11,107 Depending on the cleavage site, PARs can be activated or inactivated by these proteases. Further, the alternative cleavage sites can generate novel tethered ligands that initiate specific signaling pathways. The panel of proteases activating PARs has been described for a variety of cell types. Here, we will focus on protease cleavage of PAR1 and PAR4 in the context of platelet function. The reader is directed to recent comprehensive reviews for other contexts.10,11,108,109 Matrix metalloproteinase-1 and 2 (MMP-1, MMP2) directly activate platelets via PAR1.107,110–112 MMP-1 cleaves PAR1 at a non-canonical site, Asp39 (TLD39PRSFLLRN).112,113 In human platelets, the cleavage of PAR1 at Asp39 by MMP-1 induces G12/ 13-Rho, p38 MAPK pathways, and shape change. However, intracellular calcium mobilization and platelet aggregation in response to MMP-1 are less potent than with thrombin.112,113 In addition to Asp39, MMP-1 has been reported to cleave at the canonical thrombin site (Arg41) and Leu44.110,111 MMP-2 cleaves PAR1 at a non-canonical site, Lys38 to generate the tethered ligand (D39PRSFLLRN), which increases the sensitivity of platelets to multiple agonists.107 Plasmin cleaves both PAR1 and PAR4 on platelets, but with distinct outcomes. There are four plasmin cleavage sites on PAR1 (Arg41, Arg70, Lys76, and Lys82).114 In these studies, Kuliopulos and colleagues showed that although

plasmin is capable of cleaving PAR1 at the canonical thrombin site, the predominant result is inactivation of PAR1 by truncating the tethered ligand at the distal sites. Plasmin cleaves PAR4 at canonical thrombin cleavage site Arg47 (…LPAPR47GYPGQV…) to generate PAR4 tethered ligand peptide (GYPGQV).115 Cathepsin G is a serine protease found in the dense granules of neutrophils and is secreted upon neutrophil activation that cleaves human PAR1 and PAR4.116,117 Cathepsin G is inhibitory for PAR1 due to cleavage at Phe55, which leads to a loss of the PAR1 tethered ligand.118 In contrast, cathepsin G leads to activation of PAR4 and induces calcium mobilization in human platelets and in transfected fibroblasts.117 In mice, cathepsin G does not activate PAR4, but does cleave PAR3 and prevents it from acting as a cofactor for PAR4.119

PHARMACOLOGICALLY BLOCKING PAR SIGNALING At first look, the dual-receptor system for thrombin signaling on human platelets creates challenges for developing inhibitors, since targeting both receptors is required to block platelets’ response to thrombin. However, the unique signaling kinetics for PAR1 and PAR4 present an opportunity to fine-tune thrombin signaling by individually targeting PAR1 or PAR4.120 The major hurdles for developing PAR antagonists are competing with the tethered ligand and the irreversible activation mechanism (Fig. 13.1).88

Protease-Activated Receptor 1 Antagonists The clinical aspects of PAR1 antagonists are discussed in detail in Chapter 53. Here, we provide a framework for their mode of action as it relates to PAR1 structure and function. Given that PAR1 is the most sensitive thrombin receptor, several therapeutic approaches have been explored.88 PAR1 antagonists (Table 13.2) can be divided into three groups based on their mechanism of action: (1) orthosteric inhibitors that directly compete with the tethered ligand; (2) allosteric inhibitors that modulate downstream signaling; and (3) intracellular antagonists that interfere with PAR1 signal transduction. The orthosteric inhibitor vorapaxar (Zontivity) was approved by the FDA in 2014 as the first-in-class PAR1 antagonist.121–124 The development of vorapaxar met the need of reducing the rate of mortality caused by future cardiovascular events of the patients with a prior history of myocardial infarction (MI) or peripheral artery disease (PAD).124 Vorapaxar is contraindicated for patients with a history of stroke, transient ischemic attack, intracranial hemorrhage, or bodyweight <60 kg due to the increased risk of bleeding that outweighs the potential therapeutic benefits.124 Vorapaxar potently inhibits ex vivo thrombin-induced platelet aggregation (IC50 ¼ 47 nM) and ex vivo human TRAP (SFLLRN)-induced platelet aggregation (IC50 ¼ 25 nM).132 In cynomolgus monkeys, vorapaxar was able to disturb agonist induced platelet aggregation at 0.1 mg/kg with >24 h of activity.132 Flaumenhaft and colleagues have identified allosteric modulators of PAR1 via a high throughput screen of 16,320 compounds.125 Mechanistically, this family of compounds binds to the C-terminal 8th helix of PAR1,125 which is anchored to the plasma membrane via palmitoylated cysteines. The major difference between orthosteric inhibitors such as vorapaxar and parmodulins is the selective inhibition of downstream signaling. In platelets, the current generation of parmodulins selectively block aggregation at 10 μM, but do not impede platelet shape change at this concentration.125 These differences are due to the parmodulins’ selective inhibition of Gq activation and resultant calcium signaling while leaving G13 pathways intact.125,126 In endothelial cells, parmodulins limit

Protease-Activated Receptors

251

TABLE 13.2 PAR1 and PAR4 Antagonists Name

Chemical Structure

Mechanism of Action

Comments

Reference

Irreversible orthosteric antagonist, compete directly with endogenous tethered ligand

FDA approved (2014)

121–124

Allosteric modulator

Block Gq over G12/13 pathway, less potent than orthosteric inhibitor

125, 126

PZ-128

Lipidated peptides, Pepducin

Mimic PAR1 ICL3 sequence

127–129

PAR4 antagonists YD-3

Competitive antagonist

First non-peptide PAR4 antagonist, IC50 ¼ 130 nM

130,131

ML354

Allosteric modulator

IC50 ¼ 140 nM

150

BMS-986120

Reversible competitive antagonist

Phase I trial completed

44,99

Reversible competitive antagonist

Phase II trial completed

44

PAR1 antagonists Vorapaxar

Parmodulin 1

Parmodulin 2

BMS-986141

Undisclosed

prothrombotic and proinflammatory signaling downstream of thrombin-activated PAR1. However, the cytoprotective effects of APC-mediated PAR1 activation are not blocked.126 More recently, it was identified that the protective effects of parmodulins can partially be attributed to agonist activity via Gβγ leading downstream activation of Akt.133 Although this class of compounds will impact platelet activation, their primary benefit may be in targeting PAR1 on endothelial cells. The third class of PAR1 inhibitors, pepducins, are of Npalmitoylated peptides that can integrate into the plasma membrane and interact with PARs or other targeted GPCRs.127–129 Functioning as a dominant negative antagonist, P1pal-12 interrupts the interaction between ICL3 of PAR1 and its corresponding G-proteins to inhibit PAR1-mediated signaling. The pepducin PZ128 has been developed further and is currently in Phase 2 clinical trials for prevention of acute thrombosis complications for percutaneous coronary intervention (NCT01806077). PZ128 has favorable pharmacokinetics, pharmacodynamics, and safety tolerance.134 Pepducins are reversible antagonists with full recovery after with no impact on bleeding, coagulation, or electrocardiogram (ECG).134 PZ128 completely obstructs ex vivo SFLLRN-induced human platelet aggregation with an IC50 of 0.5 μM/L.135 Intravenous infusion of PZ-128 at 6 mg/kg dose in baboon inhibited PAR1-dependent platelet aggregation completely at 1 and

2 h, but fully recovered at 24 h.135 Administration of PZ-128 intravenously at 1–2 mg/kg prevented 80% to 100% PAR1induced human platelet activation at 30 min to 6 h postdosing.134

Protease-Activated Receptor 4 Antagonists PAR4 is gaining attention as a therapeutic target.57,88,120 As described above, PAR4 requires higher thrombin concentrations to be activated and promote sustained signaling and activation. Blocking PAR4 would spare the initial thrombin response from PAR1, which has the potential to minimize bleeding complications.120 Initial efforts have led to the discovery several compounds (Table 13.2), such as YD-3 and ML354, that have a similar indazole core structure and have been used as preclinical tools to study PAR4 function.88,130,131,136 The low potency and selectivity to PAR4 over PAR1 has limited their development. In 2017, Bristol-Myers-Squibb described BMS986120, a reversible PAR4 antagonist with high selectivity to PAR4 over PAR1.44 This compound was able to block human platelet activation in platelet rich plasma (PRP) stimulated by γ-thrombin and PAR4 activation peptide (AYPGKF) with an IC50 < 10 nM.44 A direct comparison of BMS-986120 to clopidogrel on cynomolgus monkey thrombosis model demonstrated that under the same dosage (1 mg/kg, oral

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administration) that achieved 80% thrombus weight deduction, clopidogrel led to >eightfold increase in bleeding comparing to BMS-986120.44,99 The preclinical studies in nonhuman primate models and the Prospective Randomized Open Blinded End-point (PROBE) designed phase I clinical trial of the BMS-986120 (NCT02439190) in humans showed its potential as a potent PAR4 antagonist with a lower bleeding risk and a wider therapeutic window compared to the standard antiplatelet care, clopidogrel with or without aspirin.44,99 A phase 2 trial with another compound from the same BMS library, BMS-986141, was completed in March 2017 (NCT02671461); no results have been reported.

STRUCTURAL STUDIES ON PARs High-resolution crystal structures provide key insights to understanding a protein’s function and, in some cases, the molecular contacts of agonists or antagonists. Technological advancements have dramatically increased the structural information available for GPCRs since the first structure was solved in 2000.137,138 Using this technology, a high-resolution crystal structure of PAR1 bound to the antagonist vorapaxar at 2.2 Å resolution was solved in 201243 (Fig. 13.8). The general overall structure was similar to many other GPCRs. However, when the PAR1 structure is compared to other class A GPCRs, transmembrane helix 7 was structurally similar to activated receptors, which is surprising for an antagonist bound receptor. The structure of PAR1-vorapaxar complex also revealed how it binds tightly to PAR1 and inhibits the receptor activation mediated by its tethered ligand. Other GPCRs have ligand-binding sites that are deep, but solvent-exposed. In contrast, vorapaxar interacts with PAR1 near the surface, but with limited aqueous accessibility (Fig. 13.8). Despite the benefits for further PAR1 antagonist development, little structural information was gleaned regarding how the native ligand interacts with the ligand-binding site. This is experimentally challenging for PARs, because crystallography studies with GPCRs require the insertion of stabilizing sequences and truncation of the N- and C-termini.138 Truncating the N-terminus of PARs removes the tethered ligand. However, molecular simulations based on the PAR1 structure suggests the ligand interacts on the surface of the receptor rather than deep within the transmembrane bundle.43 These modeling studies are consistent with earlier studies mapping the ligand-binding site to specific residues at the extracellular surface (Leu 96, Asp 256 Glu 260, and Glu 347).20,43

Fig. 13.8 Crystal structure of PAR1 bound to vorapaxar. Vorapaxar binds near the extracellular surface of PAR1. This is in contrast to most GPCR agonists, which typically bind more deeply in the transmembrane helical bundle. See Zhang et al. Nature 2012;492:387–92 for detailed information on the molecular contacts between PAR1 and vorapaxar. The PDB file used to generate the model in PyMol was 3VW7.

Structural approaches have also improved the understanding of the PAR activation mechanism. Simplistically, PARs are activated when, upon enzymatic cleavage, the tethered ligand bends over to interact with the receptor and bind extracellular loop 2 (Fig. 13.1A). However, using NMR and mutagenesis studies with the PAR1 exodomain, Seeley et al. demonstrated that it forms a secondary structure that facilitates receptor activation.139 Alsteens and colleagues developed a modification of atomic force microscopy to probe the surface of PAR1 to determine the free-energy landscape and uncover the nature of the ligand-binding site.140,141 Based on their studies, the authors proposed that PAR1 activation follows a two-step fashion in which the tethered ligand first interacts with the receptor in a low-affinity mode, then transitions to a high-affinity mode. The specific structural rearrangements that occur following activation by the tethered ligand, and how cofactors and heterodimers influence the receptor allostery, are remaining questions that will require alternative techniques such as structural mass spectrometry and the emerging cryo-electron microscopy.142,143 Finally, as described above, PARs have limited sequence identity (34%–41%) (Fig. 13.3), making it likely that each family member will have unique structural requirements and properties.

POLYMORPHISMS AND SEQUENCE VARIANTS Single nucleotide polymorphisms (SNPs) have been described for both PAR1 and PAR4. SNPs present the challenge of establishing a direct link from the identified polymorphisms to receptor expression or function and ultimately to a physiological output. One of the first SNPs described was a PAR1 polymorphism in an intron that affects PAR1 density on platelets and decreases platelet response to PAR1 agonists in individuals.144 However, in a recent clinical study with 660 patients who underwent percutaneous coronary intervention (PCI), there was no evidence of increased major adverse cardiovascular events (MACE) or bleeding risk correlated with the polymorphism.145 The heritable inter-individual variation in platelet reactivity has been directly linked to PAR4.70,146–148 The Platelet RNA And eXpression 1 (PRAX1) study was designed to examine mRNAs and microRNAs associated with this difference in 154 healthy individuals who self-identify as black or white.147 In this population, Edelstein et al. showed that the black individuals had increased platelet response to PAR4 stimulation, higher expression of phosphatidylcholine transfer protein

Protease-Activated Receptors

(PC-TP), and lower levels of miR-376c.147 The opposite was observed in the white individuals. Other platelet agonists, including PAR1, were not different between the groups. A second study identified two additional polymorphisms that alter amino acids in PAR4 at positions 120 (Ala/Thr) and 296 (Phe/ Val).70 The polymorphism at 120 is common and is distributed by race. PAR4-120A exhibited a lower reactivity and was found in 81% of white individuals compared to 37% of black individuals. In contrast, PAR4-120 T was hyperreactive to agonists, resistant to a PAR4 antagonist, and found in 63% of blacks compared to 19% of whites.70 The frequency of Val at 296 was low and had low reactivity regardless of the amino acid at 120 suggesting that it is a dominant negative receptor. The mechanism by which the PAR4 variants elicit their distinct response to affect platelet function is not known. These polymorphisms may change the interaction of PAR4 with the membrane, allosterically alter ligand binding, or influence the transition of the receptor to an active state.149 Structural studies examining the differences between the PAR4 sequence variants are necessary to determine the molecular basis for the differences in reactivity.

FUTURE DIRECTIONS Although PAR1 was discovered >25 years ago, recently developed therapeutic agents targeting PAR1 and PAR4 make this an exciting time for PAR research. The PAR1 antagonist vorapaxar was approved by the FDA in 2014, and new PAR4 antagonists show great promise in preclinical models. At this point, it is unclear how these new therapeutics will shape clinical practice. For example, the necessary large clinical trials for the PAR4 antagonists will need to determine the target patient population, if and how PAR4 polymorphisms impact effectiveness, and how these new agents will be used in conjunction with existing antiplatelet and anticoagulation therapies. In this chapter, we have focused on PAR signaling in platelets. As discussed above, PARs are expressed in a number of tissues in addition to platelets. An important consideration for targeting PARs therapeutically is their physiological and pathophysiological roles in other tissues and the impact of disrupting these signaling pathways. It is known that PAR1 has protective effects in endothelial cells when activated by APC. Since most PAR antagonists bind to orthosteric sites that block all activity of the receptor, inhibiting potentially protective signaling pathways needs to be balanced with inhibiting detrimental pathways. As we uncover more proteases capable of activating PARs, it is likely that the local environment and cellular cofactors will result in distinct cellular responses through biased agonism of PAR1 and potentially other PARs. The recent advances in cryoelectron microscopy (cryo-EM) and mass spectrometry have made it possible to determine the structure of smaller protein complexes. These advanced structural approaches will provide insight into the dynamics and stoichiometry of the molecular complexes required for PAR activation, and have the potential to elucidate the molecular basis for biased signaling in platelets and other cells. Finally, regardless of their clinical development, the new PAR antagonists will allow investigators to reveal further the specific roles of PAR1 and PAR4 in human platelets. Until now, the lack of good pharmacological tools has limited the full understanding of the individual contributions of PAR1 and PAR4. Investigations into the mechanisms and consequences of the short signaling from PAR1 and the prolonged signaling from PAR4 may lead to a greater separation of the surveillance and thrombus generating roles of platelets. This may move us closer to the long-standing goal for antiplatelet agents that allow platelets to mediate hemostasis while limiting thrombosis.

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