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CARDIOVASCULAR

Blockade of the Thrombin Receptor Protease-Activated Receptor-1 with a Small-Molecule Antagonist Prevents Thrombus Formation and Vascular Occlusion in Nonhuman Primates

Johnson & Johnson Pharmaceutical Research and Development, Spring House, Pennsylvania (C.K.D., B.P.D., M.F.A., A.L.D., M.R.D., H.-C.Z., B.E.M., P.A.-G.); and Charles River Laboratories, Worcester, Massachusetts (M.N.)

Received August 12, 2002; accepted October 24, 2002.

	Abstract

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Although it is well recognized that human platelet responses to -thrombin are mediated by the protease-activated receptors PAR-1 and PAR-4, their role and relative importance in plateletdependent human disease has not yet been elucidated. Because the expression profile of PARs in platelets from nonprimates differs from humans, we used cynomolgus monkeys to evaluate the role of PAR-1 in thrombosis. Based on reverse transcription-polymerase chain reaction, PAR expression in platelets from cynomolgus monkeys consisted primarily of PAR-1 and PAR-4, thereby mirroring the profile of human platelets. We probed the role of PAR-1 in a primate model of vascular injury-induced thrombosis with the selective PAR-1 antagonist (S)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]--[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide (RWJ-58259). After pretreatment with RWJ-58259 or vehicle, both carotid arteries of anesthetized monkeys were electrolytically injured and blood flow was monitored for 60 min. Time to occlusion was significantly extended after RWJ-58259 administration (27 ± 3 to 53 ± 8 min; p < 0.048). Vessels from three of the five treated animals remained patent. Ex vivo platelet aggregation measurements indicated complete PAR-1 inhibition, as well as an operational PAR-4 response. Immunohistochemical staining of mural thrombi with antibodies to the platelet marker CD61 and fibrinogen indicated that RWJ-58259 significantly reduced thrombus platelet deposition. Drug treatment had no effect on key hematological or coagulation parameters. Our results provide direct evidence that PAR-1 is the primary receptor that mediates -thrombin's prothrombotic actions in primates and suggest that PAR-1 antagonists may have potential for the treatment of thrombotic disorders in humans.

Thrombin stimulates human platelets through the activation of two protease-activated G protein-coupled receptors, PAR-1 and PAR-4 (Vu et al., 1991; Kahn et al., 1998; Xu et al., 1998). Since the discovery of PAR-1 and PAR-4 in human platelets, there has been a keen interest to dissect the specific involvement of each receptor in the process of platelet aggregation. In fact, transgenic mice deficient in these receptors have been obtained to probe for the contributions of different thrombin-sensitive PARs in platelet physiology (Connolly et al., 1996; Darrow et al., 1996; Nakanishi-Matsui et al., 2000; Sambrano et al., 2001). However, these studies have only led to indirect evidence to suggest that PAR-1 may be the primary activation mechanism for initiating human platelet activation. To examine the role of PAR-1 in arterial thrombosis, as well as to elucidate a potential role for PAR-4, we have developed selective PAR-1 antagonists, exemplified by RWJ-58259 (Andrade-Gordon et al., 2001; Zhang et al., 2001). Unfortunately, the evaluation of RWJ-58259 in small-animal models of thrombosis has been hindered by the species-dependent expression of PAR-1 in platelets (Connolly et al., 1994, 1996; Derian et al., 1995; Darrow et al., 1996). Because guinea pigs were identified as the only small animal possessing PAR-1 on their platelets, we studied the effect of PAR-1 inhibition with RWJ-58259 in two guinea pig models of arterial thrombosis (Andrade-Gordon et al., 2001). After our observation of marginal in vivo activity with RWJ-58259, we discovered that guinea pig platelets have a triple-PAR profile, which undoubtedly contributed to the lack of efficacy. Thus, there is a dearth of small-animal models for assessing the role of PAR-1 in thrombosis, due to the absence of platelet PAR-1 expression (Connolly et al., 1994; Derian et al., 1995) and/or species-dependent platelet PAR profiles (Kahn et al., 1998; Andrade-Gordon et al., 2001). We have now investigated the effect of the selective PAR-1 antagonist RWJ-58259 on thrombus formation subsequent to electrolytic injury in nonhuman primates. Our results demonstrate for the first time that selective in vivo blockade of PAR-1 attenuates thrombotic activity, even in the presence of platelet PAR-4, and suggest that PAR-1 antagonists have the potential for therapeutic utility in human thrombotic diseases.

	Materials and Methods

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Polymerase Chain Reaction (PCR) Analysis for PAR-1 and PAR-4. A PCR analysis was performed essentially as described previously (Andrade-Gordon et al., 2001) except that RNA samples were pretreated with RQ1 RNase-free DNase (Promega, Madison, WI) before cDNA synthesis. The sense (U) and antisense (L) primers for the amplification of PAR sequences were as follows: PANP1-U, 5'-CATAAGCATTGACCGGTTCCTGGC-3'; PANP1-L, 5'-CAAAGCAGACGATGAAGATGCAGA-3'; PANP3-U, 5'-CAATGGCAACAAC TGGGTATTTGG-3'; PANP3-L, 5'-AAAATCACAAGGATGAGGAG-3'; PANP4-U, 5'-GCCAATGGGCTGGCGCTGTG-3'; PANP4-L, 5'-GCCAGGCAGATGAAGGCCGG-3'; -actin PTP-U, 5'-AGGCCAACCGCGAGAAGATG-3'; and -actin PTP-L, 5'-CTCGGCCGTGGTGGTGA AGC-3'. Reactions (50 µl) were subjected to 25 cycles of 94°C for 30 s/60.1°C for 30 s/68°C for 48 s for PAR-1; 30 cycles of 94°C for 30 s/54.4°C for 30 s/68°C for 56 s for PAR-3; 25 cycles of 94°C for 30 s/63.1°C for 30 s/68°C for 56 s for PAR-4; and 25 cycles of 94°C for 30 s/60.4°C for 30 s/68°C for 45 s for -actin. The products of each reaction were electrophoresed through 2% agarose gels and transferred to Hybond N⁺ membranes (Amersham Bioscience, Inc., Piscataway, NJ). The appropriate oligonucleotide primer probes, corresponding to nested sequences within the respective PAR PCR product, were digoxigenin-labeled, hybridized, and detected using the Genius nucleic acid detection system (Roche Diagnostics, Indianapolis, IN). The sequences used in this group of nested primer probes were as follows: PANP1PP-L, 5'-CAGAGTGCGCCAGGACAGGGACTGGATGGGGTACACCAC-3' for PAR-1; PANP3PP-L, 5'-CCTGCTTCAGGATGACAAAGGGCAGCATGTATAAGAAAAC-3' for PAR-3; PANP4PP-L, 5'-CCAGCAGCAACACTGAACCATACATGTGGCCATAGAG-3' for PAR-4; and Actin PP-L, 5'-TGGGCACAGTGTGGGTGACCCCGTCACCGGAGTCCA TC AC-3' for -actin.

Carotid Injury Model of Thrombosis. We used an electrolytic injury-induced model of thrombosis in primates based on modifications to a similar model in dogs (Rote et al., 1994; Rebello et al., 1997). Ten cynomolgus monkeys of either sex, weighing 3 to 6 kg, were used in this study. All procedures involving the use of animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals (1996) and the Animal Care and Use Committees at Charles River Laboratories and Johnson & Johnson Pharmaceutical Research and Development.

Animals were preanesthetized with ketamine hydrochloride, 10 mg/kg i.m., plus atropine sulfate, 0.04 mg/kg i.m., and immediately intubated and ventilated with a respirator. Anesthesia was maintained with isoflurane. End tidal CO₂ was monitored and maintained within individual physiological ranges. An intravenous catheter was placed in a peripheral vein for administration of lactated Ringer's solution at a rate of 5 to 10 ml/kg/h. Catheters were placed in a femoral vein and femoral artery for blood collection and monitoring blood pressure, respectively. Drug or vehicle was administered at a separate distant venous site. Both common carotid arteries were exposed and isolated via blunt dissection. A Doppler flow probe was placed around each carotid artery at a point proximal to the insertion of the intra-arterial electrode. Flow velocity was monitored continuously with a flowmeter (Transonic Systems, Inc., Ithaca, NY). A stenosis was created in each carotid using a vessel occluder. The degree of stenosis was adjusted so as to eliminate the hyperemic response after brief occlusion. Blood flow in the carotid arteries was monitored and recorded continuously throughout the observation periods using a physiological data acquisition and analysis system (Biopac, Santa Barbara, CA). An intravascular electrode (27-gauge needle) was inserted into each carotid at the point of stenosis. Electrolytic injury to the intimal surface of each carotid artery was induced by delivering continuous current (100 µA) from the positive pole of a model CCUI constant current unit (Grass/Astro-Med, West Warwick, RI). The negative terminal was connected to a distant subcutaneous site.

RWJ-58259 (Fig. 1; Zhang et al., 2001) was dissolved in a solution of 5% dextrose in water at a concentration to yield 1-ml/kg dose volumes for the bolus and infusion doses. The solution was administered intravenously as a bolus 5-min infusion at 3 mg/kg followed immediately by a continuous infusion for 65 min at 0.123 mg/kg/min. Control animals were treated with dose-equivalent volumes of 5% dextrose in water. Ten minutes after the start of treatment, anodal current was applied to the intra-arterial electrodes in both carotid arteries. Current was maintained for 60 min or until 15 min after the formation of a thrombotic occlusion in both vessels. Blood flow was monitored continuously before, during, and after induction of vessel injury. Occlusion was defined as flow of less than 1 ml/min. The incidence of occlusion and the time to occlusion (t) for the left and right carotids were recorded. If the arteries were patent at 60 min after electrical stimulation, a value of 60 min was used for statistical analysis. Blood samples were obtained before and after dosing for evaluation of platelet aggregation, hematology, clinical chemistry, coagulation profiles, and plasma drug concentration. At the termination of the experiment, each carotid was ligated proximal and distal to the point of injury, removed, and placed in fixative for histological analysis. At the end of the study, animals were euthanized with an overdose of sodium pentobarbital, performed in accordance with accepted American Veterinary Medical Association guidelines.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Chemical structure of RWJ-58259.

Platelet Aggregation and Coagulation Profiles. For in vitro aggregation studies, blood samples were obtained and platelet-rich plasma (PRP) was prepared as described previously (Derian et al., 1995). Platelets were pretreated with RWJ-58259 for 5 min before agonist stimulation. For ex vivo platelet aggregation studies, PRP prepared from treated and untreated animals was used as indicated above. The PRP aggregation assay was performed in the presence of 4 mM H-Gly-Pro-Arg-Pro-NH₂ to inhibit fibrin polymerization, specifically when -thrombin was the agonist. The concentrations of -thrombin used to stimulate PRP aggregation are consistent with the presence of endogenous antithrombins in plasma. The doses used were determined from preliminary studies to achieve maximal aggregation. The presence of H-Gly-Pro-Arg-Pro-NH₂ did not affect aggregation induced by peptide agonists. Aggregation was measured using an aggregation profiler (model PAP-4; Bio/Data, Horsham, PA).

Activated clotting time was determined using a Hemochron whole blood coagulation system model 801 (ITC, Edison, NJ). Prothrombin and activated partial thromboplastin times were determined using a hand-held monitor and cartridges (CoaguChek Plus system; Roche Diagnostics).

Histological Assessment of Carotid Thrombus. Formalin-fixed segments of injured carotids were embedded in paraffin and vessel cross sections were mounted and stained with hematoxylin and eosin to assess thrombus area. Thrombus composition was determined by single or double immunohistological staining of fibrin (anti-fibrin/fibrinogen; DAKO, Carpinteria, CA) and/or platelets (anti-CD61; DAKO) as described previously (D'Andrea et al., 2001). Fibrin-rich areas and platelet-rich areas were quantified by using ImagePro Image analysis software (Media Cybernetics, Silver Spring, MD). The total labeled area was expressed as a percentage of the total thrombus cross-sectional area. A representative section from each vessel was analyzed.

Data Analysis. All results are presented as mean ± S.E. Wilcoxon rank sum test was used for statistical analysis of occlusion times. One-way analysis of variance followed by Tukey's multiple comparison test was used for the platelet aggregation data. Student's t tests were used for the hematological and coagulation data and the histological platelet content data. Values were considered statistically significant when p < 0.05.

	Results

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Expression of PARs in Platelets of Cynomolgus Monkeys. We used the sensitive reverse transcriptase (RT)-PCR methodology to document the expression of PAR-1, PAR-3, and PAR-4 in platelets from cynomolgus monkeys. Total RNA was isolated from both monkey and human gel-filtered platelets, DNase treated, converted to cDNA by using RT, and subjected to PCR amplification. The sequences of the primers used to generate the respective PAR amplification products were designed from the conserved nucleic acid sequences of the known species PAR subtypes. The products of each PCR amplification were detected by Southern blot analysis using a nested oligonucleotide primer probe, corresponding to the appropriate PAR subtype. Transcripts for both PAR-1 and PAR-4 were detected in both human and monkey platelet RNA (Fig. 2). Under these conditions, the expression of PAR-3 was not detected in either human or monkey platelet RNA.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Platelet PAR profile for cynomolgus monkeys. RNA extracted from monkey (M) and human (H) platelets was subjected to PCR analysis after treatment with (+) or without (-) RT. Samples were examined for -actin to indicate that roughly equal amounts of RNA were analyzed. Platelets were positive for PAR-1 and PAR-4 by RT-PCR. The arrowhead denotes the expected location for PAR-3.

Arterial Thrombosis Injury Model. Based on the PCR profile of PARs, the cynomolgus monkey was deemed a suitable species to assess the importance of PAR-1 blockade on thrombotic occlusion. We used an experimental model of thrombosis involving electrolytic injury to the carotid arteries and subsequent thrombus formation, as assessed by absence of blood flow. The model was based on a modification of a similar model in dogs (Rote et al., 1994; Rebello et al., 1997). After intravenous infusion of RWJ-58259 or vehicle, the degree of vessel stenosis induced by electrolytic injury to each carotid artery independently was characterized by the incidence of occlusion and time to occlusion. Ten vessels from five vehicle-treated animals and nine vessels from five drug-treated animals were studied. One of the vessels in the RWJ-58259 group spontaneously occluded before treatment or electrolytic injury and was excluded from analysis. Application of anodal current to both carotid arteries of vehicle-treated animals led to total thrombotic occlusion in all vessels. The mean time to occlusion (t) was 27 ± 3 min (Fig. 3). RWJ-58259 significantly delayed or prevented occlusion in all of the vessels in five treated animals (t > 30 min) (Fig. 3). All carotid arteries from three of the five animals in the RWJ-58259 group remained patent during the 60-min observation period, despite continued application of current. Independent statistical comparisons between the right carotid artery (p < 0.004) and left carotid artery (p < 0.048) results from the vehicle and drug-treated groups indicated a significant effect of RWJ-58259 treatment on the prolonged time to occlusion.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Antithrombotic effect of RWJ-58259. Time-to-occlusion measurements are shown for individual animals after treatment with vehicle or RWJ-58259. The experiment was terminated at 60 min; thus, 60 min was used for calculations of mean time to occlusion in the insert. Solid columns represent the right carotid artery and hatched columns represent the left carotid artery. *, indicates no data. Inset, mean ± S.E. from all vessels.

In ex vivo platelet aggregation studies, aggregation in response to the PAR-1 agonist peptide SFLLRN-NH₂ and a low concentration of thrombin (38 nM) were completely inhibited both after the bolus dose and at the end of the observation period (Fig. 4). However, increasing concentrations of thrombin, which fully aggregated platelets in the absence of RWJ-58259, gradually overcame its inhibitory effect, consistent with its PAR-1 selectivity and the expression of PAR-4 on the platelets (Andrade-Gordon et al., 2001). Plasma levels of RWJ-58259 were 90 ± 15 µM at the end of the bolus infusion and 12 ± 3 µM (n = 5) at the end of the observation period. The latter concentration is consistent with inhibitory concentrations for PRP aggregation previously determined in human and guinea pig platelets (Andrade-Gordon et al., 2001). The PAR-1 selectivity of RWJ-58259 was confirmed in additional in vitro aggregation studies using monkey platelets. Agonist peptides for PAR-1 (SFLLRN) and PAR-4 (GYPGKF) stimulated full aggregation of monkey platelets (Fig. 5). RWJ-58259 completely inhibited SFLLRN-induced aggregation at a concentration of 1 µM, whereas doses as high as 20 µM had no effect on GYPGKF-induced aggregation, in agreement with its PAR-1 selectivity.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Effect of RWJ-58259 administration on ex vivo platelet aggregation. Aggregation studies were performed with PRP samples obtained after the bolus dose (solid columns) and at the termination of the study (hatched columns). Platelet aggregation was induced by 30 µM SFLLRN-NH2 or increasing concentrations of -thrombin. Results are mean ± S.E. for four animals. *, p < 0.01.

View larger version (9K): [in this window] [in a new window]   Fig. 5. RWJ-58259 selectively blocks PAR-1 in cynomolgus monkey platelets. PRP was stimulated with 10 µM SFLLRN (PAR-1-selective peptide) or 300 µM GYPGKF (PAR-4-selective peptide) to achieve maximal aggregation in the absence or presence of RWJ-58259, as described under Materials and Methods.

Thrombus Histology. Histological evaluation of the thrombi formed in the absence or presence of RWJ-58259 indicated significant differences in the composition of mural thrombi. Based on immunolabeling for the platelet marker CD61, there was a significant lack of platelet deposition in the existing thrombus of RWJ-58259-treated animals (Fig. 6, a and b). Immunolabeling of fibrinogen revealed the predominance of fibrinogen in the RWJ-58259 thrombi (Fig. 6, c and d). Representative sections from thrombi of vehicle and RWJ-58259-treated animals were subjected to image analysis to assess the overall composition of the thrombus. The platelet composition was reduced from 50 ± 3% (n = 5 vessels) in control-derived thrombi to 16 ± 8% (n = 5 vessels; p < 0.01) in RWJ-58259-derived thrombi. Thus, there seemed to be a switch from platelet-rich to platelet-poor thrombi in the presence of RWJ-58259

View larger version (82K): [in this window] [in a new window]   Fig. 6. Immunohistochemical staining of thrombus. a and c, representative thrombus from vehicle-treated animal; b and d, representative thrombus from RWJ-58259-treated animal. a and b, CD61 staining for platelets in thrombus represented by brown immunostaining. Scale bar, 250 µm. c and d, double immunohistochemical staining for platelets (CD61) and fibrin represented by brown and red staining, respectively. Scale bar, 100 µm.

Hematology Profile. We compared the hematological profile as well as several coagulation parameters at baseline and at the termination of the study in both control and RWJ-58259-treated animals. There were no significant differences in red blood cell count, hematocrit, white blood cell count, or platelet counts between the two treatment groups at the start of the experiment and no significant changes in these parameters over the course of the experiment in either group (Table 1). Coagulation parameters, including activated clotting time, prothrombin time, and activated partial thromboplastin time were also within normal ranges and comparable in control and treated animals (Table 2). In addition, there were no significant changes in these parameters over the course of the experiment in either treatment group. Standard clinical chemistry assessments were also unaffected (data not shown).

	Discussion

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Thrombin plays a pivotal role in the development of arterial thrombi through activation of platelets and formation of fibrin (Fenton et al., 1993; Harker et al., 1995). The entrapment of thrombin within the fibrin meshwork of a thrombus provides a local elevation of thrombin protected from endogenous plasma-associated inhibitors such as antithrombin-III and heparin cofactor-II. The microenvironment of the growing thrombus presents a protected milieu for activation of platelets and other cell types such as leukocytes, which are known to reside within the thrombus. Direct thrombin inhibitors such as hirudin, bivalirudin, argatroban, and ximelagatran have progressed clinically on the basis that they can penetrate and inactivate clot-bound thrombin in contrast to heparins (Bates and Weitz, 1998; Mehta et al., 1998). However, the targeting of thrombin's enzymatic activity leads to both cellular and noncellular perturbations, which affect thrombin's procoagulant and anticoagulant activities. Indeed, clinical studies with these agents indicate positive outcomes in limiting arterial thrombosis with moderate changes in coagulation parameters such as activated partial prothromboplastin time (Badimon et al., 1994; Gallo et al., 1999; Weitz and Buller, 2002). Nonetheless, one of the most critical parameters still to be addressed is the potential for bleeding associated with this treatment modality. Thrombin's cellular actions, which are mediated through protease-activated receptors, play a critical role in arterial thrombosis. Thus, selectively blocking thrombin's cellular actions while sparing its normal hemostatic functions provides an opportunity to intervene more specifically during the formation and growth of a thrombus. The results reported herein demonstrate that nonhuman primates, exemplified by cynomolgus monkeys, offer a viable model to assess the potential efficacy of a PAR-1 antagonist for the prevention of arterial thrombosis in humans. We have determined that antagonism of PAR-1 in nonhuman primates provides significant protection against vascular injury-induced thrombosis.

The primate model of thrombosis used in this study is based on and adapted from a similar model in dogs in which electrolytic injury is used to induce thrombosis in a carotid artery (Rote et al., 1994; Toombs et al., 1995; Rebello et al., 1997). A number of antithrombotic and antiplatelet compounds have been studied and shown to be effective in this model, including direct and indirect thrombin inhibitors (Sudo and Lucchesi, 1996; Rebello et al., 1997) and platelet GPIIb/IIIa antagonists (Rote et al., 1994; Sudo et al., 1995). Thus, the model in the dog seems to be both platelet- and thrombin-dependent. Because it is likely that the mechanisms for thrombosis caused by electrolytic injury in dog and primate are similar, the antithrombotic activity of the thrombin receptor antagonist in our primate model is consistent with its antiplatelet and antithrombin mechanism of action. Another primate model of thrombosis involves mechanical injury, in which cyclic flow reductions occur as a result of cyclic formation of occlusive thrombi (Cook et al., 1995; Bellinger et al., 1998). In this model, both a GPIIb/IIIa antagonist and a direct thrombin inhibitor are effective in inhibiting cyclic flow reductions (Cook et al., 1995).

The therapeutic potential of a PAR-1 antagonist as an antithrombotic agent was first reported in a study with an antibody directed to the extracellular PAR-1 domain that binds thrombin's exosite region with high affinity (Cook et al., 1995). In this model of mechanical injury in African Green monkeys, the disruption of thrombin/PAR-1 binding effectively limited experimental thrombosis. More recently, two small peptides were reported to exert antithrombotic actions via PAR-1 antagonism. The heptapeptide AFLARAA inhibited arterial thrombosis in a rabbit model of electrolytic injury (Pakala et al., 2000), and the peptide RPPGF delayed coronary occlusion in a canine model using electrolytic injury (Hasan et al., 1996, 1999). In both cases, however, the mechanism of action of these peptides is unclear because PAR-1 is not expressed in either rabbit or canine platelets (Connolly et al., 1994; Derian et al., 1995). The development of a selective, small-molecule PAR-1 antagonist, such as RWJ-58259, which directly blocks the tethered ligand of PAR-1, provides a distinct interventional approach to thrombin-induced PAR-1 activation (Zhang et al., 2001).

We previously reported that RWJ-58259 does not effectively block arterial thrombosis in two separate models of thrombosis in guinea pigs (Andrade-Gordon et al., 2001). The lack of antithrombotic efficacy associated with RWJ-58259 in guinea pigs, one of the few species with PAR-1 on its platelets, relates to the triple PAR profile of these platelets, which could provide two fully functional thrombin-activating pathways, PAR-1 and PAR-3/PAR-4 (Nakanishi-Matsui et al., 2000; Andrade-Gordon et al., 2001). This curious outcome compels the use of a higher mammalian species that has PAR-1 on its platelets, in a manner consistent with human platelets. Our RT-PCR results indicate that PAR-1 and PAR-4 are expressed in the platelets of cynomolgus monkeys; however, no detectable PAR-3 was noted. These results are consistent with previous reports indicating relatively low expression of PAR-3 in human platelets based on both RT-PCR and protein expression (Schmidt et al., 1998; Kahn et al., 1999). The PAR-1 selectivity of RWJ-58259 demonstrated from our aggregation studies in cynomolgus monkeys coupled with our antithrombotic study indicates that blockade of PAR-1 is sufficient to protect against a significant vascular insult even when functional PAR-4 is present on platelets.

The physiological role of PAR-4 is not well understood because it is activated by relatively high concentrations of thrombin. In vitro studies with human platelets suggest its activation may serve as a secondary mechanism to ensure an antiplatelet response during vascular injury where thrombin concentrations are significantly elevated (Kahn et al., 1999). Although the complement of PAR receptors on murine and human platelets is distinct, some parallels can be drawn from studies using PAR-3- and PAR-4-deficient mice and the potential effects of a PAR-1 antagonist. Platelets isolated from PAR-3-deficient mice are less sensitive to thrombin stimulation compared with wild-type platelets, similar to the higher concentrations of thrombin needed to activate human platelet PAR-4 (Kahn et al., 1998). Hence, PAR-3 deficiency mirrors the potential effect a PAR-1 antagonist might have on human platelet responses to thrombin where PAR-4 signaling remains intact. In contrast, PAR-4 deficiency in murine platelets renders platelets totally unresponsive to thrombin consistent with its primary role as the thrombin-sensitive receptor and the cofactor role of PAR-3 (Sambrano et al., 2001). In vivo studies with PAR-4-deficient mice indicate that disabling of this primary thrombin-sensitive PAR provides protection from arteriolar thrombosis. This observation is consistent with blockade of PAR-1 on monkey or human platelets where PAR-1 is the primary thrombin-sensitive receptor and supports the concept that PAR-1 antagonism will provide sufficient antithrombotic efficacy after vascular injury. More recently, in vivo studies in PAR-3-deficient mice have shown a similar outcome (Weiss et al., 2002). The protection in these mice may again be attributed to the loss of platelet sensitivity to low nanomolar concentrations of thrombin, thus potentially shifting the response to one analogous to a human PAR-4 activation. Although direct associations cannot be made between the murine and primate studies, it is clear that thrombin's prothrombotic capacity is dependent on the complement and characteristics of the PAR profile.

There is increased bleeding in PAR-4-deficient mice, which indicates that PAR-4 plays a significant role in normal hemostasis. The lack of a secondary thrombin-activating pathway in murine platelets compared with human platelets, which have PAR-1 and PAR-4 acting independently, suggests that PAR-4 contributes to the maintenance of normal hemostasis in monkeys and humans. A potential role for PAR-4 in stabilizing platelet aggregates was revealed in studies with platelets from a patient with Hermansky-Pudlak Syndrome, a storage-pool deficiency (Covic et al., 2002). The mild bleeding diathesis observed in this patient was attributed to PAR-4 activation as a compensatory mechanism for ADP deficiency, resulting in relatively normal platelet aggregation. The ability to activate PAR-4 under conditions of exceptionally elevated thrombin concentrations thus allows for a fail-safe mechanism for maintaining hemostasis. Accordingly, we did not observe any effects on various coagulation parameters after blockade of PAR-1 with RWJ-58259. On the basis of our results, an avenue is now available to explore the role of platelet PAR-1 in human vascular occlusive disease. In this light, we propose that a selective PAR-1 antagonist has the potential for significant utility in cardiovascular therapeutics.

	Footnotes

 
DOI: 10.1124/jpet.102.042663.

ABBREVIATIONS: PAR, protease-activated receptor; PCR, polymerase chain reaction; PRP, platelet-rich plasma; RT, reverse transcriptase; RWJ-58259, (S)-N-[(1S)-3-amino-1-[[(phenylmethyl)amino]carbonyl]propyl]--[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide.

Address correspondence to: Dr. Patricia Andrade-Gordon, Vice President, Drug Discovery, Johnson Johnson Pharmaceutical Research and Development, Welsh and McKean Rds., P.O. Box 776, Spring House, PA 19477. E-mail: pandrade{at}prdus.jnj.com

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