Introduction

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-Thrombin is a powerful agonist for a variety of cellular responses, and these actions are mediated by a special type of G protein-coupled transmembrane receptor known as a protease-activated receptor (PAR). Important biological effects of thrombin are mediated by such PARs in platelets, fibroblasts, monocytes, neutrophils, osteoblast-like cells, smooth muscle cells, nerve cells, and endothelial cells (Coughlin, 1994; Dennington and Berndt, 1994; Ogletree et al., 1994; Van Obberghen-Schilling et al., 1995). Perhaps the best-characterized receptor function of thrombin is the activation of platelets, which is a crucial process in thrombosis and hemostasis. Thrombin is the most potent stimulator known of platelet aggregation and degranulation, and it may also be the most significant mediator of platelet recruitment during arterial thrombus formation. Thrombin-induced aggregation of human platelets is mediated by two PARs, PAR-1 and PAR-4 (Kahn et al., 1999), whereas PAR-1 is not relevant to thrombin-induced aggregation of rat or mouse platelets (Connolly et al., 1994; Derian et al., 1995). This species dependence makes it problematic to derive a good understanding of the in vivo physiology associated with different PARs.

The role of thrombin receptor activation in thrombosis and hemostasis could be demonstrated more clearly with specific pharmacological agents that can interrupt receptor function. Recently, we identified a series of potent, indole-based peptide-mimetic PAR-1 antagonists, represented by RWJ-56110, the biological function of which was characterized in vitro (Andrade-Gordon et al., 1999). This antagonist was very selective in blocking the actions of PAR-1 over the actions of PAR-2, PAR-3, or PAR-4. Interestingly, we found that the inhibitory effect of RWJ-56110 in thrombin-induced human platelet aggregation is attenuated at high enzyme levels (e.g., 8 nM), consistent with PAR-4 playing a role in thrombin signaling (at the elevated concentrations). This scenario raises a serious question about the ability of a PAR-1 antagonist to serve effectively as an antithrombotic agent, an issue of keen interest since the identification of PAR-1 in 1991 (Vu et al., 1991). We report herein the first in vivo investigation of the antithrombotic effects of a potent peptide-mimetic PAR-1 antagonist, RWJ-58259, by using two standard animal models. Since thrombin is implicated in the proliferative and inflammatory events associated with restenosis, we have also investigated the effects of RWJ-58259 in a rat model of vascular injury. Our results clearly suggest that a PAR-1 antagonist has the potential for therapeutic utility in restenosis following balloon angioplasty.

	Experimental Procedures

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Materials. RWJ-58259 was synthesized in our laboratories, purified by flash-column chromatography, and isolated as a dihydrochloride dihydrate (off-white powder). Details on the synthesis and isolation will be published separately. The structure of RWJ-58259 was confirmed by NMR spectroscopy and mass spectrometry; the purity was established by elemental microanalysis and reverse-phase high-pressure liquid chromatography.

Platelet Aggregation. Human platelet-rich plasma concentrate containing the anticoagulant acid-citrate dextrose (Biological Specialty Corp., Colmar, PA) was gel-filtered (Sepharose 2B, Amersham Pharmacia Biotech Inc., Piscataway, NJ) in Tyrode's buffer (140 mM NaCl, 2.7 mM KCl, 12 mM NaHCO₃, 0.76 mM Na₂HPO₄, 5.5 mM dextrose, 5.0 mM Hepes, and 2 mg/ml bovine serum albumin, pH 7.4). Gel-filtered platelets were diluted with Tyrode's buffer (143,000 platelets/µl per well), compound solution in buffer, and 2 mM CaCl₂ in a 96-well microtiter plate. All fresh blood samples were obtained using sodium citrate (0.38% final concentration) as the anticoagulant. For platelet-rich plasma studies, human blood was obtained by venipuncture from healthy volunteers who were drug free for a minimum of 10 days. Guinea pigs (Hartley; Covance Inc., Denver, PA) or rats (Sprague-Dawley, Charles River, Raleigh, NC) were anesthetized and blood drawn via an intra-arterial catheter. Platelet-rich plasma was prepared by centrifugation at 200g for 10 min. Platelet-rich plasma aggregation was performed in the presence of 4 mM H-Gly-Pro-Arg-Pro-NH₂ to inhibit fibrin polymerization. Platelet aggregation was initiated by addition of an agonist shown to achieve 80% aggregation. The -thrombin concentrations for gel-filtered platelet and platelet-rich plasma aggregation studies were 0.15 and 7.5 nM, respectively. The SFLLRN-NH₂ concentration used was 2 µM. The assay plate was gently mixed constantly. Aggregation was monitored at 0 and 5 min after agonist addition in a microplate reader by optical density at 650 nm (Molecular Devices, Sunnyvale, CA). Aggregation was calculated as the decrease in optical density between the two measurements. All samples were tested in duplicate wells on the same plate.

Cell Cultures. Human aortic smooth muscle cells and growth media were obtained from Cascade Biologics (Portland, OR). Rat aortic smooth muscle cells were obtained from Cell Applications (San Diego, CA) and were cultured as described (Owens et al., 1986).

Calcium Mobilization. Intracellular calcium mobilization was measured using a fluorescence technique. Rat aortic smooth muscle cells in 96-well microtiter plates were loaded with 5 µM fluo-3-AM (Molecular Probes, Eugene, OR) for 90 min. Plates were washed five times to remove unincorporated dye. Subsequent steps were performed using a fluorometric imaging plate reader (FLIPR, Molecular Devices). Test compounds were added and cells were monitored for 5 min to detect any inherent agonist activity. Thrombin (2 nM) was added and the fluorescence signal was recorded for 3 min. Net peak calcium, expressed in arbitrary fluorescence units, was measured automatically.

DNA Synthesis. Cell proliferation was measured by [¹⁴C]thymidine incorporation. Rat aortic smooth muscle cells were plated on Cytostar scintillating plates (Amersham). After 4 days of growth, cells were depleted of serum for 4 days (Owens et al., 1986). Thrombin (0.8 nM) was added in fresh media and cells were incubated for 24 h. [¹⁴C]Thymidine was added and incubation was continued for 24 h. [¹⁴C]Thymidine incorporation was measured in a Wallac MicroBeta counter (Wallac, Gaithersburg, MD) without additional processing steps.

Interleukin-6 Assay. For measurement of interleukin-6 release, human aortic smooth muscle cells plated on 96-well microtiter plates were quiesced in Medium 231 (Cascade) containing 0.5% fetal bovine serum for 3 days. Thrombin (2 nM) was added in fresh serum-free Medium 231 and supernatants were collected after overnight incubation. Samples were analyzed by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN).

PCR Analysis for PAR-1 and PAR-4. Total RNA was isolated from guinea pig washed platelets using Trizol Reagent (Life Technologies, Grand Island, NY). For conversion of RNA to first-strand cDNA, samples were incubated with random primers in the presence or absence (minus RT for negative controls) of Superscript II reverse transcriptase (Life Technologies) according to the manufacturer's recommendations. PCR reactions were carried out on ca. 50 ng of cDNA, or equivalent amounts of RNA in the RT reactions, using the Advantage-GC cDNA polymerase mix (CLONTECH, Palo Alto, CA). Primers to generate and detect the respective guinea pig PAR amplicons were designed using the nucleic acid alignments of the known species for PAR-1 and PAR-3. However, numerous attempts to use this strategy to detect guinea pig PAR-4 were unsuccessful. Thus, the sequences used to amplify and detect the guinea pig PAR-4 PCR product were designed from the partial sequence analysis of the guinea pig PAR-4 gene (manuscript in preparation). The sense and antisense primers used for the amplification of PAR sequences were: PANP1-U, 5'-CATAAGCATTGACCGGTTCCTGGC-3'; PANP1-L, 5'-CAAAGCAGACGATGAAGATGCAGA-3'; PANP3-U, 5'-CAATGGCAACAACTGGGTATTTGG-3'; PANP3-L, 5'-AAAATCACAAGGATGAGGAG-3'; GPPANP4-U, 5'-TGGCCGTGGGGCTGCCGGCC-AATG-3'; and GPPANP4-L, 5'-GTCAACACAGCTGTTGAGGGTGCT-3'.

Animal Models. 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 Committee, The R. W. Johnson Pharmaceutical Research Institute, Spring House, PA.

Ex Vivo Platelet Aggregation. RWJ-58259 was administered i.v. to anesthetized guinea pigs at the indicated doses as a 5 min infusion. Blood was withdrawn 5 min after dosing. Inhibition of thrombin or SFLLRN-induced platelet aggregation was assessed using platelet-rich plasma.

Guinea Pig Arteriovenous Shunt Thrombosis Model. Adult male guinea pigs (Hartley, 600-750 g) were anesthetized with a ketamine hydrochloride/xylazine hydrochloride solution i.m. The left jugular vein was cannulated (PE-50) for drug administration. The left carotid artery and right jugular vein were cannulated with silicon treated (Sigmacote, Sigma Chemical, St Louis, MO), saline-filled polyethylene tubing (PE-60) and connected with a 6-cm section of silicon-treated tubing (PE-190) to form an extracorporeal arteriovenous shunt. Shunt patency was monitored using a Doppler flow system (model VF-1, Crystal Biotech Inc., Hopkinton, MA) and flow probe (1.0 mm, Titronics, Iowa City, IA) placed proximal to the shunt.

Intravascular Photoactivation Model. Male guinea pigs (Hartley, 375-700 g) were anesthetized with ketamine/xylazine (90/12 mg/kg, i.m.) and the right carotid artery gently isolated from the surrounding connective tissue. A 1-mm ultrasonic Doppler flow probe was secured around the artery proximal to the occlusion area and flow was continuously measured. Rose Bengal (Sigma), a photoactive dye, was dissolved in saline and infused i.v. at 20 mg/kg over 10 min. A green, heat-filtered xenon light source, positioned 0.5 cm from the artery to illuminate a 1-cm length of the vessel, was turned on 5 min before Rose Bengal infusion and remained on for 15 min. Arterial flow was monitored for a total of 30 min following the start of the Rose Bengal infusion. RWJ-58259 was administered at a total dose of 10 mg/kg, i.v., split into a 5 mg/kg infusion for 10 min prior to Rose Bengal and 5 mg/kg infusion starting after the conclusion of the Rose Bengal infusion for the remaining 20 min of the 30-min observation period. Recombinant hirudin (Hoechst Marion Roussel, Kansas City, MO) was infused at either 1 or 3 mg/kg i.v. for 10 min prior to the Rose Bengal infusion. RWJ-58259 was dissolved in 5% dextrose and r-hirudin was dissolved in saline. In a separate group of RWJ-58259-treated guinea pigs, not exposed to Rose Bengal or light, the animals were exsanguinated, platelet-rich plasma was prepared, and ex vivo platelet aggregation to -thrombin and SFLLRN-NH₂ was measured.

Rat Restenosis Model. Vascular injury was induced by balloon-catheter inflation of the rat common carotid artery. A 2F embolectomy catheter was inserted via the external carotid into the left common carotid of male Sprague-Dawley rats (350-450 g) anesthetized with ketamine/xylazine (75/5 mg/kg, i.m.). The balloon tip was advanced to the aorta, inflated to 35 psi, and slowly withdrawn a total of three times. RWJ-58259 (1, 5, or 10 mg) was suspended in 150 µl of a polymer gel consisting of 50% capryolate and 50% glycolate and applied to the adventitia of the left common carotid. This polymer was shown not to affect the vascular injury response in this model. Perivascular treatment was used for these studies because RWJ-58259 is not orally active. Required intravenous infusion rates were not practical via minipump. This particular polymer has been successfully used for slow release of compounds. Since the material is absorbed slowly, we anticipated that RWJ-58259 would be released slowly over a period of time. Release kinetics were not performed for these studies. However, material tends to stay where placed and compound concentrations are expected to be high locally, likely resulting in significant levels reaching the luminal edge of the vessel. Fourteen days after injury, rats were anesthetized and perfusion-fixed with buffered formalin. Eight left carotid tissue sections (5 µm, 100 µm apart) were stained for elastin and used for morphometric analysis (Cheung et al., 1999). Medial and intimal area and thickness were measured using image analysis software. Percent stenosis was computed as intimal area as a percentage of the total area within the internal elastic lamina.

Data Analysis. All results are presented as mean ± S.E. Statistical analysis was performed either by the Student's t test or one-way analysis of variance where indicated. Mean values were considered statistically significant when P < 0.05.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

RWJ-58259 Is a Potent PAR-1 Antagonist. We recently described a series of indole-based peptide mimetics represented by RWJ-56110, which inhibits thrombin-induced PAR-1 activation in human platelets and vascular cells (Andrade-Gordon et al., 1999). Replacement of the indole template with an indazole template afforded an improved chemical series, represented by RWJ-58259 (Fig. 1). We selected this PAR-1 antagonist for animal studies because of its good potency, PAR-1 selectivity, and particularly, in vivo safety profile.

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

Effects of RWJ-58259 on Guinea Pig Platelets. Guinea pig platelets have been widely used to test for PAR-1 action in platelet aggregation because they are responsive to the PAR-1-activating peptide SFLLRN-NH₂ (Connolly et al., 1994; Derian et al., 1995), which indicates the presence of functional PAR-1 on the cell surface. Since guinea pig platelets have a lot in common functionally with human platelets, we chose this small animal to explore PAR-1 antagonism in vivo. Our previous findings with the PAR-1 antagonist RWJ-56110 (Andrade-Gordon et al., 1999) confirmed the dual PAR activation system on human platelets. Given this background, we evaluated RWJ-58259 with guinea pig platelets for a similar mode of action. RWJ-58259 inhibited 7.5 nM -thrombin-induced platelet-rich plasma aggregation with an IC₅₀ of 7.4 ± 1.4 µM (n = 5), consistent with results from human platelet-rich plasma studies. Moreover, at a 10-fold higher concentration of -thrombin, no inhibition was observed up to 100 µM RWJ-58259. In contrast, RWJ-58259 fully inhibited supramaximal concentrations of SFLLRN-NH₂ (100 µM)-mediated aggregation at a concentration of 10 µM. These results are indicative of another thrombin-sensitive receptor on guinea pig platelets in addition to PAR-1, as noted previously for human platelets. Because of the similarities between the in vitro behavior of RWJ-58259 in both human and guinea pig platelets, we considered this to be a suitable animal model for the investigation of PAR-1 physiology.

RWJ-58259 Inhibits ex Vivo Guinea Pig Platelet Aggregation. RWJ-58259 was first evaluated in a model of ex vivo platelet-rich plasma aggregation to determine the appropriate in vivo concentration ranges for further studies. RWJ-58259, administered to guinea pigs (0.3-3 mg/kg), inhibited -thrombin-induced platelet-rich plasma aggregation in a concentration-dependent manner (Fig. 2). However, as -thrombin concentrations were raised, RWJ-58259 became less effective, indicating that its ability to inhibit thrombin-mediated responses in vivo is dependent on the thrombin concentration. RWJ-58259-inhibited SFLLRN-induced aggregation under all conditions (results not shown). A dose of 10 mg/kg was chosen for further evaluations based on these results as well as pilot studies with earlier analogs including RWJ-56110, which was ineffective at 6 mg/kg. This dose was the maximally tolerated intravenous dose for RWJ-58259.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects on ex vivo platelet-rich plasma aggregation after i.v. administration of RWJ-58259. Platelet-rich plasma aggregation in response to increasing concentrations of thrombin was inhibited after 0.3, 1, and 3 mg/kg of RWJ-58259.

Guinea Pig Arteriovenous Shunt Thrombosis Model. In this thrombosis model, a thrombus comprised of platelets, fibrin, and red blood cells forms on a section of cotton thread placed in an extracorporeal shunt between the carotid artery and jugular vein. Antithrombotic efficacy is indicated by decreases in the weight of thrombus accumulated during 15 min of exposure to flowing blood. Intravenous administration of RWJ-58259 (10 mg/kg) did not reduce thrombus weight (42 ± 4 mg, n = 2) when compared with a control group (43 ± 2 mg, n = 15) even though -thrombin and SFLLRN-induced platelet-rich plasma aggregation were completely inhibited (Fig. 3A). The direct thrombin inhibitor inogatran (1 mg/kg, i.v.) or aspirin (100 mg/kg, i.v.) significantly decreased thrombus weight to 18 ± 3 mg (n = 6) and 16 ± 1 mg (n = 4), respectively. In a separate group of guinea pigs, RWJ-58259 was administered directly into the shunt just proximal to the thread in a protocol to maximize potential antithrombotic efficacy. An infusion rate of 0.1 mg/kg/min (2.0 mg/kg total dose) decreased thrombus weight slightly from a control of 35 ± 2 mg (n = 5) to 28 ± 4 mg (n = 4) (Fig. 3B). Increasing the infusion rate to 0.3 mg/kg/min (6.0 mg/kg total dose) further decreased thrombus weight to 24 ± 4 mg (n = 3). In these studies, the drug concentration (22 µM and 66 µM, respectively) was high enough to effectively inhibit -thrombin and SFLLRN-induced platelet-rich plasma aggregation. Higher doses of RWJ-58259 could not be evaluated due to a combination of drug solubility and infusion volume. Bleeding times and activated clotting times were not changed. By comparison, administration of inogatran directly into the shunt at a rate of 0.01 mg/kg/min (0.2 mg/kg total dose) significantly decreased thrombus weight to 14 ± 2 mg (n = 3).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of RWJ-58259 (RWJ) in the guinea pig A-V shunt antithrombotic model. A, intravenous administration of RWJ-58259; B, intrashunt administration of RWJ-58259. Left panels, antithrombotic efficacy measured as reduction of thrombus weight compared with vehicle control; right panels, ex vivo platelet-rich plasma aggregation induced by 35 nM -thrombin or 50 µM SFLLRN-NH2. *P < 0.05 compared with vehicle treated by Student's paired t test.

Guinea Pig Photoactivation Thrombosis Model. Intravascular photoactivation of the dye Rose Bengal with a green, heat-filtered xenon light results in endothelial damage that stimulates platelet adhesion to the vessel wall and generalized initiation of a platelet-rich thrombo-occlusive event. Antiplatelet agents and to a lesser extent, anticoagulants are effective in this model. Figure 4 (top panel) depicts carotid arterial perfusion and occlusion, as measured by Doppler flow, with each bar representing an individual animal. Initial occlusion times for the saline- and dextrose-treated animals averaged approximately 15 min. In all vehicle-treated animals except one, the arterial occlusion remained stable, whereas in the drug-treated groups the occlusion was unstable with intermittent flow observed over 30 min. At 1 mg/kg of the thrombin inhibitor r-hirudin, three of six treated animals were flowing at 30 min, and two of six did not experience occlusion. Two of eight RWJ-58259 treated animals were flowing at 30 min and one of eight did not experience occlusion. Total cumulative perfusion times (Fig. 4, middle panel) were significantly extended by r-hirudin at 1 and 3 mg/kg. RWJ-58259 at 10 mg/kg tended to increase perfusion times but this effect was not significant. RWJ-58259 significantly inhibited thrombin and SFLLRN-NH₂-induced platelet aggregation ex vivo (Fig. 4, bottom panel). Aggregation to low concentrations of -thrombin (7-25 nM) was significantly inhibited, whereas aggregation at higher concentrations was much more variable and was determined not to be significantly different from that in the untreated animals. Aggregation to SFLLRN-NH₂ was completely inhibited by RWJ-58259 at all concentrations evaluated.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of RWJ-58259 in the guinea pig photoactivation model. Top panel, carotid arterial perfusion with each bar representing individual animals treated with vehicle, r-hirudin, or RWJ-58259; light shaded areas, flow; dark areas, no flow. Middle panel, average total, cumulative, perfusion time for each group. *P < 0.05 compared with vehicle control by Student's paired t test. Bottom panel, ex vivo aggregation of platelets prepared from control (, n = 3) and RWJ-58259-treated (, n = 6) guinea pigs.

Guinea Pig Platelet PAR Profile. In vitro studies with our selective PAR-1 antagonist RWJ-58259 indicated the presence of more than one thrombin receptor on both human and guinea pig platelets. Furthermore, results from the guinea pig in vivo thrombosis models suggested that another thrombin receptor, possibly PAR-4, plays a role in platelet-dependent thrombosis. Therefore, it was necessary to characterize the thrombin-receptor profile of guinea pig platelets. Although PAR-1 has been cloned from several species, only human and murine PAR-4 have been cloned and characterized (Kahn et al., 1998; Xu et al., 1998). Thus, we tested human PAR-1 (SFLLRN-NH₂)-, human PAR-4 (GYPGQV-NH₂)-, and murine PAR-4 (GYPGKF-NH₂)-activating peptides on human, rat, and guinea pig platelets. Whereas the human PAR-1 and PAR-4 peptides induced human platelet aggregation and the human and murine PAR-4 peptides induced rat platelet aggregation (no PAR-1 in rat platelets), only the PAR-1 peptide induced guinea pig platelet aggregation (not the PAR-4 peptides; results not shown). This outcome agrees with a recent communication by Nishikawa et al. (2000), in which washed guinea pig platelets do not respond to the murine PAR-4 peptide up to a concentration of 1 mM. To follow up on this observation, we examined the constitution of PARs in isolated guinea pig platelets by RT-PCR and were able to detect the mRNAs corresponding to PAR-1, PAR-3, and PAR-4 (Fig. 5). The apparent paradox of guinea pig platelets containing the message for PAR-4, but failing to respond to the human or murine PAR-4 peptides, was probed by isolating the guinea pig PAR-4 gene and characterizing the second exon.¹ Like the genomic organization of other so-characterized PARs, exon 2 of the guinea pig PAR-4 gene contains the coding sequences of the entire receptor, without the initiation codon and signal sequence. Interestingly, sequence analysis revealed that guinea pig PAR-4 contains the activation motif SFPGQA, which diverges from the motifs in human (GYPGQV) or murine (GYPGKF) PAR-4. We synthesized SFPGQA-NH₂ and found that it does induce the aggregation of guinea pig platelets with an EC₅₀ of 131 µM. This result illustrates a notable flexibility in the evolution of the PAR-4 gene. In the final analysis, guinea pig platelets possess two functional thrombin-responsive systems, PAR-1 and PAR-3/PAR-4.

View larger version (96K): [in this window] [in a new window]   Fig. 5.   PAR profile in guinea pig platelets. RT-PCR was used to examine the presence of PAR-1, PAR-3, and PAR-4 mRNA in guinea pig platelets. The predicted sizes of the amplicons, detected following hybridization with the appropriate nested primer probe, are as follows: PAR-1, 395 bp; PAR-3, 468 bp; and PAR-4, 746 bp.

Effects of RWJ-58259 in a Rat Restenosis Model. Since -thrombin-mediated vascular smooth muscle cell responses associated with vascular injury (inflammatory cytokine release and cell proliferation) were inhibited by RWJ-58259, this agent would be a good candidate to assess the role of PAR-1 in a rat balloon angioplasty model of vascular injury. Furthermore, since rat platelet aggregation stimulated by -thrombin was not inhibited by RWJ-58259, confirming the lack of PAR-1 on these cells as well as the PAR-1 selectivity of RWJ-58259, this in vivo model would reflect effects directly on the vasculature. Perivascular treatment (1, 5, 10 mg) with RWJ-58259 produced dose-related reductions in intimal area and thickness, and a decrease in percent stenosis (Table 1), which became statistically significant at the 10 mg dose. Medial area and thickness were not changed, resulting in a significant reduction in the intimal to medial ratio. There was no evidence of an effect on remodeling. There was a trend toward increased lumen area at the 10 mg dose, but this was not significant. There was no significant difference in the vessel size among the treatment groups. An example of the effect of RWJ-58259 on vascular injury is shown in Fig. 6. Thus, there is a clear reduction in neointimal thickness in the section from a rat treated with RWJ-58259 compared with a section from a rat treated with vehicle. These results indicate that inhibition of thrombin-induced activation of PAR-1 in vivo can reduce the vascular injury response.

View larger version (107K): [in this window] [in a new window]   Fig. 6.   Examples of rat carotid artery changes 14 days after vascular injury. A, uninjured, vehicle-treated; B, injured, vehicle-treated; C, uninjured, RWJ-58259-treated; and D, injured, RWJ-58259-treated. Scale bar, 100 µm.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The thrombin receptor PAR-1 has been implicated in a variety of cellular events mediated by thrombin, including those associated with thrombosis and vascular injury. In this report, we have demonstrated that PAR-1 is involved in the restenotic events associated with balloon angioplasty in rats by using a potent, selective PAR-1 antagonist, RWJ-58259. Furthermore, results with RWJ-58259 in two different guinea pig thrombosis models reveal that PAR-1 may partially mediate platelet-dependent thrombus generation; however, there are serious concerns about the suitability of this, and other, species for such antithrombotic studies.

Antithrombotic Effect of RWJ-58259. The presence of divergent thrombin-receptor profiles for platelets of different species was first recognized in studies employing the PAR-1 agonist peptide SFLLRN (Connolly et al., 1994; Derian et al., 1995). Platelets isolated from the blood of humans, primates, and guinea pigs, but not rabbits, rodents, and dogs, were responsive to SFLLRN, although all of the species responded to thrombin. Based on the species studies, we reasoned that the guinea pig would provide an appropriate small-animal model to assess platelet PAR-1-dependent responses associated with thrombosis. In both models evaluated, inhibition of thrombin's proteolytic activity resulted in significant antithrombotic effects, confirming a significant role for thrombin-mediated thrombus formation. Our results with RWJ-58259 revealed just a modest effect on thrombus formation in the two guinea pig models, raising the distinct possibility that PAR-1 is not a significant contributor to platelet thrombus formation. Our in vitro and in vivo platelet aggregation results with RWJ-58259 indicated that it is an effective antagonist of guinea pig PAR-1; however, its effectiveness was dependent on thrombin concentration. Complete antagonism of thrombin in vitro was achieved at low thrombin concentrations, but the effect diminished as the thrombin levels rose above 10 nM. Thus, we hypothesized that another thrombin-responsive receptor existed on guinea pig platelets.

Antirestenotic Action of RWJ-58259. Vascular injury associated with angioplasty procedures results from both thrombotic and restenotic components. While our results with RWJ-58259 in the thrombosis models did not conclusively determine the impact of PAR-1 antagonism on thrombotic processes, RWJ-58259 showed significant inhibition of neointimal thickening in the rat model of vascular injury, consistent with a direct effect on PAR-1-mediated vascular smooth muscle function. These results are highly significant, since rat platelets are fully responsive to thrombin through PAR-3/PAR-4 activation. Our results are consistent with a recent study that showed a reduced vascular injury response in rats treated with an antibody to PAR-1 (Takada et al., 1998). We have also found that the vascular injury response is reduced in mice deficient in PAR-1 compared with wild-type mice (Cheung et al., 1999).