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CARDIOVASCULAR

The Thromboxane Receptor Antagonist PBT-3, a Hepoxilin Stable Analog, Selectively Antagonizes the TP Isoform in Transfected COS-7 Cells

Programme in Integrative Biology, Research Institute, the Hospital for Sick Children, Toronto, Canada (N.Q., D.R., P.D., C.R.P.-A.); Departments of Cell and Molecular Pharmacology and Experimental Therapeutics and Medicine, Medical University of South Carolina, Charleston, South Carolina (P.H.); and Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto, Canada (C.R.P.-A.)

Received for publication July 7, 2003
Accepted September 8, 2003.

	Abstract

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The hepoxilin analog PBT-3 [10(S)-hydroxy-11,12-cyclopropyleicosa-5Z,8Z,14Z-trienoic acid methyl ester] was previously shown to inhibit the aggregation of human platelets and to antagonize the binding of the thromboxane receptor agonist I-BOP [[1S-[1,2 (Z),3(1E,3S*),4]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid] in human platelets (Pace-Asciak et al., 2002). We show herein that PBT-3 inhibits, to different degrees, binding of the TP receptor antagonist [³H]SQ 29,548 [[1S-[1,2 (Z),3,4]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2. 1]hept-2-yl]-5-heptenoic acid], to the TP receptor isoforms in TP- and TP-transfected COS-7 cells. These isoforms possess a different tail length, the being shorter than the isoform. In contrast, SQ 29,548 shows no selection for the two TP isoforms. The IC₅₀ value for PBT-3 = 2.0 ± 0.3 x 10^–7 M was observed for TP, whereas this was one-sixth less active on the TP isoform (IC₅₀ = 1.2 ± 0.2 x 10^–6 M), suggesting selectivity for the TP isoform. To investigate whether the tail contributes to the difference in competition binding by PBT-3, we investigated the tailless TP isoform expressed in transfected COS-7 cells. Its IC₅₀ was similar to that of the TP isoform. In additional studies, we investigated the effect of PBT-3 on the collagen and I-BOP evoked intracellular calcium release and on the collagen and I-BOP evoked phosphorylation of pleckstrin. PBT-3 blocked both pathways further demonstrating its TP receptor antagonist activity. These results demonstrate that the action of PBT-3 in inhibiting platelet aggregation is mediated via inhibition of the TP isoform of the thromboxane receptor and that the tail may play an important role in recognition of this TP receptor antagonist.

Activation of TXA₂ (TP) receptors induces platelet aggregation, constriction of vascular and bronchiolar smooth muscle cells, as well as mitogenesis and hypertrophy of vascular smooth muscle cells (Moncada and Vane, 1979; Packham, 1993). TXA₂ formation is increased in thrombotic disorders (Fitzgerald et al., 1987) and has been implicated in a variety of cardiovascular diseases (Smith, 1992).

TXA₂ exerts intracellular effects by interaction with selective members of the G protein-coupled receptor family (Coleman et al., 1994; Watson and Arkinstall, 1994). The receptor seems to be encoded by a single gene that can be alternatively spliced in the carboxyl-terminal tail (C-tail), leading to two variants, termed TP and TP, which share the first 328 amino acids. Complementary DNAs for the 343-amino acid TP were cloned from placental and megakaryocytic sources (Hirata et al., 1991), whereas a cDNA for the 407-amino acid TP was isolated from a vascular endothelial library (Raychowdhury et al., 1994). The receptors have been shown to have different C-tails that are likely to play a role in G protein-coupling specificity and different desensitization characteristics (Hirata et al., 1996).

The TP receptor is abundantly expressed at both the mRNA and protein level in tissues of relevance to TXA₂ biology, such as platelets, vascular and uterine smooth muscle, uterus and placental tissue, endothelium, epithelium, trophoblasts, thymus, liver, and small intestine with TP expression predominating in most tissues examined (Miggin and Kinsella, 1998). There is evidence that mRNA for both isoforms is coexpressed in platelets, endothelial cells, and a number of other cell/tissue types with significantly greater levels of TP than TP expressed. Isoform-selective antibodies permitted detection of TP, but not TP, in human platelets, leading to the suggestion that TP may be the predominant isoform in platelets, despite the presence of mRNA for both isoforms in these cells (Miggin and Kinsella, 1998).

TP receptors have also been cloned from K562 cells, HEL cells, endothelial cells, mouse lung, rat kidney, and rat astrocytes (Namba et al., 1992; D'Angelo et al., 1994; Abe et al., 1995; Kitanaka et al., 1995; Allan et al., 1996). The cloned receptor has been expressed in several cell lines, including HEK293, Chinese hamster ovary cells, COS-1, and COS-7 cells (Ushikubi et al., 1989; Funk et al., 1993). These cell lines have been used in the past to express and characterize cloned receptors. COS-7 cells are a simian virus 40-transformed African Green monkey kidney cell line that has been used extensively for the transient expression of cloned receptors (Becker et al., 1998) and can produce desirable expression efficiency.

Hepoxilins are hydroxy epoxide metabolites of arachidonic acid formed via the 12-lipoxygenase pathway (Pace-Asciak et al., 1995). We have previously shown that the hepoxilins possess a variety of biological actions in vitro as well as in vivo, namely, they cause the release of intracellular calcium from calcium stores in human neutrophils (Dho et al., 1990; Laneuville et al., 1993), and the release of insulin from pancreatic beta cells in vitro (Pace-Asciak and Martin, 1984) and into the circulation of rats in vivo (Pace-Asciak et al., 1999). Because the native hepoxilins are rather unstable, we prepared a series of chemically stable analogs for in vivo use (Demin and Pace-Asciak, 1993). During more comprehensive screening for in vitro biological actions, we discovered that one of these analogs, PBT-3, potently inhibited collagen-evoked aggregation of human platelets (Pace-Asciak et al., 2002).

The present study was designed to further investigate PBT-3 binding to TP receptor isoforms transiently expressed in COS-7 cells. Our results demonstrate significant selectivity of PBT-3 to the TP isoform, which is the isoform most abundant in human platelets.

	Materials and Methods

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Materials. PBT-3 was prepared in our laboratory as described previously (Demin and Pace-Asciak, 1993). [1S-[1,2 (Z),3(1E,3S*),4]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP), iodophenyl sulfonyl amino pinane thromboxane A₂ (I-SAP), 9,11-(dimethyl) methylene-15S-hydroxy-11a-deoxy-11a-methylene-thromba-5Z,13E-dien-1-oic acid (pinane thromboxane A₂), and [1S-[1,2 (Z),3,4]]-7-[3-[[2-[(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ 29,548) were purchased from Cayman Chemicals (Ann Arbor, MI), and indo-1-AM and ionomycin were from Calbiochem (La Jolla, CA). All reagent-grade chemicals for buffers were purchased from Sigma-Aldrich (Oakville, ON, Canada). [³²P]Orthophosphate (400–800 mCi/ml) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Anti-rabbit IgG, horseradish peroxidase-linked whole antibody, enhanced chemiluminescence (ECL) Western blotting detection reagents were purchased from Amersham Biosciences Inc. (Baie d'Urfe, QC, Canada). Prestained SDS-PAGE marker broad range was from New England Biolabs (Mississauga, ON, Canada). Rabbit serum containing a polyclonal anti-human pleckstrin antibody was prepared and kindly supplied by Dr. Richard J. Haslam (McMaster University, Hamilton, ON, Canada) (Sloan et al., 2002). [³H]SQ 29,548 and SQ 29,548 were from Cayman Chemicals. DEAE-dextran, chloroquine, indomethacin, and HEPES were obtained from Sigma-Aldrich. dRhodamine terminator cycle sequencing kit was from Applied Biosystems (Foster City, CA). TP, , and tailless cDNA were as described previously (Allan et al., 1996; Becker et al., 1998). The tailless cDNA is truncated at amino acid 328.

Cell Culture and Transient Transfection. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Hyclone Laboratories, Logan, UT) supplemented with 10% fetal bovine serum and 1% of solution containing 10,000 units/ml penicillin G, 10,000 µg/ml streptomycin, and 25 µg/ml amphotericin (Cellgro; Mediatech, Herndon, VA). Cells were grown at 37°C in a humidified atmosphere of 95% O₂ and 5% CO₂. cDNAs for the TP and TP were subcloned into pcDNA3, the resultant plasmid, pcDNA3:TP and pcDNA3:TP were introduced into COS-7 cells (5 µg/100 mm plate) by the DEAE-dextran/chloroquine method. After 48 h post-transfection, cells were harvested by centrifugation at 500g for 5 min and washed three times in phosphate-buffered saline. Protein concentrations were determined using the Bradford assay (Bradford, 1976). Cells were resuspended in buffer containing 25 mM HEPES/125 mM NaCl/10 µM indomethacin, pH 7.4, and kept on ice for the binding study. TP expression was determined according to Miggin and Kinsella (1998) using [³H]SQ 29,548 (10 nM) and a single saturating amount of SQ 29,548 (10 µM).

Radioligand Binding Assay. Competition binding curves were carried out on COS-7 cells expressing TP receptor isoforms. Binding reactions were carried out on 5 x 10⁵ cells in a total volume of 0.2 ml in the above buffer with 10 nM [³H]SQ 29,548 added to all tubes in triplicate, containing various concentration of PBT-3 (10^–8 to 10^–5 M) in 1 µl of ethanol. Additional tubes containing excess unlabeled SQ 29,548 (10 µM) were included to assess the extent of nonspecific binding. Binding was allowed to take place for 30 min at 37°C; free radioligand was removed by rapid vacuum filtration through Whatman (Maidstone, UK) GF/B glass fiber filters prewashed with the cell suspension buffer. The tubes and the filters were rapidly washed with ice-cold buffer (three times with 3 ml). The radioactivity on the filters containing the ligand-receptor complexes was counted in 10 ml of Ecolite scintillation fluid (ICN, St. Laurent, QC, Canada) in a Beckman (model LS 3800) liquid scintillation counter.

Sequencing Analysis. TP, TP, and TPtailless sequences were confirmed on both strands using the ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit, and the products were resolved on an ABI Prism 310 genetic analyzer (PerkinElmer/Applied Biosystems). The sequences were assembled and analyzed using the ClastalW Sequence analysis.

Preparation of Human Platelet Suspension. Blood samples were collected from healthy volunteers who had not taken nonsteroidal anti-inflammatory drugs for at least 2 weeks. Blood was drawn in 60-ml plastic syringes containing 1.36% citric acid, 2% glucose, 2.5% trisodium citrate dihydrate in the absence or presence of aspirin (ASA, 1 mM) (ratio: 9 volumes of blood for 1 volume of 1.36% citric acid, 2% glucose, 2.5% trisodium citrate dihydrate) and centrifuged at 200g for 15 min at room temperature. The platelet-rich-plasma supernatant was used for dye loading cells (next paragraph) or further centrifuged at 400g for 5 min to prepare platelet suspension. The resulting platelet pellet was resuspended in fresh medium containing 137 mM NaCl, 1 mM KCl, 0.4 mM NaH₂PO₄, 5.5 mM glucose, and 20 mM HEPES, pH 7.4.

Measurement of Intracellular Calcium ([Ca²⁺]_i) Mobilization in Platelets. The method previously described with human neutrophils was modified to utilize human platelets (Dho et al., 1990). Briefly, platelet-rich-plasma was incubated with 3 mM (final concentration 3 µM) of the acetoxymethyl ester precursor of the calcium indicator indo-1 during 45 min at 37°C. The platelets were centrifuged (400g for 5 min). The supernatant containing excess dye was removed and the dye-loaded platelet pellet was gently resuspended in the same fresh medium (containing no CaCl₂). Aliquots of 350 x 10⁶ cells were placed in a plastic cuvette (Diamed Lab, Toronto, ON, Canada) and equilibrated, when necessary, with 1 mM CaCl₂ or EGTA. The cell suspension was continuously stirred magnetically and the temperature was controlled at 37°C. Intracellular calcium concentrations were monitored with a PerkinElmer fluorescence spectrophotometer (model 650-40) and recorded on a chart recorder (LKB model 2210) set at 1 cm/min. The excitation wavelength was set at 331 nm, the emission wavelength at 410 nm, with slits of excitation and emission set at 3 and 15 nm, respectively. Each sample was stirred for 1 min in the spectrofluorometer before any addition. Typical measurement was initiated by addition of 1 µl of glass distilled ethanol or test compound in ethanol followed 2 min later by collagen (2 µg) or I-BOP (2 ng). The resulting effect was recorded for the next 6 min. At the end of the test, a calibration was carried out to determine the maximal fluorescence by adding ionomycin at 1 mM (final concentration 1 µM) and minimal fluorescence by adding MnCl₂ (final concentration 3 mM).

Platelet Labeling and Stimulation. Platelets were prepared as described above. They were resuspended at 7 x 10⁸ cells/ml in a phosphate-free HEPES Tyrode's buffer without calcium (20 mM HEPES, 1 mM KCl, 5.5 mM glucose, 125 mM NaCl, pH 7.4), and labeled with 250 µCi/ml [³²P]orthophosphate for1hat room temperature (Markus et al., 1999). For stimulation, unless otherwise indicated, the ³²P-labeled platelet suspension was incubated (final 1 mM calcium concentration) at 37°C with vigorous shaking. Where appropriate, SQ 29,548, PBT-3, or vehicle was added before stimulation for 5 min. Thereafter, selective agonists or vehicle were added for 2 to 5 min. At the time indicated, the reaction was terminated by transferring an aliquot to a denaturing solution (6% SDS, 2% 2-mercaptoethanol, 30% glycerol, 3 mM EDTA, 12 mM EGTA, 0.03% bromphenol blue, 450 mM Tris; pH 6.8). The samples were denatured by boiling for 3 min and separated on a 12% SDS-PAGE gel for autoradiography and Western blot analysis of pleckstrin (Habib et al., 1999).

SDS-PAGE, Western Immunoblotting. Platelet proteins were analyzed on 12% SDS-PAGE for the separating gel in the presence of prestained protein markers. The gels were transferred to a Trans-Blot Nitrocellulose membrane (Bio-Rad, Hercules, CA). Visual protein bands on the nitrocellulose membranes were checked with Ponceau S-staining to ensure equivalent protein loading/transfer comparing different samples. Membranes were blocked with nonfat dry milk [5% (w/v)] in phosphate-buffered saline containing 0.5% (v/v) Tween 20 for 1 h at room temperature and then incubated with 1:20,000 dilution of anti-pleckstrin antibody overnight at 4°C. The secondary antibody of horseradish peroxidase anti-rabbit antibody was used at 1:2000 dilution. Bound antibodies were detected using ECL kit and exposed to Hyperfilm.

Autoradiography and Densitometric Analysis. The above-mentioned membrane exposed to ECL was kept in strong light overnight to eliminate the chemiluminescence and then it was exposed to Hyperfilm at –70°C for autoradiography. The intensity of the autoradiograph bands was analyzed using FluorChem software. The areas were integrated using the same program, and the results were expressed in arbitrary units as percentage of control (untreated) bands.

Statistical Analysis. Values stated are the mean ± S.D. of the number of observations (n) indicated. Analysis of statistical significance was performed using Student's t test using a Macintosh Stat-View software program. Competition binding were fitted to a line of best fit through a Michaelis-Menten-like hyperbolic treatment with a Kaleidagraph statistical software package. The TP expression rates are expressed as picomoles of [³H]SQ 29,548 incorporated per milligram of cell protein ± S.D., where n = 3 to 5.

	Results

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PBT-3 Dose Dependently Inhibits Intracellular Calcium Release Evoked by collagen and I-BOP in Human Platelets in Vitro
Free Cytoplasmic Ca²⁺ concentrations in human platelets were measured by the indo-1-AM fluorescent dye method. The experiments were carried out in the absence of extracellular calcium ions to demonstrate that the observed [Ca²⁺]_i transients were exclusively derived from intracellular stores.

Effects on Collagen. Dose-response curves comparing the actions of PBT-3 and two TP receptor antagonists, SQ 29,548 and pinane thromboxane A₂, on [Ca²⁺]_i release evoked by collagen are shown in Fig. 1A. IC₅₀ values for inhibition by these three compounds are 6.8 x 10^–8 M, 1.2 x 10^–9 M, and 6.5 x 10^–8 M, respectively, for the three agents. Hence, PBT-3 seems to have similar potency to pinane thromboxane A₂, but SQ 29,548 is about 50-fold more active in inhibiting [Ca²⁺]_i release evoked by collagen (Table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Dose responses showing inhibition by PBT-3 of [Ca2+]i release induced by collagen (A) and I-BOP (B). Also shown is a comparison of the effects of PBT-3 with those of the TP receptor antagonists SQ 29,548 and pinane thromboxane A2 (A), and with SQ 29,548 and I-SAP (B). Inhibition data were fitted to a line of best fit through a Michaelis-Menten-like hyperbolic treatment using Kaleidagraph statistical software package for Macintosh. IC50 values represent data from three separate experiments ± S.D.

Effects on I-BOP. I-BOP (8 x 10^–9 M) evoked a fast rise in [Ca²⁺]_i from baseline (152 ± 5 nM), reaching a maximum (564 ± 10 nM above baseline) within seconds before decreasing back to the baseline level. This I-BOP-evoked increase in [Ca²⁺]_i was markedly inhibited by PBT-3 (2.85 x 10^–7 M), added 2 min before I-BOP and this inhibition was dose related as shown in Fig. 1B. The estimated IC₅₀ value for the inhibition of [Ca²⁺]_i release evoked by I-BOP was 7.0 x 10^–9 M. The TP receptor antagonists SQ 29,548 and I-SAP dose dependently inhibited [Ca²⁺]_i release evoked by I-BOP (Fig. 1B) with IC₅₀ values estimated at 0.5 x 10^–9 and 0.6 x 10^–9 M for SQ 29,548 and I-SAP, respectively (Table 1). These results show that PBT-3 is about one-tenth less active as an antagonist of I-BOP evoked [Ca²⁺]_i release than SQ 29,548 or I-SAP.

PBT-3 Inhibits Phosphorylation of Pleckstrin
Effects of Collagen. Collagen at 4 µg/ml caused the incorporation of ³²P into a P47 protein (pleckstrin) in human platelets. A reduction in ³²P incorporation took place in the presence of ASA, indicating that pleckstrin phosphorylation followed TP receptor activation by the endogenous thromboxane formed during collagen challenge (Fig. 2). PBT-3 blocked the collagen-evoked phosphorylation, both in the presence and absence of ASA. Figure 2A shows an autoradiogram of newly formed phosphorylated proteins separated on 12% SDS-PAGE gel, with total pleckstrin protein (a measure of loading) being detected by a rabbit anti-human pleckstrin-selective antibody (bottom). Because the collagen effect is a thromboxane-mediated event, the presence of ASA reduced the incorporation of ³²P into phosphorylated pleckstrin (P47) as shown by comparing the left panels with the right panels. PBT-3 blocks this event quite well. Note that pleckstrin expression did not change by the various treatments, only the extent of newly phosphorylated pleckstrin did (X-ray film). The inhibitory actions of PBT-3 were dose-dependent (Fig. 3) within the concentration range of 0.06 to 6 µM. The IC₅₀ value was estimated to be 3 x 10^–7 M (Table 1).

View larger version (31K): [in this window] [in a new window]   Fig. 2. PBT-3 inhibition of collagen-induced phosphorylation of pleckstrin in the presence or absence of ASA treatment. Platelets were labeled with 32P, pretreated with or without ASA, and were incubated with PBT-3 (2 µg/ml) for 5 min at 37°C. They were stimulated with collagen (4 µg/ml) for 3 to 5 min. Protein in the samples was resolved on 12% SDS-PAGE and then it was transferred to a nitrocellulose membrane. A, the membrane was autoradiographed, the arrowhead indicating phosphorylated pleckstrin (47 kDa, P47). Densitometry analysis of the 32P incorporated into pleckstrin is reported by the bar diagram as mean ± S.D. (n = 3). B, pleckstrin immunoblotting analysis showing similarity in the amount of pleckstrin protein loaded in each well.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Dose responses showing inhibition by PBT-3 of the collagen (4 µg/ml)-evoked phosphorylation of pleckstrin (P47) in human platelets in vitro. Experimental conditions were described in Fig. 2.

Effects of I-BOP. I-BOP at 4 ng/ml strongly induced the incorporation of ³²P into pleckstrin (Fig. 4, autoradiogram, top, A). PBT-3 effectively blocked this phosphorylation within the tested concentration range of 0.0006 to 6 µM. The expression levels of the P47 phosphorylated pleckstrin protein were unchanged by the various treatments (Fig. 4, bottom, B) as determined by Western blot analysis and densitometry. The IC₅₀ value is estimated to be 1 x 10^–8 M (Table 1).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Dose responses showing inhibition by PBT-3 of the I-BOP (8 x 10–9 M) evoked phosphorylation of pleckstrin (P47) in human platelets in vitro. Experimental conditions were described in Fig. 2.

Inhibition of Binding by PBT-3 of [³H]SQ 29,548 to TP, TP, and TPtailless Transfected COS-7 Cells. Because the message for the TP isoform has been found in platelets, there may be a low expression of the receptor. Hence, we decided to determine the potency of PBT-3 on the two receptors. Additionally, we decided to investigate the effect of the TPtail on PBT-3 antagonism of binding of SQ 29,548 by expressing TPtailless in COS-7 cells. To establish the efficiency of transfection and to confirm sustained protein expression at 48 h post-transfection, for each independent experiment COS-7 cells were transfected with the vector only. Routinely, TP expression was 1.6 ± 0.1, TP was 1.2 ± 0.1, and TPtailless was 1.8 ± 0.2 pmol of [³H]SQ 29,548/mg cell protein, respectively. PBT-3 effectively competed with [³H]SQ 29,548 for binding to the TP receptor isoforms. PBT-3 displaced SQ 29,548 with different potencies depending on which isoform was studied. Competition curves are shown for TP and TP, from which the IC₅₀ values were derived (Fig. 5). PBT-3 was about 6 times more potent in competing with [³H]SQ 29,548 binding for the TP than TP. The IC₅₀ value for TP was 2.0 ± 0.3 x 10^–7 M, for TP was about 1.2 ± 0.2 x 10^–6 M. Due to the different potency for the two isoforms, we decided to investigate whether the tail was responsible for the difference, by removing it. The IC₅₀ for the TPtailless was 9.1 ± 0.2 x 10^–8 M, similar to the TP isoform that possesses a short tail.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Competition dose-response curves comparing the inhibition by PBT-3 of the binding of [3H]SQ 29,548 to TP, TP, and TPtailless transfected COS-7 cells. Note the greater selection by PBT-3 for TP than TP. The similarity in competition between the TP isoform and the TPtailless is suggestive that the tail length may play an important role in PBT-3 inhibition of binding. n = 3 separate experiments.

	Discussion

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The results demonstrate that PBT-3 is an effective antagonist to the effects of collagen and I-BOP in stimulating intracellular calcium release and phosphorylation of pleckstrin. Stimulation of the phosphorylation of pleckstrin via the TP receptor represents a new intracellular event coupled to this receptor. The TP receptor is abundantly expressed at both the mRNA and protein level in tissues of relevance to TXA₂ biology, such as platelets, vascular and uterine smooth muscle, uterus and placental tissue, endothelium, epithelium, trophoblasts, thymus, liver, and small intestine with TP expression predominating in most tissues examined (Miggin and Kinsella, 1998). There is evidence that mRNA for both isoforms is coexpressed in platelets, endothelial cells, and a number of other cell/tissue types with significantly greater levels of TP than TP expressed. Isoform-selective antibodies permitted detection of TP, but not TP, in human platelets, leading to the suggestion that TP may be the predominant isoform in platelets, despite the presence of mRNA for both isoforms in these cells (Miggin and Kinsella, 1998). From our previous work, we demonstrated that PBT-3 is an antagonist of the human platelet TP receptor resulting in the inhibition of aggregation (Reynaud et al., 2001). We further show herein that the effect of PBT-3 in inhibiting platelet aggregation may involve inhibition of pleckstrin phosphorylation and, in addition, inhibition of intracellular calcium release.

TP and TP display similar binding affinities for the selective TP receptor antagonist SQ 29,548, with respect to K_d values of 11.2 ± 1.4 and 12.4 ± 1.8 nM, indicating that the C-tails do not affect the binding properties of these receptors to this TP receptor antagonist (Parent et al., 1999). In contrast, the inhibition of binding of both isoforms show significant difference for PBT-3 with 2.0 ± 0.3 x 10^–7 M for TP, and 1.2 ± 0.2 x 10^–6 M for TP. Even at the high concentration of PBT-3 (200 µM), the inhibition of binding of TP only reached 76% in comparison with 100% inhibition of TP at 10 µM. These results raise the interesting possibility that the tail of the TP receptor participates in the selective binding of this receptor antagonist. Previous studies of the TP receptor have raised the possibility that the binding of agonists and antagonists may be at different sites, but not in the tail. The different tails of the TP and TP receptors have been thought to confer differences in desensitization and internalization but not binding (Spurney and Coffman, 1997; Spurney, 1998; Parent et al., 1999). Indeed, previous studies have focused on either intramembraneous domains or extracellular domains as potential ligand binding sites (Dorn et al., 1997; Turek et al., 2002). Because PBT-3 may have different inhibition mechanisms on the two isoforms, studies on the signaling pathways affected by PBT-3 warrants further investigation. Because the distribution of the TP and TP receptor isoforms differs, selective inhibition of the TP isoform in the platelets may provide an important selectivity for a potential therapeutic benefit that has not been seen with other TP receptor antagonists.

New drugs that act both as TXA₂ synthase inhibitors and as TP receptor antagonists have been developed, such as picotamide or ridogrel. These compounds were noted to have good clinical efficacy in patients with thrombotic disorders (Keith et al., 1994; Neirotti et al., 1994). Terbogrel exhibits an equipotent (IC₅₀ of about 10 nM) activity as TXA₂ synthase inhibitor and TP receptor antagonist (Muck et al., 1998). In our study, we demonstrated that PBT-3, a novel combined TXA₂ synthase inhibitor/TP receptor antagonist (Pace-Asciak et al., 2002), selectively inhibits TXA₂-dependent platelet activation but exhibits a more potent activity as TP receptor antagonist (IC₅₀ for inhibition of ¹²⁵I-BOP binding, intracellular calcium release, platelet aggregation are 8, 7, and 56 nM, respectively) compared with its activity as TXA₂ synthase inhibitor (IC₅₀ for inhibition of TXA₂ formation and platelet aggregation evoked by collagen are 0.4 and 0.64 µM). In this study we have found that PBT-3 is selective toward the TP isoform present in platelets. PBT-3 may therefore be an excellent candidate for further in vivo testing as a novel therapeutic in the treatment of thrombotic disorders or thromboxane-mediated diseases.

	Acknowledgements

 
We thank Prof. Richard Haslam for reading an earlier version of this article and for constructive suggestions.

	Footnotes

 
This study was supported by funds from the Canadian Institutes of Health Research (MT-4181; to C.R.P.-A.) and from the National Institutes of Health (HL36838; to P.V.H.).

DOI: 10.1124/jpet.103.056705.

ABBREVIATIONS. TP, thromboxane receptor; TXA₂, thromboxane A₂; PBT-3, 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methyl ester; ECL, enhanced chemiluminescence; PAGE, polyacrylamide gel electrophoresis; ASA, aspirin.

Address correspondence to: Prof. Cecil R. Pace-Asciak, Research Institute, The Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8. E-mail: pace{at}sickkids.ca

	References

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