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Keywords:

  • P2Y receptors;
  • P2Y1 receptor antagonist;
  • 2-chloro N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate;
  • phospholipase C;
  • adenylyl cyclase;
  • platelet aggregation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y1 receptor and that modification of the ribose in these analogues is tolerated in the P2Y1 receptor binding pharmacophore.

  • Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety.

  • MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pKB=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y1 receptor (pKB=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y1 receptor since MRS2279 had no effect on activation of the human P2Y2, P2Y4, P2Y6, or P2Y11 receptors by their cognate agonists.

  • MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation.

  • In contrast, the P2Y1 receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affnity (pKB=8.05) similar to that observed at the human P2Y1 receptor heterologously expressed in 1321N1 cells.

  • Taken together these results illustrate selective high affinity antagonism of the P2Y1 receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.

British Journal of Pharmacology (2002) 135, 2004–2010; doi:10.1038/sj.bjp.0704673


Abbreviations:
2MeSADP

2-Methylthioadenosine 5′-diphosphate

DMEM

Dulbecco's Modified Eagle's Medium

EGTA

Ethylene glycol-bis(beta-aminoethyl ether)- N,N,N′,N′-tetraacetic acid

GTPγS

Guanosine 5′-O-(3-thiotriphosphate)

HEPES

4-(2-Hydroxy-ethyl)-piperazine-1-ethane-sulphonic acid

MRS2179

2′-deoxy-N6-methyladenosine-3′,5′-bisphosphate

MRS2216

2-chloro-2′-deoxy-N6-methyladenosine-3′,5′-bisphosphate

MRS2279

2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosione-3′,5′-bisphosphate

MRS2286

2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy (diaminophosphate)

MRS2298

Phosphoric acid mono-[2-(2-chloro-6-methylamino-purin-9-ylmethyl)-3-phosphonooxy-propyl] ester

PLC-βt

Turkey phospholipase C-beta

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Appreciation of the importance of adenine and uridine nucleotides as extracellular signalling molecules has been heightened during the past decade by identification of up to 15 different mammalian receptors for nucleotides (Fredholm et al., 1997; Ralevic & Burnstock, 1998). These receptors are readily subdivided into the ligand-gated ion channel P2X receptors and the G protein-coupled P2Y receptors. The metabotropic P2Y receptors are essentially ubiquitously expressed and regulate myriad physiological effects ranging from muscle contraction and epithelial cell Cl secretion to immunological/inflammatory responses of circulating lymphocytes and platelet aggregation (Dubyak & El-Moatassim, 1993; Ralevic & Burnstock, 1998).

Whereas a central role for extracellular nucleotides in many physiological effects is well-accepted, few nucleotide-regulated responses have been aligned unambiguously with a given P2 receptor subtype. This follows from several problems associated with the study of nucleotide-regulated responses. First, a complex array of ectoenzymes hydrolyze and interconvert extracellular nucleotides (Zimmermann, 1996; Harden et al., 1997). Given that the P2Y receptor subtypes are differentially activated by adenine and uridine tri- and diphosphates and selective stable agonists for these receptors are not available, determination of P2Y receptor identity in tissues using agonists alone has proven difficult. Second, few selective antagonists for the P2Y receptors have been available, and many molecules utilized as antagonists to date not only block certain of the P2Y receptors but also interact with P2X receptors and many other proteins (Harden et al., 1995, 1998).

Our laboratory has focused on the P2Y1 receptor as a potentially important therapeutic target. This receptor is relatively broadly distributed in the central nervous system and in peripheral tissues (Ralevic & Burnstock, 1998; Webb & Barnard, 1999), and its role in the platelet aggregation response to ADP has been a topic of increased interest (Leon et al., 1997, 1999; Jin et al., 1998; Hechler et al., 1998; Fabre et al., 1999). Our initial observation that adenosine bisphosphate molecules are competitive antagonists of the P2Y1 receptor (Boyer et al., 1996a) has been followed by directed chemical syntheses of new molecules exhibiting potential as selective competitive antagonists of the P2Y1 receptor (Camaioni et al., 1998; Kim et al., 2000). We recently reported the synthesis of a series of ribose-modified-2′-deoxyadenosine bisphosphate analogs (Nandanan et al., 2000). Fusion of a cyclopropane bridge into a cyclopentane ring indicated that a constrained carbocyclic ring was tolerated in place of the ribose moiety in molecules that retained relatively high affinity for the P2Y1 receptor of turkey erythrocytes. We now report the pharmacological properties of the most promising of these molecules, 2-chloro-N6- methyl-(N)-methanocarba -2′-deoxyadenosine-3∼prime;,5′-bisphosphate (MRS2279). Our results reveal that a non-nucleotide bisphosphate molecule exhibits very high affinity and selectivity among the P2Y receptors for the P2Y1 receptor. This molecule does not bind to the Gi/adenylyl cyclase-linked P2Y receptor of platelets, and therefore, can be utilized as a high affinity probe for differentiating the role of the P2Y1 receptor versus a similar receptor for ADP found in platelets and other tissues.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Synthesis of N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate

The IUPAC name of N6-mehyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279) is (1R,2S,4S,5S)-1-[(phosphato)methyl]-4-(2-chloro-6-methylaminopurin-9-yl) bicyclo [3.1.0]-hexane-2-phosphate. MRS2279 was synthesized as previously described in detail (Nandanan et al., 2000).

Assay of P2Y1 receptor-promoted inositol lipid hydrolysis in turkey erythrocyte membranes

P2Y1-receptor-promoted activation of phospholipase C was studied in turkey erythrocyte membranes as we have described (Boyer et al., 1989, 1996a). Briefly, erythrocytes were incubated in inositol-free medium (DMEM; Gibco, Gaithersburg, MD, U.S.A.) with 0.5 mCi of 2-[3H]-myo-inositol (20 Ci/mmol; American Radiolabeled Chemicals, Inc., St. Louis, MO, U.S.A.) for 18 – 24 h in a humidified atmosphere of 95% air/5% CO2 at 37°C. Membranes were prepared and phospholipase C activity was measured in 25 μl of 3H-inositol-labelled membranes (approximately 175 μg of protein, 200 – 500,000 c.p.m. per assay) in a medium containing (mM): CaCl2 0.424, MgSO4 0.91, EGTA 2, KCl 115, KH2PO4 5, and HEPES 10, pH 7.0. Assays (200 μl final volume) contained 1 μM GTPγS and the indicated concentrations of nucleotide analogues. Membranes were incubated at 30°C for 5 min, and total [3H]-inositol phosphates were quantified by anion-exchange chromatography. Typical values for inositol phosphate accumulation were approximately 200 – 300 c.p.m. (basal), 2000 – 3000 c.p.m. (1 μM GTPγS alone), and 15,000 – 20,000 c.p.m. (30 nM 2MeSADP+1 μM GTPγS). The range of triplicate values was within 10% of the mean.

Assay of inositol phosphate accumulation in P2Y receptor-expressing 1321N1 cells

The P2Y1, P2Y2, P2Y4, P2Y6, or P2Y11 receptors were stably expressed in 1321N1 human astrocytoma cells using retroviral vectors as previously described (Schachter et al., 1996). Cells were labelled overnight with [3H]-inositol and agonist-promoted [3H]-inositol phosphate accumulation was quantitated after a 10 min incubation in the presence of 10 mM LiCl and the cognate agonist for each receptor. The agonists used were 2MeSADP or ADP for P2Y1 receptor expressing cells, UTP for P2Y2 receptor-expressing cells, UTP for P2Y4 receptor-expressing cells, UDP for P2Y6 receptor-expressing cells, and ATP for P2Y11 receptor-expressing cells. Typical values for inositol phosphate accumulation were 500 – 700 c.p.m. for basal and 4000 – 10,000 c.p.m. for accumulation in the presence of a maximally effective concentration of cognate agonist for each receptor. The range of triplicate values was within 10% of the mean.

Preparation of washed platelets

Venous blood was obtained from healthy volunteers and mixed with 20% of the final volume of acid/citrate/dextrose. The blood was centrifuged 180×g for 20 min, and the platelet-rich plasma was removed and incubated for 1 h in the presence of 1 mM aspirin. The platelets were centrifuged at 1000×g and resuspended to a density of 2×108 platelets ml−1 in HEPES-buffered Tyrode solution containing 0.2% BSA and 0.05 U ml−1 apyrase.

Assay of cyclic AMP accumulation in human platelets

Cyclic AMP accumulation was measured as described previously (Meeker & Harden, 1982). Briefly, platelets isolated from 50 ml of blood were labelled with 1 μCi ml−1 [3H]-adenine for 1 h at 37°C. The platelets were then washed and resuspended in (mM): NaCl 137 , KCl 2.7, MgCl2 1, NaH2PO4 3, glucose 5 and HEPES 10, pH 7.4. Incubations were for 10 min in the presence of 200 μM 3-isobutyl-1-methyl xanthine, and the reaction was stopped with 10% trichloroacetic acid. [3H]-Cyclic AMP was quantitated after chromatography over Dowex and alumina columns.

Platelet aggregation

Platelet aggregation was measured using a four-channel Chrono-Log aggregometer (Haverton, PA, U.S.A.). Briefly, a 500 μl aliquot of washed platelets, supplemented with 2 mM CaCl2 and 1 mg ml−1 fibrinogen, was stirred at 37°C and the indicated concentrations of ADP were added and aggregation monitored during an 8 min incubation. Antagonist effects of MRS2279 were studied by preincubating platelets for 2 min with the P2Y1 antagonist prior to addition of ADP. The baseline for the aggregation response was set using 500 μl of HEPES-buffered Tyrode solution.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We recently reported the synthesis of a series of methanocarbocyclic 2′-deoxyadenosine bisphosphate analogues (Nandanan et al., 2000). The fused cyclopropane ring, depending on its position on the cyclopentane ring, fixes the carbocyclic nucleoside into a rigid Northern (N) or Southern (S) envelope conformation. Our preliminary data indicated that the N conformation is favoured in recognition at the P2Y1 receptor (Nandanan et al., 2000), and we have chosen one such molecule, 2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279; Figure 1) to study as a potentially useful high affinity non-nucleotide antagonist of the P2Y1 receptor.

image

Figure 1. Structure of 2-chloro N6-methyl-(N)-methanocarbaxyadenosine-3′, 5′-bisphosphate (MRS2279).

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Turkey erythrocyte membranes have been developed as a cell-free model for the study of a P2Y receptor and its associated signalling cohorts (Harden et al., 1987; Boyer et al., 1989). The involved receptor (P2Y1 receptor), G protein (Gα11), and phospholipase C (PLC-βt) all were identified, purified, and cloned (Boyer et al., 1996b). This model system has been highly useful for identification of P2Y1 receptor agonists (Boyer et al., 1995, 1996c) and antagonists (Nandanan et al., 2000; Camaioni et al., 1998; Kim et al., 2000). Our preliminary studies suggested high affinity of MRS2279 for the P2Y1 receptor of turkey erythrocytes (Nandanan et al., 2000). Therefore, the activity of MRS2279 was examined in detail by generating a series of concentration effect curves for the agonist 2MeSADP in the presence of increasing concentrations of MRS2279 (Figure 2). MRS2279 alone had no effect on inositol phosphate accumulation in the turkey erythrocyte membrane preparation. However, coaddition of MRS2279 with 2MeSADP resulted in a shift to the right of the 2MeSADP activation curve that was dependent on the concentration of MRS2279. Schild analysis revealed that the antagonism observed with MRS2279 was competitive (slope=0.97), and the calculated pKB (pKB=7.75±0.10; n=3 experiments) indicated a very high binding affinity of MRS2279 for the avian receptor.

image

Figure 2. Competitive antagonism of 2MeSADP stimulated phospholipase C activity in turkey erythrocyte membranes. (A), phospholipase C activity was measured in turkey erythrocyte membranes as described in Methods in the presence of the indicated concentrations of 2MeSADP in the absence or presence of the indicated concentrations of MRS2279. The data are presented from duplicate determinations from an experiment that is representative of results obtained in four separate experiments. (B), a Schild plot is presented of the data presented in (A).

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The P2Y1 receptor of turkey erythrocyte membranes has proven to be an excellent predictor of activity of agonist and antagonist molecules at the human P2Y1 receptor (Boyer et al., 1996a, 1996b; 1998). As illustrated in Figure 3, this trend also holds for MRS2279. Inositol phosphate accumulation was measured in 1321N1 cells stably expressing the human P2Y1 receptor. MRS2279 alone had no effect on inositol phosphate accumulation. However, this bisphosphate analogue shifted to the right the concentration effect curve of 2MeSADP for stimulation of inositol phosphate accumulation. Schild analysis revealed that antagonism was competitive, and the pKB (8.10±0.19; n=4 experiments) was similar to that observed at the avian receptor. The affinity observed with MRS2279 indicates that this molecule is the most potent competitive antagonist of the human P2Y1 receptor reported to date.

image

Figure 3. Competitive antagonism of 2MeSADP-stimulated phospholipase C activity in 1321N1 cells stably expressing the human P2Y1 receptor. (A), P2Y1 receptor-expressing 1321N1 cells were labelled with 3H-inositol and 3H-inositol phosphate accumulation was measured as described in Methods. Assays were in the presence of the indicated concentrations of 2 MeSADP alone or 2MeSADP in the presence of the indicated concentrations of MRS2279. The data are presented as the average of triplicate determinations from an experiment that is representative of three separate experiments.

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To establish the P2Y receptor selectivity of MRS2279, we studied the inositol phosphate response to cognate agonists of the human P2Y2 receptor (UTP), the human P2Y4 receptor (UTP), the human P2Y6 receptor (UDP), and the human P2Y11 receptor (ATP) all stably expressed in 1321N1 cells. As illustrated in Figure 4, 30 μM MRS2279 completely antagonized the stimulatory effects of 2MeSADP at the P2Y1 receptor but had no effect on the capacity of the cognate agonists of the P2Y2, P2Y4, P2Y6, or P2Y11 receptors to promote inositol phosphate accumulation. Therefore, the high affinity antagonism of the human P2Y1 receptor exhibited by MRS2279 also is accompanied by high selectivity for this P2Y receptor.

image

Figure 4. Selectivity of antagonism of the human P2Y1 receptor by MRS2279. 1321N1 human astrocytoma cells stably expressing the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptor were incubated, respectively, with 10 μM 2MeSADP, 10 μM UTP, 10 μM UTP, 10 μM UDP, or 10 μM ATP without (−) or with (+) 30 μM MRS2279. The data are presented as mean±s.e.mean of three separate experiments with stimulation with agonist alone given a value of 100% of maximum in each experiment.

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The physiological effects of ADP on platelet secretion and aggregation apparently occur due to the action of two different G protein-coupled receptors: the Gq/phospholipase C-linked P2Y1 receptor and the recently cloned (Hollopeter et al., 2001) P2Y12 receptor, which couples through Gi to inhibit adenylyl cyclase (Jin et al., 1998; Hechler et al., 1998; Leon et al., 1999; Fabre et al., 1999). The presence of these two receptors exhibiting similar agonist selectivity has complicated both the study of the physiological effects of ADP in regulation of platelet aggregation and resolution of the relative role of the adenylyl cyclase-linked P2Y12 receptor versus the P2Y1 receptor in the action of ADP. As such, the synthesis of MRS2279 may provide an additional high affinity tool to study the role of ADP in platelet physiology. As illustrated in Figure 5, ADP inhibited the capacity of PGE1+forskolin to stimulate cyclic AMP accumulation in intact human platelets. In contrast to its potent activity at the ADP-activated P2Y1 receptor, MRS2279 exhibited no effect on ADP-promoted inhibition of cyclic AMP accumulation through the Gi/adenylyl cyclase-linked P2Y receptor of platelets (Figure 5).

image

Figure 5. Effect of MRS2279 on 2MeSADP-promoted inhibition of adenylyl cyclase activity in human platelets. Cyclic AMP accumulation was measured as described in Methods. Assays were in the presence of 10 μM PGE1 and 50 μM forskolin (solid circle) or 10 μM PGE1, 50 μM forskolin, and 10 nM 2MeSADP in the presence of the indicated concentrations of MRS2279 (solid square). The data are presented as the mean±s.e.mean of three separate experiments.

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Given the activity of MRS2279 in blocking P2Y1 receptors and its lack of activity in blocking the platelet adenylyl cyclase-linked P2Y12 receptor, we examined the capacity of MRS2279 to block ADP-induced platelet aggregation. As illustrated in Figure 6A, MRS2279 antagonized the action of ADP in inducing aggregation. Schild analyses of these data indicated that antagonism was competitive (Figure 6B), and the pKB of this effect (pKB=8.05±0.10; n=3 experiments) was similar to that observed for MRS2279 at either the avian or recombinant human P2Y1 receptor. ADP-induced shape change of platelets was inhibited by MRS2279 with the same apparent affinity as that illustrated for inhibition of aggregation (data not shown). Thus, as has been previously illustrated pharmacologically (Jin et al., 1998; Hechler et al., 1998; Savi et al., 1998) and by P2Y1 receptor gene disruption (Leon et al., 1999; Fabre et al., 1999), these data with MRS2279 illustrate that the P2Y1 receptor plays an obligatory role in ADP-promoted platelet aggregation.

image

Figure 6. Effect of MRS2279 on ADP-promoted platelet aggregation. (A), platelet aggregation was measured as described in Methods. Assays were in the presence of the indicated concentrations of ADP alone or the indicated concentrations of ADP with the indicated concentrations of MRS2279. The cyclic AMP response to PGE1+forskolin in the absence of ADP also is indicated (solid circle). The data are the mean of duplicate determinations and are representative of results from three experiments. (B), a Schild plot of the data in (A) is presented.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The results described here illustrate very high affinity antagonism of the P2Y1 receptor by 2-chloro N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate. Moreover, this antagonism is specific for the P2Y1 receptor among the metabotropic P2Y receptors. No effect was observed on the four other cloned human Gq/phospholipase C linked P2Y receptors, and MRS2279 also did not block the P2Y12 receptor for ADP on platelets that couples in an inhibitory fashion to adenylyl cyclase. Thus, we have developed a non-nucleotide high affinity P2Y1 receptor antagonist that exhibits notable selectivity.

The work described here follows from our original observation that bisphosphate-substitution of adenosine results in molecules that act as competitive antagonists at the P2Y1 receptor (Boyer et al., 1996a). A broad series of bisphosphate analogues subsequently was synthesized (Camaioni et al., 1998; Boyer et al., 1998) including the widely utilized P2Y1 receptor antagonist, 2′-deoxy-N6-methyladenosine-3′,5′-bisphosphate (MRS2179), and its 2-chloro analogue (MRS2216). Both of these antagonists are adenine-9-riboside 3′,5′-bisphosphate derivatives that exhibit Ki values for the P2Y1 receptor in the 100 – 300 nM range. We have moved away from riboside nucleotides in recent chemical synthesis (Nandanan et al., 2000; Kim et al., 2000, 2001), and the carbocyclic nucleotide analogue MRS2279 studied here is the most potent P2Y1 receptor antagonist reported to date. Similarly, phosphoric acid mono-[2-(2-chloro-6-methylamino-purin-9-ylmethyl)-3-phosphonooxy-propyl] ester (MRS2298) was almost as potent as the riboside MRS2179 for inhibition of P2Y1 receptor-mediated activation of phospholipase C (Kim et al., 2001). Although the widely used riboside bisphosphate MRS2179 exhibits no agonist activity at P2Y1 receptors, several other riboside bisphosphates are partial agonists. P2Y1 receptor agonist activity was not observed with MRS2279 or any other acyclic bisphosphate molecule we have studied.

The adenosine bisphosphate backbone of MRS2279 contains four additional modifications that in composite enhance P2Y1 receptor selectivity and affinity by 3 – 4 orders of magnitude (Camaioni et al., 1998; Nandanan et al., 2000). These include a methyl group addition at the N6-position of the adenine base, a 2-chloro substitution of the adenine base, removal of the 2′-hydroxyl of the ribose, and a cyclopropane (methanocarba) bridge in a cyclopentane ring replacing the ribose. The methanocarba substitution of MRS2279 constrains the pseudo ribose in an envelope (N)-(2′-exo) conformation. We previously reported that a (S)-(2′-endo) conformation was many fold less favourable for P2Y1 receptor binding (Nandanan et al., 2000). Molecular modelling of the P2Y1 receptor pharmacophore based on the 2.8 A three dimensional structure of rhodopsin and our previous mutational analyses of the P2Y1 receptor indicated that the (N)-conformation maximizes electrostatic interactions between the negatively charged bisphosphates and positively charged amino acids of the binding pocket (Kim et al., 2000).

This and other work on acyclic analogues (Kim et al., 2000) indicate that the ribose per se is not necessary for ligand binding, and a non-ribose spacer of similar length between the N position of the adenine base and the two phosphates will suffice for full receptor recognition. This observation is important because retention of P2Y1 receptor binding in molecules lacking a ribose essentially assures that binding will not occur to the thousands of other nucleotide binding proteins known to exist in the human genome. Therefore, receptor selectivity is highly likely and development of an MRS2279 radioligand is underway to take advantage of this evolution of a high affinity non-nucleotide molecule. We have not tested the activity of MRS2279 at any of the ATP-activated ionotropic P2X receptors, although a closely related acyclic derivative, 2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy (diammonium-phosphate) (MRS2286), was inactive at the rat P2X1 receptor (Brown et al., 2000). The earlier bisphosphate nucleotide analogue MRS2179 developed by our laboratory as a high affinity antagonist for the P2Y1 receptor (Boyer et al., 1996c), interacted with the P2X1 receptor but with 20 – 40 fold lower affinity than at the P2Y1 receptor (Brown et al., 2000).

The work of several groups (Hechler et al., 1998; Jin et al., 1998; Kunapuli, 1998; Savi et al., 1998; Fagura et al., 1998) has amplified the idea that both the Gq-coupled P2Y1 receptor and the recently cloned (Hollopeter et al., 2001) adenylyl cyclase-linked P2Y12 receptor are involved in ADP action in platelets. This concept was initially based in part on utilization (Hechler et al., 1998; Jin et al., 1998; Savi et al., 1998) of the adenosine bisphosphate molecules that were identified as the lead molecules in the eventual development of MRS2279 (Boyer et al., 1996a). As we illustrate here MRS2279 now provides a very high affinity antagonist for the platelet P2Y1 receptor that does not bind to the second ADP-activated receptor on the platelet, the P2Y12 receptor. MRS2279 blocked ADP-promoted aggregation of human platelets with a Ki identical to its Ki for antagonism of the recombinant human P2Y1 receptor. Thus, as previously concluded by others the P2Y1 receptor is indispensable in the platelet aggregation response to ADP.

The high affinity and absolute specificity of MRS2279 for the P2Y1 receptor over the other metabotropic P2Y receptors provides a molecule that should be widely useful in delineation of the physiological role of the P2Y1 receptor in the central nervous system and in peripheral tissues. Binding affinity in the low nanomolar range also provides an attractive non-nucleotide antagonist molecule that can be radiolabelled as a probe for P2Y1 receptor quantification and characterization.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This research was supported by United States Public Service Grants HL54889 and GM38213. The authors are indebted to Savitri Maddileti for technical support and Meredith Jones and David Rinker for assistance in preparing the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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