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Keywords:

  • P2Y1-receptors;
  • Jurkat cells;
  • human washed platelets;
  • receptor reserve;
  • intracellular calcium measurements;
  • ATP;
  • ADP;
  • AR-C66096;
  • adenosine-3′-phosphate-5′-phosphate

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  • In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1-receptor.

  • The P2Y1-receptor antagonist, adenosine-3′-phosphate-5′-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pKB of 5.8 and 6.0, respectively).

  • The selective P2YADP antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an affinity (pKA) of 4.9. This value is consistent with its known P2Y1-receptor activity. In platelets, AR-C66096 at a concentration (0.1 μM) approximately 100 fold greater than its known P2YADP receptor affinity, had no effect on ADP-induced increases in [Ca2+]i.

  • The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2-MeSADP (8.3)>2-ClATP (7.8)>ADP (7.5)=2-MeSATP (7.4)>ATPγS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist.

  • The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPγS and ATP were lower in platelets.

  • The operational model of agonism was used to test whether the agonist concentration-effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1-receptor system.

  • The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1-receptors.

  • In summary, the results of this study indicate that human washed platelets contain P2Y1-receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2YADP-receptors.


Introduction

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

It is well established that extracellular nucleotides are able to mediate a number of biological actions via P2-receptors. As a result, this receptor family has been the focus of much investigation over recent years. With advances in both molecular and pharmacological approaches, these receptors have now been divided into ionotropic (P2X1–7) and G-protein linked receptors (P2Y1–8) (Burnstock & King, 1996). A further G-protein linked receptor which mediates adenosine 5′-diphosphate (ADP)-induced platelet aggregation, remains elusive to cloning, with attempts being confounded by the fact that this receptor appears to be unique to the platelet which is anucleonic.

This receptor, at which ADP and its 2-substituted analogues (e.g. 2-methylthioADP (2-MeSADP)) act as agonists, whilst adenosine 5′-triphosphate (ATP) and its 2-substituted analogues (e.g. 2-methylthioATP (2-MeSATP)) act as low affinity antagonists (Hall & Hourani, 1993; Cusack & Pettey, 1996) has previously been referred to as the P2T receptor. Recently, development of the potent, selective and competitive P2T- receptor antagonist, AR-C66096 (formerly known as FPL 66096; Humphries et al., 1994) has allowed the definitive pharmacological characterization of this receptor. However, as recommended by the IUPHAR committee (Fredholm et al., 1997) the nomenclature used for the P2T-receptor throughout the remainder of this paper is P2YADP.

ADP elevates intracellular calcium concentration ([Ca2+]i) in platelets by mobilizing internal stores, increasing extracellular influx (Sargeant & Sage, 1994) and inhibiting adenosine 3′,5′-cyclic monophosphate cyclic AMP production (Cusack & Hourani, 1982). In terms of platelet function, ADP causes adhesion, degranulation, shape change and aggregation (Gear, 1993). The diversity of these effects, with differing agonist and antagonist potency orders, has led some workers to propose the existence of several ADP-recognizing receptors on the surface of platelets (for review see Hourani & Hall, 1994). Indeed, more recent studies have suggested that human platelets possess P2YADP- (Humphries et al., 1994), P2Y1-(Léon et al., 1997) and P2X1-receptors (Mackenzie et al., 1996). However, several investigators (Nicholas et al., 1996; Léon et al., 1997) have recently highlighted the fact that the use of adenine nucleotides and their analogues in classification studies can be complicated by the presence of active impurities, in commercially available samples. Léon et al., (1997) showed that when ATP and 2-MeSATP are purified to remove contaminating diphosphates they no longer exhibited agonism at P2Y1-receptors, but instead behaved as antagonists. Thus, when purified triphosphate analogues are used, the pharmacological profile observed at P2Y1-receptors changes and more closely resembles that found for the P2YADP-receptor (Léon et al., 1997). These observations have led to the suggestion that the P2Y1-receptor may in fact be the P2YADP-receptor.

The aim of this study was to classify the receptor(s) present on human platelets responsible for increasing [Ca2+]i and to determine if the P2Y1-receptor is indeed the P2YADP-receptor. To this end, we have generated agonist potency information for a series of adenine nucleotides and examined the effects of the P2YADP-receptor antagonist AR-C66096 (pA2= 9.1; Tomlinson et al., 1997) and the P2Yi-receptor antagonist, adenosine-3′-phosphate-5′-phosphate (A3P5P; pKB= 6.0; Boyer et al., 1996). These studies were conducted under conditions where receptor desentitization would exclude any contribution to [Ca2+]i elevation resulting from P2X1-receptor activation (Mackenzie et al., 1996).

A preliminary account of part of this study was presented to the British Pharmacological Society (Dainty et al., 1997).

Methods

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

Cell culture

Jurkat cells transfected with a cloned bovine P2Y1-receptor (Henderson et al., 1995) were cultured in RPMI growth media supplemented with 10% foetal calf serum, 5% 1-glutamine and 10% penicillin/streptomycin. Cultures were grown at 0.2–1.2 ± 106 cells ml−1 in a humidified atmosphere of 5% CO2 in air and maintained at 37°C.

Loading of Jurkat cells with fura 2-AM

To remove any growth media, the Jurkat cells were centrifuged (5 min, 1000 r.p.m.) and re-suspended in basal salt solution (BSS, composition (mM): NaCl 125.0, KCl 5.0, MgCl2 1.0, CaCl2 1.5, HEPES 25.0, D-glucose 5.0 and 1 mg ml−1 bovine serum albumin, pH 7.3). In a loading volume of BSS (3 ml), the washed Jurkat cells were incubated with fura 2-AM (17 μM) and apyrase (2 u ml−1) at 37°C with shaking for 45 min. Apyrase was included to prevent tonic desensitization of receptors by ATP or ADP (Dainty et al., 1997). Cells were then centrifuged (5 min, 1000 r.p.m.), the supernatant discarded and the pellet re-suspended in BSS to a final concentration of 1 ± 106 cells ml−1.

Preparation of human washed platelets

Suspensions of human washed platelets were prepared as previously described by Humphries et al., (1994). Venous blood was obtained from healthy male and female volunteers and anti-coagulated with 1/10 volume, 3.2% trisodium citrate. The blood was centrifuged (15 min, 240 g) to obtain platelet rich plasma (PRP) to which prostacyclin (PGI2; 300 ng ml−1) was added to stabilize platelets during the washing procedure. Red cell-free PRP was obtained by centrifugation (10 min, 125 g), and following further centrifugation (15 min, 640 g), the supernatant was discarded and the pellet re-suspended in 20 ml calcium-free Tyrode solution (CFT, composition (mM): NaCl 137.0, NaHCO3 11.9, NaH2PO4 0.38, KCl 2.68, MgCl2 1.05, D-glucose 5.55, gassed with 95% O2/5% CO2 and maintained at 37°C) containing 300 ng ml−1 PGI2 to give washed platelets.

Loading of platelets with fura 2-AM

Washed platelets (3 ml) were incubated with fura-2 AM (17 μM) and apyrase (0.06 u ml−1) at 37°C for 45 min. Suspensions were then centrifuged (15 min, 640 g), the supernatant was discarded and the pellet re-suspended in BSS to a final platelet count of 1 ± 107 cells ml−1.

Experimental protocols

Fura-2 fluorescence measurements were made from stirred cell suspensions by use of a SPEX FluoroMax fluorimeter. Light emission was measured at 510 nm and the ratio of the light emissions when samples were excited at 340/380 nm was used as a measurement of [Ca2+]i. Aliquots (18 ml) of loaded cells (Jurkat cells or platelets) were incubated at 37°C for 15 min before the measurement of [Ca2+]i. Fluorescence was measured in cuvettes containing 2 ml of cells for 5 s before the addition of an agonist. Agonist concentration-effect (E/[A]) curves were constructed with each cuvette used for one agonist concentration only. When antagonists were studied, they were added to the cuvette for 15 s before the addition of 2-MeSATP (Jurkat cells) or ADP (platelets). At the end of each E/[A] curve, a maximally effective concentration of 2-MeSATP (Jurkat cells) or ADP (platelets) was tested and all responses were subsequently expressed as a percentage of these values.

Drugs

Adenosine 5′-diphosphate (sodium salt; ADP), adenosine 5′-triphosphate (disodium salt; ATP), adenosine 5′-O-(3-thiotri-phosphate) (tetralithium salt; ATPyS), adenosine 3′-phosphate-5′-phosphate (monosodium salt; A3P5P), prostacyclin, apyrase (grade V) and Fura 2-AM were obtained from the Sigma Chemical Co. (Poole, U.K.). 2-pro-pylthio-D-β,γ-difluoromethylene ATP (trisodium salt AR-C66096) was synthesized in the Medicinal Chemistry Department, Astra Charnwood; 2-methylthio-adenosine 5′-di-phosphate (trisodium salt; 2-MeSADP), 2-methylthio-adenosine 5′-tri-phosphate (tetrasodium salt; 2-MeSATP) and 2-chloro-adenosine 5′-tri-phosphate (tetrasodium salt; 2-ClATP) were supplied by Research Biochemicals Inc. (St. Albans, U.K.). Fura 2-AM was dissolved in dimethylsulphoxide (DMSO); all other drugs were dissolved and diluted in distilled water.

Heat inactivated foetal calf serum, RPMI 1640, L-glutamine (200 mM) and penicillin/streptomycin (5000 iu ml−1, 5000 ug ml−1) were obtained from Gibco BRL (Paisley, U.K.).

Data analysis

Logistic curve fitting Individual agonist E/[A] curve data were fitted to the following form of the Hill equation:

  • image(1)

in which α, [A]50 and nH are the asymptote, location and slope parameters, respectively. [A]50 values were assumed to be log-normally distributed and quoted as p[A]50 (-log[A]50) values.

Antagonist affinity estimation [A]50 data obtained in antagonist experiments were fitted to the following linear form of the Schild equation (Trist & Leff, 1985):

  • image(2)

where [A]c50 is the estimated control [A]50 value, [B] is the concentration of the antagonist, KB is the antagonist equilibrium constant and n is equivalent to the Schild plot slope parameter (unity for simple competition). If n was not significantly different from unity, it was constrained to unity for pKB estimation.

Operational model-fitting The affinity and efficacy of the partial agonist, AR-C66096 (in bovine P2Y1-receptor trans-fected Jurkat cells), was estimated by use of the operational model of agonism (Black & Leff, 1983; Black et al., 1985):

  • image(3)

in which E and [A] are the pharmacological effect and agonist concentration respectively; Em is the maximum possible effect; KA is the agonist dissociation constant (estimated as the negative logarithm, i.e. pKA); T is the efficacy of the agonist (estimated as a logarithm) and n determines the steepness of the occupancy-effect relation.

The analysis was performed with the comparative method, in which the partial agonist (AR-C66096) E/[A] curve data were fitted to the operational model (Equation 3) simultaneously to fitting the full agonist (ADP) E/[A] curve data to a Hill equation of the form:

  • image(4)

where Em and n are as defined above and [A]50 is the location parameter of the full agonist curve. This allows KA and τ for the partial agonist to be estimated as well as Em and n from each pair of curves (see Leff et al., 1990 for details).

Theoretical analysis of the effect of ATP contamination by ADP Increases in [Ca2+]i induced by commercially obtained‘ATP’ may actually be attributed to the agonist effects of contaminating ADP. To determine whether the partial agonist effects of ATP observed in the present study were the result of receptor activation by ADP and concomitant receptor blockade by ATP, we fitted the ADP and ATP E/[A] curve data from human washed platelets simultaneously to the following equation:

  • image(5)

in which ADP has been assumed to be a full agonist and ATP to be a competitive antagonist; X represents the total concentration of agonist (A) and antagonist (B), that is, ([A] + [B]) in the sample; q represents the fraction of the total sample that is antagonist (q = [B]/([A] + [B]) and therefore (1-q) represents the fraction that is agonist; Em is the maximum possible effect; n is the slope index of the occupancy-effect function; [A]50 is the midpoint location parameter and KB is the antagonist equilibrium dissociation constant.

q was measured for the ATP sample by high performance liquid chromatography (h.p.l.c.) whereas q was assumed to be zero for the ADP sample. The analysis was carried out by fitting data from individual donors which allowed the estimation of KB (as well as Em, n and [A]50) for each donor.

All of the data fitting procedures and simulations were carried out with Microsoft Excel. Results are expressed and plotted as mean values ± s.e. Statistical differences were assessed by the use of Student's t test and considered significant at the level P < 0.05.

Results

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

Estimation of antagonist affinities

Exposure of P2Y1-receptor transfected Jurkat cells to A3P5P (10–100 μM) caused small decreases (∼ 15 nM) in [Ca2+]i. The cause of these decreases in [Ca2+]i remains unclear and were not observed in platelets. A3P5P (3–100 μM) caused concentration-dependent, parallel, rightward displacements of 2-MeSATP E/[A] curves in Jurkat cells and of ADP E/[A] curves in platelets (Figure 1a and b respectively). Analysis of the computed [A]50 values from Equation 2 for the two sets of data indicated that the interactions between A3P5P and the agonists conformed to simple competition in each case. Figure 1c and d show the [A]50 data for Jurkat cells and platelets in Clark plot form (Stone & Angus, 1978). The Schild slope parameters were 1.1 ± 0.13 and 1.0 ± 0.10, respectively. Neither value was significantly different from unity and the resulting pKB estimates wre 5.8 ± 0.1 (n= 4) and 6.0 ± 0.2 (n= 4). Statistical analysis of the resulting pKB values showed that the affinities of A3P5P were not significantly different in the two systems. The selective P2YADP-receptor antagonist, AR-C66096, exhibited partial agonism in Jurkat cells (n= 4, Figure 2a). Comparative analysis of these data from the operational model of agonism gave an affinity estimate (pKA) of 4.9 ± 0.2 (n= 4) and an efficacy estimate (τ) of 1.1 (log τ= 0.04 ± 0.05). In contrast, AR-C66096 (100 μM) showed no agonism in platelets. Furthermore, at a concentration, approximately 100 fold greater than its published P2YADP receptor affinity (see Table 1), AR-C66096 (0.1 μM) had no effect on ADP E/[A] curves in this system (n= 4; Figure 2b).

image

Figure 1. Antagonist effects of A3P5P (a) on 2-MeSATP-induced increases in [Ca2+]i in bovine P2Y1-receptor transfected Jurkat cells and (b) on ADP-induced increases in [Ca +]i in human washed platelets: E/[A] curves to 2-MeSATP were obtained in the absence and presence of A3P5P (10–100 μM); E/[A] curves to ADP were obtained in the absence and presence of A3P5P (3–30 μM). The data are the averages of 4 replicate E/[A] curves with vertical lines indicating s.e. The lines drawn through the data are the results of curve-fitting with Equation 1 (see Methods). (c) and (d) Illustrate the corresponding [A]50 data in Clark plot form. The adherence of the data with the unit slope line drawn through them indicates consistency with simple competition. The pKB values estimated by fitting the [A]50 data to Equation (2) were 5.8 ± 0.1 and 6.0 ± 0.2 for the Jurkat system and platelets, respectively.

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image

Figure 2. Effects of AR-C66096 on (a) E/[A] curves for agonist-induced increases in [Ca2+]i to ADP and AR-C66096 in bovine P2Y1-receptor transfected Jurkat cells. (b) E/[A] curves to ADP were obtained in the absence and presence of AR-C66096 (0.1 μM) in human washed platelets. The data are the mean of 4 experiments with vertical lines indicating s.e. The lines drawn through the data are the results of operational model-fitting in (a) and curve-fitting with Equation 1 (see Methods) in (b).

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Table 1.  Comparison of the affinity estimations for A3P5P and AR-C66096 in bovine P2Y1-receptor transfected Jurkat cells and human washed platelets with published values at P2Y1- and P2YADP- receptors.
 Jurkat cellsPlateletsP2YADP-receptorP2Y1-receptor
  1. Results are presented as mean ± s.e. of 4 experiments. *Affinity estimations were not significantly different between systems. Published values taken from aBoyer et al., (1996), bTomlinson et al., (1997) and cHumphries et al., (1994).

A3P5PpKB= 5.8 ± 0.1*pKB= 6.0 ± 0.2*pKB= 6.0a
AR-C66096pKA= 4.9 ± 0.2No effect at 0.1 μMpA2= 9.1bpA2= 4.7c

Agonist profiles

The ability of a series of adenine nucleotide analogues to modulate [Ca2+]i in Jurkat cells and platelets was investigated. Addition of 2-MeSADP, 2-ClATP, ADP, 2-MeSATP, ATPγS or ATP induced concentration-dependent increases of [Ca2+]i in both systems (Figure 3a and b). Whilst the potency order obtained was the same in both systems, the absolute potencies of all the agonists and the intrinsic activities of ATPyS and ATP were significantly lower in platelets (see Table 2). In order to test the hypothesis that platelets may contain a low reserve (low density or poorly coupled) P2Y1-receptor population, we employed the operational model of agonism (see Figure 3 legend for details). The lines in Figure 3a and b are the result of this simulation and show a close correspondence to the experimental data points.

image

Figure 3. E/[A] curves for agonist-induced increases in [Ca2+]i to 2-MeSADP, 2-ClATP, ADP, 2-MeSATP, ATPyS and ATP in (a) bovine P2Y1-receptor transfected Jurkat cells and (b) human washed platelets. The data are the mean of 4–10 experiments with vertical lines indicating the s.e. Lines drawn through the data are the result of simultaneously fitting all the experimental data to the operational model of agonism (see Equation 3, Methods). This procedure assumed that responses were mediated by the same receptor in the two systems but that the systems have different receptor reserves. Accordingly, the affinities (KA) of the agonists were kept constant but the efficacies (τ) were allowed to differ by a fixed value (estimated by the fitting-procedure). The parameters used for the fitting-procedure were as follows:

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Table 2.  Summary of E/[A] curve parameters for adenine nucleotide analogues in bovine P2Y1-transfected Jurkat cells and human washed platelets
 2-MeSADP2-CIATPADP2-MeSATPATPγSATP
  1. Results are presented as mean ± s.e. of 4–10 experiments.

P2Y1-receptor transfecteä Jurkat cells
α95.0 ± 3.599.4 ± 6.697.1 ± 5.3103.7 ± 5.091.2 ± 3.075.5 ± 3.1
nH1.1 ± 0.31.0 ± 0.040.9 ± 0.10.7 ± 0.10.8 ± 0.10.7 ± 0.04
p[A]508.3 ± 0.17.8 ± 0.17.5 ± 0.27.4 ± 0.16.5 ± 0.16.2 ± 0.1
Human washed platelets
α103.6 ± 1.196.0 ± 2.9100.6 ± 2.188.3 ± 3.254.4 ± 10.517.4 ± 1.2
nH1.5 ± 0.11.2 ± 0.11.3 ± 0.11.2 ± 0.11.1 ± 0.11.1 ± 0.3
p[A]507.5 ± 0.17.1 ± 0.16.4 ± 0.16.4 ± 0.045.6 ± 0.25.0 ± 0.1

Theoretical analysis of the effect of ATP contamination by ADP

H.p.l.c. analysis of the commercially obtained‘ATP’ sample used in this study showed it to be approximately 99% pure with ADP being the major impurity (data not shown). Computer fitting of the experimentally obtained data for‘ATP’ in human washed platelets to Equation 5, with a q value of 0.99, gave a close fit to this model (Figure 4) and yielded an average pKB value for ATP of 5.2 ± 0.04 (n= 4).

image

Figure 4. Theoretical analysis of the effect of ATP contamination by ADP in human washed platelets. Shown are representative E/[A] curve data observed for ADP and ATP in a single donor. The solid lines drawn through the data are the result of simultaneously fitting the ADP and ATP E/[A] curves to Equation 5 respectively (see Methods). The fitting procedure for ATP assumed: the partial agonist effects of commercially obtained‘ATP’ were solely due to a 1% contamination with ADP and that ATP acted as a competitive antagonist of these responses. The dashed line indicates the expected position of the‘underlying’ ADP E/[A] curve resulting from this 1% contamination of ATP (100 fold to the right of the observed ADP E/[A] curve). The estimated affinity (pKB) of ATP from this set of data was 5.1.

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2-MeSADP2-CIATPADP2-Me-SATPATPγSATP 
  1. Jurkats: Em = 106.4, n= 0.7 for all agonists; platelets: Em= 110.2, n= 1.4 for all agonists. *Receptor reserve approximately 8 fold lower in platelets.

Jurkat/platelets6.56.35.76.05.55.2
Jurkats58.538.839.022.97.62.7
Platelets*7.55.05.02.91.00.3

Discussion

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

The objective of this study was to classify the receptor(s) mediating increases in [Ca2+]i in human washed platelets and to compare the pharmacological profile obtained with that observed in Jurkat cells stably transfected with the bovine P2Y1-receptor.

Competitive antagonist studies

Recently, Boyer et al., (1996) have shown that A3P5P is a competitive antagonist in human P2Y1-receptor transfected 1321N1 cells, with a pKB value of 6.0. This value is similar to the pKB estimate of 5.8 obtained in our study with bovine P2Y1-receptor transfected Jurkat cells. The similarity of this value to the antagonist affinity obtained in human washed platelets (Table 1) suggests that ADP-induced increases in [Ca2+]i in platelets are elicited by P2Y1-receptor activation.

The affinity of A3P5P for P2YADP-receptors, mediating platelet aggregation has not been demonstrated. Therefore, it could be argued that the above results do not definitively ascribe the observed ADP-induced increases in [Ca2+]i in platelets to P2Y1-receptor activation. To address this concern, we determined the affinity of the potent and selective P2YADP-receptor antagonist, AR-C66096 (pA2= 9.1 ± 0.1; Tomlinson et al., 1997) in both Jurkat cells and platelets. In the Jurkat cells, AR-C66096 exhibited partial agonism with an affinity estimate (pKA) of 4.9, which is consistent with its P2Y1-receptor affinity in the guinea-pig isolated aorta (pA2= 4.7 ± 0.2; Humphries et al., 1994). The agonist effect of AR-C66096 in Jurkat cells cannot be explained by its metabolism to an active compound since pre-incubation of this compound with Jurkat cells did not affect its subsequent affinity determined by a platelet aggregation bio-assay (data not shown). In platelets, AR-C66096 (0.1 μM; a concentration approximately 100 fold greater than its P2YADP-receptor affinity), had no effect on ADP-induced increases in [Ca2+]i (Table 1). The data obtained with this selective P2YADP-receptor antagonist provides compelling evidence that, under the experimental conditions used, ADP-induced [Ca2+]i increases in platelets are not mediated by P2YADP-receptors.

Agonist profiles

Our hypothesis that ADP-induced increases in [Ca2+]i in platelets, are mediated by P2Y1-receptors is further strengthened by the observation that agonist profiles obtained in both the transfected Jurkat system and human washed platelets were consistent with those found previously in other P2Y1-receptor assays (Filtz et al., 1994; Boyer et al., 1995). In all these systems the most potent compounds were the 2-substituted analogues, with the diphosphates being more potent than their triphosphate equivalents (Table 2). However, although the rank order of potencies were identical in both systems, the absolute potencies of the agonists were lower in platelets. These results might indicate that platelets possess a low reserve P2Y1-receptor population. To test this hypothesis, we fitted the data obtained in platelets and Jurkat cells to the operational model of agonism, assuming that the same receptor was activated in both systems but the receptor reserve differed (see Figure 3 legend). This analysis led us to conclude that the agonist data observed in this study could be accommodated by platelets having an 8 fold lower receptor reserve than the P2Y1-receptor transfected Jurkat cells (Figure 3a and b).

One criticism of this conclusion could be that some of the ligands used are broken down by ecto-ATPases and the effects seen are not those of the parent compound. This is unlikely to be the case since transfected Jurkat cells have been shown to have minimal ecto-ATPase activity (Dainty et al., 1997) and in washed platelets, nucleoside diphosphokinase activity predominates over ecto-ATPases activity (Hourani et al., 1991). Moreover, the kinetics of the responses to equi-effective concentrations of di- and triphosphates were identical (data not shown), which would not be expected if the observed responses were due to the breakdown of tri- to diphosphates (Léon et al., 1997).

A further criticism of this conclusion could be that the agonists used in this study were from commercial sources and unlikely to be chemically pure. Others have shown that when purified ligands are used, triphosphates appear to be devoid of agonist activity at P2Y1-receptors (Léon et al., 1997) or even act as antagonists of ADP-induced [Ca2+]i increases in platelets (Hall & Hourani, 1993). In theory, the similarity of the agonist profiles in the Jurkat system and platelets may be the fortuitous result of activation of different receptors in these two systems, by the parent compounds and/or their active impurities. However, if this was the case, it seems highly unlikely that our data could be described so accurately by the fitting procedure adopted. Furthermore, even if the data obtained with the triphosphates were discounted, the lower potencies of ADP and 2-MeSADP in human washed platelets, compared to the transfected Jurkat cells, still indicate that platelets have a lower P2Y1-receptor reserve.

The possibility of a complicating influence by the stimulation of P2X1-receptors on agonist potencies in platelets (Mackenzie et al., 1996) can also be excluded; firstly, the diphosphates analogues used are known to be highly selective for P2Y1- and P2YADP-receptors in comparison to P2X1-receptors; secondly, P2X1-receptor-mediated [Ca2+]i increases in platelets are only observed under conditions where receptor desensitization, by release of endogenous adenine nucleotides, is prevented by apyrase treatment during the measurement of [Ca2+]i (Mackenzie et al., 1996).

A number of haematopoietic cell lines exhibiting megakaryocytic characteristics have been described in the literature and possess similar agonist profiles to that obtained in the present study (Murgo & Sistare, 1992; Sistare et al., 1994; Murgo et al., 1994; see Table 3). However, in contrast to the results observed in our study, ATP acted as an antagonist in these cell lines. This, together with the observation that the diphosphates were agonists led the authors to conclude that they had identified a novel ADP-recognizing receptor. However, we suggest that these haematopoietic cell lines contain a low reserve P2Y1-receptor population and the ATP activity observed in this study may be due to other phenomena discussed below.

Table 3.  Comparison of agonist potencies (p[A]50) in bovine P2Y1-receptor transfected Jurkat cells and human washed platelets with published values in megakaryocyte-like cell lines
 Jurkat cellsPlateletsMegakaryocyte-like cells*
  1. *Potency estimations in Dami, and K562 megakaryocyte-like cell lines (from Table 2 of Murgo et al., 1994). A similar agonist profile has been obtained in UMR106 cells (Sistare et al., 1994).

2-MeSADP8.37.57.4
2-C1ATP7.87.17.0
ADP7.56.46.4
2-MeSATP7.46.46.4

Theoretical analysis of ATP contamination

ATP has been widely used as a tool for assigning numerous platelet functions to the P2YADP-receptor. Apparently without antagonist effects at other P2-receptors, ATP was shown to antagonize ADP-induced effects on platelet aggregation, [Ca2+]i and adenylyl cyclase activity with similar affinity (pA2 values of 4.6–5.2; Cusack & Hourani, 1982; Hall & Hourani, 1993). These observations led the authors to conclude that the same ADP-recognizing receptor was responsible for all of these platelet functions. However, Léon et al., (1997) have recently shown that purified ATP is a competitive antagonist at human P2Y1-receptors. This brings into question the use of ATP as a selective antagonist of P2YADP-receptors.

In the present study, ATP exhibited an apparent partial agonism in both the Jurkat system and in platelets (Figure 3a and b). However, using computer fitting of the data in human washed platelets, we have demonstrated that these results are consistent with the supposition that ATP acts as a P2Y1-receptor antagonist, blocking the effects of ADP which is an impurity of commercially available ‘ATP’. Whilst we have not directly shown that ATP is devoid of agonist activity at P2Y1-receptors, the estimated pKB value of 5.2 obtained from this analysis is similar to the pA2 value of 4.5 which can be derived from the data of Léon et al., (1997). In light of these observations, studies showing ATP exhibiting partial agonism at P2Y1-receptors may now need to be re-evaluated (Feolde et al., 1995; Dainty et al., 1997). Furthermore, these studies serve to highlight the limited use of ATP as a pharmacological tool for the characterization of ADP-recognizing receptors.

Is the P2Y1-receptor the elusive P2YADP-receptor?

Whilst the successful cloning of P2YADP-receptor remains unaccomplished, mRNA for P2Y1-receptors have been found in both human platelets and megakaryoblastic cell lines (Conley et al., 1996; Léon et al., 1997). When purified ligands are used, the agonist profile at human P2Y1-receptors is similar to that expected at P2YADP-receptors. These findings have led Léon et al., (1997) to propose that the P2Y1-receptor may in fact be the P2YADP-receptor.

The results of our study provide evidence that human platelets possess P2Y1-receptors mediating increases in [Ca2+]i. Furthermore, using the selective P2YADP-receptor antagonist, AR-C66096, and the P2Y1-receptor antagonist, A3P5P, we have conclusively demonstrated that these ADP recognizing P2Y1-receptors are pharmacologically distinct from the P2YADP-receptors which mediate platelet aggregation (Humphries et al., 1994).

Other lines of evidence also suggest that there is more than one ADP-recognizing receptor on human platelets. The antithrombotic agents clopidogrel and ticlopidine inhibit ADP-induced aggregation but do not prevent ADP-induced [Ca2+]i increase or shape change (Gachet et al., 1990). Additionally, in patients with a congenital deficiency of ADP-induced platelet aggregation, the shape change response to ADP remains intact (Cattaneo et al., 1992). These observations led Gachet and colleagues (1995) to propose a model for the existence of two distinct ADP receptor subtypes on platelets: one receptor mediating aggregation and the other mediating shape change and calcium influx. More recently, Gachet and colleagues (1997) have incorporated the work of Léon et al., (1997) into this model. They now propose aggregation is mediated by P2Y1-receptors and shape change/calcium influx is mediated by P2X1-receptors.

In the present study, we have shown that ADP-induced [Ca2+]i increases are mediated by P2Y1-receptors which are pharmacologically distinct from P2YADP-receptors. P2YADP-receptors have been shown to mediate ADP-induced platelet aggregation, defined pharmacologically by AR-C66096 (Humphries et al., 1994). However, the full functional role of P2Y1-receptors and P2X1-receptors (which also cause [Ca2+]i elevation in platelets) remains to be defined. Sanderson et al., (1996) have shown that AR-C66096 antagonizes ADP-induced platelet aggregation but not shape change in whole blood. Furthermore, P2Y1-receptors have been found to mobilize [Ca2+]i from intracellular stores by activation of phospholipase C and formation of inositol trisphosphate, possibly via coupling to Gαq (Schachter et al., 1997). Studies showing deficiencies in Gαq have shown impaired shape change and aggregation responses in platelets (Gabberta et al., 1997; Offermanns et al., 1997). On this basis, it is likely that the ADP-recognizing receptor mediating shape change in the platelet is the P2Y1-receptor. However, these studies also suggest P2YADP-receptors and P2Y1-receptors may both play a role in ADP-induced platelet aggregation. The precise mechanism of ADP-induced platelet aggregation therefore requires further study.

Conclusions

The results of the present study demonstrate that human washed platelets contain a low reserve P2Y1-receptor population that mediates ADP-induced increases in [Ca2+]i. This receptor population is pharmacologically distinct from the P2YADP-receptor. Furthermore, the endogenous activator of platelets, ADP, appears to be non-selective and is capable of activating several receptors on the surface of platelets, namely P2YADP, P2Y1 and P2X1.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  • Black, J.W. & Leff, P. (1983). Operational models of pharmacological agonism. Proc. R. Soc. B., 220, 141162.
  • Black, J.W., Leff, P., Shankley, N.P. & Wood, J. (1985). An operational model of agonism: the effect of E/[A] curve shape on agonist dissociation constant estimation. Br. J. Pharmacol., 84, 561571.
  • Boyer, J.L., O'Tuel, J.W., Fischer, B., Jacobsen, K.A. & Harden, T.K. (1995). Potent agonist action of 2-thioether derivatives of adenine nucleotides at adenylyl cyclase-linked P2Y-receptors. Br. J. Pharmacol., 116, 26112616.
  • Boyer, J.L., Robero-Avila, T., Schachter, J.B. & Harden, T.K. (1996). Identification of competitive antagonists of the P2Y1-receptor. Mol. Pharmacol., 50, 13231329.
  • Burnstock, G. & King, B.F. (1996). Numbering of cloned P2 receptors. Drug Dev. Res., 38, 6771.
  • Cattaneo, M., Lecchi, A., Randi, A.M., Mcgregor, J.L. & Mannuci, P.M. (1992). Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate. Blood, 80, 27872796.
  • Conley, P., Vincent, D., Tai, A., Bhaskar, V., Li, G., Chiu, E., Kartik, A. & Jantzen, H.M. (1996). Cloning of two receptors from a human megakaryocytic cell line. Drug Dev. Res., 37, 111.
  • Cusack, N.J. & Hourani, S.M.O. (1982). Adenosine 5′-diphosphate antagonists and human platelets: no evidence that aggregation and inhibition of stimulated adenylate cyclase are mediated by different receptors. Br. J. Pharmacol., 76, 221227.
  • Cusack, N.J. & Pettey, C.J. (1996). Effects of phosphate-modified analogs of adenosine 5′-diphosphate and adenosine 5′-triphosphate at P2T-receptors mediating human platelet activation by ADP. Drug Dev. Res., 37, 212222.
  • Dainty, I.A., Dougall, I.G., Mckay, G.D. & Leff, P. (1997). Pharmacological characterisation of a cloned bovine P2Y1-purinoceptor transfected into Jurkat cells. Br. J. Pharmacol., 120, 299P.
  • Feolde, E., Vigne, P., Breittmayer, J.P. & Frelin, C. (1995). ATP, a partial agonist of atypical P2Y receptors in rat brain microvascular endothelial cells. Br. J. Pharmacol., 115, 11991203.
  • Filtz, T.M., Li, Q., Boyer, J.L., Nicholas, R.A. & Harden, T.K. (1994). Expression of a cloned P2Y purinergic receptor that couples to phospholipase C. Mol. Pharmacol., 46, 814.
  • Fredholm, B.B., Abbracchio, M.P., Burnstock, G., Dubyak, G.R., Harden, T.K., Jacobson, K.A., Schwabe, U. & Williams, M. (1997). Towards a revised nomenclature for P1 and P2 receptors. Trends Pharmacol. Sci., 18, 7982.
  • Gabberta, J., Yang, X., Kowalska, M.A., Sun, L. & Dhanasekaran, N. (1997). Platelet signal transduction defect with Gα subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses. Proc. Natl. Acad. Sci. U.S.A., 94, 87508755.
  • Gachet, C., Cattaneo, M., Ohlmann, P., Hechler, B., Lecchl, A., Chevalier, J., Cassel, D., Mannuccio, P.M. & Cazenave, J.P. (1995). Receptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors. Br. J. Haematol., 91, 434444.
  • Gachet, C., Cazenave, J-P., Ohlmann, P., Bouloux, C., Defreyn, G., Driot, F. & Maffrand, J.P. (1990). The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1. Biochem. Pharmacol., 40, 26832687.
  • Gachet, C., Hechler, B., Leon, C., Vial, C., Leray, C., Ohlmann, P. & Cazenave, J. (1997). Activation of ADP receptors and platelet function. Thromb. Haemost., 78, 271275.
  • Gear, A.R.L. (1993). Platelet adhesion, shape change and aggregation: rapid initiation and signal transduction events. Can. J. Physiol. Pharmacol., 72, 285294.
  • Hall, D.A. & Hourani, S.M.O. (1993). Effects of analogues of adenine nucleotides on increase in intracellular calcium mediated by P2T-receptors on human blood platelets. Br. J. Pharmacol., 108, 728733.
  • Henderson, D.J., Elliot, D.G., Smith, G.M., Webb, T.E. & Dainty, I.A. (1995). Cloning and characterisation of a bovine P2Y receptor. Biochim. Biophys. Res. Commun., 212, 648656.
  • Hourani, S.M.O. & Cusack, N.J. (1991). Pharmacological receptors on blood platelets. Pharmacol. Rev., 43, 243298.
  • Hourani, S.M.O. & Hall, D.A. (1994). Receptors for ADP on human blood platelets. Trends Pharmacol. Sci., 15, 103108.
  • Humphries, R.G., Tomlinson, W., Ingall, A.H., Cage, P.A. & Leff, P. (1994). FPL 66096LT: a novel, highly potent and selective antagonist at human platelet P2T-receptors. Br. J. Pharmacol., 113, 10571063.
  • Leff, P., Prentice, D.J., Giles, H., Martin, G.R. & Wood, J. (1990). Estimation of agonist affinity and efficacy by direct, operational model-fitting. J. Pharmacol. Methods, 23, 225237.
  • Léon, C., Hechler, B., Vial, C., Leray, C., Cazenave, J-P. & Gachet, C. (1997). The P2Y1 receptor is an ADP receptor antagonised by ATP and expressed in platelets and megakaryo-blastic cells. FEBS Lett., 403, 2630.
  • Mackenzie, A.M., Mahaut-Smith, M.P. & Sage, S.O. (1996). Activation of receptor-operated cation channels via P2X1 not P2T receptors in human platelets. J. Biol. Chem., 271, 28792881.
  • Murgo, A.J., Contrera, J.G. & Sistare, F.D. (1994). Evidence for separate calcium-signaling P2T and P2U receptors in human megakaryocytic Dami cells. Blood, 83, 12581267.
  • Murgo, A.J. & Sistare, F.D. (1992). K562 leukemia cells express P2T (adenosine di-phosphate) purinergic receptors. J. Pharmacol. Exp. Ther., 261, 580585.
  • Nicholas, R.A., Watt, W.C., Lazarowski, E.R., Li, Q. & Harden, T.K. (1996). Uridine nucleotide selectivity of three phospholipase C-activating P2-receptors: Identification of a UDP-selective, a UTP-selective and an ATP- and UTP-specific receptor. Mol. Pharmacol., 50, 224229.
  • Offermanns, S., Toombs, C.F., Hu, Y. & Simon, M. (1997). Defective platelet activation in Gαq-deficient mice. Nature, 389, 183186.
  • Sanderson, H.M., Hepinstall, S., Vickers, J. & Losche, W. (1996). Studies on the effects of agonists and antagonists on platelet shape change and platelet aggregation in whole blood. Blood Coag. Fibrinol., 7, 245248.
  • Sargeant, P. & Sage, S.O. (1994). Calcium signalling in platelets and other nonexcitable cells. Pharmacol. Ther., 64, 395443.
  • Schachter, J.B., Boyer, J.L., Li, Q., Nicholas, A. & Harden, T.K. (1997). Fidelity in functional coupling of the rat P2Y1 receptor to phospholipase C. Br. J. Pharmacol., 122, 10211024.
  • Sistare, F.D., Rosenzweig, B.A., Contrera, J.G. & Jordan, B. (1994). Separate P2T and P2U purinergic receptors with similar second messenger signalling pathways in UMR-106 osteoblasts. J. Pharmacol. Exp. Ther., 269, 10491061.
  • Stone, M. & Angus, J.A. (1978). Development of computer-based estimation of pA2 values and associated analysis. J. Pharmacol. Exp. Ther., 207, 705718.
  • Tomlinson, W., Humphries, R.G., Robertson, M.J. & Leff, P. (1997). ARL 67085 and ARL 66096 are slowly dissociating competitive P2T-purinoceptor antagonists. Br. J. Pharmacol., 120, 131P.
  • Trist, D.J. & Leff, P. (1985). Quantification of H2-agonism by clonidine and dimaprit in an adenylate cyclase assay. Agents Actions, 16, 222226.