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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 152-159
The P2Y1 Receptor Is Necessary for Adenosine
5 -Diphosphate-Induced Platelet Aggregation
By
Béatrice Hechler,
Catherine Léon,
Catherine Vial,
Paul Vigne,
Christian Frelin,
Jean-Pierre Cazenave, and
Christian Gachet
From INSERM U.311, Etablissement de Transfusion Sanguine de
Strasbourg, Strasbourg, Cédex, France; and Institut
de Pharmacologie Moléculaire et Cellulaire, CNRS UPR 411, Valbonne, France.
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ABSTRACT |
The human P2Y1 receptor heterologously expressed in
Jurkat cells behaves as a specific adenosine 5 -diphosphate (ADP)
receptor at which purified adenosine triphosphate (ATP) is an
ineffective agonist, but competitively antagonizes the action of ADP.
This receptor is thus a good candidate to be the elusive platelet P2T receptor for ADP. In the present work, we examined the
effects on ADP-induced platelet responses of two selective and
competitive P2Y1 antagonists,
adenosine-2-phosphate-5-phosphate (A2P5P) and
adenosine-3-phosphate-5-phosphate (A3P5P). Results were compared with those for the native P2Y1 receptor expressed
on the B10 clone of rat brain capillary endothelial cells (BCEC) and
for the cloned human P2Y1 receptor expressed on Jurkat
cells. A2P5P and A3P5P inhibited ADP-induced platelet shape change and aggregation (pA2 = 5) and competitively antagonized
calcium movements in response to ADP in fura-2-loaded platelets, B10
cells, and P2Y1-Jurkat cells. In contrast, these compounds
had no effect on ADP-induced inhibition of adenylyl cyclase in
platelets or B10 cells, whereas known antagonists of platelet
activation by ADP such as Sp-ATP S were effective. These identical
signaling responses and pharmacologic properties suggest that platelets and BCEC share a common P2Y1 receptor involved in
ADP-induced aggregation and vasodilation, respectively. This
P2Y1 receptor coupled to the mobilization of intracellular
calcium stores was found to be necessary to trigger ADP-induced
platelet aggregation. The present results, together with data from the
literature, also point to the existence of another as yet unidentified
ADP receptor, coupled to adenylyl cyclase and responsible for
completion of the aggregation response. Thus, the term, P2T, should no
longer be used to designate a specific molecular entity.
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INTRODUCTION |
EXTRACELLULAR ADENINE nucleotides induce
various physiologic responses in many tissues. In the cardiovascular
system, adenine nucleotides released from damaged cells, especially
from red blood cells, endothelial cells, or aggregating platelets
contribute to the control of vascular tone and hemostasis.1
The central role of adenosine 5-diphosphate (ADP) as an
aggregating agent,2,3 not only in the physiologic processes
of hemostasis but also in the development and extension of arterial
thrombosis,4 has been established for a long time. Patients
who display a rare congenital thrombopathy resulting in a selective
defect in ADP-induced platelet activation have been
described.5,6 This would suggest a potential
clinical importance of the platelet ADP receptor.7-9 This
receptor, called P2T, belongs to the nucleotide receptor or P2
superfamily, as distinct from the P1 receptor family specific for
adenosine.10 P2 receptors are divided into two main groups: the G protein coupled receptor or "metabotropic" superfamily
termed P2Y and the ligand gated ion channel receptor or
"ionotropic" superfamily termed P2X. Seven subtypes of P2X and 11 subtypes of P2Y receptor have been identified.11 To date,
the P2T receptor has been characterized by second messenger signaling
and pharmacologic data, ADP, and related diphosphate analogues being
agonists as opposed to adenosine 5-triphosphate (ATP) and
related triphosphate nucleotides such as Sp-ATPS, 2ClATP, and
  MeATP, which are competitive antagonists.8
Stimulation of platelets by ADP leads to a transient increase in
intracellular calcium ([Ca2+]i) due to both
rapid calcium influx and mobilization of internal stores12
and simultaneously to inhibition of adenylyl cyclase.8,9,13 The question of the presence of one or more receptors separately mediating these effects of ADP on calcium movements and adenylyl cyclase has been debated for a long time8,9,13 and still remains open. A good correlation between the effects of agonists and
antagonists on aggregation, inhibition of adenylyl cyclase, and
[Ca2+]i increases argues for a single ADP
receptor mediating these processes.13 However, studies
using selective inhibitors of ADP-induced platelet aggregation such as
the thienopyridines, ticlopidine and clopidogrel,14 which
block ADP-induced inhibition of adenylyl cyclase15 and G
protein activation,16 but inhibit only partially the
binding of radiolabeled 2MeSADP to intact platelets17-19 without inhibiting shape change or the ADP-induced
[Ca2+]i increase,15,19 strongly
suggest the existence of two receptors separately mediating
[Ca2+]i increases and inhibition of adenylyl
cyclase. Finally, platelets also exhibit a nonselective cation channel
responsible for the rapid calcium influx component of the
[Ca2+]i increase unique to ADP
stimulation.20 Although this has been shown to be a
P2X1 receptor,21,22 its role in the complex
process of ADP-induced platelet activation remains to be
assessed.23
Recently, we reported cloning24 of the human
P2Y1 purinoceptor and its pharmacologic characterization
through heterologous expression in Jurkat cells.25 It was
demonstrated that this receptor, contrary to common
knowledge,26-30 is not an ATP receptor, but an ADP receptor
for which adenosine triphosphate nucleotides are competitive
antagonists. This pharmacologic profile closely resembles that of the
still unidentified platelet ADP receptor. Furthermore, using reverse
transcriptase-polymerase chain reaction (RT-PCR) amplification, we
found the P2Y1 receptor to be present on blood platelets
and megakaryoblastic cell lines. Thus, these results strongly suggested
the P2Y1 receptor to be the elusive P2T receptor. The
P2Y1 receptor, the first P2 receptor subtype to be
cloned,26 has a broad tissue distribution.11
Rat brain capillary endothelial cells (BCEC) have been shown to express a specific ADP receptor,31-33 which was more recently
identified as a P2Y1 receptor using RT-PCR in a subclone of
BCEC termed B10.34 This receptor is linked to the
mobilization of internal calcium stores and negatively to adenylyl
cyclase, thus bearing a striking resemblance to the ADP receptor of
platelets.
The aim of the present study was to further address the question of the
molecular identity of the platelet ADP receptor and in particular the
possibility of its being of the P2Y1 type. In this
objective, we compared the effects of two selective P2Y1 antagonists, adenosine-2-phosphate-5-phosphate (A2P5P)
and adenosine-3-phosphate-5-phosphate (A3P5P),35 on ADP-induced platelet activation, on the
native P2Y1 receptor expressed on the B10 clone of rat BCEC
and on the cloned human P2Y1 receptor heterologously
expressed in Jurkat cells. Platelets and BCEC are found to share a
common P2Y1 receptor coupled to the mobilization of
intracellular calcium stores, which is necessary to allow ADP-induced
platelet aggregation. These results, together with data from the
literature, support the hypothesis that an ADP receptor coupled to
adenylyl cyclase is responsible for completion of the aggregation
response.
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MATERIALS AND METHODS |
Materials.
Adenosine 5-O-(1-thiotriphosphate) (Sp-isomer) (Sp-ATPS) was
from Boehringer (Mannheim, Germany) and 2-methylthio-adenosine 5-diphosphate (2MeSADP) from Research Biochemicals Inc
(Natick, MA). ADP, ATP, A2P5P, A3P5P, isobutyl methyl
xanthine (IBMX), U46619, thrombin, epinephrine, prostaglandin
E1 (PGE1), bovine collagen type I, and
essentially fatty acid-free human serum albumin were purchased from
Sigma (Saint Quentin-Fallavier, France) and human fibrinogen from Kabi
(Stockholm, Sweden). Fura-2/acetoxymethyl ester (fura-2/AM) and
indo-1/AM were from Calbiochem (Meudon, France). The cyclic adenosine
3-5-monophosphate (cAMP) dosage kit was purchased from
Amersham (Les Ulis, France) and apyrase was purified from potatoes as
previously described.36 A2P5P and A3P5P were checked for
purity by high performance liquid chromatography (HPLC) analysis on a
Partisil 10 µm SAX column (Interchrom, Interchim, Monluçon,
France) eluted with a linear gradient of 0 to 1 mol/L ammonium
phosphate buffer, pH 3.8.
Cell cultures.
Jurkat E6.1 cells (ECACC No. 88042803, Cerdic, France) stably
expressing the human P2Y1 receptor were grown in RPMI-1640
medium supplemented with 10% (vol/vol) heat inactivated fetal calf
serum, 2 mmol/L glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 1 mg/mL geneticin. B10 clone cells from rat BCEC were grown in
Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) heat inactivated fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cultures were kept at 37°C
in a humidified atmosphere containing 5% CO2 and cells
were subcultured every 3 days so as to maintain a density of
approximately 5 × 105 cells/mL.
Preparation of washed human platelets.
Washed human platelets were prepared as previously
described.36 Briefly, fresh blood obtained from healthy
donors was centrifuged at 175g for 15 minutes at 37°C and
platelet-rich plasma was removed and centrifuged at 1,570g for
15 minutes at 37°C. The platelet pellet was washed twice in
Tyrode's buffer (137 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L
NaHCO3, 0.3 mmol/L NaH2PO4, 1 mmol/L MgCl2, 5.5 mmol/L glucose, 5 mmol/L HEPES, pH 7.3)
containing 0.35% human serum albumin and finally resuspended at a
density of 3 × 105 platelets/µL in the same buffer
in the presence of 0.02 U/mL of the ADP scavenger apyrase (adenosine
5-triphosphate diphosphohydrolase, EC 3.6.1.5), a concentration
sufficient to prevent desensitization of platelet ADP receptors during
storage. Platelets were kept at 37°C throughout all experiments.
Platelet aggregation studies.
Aggregation was measured at 37°C by a turbidimetric method in a
dual-channel Payton aggregometer (Payton Associates, Scarborough, Ontario, Canada). A 450-µL aliquot of platelet suspension was stirred
at 1,100 rpm and activated by addition of different agonists, in the
presence or absence of A2P5P or A3P5P at varying concentrations and in
the presence of human fibrinogen (0.8 mg/mL), in a final volume of 500 µL. The extent of aggregation was estimated quantitatively by
measuring the maximum curve height above baseline level. ADP-induced shape change was determined turbidimetrically in the presence of 5 mmol/L ethylenediaminetetraacetic acid (EDTA).
[Ca2+]i measurements.
After centrifugation of human platelet-rich plasma at 1,570g
for 15 minutes at 37°C, the platelet pellet was resuspended in Tyrode's buffer containing no albumin or calcium at a density of about
6 × 105 platelets/µL. Platelets were loaded with
2 µmol/L fura-2/AM for 45 minutes at 37°C in the dark,
washed in Tyrode's buffer containing 0.35% human serum albumin, and
finally resuspended at 37°C at a density of 3 × 105 platelets/µL in Tyrode's buffer containing apyrase
and 0.1% essentially fatty acid-free human serum albumin.
Jurkat cells stably expressing the human P2Y1 receptor were
washed in basal salt solution (BSS: 25 mmol/L HEPES, pH 7.3, 125 mmol/L
NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 5 mmol/L glucose, 0.1% human serum albumin) containing 2 mmol/L CaCl2. After
centrifugation at 100g for 5 minutes, the cells were
resuspended in BSS without calcium at a concentration of 15 × 106 cells/mL and loaded with 5 µmol/L fura-2/AM
at 37°C for 30 minutes in the dark. The cells were then pelleted
and resuspended at a density of 2 × 106 cells/mL in
BSS containing either 2 mmol/L calcium or no calcium (0.2 mmol/L
ethylene glycol-bis[-aminoethyl ether]N,N,N',N'-tetraacetic acid
[EGTA]). Aliquots of fura-2-loaded platelets or cells were transferred to a 10 × 10-mm quartz cuvette maintained at 37°C and fluorescence measurements were performed under continuous stirring,
using a PTI deltascan spectrofluorimeter (Photon Technology International Inc, South Brunswick, NJ). The excitation
wavelength was alternately fixed at 340 or 380 nm and fluorescence
emission was determined at 510 nm.
B10 cells were loaded with 5 µmol/L indo-1/AM for 2 hours in complete
culture medium at 37°C. After dissociation from the culture dishes,
the cells were centrifuged at low speed and resuspended in Earle's
salt solution (25 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, 5 mmol/L KCl, 1.8 mmol/L CaCl2, 0.8 mmol/L
MgSO4, 5 mmol/L glucose). Flow cytometric analysis of
indo-1 fluorescence was performed as previously described32
using a FacStar Plus apparatus (Becton Dickinson, San
Jose, CA). The indo-1 fluorescence ratio was measured in 5,000 single
cells and collected in real time at a rate of 500 cells/second.
Measurement of adenylyl cyclase activity.
A 450-µL aliquot of washed platelets was stirred at 1,100 rpm in an
aggregometer cuvette, and the following reagents were added at
30-second intervals: 1 µmol/L PGE1, 100 µmol/L A2P5P or A3P5P, and 5 µmol/L ADP or vehicle (Tyrode's
buffer containing no Ca2+ or Mg2+). One minute
later, the reaction was stopped by addition of 50 µL of ice-cold 6.6 N perchloric acid. B10 cells grown in 6-well tissue culture clusters
were first incubated in BSS supplemented with 10-4 mol/L
IBMX for 10 minutes at 37°C. Forskolin (1 µmol/L) and/or nucleotides were added (final volume 1 mL per well) and incubation was
continued for 5 minutes at 37°C, after which the incubation solution was removed by aspiration and the cells extracted in 10%
(vol/vol) ice-cold 6.6 N perchloric acid. The same procedure was
applied to Jurkat cells except that the incubation solution was
eliminated by centrifuging each tube at 200g for 30 seconds before extracting the cells in perchloric acid. Perchloric acid extracts were centrifuged at 11,000g for 5 minutes to eliminate protein precipitate and cyclic AMP was isolated from the supernatants as described by Khym37 using a mixture of trioctylamine and freon (28/22, vol/vol). The upper aqueous phase was lyophilized and the
dry residue dissolved in the buffer provided with the commercial
radioimmunoassay kit for cyclic AMP measurement.
Data analysis.
Agonist potencies and apparent dissociation constants of inhibitors
(pA2 =  log KD) were calculated using the
GraphPad software package (GraphPad, San Diego, CA).
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RESULTS |
P2Y1 antagonists noncompetitively inhibit ADP-induced
platelet aggregation.
The adenine nucleotide derivatives A2P5P and A3P5P induced no
aggregation or shape change of washed human platelets, even at high
concentrations (up to 100 µmol/L). On the other hand, ADP-induced platelet aggregation was inhibited by both A2P5P and A3P5P
(Fig 1A). The two P2Y1 receptor
antagonists were also able to inhibit ADP-induced platelet shape
change, as was demonstrated in the presence of 5 mmol/L EDTA,
an agent that blocks aggregation by preventing the binding of
fibrinogen to platelets (Fig 1B). This effect was selective, as these
antagonists did not inhibit the aggregation induced by 0.1 U/mL
thrombin or 2 µmol/L U46619 under conditions where the participation
of ADP secreted from platelet dense granules was precluded by addition
of 0.2 U/mL apyrase, a concentration sufficient to block the
aggregation induced by 5 µmol/L ADP (Fig 1C and D). A3P5P produced a
parallel concentration-dependent shift to the right of the
dose-response curve for ADP (Fig 2). The
50% efficacy concentrations (EC50) of ADP-induced platelet aggregation were 5.2 ± 4.0 µmol/L, 8.5 ± 5.1 µmol/L, 10.2 ± 6.3 µmol/L, 14.8 ± 10.2 µmol/L, and 20.8 ± 9.7 µmol/L in the presence of 0, 3, 10, 30, and 100 µmol/L A3P5P,
respectively. Schild analysis of the inhibition by A3P5P resulted in a
pA2 value of 5 and a slope of 0.53, which suggests that the
antagonism by A3P5P of ADP-induced platelet aggregation is
noncompetitive. The isomer A2P5P produced a similar right-hand shift of
the dose-response curve for ADP. EC50 of ADP-induced
platelet aggregation were 4.4 ± 1.2 µmol/L, 6.2 ± 1.6 µmol/L, 8.1 ± 4.1 µmol/L, 16.7 ± 3.5 µmol/L, and 34.4 ± 14.8 µmol/L in the presence of 0, 1, 3, 30, and 100 µmol/L
A2P5P, respectively. Schild analysis of the inhibition by A2P5P gave a
pA2 value of 5 and a slope of 0.55, which likewise suggests
that the antagonism by A2P5P of ADP-induced platelet aggregation is
noncompetitive.
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| Fig 1.
Effects of A3P5P on ADP-induced aggregation of washed
human platelets. (A) Platelet aggregation was induced by 10 µmol/L
ADP alone (1) or in the presence of 100 µmol/L A3P5P (2). (B)
Platelet shape change induced by 0.3 µmol/L ADP in the presence of 5 mmol/L EDTA (1) was inhibited by 100 µmol/L A3P5P (2). (C and D)
A3P5P (100 µmol/L) (2) did not inhibit platelet aggregation induced by 0.1 U/mL thrombin (1, C) or 2 µmol/L U46619 (1, D) in the presence of 0.2 U/mL apyrase.
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| Fig 2.
Inhibition by A3P5P of ADP-induced platelet aggregation.
Aggregation was induced by increasing concentrations of ADP alone ( )
or in the presence of increasing concentrations of A3P5P: ( ), 3 × 10 6 mol/L; ( ), 105 mol/L; ( ), 3 × 105 mol/L; ( ), 104 mol/L. Curves each
represent the mean of three independent experiments and bars show the
standard error of mean (SEM).
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P2Y1 antagonists competitively inhibit ADP-induced
[Ca2+]i increases in platelets, B10 cells,
and P2Y1-transfected cells.
A3P5P (100 µmol/L) had no effect on intracellular calcium levels in
fura-2-loaded washed human platelets, but produced a parallel concentration-dependent shift to the right of the dose-response curve
for ADP-induced [Ca2+]i increases in washed
platelets resuspended in Tyrode's buffer containing 0.35% human
albumin and either 2 mmol/L calcium (Fig 3A) or no calcium (0.2 mmol/L EGTA) (data not shown). EC50
values for ADP were 0.29 ± 0.1 µmol/L, 0.55 ± 0.14 µmol/L,
1.2 ± 0.4 µmol/L, 4.6 ± 0.5 µmol/L, and 12.1 ± 3 µmol/L in the presence of 0, 3, 10, 30, and 100 µmol/L A3P5P,
respectively. Schild analysis of these data gave an apparent
pA2 value of 5.3 (KD 5 µmol/L) and a slope of
1.1, which suggests competitive antagonism by A3P5P of ADP-induced
[Ca2+]i increases in platelets. Identical
inhibition of ADP-induced [Ca2+]i increases
was obtained using A2P5P. EC50 values for ADP were 0.54 ± 0.4 µmol/L, 2.4 ± 1.5 µmol/L, 4.5 ± 1.8 µmol/L, and
13.3 ± 5.7 µmol/L in the presence of 0, 10, 30, and 100 µmol/L
A2P5P, respectively. Schild analysis of these data led to an apparent pA2 value of 5.5 (KD 3 µmol/L) and a slope of
0.9, which again suggests competitive antagonism by A2P5P of
ADP-induced [Ca2+]i increases in platelets.
The two nucleotide analogues had, on the contrary, no effect on the
[Ca2+]i increases induced by 2 µmol/L
U46619 or 0.1 U/mL thrombin in platelets (Fig 3D).
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| Fig 3.
Competitive inhibition by A3P5P of ADP-induced
[Ca2+]i increases in washed human platelets
(A) and in Jurkat cells stably expressing the human P2Y1
receptor (B). [Ca2+]i was stimulated by ADP
alone or in the presence of increasing concentrations of A3P5P, in the
presence of 2 µmol/L external calcium. (C) Effects of increasing
concentrations of A2P5P and A3P5P on
[Ca2+]i increases induced by 1 µmol/L ADP
in B10 cells. (D) A3P5P (100 µmol/L) (2) did not inhibit
[Ca2+]i increases induced by 2 µmol/L
U46619 (1, left] or 0.1 U/mL thrombin (1, right) in washed human
platelets. Curves each represent the mean of three independent
experiments and bars show the SEM.
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A3P5P also produced a parallel right-hand shift of the dose-response
curve for ADP-induced [Ca2+]i increases in
Jurkat cells stably expressing the human P2Y1 receptor,
either in the presence of 2 mmol/L external calcium (Fig 3B) or in its
absence (0.2 mmol/L EGTA) (data not shown). EC50 values for
ADP were 0.11 ± 0.07 µmol/L, 0.13 ± 0.04 µmol/L, 0.28 ± 0.07 µmol/L, 0.6 ± 0.07 µmol/L, and 2.3 ± 0.6 µmol/L in the presence of 0, 3, 10, 30, and 100 µmol/L A3P5P, respectively. Schild analysis gave an apparent pA2 of 5.1 (KD
8 µmol/L) and a slope of 1.1, suggesting competitive antagonism of
A3P5P at the P2Y1 receptor. Similar results were obtained
using A2P5P. EC50 values for ADP were 0.10 ± 0.05 µmol/L, 0.75 ± 0.03 µmol/L, 1.75 ± 0.08 µmol/L, and 7.6 ± 0.6 µmol/L in the presence of 0, 10, 30, and 100 µmol/L
A2P5P, respectively. Schild analysis gave an apparent pA2
of 5.5 (KD 3 µmol/L) and a slope of 1, likewise suggesting competitive antagonism of A2P5P at the P2Y1
receptor. Once again, the two nucleotide analogues had no antagonistic
effect on the [Ca2+]i increase induced by 1 U/mL thrombin (data not shown). In the case of the B10 clone of rat
BCEC, A3P5P, and A2P5P both inhibited the
[Ca2+]i increase in response to stimulation
by 1 µmol/L ADP, with 50% inhibitory concentrations
(IC50) of 6.6 ± 0.1 µmol/L and 10.3 ± 0.4 µmol/L, respectively (Fig 3C), corresponding to Ki values of 3.1 µmol/L and 4.8 µmol/L, respectively. At higher concentration (100 µmol/L), the two nucleotide analogues completely abolished the action
of 1 µmol/L ADP. However, they once again had no antagonistic effect
on the [Ca2+]i increase induced by 0.1 U/mL
thrombin (data not shown) in B10 cells.
Lack of effect of P2Y1 antagonists on ADP-induced
inhibition of adenylyl cyclase activity.
A2P5P and A3P5P (100 µmol/L) had no impact on basal levels of cyclic
AMP in human platelets (data not shown). A3P5P likewise had no
influence on the increased cyclic AMP levels induced by 1 µmol/L
PGE1 (data not shown), whereas 5 µmol/L ADP produced 65%
inhibition of the PGE1 response
(Fig 4A). A3P5P or A2P5P (100 µmol/L) had
no effect on this ADP-induced inhibition of PGE1
stimulation, as opposed to Sp-ATPS (100 µmol/L), which totally
reversed the effects of 1 µmol/L ADP on the cyclic AMP levels
produced by PGE1 (Fig 4A). Sp-ATPS is a well-known
antagonist of the ADP receptor inhibiting ADP-induced platelet
aggregation, intracellular calcium increases, and adenylyl cyclase
inhibition.8,13
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| Fig 4.
Effects of A3P5P on cAMP levels in washed human platelets
(A) and in B10 cells (B). (A) ( ), Vehicle; ( ), PGE1 1 µmol/L; ( ), +ADP 5µmol/L; ( ), +ATPS 100 µmol/L / ADP
5 µmol/L; (), + A3P5P 100 µmol/L / ADP 5 µmol/L; ( ),
+A2P5P 100 µmol/L / ADP 5 µmol/L. (B) (), Vehicle; (),
forskolin 1 µmol/L; (), +ADP 1 µmol/L; (), +ATPS 100 µmol/L / ADP 1 µmol/L; (), +A3P5P 100 µmol/L / ADP 1 µmol/L; (), +A2P5P 100 µmol/L / ADP 1 µmol/L. Values are
means (±SEM) from one experiment performed in triplicate, representative of three separate experiments giving identical results.
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In B10 cells, A3P5P (100 µmol/L) had no influence on basal levels of
cyclic AMP. Cyclic AMP increased fourfold in the presence of 1 µmol/L
forskolin, and A3P5P had no effect on this stimulation (data not
shown). Conversely, addition of 1 µmol/L ADP to forskolin-stimulated cells resulted in a 40% reduction in cyclic AMP levels (Fig 4B). A3P5P
or A2P5P (100 µmol/L) did not modify the inhibitory effect of ADP on
adenylyl cyclase activity (Fig 4B), whereas under the same conditions,
Sp-ATPS (100 µmol/L) totally reversed the inhibitory action of
1 µmol/L ADP on cyclic AMP levels (Fig 4B). Finally, in
Jurkat cells stably expressing the P2Y1 receptor, no
positive or negative influence of A2P5P or A3P5P on adenylyl cyclase
activity could be detected (data not shown).
Inhibition of ADP-induced aggregation by P2Y1 antagonists
is not reversed by epinephrine.
Epinephrine potentiates platelet aggregation induced by low
concentrations of ADP (Fig 5A). In the
presence of 100 µmol/L A3P5P, which completely inhibited aggregation
and shape change, epinephrine could no longer potentiate any platelet
response (Fig 5B). Cyclic AMP formation was examined under the same
conditions and was found to be inhibited by both ADP and epinephrine
(Fig 5C).
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| Fig 5.
Effects of epinephrine on ADP-induced platelet
aggregation. (A) Aggregation was induced by 0.5 µmol/L ADP alone
(top) or the presence of 1 µmol/L epinephrine (bottom). (B)
Aggregation induced by 0.5 µmol/L ADP was inhibited by 100 µmol/L
A3P5P in the absence (top) or the presence (bottom) of 1 µmol/L
epinephrine. (C) Effects of A3P5P on cAMP levels in the absence (top)
or the presence (bottom) of 1 µmol/L epinephrine.
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| |
DISCUSSION |
In a previous report, we presented the pharmacologic characteristics of
the human P2Y1 receptor heterologously expressed in Jurkat
cells.25 ADP was shown to be a selective agonist of this receptor, while freshly purified ATP was an ineffective agonist, but
competitively antagonized the action of ADP. Because P2Y1 receptor transcripts were found to be present in platelets and megakaryoblastic cell lines, we suggested that the P2Y1
receptor could be similar to the platelet P2T receptor for ADP.
However, 2MeSATP and 2ClATP were still found to be agonistic to the
P2Y1 receptor-transfected cells, contrary to their known
antagonistic action on platelets.13 It was suggested that
the triphosphate analogues could have been metabolized into the
corresponding diphosphates by ectoenzymes, thus explaining their
apparent agonistic effect. This was later confirmed by the finding that
when the purity of adenine triphosphate nucleotides was controlled with
a creatine phosphokinase/creatine phosphate ATP regenerating system,
all triphosphate nucleotide derivatives were antagonists to the
P2Y1 receptors on Jurkat cells and on the B10 clonal cell
line of rat BCEC.38 These data thus support the hypothesis
that the P2Y1 receptor common to platelets and endothelial
cells could be the P2T receptor.
In the present work, we further addressed the question as to whether
the P2Y1 receptor could be the elusive P2T receptor. As
probes, we chose A3P5P and A2P5P, two adenine nucleotide isomers recently demonstrated to be competitive and selective antagonists of
the P2Y1 receptor.35 The effects of these
compounds on human platelets were compared with their effects on the
heterologously expressed human P2Y1 receptor and on the
P2Y1 receptor endogenously expressed on the B10 clonal cell
line of rat BCEC. This clone is useful for studies of the
P2Y1 receptor, as in contrast to other endothelial cell
lines, which express both P2Y1 and P2Y2 and
thus display confusing ligand structure-activity relationships, the B10
clone expresses only the P2Y1 receptor.33,34
A3P5P and its isomer A2P5P both specifically inhibited ADP-induced
platelet shape change and aggregation, demonstrating the critical role
of the P2Y1 receptor in these events. Inhibition of
ADP-induced aggregation was observed at relatively low concentrations of A2P5P and A3P5P and these compounds exhibited potencies
(pA2 = 5) similar to that of the natural competitive
antagonist ATP (pA2 = 4.6).13 However, this
effect on aggregation was found to be noncompetitive, suggesting that
more than one receptor could be involved in a highly complex process.
A2P5P and A3P5P were nevertheless specific and competitive antagonists
of the [Ca2+]i increases induced by ADP in
human platelets, in B10 cells, and in Jurkat cells stably expressing
the human P2Y1 receptor, which clearly demonstrates the
essential role of the calcium pathway triggered by the P2Y1
receptor in ADP-induced platelet aggregation. In further support of
this view, it was recently shown that platelets from mice lacking the
Gq subunit of the phospholipase C activating G-protein
Gq are unable to aggregate in response to ADP, while Inositol-1,4,5-triphosphate formation and calcium signaling are totally
abolished.39
In contrast to their inhibitory effect on calcium mobilization, A2P5P
and A3P5P had no influence on the inhibition by ADP of stimulated
adenylyl cyclase activity in platelets or B10 cells, even at high
concentration (100 µmol/L). This indicates that under conditions
where platelet aggregation was blocked, ADP could still promote the
inhibition of adenylyl cyclase. It is well established that inhibition
of adenylyl cyclase is not alone sufficient to support platelet
aggregation.40 This is the case, for instance, when
platelets are stimulated by epinephrine, which by inhibiting adenylyl
cyclase in the absence of an increase in intracellular calcium, does
not induce platelet aggregation, but potentiates the response to all
other aggregating agents.41 Figure 5 shows that under
conditions where the P2Y1 receptor was completely
antagonized, epinephrine could no longer potentiate any response, even
though adenylyl cyclase was still inhibited by ADP. Thus
P2Y1 is absolutely necessary for ADP to induce aggregation,
and inhibition of adenylyl cyclase by ADP or epinephrine is not
sufficient to promote an aggregation response.
In Jurkat cells stably expressing the human P2Y1 receptor,
it was not possible to detect any positive or negative coupling of the
receptor to adenylyl cyclase. Although this might be due to weak
expression of the P2Y1 receptor in these cells, it more probably reflects the fact that the P2Y1 receptor is
selectively coupled to calcium mobilization rather than to adenylyl
cyclase inhibition.42 The specific inhibition by A2P5P and
A3P5P of the intracellular calcium increases induced by ADP in
platelets and endothelial cells, in the absence of any inhibition of
the effects of ADP on adenylyl cyclase in these cells, points to the existence of two ADP receptors, the P2Y1 receptor coupled
to calcium movements and an as yet unidentified receptor coupled to
adenylyl cyclase inhibition. Other lines of evidence reinforce this
hypothesis. Thus, thienopyridines inhibit ADP-induced platelet
aggregation and inhibition of adenylyl cyclase without affecting the
concomitant ADP-induced [Ca2+]i
increase.14,15 Clopidogrel, in particular, inhibits only 70% of the binding of radiolabeled 2MeSADP, leaving residual binding sites even at the highest doses giving maximal blockade of platelet aggregation.18,19 The compound, ARL66096, an ATP analogue
that has been proposed as a selective P2T antagonist on the basis of its inhibitory effect on ADP-induced platelet
aggregation,43 has been recently reported to block
ADP-induced inhibition of adenylyl cyclase without affecting
ADP-mediated [Ca2+]i increases or shape
change.44
Overall, these data strongly support the view that a full aggregation
response when platelets are stimulated with ADP involves the
P2Y1 receptor, which triggers calcium signaling, shape
change, and initial aggregation, while another ADP receptor coupled to the inhibition of adenylyl cyclase potentiates and completes the initial response. This amplification pathway would also be involved in
aggregation induced by other agonists when ADP is released from
platelet dense granules. One can then speculate that the antithrombotic
properties of clopidogrel and ARL66096 are due to blockade of this ADP
pathway whatever the original stimulus. In earlier work, we reported
that a full aggregation response could be restored in the platelets of
ticlopidine-treated humans by inhibiting adenylyl cyclase with
epinephrine.15 Similarly, in the case of the specific
defect of ADP-induced platelet aggregation described in two
patients5,6 who display a "ticlopidine-like syndrome",19 one may anticipate that the defect should
lie on the receptor coupled to adenylyl cyclase. Indeed, the calcium response is almost normal in these patients and shape change is not
abolished, whereas 2MeSADP binding sites are reduced and adenylyl cyclase inhibition is blocked. Sequencing of the P2Y1
receptor gene and platelet mRNA along with functional studies using
selective P2Y1 antagonists will be required to resolve this
question.
The molecular identity of the ADP receptor coupled to inhibition of
adenylyl cyclase should be of the P2Y type, as we have previously shown
that ADP activates the Gi2 heterotrimeric G-protein in
human platelet membranes.45 Such a receptor should exhibit a pharmacologic profile identical to that of the P2Y1
receptor, ADP being an agonist and triphosphate nucleotides competitive antagonists, but with subtle differences in the selectivity of a number
of ligands. A2P5P and A3P5P, in particular, do not appear to interact
with this receptor. Conversely, two other adenine nucleotide
derivatives, 2-methylthioadenosine 5-,
-methylenetriphosphonate (2MeSAMPPCP) and 2-ethylthioadenosine
5-monophosphate (2EtSAMP), reported to selectively and
competitively inhibit the effects of ADP on adenylyl cyclase in
platelets while only partially inhibiting ADP-induced platelet
aggregation,46 should interact with this receptor. Other
compounds have long been known to display specificity for shape change
and aggregation or for inhibition of adenylyl cyclase. Thus, ADPS
induces platelet aggregation without affecting adenylyl cyclase,
whereas the thiol reagent p-mercurybenzoylsulfate (pCMBS) blocks
inhibition of adenylyl cyclase, but not shape change (extensively
reviewed in Mills8). Altogether, the currently available
data best fit a model of two P2Y receptors mediating the effects of ADP
on platelet aggregation.
Because of the key pharmacologic feature of agonism by ADP and
antagonism by ATP, an adenylyl cyclase-coupled P2Y receptor (P2Ycyc)
should display high molecular identity with the P2Y1 receptor and be detectable by RT-PCR using wide range primer sets covering the known P2Y receptors. However, such experiments only allowed detection of the P2Y1 receptor of B10
cells,34 leaving open the search for P2Ycyc. A P2Y receptor
of this type has been reported to be present in a subclone of glioma
cells termed C6-2B,47 which has the advantage of expressing
no other P2 receptors, but whether this receptor is the same as that of
platelets remains to be established. One would wish to know the effects
of compounds like ARL66096 on both C6-2B and B10 cells to clarify this
point and such compounds are unfortunately not yet commercially
available.
In conclusion, our results demonstrate that platelets and endothelial
cells share a common P2Y1 receptor involved in ADP-induced vasodilation and platelet shape change and aggregation and that this
receptor is necessary to trigger ADP-induced platelet aggregation. Our
findings and other data from the literature also strongly suggest the
existence of another as yet unidentified P2Y receptor coupled to the
inhibition of adenylyl cyclase. This means that the receptor previously
known as "P2T" is probably composed of three distinct receptors,
the P2Y1 receptor, the P2Ycyc receptor, and the
P2X1 receptor, the role of which appears to be discrete. Thus, the term "P2T" should no longer be used to designate a
specific molecular entity. In the near future, it should be possible to establish the respective contributions of each of these receptors not
only to platelet aggregation induced by ADP itself, but also to the
potentiation of platelet activation by ADP released in different
physiologic situations such as adhesion or aggregation in response to
thrombin or other strong platelet agonists.
| |
FOOTNOTES |
Submitted September 16, 1997;
accepted February 16, 1998.
Address reprint requests to Christian Gachet, MD, PhD,
INSERM U. 311, Etablissement de Transfusion Sanguine de Strasbourg, 10, rue Spielmann, BP N° 36, 67065 Strasbourg, Cédex, France; e-mail: christian.gachet{at}etss.u-strasbg.fr.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank D. Cassel for expert technical assistance and J.N.
Mulvihill for reviewing the English of the manuscript.
| |
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J. W M Heemskerk, G. M Willems, M. B Rook, and S. O Sage
Ragged spiking of free calcium in ADP-stimulated human platelets: regulation of puff-like calcium signals in vitro and ex vivo
J. Physiol.,
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535(3):
625 - 635.
[Abstract]
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R. A. Nicholas
Identification of the P2Y12 Receptor: A Novel Member of the P2Y Family of Receptors Activated by Extracellular Nucleotides
Mol. Pharmacol.,
September 1, 2001;
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J. Takasaki, M. Kamohara, T. Saito, M. Matsumoto, S.-i. Matsumoto, T. Ohishi, T. Soga, H. Matsushime, and K. Furuichi
Molecular Cloning of the Platelet P2TAC ADP Receptor: Pharmacological Comparison with Another ADP Receptor, the P2Y1 Receptor
Mol. Pharmacol.,
September 1, 2001;
60(3):
432 - 439.
[Abstract]
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G. Vassort
Adenosine 5'-Triphosphate: a P2-Purinergic Agonist in the Myocardium
Physiol Rev,
April 1, 2001;
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[Abstract]
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C. Leon, M. Freund, C. Ravanat, A. Baurand, J.-P. Cazenave, and C. Gachet
Key Role of the P2Y1 Receptor in Tissue Factor-Induced Thrombin-Dependent Acute Thromboembolism : Studies in P2Y1-Knockout Mice and Mice Treated With a P2Y1 Antagonist
Circulation,
February 6, 2001;
103(5):
718 - 723.
[Abstract]
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The Clopidogrel in Unstable angina to prevent Recurrent Events (CURE) trial programme. Rationale, design and baseline characteristics including a meta-analysis of the effects of thienopyridines in vascular disease
Eur. Heart J.,
December 2, 2000;
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[Abstract]
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M.-P. Gratacap, J.-P. Herault, C. Viala, A. Ragab, P. Savi, J.-M. Herbert, H. Chap, M. Plantavid, and B. Payrastre
Fcgamma RIIA requires a Gi-dependent pathway for an efficient stimulation of phosphoinositide 3-kinase, calcium mobilization, and platelet aggregation
Blood,
November 15, 2000;
96(10):
3439 - 3446.
[Abstract]
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M. Cattaneo, A. Lecchi, R. Lombardi, C. Gachet, and M. L. Zighetti
Platelets From a Patient Heterozygous for the Defect of P2CYC Receptors for ADP Have a Secretion Defect Despite Normal Thromboxane A2 Production and Normal Granule Stores : Further Evidence That Some Cases of Platelet 'Primary Secretion Defect' Are Heterozygous for a Defect of P2CYC Receptors
Arterioscler Thromb Vasc Biol,
November 1, 2000;
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e101 - e106.
[Abstract]
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P. Ohlmann, A. Eckly, M. Freund, J.-P. Cazenave, S. Offermanns, and C. Gachet
ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galpha q
Blood,
September 15, 2000;
96(6):
2134 - 2139.
[Abstract]
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Y. Kawashima, T. Nagasawa, and H. Ninomiya
Contribution of ecto-5'-nucleotidase to the inhibition of platelet aggregation by human endothelial cells
Blood,
September 15, 2000;
96(6):
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[Abstract]
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I. M. B. Francischetti, J. M. C. Ribeiro, D. Champagne, and J. Andersen
Purification, Cloning, Expression, and Mechanism of Action of a Novel Platelet Aggregation Inhibitor from the Salivary Gland of the Blood-sucking Bug, Rhodnius prolixus
J. Biol. Chem.,
April 21, 2000;
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[Abstract]
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C. Trumel, B. Payrastre, M. Plantavid, B. Hechler, C. Viala, P. Presek, E. A. Martinson, J.-P. Cazenave, H. Chap, and C. Gachet
A Key Role of Adenosine Diphosphate in the Irreversible Platelet Aggregation Induced by the PAR1-Activating Peptide Through the Late Activation of Phosphoinositide 3-Kinase
Blood,
December 15, 1999;
94(12):
4156 - 4165.
[Abstract]
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M. J. Quinn and D. J. Fitzgerald
Ticlopidine and Clopidogrel
Circulation,
October 12, 1999;
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[Abstract]
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M. Cattaneo and C. Gachet
ADP Receptors and Clinical Bleeding Disorders
Arterioscler Thromb Vasc Biol,
October 1, 1999;
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[Abstract]
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M. Bauer, M. Retzer, J. I. Wilde, P. Maschberger, M. Essler, M. Aepfelbacher, S. P. Watson, and W. Siess
Dichotomous Regulation of Myosin Phosphorylation and Shape Change by Rho-Kinase and Calcium in Intact Human Platelets
Blood,
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[Abstract]
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J. Geiger, J. Brich, P. Honig-Liedl, M. Eigenthaler, P. Schanzenbacher, J. M. Herbert, and U. Walter
Specific Impairment of Human Platelet P2YAC ADP Receptor–Mediated Signaling by the Antiplatelet Drug Clopidogrel
Arterioscler Thromb Vasc Biol,
August 1, 1999;
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C. L. Yap, S. C. Hughan, S. L. Cranmer, W. S. Nesbitt, M. M. Rooney, S. Giuliano, S. Kulkarni, S. M. Dopheide, Y. Yuan, H. H. Salem, et al.
Synergistic Adhesive Interactions and Signaling Mechanisms Operating between Platelet Glycoprotein Ib/IX and Integrin alpha IIbbeta 3. STUDIES IN HUMAN PLATELETS AND TRANSFECTED CHINESE HAMSTER OVARY CELLS
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L. M. Schwiebert, W. C. Rice, B. A. Kudlow, A. L. Taylor, and E. M. Schwiebert
Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells
Am J Physiol Cell Physiol,
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Abstract
Introduction
Abstract