Acting through cell surface receptors, ADP
activates platelets resulting in shape change, aggregation, thromboxane
A2 production, and release of granule contents. ADP
also causes a number of intracellular events including inhibition of
adenylyl cyclase, mobilization of calcium from intracellular stores,
and rapid calcium influx in platelets. However, the receptors that
transduce these events remain unidentified and their molecular
mechanisms of action have not been elucidated. The receptor responsible
for the actions of ADP on platelets has been designated the P2T
receptor. In this study we have used ARL 66096, a potent antagonist of
ADP-induced platelet aggregation, and a P2X ionotropic receptor
agonist,
,
-methylene adenosine 5
-triphosphate, to distinguish
the ADP-induced intracellular events. ARL 66096 blocked ADP-induced
inhibition of adenylyl cyclase, but did not affect ADP-mediated
intracellular calcium increases or shape change. Both ADP and
2-methylthio-ADP caused a 3-fold increase in the level of inositol
1,4,5-trisphosphate over control levels which peaked in a similar
fashion to the Ca2+ transient. The increase in inositol
1,3,4-trisphosphate was of similar magnitude to that of inositol
1,4,5-trisphosphate.
,
-Methylene adenosine 5
-triphosphate did
not cause an increase in either of the inositol trisphosphates. These
results clearly demonstrate the presence of two distinct platelet ADP
receptors in addition to the P2X receptor: one coupled to adenylyl
cyclase and the other coupled to mobilization of calcium from
intracellular stores through inositol trisphosphates.
 |
INTRODUCTION |
ADP was the first low molecular weight agent recognized to cause
platelet aggregation (1, 2). It is stored in the dense granules of
human platelets and is an important platelet agonist as evidenced by
the fact that patients with defective ADP storage have bleeding
tendencies (3, 4). Activation of platelets by ADP follows a defined
sequence. The first event, shape change, occurs when discoid shaped
resting cells are rapidly converted to spiculated spheres. Shape change
is followed by platelet aggregation and granule secretion which
releases more ADP as well as many other substances (5). Acting
extracellularly, ADP causes a number of intracellular events including
rapid calcium influx (6, 7), mobilization of intracellular calcium
stores (8), and inhibition of adenylyl cyclase (9). An increase in
intracellular Ca2+ may be due to an increase in inositol
trisphosphate (10-13) but this finding remains controversial (14, 15).
In addition, arachidonic acid, liberated from platelet membranes due to
activation of phospholipase A2, is converted to thromboxane
A2, itself a powerful platelet agonist. Despite this
knowledge the exact identity of platelet ADP receptors responsible for
functional responses of ADP are not fully defined and the mechanism by
which aggregation occurs is still under investigation.
The idea that ADP's effects on platelets are receptor-mediated was
indicated by the finding that ATP shows true competitive inhibition of
ADP-induced aggregation and adenylyl cyclase inhibition (16). Several
other nucleoside triphosphates are also competitive antagonists of ADP
and have similar activity ratios for inhibition of aggregation and for
inhibition of ADP's effect on adenylyl cyclase (17). Evidence that an
ADP receptor couples to G-proteins is based on the fact that ADP
stimulates the binding of
GTP
S1 to platelet
membranes in humans and rats (18). The receptors activated by
extracellular nucleotides have been classified as P2 receptors and are
divided into two subclasses: P2X intrinsic ion channels and P2Y
metabotropic receptors coupled to heterotrimeric G-proteins (19). These
receptor subtypes are numbered in the order of cloning and to date
eight subtypes of P2X receptors and six subtypes of P2Y receptors have
been cloned (20). Because of its pharmacologic profile, the identity of
platelet ADP receptor(s) could not be assigned to any cloned member of
the P2Y receptor family and hence has been designated P2T (21, 22).
Several molecules have been proposed to be the platelet ADP receptor, including aggregin, a 100-kDa protein that covalently binds to 5
-p-fluorosulfonyl benzoyladenosine (23), and platelet
glycoprotein IIb, a component of the fibrinogen receptor on platelets
(24), but none has been conclusively demonstrated to be a platelet ADP receptor.
Using
,
-MeATP as the agonist, MacKenzie et al. (7)
demonstrated an ADP receptor in platelets, suggested to be the P2X1 receptor, that could cause a rapid calcium influx but not internal Ca2+ mobilization. However, it is unclear whether
,
-MeATP can activate any physiological platelet responses (7).
Léon et al. (25) detected mRNA for the P2Y1
receptor, a metabotropic receptor, in platelets. They expressed this
receptor in Jurkat cells and demonstrated that ATP is an antagonist at
this receptor and postulated that the P2Y1 receptor is the
platelet ADP receptor coupled to adenylyl cylase inhibition (25,
26).
In this report, we provide evidence that there are two separate
receptors for ADP on the platelet surface; one coupled to inhibition of
adenylyl cyclase, designated P2TAC, and a second coupled to
mobilization of intracellular calcium stores through inositol phosphate
production, designated P2TPLC, in addition to the intrinsic
ion channel P2X, coupled to rapid calcium influx previously
demonstrated by MacKenzie et al. (7). ATP is an antagonist
at both the receptor coupled to inhibition of adenylyl cyclase and that
coupled to increases in inositol phosphates.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Apyrase (Type V), ADP, ATP, fibrinogen, and
bovine serum albumin (fraction V) were from Sigma. Fura-2 was from
Molecular Probes (Eugene, OR). ARL 66096 was a gift from Fisons (now
known as Astra Research Laboratories, Loughborough, United Kingdom).
RGDS was from Bachem Bioscience Inc. (King of Prussia, PA).
[32P]PO4,
[3H]1,3,4-IP3,
[3H]1,4,5-IP3, [3H]adenine, and
[14C]cAMP were from NEN Life Science Products (Boston,
MA). 2-MeSADP and
,
-MeATP were from Research Biologics, Inc.
(Natick, MA).
Preparation of [32P]PO4, Fura-2-labeled
Platelets--
Human blood was collected from informed healthy
volunteers in acid/citrate/dextrose. Platelet-rich plasma was obtained
by centrifugation at 180 × g for 15 min at ambient
temperature and was recentrifuged (800 × g for 15 min,
ambient temperature). The platelet pellet was resuspended in 0.5 volumes of autologous plasma and incubated with
32PO4 (0.25 mCi/ml) at 37 °C. After 15 min,
fura-2 (3 µM) and aspirin (1 mM) were added
and the incubation at 37 °C continued for another 45 min. Platelets
were isolated from the incubation medium by gel filtration as described
previously (27). The final buffer consisted of 137 mM NaCl,
2.7 mM KCl, 1 mM MgCl2, 3.0 mM NaH2PO4, 5 mM
glucose, 10 mM HEPES (pH 7.4), 0.2% bovine serum albumin, and 20 µg/ml apyrase. The platelet count was adjusted to 2 × 108 cells/ml. All experiments were carried in buffers with
no added Ca2+ unless otherwise noted.
Measurement of Ca2+ with Fura-2 and Platelet
Activation--
Platelets (1.0 ml) were activated in a Perkin-Elmer
LS-5 spectrofluorimeter in a water jacketed cuvette maintained at
37 °C with stirring. Fura-2 fluorescence was monitored continuously using settings of 340 nm (excitation) and 510 nm (emission). Fura-2 fluorescence signals were calibrated as described by Pollock et al. (28). Reactions were stopped by the addition of 1:10 volume of
ice-cold 6.6 N HClO4 and kept on ice until
further processing.
Measurement of Inositol Trisphosphates--
After addition of
HClO4, 0.02 µCi each of
[3H]Ins(1,4,5)P3 and
[3H]Ins(1,3,4)P3 were added to each sample
and the samples were centrifuged. Supernatants were adjusted to pH
7.5-8.0 by addition of 2 M K2CO3
and the precipitate of KClO4 was removed by centrifugation. The resulting supernatant was treated at 37 °C overnight with inorganic pyrophosphatase (10 units/ml) in the presence of 5 mM MgCl2, 10 mM Tris-HCl (pH 8.0).
Each sample was treated with 100 mg of charcoal (Darco, G-60) for 5 min
on ice to remove nucleotides. Charcoal was removed by centrifugation (3 min at 5000 × g) followed by filtration through Rainin
microfilterfuge tubes. Separation of inositol phosphates was
accomplished using a Rainin Rabbit HP HPLC and a Whatman Partisil 10 SAX column. The column was eluted at a flow rate of 1.5 ml/min with a
45-min gradient of water as initial buffer and 1.5 M
ammonium formate adjusted to pH 3.7 with H3PO4,
as final buffer. Fractions were collected and counted in a LKB liquid
scintillation counter with windows set to separate 3H and
32P. The [3H]Ins(1,4,5)P3 and
[3H]Ins(1,3,4)P3 standards were used to
indicate the positions of their 32P counterparts and to
correct for recovery.
Platelet Aggregation--
For aggregation experiments, aspirated
platelets were isolated from plasma by centrifugation at 800 × g for 15 min and resuspended in the same buffer that was
used for gel filtration. Platelet shape change and aggregation (0.5-ml
samples) were measured in a Chronolog lumi-aggregometer with stirring
at 37 °C. Fibrinogen (400 µg/ml) was added to all samples. The
base line was set using the platelet suspension diluted 1:1 with
platelet suspension buffer to increase the gain of the aggregometer
output.
Measurement of Cyclic AMP Formation in Intact
Platelets--
Platelet-rich plasma was incubated with 2 µCi/ml
[3H]adenine and aspirin (1 mM) for 1 h
at 37 °C (29). Platelets were isolated from plasma by centrifugation
at 800 × g for 15 min and resuspended in the same
buffer as was used for gel filtration. Reactions were stopped with 1 M HCl and 4000 disintegrations/min of
[14C]cAMP as recovery standard. Cyclic AMP was determined
by the method of Salomon (30) and expressed as percentage of total [3H]adenine nucleotides.
 |
RESULTS |
Effect of ADP and 2-MeSADP on Inositol Phosphate Metabolism in
Platelets--
The time course of ADP and 2-MeSADP-induced increases
in [32P]Ins(1,4,5)P3 and
[3H]Ins(1,3,4)P3 are compared with the
changes in intracellular Ca2+ in Fig.
1, A and B,
respectively. Both agonists caused a rapid increase in
[32P]Ins(1,4,5)P3 that reached peak levels at
about 3-fold over basal at the earliest measured time of 2 s.
[32P]Ins(1,3,4)P3 also increased rapidly.
Intracellular Ca2+ also increased rapidly and decreased to
near control levels at 30 s.

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Fig. 1.
Time course comparing intracellular
Ca2+ mobilization to IP3 formation.
Gel-filtered human platelets at 37 °C with constant stirring were
activated with either 10 µM ADP (Panel A) or
10 µM 2-MeSADP (Panel B). Samples were stopped
at the indicated times by addition of 0.1 volume of 6.6 N
HClO4. Symbols indicate the agonist-induced
changes in intracellular free Ca2+ in nM ( ),
the 32P disintegrations/min associated with
Ins(1,3,4)P3 ( ), and 32P disintegrations/min
associated with Ins(1,4,5)P3 ( ). Data are the average
and standard deviation of three determinations from the same platelets
and are representative of five experiments.
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|
Concentration Dependence of ADP and 2-MeSADP-induced Inositol
Phosphate Formation--
The concentration dependence of ADP and
2-MeSADP-induced inositol phosphate formation and intracellular
[Ca2+] mobilization is shown in Fig.
2, A and B,
respectively. Both agonists caused a
concentration-dependent increase in the two isomers of
IP3. There was a good correlation between the concentration dependence of Ca2+ mobilization and IP3
metabolism for both agonists. The EC50 for ADP was about 1 µM, which is comparable with published results for
induction of shape change and Ca2+ mobilization (21). The
EC50 for 2-MeSADP was about 4-fold lower which is in
agreement with the published activity of 2-MeSADP for induction of
platelet Ca2+ mobilization (21).

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Fig. 2.
Comparison of concentration dependence of
intracellular Ca2+ mobilization to IP3
formation. Samples were taken after stimulation with indicated
concentrations of either ADP (Panel A) or 2-MeSADP (Panel B). Symbols and conditions are as indicated Fig. 1
with the exception that Ins(1,3,4)P3 and
Ins(1,4,5)P3 data are expressed as change from basal. The
curves represent hyperbolae that best fit the data using the
program Kaleidagraph (Synergy Software, Reading, PA).
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|
Effect of
,
-MeATP on Inositol Phosphate Formation and
Adenylyl Cyclase Inhibition--
,
-MeATP induced a small
Ca2+ transient similar to that shown by MacKenzie et
al. (7) but only if the suspension buffer contained 1 mM Ca2+. However, it did not induce a
significant increase in Ins(1,4,5)P3 formation (Table
I). In addition,
,
-MeATP did not
inhibit forskolin-stimulated adenylate cyclase activity. An increase in
external Ca2+ from nominal (approximately 10 µM) to 1 mM had no effect on the amount of
Ins(1,4,5)P3 or cyclic AMP formed when platelets were stimulated by ADP (Table I).
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Table I
Comparison of the effect of ADP and , -MeATP on platelet adenylyl
cyclase activity and Ins(1,4,5)P3 formation
For cyclic AMP determination, forskolin (100 µM) was
added 1 min 30 s prior to the addition of ADP or , -MeATP.
Incubations were continued for 2 min after ADP addition after which
stopping solution was added. For [32P]Ins(1,4,5)P3
determination, all reactions were stopped by addition of 0.1 volume of
6.6 N HClO4 at 5 s. In both columns values are given as average of three determinations with standard deviation.
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|
Effect of ARL 66906 on Platelet Aggregation and Shape
Change--
ARL 66096 is an ATP analog that is a highly potent and
selective inhibitor of ADP-induced platelet aggregation (31). As shown
in Fig. 3, ARL 66096 was an effective
inhibitor of ADP-induced platelet aggregation, resulting in complete
inhibition of aggregation at 100-300 nM. However, ARL
66096 failed to block shape change even when a concentration of 1 µM was used (Fig. 3).

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Fig. 3.
The effect of ARL 66096 on ADP-induced
platelet aggregation and shape change. Platelet aggregation was
measured as described. The ordinate represents apparent
changes in optical density due to light scattering by the platelets.
The first arrow indicates the addition of ARL 66096 or
solvent; the second arrow indicates the addition of 10 µM ADP. This scale was set to give a full response for
control ADP-induced aggregation.
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|
Effect of ARL 66096 on Ca2+ Mobilization,
IP3 Metabolism, and Adenylyl Cyclase--
Since ARL 66096 inhibited ADP-induced platelet aggregation, we expected that it would
be equally effective as an inhibitor of ADP-induced Ca2+
mobilization and IP3
metabolism. However, concentrations known to effectively inhibit
aggregation had no effect on either Ca2+ mobilization (Fig.
4) or IP3 formation (Table
II). Since inhibition of adenylyl cyclase
is another major intracellular effect of ADP on platelets, we
investigated the ability of ARL 66096 to block the ADP-induced
inhibition of forskolin-stimulated adenylyl cyclase activity (Fig.
5). ARL 66096 inhibited this response in
the same concentration range as it inhibited aggregation. In contrast
to ARL 66096, ATP blocked ADP-induced Ca2+ mobilization,
shape change, and aggregation within the same concentration range (data
not shown).

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Fig. 4.
The effect of ARL 66096 on ADP-induced
Ca2+ mobilization. Aspirinated platelets labeled with
fura-2 were treated as indicated in a cuvette which was stirred and
maintained at 37 °C.
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Table II
The effect of ARL 66096 on ADP-induced Ins(1,4,5)P3 formation
All reactions were stopped by addition of 0.1 volume of 6.6 N HClO4 at 3 s. Values are given as average of
three determinations with standard deviation.
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Fig. 5.
The effect of ARL 66096 on the inhibition of
adenylyl cyclase by ADP. Washed platelets were stirred at
37 °C. Forskolin (100 µM) was added 1 min 30 s
prior to the addition of ADP (10 µM). The indicated
concentrations of ARL 66096 were added 30 s prior to ADP.
Incubations were continued for 2 min after ADP addition after which
stopping solution was added. Forskolin-stimulated cyclic AMP level in
the absence of ADP was 4.2% of total [3H]adenine
nucleotide. The curve represents the hyperbola that best fit the data
using the program Kaleidagraph (Synergy Software, Reading PA).
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 |
DISCUSSION |
ADP has been known to be a platelet agonist for more than three
decades and the ADP-induced physiological responses and several intracellular responses are well characterized. However, the molecular mechanisms of the effect of ADP on platelets remain obscure. It is well
accepted that the effects of ADP in platelets are receptor-mediated and
this receptor has been designated P2T receptor. However, several observations have remained unexplained. First, 2-MeSADP is at least
100-fold more potent than ADP in causing platelet aggregation and
adenylyl cyclase inhibition, but only 4-fold more potent in mobilization of calcium from intracellular stores (21). Second, the
thienopyridine analogs ticlopidine and clopidogrel when used in
vivo are potent and specific inhibitors of ADP-induced platelet aggregation and antagonize ADP inhibition of prostaglandin
E1-activated adenylyl cyclase in intact platelets (21).
However, these compounds do not block ADP-induced shape change or
intracellular calcium mobilization (32) nor do they affect ADP-induced
phosphorylation of myosin light chain and pleckstrin in platelets (33).
Third, a congenital defect in platelet function attributed to an ADP receptor defect has been described by Nurden et al. (34).
Platelet aggregation was abnormal and ADP-induced inhibition of
adenylyl cyclase activity was abolished. However, ADP-induced shape
change was unaltered. Finally, several megakaryocytic cell lines and human erythroleukemia cells have been reported to express platelet ADP
receptors (35-37) but ADP does not inhibit adenylyl cyclase in these
cell lines (38).
Hourani and Hall (22) have proposed a two-receptor model to explain the
actions of ADP on platelet function. In this model, rapid calcium
influx is mediated by a receptor-operated Ca2+ channel
while adenylyl cyclase inhibition and intracellular Ca2+
mobilization are modulated by the P2T receptor through multiple G-proteins. A similar model was advocated by Gachet and co-workers (26), who proposed that the P2Y1 receptor is the P2T receptor mediating
inhibition of adenylyl cyclase. They propose that calcium entering
through P2X receptor activation could cause mobilization of calcium
from intracellular stores by a calcium-induced calcium release
mechanism. Severe deficiencies of the latter model are first that the
P2X receptor agonist,
,
-MeATP, does not produce either the extent
or duration of Ca2+ mobilization that ADP produces (7) and
second that ADP can cause intracellular Ca2+ mobilization
in the absence of external Ca2+.
Although phospholipase C activation and inositol phosphate formation
have been shown to be important in mobilization of calcium from
intracellular stores in many cellular systems, this intracellular effect of ADP-induced platelet activation has been controversial. In
this paper, we have confirmed our earlier findings that ADP causes an
increase in inositol trisphosphates using high performance liquid
chromatography to separate inositol trisphosphate isomers and show that
there is a significant, 3-fold increase in Ins(1,4,5)P3, which is the isomer that causes release of intracellular stores of
Ca2+ (39). Overall the increases in
Ins(1,4,5)P3 correlate well with changes in intracellular
Ca2+. However, Ca2+ levels start to decline at
10 s while IP3 levels remain relatively high. This
suggests that the initial release of Ca2+ may trigger
resequestration mechanisms that rapidly lower intracellular Ca2+ levels. Levels of intracellular Ca2+, even
through much lower than the peak concentration, remain above basal for
30 s (Fig. 1), especially in the presence of 1 mM
external Ca2+ (7).
We were surprised to find that ARL 66096 had no effect on ADP-induced
intracellular Ca2+ mobilization, in the range where it is
known to inhibit ADP-induced platelet aggregation (31). The possibility
that the effects of ADP on platelets are mediated through at least two
different receptors has long been the subject of conjecture (21, 22). Using ARL 66096, we can clearly separate two distinct responses of
platelets to ADP. ARL 66096 was able to completely inhibit ADP-induced
aggregation at a concentration as low as 100 nM while it
had no effect platelet shape change at concentrations over 10-fold
higher. ARL 66096 also did not inhibit ADP-induced Ca2+
mobilization or IP3 production in this range.
MacKenzie et al. (7) have shown a receptor of the P2X
family, P2X1, on platelets that is coupled to rapid Ca2+
influx.
,
-MeATP, a P2X receptor agonist, while causing a small transient increase in intracellular Ca2+ of a few seconds
duration, failed to cause prolonged mobilization of intracellular
calcium, IP3 formation, or inhibition of adenylyl cyclase
(Fig. 3). Thus IP3 formation and inhibition of adenylyl cyclase are independent of the P2X receptor activation.
These data can best be explained if there are two separate receptors
for ADP on platelets apart from the P2X1 receptor on platelets that is
coupled to rapid Ca2+ influx, one coupled to inhibition of
adenylyl cyclase and the other coupled to phospholipase
C-Ca2+ mobilization. Thus, we propose a three-receptor
model to explain the effects of ADP on platelet activation. This model
explains several of the inconsistencies that two receptors models fail to explain. We propose that 2-MeSADP has relatively higher affinity at
the ADP receptor coupled to adenylyl cyclase inhibition, that thienopyridines selectively inhibit signal transduction through this
same receptor, and that the patient described by Nurden et al. (34) has a defect in this receptor but not in the ADP receptor coupled to activation of phospholipase C. Finally, hemopoetic cell
lines have been shown to lack the ADP receptor coupled to adenylyl
cyclase inhibition but express the ADP receptor coupled to activation
of phospholipase C.
Currently the platelet ADP receptor has been designed P2T (21, 22).
Since this nomenclature does not distinguish between the two
physiological receptors that we have defined, we propose a new
nomenclature that will functionally distinguish the platelet ADP
receptors; a P2TAC receptor coupled to inhibition
of adenylyl cyclase, a P2TPLC
receptor coupled to activation of phospholipase C, and the P2X1
receptor responsible for rapid Ca2+ influx (Fig. 6 and
Table III). The IUPHAR committee has
recommended that the uncloned ADP receptor on platelets should be
designated P2YADP, as the P2Y receptor at which ADP is an
agonist (20). However, since both the P2TPLC and
P2TAC receptors respond to ADP and are not cloned at this
stage, the name P2YADP would not distinguish the two
receptors that we have defined. After cloning, these receptors will be
designed with standard P2Y numbering. The accompanying paper (40)
describes the molecular identity of the P2TPLC receptor and
its functional role in platelet activation. The fact that P2Y1 has been
shown to couple through a G-protein dependent mechanism to
phospholipase C activation, IP3 formation, and
Ca2+ mobilization (25, 41, 42) is fully consistent with our observations that ADP produces IP3 through activation of
this receptor on platelets.
We thank Drs. David C. B. Mills,
A. Koneti Rao, and Robert W. Colman, The Sol Sherry Thrombosis Research
Center, for helpful discussions.