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Platelets, November 2011; 22(7): 504–515
Copyright � 2011 Informa UK Ltd.
ISSN: 0953-7104 print/1369-1635 online
DOI: 10.3109/09537104.2011.576284
ORIGINAL ARTICLE
P2Y12 and EP3 antagonists promote the inhibitory effects of naturalmodulators of platelet aggregation that act via cAMP
DAVID IYU, JACKIE R. GLENN, ANN E. WHITE, SUE C. FOX,
NATALIA DOVLATOVA, & STAN HEPTINSTALL
Cardiovascular Medicine, University of Nottingham, Queen’s Medical Centre, Nottingham, UK
AbstractSeveral antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y12 and EP3receptors, act as antagonists at Gi-coupled receptors, thus preventing a reduction in intracellular cyclic adenosinemonophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function byraising intracellular cAMP. Agents that act as antagonists at Gi-coupled receptors might be expected to promote theinhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y12 antagonists cangrelor, ticagrelor andprasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators ofplatelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response tothe TXA2 mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulatedphosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased thepotency of inhibitory agents that act by raising cAMP (PGI2, iloprost, PGD2, adenosine and forskolin). Ticagrelor and PAMacted similarly to cangrelor. DG-041 increased the potency of PGE1 and PGE2 as inhibitors of aggregation, and cangrelorand DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability ofagents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y12 antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellularcAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.
Keywords: DG-041, EP3 receptor, platelets, prostaglandins, P2Y12 antagonists
Introduction
There are several drugs that modify platelet function
which are used routinely as antithrombotic therapy
or are under investigation for future use. These
include drugs that act as antagonists at P2Y12
receptors on platelets and also drugs that act as
antagonists at EP3 receptors. Drugs already in use as
antithrombotic agents are clopidogrel [1] and prasu-
grel [2], both of which are converted into active
metabolites that act as antagonists at P2Y12 receptors
following administration to man [3–5]. Those in
development include cangrelor [6, 7] and ticagrelor
[8, 9], which are direct-acting P2Y12 antagonists. All
these agents block promotion of platelet function by
adenosine diphosphate (ADP) secreted from plate-
lets [10]. The ADP interacts with P2Y12 receptors
which are linked through Gi to adenylate cyclase and
inhibition of this enzyme results in a lowering of
intracellular cyclic adenosine monophosphate
(cAMP). P2Y12 antagonists prevent this effect of
ADP on platelets [11–13]. EP3 antagonists are also
being developed for potential use as antithrombotic
agents and these include DG-041 [14–17]. The
potential benefit may arise from the ability of EP3
antagonists to prevent promotion of platelet function
by prostaglandin E2 (PGE2) released from athero-
sclerotic plaque [16, 17]. Like P2Y12 receptors, EP3
receptors are also linked through Gi to adenylate
cyclase.
In addition to these pharmacological agents, there
are many other agents that inhibit platelet function.
Some are produced naturally within the vasculature
and there are also some synthetic derivatives of these
agents. Such agents inhibit platelet function by
interacting with Gs-linked receptors and thereby
stimulating the activity of adenylate cyclase and
Correspondence: S. Heptinstall, Cardiovascular Medicine, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH, UK. Tel: 44
115 823 1013. Fax: 44 115 823 1017. E-mail: [email protected]
(received 3 March 2011; accepted 25 March 2011)
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raising the intracellular levels of cAMP. These
include prostaglandin I2 (prostacyclin, PGI2) and
the PGI2 mimetic iloprost which act at IP receptors
[18–20], prostaglandin D2 (PGD2) which acts at DP
receptors [18–20] and adenosine which acts mainly
at A2A receptors [21–23]. Another agent, forskolin,
stimulates adenylate cyclase directly rather than via a
Gs-linked receptor [24]. Prostaglandin E1 (PGE1)
and PGE2 have more complex effects on platelet
function. They have the ability to both promote
platelet function through interaction with EP3 recep-
tors, and to inhibit platelet function through inter-
action with receptors linked to Gs, with the overall
effect being the balance between these opposing
actions. PGE1 inhibits platelet function acting via Gs-
linked IP receptors [25] and PGE2 inhibits platelet
function mainly via EP4 receptors [25–28].
Since P2Y12 antagonists and EP3 antagonists
block the effects of platelet agonists that act via Gi
and result in a lowering of cAMP in platelets, they
might also be expected to enhance the inhibitory
effects of natural or other agents that raise cAMP.
Here, we have investigated this possibility. We
measured the platelet aggregation that occurred in
whole blood in response to the TXA2 mimetic
U46619, to thrombin receptor activating peptide
(TRAP) and to both of these agents added together,
and determined the ability of cangrelor to promote
inhibition of platelet aggregation by a number of
different agents. Some experiments were also per-
formed in which cangrelor was replaced by ticagrelor
or prasugrel active metabolite (PAM). We also
looked at the ability of the EP3 antagonist DG-041
to modify the effects of PGE1 and PGE2 on platelet
aggregation, both alone and in combination with
cangrelor. The effects on the action of prostaglandin
E3 (PGE3) were also investigated.
The results of our investigations demonstrate the
ability of antagonists that act at Gi-linked receptors
such as P2Y12 and EP3 to enhance the inhibitory
effects of natural and other modulators of platelet
function that act via cAMP. Our results demonstrate
potentiation of inhibition of aggregation even under
circumstances where the antagonists have little direct
effect on the aggregation response. Indeed, potenti-
ation by P2Y12 and/or EP3 antagonists of inhibition
of aggregation by natural agents should be seen as an
additional mechanism through which such antago-
nists may provide clinical benefit.
Materials and Methods
Materials
Hirudin (recombinant desulphato-hirudin, RevascTM)
was a gift from Novartis (Basel, Switzerland).
Cangrelor was a gift from The Medicines Company
(Abingdon, Oxfordshire, UK) and was dissolved in
saline. Ticagrelor and PAM were gifts from
AstraZeneca (R&D Molndal, Sweden) and were
dissolved in DMSO. PGE1, PGE2, U46619, TRAP
6, ADP, adenosine deaminase (ADA), adenosine,
forskolin and dipyridamole were from the Sigma
Chemical Co (Poole, UK). PGI2, PGE3, PGD2 and
iloprost were from Cayman Chemical (Ann Arbor,
MI, USA). Polyfusor saline 0.9% was obtained from
Fresenius Kabi Ltd (Cheshire, UK). The fixative
solution was 150 mM sodium chloride containing
4.6 mM EDTA, 4.5 mM disodium hydrogen phos-
phate (Na2HPO4), 1.6 mM potassium dihydrogen
phosphate (KH2PO4) and 0.16% (w/v) formaldehyde,
pH 7.4. For the Vasodilator-stimulated phosphopro-
tein phosphorylation (VASP-P) measurements, the
Functional Bead Conjugation Buffer Set and Cell
Signaling Master Buffer Kit were obtained from
Becton Dickinson (Oxford, UK). IE273 anti-VASP
was from Alexis Biochemicals (San Diego, CA, USA).
FITC-conjugated antibody 5C6 anti-VASP pSer 157
was from Acris Antibodies (Insight Biotechnology,
Wembley, UK). Lysis buffer consisted of Tris buff-
ered saline, pH 7.6 from the Sigma Chemical Co
(Poole, UK) containing Triton X-100 (1%), the ionic
detergent sodium deoxycholate (0.25%) and phenyl-
methanesulfonyl fluoride (1 mM). A protease inhib-
itor tablet (Complete Mini, Roche Diagnostics
GmbH, Manheim, Germany) and phosphatase inhib-
itor (Cocktail Set II, Calbiochem, Darmstadt,
Germany) were also added to the lysis buffer accord-
ing to the manufacturers’ instructions.
Methods
Blood collection. Venous blood was obtained follow-
ing informed consent from healthy volunteers who
denied taking any medication. For aggregation stud-
ies, blood was taken into hirudin (50 mg/ml) contain-
ing either cangrelor or vehicle, and maintained at
37�C for 30 minutes prior to experimentation.
Sometimes, ticagrelor or PAM was used in place of
cangrelor, in which case the vehicle was DMSO
(0.03%). For studies of VASP-P, blood was taken
into hirudin (50mg/ml) and platelet-rich plasma
(PRP) prepared.
Preparation of PRP. Freshly collected blood was
centrifuged (180g, 10 minutes) and the supernatant
PRP removed. The remainder of the blood was re-
centrifuged (1500g, 10 minutes) to obtain platelet-
poor plasma (PPP) and the PRP diluted using
autologous PPP to a standard platelet count of
300� 109/l. The PRP was incubated with ADA
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(1.2 U/ml) at 37�C for 10 minutes prior to
experimentation.
Platelet aggregation. Aliquots of whole blood (final
volume 495ml) were dispensed into pre-warmed
tubes containing the agent(s) under investigation
and a stirrer bar. The tubes were placed in the
stirring wells of a Multi-Sample Agitator (MSA,
University of Nottingham, UK) operating at
1000 rpm, 37�C. After 1 minute, an aliquot (15 ml)
of the sample was removed and mixed with fixative
solution (30 ml) to provide a ‘starting’ platelet count.
After a further minute, 40 ml of a solution of U46619,
TRAP or a combination of the two was added.
Aliquots (15 ml) of the sample were removed at 1, 2
and 4 minutes and mixed with fixative solution
(30ml). The platelet count of the fixed samples was
determined using the Ultra-Flo 100 whole blood
platelet counter. Platelet aggregation was calculated
as the percentage loss of single platelets compared to
the ‘starting’ platelet count of whole blood. The data
are presented as area under the curve (AUC),
measured over a 4-minute period and calculated
using the trapezium rule.
Measurement of phosphorylation of VASP. Aliquots of
PRP (final volume 250 ml) were dispensed into pre-
warmed (37�C) tubes containing each of the agents
under investigation or vehicle. After 5 minutes, the
reaction was stopped by the addition of ice-cold PBS
containing 4 mM EDTA (1 ml) and the samples were
placed on ice and transferred immediately to cold
Eppendorf tubes. The samples were then centrifuged
(11600g, 10 seconds), the supernatant discarded and
the resulting platelet pellet lysed by vigorous resus-
pension in lysis buffer (100ml). Samples were then
frozen and stored at �20�C prior to assay. The
amounts of the phosphorylated form of VASP
(VASP-P) present in the lysates were measured
using a flow cytometric bead assay which has been
described previously [25, 27–29]. Briefly, platelet
lysates (25ml) were incubated with APC-fluorescent
beads (Becton Dickinson) (25ml) coated with a
monoclonal antibody to VASP (mAb IE273 anti-
VASP conjugated using the Functional Bead
Conjugation Buffer Set). Bound VASP-P was
detected by addition of a second fluorescent antibody
(FITC-conjugated antibody 5C6 anti-VASP pSer
157, 10 mg/ml) (7.5 ml). Samples were incubated in
the dark at room temperature for 2 hours prior to
analysis. Samples were then resuspended in 150 ml
wash buffer (Cell Signaling Master Buffer Kit) and
flow cytometry performed on a Becton Dickinson
LSRII flow cytometer using FACSDiva acquisition
software using both the blue (488 nm) and red laser
(17 mW power, excitation 633 nM). Capture beads
were identified by their associated APC fluorescence
and 300 bead events were collected. Phosphorylated
VASP was detected as FITC fluorescence expressed
as median fluorescence (MF).
Data presentation. Data are presented as
mean�SEM and concentration-response curves
were compared by repeated measures using SPSS
16 statistical software. Actual numbers of the differ-
ent experiments performed are provided in the
Figure captions.
Results
Effects of cAMP-elevating agents on platelet
aggregation in the absence or presence of the P2Y12
antagonist cangrelor
We determined the effects of several different cAMP-
elevating agents on the extent of the platelet aggre-
gation that occurred in response to U46619, TRAP
and the combination of the two agents, in the
absence and presence of the P2Y12 antagonist
cangrelor. We chose 10 mM U46619, 20mM TRAP
or the combination of 10 mM U46619 and 20 mM
TRAP as the aggregating agents because these
agonists at the concentration used were always
found to produce a substantial aggregation response
that was unaffected by Gi antagonists when used on
their own. We chose to use cangrelor at a concen-
tration of 1mM as this is known to provide effective
inhibition of the effects of ADP at P2Y12 receptors
on platelets [28]. In each case, the effects of a range
of concentrations of the cAMP-elevating agent were
tested.
The effects of PGI2 are presented in Figure 1(a)–
(c) and Table I. PGI2 inhibited the platelet
aggregation induced by U46619 (Figure 1(a)),
TRAP (Figure 1(b)) and the combination of
U46619 and TRAP (Figure 1(c)) in a concentra-
tion-dependent way, and cangrelor markedly
enhanced the ability of the PGI2 to inhibit the
aggregation response. The effect of cangrelor was to
reduce the IC50 values for inhibition of aggregation by
PGI2 from (1) 2.8� 0.6 to 0.8� 0.2 nM, (2) 6.0� 1.0
to 1.1� 0.4 nM and (3) 49� 23 to 4.3� 1.2 nM
(Table I).
Similar effects of cangrelor were also seen when
forskolin was used as the cAMP-elevating agent
(Figure 2, Table I). Forskolin itself had little effect
on the platelet aggregation induced by U46619
(Figure 2(a)), TRAP (Figure 2(b)) and the combi-
nation of U46619 and TRAP (Figure 2(c)) but
cangrelor markedly enhanced the ability of forskolin
to inhibit the aggregation response. The effect of
cangrelor was to reduce the IC50 values for inhibition
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of aggregation by forskolin from 410mM to (1)
1.9� 0.5 mM, (2) 3.2� 1.3 mM and (3) 6.4� 2.0mM
(Table I).
Very similar results were obtained using the
PGI2 mimetic iloprost or PGD2 in place of PGI2
(Table I). Similar results were also obtained
using adenosine as the cAMP-elevating agent
(it should be noted that the effects of adenosine
were determined in the presence of dipyridamole
[10mM] which prevents uptake of adenosine into red
cells, thereby allowing the adenosine to interact with
platelets).
0
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400
0 1 3 10 30 100
0 1 3 10 30 100
0 1 3 10 30 100
Agg
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(AU
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PGI2 (nM)
*
0
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Agg
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(AU
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PGI2 (nM)
*
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Agg
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(AU
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PGI2 (nM)
*
U46619
TRAP
U46619 + TRAP
(a)
(b)
(c)
Vehicle
Cangrelor
Figure 1. Cangrelor promotes the ability of PGI2 to inhibit platelet aggregation. Aggregation was induced by (a) U46619; (b) TRAP; and
(c) U46619 in combination with TRAP, and was determined in whole blood by single platelet counting. Data is presented as AUC
calculated over a 4-minute period. The results shown are the mean�SEM of three experiments. *p50.05 cangrelor cf vehicle.
Mechanisms in inhibition of platelet aggregation 507
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Effects of PGEs on platelet aggregation in the absence
or presence of the P2Y12 antagonist cangrelor and the
EP3 antagonist DG-041
We determined the effects of PGE1, PGE2 and PGE3
on the extent of the platelet aggregation that
occurred in response to U46619, TRAP and the
combination of the two agents, in the absence and
presence of the P2Y12 antagonist cangrelor and also
in the absence and presence of the EP3 antagonist
DG-041. Again, we chose 10mM U46619, 20mM
TRAP or their combination as the primary agonists
and used cangrelor at a concentration of 1mM. We
chose to use DG-041 at a concentration of 3mM
which has been demonstrated to provide effective
EP3 receptor blockade [14]. The effects of a range of
concentrations of PGEs were tested; the actual
concentrations used were different for each depend-
ing on efficacy.
The effects of PGE2 are presented in Figure 3, and
the IC50 values for all three PGEs are presented in
Table II. PGE2 used alone provided little or no
inhibition of platelet aggregation but did so after
blockade of either the P2Y12 receptor with cangrelor
or the EP3 receptor with DG-041. Blockade of both
receptors enhanced the ability of PGE2 to inhibit
platelet aggregation very effectively indeed, with IC50
values reduced from 410 mM to (1) 0.2� 0.0 mM
when U46619 was used as the agonist, to (2)
0.5� 0.2 mM when TRAP was used as the agonist
and to (3) 2.0� 0.6 mM using the combination of
U46619 and TRAP.
PGE1 was much more potent than PGE2 and
provided inhibition of aggregation when used alone
with IC50 values of 167� 59 and 173� 50 nM for
U46619 and TRAP, respectively. Again, cangrelor
and DG-041 enhanced the ability of the prostaglan-
din to inhibit platelet aggregation, and the combina-
tion of cangrelor and DG-041 provided further
inhibition (Table II).
PGE3 was similar to PGE2 in that when used
alone it produced little inhibition of platelet aggre-
gation. In the presence of cangrelor, however,
concentration-dependent inhibition by PGE3 of
aggregation induced by U46619 or TRAP was
evident with IC50 values of 0.3� 0.1 mM in both
cases. These appeared to be reduced even further
when DG-041 was also present (Table II).
Effects of PGE1 on platelet aggregation in the presence
of a range of concentrations of the P2Y12 antagonist
cangrelor
We determined the effects of a range of concentra-
tions of cangrelor on the ability of PGE1 to inhibit the
aggregation induced by 10 mM U46619 and 20 mM
TRAP. The results are presented in Figure 4. The
results clearly show that the effects of cangrelor are
concentration-dependent.
Effects of PGI2 on platelet aggregation in presence of
the P2Y12 antagonists cangrelor, ticagrelor or PAM,
with or without aspirin
The main purpose of this experiment was to compare
the effects of cangrelor with those of other P2Y12
antagonists. The comparative agents that were
chosen were ticagrelor and PAM. The abilities of
these three agents to affect the inhibition by PGI2 of
the platelet aggregation induced by 20mM
TRAP were compared. The concentrations of
cangrelor, ticagrelor and PAM used were 1, 10
and 10 mM, respectively, all concentrations being
Table I. IC50 values for various cAMP-elevating agents in the absence and presence of cangrelor.
cAMP-elevating agent Aggregating agent Vehicle Cangrelor
PGI2 (nM) U46619 2.8� 0.6 0.8�0.2
TRAP 6.0� 1.0 1.1�0.4
U46619þTRAP 49� 22.6 4.3�1.2
Iloprost (nM) U46619 3.3� 1.3 0.9�0.1
TRAP 4.5� 1.1 1.0�0.0
U46619þTRAP 19.7� 0.3 2.1�0.1
PGD2 (nM) U46619 33� 13 19�5
TRAP 117� 62 15�8
U46619þTRAP 413� 213 43�24
Adenosine (mM) U46619 410 0.7�0.1
TRAP 410 2.3�0.4
U46619þTRAP 410 410
Forskolin (mM) U46619 410 1.9�0.5
TRAP 410 3.2�1.3
U46619þTRAP 410 6.4�2
Notes: IC50 values were determined in response to aggregation induced by U46619, TRAP or a
combination of U46619 and TRAP in whole blood. For determination of the IC50 value for adenosine,
experiments were performed in the presence of dipyridamole.
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those expected to provide high levels of P2Y12
receptor blockade [28]. The results of this investiga-
tion are shown in Figure 5, where it can be seen that
all three agents were equally effective in enhancing
the ability of PGI2 to inhibit the aggregation
response.
Parallel experiments were also performed to find
out whether the presence of aspirin affected the
0
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400
Agg
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(AU
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*
0
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400
Agg
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(AU
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*
0
100
200
300
400
0 0.1 0.3 1 3 10
0 0.1 0.3 1 3 10
0 0.1 0.3 1 3 10
Agg
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(AU
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Forskolin (mM)
Forskolin (mM)
Forskolin (mM)
*
U46619
TRAP
U46619 + TRAP
(a)
(b)
(c)
Vehicle
Cangrelor
Figure 2. Cangrelor promotes the ability of forskolin to inhibit platelet aggregation. Aggregation was induced by (a) U46619; (b) TRAP;
and (c) U46619 in combination with TRAP, and was determined in whole blood by single platelet counting. Data is presented as AUC
calculated over a 4-minute period. The results shown are the mean�SEM of three experiments. *p50.05 cangrelor cf vehicle.
Mechanisms in inhibition of platelet aggregation 509
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results obtained. It can be seen that aspirin had
virtually no effect (Figure 5).
Effects of the P2Y12 antagonist cangrelor and the EP3
antagonist DG-041 on VASP-P
The purpose of these experiments was to con-
firm that both the P2Y12 antagonist cangrelor
and the EP3 antagonist DG-041 are able to
promote cAMP production in platelets as measured
using VASP-P. We show in Figure 6(a) that
cangrelor promotes the ability of the cAMP-
elevating agent iloprost to increase VASP-P when
ADP is present. We show in Figure 6(b) that
DG-041 promotes the ability of PGE2 to increase
VASP-P.
0
100
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300
400
Agg
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(AU
C)
U46619
TRAP
U46619 + TRAP
0
100
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400
0 0.1 0.3 1 3 10
0 0.1 0.3 1 3 10
0 0.1 0.3 1 3 10
Agg
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(AU
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PGE2 (mM)
PGE2 (mM)
PGE2 (mM)
Vehicle
Cangrelor
DG-041
DG-041 + Cangrelor
0
100
200
300
400
Agg
rega
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(AU
C)
*
#
§
#
§
*
§
(a)
(b)
(c)
Figure 3. Cangrelor and the EP3 antagonist DG-041 promote the ability of PGE2 to inhibit platelet aggregation. Aggregation was induced
by (a) U46619; (b) TRAP; and (c) U46619 in combination with TRAP, and was determined in whole blood by single platelet counting.
Data is presented as AUC calculated over a 4-minute period. The results shown are the mean�SEM of three experiments. *p50.05
cangrelor cf vehicle, #p50.05 DG-041 cf vehicle, xp50.05 DG-041þ cangrelor cf cangrelor.
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U46619
TRAP
0
100
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400
Agg
rega
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(AU
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PGE1 (mM)
PGE1 (mM)
0
0.001
0.01
0.1
1
Cangrelor (mM)
0
100
200
300
400
0 0.01 0.03 0.1 0.3 1
0 0.01 0.03 0.1 0.3 1
Agg
rega
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(AU
C)
0
0.001
0.01
0.1
1
Cangrelor (mM)
(a)
(b)
Figure 4. The ability of cangrelor to promote inhibition of platelet aggregation by PGE1 is concentration-dependent. Aggregation was
induced by (a) U46619 and (b) TRAP, and was determined in whole blood by single platelet counting. Data are presented as AUC
calculated over a 4-minute period. The results shown are the mean�SEM of three experiments.
Table II. IC50 values for various cAMP-elevating agents in the absence and presence of cangrelor and/or the EP3 antagonist, DG-041.
cAMP-elevating agent Aggregating agent Vehicle Cangrelor DG-041 CangrelorþDG-041
PGE1 (mM) U46619 0.167� 0.059 0.015� 0.005 0.023� 0.003 0.011� 0.005
TRAP 0.173� 0.050 0.042� 0.029 0.077� 0.038 0.010� 0.005
U46619þTRAP 41 0.120� 0.062 0.157� 0.043 0.032� 0.009
PGE2 (mM) U46619 410 6.1� 1.7 4.4� 1.9 0.2� 0.0
TRAP 410 5.8� 1.1 4.0� 1.6 0.5� 0.2
U46619þTRAP 410 410 410 2.0� 0.6
PGE3 (mM) U46619 41 0.3� 0.1 41 0.05� 0.01
TRAP 41 0.3� 0.1 41 0.1� 0.06
U46619þTRAP 41 41 41 41
Note: IC50 values were determined in response to aggregation induced by U46619, TRAP or a combination of U46619 and TRAP in
whole blood.
Mechanisms in inhibition of platelet aggregation 511
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Discussion
In these experiments, we consistently saw marked
potentiation of the inhibitory effects of natural and
other modulators of platelet function by a P2Y12
antagonist. Cangrelor increased the inhibitory
potency of PGI2, the PGI2 mimetic iloprost, PGD2,
PGE1, PGE2, PGE3, adenosine and forskolin. The
degree of potentiation of inhibition was dependent
on whether aggregation was induced by a single
agent (U46619 or TRAP), or both in combination.
The effect depended on the concentration of
cangrelor that was used. Other P2Y12 antagonists
0
10000
20000
30000
40000
0 0.1 0.3 1 3 10
VA
SP
-P (m
f)
Iloprost (nM)
ADP
ADP + cangrelor
0
10000
20000
30000
40000
50000
0 0.1 0.3 1 3 10
VA
SP
-P (m
f)
PGE2 (mM)
Vehicle
DG-041
Iloprost
PGE2
*
#
(a)
(b)
Figure 6. Cangrelor promotes the ability of iloprost to increase platelet VASP-P in the presence of ADP ((a). Similarly, the EP3 antagonist
DG-041 promotes the ability of PGE2 to increase VASP-P (b). VASP-P was determined in PRP by flow cytometric bead assay and
is presented as MF. The results shown are the mean�SEM of three experiments. *p50.05 ADPþ cangrelor cf ADP, #p50.05
DG-041 cf. vehi.
0
100
200
300
400
0 1 3 10 30 100
Agg
rega
tion
(AU
C)
PGI2 (nM)
Vehicle
Cangrelor
Ticagrelor
PAM
Aspirin
Cangrelor + Aspirin
Ticagrelor + Aspirin
PAM + Aspirin
Figure 5. Cangrelor, ticagrelor and PAM promote the ability of PGI2 to inhibit platelet aggregation in both the absence and presence of
aspirin. Aggregation was induced by U46619 and was determined in whole blood by single platelet counting. Data are presented as AUC
calculated over a 4-minute period. The results shown are the mean�SEM of three experiments.
512 D. Iyu et al.
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ticagrelor and the PAM produced effects that were
very similar to those of cangrelor.
We must point out that potentiation by P2Y12
antagonists of the inhibitory effects of PGE1 on
platelet function has been described before. Fox et al.
[30] described the effects of cangrelor (AR-C69931,
used in vitro) and of clopidogrel (effects measured
ex vivo) on ADP-induced platelet aggregation and
Ca2þ mobilization in human platelets in the absence
and presence of PGE1. They concluded that both
P2Y12 antagonists acted in synergy with PGE1 to
reduce platelet function. There has also been an
investigation of the effects of cangrelor and PGI2 on
thrombin-induced platelet aggregation and this also
demonstrated an interaction between these agents
[31]. Our own studies both confirm and extend these
observations.
Our results also show that, in the case of PGE1
and PGE2, inhibition of platelet aggregation is also
enhanced by the EP3 receptor antagonist DG-041.
Further, the combination of a P2Y12 antagonist and
DG-041 produces even more inhibition of platelet
aggregation. PGE1 affects platelet aggregation via
interaction with the Gs-linked IP receptor on plate-
lets, and with the Gi-linked EP3 receptor. The
overall effect is inhibition of platelet aggregation
because the effect at IP outweighs the effect at EP3.
Nevertheless, as demonstrated here and previously
[25], the EP3 receptor antagonist DG-041 enhances
the overall inhibitory effect of PGE1 on platelet
aggregation. The new information provided here is
that the combination of DG-041 and a P2Y12
antagonist which together block the effects of both
PGE1 and ADP at their Gi-linked receptors, pro-
duces even further inhibition of platelet function.
PGE2 affects platelet aggregation via interaction
with the Gs-linked EP4 receptor on platelets, and
also with the Gi-linked EP3 receptor [25, 26, 32, 33].
In this case, the effects at these two receptors
counterbalance each other and the overall effect of
PGE2 on platelet function is quite neutral. When the
effect of PGE2 at EP3 receptors is blocked using DG-
041, only the effects of PGE2 at EP4 receptors are
evident and inhibition of platelet aggregation is
clearly seen [25–27]. Also, as with PGE1, the
combination of DG-041 and a P2Y12 antagonist
produces even further inhibition of platelet function.
Our data also show that similar results are obtained
using PGE3 in place of PGE2.
In a previous publication [29], we have already
shown that P2Y12 antagonists enhance the inhibitory
effects of adenosine. Indeed, concentration-depen-
dent inhibition of platelet function was also seen with
ADP in the presence of a P2Y12 antagonist conse-
quent to conversion of the ADP to adenosine.
However, this was only seen in experiments per-
formed in PRP; it did not occur in whole blood
because of the rapid uptake of adenosine into red
cells via the equilibrative nucleotide transporter
(ENT). However, in the presence of dipyridamole,
an inhibitor of ENT [34–37], both adenosine and
ADP brought about inhibition of platelet function as
in PRP. That adenosine is indeed the mediator of
inhibition of platelet function was confirmed using
ADA which prevented the inhibitory effects of
adenosine and ADP [29]. In the experiments
described here, we looked at the potency of adeno-
sine acting as an inhibitor of platelet aggregation in
whole blood containing dipyridamole, and showed
that in the presence of a P2Y12 antagonist that
potency was increased in the same way as for the
prostaglandins that we investigated.
In the current investigation, we also demonstrate
that the P2Y12 antagonist cangrelor potentiates the
effects of forskolin on platelet function. Thus, the
mechanism through which agents increase cAMP is
immaterial; involvement of Gs-linked receptors in
increasing cAMP is not a requirement in order for an
antagonist acting at a Gi-linked receptor to enhance
inhibition of platelet aggregation.
In all the experiments described in this article,
cangrelor or another P2Y12 antagonist when used
alone had no or only a minor effect on the aggrega-
tion induced by U46619, TRAP or the combination
of these. Also, aspirin, which is commonly adminis-
tered with P2Y12 antagonists as a means of providing
additional inhibition of platelet function, did not add
to the inhibition of aggregation. Despite this, there
was always potentiation by the P2Y12 antagonists of
the inhibitory effects of agents that inhibit platelet
function by raising cAMP. Thus, the increase in
inhibitory potency of natural modulators of platelet
function achieved in the presence of a P2Y12 antag-
onist appears to be more important than the direct
effects on platelet function of either a P2Y12 antag-
onist or aspirin.
It is interesting to consider whether the findings
reported above are relevant to the clinical use of
drugs that act at Gi-linked receptors. For example,
the current practice is to administer a P2Y12 antag-
onist in combination with aspirin. But is this a good
practice given that aspirin, at least when used in
higher doses, will inhibit synthesis of natural mod-
ulators of platelet function such as PGI2, PGD2 and
PGE prostaglandins? Similarly, the ability of a P2Y12
antagonist to interact with adenosine may lead to a
re-appraisal of the potential for joint therapies that
include dipyridamole, and also possibly cilostazol
[38] given the ability of these antithrombotic drugs to
prevent uptake of adenosine into red cells.
Regarding the advisability of using a P2Y12
antagonist with aspirin for antithrombotic therapy,
it is interesting to note the apparent reduction in
therapeutic value of the P2Y12 antagonist ticagrelor
Mechanisms in inhibition of platelet aggregation 513
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Page 11
compared with clopidogrel in the North America
subset of data in the PLATO study, compared with
the clear superiority of ticagrelor in reducing vascular
events in other parts of the world [39]. The North
America subset is a group in whom aspirin was used
at a higher dose than in other study participants.
Presumably, a high dose of aspirin would have
prevented synthesis of circulating inhibitory prosta-
glandins resulting in no possibility of further inhibi-
tion of platelet function via the interaction between
the P2Y12 antagonists used in PLATO and vascular
prostaglandins. This could be a consideration for
those investigating the reasons for the different
results in the North America subgroup [40].
Regarding the possible future use of an EP3
antagonist such as DG-041 as antithrombotic ther-
apy based on its ability to improve inhibition of
platelet aggregation brought about by prostaglandins
of the E series [25–27], account should now be taken
of the further inhibition of platelet function seen
when used in combination with a P2Y12 antagonist,
as demonstrated here. Interestingly, there is evidence
that omega-3 fatty acids, which are precursors of
PGE3, potentiate platelet inhibition associated with
clopidogrel in patients undergoing percutaneous
coronary intervention [41]. The possibility of achiev-
ing additional clinical benefit through the use of DG-
041 may warrant further investigation.
In conclusion, we believe that the ability of
antagonists that act at receptors linked to Gi such
as P2Y12 and EP3 antagonists to enhance the
inhibitory effects of natural modulators of platelet
function could have major implications for their
clinical application that will need to be assessed in
future clinical trials.
Acknowledgements
The authors are grateful to the following: The
Medicines Company (Abingdon, Oxfordshire, UK)
for the gift of cangrelor; AstraZeneca (R&D
Molndal, Sweden) for ticagrelor and PAM;
deCODE genetics (Reykjavik, Iceland) for DG-041.
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