European Heart Journal (2016) 37, 3347–3356
doi:10.1093/eurheartj/ehv551
BASIC SCIENCE
Inhibition of the platelet P2Y12 receptor for
adenosine diphosphate does not impair the
capacity of platelet to synthesize thromboxane A2
Mariangela Scavone1†, Eti Alessandra Femia 1†, Vera Caroppo 1,
and Marco Cattaneo 1,2*
1
Dipartimento di Scienze Della Salute, Università Degli Studi di Milano, Milano, Italy; and 2Unità di Medicina 3, Azienda Ospedaliera San Paolo, Via di Rudinı̀, 8, 20142 Milano, Italy
Received 20 April 2015; revised 21 August 2015; accepted 25 September 2015; online publish-ahead-of-print 29 October 2015
Aims
Patients with acute coronary syndromes (ACSs) are treated with acetylsalicylic acid (ASA) and antagonists of the P2Y12
receptor (P2Y12R) for adenosine diphosphate (ADP). Based on the demonstration that P2Y12R antagonists inhibit
thromboxane A2 (TxA2) production (target of ASA), it was surmised that ACS patients might be treated with
P2Y12R antagonists only. However, this demonstration contrasts with the results of previous studies. The aim of this
study was to test whether P2Y12R antagonists have off-target/indirect inhibitory effects on platelet TxA2 production.
.....................................................................................................................................................................................
Methods
We studied 3 patients with inherited P2Y12R deficiency and 33 healthy subjects. Serum TxB2 (TxA2 metabolite) levels
and results
were similar in P2Y12R-deficient patients and healthy subjects and were not decreased by P2Y12R antagonists in vitro.
Serum TxB2 levels did not decrease in 20 patients treated with prasugrel (10 mg q.i.d.) or placebo for 14 days. Arachidonic acid- and collagen-induced platelet aggregation (PA) and TxB2 production in platelet-rich plasma (PRP) of healthy
subjects were inhibited in vitro by P2Y12R antagonists. However, P2Y12R antagonists did not inhibit TxB2 production
when PA was prevented by avoiding the stirring of PRP in the aggregometer. The P2Y1 ADP-receptor antagonist
MRS2500 had similar effects on PA and TxB2 production as P2Y12R antagonists. Acetylsalicylic acid inhibited TxB2 production more effectively than a P2Y12R antagonist; only the combination of ASA and a P2Y12R antagonist inhibited PA
induced by high concentration of collagen.
.....................................................................................................................................................................................
Conclusion
Inherited deficiency or pharmacological inhibition of P2Y12R does not affect the platelet capacity to synthesize TxA2.
There is no pharmacological evidence that ACS patients may be safely treated with P2Y12R antagonists without ASA.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
Antiplatelet drugs † Aspirin † Myocardial infarction † P2Y12 receptor † Platelet aggregation † Thromboxane A2
Translational perspective
Based on the demonstration that antagonists of the platelet P2Y12R for ADP inhibit the platelet production of TxA2, it has been surmised
that patients with ACSs, who are generally treated by double antiplatelet therapy with ASA (which almost completely inhibits the production of TxA2) and a P2Y12R antagonist, may be treated with the latter compound only. The present study shows that P2Y12R antagonists do
not inhibit the capacity of platelets to synthesize TxA2. The reduction of TxA2 production by P2Y12R antagonists that were shown in recent
studies is partial (therefore, of very limited clinical significance) and mediated by inhibition of PA; as such, it is not distinctive feature of
P2Y12R antagonists, but common to other inhibitors of PA, independently of their mechanism of action. The study also shows that only
the combination of ASA and a P2Y12R antagonist efficiently inhibit PA induced by high concentration of collagen. Therefore, there is no
pharmacological rational for suggesting to treat ACS patients with a P2Y12R-receptor antagonist only.
* Corresponding author. Tel: +39 0250323095, Fax: +39 0250323089, Email: [email protected]
†
These authors contributed equally.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected].
3348
Introduction
Dual antiplatelet therapy (DAPT) with acetylsalicylic acid (ASA)
and an inhibitor of the platelet P2Y12 receptor (P2Y12R) for adenosine diphosphate (ADP) is indicated for treatment of patients
with acute coronary syndromes (ACSs) or undergoing percutaneous coronary intervention (PCI).1 Dual antiplatelet therapy inhibits two major amplification pathways of platelet aggregation
(PA): the arachidonic acid (AA) – thromboxane A2 (TxA2) pathway (inhibited by ASA) and the ADP/P2Y12R pathway. This double mechanism of action explains the superiority of DAPT,
compared with ASA alone, in preventing major adverse cardiovascular events.1
The need for DAPT has recently been questioned, based on the
observation that inhibition of P2Y12R decreases the platelet production of TxA2,2 – 4 thus rendering the use of ASA superfluous or
even detrimental, considering its side effects and that it can inhibit
the production of the natural antithrombotic prostacyclin.5 Based
on these findings, it has been speculated that patients with ACS
may be treated with P2Y 12 R antagonists only. 5 As a matter of
fact, a trial comparing the use of ticagrelor alone with ticagrelor
plus ASA after treatment with ticagrelor and ASA for the first
month after PCI is ongoing (ClinicalTrials.gov identifier:
NCT01813435).
However, the findings of inhibition of TxA2 production by
P2Y12R antagonists contrast with previous demonstrations that patients with inherited P2Y12R deficiency have normal serum TxB2 levels6,7 and that ADP does not elicit the formation of TxA2 by
platelets.8,9 In order to try and shed some light on this issue, we
tested whether P2Y12R antagonists have off-target or indirect effects on TxA2 production. Moreover, we tested whether the combination of ASA and P2Y12R inhibition is superior to either drug
alone in inhibiting PA and TxA2 production.
Methods
Study populations
M. Scavone et al.
Sweden). Cangrelor was provided by The Medicines Company, USA.
MRS2500 was from Tocris Bioscience (Bristol, UK). The lysine salt of
ASA (Flectadol, Sanofi-Aventis, Milano, Italy) and cangrelor was dissolved in saline, the remaining P2Y12R antagonists were dissolved in
dimethylsulfoxide.
Blood sampling
Venous blood samples were collected using a 21-gauge butterfly needle with minimal stasis. The first 3 mL was collected into K-EDTA and
analysed by coulter haematology analyser (Beckman Coulter, Milano,
Italy); 60 mL was collected into trisodium citrate (109 mM, 1:9, v/v) or
400 ATU/mL lepirudin (Verum Diagnostica, Munich, Germany), gently mixed, allowed ‘to rest’ at room temperature (RT) for 15 min, and
centrifuged to obtain platelet-rich plasma (PRP).12 Blood samples
for preparation of serum were collected into tubes without
anticoagulant.
Platelet aggregation studies
Platelet aggregation was measured in PRP by light transmission aggregometry (LTA) using Chrono-Log 560 (Havertown, PA, USA) or Platelet
Aggregation Profiler-8E (Biodata, Horsham, PA, USA) and, in whole
blood, by impedance aggregometry (MultiplateTM , Roche Diagnostics,
Monza, Italy).
Light transmission aggregometry
Platelet-rich plasma was obtained by centrifuging blood samples at
200 g at RT for 10 min.12,13 Platelet count of PRP was not adjusted
to a pre-determined value.12,14 Platelet-rich plasma was placed into aggregation cuvettes and incubated with vehicle, ASA (500 mM), a
P2Y 12 R antagonist (10 mM), or the P2Y 1 R-antagonist MRS2500
(10 mM) at 378C for 10 min, and then stimulated with AA (1 mM),
ADP (5 or 10 mM), epinephrine (1 mM), or collagen (0.5 or 10 mg/
mL). After stimulation, PRP was stirred at 1000 rpm to allow PA to
occur,12 or left unstirred, to prevent PA to occur. Changes in light
transmission (caused by the formation of platelet aggregates) were
recorded for 3 min.
Impedance aggregometry
Citrate blood (300 mL) was mixed with 300 mL of saline and CaCl2
(3 mM) and incubated with vehicle or platelet inhibitors at 378C for
10 min. Then, platelet agonists were added under stirring and nonstirring conditions, and platelet aggregates formation was recorded.
For in vitro studies of the effects of P2Y12R antagonists on PA and TxB2
production, we enrolled 33 healthy subjects (15 men, age range: 20 – 63
years) and patients with inherited homozygous (n ¼ 2) or heterozygous
(n ¼ 1) P2Y12R deficiency.6,7,10
To study the effect of in vivo administration of the P2Y12R-antagonist
prasugrel on serum TxB2 levels, we used serum samples from 20 patients with bronchial asthma who had been enrolled in PRasugrel IN
Asthma (PRINA), a randomized, cross-over, double-blind, placebocontrolled trial,11 in which patients were randomized to treatment
with 10 mg prasugrel q.i.d. or placebo for 15 days and, after a washout
of ≥15 days, to the alternative treatment.11
All studied subjects abstained from drugs known to affect platelet
function for at least 10 days before enrolment.12 The study was approved by the institutional review committee of Azienda Ospedaliera
San Paolo, Milano, IT; all subjects signed their informed consent.
Non-anticoagulated blood samples (3 mL) were allowed to clot at 378C
for 1 h and then centrifuged at 1400 g at RT for 15 min.15,16 For studies
of in vitro effects of inhibitors, 1 mL aliquots from a blood sample of 7 mL
were mixed with vehicle or each P2Y12R antagonist (10 and 100 mM)
and processed as above. Careful attention was paid to mix samples
very gently, to avoid the formation of platelet aggregates by endogenous
thrombin.
Reagents
Supernatants of samples stimulated in the aggregometer
Arachidonic acid, epinephrine, ADP, and indomethacin were from Sigma
Aldrich (Milano, Italy). Horm collagen from Mascia Brunelli (Milano,
Italy). Ticagrelor, clopidogrel active metabolite (CAM), and prasugrel active metabolite (PAM) were provided by AstraZeneca R&D (Mölndal,
At the end of PA recording in the aggregometer, samples were mixed
with indomethacin (20 mM) and centrifuged at 13 000 g in an Eppendorf
microcentrifuge at RT for 3 min. All supernatants were stored at 2808C
until assay.
Preparation of samples for TxB2
measurements
Serum samples
3349
P2Y12R antagonism and platelet production of TxA2
Results
Measurement of TxB2
TxB2, the stable metabolite of TxA2, was measured by selective, competitive enzyme immunoassay (Thromboxane B2 EIA kit, Cayman Chemicals, Ann Arbor, MI, USA). Samples were diluted 1:5 – 1:1000 with
buffer and tested in duplicate (detection limit ¼ 11 pg/mL). When measurements fell outside the range of the standard curve, samples were reassayed using more appropriate dilutions. Results are expressed as
pmol/108 platelets.
Statistical analysis
Statistical analysis was performed using GraphPad Prism version 5.0
(GraphPad Software Inc., San Diego, CA, USA). Data distribution
was evaluated by D’Agostino-Pearson test. Parametric or nonparametric tests were used as appropriate: the in vitro effects of
P2Y12R antagonists on serum TxB2 levels in healthy subjects were analysed by one-way ANOVA for repeated measurements; the effects on
serum TxB2 levels of the in vivo administration of prasugrel or placebo
to patients with bronchial asthma were analysed by comparing the
differences of TxB2 levels between day 0 and day 15 of treatment, placebo vs. prasugrel, using paired t-test; the in vitro effects of P2Y12R antagonists and ASA on PA and TxB2 production in PRP or whole blood
were analysed by Friedman test followed by Dunn’s post hoc test;
the effects of MRS2500 on PA and TXB2 production were analysed
using the exact Wilcoxon rank-sum for comparison between two
groups of matched data with small sample sizes.
All tests were two-tailed and P , 0.05 was chosen as the cut-off level
for statistical significance.
No statistical analysis was performed of data obtained in patients with
the rare inherited P2Y12R deficiency, due to the limited number of patients studied. These data should be considered descriptive of a condition that represents the ideal natural model of lack of P2Y12R-related
platelet function.
Effects of P2Y12 receptor antagonists
on serum TxB2 levels
Serum TxB2 levels in healthy subjects and patients with
P2Y12 receptor deficiency in the presence and absence of
P2Y12 receptor antagonists
Serum TxB2 levels in patients with inherited P2Y12R deficiency were
similar to those of seven healthy subjects, and comprised within the
normal range of our laboratory (Table 1). High concentrations of
CAM, cangrelor, PAM, or ticagrelor (10 and 100 mM) did not decrease serum levels of TxB2 in any subject (Table 1).
Serum TxB2 levels in patients on daily treatment with
prasugrel or placebo
Serum TxB2 levels in patients enrolled in the PRINA study,11 measured 15 days after treatment with placebo or prasugrel, were almost identical to those measured at baseline (P ¼ 0.2164)
(Figure 1), despite the fact that prasugrel, but not placebo, effectively
inhibited P2Y12R function, as measured by vasodilator-stimulated
phosphoprotein phosphorylation assay.11 Changes in serum TxB2 vs.
baseline after treatment with prasugrel (mean, +3.09 pmol/108 platelets; 95% CI, 249.78 to +55.95) or placebo (mean, +0.09; 95% CI,
234.5 to +34.7) were not significantly different (P ¼ 0.9314).
Effects of P2Y12 receptor antagonists on
platelet aggregation and TxB2 production
Studies in healthy subjects
In the presence of any of the P2Y12R antagonists under the study, PA
(LTA) in citrate-PRP induced by ADP (10 mM) was severely
Table 1 Serum TxB2 levels in patients with inherited P2Y12 receptor deficiency and healthy subjects, in the presence/
absence of P2Y12 receptor antagonists added in vitro
TxB2 (pmol/108 platelets)
.........................................................................................................................................................
Patient 1 homozygous
P2Y12R deficiency
Patient 2 homozygous
P2Y12R deficiency
Patient 3 heterozygous
P2Y12R deficiency
Healthy subjects
(n 5 7)a
Normal range
(n 5 56)
262.7
158.2
112.1
233.1 + 139.3
64.9–482.3
325.5
275.8
190.6
229.5
–
–
221.2 + 100.8
255.9 + 137.3
–
–
252.8
200.2
192.4
166.4
125.7
77.4
270.3 + 131.0
191.9 + 79.4
–
–
277.2
288.3
196.2
181.8
76.3
75.5
234.0 + 126.3
272.2 + 178.3
–
–
266.6
248.0
171.9
154.6
98.2
70.6
200.7 + 106.0
260.4 + 156.5
–
–
...............................................................................................................................................................................
Vehicle
Cangrelor
10 mM
100 mM
Ticagrelor
10 mM
100 mM
CAM
10 mM
100 mM
PAM
10 mM
100 mM
CAM, clopidogrel active metabolite; PAM, prasugrel active metabolite.
a
Mean + SD. The observed differences in the presence and absence of P2Y12R antagonists in seven healthy subjects were not statistically significant (P ¼ 0.09, one-way ANOVA for
repeated measures).
3350
M. Scavone et al.
secondary to the induced inhibition of PA, which amplifies TxB2
production by stimulated platelets.9
Effects of MRS2500, a P2Y1R antagonist,
on platelet aggregation and TxB2
production
Figure 1 Serum TxB2 levels before and 15 days after treatment
with prasugrel or placebo. Samples were obtained from 20 patients with bronchial asthma treated with prasugrel (10 mg q.i.d.)
or placebo for 15 days in a cross-over, double-blind, randomized
trial (PRINA study).11 Data are represented as box-and-whiskers
(10th– 90th percentile) plots. One-way ANOVA test for repeated
measures.
impaired, comparable with that of patients with severe P2Y12R deficiency17 (not shown).
Under stirring conditions (which allow the formation of platelet
aggregates), all tested P2Y12R antagonists caused a statistically significant inhibition of AA- and collagen-induced PA (Figure 2A and C),
which was paralleled by a statistically significant reduction of TxB2
formation (Figure 2B and D). Under non-stirring conditions, no PA
was detectable and the platelet production of TxB2 was lower
than under stirring conditions and not inhibited by any of the tested
P2Y12R antagonists (Figure 2B and D).
Experiments in whole blood (impedance aggregometry) gave
similar results as those obtained by LTA in PRP (not shown).
Studies in patients with inherited P2Y12 receptor
deficiency
Experiments performed by LTA under stirring conditions showed
that, compared to healthy controls, the extent of aggregation of
P2Y12R-deficient platelets was severely impaired and was not
further reduced by the in vitro addition of any of the four P2Y12R
antagonists (Figure 2A and C ). Moreover, TxB2 production by P2Y12R-deficient platelets was comparable with that by normal platelets in
the presence of P2Y12R antagonists and was not further reduced by
P2Y12R antagonists (Figure 2B and D). Under non-stirring conditions,
no aggregation of P2Y12R-deficient platelets was detectable (not
shown) and TxB2 production was comparable with that of normal
platelets under non-stirring conditions, both in the presence and absence of P2Y12R antagonists (Figure 2B and D).
The results of the experiments described so far suggest that the
inhibition of platelet TxB2 production by P2Y12R antagonists is
In order to test further the hypothesis that the inhibition of platelet
TxB2 production by P2Y12R antagonists is mediated by inhibition
of PA, we tested the effects of MRS2500, an inhibitor of PA that
antagonizes the other platelet ADP receptor, P2Y1 (P2Y1R). Under stirring conditions, MRS2500 inhibited collagen (0.5 mg/
mL)-induced PA and TxB 2 production (Figure 3A). Under nonstirring conditions, the platelet production of TxB2 was dramatically lower than that under stirring condition and was not significantly inhibited by MRS2500 (Figure 3A). Similar effects, albeit
much less pronounced, were observed using collagen at high concentration (10 mg/mL) (Figure 3B). Therefore, inhibition by MRS2500
of the ADP-dependent, P2Y1R-mediated PA induced by collagen affected the platelet TxB2 production in a similar way as inhibition by
P2Y12R antagonists.
Effects of epinephrine on platelet
aggregation and TxB2 production in the
presence of the P2Y12 receptor antagonist
cangrelor
If the inhibitory effect on TxB2 production observed with P2Y12R
(and P2Y1R) antagonists is mediated by the induced inhibition of
PA, then restoring PA in the presence of P2Y12R antagonists should
increase TxB2 production. In order to test this hypothesis, we studied the potentiating effects on PA by epinephrine in hirudin-PRP: this
choice was based on the demonstration that epinephrine does not
by itself induce PA and TxB2 production in hirudin-PRP.18 As previously shown,19 epinephrine (1 mM) restored PA induced by ADP
(5 mM) in the presence of any P2Y12R antagonist, because, like
ADP via P2Y12R, it stimulates a G-inhibitory (Gi)-protein via a2a receptors20; the effect of epinephrine was independent of the type of
P2Y12R antagonist used (not shown). Epinephrine (1 mM) increased
PA and TxB2 production induced by collagen (0.5 mg/mL) in the
presence of cangrelor (Table 2).
Inhibition by acetylsalicylic acid and
cangrelor, alone and in combination, of
collagen-induced platelet aggregation and
TxB2 production
We explored the individual and combined in vitro effects of ASA and
P2Y12R antagonism by cangrelor on PA and TxB2 production induced by collagen (0.5 and 10 mg/mL) both in citrate-PRP (low plasma [Ca2+]) and hirudin-PRP (physiological plasma [Ca2+])
(Figure 4). As expected, the extent of TxB2 production was greater
in citrate-PRP than in hirudin-PRP (Figure 4). In citrate-PRP, ASA inhibited PA and TxB2 production induced by collagen 0.5 mg/mL
more effectively than cangrelor; ASA plus cangrelor in combination
did not inhibit PA and TxB2 production further, compared with ASA
alone (Figure 4A). In hirudin-PRP, ASA, and cangrelor, alone or in
combination, almost completely abolished PA induced by collagen
P2Y12R antagonism and platelet production of TxA2
3351
Figure 2 In vitro effects of P2Y12 receptor antagonists on platelet aggregation and TxB2 production of citrate-platelet-rich plasma from healthy
subjects and two patients with inherited homozygous P2Y12 receptor deficiency. Platelet-rich plasma was stimulated by arachidonic acid (1 mM)
or collagen (0.5 mg/mL) in the presence and absence of P2Y12 receptor antagonists (10 mM) under stirring (1000 rpm) and non-stirring conditions:
under non-stirring conditions, platelet aggregation did not occur (see ‘Methods’ section for details). Data (top panels) are represented as
box-and-whiskers (10th – 90th percentile) plots and analysed by Friedman plus Dunn’s multiple comparison post-test (n ¼ 13). Internal contrasts:
(A) platelet aggregation—‘with stirring’: P2Y12 receptor antagonist vs. vehicle, P , 0.05; (B) TxB2 production—‘with stirring’: each P2Y12 receptor
antagonist vs. vehicle, P , 0.05; (C ) platelet aggregation—‘with stirring’: each P2Y12 receptor antagonist vs. vehicle, P , 0.001; (D) TxB2 production—‘with stirring’: each P2Y12 receptor antagonist vs. vehicle, P , 0.001; the remaining contrasts were not statistically significant. Cang, cangrelor; TIC, ticagrelor; CAM, active metabolite of clopidogrel; PAM, active metabolite of prasugrel.
3352
M. Scavone et al.
Figure 3 Effects of the P2Y1R antagonist MRS2500 on collagen-induced platelet aggregation and TxB2 production. Platelet aggregation (under
stirring conditions) and TxB2 production (under stirring and non-stirring conditions) were induced by collagen 0.5 mg/mL (n ¼ 8) or 10 mg/mL
(n ¼ 6) in citrate-platelet-rich plasma of healthy subjects after incubation with MRS2500 (10 mM), an antagonist of the platelet P2Y1R for adenosine diphosphate, or vehicle. Data are represented as box-and-whiskers (10th– 90th percentile) plots and analysed by Wilcoxon test.
Table 2 Effect of epinephrine on collagen-induced platelet aggregation and increase in TxB2 production in
hirudin-platelet-rich plasma from eight healthy subjects in the presence of the P2Y12 receptor antagonist cangrelor
Vehicle
Cangrelor
Cangrelor 1 epinephrine
P-Valuesa
...............................................................................................................................................................................
Platelet aggregation (%)
TxB2 (pmol/108 platelets)
49 + 34
1+1
46 + 28
0.0011
9.64 + 5.80
5.16 + 2.62
7.75 + 4.62
0.0179
Mean + SD.
a
Friedman test. Internal contrasts (Dunn’s post-test), platelet aggregation: cangrelor vs. vehicle or cangrelor + epinephrine, P , 0.05; vehicle vs. cangrelor + epinephrine, P . 0.05;
TxB2 production: vehicle vs. cangrelor, P , 0.05; cangrelor + epinephrine vs. vehicle or cangrelor, P . 0.05. Collagen, 0.5 mg/mL; epinephrine, 1 mM; cangrelor, 10 mM.
0.5 mg/mL; in contrast, TxB2 production was only slightly affected by
cangrelor, while it was completely abolished by ASA and ASA plus
cangrelor (Figure 4B). The inhibitory effects of ASA and cangrelor on
PA induced by 10 mg/mL collagen were weaker, both in citrate-PRP
and in hirudin-PRP. The two drugs in combination accomplished
stronger inhibition of PA than each drug alone (Figure 4C and D).
Cangrelor inhibited TxB2 production to a lesser extent than ASA,
which almost completely abolished it; the combination of ASA
and cangrelor did not further inhibit TxB2 production, compared
with ASA alone.
P2Y12R antagonism and platelet production of TxA2
3353
Figure 4 Inhibitory effects of cangrelor or ASA on collagen-induced platelet aggregation and TxB2 production. Citrate- or hirudin-platelet-rich
plasma of healthy subjects was stimulated by collagen (0.5 or 10 mg/mL) under stirring conditions in the presence or absence of cangrelor (10 mM)
and ASA (500 mM), alone or in combination. Data are represented as box-and-whiskers (10th– 90th percentile) plots and analysed by Friedman
plus Dunn’s multiple comparison post hoc test (healthy subjects, n ¼ 5). Internal contrasts: (A) platelet aggregation: vehicle vs. cangrelor + ASA,
P , 0.01; TxB2 production: vehicle vs. ASA and vs. cangrelor + ASA, P , 0.05; (B) TxB2 production: vehicle vs. ASA and vs. cangrelor + ASA,
P , 0.05; (C) platelet aggregation: vehicle vs. cangrelor + ASA, P , 0.01; TxB2 production: vehicle vs. ASA and vs. cangrelor + ASA, P , 0.05; (D)
platelet aggregation: vehicle vs. cangrelor + ASA, P , 0.01; TxB2 production: vehicle vs. ASA and vs. cangrelor + ASA, P , 0.05; the remaining
contrasts were not statistically significant. Cang, cangrelor; ASA, acetylsalicylic acid.
3354
Discussion
This study was designed to test whether or not pharmacological antagonism of P2Y12R inhibits the ability of platelets to synthesize
TxA2. Inhibition of the platelet TxA2 production by ASA, through
acetylation of COX-1, represents the main mechanism by which
ASA reduces the incidence of cardiovascular events.21 Dual antiplatelet therapy with ASA and a P2Y12R antagonist provides greater
clinical efficacy than ASA alone and is the recommended treatment
of patients undergoing PCI and ACS patients.1
Based on the demonstration that P2Y12R antagonists inhibit in vitro the platelet production of TxA2 (measured as its stable metabolite TxB2) and in vivo the urinary excretion of the TxB2 metabolite
11-dehydro-TxB2,3,4,22 – 24 it has been postulated that patients
with ACS may be treated with P2Y12R antagonists only, as inhibition
of TxA2 production by ASA may result superfluous, or even detrimental, because it can inhibit the synthesis of the natural antithrombotic prostacyclin.5 This might be particularly relevant for patients
treated with ticagrelor or prasugrel, which more effectively inhibit
P2Y12R.1
However, the extent of the contribution of P2Y12R in the platelet
production of TxA2 is controversial. First of all, ADP does not cause
platelet production of TxA2.8 When ADP-stimulated platelets are
allowed to aggregate, it is indeed PA, and not receptor occupancy
by ADP, that triggers platelet TxA2 production.8,9 This characteristic
distinguishes ‘weak agonists’, such as ADP and epinephrine, from
‘strong agonists’, such as thrombin and collagen, which directly induce platelet TxA2 production and granules secretion independently of PA.9 Typically, the aggregation-dependent TxA2 production
and platelet secretion are clearly detectable under conditions of
low concentration of external ionized calcium ([Ca2+]o), such as
in citrate-PRP.8,9 When [Ca2+]o is maintained in its physiological
range, by using in vitro anticoagulants that do not chelate Ca2+
(e.g. hirudin), the aggregation-dependent TxA 2 production and
platelet secretion does not usually occur, except for few subjects
in whom it may be slightly detectable.9,25 The potentiating effect
of PA on platelet secretion and TxA2 production is observed also
when platelets are stimulated by strong agonists at low concentrations (strong agonists at low concentrations behave like weak
agonists).9,26
Based on this background, it is difficult to hypothesize that antagonists of platelet ADP receptors inhibit the platelet TxA2 production. Moreover, we found that patients with inherited P2Y12R
deficiency have normal serum TxB2 levels.6,7
Deficiency or inhibition of P2Y12 receptor
does not impair the platelet capacity to
synthesize thromboxane A2
In the present study, we confirmed that patients with inherited
P2Y12R deficiency have normal serum TxB2 levels, which is the
most accurate parameter of platelet COX-1 function,15,16 and
that four different P2Y12R antagonists, added in vitro at extremely
high concentrations, do not decrease serum TxB2 levels both in normal subjects and patients with P2Y12R deficiency. Moreover, the administration of 10 mg prasugrel daily to patients with allergic
bronchial asthma did not decrease serum TxB2 levels compared
M. Scavone et al.
with placebo, in a double-blind, placebo-controlled crossover
study.11 Therefore, we can safely conclude that inherited deficiency
or inhibition of P2Y12R does not impair the ability of platelets to synthesize TxA2 and that four different P2Y12R antagonists do not display off-target effects on platelet TxA2 production.
As the production of TxA2 in serum occurs under conditions in
which endogenous thrombin stimulates platelets without inducing
the formation of platelet aggregates, we tested the hypothesis that
the reported inhibitory effects of P2Y12R antagonists on platelet
TxA2 production2 – 4 are secondary to inhibition of PA.
P2Y12 receptor antagonists, like any other
antiplatelet agent, inhibit the
aggregation-dependent platelet
production of thromboxane A2
Inhibition of AA- or collagen-induced PA by P2Y12R antagonists was
paralleled by inhibition of platelet TxA2 production. When experiments were performed under non-stirring conditions in order to
prevent PA, the platelet production of TxA2 was lower and was
not inhibited by P2Y12R antagonists. The dependency of the inhibition of TxA2 production by P2Y12R antagonists on the induced inhibition of PA was confirmed by two other sets of experiments: (i)
MRS2500, a potent and selective antagonist of the other platelet
ADP receptor, P2Y1R,27 also inhibited collagen-induced PA and
TxA2 production; however, like P2Y12R antagonists, it did not inhibit
TxA2 production by platelets that were unable to aggregate when
they were stimulated under non-stirring conditions; (ii) epinephrine,
whose alpha2 receptors on platelets, like P2Y12R, are associated
with a Gi-protein,20 restored collagen-induced PA that had been inhibited by the P2Y12R antagonist cangrelor and concurrently increased collagen-induced production of TxA2. These effects of
epinephrine were observed in hirudin-PRP, despite the fact that epinephrine alone induced no or minimal PA and TxA2 production in
hirudin-PRP, confirming previous results.18
It is unlikely that the observed indirect inhibition of TxA2 production by P2Y12R antagonists in vitro, which is shared by any type of
drugs inhibiting PA, has relevant in vivo consequences. The lack of
measurement of urinary TxB2 metabolite might be considered a
limitation of our study. However, the demonstration that the
in vivo administration of P2Y12R antagonists decreases the urinary
excretion of 11-dehydro-TxB222 was not confirmed in a subsequent
study2 and, at any rate, should not be interpreted as a demonstration
that P2Y12R antagonists inhibit the platelet production of TxA2
in vivo. The relationship between inhibition of serum TxB2 and
that of urinary levels of 11-dehydro-TxB2 is non-linear28; 30%
of urinary 11-dehydro-TxB2 is actually generated by COX-2 in
macrophages and other inflammatory cells,21 which is not inhibited
by ASA at the low, antithrombotic doses used in patients at risk of
thrombosis. Because P2Y12R antagonists display anti-inflammatory
properties,29 the reduction of urinary levels of 11-dehydro-TxB2
by these drugs may simply represent a reflection of their antiinflammatory effects.
Finally, it must be emphasized that the observed, indirect inhibition of platelet production of TxA2 not only is not a distinctive feature of P2Y12R antagonists, being shared by any drug inhibiting PA, it
is also much weaker than that accomplished by ASA, and likely
P2Y12R antagonism and platelet production of TxA2
devoid of any clinical relevance. In fact, highly effective inhibition of
TxA2 production is necessary to decrease the cardiovascular risk,
because TxA2, even at low concentrations, may synergize in vivo
with other platelet agonists to amplify PA.30
Acetylsalicylic acid abolished
thromboxane A2 production and, in
combination with a P2Y12 receptor
antagonist, inhibited platelet aggregation
more potently than either drug alone
The effective cooperation of P2Y12R antagonist and ASA in inhibiting platelet function was demonstrated in experiments in which platelets (both in citrate-PRP and hirudin-PRP) were stimulated by high
concentrations of collagen. Under these experimental conditions,
which simulate more closely the stimulation of platelets interacting
with the sub-endothelium, neither ASA nor cangrelor effectively inhibited PA. It was only when the two drugs were used in combination that strong inhibition of PA was observed. The effects on
TxA2 production did not quite parallel the effects on PA, compatibly
with the notion that high concentrations of collagen induce PA that
is mostly independent of TxA2 production. Cangrelor inhibited
TxA2 production by 50%, while ASA, both alone and in combination with cangrelor, inhibited it by .98%.
Conclusion
Based on the results of our experiments, we conclude that P2Y12R
antagonists do not inhibit the platelet capacity to synthesize TxA2
and that, to date, there is no clinical, nor pharmacological/physiological evidence that DAPT in ACS patients should be replaced by
treatment with a P2Y12R antagonist alone. Only the results of ongoing clinical studies will tell whether this is a safe and effective
approach.
Authors’ contributions
M.S. and E.A.F.: performed statistical analysis and drafted the manuscript; M.C.: handled funding and supervision, and conceived and designed the research; M.S., E.A.F., and V.C.: acquired the data; M.S.,
E.A.F., and M.C.: made critical revision of the manuscript for key intellectual content.
Funding
The study was partially funded by an unrestricted research grant from
AstraZeneca, Sweden.
Conflict of interest: M.C. received honoraria for lectures and advisory board meetings by AstraZeneca, Eli Lilly, Daiichi-Sankyo,
Sanofi-Aventis, and The Medicines Company, and research grants by AstraZeneca, Eli Lilly, and Daiichi-Sankyo.
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CARDIOVASCULAR FLASHLIGHT
doi:10.1093/eurheartj/ehw288
Online publish-ahead-of-print 1 July 2016
.............................................................................................................................................................................
Two hearts, one soul: heterotopic heart transplantation follow-up with
cardiac computed tomography
Charles Roux1,2*, Shaida Varnous3, Pascal Leprince3, and Philippe Cluzel1,2
1
Département d’imagerie cardiovasculaire, Hôpital de la Pitié Salpêtrière, Paris, France; 2Laboratoire d’imagerie biomédicale, INSERM/CNRS UMR-S1146, UMR 7371, Paris,
France; and 3Service de chirurgie cardio-vasculaire, Hôpital de la Pitié Salpêtrière, Paris, France
* Corresponding author. Tel: +33 689353546, Email: [email protected]
A 68-year-old asymptomatic man with a history
of heart transplantation for advanced heart failure
from ischaemic cardiomyopathy was referred to
our institution for scheduled routine follow-up.
Retrospectively gated cardiac computed tomography, performed on a 256-slice dual source
CT with multiphase reconstruction, revealed heterotopic cardiac transplantation (HHT). The explanted heart from a 12-year-old donor was
transplanted 25 years ago in the right side of
the chest next to the recipient’s native heart
(Supplementary material online, Video S1). Dynamic cine analysis showed asynchronous contraction of both hearts with independent donor
and recipient heart rhythms.
The transplant right atrium was connected to
the superior vena cava and recipient right atrium
remained connected to the inferior vena cava (Panel A). Left atria were connected (Panel B) as well
as both ascending aortas using an end-to-side
anastomosis (Panels C and D) and pulmonary arterial trunks with a Dacron graft.
Heterotopic heart transplantation has been
used since 1974 for highly selected patients
such as irreversible high pulmonary vascular resistance and body weight mismatch (over 20%) between recipient and donor. Xenotransplant heterotopic grafts have also been
proposed to be used in reversible heart failure as a bridge to recovery.
However, drawbacks include well-recognized complications in HHT such as native heart angina, ventricular fibrillation, and thromboembolics events as well as complex surgery.
Cardiac CT and MR are imaging modalities of choice for the follow-up and optimal workup of patients with complex cardiovascular
anatomy including patients with HHT.
Supplementary material is available at European Heart Journal online.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2016. For permissions please email: [email protected].