L. Slavík 1; J. Úlehlová 1; V. Krcova 1; A. Hlusi 1; J. Indráková 1; M. Hutyra 2; J. Galuszka 2; K. Indrák 1
Coagulation laboratory: Department of Haemato-Oncology, University Hospital, Olomouc, Czech Republic
1; Department of Internal Medicine I, University Hospital, Olomouc, Czech Republic
Transfuze Hematol. dnes,17, 2011, No. 2, p. 92-96.
Comprehensive Reports, Original Papers, Case Reports
Antiaggregation therapy is still the most frequently used approach to prevent thrombotic events in cardiovascular disease. It has a good clinical effect but there is increasing evidence of high residual platelet aggregation activity in a number of patients. Laboratory methods only allow us to detect clopidogrel “non-responders” or “low responders”. Recent methods are based on monitoring residual platelet aggregation activity (aggregation methods) or detecting the number of free epitopes for binding a specific monoclonal antibody such as vasodilator-stimulated phosphoprotein phosphorylation (VASP). The aim of our study was the comparison of light transmission aggregometry (LTA) and multiple electrode platelet aggregometry (MEA) with induction by ADP at concentrations of 20 μmol/L with or without prostaglandin E1 (PGE1). In the studied group of 84 patients with cardiovascular disease (CAD), an impaired individual response to clopidogrel therapy was detected by MEA and LTA in 11.9% and 10.7%, respectively. The LTA and MEA methods with induction by ADP with PGE1 and without PGE1 were statistically compared using Spearman’s nonparametric correlation analysis. Both methods using PGE1 have a positive significant correlation (P=0.003) in contrast to the results without PGE1 with no significant correlation (P=0.732).
The sensitivity of clopidogrel resistance detection correlates well with other data in literature suggesting that there are 5% - 30% of clopidogrel low-responders depending on the type of platelet function assay used and the criteria for defining a low-responder. These results favour implementation of the ADP test with PGE1 by MEA specifically for the identification of low-responders on clopidogrel.
Antiaggregation therapy remains the most frequently used
approach to prevent thrombotic events in cardiovascular disease. It
has a good clinical effect but there is increasing evidence of
high residual platelet aggregation activity in a number of
In current literature, there are multiple definitions of
resistance to antiplatelet drugs. The most common is resistance to
a specific agent best indicated by persistent activity of the
agent’s target despite treatment. This definition also assumes
sufficient dosage to provide optimal levels for target inhibition.
However, thrombosis is a multifactorial process involving
multiple pathways of platelet activation and factors other than
platelets. For this reason, the occurrence of clinical events during
treatment with a specific antiaggregation agent cannot be
interpreted solely as “resistance” to the therapeutic agent (1).
On the basis of the above definition, laboratory methods
only allow us to detect clopidogrel “non-responders” or “low
responders”. An optimal laboratory method is the key for good
clinical correlation. Laboratory methods arise from knowledge of the
metabolic conversion of thienopyridine pro-drugs. As a typical
thienopyridine agent, clopidogrel is a pro-drug that requires
hepatic conversion to an active metabolite in order to inhibit
platelet function. Clopidogrel is absorbed in the intestine and
extensively metabolized by hepatic cytochrome P450 (CYP3A4) to an
active thiol metabolite (2, 3). This metabolite irreversibly binds to
the P2Y12 receptor for the lifetime of the platelet. In addition to
inhibiting adenosine diphosphate (ADP) - induced platelet
aggregation, clopidogrel also inhibits ADP-stimulated P-selectin and
CD40L expression (4–7).
Based on this explanation, a number of authors have
optimized laboratory methods for monitoring clopidogrel therapy.
These methods are based on monitoring residual platelet aggregation
activity (aggregation methods) or detecting the number of free
epitopes for binding a specific monoclonal antibody such as
vasodilator-stimulated phosphoprotein phosphorylation (VASP). For
many years, the gold standard for monitoring clopidogrel therapy
action was the VASP method, while aggregation methods were considered
less suitable. However, the costs of VASP and the need for
experience with flow-cytometry substantially limit the use of this
assay. These limitations formed the background of the present
research on aggregation methods.
Numerous studies have examined the effects of
antiaggregation therapy on ex-vivo assays of platelet function
variability as the degree of inhibition of platelet function observed
in individuals taking acetylsalicylic acid (ASA) or clopidogrel or
There are a number of modifications of the
laboratory methods used for monitoring clopidogrel treatment on the
basis of measuring residual platelet aggregation activity.
Aggregation is induced by ADP in concentrations of 20 μmol
/L with or without prostaglandin E1 (PGE1). A more detailed
knowledge of the aetiopathology of platelet activation is needed for
understanding these different results.
Platelet aggregation induced by ADP is preceded by rapid
increase in the concentration of Ca2+
ions in the cell cytoplasm (8). The increasing Ca2+
concentration is caused by ADP acting at the P2Y1receptor. This receptor is a Gq-coupled
receptor linked to phospholipase C and the generation of inositol
1,4,5-triphosphate. Rapid increase in intracellular Ca2+
occurs mainly through release of Ca2+
into the cytoplasm from intracellular stores and additionally by
influx (9). The released Ca2+
leads to cell activation followed by initiation of aggregation. It is
already known that mobilization of Ca2+
from intracellular stores is inhibited by raised levels of cAMP via
inhibition of phospholipase C activation. Agents that raise the level
of cAMP in platelets thus reduce [Ca2+]i
and this leads to inhibition of platelet aggregation. One such agent
is PGE1 which increases cAMP by stimulating adenylate cyclase. Based
on this knowledge, an optimal laboratory method can be proposed with
good clinical correlation for measuring the effect of a specific
antiaggregation agent such as clopidogrel on receptors like the P2Y1
Platelet aggregation can also be regulated by
endothelial functions. The endothelium is involved in a multitude
of physiological processes including the control of cellular
trafficking, the regulation of vasomotor tone and the maintenance of
blood fluidity. Endothelial cells (ECs) possess surface receptors for
a variety of physiological substances, for example thrombin and
angiotensin II, which may influence vascular tone directly or
indirectly through various haemostasis-related events. Once
activated, ECs express on their surface, and in some cases release
into the plasma, a variety of intracellular adhesion molecules
(e.g. vascular cell adhesion molecule, E-selectin, P-selectin, and
von Willebrand factor, vWf), which modulate leukocyte and platelet
adhesion, inflammation, phagocytosis and vascular permeability.
Intact ECs exert a powerful inhibitory effect on haemostasis by
virtue of the factors that they synthesize and release or express on
their surface. The endothelium can affect the methods for detecting
the effect of antiplatelet therapy by potentiating residual platelet
Dual antiaggregation therapy (ASA and clopidogrel) is
the treatment of choice for preventing thrombotic complications in
patients undergoing percutaneous coronary intervention (PCI).
Clopidogrel with a single loading dose as thrombotic prevention
has been shown to be optimal, but recent studies have demonstrated
that the response to clopidogrel varies widely. This highlights the
need to incorporate a method for monitoring the effects of
clopidogrel therapy in clinical practice.
Clopidogrel-mediated platelet inhibition was evaluated
after obtaining signed, written informed consent from 84 patients (67
males, 17 females) with cardiovascular disease (coronary, peripheral,
or carotid artery disease) after coronary stent implantation. The
median age of this cohort of patients was 60.5 years and a family
history of cardiovascular disease was present in 56% of the patients.
The associated diseases in this group were hypertension in 68%,
diabetes mellitus in 26.5% and dyslipidemia in 80.9%.
The control group consisted of 40 healthy blood donors
with a comparable sex ratio and a median age of 32.5 years
in order to establish cut-off values for both tests.
All patients received a loading dose of 300 mg
clopidogrel 24 hours (h) prior to intervention followed by
a once-daily dose of 75 mg clopidogrel. The exclusion
criteria were known ASA or thienopyridine intolerance (allergic
reaction, gastrointestinal bleeding), treatment with vitamin
K antagonists (warfarin, phenprocoumon, acenocoumarol),
treatment with dipyridamol or non-steroidal anti-inflammatory drugs,
a family or personal history of bleeding disorders, malignant
paraproteinemias, myeloproliferative disorders or heparin-induced
thrombocytopenia, severe hepatic failure, known qualitative defects
in thrombocyte function, a major surgical procedure within one
week before inclusion, a platelet count < 100,000 or >
and a haematocrit < 0.30.
The samples for laboratory testing were taken at least
72 hours after receiving a loading dose of clopidogrel treatment
for the maximum antiaggregation effect.
Blood was withdrawn from the antecubital vein using
a 21-gauge butterfly needle (0.8 x 19 mm; Greiner Bio-One,
Kremsmünster, Austria) 72 h after percutaneous intervention. After
the initial 3 ml of blood had been discarded to reduce
procedurally induced platelet activation, blood was drawn into a 3.8%
sodium citrate Vacuette tube (Greiner Bio-One; 9 parts of whole
blood, 1 part of sodium citrate 0.129 M/L) for evaluation by light
transmission aggregometry, into a 3.2% sodium citrate Vacuette
tube (Greiner Bio-One; 9 parts whole blood, 1 part sodium citrate
0.109 M/L) and into a Vacuette tube containing hirudin (15
IU/mL) for determination by multiple electrode platelet aggregometry.
To avoid procedural deviations, all blood samples were
taken by the same team using the same method. The blood samples were
mixed adequately by gently inverting the tubes. To avoid
investigator-related variation of results, each of the different
tests was performed by just one single blind operator. The results of
all assays were available to all patients (10).
transmission aggregometry (LTA)
LTA was performed on the APACT 4004 aggregometer
(LABiTec, Ahrensburg, Germany). Citrate-anticoagulated whole blood
was centrifuged at 150 x g for 10 minutes (min) at room
temperature to obtain platelet-rich plasma (PRP). Platelet-poor
plasma (PPP) was obtained from the remaining specimen by
re-centrifugation at 2,000 x g for 10 min. Platelet counts were
not adjusted with a median platelet count of 250 x 1012/L
(range 225–278 x 1012/L).
The baseline optical density was set with PPP. Aggregation was
performed using ADP (Helena Biosciences, United Kingdom) at a final
concentration of 10 μM
and a final concentration of 30 μmol/L
and optical density changes were recorded photoelectrically for 6 min
as platelets began to aggregate. The maximal aggregation response was
registered and used to differentiate between patients with and
without residual ADP-inducible platelet aggregation (11 – 13).
electrode platelet aggregometry (MEA)
Whole blood impedance aggregometry was performed with
the Multiplate analyzer (10-15) (Dynabyte, Munich, Germany). One
Multiplate test cell contains two independent sensor units and one
unit consists of two silver-coated highly conductive copper wires
3.2 mm long. After dilution (1:2 with 0.9% NaCl solution) of
hirudin-anticoagulated whole blood and stirring in the test cuvettes
for 3 min at 37 °C, ADP (Dynabyte, Munich, Germany, final
concentration of 6.4 μM)
was added and the aggregation was continuously recorded for 6 min.
The adhesion of activated platelets to the electrodes was initiated
using ADP (Dynabyte, Germany) to a final concentration of 10 μM
and a final concentration of 30 μmol/L
and the adhesion was monitored as an increase in impedance detected
for each sensor unit separately and transformed to aggregation units
(AU)*min that were plotted against time (14, 15).
In the studied group of 84 patients with cardiovascular
disease (CVD), an impaired individual response to clopidogrel therapy
was detected by MEA and LTA in 11.9% and 10.7%, respectively.
In our opinion, the crux of the problem lies in the
definition of cut-off values and the implementation of these methods.
Our locally determined reference ranges in a population of 40
healthy blood donors were 298 – 711 AUC/min. for ADP HS with PGE1
using MEA and 47 – 99% for ADP with PGE1
using LTA. The cut-off values for failure of clopidogrel therapy for
methods with PGE1were determined as 298 AUC/min. for MEA and
47 % for LTA. The cut-off values for methods without PGE1
could not be determined, because the statistical distribution of
results was without any relationship. PGE1reduce the activation contribution from ADP
binding to P2Y1
receptors, thus making the assay using PGE1
specific for the effects of ADP mediated by P2Y12.
The test using PGE1as activator is designed to measure the
receptor blockade – specific effect of clopidogrel treatment.
The difference between detecting systems minimally
influenced the result for resistance to antiaggregation therapy. Both
methods offer comparable results of 11.9% and 10.7%, respectively in
resistant patients, although LTA has more marginal results (11%).
The statistical comparison of MEA and LTA methods with a specific
blocker of the P2Y1
receptor by Spearman non-parametric analysis showed a positive
significant correlation (p=0.01) in the group of non-resistant
patients. The statistical comparison methods without a specific
blocker of the P2Y1receptor failed to provide any significant
correlation by Spearman non-parametric analysis.
The two methods used here offer comparable results but
the different detection methods increase the variability of these
results. However, the increase in variability is only a few
percent and this is minimal in comparison with published results
(resistance detected ranges from 0 to 45% in the literature).
The general problem of laboratory detection of
resistance to thienopyridine drugs lies in the variability of blood
sampling, test implementation and result interpretation.
Blood was collected into sodium citrate 0.129 M/L as an
anticoagulant for LTA. Multiple electrode aggregometry yields better
results with samples where 15 IU/L hirudin is used as an
anticoagulant. The sensitivity is slightly higher using hirudin than
sodium citrate. A larger difference in reproducibility occurs in
measurement using MEA. The reproducibility using blood samples with
hirudin as an anticoagulant was under 5% in comparison with 12% when
sodium citrate was used.
The main problem in laboratory result interpretation is
the use of a specific blocker of the P2Y1
receptor. One specific agent is PGE1.
The use of this specific blocker of the P2Y1 stimulating reaction decreased aggregation
by ADP from 1 – 49% and 6 – 608 AUC*min, respectively in a group
of patients treated with clopidogrel. When we look at the results
without using this specific blocker (graph 2), we cannot define the
cut-off value for either LTA or MEA. The use of this specific blocker
has unique possibilities for defining the cut-off values for both
Clopidogrel is a pro-drug that needs to be
metabolized to the active thiol metabolite by the cytochrome P450
(CYP) system. This activation is a source of significant
inter-individual variability in clopidogrel responsiveness (16, 17).
The presence of CYP3A4, CYP3A5 and CYP2C19 polymorphisms can reduce
the formation of the active metabolite of clopidogrel, resulting in
less platelet inhibition (18). The polymorphisms in the genes
encoding P-glycoprotein (an efflux transporter) and purinergic
receptor P2Y(12) (the active site for clopidogrel) have been studied
for their role in clopidogrel responsiveness (19). Polymorphisms of
platelet receptors, GP Ia (807C>T, rs1126643), GP VI (13254T>C,
rs1613662), GP IIIa (HPA-1, rs5918), PAR-1 (IVS-14A>T, rs168753),
P2Y(12) (34C>T, rs6785930 and H1/H2 haplotype, rs2046934), and
genetic variations of the gene coding for cyclooxygenase-1 (COX-1)
(-842A>G, rs10306114 and 50C>T, rs3842787) were studied, but
only for their role in the risk of bleeding associated with
clopidogrel therapy (20).
The sensitivity of detection of clopidogrel resistance
correlates well with other data in literature suggesting that there
are 5% – 30% of clopidogrel low-responders
depending on the type of platelet function
assay used and the criteria for defining a low-responder (21 –
23). These results favour implementation of the ADP test with PGE1
by MEA specifically for the identification of low-responders on
clopidogrel (confirmed by a significant statistical
correlation). We also determined aggregation using LTA, APACT 4004
(LABiTec, Ahrensburg, Germany) on citrated platelet-rich plasma,
which demonstrates very similar results to MEA. However this method
produces more results very near the cut-off limit, representing 11%
of detected samples and misrepresenting the true resistance to
by the Czech Ministry of Health grant projects IGA NH NS 10319-3/2009
86-14 Supported by the project MSM 6198959205 of the MSMT Czech Rep. Supported
by the UP LF-2011-006 grant project
Slavík, Ph.D. koagulační
Olomouc I. P. Pavlova
do redakce: 7. 1. 2011 Přijato
po recenzi: 27. 5. 2011
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