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High on-treatment platelet reactivity
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Theme Issue Article
High on-treatment platelet reactivity – definition and measurement
Marco Cattaneo
Unità di Medicina 3, Ospedale San Paolo, Dipartimento di Scienze della Salute. Università degli Studi di Milano, Milan, Italy
Summary
In the last decade, several studies revealed inter-patient response
variability to antiplatelet agents: patients who display negligible or no
responses to these drugs are considered poor responders, or “resistant” to treatment. In order to identify poor responders to an antiplatelet drug, laboratory tests of platelet function that specifically explore
the platelet activation pathway that is targeted by the drug should be
utilised. In addition, they should be performed both at baseline and
during treatment: however, most studies explored platelet function
during antiplatelet treatment, in order to identify those patients with
“high on-treatment platelet reactivity” (HPR), which exposes them to
Correspondence to:
Marco Cattaneo, MD
Unità di Medicina 3, Ospedale San Paolo
Dipartimento di Scienze della Salute – Università degli Studi di Milano
Via di Rudinì 8, 20142 Milano, Italy
Tel.: +39 0250323095, Fax: +39 0250323089
E-mail: [email protected]
Introduction
Acetylsalicylic acid (aspirin) and inhibitors of the platelet P2Y12 receptor for adenosine diphosphate (ADP) are antiplatelet drugs that
decrease the risk of major adverse cardiovascular events (MACE).
They are used during coronary interventions, in the medical management of acute coronary syndromes, and in long-term secondary prevention of cardiovascular and cerebrovascular events.
Both aspirin and P2Y12 antagonists selectively inhibit a single
pathway of platelet activation: aspirin affects the thromboxane A2
(TxA2) pathway by irreversibly inhibiting cyclo-oxygensase-1
(COX-1), while P2Y12 antagonists affect the ADP pathway, by antagonizing one of the two platelet ADP receptors irreversibly (thienopyridines) or reversibly (ticagrelor) (1, 2). The good anti-thrombotic efficacy of these drugs, despite their selective mechanism of
action, is explained by the fact that both the TxA2 pathway and the
ADP pathway contribute to the amplification of platelet activation
and are essential for the full aggregation response of platelets (1).
Since their introduction in clinical practice, antiplatelet drugs
have been administered to patients at standard doses, without
monitoring their pharmacological effects by means of laboratory
tests. However, in the last few years several studies revealed interpatient response variability to aspirin and the thienopyridine drug
clopidogrel: patients who display no or negligible responses to
these drugs have been considered poor responders, or “resistant”
to treatment.
increased risk of major adverse cardiovascular events (MACE). Many
tests of platelet function have been used, most of which are able to
identify patients at risk of MACE. Unfortunately, universal cut-off values for HPR have not been clearly established yet. In addition, the concordance among different tests in the identification of patients at risk
is very poor and the most effective and safe treatment for patients at
risk is still unknown.
Keywords
Antiplatelet agents, platelet pharmacology, arterial thrombosis, clopidogrel, aspirin
Received: October 18, 2012
Accepted after minor revision: January 23, 2013
Prepublished online: February 21, 2013
doi:10.1160/TH12-10-0758
Thromb Haemost 2013; 109: 792–798
Resistance (or poor responsiveness ) to
antiplatelet agents versus high on-treatment
platelet reactivity (HPR)
The term “resistance” to a drug should be used when a drug is unable to hit its pharmacological target, due to inability to reach it (as
a consequence of reduced bioavailability, in vivo inactivation,
negative interaction with other substances) or to alterations of the
target (1). Based on this definition, resistance to aspirin should be
limited to situations in which aspirin is unable to inhibit COX1-dependent TxA2 production (and, consequently, TxA2-dependent platelet functions), while resistance to clopidogrel should be limited to situations in which the drug is unable to inhibit the platelet P2Y12 receptor (and, consequently, the P2Y12-dependent platelet functions).
Although it can be easily predicted that patients who fail to
show a pharmacological response to an effective antithrombotic
drug are not adequately protected from MACE, it is incorrect to
consider “resistant” to antiplatelet agents those patients who experience MACE, despite being compliant with the therapy. This
phenomenon has been called “clinical antiplatelet resistance”, but it
should be termed “treatment failure” (1): it can be observed with
any kind of treatment and is expected to be particularly frequent
for drugs, like all anti-thrombotic agents, that are used to prevent
multi-factorial diseases, such as those associated with vascular occlusions. As previously mentioned, aspirin and clopidogrel inhibit
only one pathway of platelet aggregation; platelet aggregation is
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only one mechanism regulating thrombus formation; thrombus
formation is the most common, but not the only mechanism leading to vascular occlusion; vascular occlusion causes clinical events
that can be widely different in terms of severity, ranging from
asymptomatic in some patients to lethal in others. Given this picture, it would be unreasonable to expect an antiplatelet agent, or
any anti-thrombotic drug, to prevent clinical events in all patients
at risk. Therefore, the definition of “antiplatelet drug resistance”
that is based on clinical outcomes is certainly unacceptable (1).
Because both the TxA2 and the ADP pathways contribute to the
amplification of platelet activation and are essential for full aggregation responses of platelets, inhibition of each of these pathways
will negatively affect not only thrombus formation in vivo, but also
platelet function in vitro. Therefore, many studies used various
techniques to measure platelet function in vitro in order to evaluate the degree of its inhibition by anti-platelet treatment and, in
some instances, to predict the risk of atherothrombotic events. Although this approach is justified and rationale, one should be
aware that the relative importance of the TxA2 and P2Y12 pathways in platelet activation varies considerably among different
subjects and with the type of laboratory test used (1). Two important issues should be taken in consideration when measuring platelet function in vitro in patients on treatment with antiplatelet
agents: a) the specificity of the laboratory tests for the platelet activation pathway targeted by the drug; b) whether or not the laboratory test is performed both before and after the administration of
the drug.
Specificity of the laboratory test
Because antiplatelet drugs target one single pathway of platelet activation, in order to measure the pharmacological response to an
antiplatelet agent, tests that are very specific for the platelet activation pathway that is targeted by the drug should be used (1, 3, 4).
The use of unspecific, global tests of haemostasis, which are sensitive to several pathways that lead to the formation of platelet aggregates, may be very marginally affected by inhibition of the
pathway targeted by the drug, and therefore are not adequate for
this purpose (1, 3, 4). However, because all pathways of platelet activation are expected to be involved in thrombus formation in vivo,
global tests of hemostasis are theoretically more useful than specific tests to predict the risk of MACE (3).
“Response to treatment” versus “On-treatment
platelet reactivity”
In order to measure the pharmacological response to an antiplatelet agent one should compare the results obtained before and
after the ingestion of the drug (1). As a matter of fact, due to the
high baseline inter-individual variability, high platelet reactivity
measured during antiplatelet treatment could result either from
insufficient inhibition of “normal” baseline platelet reactivity or
from adequate inhibition of platelets that were hyper-reactive at
baseline (1). To distinguish between these two scenarios, it would
be necessary to measure platelet reactivity both before and after
treatment. However, measurement of baseline platelet reactivity
would be impractical or even impossible in many cases of patients
with acute coronary syndromes, who need to be treated with antiplatelet agents under emergency conditions. Therefore, it has become common practice to measure platelet reactivity during antiplatelet treatment (“on treatment platelet reactivity”), without having measured a reference, baseline value. Although measurement
of on-treatment platelet reactivity is a valuable parameter to consider when the aim of platelet function testing is to predict the risk
of clinical events that are dependent on platelet activation/aggregation (3), it will fail to provide information on the individual
pharmacological response to the drug. Knowing whether or not a
high level of on-treatment platelet reactivity is caused by failure to
respond to the drug is clinically relevant: treatment could be
changed increasing the dose of the drug or switching to a different
drug of the same family, in case of poor responsiveness, while it
would not be changed in case of platelet hyper-reactivity despite
good responsiveness to the drug, although additional treatments, if
available, might be taken in consideration by the treating physician.
While on-treatment platelet reactivity was initially studied
mostly with unspecific or global tests of platelet function, in the
last years it has been preferentially measured using specific tests.
Therefore, the most recent and the ongoing studies that use specific tests aim at identifying patients with HPR that is likely due to
poor responsiveness to the drug. The cut-off values for “HPR”
must be identified, based on its ability to discriminate between patients who are at increased risk of future MACE from those who
are not: this goal can be accomplished uniquely based on the results of large-scale, well-designed, ad hoc studies.
Laboratory tests of platelet function
that have been used to measure platelet
reactivity during antiplatelet treatment
Platelet function in vivo has been measured by the bleeding time,
while platelet function in vitro has been measured by several tests,
many of which have been criticised because they do not reproduce
the physiological conditions that characterise the development of
platelet aggregates in vivo. However, this potential drawback does
not necessarily decrease their accuracy in measuring the efficacy
of an anti-platelet drug to hit its pharmacological target (3). In addition, it is likely that in the future platelet tests will also focus on
platelet functions that do not seem strictly related to haemostasis
(5).
Finally, it must be emphasised that the results of platelet function is affected by many variables, including the cyrcadian rhythm,
comorbidities, physical exercise, the type and quantity of food and
drinks, etc. As a consequence, the inter-assay coefficient of variation of platelet function tests tends to be high, although this has
been only very rarely reported in published studies. For a list of
laboratory tests of platelet function that have been used to measure
platelet reactivity during antiplatelet treatment▶ Table 1.
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793
High on-treatment platelet reactivity
Cattaneo: HPR – definition and measurement
High on-treatment platelet reactivity
794
Cattaneo: HPR – definition and measurement
Bleeding time
The bleeding time is an invasive technique, which is poorly reproducible, even when carried out by experienced personnel. In addition to these drawbacks, the technique is influenced by several
variables, which include platelet function, platelet count, plasma
factors, red blood cells, the vessel wall and the skin trophism. For
this reason, it displays low sensitivity to mild/moderate abnormalities of inherited and acquired (including drug-induced) defects of
platelet function and should not be used to evaluate the effects of
anti-platelet agents (1).
Light transmission aggregometry (LTA)
LTA measures the increase in light transmission through a platelet
suspension that occurs when platelets aggregate in response to an
agonist. It is not ideal for testing platelet sensitivity to aspirin or
thienopyridines for several reasons:
• It is time-consuming and should be performed in highly
specialised laboratories only.
• Many pre-analytical and analytical variables affect the results:
even when all of them are controlled for, the accuracy and reproducibility of the technique are very poor.
• The results obtained within one lab can hardly be compared to
those obtained in a different lab due to lack of standardisation
(6): therefore, any attempt to define universal cut-off values of
platelet aggregation to identify non responders to anti-platelet
therapies would be pointless.
• Depending on the type and concentration of agonist used, and
the type of anticoagulant used for blood collection, the aggregation response is only partially and variably modulated by
TxA2, synthesised from arachidonic acid in the platelet membrane, or ADP, released from platelet granules. Even when the
two most specific platelet agonists are used, arachidonic acid
for monitoring aspirin, and ADP for monitoring P2Y12 inhibitors, the results obtained with this technique may over-estimate the prevalence of resistance to anti-platelet agents (1, 3).
Table 1: Some platelet function tests that have been used to
measure platelet reactivity during antiplatelet treatment.
Bleeding time
Light transmission aggregometry
Impedance aggregometry
Whole blood platelet aggregation measured by platelet counting
The PFA-100® system
Ultegra Rapid Platelet Function Assay (RPFA)-VerifyNow®
Flow cytometry
Cone-and-Plate(let) Analyzer (CPA)
Thromboelastography (TEG)
Serum thromboxane B2 (TxB2) levels
Urinary levels of the TxB2 metabolite, 11-dehydrothromboxane B2
The inadequacy of LTA for measuring the pharmacological effect
of aspirin was demonstrated by Ohmori et al., who showed that,
although platelet COX-1 activity seemed to be uniformly inhibited
in all studied patients, LTA studies showed great inter-individual
differences (7). Despite its limitations, the extent of maximal and/
or late platelet aggregation measured by LTA was linked to clinical
outcomes after percutaneous coronary interventions (PCI) in
some studies (8-15).
Impedance aggregometry
Impedance aggregometry measures the change in electrical impedance that occurs when platelets form an aggregate on electrodes, which are immersed in a sample of diluted whole blood,
after the addition of a platelet agonist. The instrument Multiplate®
(Verum Diagnostica, Munich, Germany) is the impedance aggregometer that has been most widely used in studies that measured
the platelet aggregation in patients on treatment with clopidogrel.
The technique is less technical demanding and time consuming
than LTA; however, some drawbacks that have been listed for LTA,
also apply to this technique. In particular, pre-analytical variables
(correctness of blood sampling and handling, for instance) will affect the aggregation response of platelets, independently of the intrument used for its measurement. According to some studies, the
technique is able not only to identify patients at risk of MACE, but
also those at risk of bleeding (16-19).
Whole blood platelet aggregation measured by
platelet counting
This is an easy and extremely sensitive method to study platelet aggregation. It is based on the principle that single platelet counts in
whole blood stimulated with a platelet agonist decrease as a function of platelet aggregate formation. It is more sensitive than LTA,
because it can detect very small aggregates, formed by 2-3 aggregated platelets. Very limited experience with this technique is
available for assessment of the effects of clopidogrel on platelet aggregation (20).
The PFA-100® system
PFA-100® (Dade Behring, Marburg, Germany) could be considered an in vitro bleeding time: a sample of anticoagulated blood is
aspirated through a capillary (mimicking the resistance of a small
artery) and a 150 μm aperture in a membrane (mimicking the injured part of the vessel wall) coated with collagen plus ADP
(C-ADP) or collagen plus epinephrine (C-EPI). A platelet plug
forms that gradually occludes the aperture; as a consequence, the
blood flow through the aperture gradually decreases and eventually stops. The time needed till blood flow interruption ("closure
time") is recorded (1, 21). The system is very easy to use, automated, quick and requires a small volume of whole blood. It is sensitive to von Willebrand Disease and to severe abnormalities of
platelet function, for example Glanzmann Thrombasthenia and
Bernard-Soulier Syndrome (22). In contrast, its sensitivity to mild/
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moderate, inherited and drug-induced defects of platelet function
is low (22): closure times (CT) of the C-EPI cartridge may be prolonged in some, but not all patients on aspirin treatment, while
variable but often normal CT have been observed on both C-ADP
and C-EPI cartridges in samples from patients receiving clopidogrel therapy (1, 22-27). The low sensitivity of the method to mild
abnormalities of platelet function can be accounted for by the fact
that it is sensitive to many variables including platelet count, red
blood cells, platelet reactivity to collagen and, above all, plasma
von Willebrand factor (28, 29), the effects of which on platelet aggregate formation can easily outweigh the effects of mild inhibition of platelet function (1). As a matter of fact, it has been
shown that both individuals with short and individuals with prolonged C-EPI closure times on aspirin treatment had very low levels of serum thromboxane B2 (TxB2), indicating that aspirin inhibited COX-1 to the same extent in the two groups of patients
(30, 31). For the above reasons the PFA-100® system is not an adequate method to measure platelet inhibition by aspirin or P2Y12
inhibitors.
A novel cartridge (Innovance PFA® P2Y, Siemens, Erlangen,
Germany), which is more sensitive to P2Y12, is under clinical
evaluation (32).
Ultegra Rapid Platelet Function Assay (RPFA)VerifyNow®
The Ultegra Rapid Platelet Function Assay (RPFA)-VerifyNow®
(Accumetrics, San Diego, CA, USA) is a simple point-of-care
whole blood test that measures the agglutination of fibrinogencoated beads by platelets stimulated by an agonist in citrated whole
blood. It was initially developed to measure the anti-platelet effects
of glycoprotein (GP)IIb-IIIa antagonists. Later, it was slightly
modified to become more sensitive and specific for the two most
widely used anti-platelet treatments: RPFA-VerifyNow® ASA, for
monitoring aspirin treatment (24, 33) and RPFA-VerifyNow® P2Y12
(34), which is a rather specific test for monitoring clopidogrel
treatment (35, 36). Results are expressed as P2Y12 reaction units
(PRU), with higher values being associated with higher platelet
reactivity. In the last years, VerifyNow® P2Y12 has become the most
widely used test for monitoring clopidogrel treatment. PRU values
have been correlated with MACE in many studies, involving a
large number of patients (37-41). Despite its widespread use, the
cut-off value for predicting MACE is still a matter of debate (42).
VerifyNow has been chosen as the laboratory test to measure platelet function in trials that were designed to test the safety and efficacy of tailored treatment with clopidogrel (37, 43), based on platelet function testing, which so far have given disappointing results. The main drawback of the assay is its very high cost.
Cone-and-Plate(let) Analyzer (CPA)
The Cone-and-Plate(let) Analyzer (CPA) is a cone-and-plate viscometer that measures platelet adhesion and aggregation under
laminar flow with uniform high shear. It was shown to be accurate
for detection of von Willebrand Disease and for monitoring treat-
ment with GPIIb/IIIa antagonists. In a small study, it could detect
poor responders to clopidogrel (44). Its clinical development has
been recently interrupted.
Flow cytometry
Flow cytometry allows the evaluation of platelet reactivity to agonists in vitro and the presence of circulating activated platelets, platelet-leukocyte aggregates and platelet-derived microparticles. Although it requires a sophisticated and expensive instrument,
which is not available to every institution, it is simple to perform
and requires very small volumes of whole blood samples. It has
been used to measure in vitro platelet activation on anti-platelet
treatment (45).
A flow cytometry-based method, the Platelet VASP®, (Biocytex,
Diagnostica Stago, Asnieres, France) measures the inhibition by
ADP of phosphorylation of vasodilator-stimulated phosphoprotein (VASP) (46), which is mediated by P2Y12 through the inhibition of adenylyl cyclase. The ratio of dephosphorylated and
phosphorylated VASP is a specific measure of P2Y12 activity, which
is expressed as “platelet reactivity index” (PRI). Therefore, it represents a most specific available assay for measuring the effects of
thienopyridines and other drugs inhibiting the platelet P2Y12 receptor (23, 47, 48): good inhibition of P2Y12 is reflected by low PRI
values. Unfortunately, the technique is not sensitive to mild abnormalities of the receptor (49, 50). There was a good correlation between ADP-induced platelet aggregation and VASP-phosphorylation assay in detecting P2Y12 inhibition (23, 51), although occasional individuals on clopidogrel treatment who appeared low responders with LTA were good responders on the more specific
VASP-phoshorylation assay (23). An important advantage of Platelet VASP® is that its endpoint is not based on platelet aggregation
and is therefore absolutely independent of GPIIb/IIIa function:
this property allows its use also in patients on treatment with
GPIIb/IIIa antagonists, which are used in some categories of ACS
patients undergoing PCI. Several studies showed that high PRI
values in patients on treatment with clopidogrel (52-55), or even
prasugrel in one case (56), correlate well with the risk of MACE.
However, the correct cut-off value has not been clearly identified
yet (55, 57), as it significantly depends on the method of calculation, the time-to-assay as well as on the lag time after processing
(58). Recently, an ELISA-based kit became available, which might
simplify the measurement of VASP phosphorylation in some circumstances.
Thromboelastography (TEG)
The method measures the viscoelastic changes that occur during
clot formation in whole blood. It is influenced by platelet function,
coagulation factors, natural inhibitors of coagulation and fibrinolysis. Some studies showed that TEG is potentially useful for
studying patients on antiplatelet treatment (59-66).
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795
High on-treatment platelet reactivity
Cattaneo: HPR – definition and measurement
High on-treatment platelet reactivity
796
Cattaneo: HPR – definition and measurement
Serum TxB2 levels
Serum TxB2 reflects the total capacity of platelets to synthesise
thromboxane A2, of which it is a stable metabolite, and is therefore
the most specific test to measure the pharmacological effect of aspirin (1). In studies of 680 patients undergoing cardiac catheterisation (45) and 96 healthy subjects (31) on aspirin treatment, only
about 1-2% had evidence of incomplete response to aspirin, based
on serum TxB2 levels, which was either due to under-dosing or
non-compliance (45), with the exception of one healthy subject
who really appeared resistant to aspirin (31). Frelinger et al. reported that a direct measure (serum TXB2 <3.1 ng/ml) but not indirect measures of residual platelet COX-1 function are associated
with subsequent MACE in aspirin-treated patients. However,
given a potential for bias based on the method used to define the
cut-off for high residual serum TXB2, the need to adjust for covariables to show a significant association between serum TXB2 and
subsequent MACE, and the failure of indirect assays of residual
platelet COX-1 function to confirm an association with MACE,
the link between residual platelet COX-1 function as reported by
serum TXB2<3.1 ng/ml and MACE should be further verified (63).
The ability of platelets to synthesize TxB2 after in vitro stimulation with an agonist, such as collagen, can also be considered a
specific test for measuring the effects of aspirin, albeit probably
less sensitive and more time consuming than serum TxB2 (7).
Urinary levels of the TxB2 metabolite, 11-dehydrothromboxane B2
Urinary levels of 11-dehydro-TxB2 represent a time-integrated
index of TxA2 biosynthesis in vivo (64). Because it is not formed in
the kidney, detection of this TxA2 metabolite in the urine reflects
systemic TxA2 formation, which largely, albeit not exclusively, occurs in the platelets. It has been calculated that about 30% of the
urinary metabolite derives from extra-platelet sources (65); however, in pathological conditions, such as in inflammatory diseases,
the contribution of extra-platelet sources may increase. Since
atherosclerosis is an inflammatory condition, high levels of urinary 11-dehydro-TxB2 may be considered a marker of inflammation, rather than a marker of poor responsiveness to aspirin
(66).
Comparison of different laboratory tests of platelet
function in the assessment of the pharmacological
responses to aspirin or P2Y12 inhibitors
Comparison of different laboratory methods in patients on aspirin
treatment (arachidonic acid-induced aggregation with LTA,
PFA-100®, Ultegra VerifyNow® ASA, serum TxB2, agonist-induced
TxB2 production, urinary 11-dehydrothromboxane B2) usually
showed very weak or no correlation in published studies (24, 31,
67, 68), indicating that they are sensitive to different parameters.
Usually, the number of individuals with residual, significant TxB2
production on aspirin was extremely low, while the prevalence of
individuals with no inhibition of platelet function measured by
other tests tended to be much higher.
Many studies compared the performance of different laboratory tests for evaluating the pharmacological effects of clopidogrel.
All studies demonstrated that the degree of agreement among different tests is unacceptably low (69-77): the same patients could be
considered poor responders by one test and good responders by
another test, and vice-versa. Therefore, because no single laboratory test has been shown to be more accurate than others in
measuring the degree of inhibition of ADP/P2Y12-dependent platelet function by clopidogrel, the ideal test for monitoring clopidogrel therapy has not been identified yet.
Conclusion
In conclusion, a clear distinction should be made between monitoring patients on anti-platelet treatment to identify “poor responders” and to identify patients with HPR. For practical reasons,
the vast majority of studies that have been performed so far in patients on antiplatelet therapy measured on-treatment platelet reactivity. Many good tests of platelet function have been used, most of
which were shown to be able to identify patients at risk of MACE.
Unfortunately, universal cut-off values for high on-treatment platelet reactivity have not be clearly established yet. In addition, the
concordance among different tests in the identification of patients
at risk is very poor, and the most effective and safe treatment for
patients at risk is still unknown. Therefore, many studies need to
be performed to answer basic questions on its clinical utility and
cost-effectiveness, before anti-platelet monitoring can be recommended in the clinical practice. While waiting for the results of
some of these studies that are ongoing, monitoring of anti-platelet
therapy should be done for investigational purposes only.
Conflicts of interest
M. Cattaneo has received honoraria and research funds from
AstraZeneca, Eli Lilly and Daiichi Sankyo.
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