Atherosclerosis 209 (2010) 504509

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Variability of residual platelet function despite clopidogrel treatment in patients

with peripheral arterial occlusive disease
Birgit Linnemann a, , Jan Schwonberg a , Stefan W. Toennes b , Helen Mani a , Edelgard Lindhoff-Last a
a
Division of Vascular Medicine, Department of Internal Medicine, J.W. Goethe University Hospital, Frankfurt/Main, Germany
b
Institute of Forensic Toxicology, Center of Legal Medicine, J.W. Goethe University Hospital, Frankfurt/Main, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Residual platelet function despite treatment with clopidogrel may predict an unfavourable cardiovascular
Received 19 January 2009 outcome. The majority of studies have investigated the effects of clopidogrel administration in conjunction
Received in revised form 8 March 2009 with aspirin in patients undergoing percutaneous coronary intervention. The primary objective of the
Accepted 3 May 2009
present study was to assess the platelet response to clopidogrel in the absence of aspirin in patients with
Available online 25 January 2010
peripheral arterial occlusive disease (PAOD) and to investigate whether non-responsiveness to clopidogrel
is reproducible during long-term follow-up. Fifty-four clinically stable PAOD patients on a maintenance
Keywords:
dose of 75 mg/d clopidogrel were enrolled in this study. Platelet function was assessed at baseline and
Platelet reactivity
Clopidogrel
after a median follow-up of 18 months using light transmittance aggregometry (LTA) with 2 M ADP as an
Non-response agonist. HPLC-coupled mass spectrometry was used to detect clopidogrel and clopidogrel carboxylic acid,
Aggregometry the main metabolite of clopidogrel. Residual platelet function, as dened by late aggregation values within
HPLC-coupled mass spectrometry the reference range (i.e., >43%), was observed in 35.2% of patients at baseline and 17.6% during follow-
Peripheral arterial occlusive disease up. During the observation period, 26.5% had switched from responder to non-responder status or vice
versa. Among non-responders, either clopidogrel or its metabolite was detected in 89.5% and 83.3% of
patients at baseline and at follow-up, respectively. We conclude that non-responsiveness to clopidogrel
as determined by ADP-induced LTA is not stable over time. This phenomenon cannot be attributed to
non-compliance alone.
2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 5 -diphosphate-(ADP-)induced platelet aggregation in citrated

Clopidogrel was approved for clinical application in 1997 after
the large phase III clinical trial, Clopidogrel versus aspirin in
patients at risk of ischemic events (CAPRIE), which involved 19,185
patients with a history of symptomatic atherosclerotic disease [1].
The CAPRIE trial demonstrated an overall benet of clopidogrel over
aspirin in the prevention of vascular ischemic events, such as stroke,
myocardial infarction, and vascular death. Clopidogrel treatment
resulted in an 8.7% relative reduction in vascular events. Subsequent
studies demonstrated a benet when clopidogrel was adminis-
tered with aspirin. This has been best characterized in patients with
unstable angina or non-ST elevation myocardial infarction or after
percutaneous coronary interventions (CURE, CREDO) [2,3].
Clopidogrel is a thienopyridine that is metabolized in the liver
to generate an active metabolite. At the recommended dosage
of 75 mg/d, a steady state is achieved after three to four days.
For safety reasons in terms of bleeding risk, this dosage was
chosen to achieve approximately 50% inhibition of adenosine
Corresponding author. Tel.: +49 69 6301 5096; fax: +49 69 6301 7219.
E-mail address: Birgit.Linnemann@kgu.de (B. Linnemann).
platelet-rich plasma [4]. Alternative dose regimens have resulted
in improved clinical outcomes in acute situations with loading
doses of clopidogrel ranging from 300 to 900 mg to achieve a more
rapid effect, which is then followed by the maintenance dose [5].
Most of the studies concerning high residual platelet func-
tion or non-responsiveness to clopidogrel have been performed in
patients with ischemic heart disease (IHD) who undergo percuta-
neous catheter intervention (PCI) [6]. In the majority of studies,
efcacy of clopidogrel treatment was assessed within 24 h after
administration of the loading dose. Most commonly, clopidogrel
has been administered in conjunction with aspirin, and it is critical
to account for this second drug when platelet function is assessed
by ADP-induced aggregation because aspirin is known to inhibit
ADP-induced platelet function.
Besides its use in co-medication with aspirin in the setting
of catheter interventional procedures, clopidogrel has also been
approved as monotherapy to reduce atherosclerotic events in
patients with recent myocardial infarction, stroke, or established
PAOD, and it is prescribed especially in the case of adverse reac-
tions or intolerance to aspirin. Therefore, the aim of the present
study was to evaluate responsiveness to chronic clopidogrel ther-
apy without concomitant aspirin use by assessing residual platelet

0021-9150/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2009.05.039
 B. Linnemann et al. / Atherosclerosis 209 (2010) 504509
function with light transmittance aggregometry (LTA) in patients
with clinically stable peripheral arterial occlusive disease (PAOD).
Moreover, we evaluated whether non-responsiveness to clopido-
grel was reproducible during long-term follow-up.
2. Methods
2.1. Study population
Fifty-four consecutive PAOD patients (29 males, 25 females)
seen in our outpatient department between July 2003 and Jan-
uary 2006 and treated with clopidogrel 75 mg daily as the only
antithrombotic drug were enrolled in this study. Patients ages
ranged from 53 to 94 years (median 68). PAOD was dened as an
ankle brachial index 0.9 as measured with Doppler ultrasound
[7]. The disease had to be in a stable condition without any clinical
deterioration within the last 3 months. Patients with a precedent
cardiovascular event (e.g., acute coronary syndrome) were assessed
at least 4 weeks after the cardiovascular event. Clopidogrel therapy
had to have been administered over a period of at least 14 days.
Patients taking additional medications known to inuence platelet
function (e.g., aspirin, NSAID) were excluded, as well as patients
with known coagulation disorders, liver cirrhosis, alcohol abuse,
end-stage renal failure, malignant disease, or other concomitant
disease with a life expectancy less than one year. A platelet count
<100 109 L, a haemoglobin <9 g/dL, or a haematocrit <28% were
additional exclusion criteria. The study was carried out according to
the principles of the Declaration of Helsinki. Our Institutional Ethics
Committee approved this study, and all patients provided written
informed consent. Using a standardized questionnaire, clinical data
detailing vascular and concomitant disease, cardiovascular risk fac-
tors, and co-medication were recorded. All patients conrmed that
they had taken clopidogrel regularly as directed during the last 14
days. The last dose had been administered within one to 24 h before
blood sampling.
2.2. Blood sampling
Blood was drawn by clean venipuncture from an antecubital
vein using a 21-gauge buttery cannula system (Multiy -Set,
21 G 1 1/2 TW, 0.8 19 mm, Sarstedt, Nmbrecht, Germany).
EDTA- and citrate-supplemented blood was collected into plas-
tic syringes (Monovette , Sarstedt, Nmbrecht, Germany). Platelet
count was measured on the Sysmex KX-21 (Roche Diagnostics,
Basel, Switzerland) automatic multi-parameter blood cell counter.
Platelet counts between 100 and 500/nl were necessitated for sub-
sequent platelet function testing. Platelet-rich plasma (PRP) was
obtained by centrifuging 0.106 M (3.2%) citrated whole blood at
room temperature at 140 g for 5 min. To obtain platelet-poor
plasma (PPP, platelet count <10/nl), citrated whole blood was cen-
trifuged at 1500 g for 15 min. The time interval between blood
sampling and testing was at least 1 h and did not exceed 3 h. All
platelet function tests were performed in duplicate.
2.3. Light transmittance aggregometry
LTA was performed on the Behring Coagulation Timer (BCT ;
Dade Behring, Ddingen, Switzerland). The BCT is a fully auto-
matic machine used for routine and special coagulation testing. The
BCT detects platelet aggregate formation in PRP by changes in light
transmission (monochromatic light; wavelength: 620 nm) at 37 C.
Platelet aggregation agonists (15 L reagent) are introduced auto-
matically into PRP (135 L plasma) with stirring at a velocity of
600 rpm. In this study, adenosine 5-diphosphate (ADP; AppliChem,
Darmstadt, Germany) in a nal concentration of 2 M was used
to stimulate aggregation. The extent of induced aggregation was
505
dened by the slope of the aggregation curve obtained from the
change in light transmission over time. Measurements were per-
formed in native PRP that was not adjusted for platelet count.
Because disaggregation is a frequent phenomenon in clopidogrel
treated patients, we measured the late aggregation response after
10 min. Light transmission was determined in PRP at the start and
end of the measurement and compared with PPP. Late aggregation
was calculated with the following formula:
Late aggregation [%]
(start aggregation of PRP end aggregation of PRP)
= 100%
(start aggregation of PRP aggregation of PPP)
In a previous investigation, we demonstrated that late aggrega-
tion induced by 2 M ADP in non-adjusted PRP offered the best
discrimination between patients on clopidogrel medication and
healthy volunteers not taking antithrombotic drugs when com-
pared with 5 M ADP with and without adjustment for platelet
count (i.e., 250 109 L) (Fig. 1) [8]. In addition, we determined
the within-day precision of LTA by drawing a blood sample from
ve healthy volunteers, performing platelet function tests on ve
consecutive days, and calculating the coefcient of variation (CV).
The CV using 2 M ADP as the agonist in non-adjusted PRP was
2.72%. In accordance with the actual recommendations given at the
53rd Annual Scientic and Standardization Committee Meeting of
the International Society of Thrombosis and Haemostasis, Geneva
2007, the 5th95th percentiles measured in duplicate in a group of
healthy volunteers (n = 20) was considered as the reference range.
Non-responsiveness to clopidogrel was dened as an aggregation
value within the reference range despite clopidogrel medication
(i.e., late aggregation (LateAggr) 42.9%).
2.4. Liquid chromatography-mass spectrometry analysis
Plasma samples were analyzed for clopidogrel and its main
metabolite clopidogrel carboxylic acid using high performance
liquid chromatography (HPLC) coupled to a time of ight mass
spectrometer as described previously [9]. The assay consisted of
an automated solid phase plasma extraction (0.5 mL that was
optimized for basic drugs and analysis by an Agilent 1100 series
liquid chromatograph interfaced to an Agilent 1100 series oa-TOF
system (Waldbronn, Germany) operated in positive electrospray
ionization mode. Chromatographic separation was achieved on
a 100 2.0 mm Polaris C18-ether 3 m column (Varian, Darm-
stadt, Germany) at 50 C using an acetonitrile/0.1% formic acid
(10100%, v/v) gradient at 0.4 mL/min for 12 min. Clopidogrel could
be detected at 0.1 g/L, and the carboxylic acid metabolite could be
detected at 10 g/L. This method was successful in our last study
[9], and the sensitivity is sufcient to detect clopidogrel up to 12 h
after the last administered dose, as seen in the work of Lainesse et
al. which measured clopidogrel kinetics after an oral dose of 150 mg
[10]. The carboxylic acid metabolite can be detected for two days
after the last clopidogrel dose (7 half-lives of 7.3 h), which is con-
sistent with the results of McEwen et al. in which the metabolite
was detected 36 h after a single oral 75 mg clopidogrel dose [11].
2.5. Follow-up
Of the 54 patients included in this study, three (5.6%) were lost
to follow-up due to a change of residence. Thus, clinical outcome
data were available for 51 cases (94.4%). The median observation
time was 17.5 months (range 10.540.4). Three patients died dur-
ing the observation period, and ve patients were in poor health
such that they could not come to our outpatient department for a
second blood sampling. The antithrombotic medication prescribed
to nine patients was changed during the observation period mainly
506 B. Linnemann et al. / Atherosclerosis 209 (2010) 504509

Fig. 1. Late aggregation (LateAggr) with ADP 2 and 5 M as agonist comparing measurements in non-adjusted and platelet count-adjusted PRP (platelet count 250 109 10%)
in healthy subjects and clopidogrel treated patients. (Outliers are illustrated as circles and extreme values as stars.).

due to an acute cardiovascular event. At the time of follow-up, Table 1

Baseline characteristics of the study cohort (n = 54): prevalence of vascular risk fac-
ve patients received combination therapy with aspirin because
tors and co-morbidities.
they had suffered a major cardiovascular event or required a surgi-
cal or catheter-guided revascularization procedure. Two patients n (%)
were switched to aspirin, and another one to phenprocoumon. Vascular risk factor
One patient became asymptomatic during the follow-up period Male gender 29 (53.7)
and refused any antithrombotic treatment. These patients were Current smoker 13 (24.1)
Arterial hypertension 42 (77.8)
excluded from the repeated assessment of platelet function. Thus,
Diabetes mellitus 18 (33.3)
34 patients (63.0%) were willing and able to provide a second blood Hyperlipidemia 44 (81.5)
sample and were still on clopidogrel monotherapy at the time of Obesity (BMI 30 kg/m2 ) 10 (22.2)
follow-up. Chronic renal failure 8 (14.8)

Peripheral arterial occlusive disease (PAOD) 54 (100.0)

Prior amputation 1 (1.9)
2.6. Statistical analyses
Prior vascular surgery 10 (18.5)
Prior foot ulcer/gangrane 5 (9.3)
Statistical analysis was performed using the Statistical Package Prior catheter intervention (PTA/stenting) 38 (70.4)
for Social Sciences (SPSS version 15.0, Chicago, IL, USA). In addi- Ischemic heart disease (IHD) 21 (38.9)
tion to descriptive statistics with frequencies, mean and standard Prior myocardial infarction 8 (14.8)
deviation, median and range, we performed the Chi-squared and Prior aorto-coronary bypass surgery 6 (11.1)
Fishers exact tests. The criterion for statistical signicance was Prior percutaneous coronary intervention (PCI) 13 (24.1)
a p-value less than 0.05. Results are also presented as box plots Cerebrovascular disease (CVD) 10 (18.5)
with the bare length indicating the interquartile range (25th75th Prior cerebral infarction 3 (5.6)
percentile). Outliers are dened as values differing 1.53.0 bare Carotid artery stenosis/occlusion 7 (13.0)
Prior carotid artery revascularization (stent-PTA or surgery) 2 (3.7)
lengths, whereas extreme values are those differing >3.0 box
lengths from the upper or lower edge of the box. In the gures,
outliers are illustrated as circles and extreme values as stars.

3. Results

The prevalence of cardiovascular risk factors and co-morbidities

of the 54 patients enrolled in this study is presented in Table 1.

3.1. Prevalence of non-responsiveness to clopidogrel

In the total cohort, 19 patients (35.2%) were non-responsive to

clopidogrel at baseline. Platelet function was measured in a sec-
ond blood sample from 34 patients after a median follow-up of 17.5
months. Thirteen of these 34 patients (38.2%) were non-responders
at baseline. At follow-up, non-responsiveness was detected in six
patients (17.6%). Nine patients (26.5%) had a change in response sta-
tus during the observation period (Fig. 2). The absolute aggregation
values are presented in Fig. 3. The gure illustrates that changes
in responsiveness were not solely due to small changes around the Fig. 2. Response to clopidogrel at baseline and at follow-up determined by LTA with
cut-off value. 2 M ADP as an agonist (n = 34).
 B. Linnemann et al. / Atherosclerosis 209 (2010) 504509 507

all responsive to clopidogrel. The prevalence of clopidogrel non-

responsiveness in the group of drop-out patients (29.2%, 7/24) was
similar to that for patients who provided a second blood sample at
the time of follow-up (38.2%, p = ns).

4. Discussion

In the present study, we observed non-responsiveness to clopi-

dogrel in 35.2% of patients with stable PAOD receiving 75 mg/d
clopidogrel as the only antithrombotic drug using conventional
ADP-induced LTA. However, the main nding of our investigation
is that non-responsiveness to clopidogrel is not stable over time.
When LTA was repeated after a median of 17.5 months, nine of 34
patients (26.5%) had switched from responder to non-responder
status or vice versa.
The concept of clopidogrel resistance or non-responsiveness
Fig. 3. Comparison of late aggregation values at baseline and follow-up (consistent
test results over time: , black lines; change of response during follow-up: , red is based on the observation that a respectable number of patients
lines). under treatment with clopidogrel display aggregation parameters
that do not differ from those of untreated healthy subjects [12]. In
3.2. Detection of clopidogrel and its main metabolite clopidogrel patients with IHD undergoing PCI with stent implantation, resid-
carboxylic acid ual platelet function is observed in 544% of patients [6]. The wide
range of non-responsiveness is considered to be due to the lack
To determine whether clopidogrel non-response was due to of a standardized methodology to quantify platelet response to
non-compliance, clopidogrel and its main metabolite clopidogrel antithrombotic therapy and the lack of a consensus denition of
carboxylic acid were determined by HPLC mass spectrometry. non-responsiveness. In the majority of investigations, platelet func-
Clopidogrel or its metabolite was detected in 17 of 19 non- tion tests were performed within 24 h after administration of a
responders (89.5%) and 34 of 35 responders (97.1%) at baseline loading dose in addition to simultaneous treatment with aspirin
and in ve of six (83.3%) and 27 of 28 (96.4%) non-responders and [6]. However, these ndings may not reect the platelet inhibition
responders, respectively, at follow-up (Table 2). Neither clopidogrel that is observed with the maintenance dose that is used dur-
nor its metabolite was detectable in two non-responders at baseline ing long-term treatment. Matetzky et al. measured ADP-induced
and in one non-responder at follow-up. When asked again, these aggregation on ve consecutive days in acute myocardial infarction
three patients stated that the last drug intake had been either 18, patients who received a loading dose of 300 mg clopidogrel before
24 or 73 h before blood sampling. PCI followed by a daily dose of 75 mg. This study observed maximal
platelet function inhibition after 24 h of the loading dose and vari-
3.3. Cardiovascular risk factors and statin use according to able partial recovery of platelet function during administration of
clopidogrel response the maintenance dose [13]. This variability has also been observed
by Serebruany et al. who performed aggregation measurements
We did not observe any differences in the distribution of repeatedly over 30 days [14].
common vascular risk factors, such as male gender, smoking, dia- Only two investigations of non-responsiveness to clopidogrel
betes mellitus, arterial hypertension, hyperlipidemia, obesity (BMI in PAOD patients have been published in the past. Cassar et al.
30 kg/m2 ), or chronic renal failure, between responders and non- analyzed platelet function by ow cytometric determination of
responders at baseline. In addition, frequency of statin use was not ADP-stimulated platelet brinogen binding and identied 9.8%
higher among non-responders compared with responders. (6/61) non-responders among patients with intermittent claudi-
cation who received clopidogrel in addition to aspirin [15]. Ziegler
3.4. Drop-out patients et al. investigated platelet function in PAOD patients undergoing
peripheral angioplasty using the CADP cartridge on the PFA-100
In a notable number of patients, a second blood sample could system [16]. Among those who were treated with clopidogrel, 32.4%
not be obtained. Among those were three patients who had died (11/34) were determined to be non-responders. However, other
and another nine in whom antithrombotic medication had been investigations have shown that this cartridge is not suitable for
changed during follow-up. The three patients who had died were monitoring clopidogrel therapy [17].
Our study is the rst that has investigated the stability of non-
responsiveness to clopidogrel during long-term follow-up. In a
Table 2
Presence of clopidogrel and its main metabolite clopidogrel carboxylic acid as previous study, Gurbel et al. observed a strong correlation (r = 0.8)
detected by HPLC mass spectrometry. between the 5- and 30-day responses to 5 and 20 M ADP in a
cohort of IHD patients undergoing elective PCI who received a
Non-responder Responder
300 mg loading dose followed by daily 75 mg doses [18,19]. The
1st Blood sampling (n = 54) n = 19 n = 35 authors concluded that the inhibitory response on platelet aggre-
Presence of
gation is stable over 30 days. However, a recently published study
Clopidogrel 10 (52.6%) 23 (65.7%)
Clopidogrel carboxylic acid 17 (89.5%) 34 (97.1%) reported that non-responsiveness to aspirin might not be stable
No drug or metabolite 2 (10.5%) 1 (2.9%) over time [20].
2nd Blood sampling (n = 34) n=6 n = 28
Compliance with the prescribed medication is a major concern.
Presence of Cassar et al. observed that six of 67 patients (8.9%) showed inad-
Clopidogrel 4 (66.7%) 25 (89.3%) equate adherence to clopidogrel therapy when compliance was
Clopidogrel carboxylic acid 5 (83.3%) 27 (96.4%) assessed by counting the number of tablets that were returned
No drug or metabolite 1 (16.7%) 1 (3.6%)
[15]. In the study of Schwartz et al. non-compliance was the main
508 B. Linnemann et al. / Atherosclerosis 209 (2010) 504509
reason for resistance to aspirin [21]. In their study, 17 of 190
patients were identied as non-responders to aspirin when assess-
ing platelet function by LTA. However, after observation of aspirin
intake and repeating LTA, only one patient who admitted having
taken an NSAR within the last 12 h presented with a persistent
non-inhibition of platelet function. Other investigators have also
described similar results, and non-compliance has been related to
a poor clinical outcome [22].
In our results, it was striking that response to clopidogrel obvi-
ously improved in eight patients when they were re-evaluated at
follow-up. One might speculate that non-compliance was the rea-
son as blood sampling at baseline was performed when patients
were recruited during a routine attendance at our outpatient
department whereas at follow-up, patients already knowing the
studys intention expected blood sampling for assessing clopido-
grel effects at follow-up. However, our data indicate that changes in
responsiveness are not only due to non-adherence to the prescribed
medication. Using HPLC mass spectrometry, we were able to detect
either clopidogrel or its main metabolite clopidogrel carboxylic acid
in 89.5% of non-responders and 97.1% of responders at baseline and
in 83.3% and 96.4% of non-responders and responders at follow-up.
As demonstrated in a previous investigation by our study group,
clopidogrel is detectable for at least 12 h after the administration
of the last dose, whereas detection of the carboxylic acid metabo-
lite can last for 48 h [9]. Patients were only included in this study
if they agreed to take the drug regularly with the last administra-
tion occurring within the preceding 24 h. Only one non-responder
with undetectable levels of clopidogrel and its metabolite admitted
that he had not taken clopidogrel regularly when confronted with
the test results. Hence, it seems unlikely that non-compliance was
a major cause of the variability of responsiveness observed during
long-term follow-up.
Several mechanisms of clopidogrel resistance have been dis-
cussed in the literature and may explain not only inter-patient
but also intra-patient variability in clopidogrel response. These
include inappropriate dosing, a variable enteric absorption and
metabolism of the prodrug in the liver and an increased platelet
turnover [12,23]. Recently, drug-to-drug interactions (e.g. omepra-
zole, calcium-channel blockers) have also been described that may
impair clopidogrel efcacy [24,25].
An association of ADP-induced platelet aggregation with the risk
of recurrent cardiovascular events in acute ST elevation myocar-
dial infarction was demonstrated for the rst time in a prospective
study by Matetzky et al. [13]. According to the results of subse-
quent prospective studies [2629], there is growing evidence that
high platelet reactivity after administration of clopidogrel is a risk
factor for recurrent ischemic events in patients undergoing PCI. The
overall number of cases in the present study is small. Therefore, this
study cannot answer the question of whether adverse outcomes in
PAOD patients can be predicted by detection of residual platelet
function by ADP-induced LTA.
Some limitations of the present study require consideration.
First, pre-treatment aggregation values are unknown because
patients were on chronic clopidogrel therapy when they were
enrolled in this study. In recent studies, the relationship between
pre-treatment aggregation, post-treatment aggregation, and clopi-
dogrel responsiveness was evaluated, and pre-treatment reactivity
did not predict clopidogrel responsiveness [30,31]. Moreover, it
appears that post-treatment platelet reactivity may be a better pre-
dictor of thrombotic risk. Second, LTA was performed using 2 M
ADP in PRP in this study and was not adjusted for platelet count.
To date, most studies utilize higher ADP concentrations (i.e., 5 and
20 M), and many laboratories still perform platelet count adjust-
ments. However, in a previous investigation, we demonstrated that
late aggregation using 2 M ADP in non-adjusted PRP provides
a good discrimination between clopidogrel treated patients and
healthy subjects [8]. Adjusting PRP for platelet count did not offer
any advantages, but, if adjustment is performed, the 5 M ADP con-
centration has to be used because healthy subjects without any
antithrombotic medication showed impaired platelet function in
response to 2 M ADP in adjusted PRP (Fig. 1). Furthermore, we
presented late aggregation values. Although maximum aggregation
is more commonly utilized, recent studies have shown that late
aggregation values may be more representative of P2Y12 receptor
signalling and have also been associated with adverse cardiovascu-
lar outcomes [26]. Third, ADP-induced LTA is a non-specic method
to measure the inhibitory effects of clopidogrel because inhibi-
tion is mediated by many factors, including multiple ADP-receptor
pathways, and only one of these receptors is inhibited by clopido-
grel. Ideally, non-responsiveness to clopidogrel should be dened
as the inability to achieve the expected inhibition of the P2Y12
receptor. A ow cytometric vasodilator-stimulated phosphopro-
tein (VASP) phosphorylation assay has been introduced to measure
more specic inhibition of clopidogrels biochemical target, the
P2Y12 receptor [32,33]. Moreover, the assay is not inuenced by
the concomitant use of aspirin as compared with ADP-induced
aggregation. Based on the analysis of measurements of intracellu-
lar VASP phosphorylation levels, non-responsiveness to clopidogrel
has been suggested as a risk factor for the occurrence of stent throm-
bosis [34,35]. However, LTA has a higher specicity and a higher
positive predictive value than the PRI (Platelet Reactivity Index) val-
ues obtained from the VASP assay [36]. Thus, LTA is still regarded
as the gold standard technique to test for antiplatelet drug effects
and remains the test against which more novel and user-friendly
assays need to be compared.
Finally, platelet function tests were repeated in only 34 of 54
patients (63%) after a median of 18 months. The drop-out rate was
high, but this can be largely explained by a change of antithrombotic
medication or serious comorbidity that prevented the patient from
returning to our facility for follow-up. A selection bias can be ruled
out insofar as the non-responsiveness to clopidogrel was similar
in prevalence among the drop-out patients and those who were
followed-up.
5. Conclusions
Despite the above mentioned limitations, the major strength of
our study was that we investigated patients on clopidogrel therapy
as the only antithrombotic drug, thus avoiding an impairment of
ADP-induced platelet aggregation by aspirin. While in the major-
ity of studies the platelet response is assessed within 24 h after
administering a loading dose, we evaluated platelet response to
the common clopidogrel maintenance dose. To our knowledge, this
is the rst study to investigate the stability of non-responsiveness
to clopidogrel after long-term follow-up. In conclusion, our results
indicate that non-responsiveness to clopidogrel is present in about
one third of stable PAOD patients who receive 75 mg clopidogrel
daily. However, the status of response is not stable with long-
term follow-up. Due to uctuations in responsiveness to clopidogrel
over time, repeated platelet function assessment may be useful
to identify patients with either consistent or intermittent non-
responsiveness. Additional work is required to answer the question
of whether routine screening for clopidogrel non-responsiveness
will provide benets in patients with PAOD and in, particular, in
high risk patients (e.g., unstable disease or critical limb ischemia).
References
[1] CAPRIE Steering Committee. A randomised, blinded, trial of clopidogrel
versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet
1996;348:132939.
[2] Yusuf S, Zhao F, Mehta SR, et al. Clopidogrel in unstable angina to prevent recur-
rent events trial investigators. Effects of clopidogrel in addition to aspirin in
 B. Linnemann et al. / Atherosclerosis 209 (2010) 504509
patients with acute coronary syndromes without ST-segment elevation. N Engl
J Med 2001;345:494502.
[3] Steinhubl SR, Berger PB, Mann JT, et al. CREDO Investigators. Clopidogrel for
the reduction of events during observation. Early and sustained dual oral
antiplatelet therapy following percutaneous coronary intervention: a random-
ized controlled trial. J Am Med Assoc 2002;288:241120.
[4] Gachet C. Regulation of platelet functions by P2 receptors. Annu Rev Pharmacol
Toxicol 2006;46:277300.
[5] Savi P, Herbert JM. Clopidogrel and ticlopidine: P2Y12 adenosine diphosphate-
receptor antagonists for the prevention of atherothrombosis. Semin Thromb
Hemost 2005;31:17483.
[6] Gurbel PA, Tantry US. Clopidogrel resistance? Thromb Res 2007;120:31121.
[7] Norgren L, Hiatt WR, Dormandy JA, et al. Inter-society consensus for the man-
agement of peripheral arterial disease (TASC II). Eur J Vasc Endovasc Surg
2007;33:S175.
[8] Linnemann B, Schwonberg J, Mani H, Prochnow S, Lindhoff-Last E. Stan-
dardization of light transmittance aggregometry for monitoring antiplatelet
therapy: an adjustment for platelet count is not necessary. J Thromb Haemost
2008;6:67783.
[9] Mani H, Toennes SW, Linnemann B, et al. Determination of clopidogrel main
metabolite in plasma: a useful tool for monitoring therapy? Ther Drug Monit
2008;30:849.
[10] Lainesse A, Ozalp Y, Wong H, Alpan RS. Bioequivalence study of clopidogrel
bisulfate lm-coated tablets. Arzneimittelforschung 2004;54:6004.
[11] McEwen J, Strauch G, Perles P, et al. Clopidogrel bioavailability: absence of
inuence of food or antacids. Semin Thromb Hemost 1999;25(Suppl 2):4750.
[12] Gachet C. P2 receptors, platelet function and pharmacological implications.
Thromb Haemost 2008;99:46672.
[13] Matetzky S, Shenkman B, Guetta V, et al. Clopidogrel resistance is associated
with increased risk of recurrent atherothrombotic events in patients with acute
myocardial infarction. Circulation 2004;109:31715.
[14] Serebruany VL, Steinhubl SR, Berger PB, et al. Variability in platelet responsive-
ness to clopidogrel among 544 individuals. J Am Coll Cardiol 2005;45:24651.
[15] Cassar K, Bachoo P, Ford I, Greaves M, Brittenden J. Variability in responsiveness
to clopidogrel in patients with intermittend claudication. Eur J Vasc Endovasc
Surg 2006;32:715.
[16] Ziegler S, Maca T, Alt E, et al. Monitoring of antiplatelet therapy with the PFA-
100 in peripheral angioplasty patients. Platelets 2002;13:4937.
[17] Pidcock M, Harrison P. Can the PFA-100 be modied to detect P2Y12 inhibi-
tion? J Thromb Haemost 2006;4:14246.
[18] Gurbel PA, BLiden KP, Hiatt BL, OConnor CM. Clopidogrel for coronary stenting:
response variability, drug resistance, and the effect of pre-treatment platelet
reactivity. Circulation 2003;107:290813.
[19] Gurbel PA, Bliden KP. Durability of platelet inhibition by clopidogrel. Am J Car-
diol 2003;91:11235.
[20] Harrison P, Segal H, Silver L, et al. Lack of reproducibility of assessment of
aspirin responsiveness by optical aggregometry and two platelet function tests.
Platelets 2008;19:11924.
509
[21] Schwartz KA, Schwartz DE, Ghosheh K, et al. Compliance as a critical considera-
tion in patients who appear to be resistant to aspirin after healing of myocardial
infarction. Am J Cardiol 2005;95:9735.
[22] Cotter G, Shemesh E, Zehavi M, et al. Lack of aspirin effect: aspirin resistance or
resistance to taking aspirin? Am Heart J 2004;147:293300.
[23] Siller-Matula J, Schroer K, Wojta J, Huber K. Thienopyridines in cardiovascu-
lar disease: focus on clopidogrel resistance. Thromb Haemost 2007;97:385
93.
[24] Siller-Matula JM, Lang I, Christ G, Jilma B. Calcium-channel blockers reduce the
antiplatelet effect of clopidogrel. J Am Coll Cardiol 2008;52:155763.
[25] Gilard M, Arnaud B, Cornily JC, et al. Inuence of omeprazole on the
antiplatelet action of clopidogrel associated with aspirin: the randomized,
double-blind OCLA (Omeprazole CLopidogrel Aspirin) study. J Am Coll Cardiol
2008;51:25660.
[26] Hochholzer W, Trenk D, Bestehorn HP, et al. Impact of the degree of peri-
interventional platelet inhibition after loading with clopidogrel on early
clinical outcome of elective coronary stent placement. J Am Coll Cardiol
2006;48:174250.
[27] Buonamici P, Marcucci R, Migliorini A, et al. Impact of platelet reactivity after
clopidogrel administration on drug-eluting stent thrombosis. J Am Coll Cardiol
2007;49:23127.
[28] Frere C, Cuisset T, Quilici J, et al. ADP-induced platelet aggregation and platelet
reactivity index VASP are good predictive markers for clinical outcomes in
non-ST elevation acute coronary syndrome. Thromb Haemost 2007;98:838
43.
[29] Marcucci R, Paniccia R, Antonucci E, et al. Residual platelet reactivity is an inde-
pendent predictor of myocardial injury in acute myocardial infarction patients
on antiaggregant therapy. Thromb Haemost 2007;98:84451.
[30] Samara WM, Bliden KP, Tantry US, Gurbel PA. The difference between clopi-
dogrel responsiveness and post-treatment platelet reactivity. Thromb Res
2005;115:8994.
[31] Michelson AD, Linden MD, Furman MI, et al. Evidence that pre-existent vari-
ability in platelet response to ADP accounts for clopidogrel resistance. J Thromb
Haemost 2007;5:7581.
[32] Aleil B, Ravanat C, Cazenave JP, et al. Flow cytometric analysis of intraplatelet
VASP phosphorylation for the detection of clopidogrel resistance in patients
with ischemic cardiovascular diseases. J Thromb Haemost 2005;3:8592.
[33] Geiger J, Teichmann L, Grossmann R, et al. Monitoring of clopidogrel action:
comparison of methods. Clin Chem 2005;51:95765.
[34] Gurbel PA, Bliden KP, Samara W, et al. Clopidogrel effect on platelet reactivity in
patients with stent thrombosis: results from the CREST study. J Am Coll Cardiol
2005;46:182732.
[35] Barragan P, Bouvier JL, Roquebert PO, et al. Resistance to thienopyridines:
clinical detection of coronary stent thrombosis by monitoring of vasodilator-
stimulated phosphorylation. Catheter Cardiovasc Interv 2003;59:295302.
[36] Angiolillo DJ, Alfonso F. Platelet function testing and cardiovascular outcomes:
steps forward in identifying the best predictive measure. Thromb Haemost
2007;98:7079.