Download Platelet Function Under Aspirin, Clopidogrel, and Both After

Platelet Function Under Aspirin, Clopidogrel, and Both
After Ischemic Stroke
A Case-Crossover Study
Armin J. Grau, MD, PhD; Sven Reiners, Cand Med; Christoph Lichy, MD;
Florian Buggle, MD; Andreas Ruf, MD, PhD
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Background and Purpose—Combined antiplatelet agents may offer additive protection over single drugs after stroke. We
investigated whether platelet activation is reduced under combined aspirin and clopidogrel compared with each drug
alone.
Methods—In a case-crossover study, 31 patients with previous atherothrombotic or lacunar stroke who were treated with
aspirin (100 to 300 mg/d) received clopidogrel (75 mg/d) and both aspirin and clopidogrel for 4 weeks. Platelet function
in whole blood was studied after each treatment period and in healthy control subjects to assess activation-dependent
antigens CD62p and CD63 by flow cytometry and collagen/epinephrine (CEPI-CT) and collagen/ADP (CADP-CT)
closure times with the platelet function analyzer PFA-100, which investigates platelet-related function under shear
stress.
Results—CD62p expression and CD63 expression were not different under the 3 treatment regimens. CD63 but not CD62p
expression was lower in control subjects than in stroke patients regardless of the antiplatelet treatment (P⬍0.05).
CEPI-CT was prolonged under aspirin and aspirin plus clopidogrel compared with clopidogrel monotherapy
(P⬍0.0001). CADP-CT was longer under combination therapy than under aspirin (P⫽0.0009) or clopidogrel
(P⫽0.0074) or in control subjects (P⫽0.0010), mainly because of strong prolongation in a patient subgroup (28%).
Conclusions—CD63 expression reflecting the release of platelet lysosomes is consistently increased after stroke and
incompletely suppressed by treatment with aspirin, clopidogrel, or both. The strong prolongation of CADP-CT under
combined aspirin and clopidogrel in a patient subgroup may indicate a lower risk of thrombosis but also a higher risk
of hemorrhage. The predictive value of platelet activation parameters requires investigation in prospective studies.
(Stroke. 2003;34:849-855.)
Key Words: inflammation 䡲 platelet aggregation inhibitors 䡲 platelets 䡲 secondary prevention
A
ntiplatelet drugs are the treatment of choice for secondary prevention after cerebral ischemia of noncardioembolic origin. However, the efficacy of presently available
drugs is limited. Compared with placebo, aspirin reduced the
relative risk of vascular events by 13% according to two
meta-analyses1,2 and stroke risk by 18% in the European
Stroke Prevention Study 2.3 Under clopidogrel, the relative
risk of a compound outcome cluster of stroke, myocardial
infarction, and vascular death was marginally, although
significantly, lower than under aspirin (⫺8.4%), whereas the
risk reduction for stroke alone was not significant.4 Aspirin
inhibits platelet aggregation by inhibition of cyclooxygenase,
whereas clopidogrel reduces platelet activation via ADP
receptor-dependent pathways. On the basis of these different
modes of action, it is an attractive concept that the combination of both drugs may have additive effects on platelet
See Editorial Comment, page 854
inhibition. The combination of aspirin and clopidogrel or
ticlopidine was shown to be a successful treatment strategy
after coronary stenting and in unstable angina pectoris5,6 and
is currently being tested in a large, randomized, placebocontrolled study on secondary prevention after stroke. However, it is unknown at present whether the combination of
aspirin and clopidogrel leads to reduced platelet activation in
patients at high risk for ischemic stroke or other vascular
events.
We performed a case-crossover study to test the hypothesis
that in patients with a history of stroke, platelet activation
parameters are reduced under combination therapy with
aspirin and clopidogrel compared with either therapy alone.
We also investigated inflammatory parameters under treat-
Received September 9, 2002; final revision received September 30, 2002; accepted November 5, 2003.
From the Neurology Department, University of Heidelberg, Heidelberg, and Center for Laboratory Medicine, Microbiology, Transfusion Medicine,
Klinikum Karlsruhe (A.R.), Karlsruhe, Germany.
Correspondence to Armin J Grau, MD, PhD, Neurologische Klinik, Städtisches Klinikum Ludwigshafen a.Rh., Bremserstr. 79, D-67063 Ludwigshafen,
Germany. E-mail [email protected]
© 2003 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000064326.65899.AC
849
850
Stroke
April 2003
ment with aspirin, clopidogrel, and both because aspirin but
not clopidogrel possesses anti-inflammatory potential and
inflammatory indexes were shown to predict the risk of
vascular events.7 Furthermore, we compared platelet activation and inflammatory parameters in patients with previous
stroke and healthy control subjects. Platelet function in whole
blood was assessed by flow cytometry and by the platelet
function analyzer (PFA-100).
Subjects and Methods
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
In the present study, patients who had suffered ischemic stroke and
were treated with aspirin for secondary prevention received clopidogrel alone or a combination of clopidogrel and aspirin for 4 weeks.
Platelet function was determined at the end of each treatment period
and included flow cytometric assessment of the expression of
p-selectin (CD62p) or lysosome-associated membrane protein
(CD63) and studies with the PFA-100 (Dade Behring) assessing
collagen/epinephrine (CEPI) and collagen/ADP (CADP) closure
time (CT).
Patients between 18 and 80 years of age were eligible for study
participation if they had suffered ischemic stroke between 6 months
and 2 years ago, if the index stroke was caused by large-artery
atherosclerosis or cerebral microangiography, and if they had received aspirin (100 to 300 mg/d) for at least 3 months. Large-artery
atherosclerosis was diagnosed by duplex studies or angiography
showing stenoses of brain-supplying arteries of ⱖ50% diameter
reduction and a typical morphology for atherosclerotic lesions.
Cerebral microangiopathy was diagnosed if neuroimaging showed
ischemic lesions of ⬍1.5 cm and clinical symptoms in accordance
with typical lacunar syndromes (pure motor stroke, pure sensory
stroke, sensorimotor stroke, dysarthria clumsy hand syndrome,
ataxic hemiparesis).
Exclusion criteria included the presence of concurrent stroke
etiologies, operation or angioplasty for symptomatic stenosis performed within 3 months or planned for the future, pregnancy or
childbirth, known allergy against or previous treatment with clopidogrel, hemorrhagic diathesis, history of gastrointestinal or other
bleedings, history of drug-induced disorders, poorly controlled
hypertension (ⱖ180/110 mm Hg), trauma or surgery within the last
3 months or any surgery planned for the next 3 months, cancer,
rheumatic diseases, intake of anticoagulants or nonsteroidal antiphlogistic drugs, severe liver (international normalized ratio ⱖ1.5) or
renal (creatinine ⱖ1.5 mg/dL) insufficiency, drug or alcohol abuse,
any blood cell abnormalities (anemia, leukopenia, thrombocytopenia, or thrombocytosis), and epileptic seizures.
Thirty-five patients were identified by chart review and were
willing to participate. Of these, 4 had to be excluded because of
anemia, thrombocytosis, severe hepatic disturbance and suspected
alcohol abuse, and recent pretreatment with clopidogrel (n⫽1,
respectively). Therefore, 31 patients were entered into the study, 20
with stroke caused by large-artery atherosclerosis and 11 with
lacunar strokes. The patients took 300 (n⫽26), 250 (n⫽3), 200
(n⫽1), and 100 (n⫽1) mg/d aspirin. Demographic data of the
patients are given in Table 1. All patients gave written, informed
consent. The study protocol was approved by the local Human
Subjects Committee. According to legal requirements, patients
received special insurance.
Patients were asked to take clopidogrel (75 mg/d) instead of
aspirin for 4 weeks and both aspirin (300 mg/d) and clopidogrel (75
mg/d) for another 4 weeks. In the first part of the study, clopidogrel
was administered, followed by the combination therapy. In the
second part, 6 patients were randomly assigned to first receive the
combination therapy and then the clopidogrel monotherapy. After
each treatment phase, the patients visited the clinic, were asked about
potential side effects, and received a physical examination. Laboratory examinations—including whole blood count, liver enzymes,
electrolytes, urea, creatinine, C-reactive protein (CRP), fibrinogen,
and partial thromboplastin time—were performed at study entry and
after both treatment periods. Clopidogrel was made available by
TABLE 1.
Demographic Data in Patients and Control Subjects
Patients
(n⫽31)
Control Subjects
(n⫽21)
Age, y (mean⫾SD)
63.0⫾9.4
62.8⫾10.4
Female sex
11 (35.5%)
7 (33.3%)
Hypertension
22 (71.0%)
䡠䡠䡠
Diabetes mellitus
8 (25.8%)
䡠䡠䡠
Current smoking
12 (38.7%)
䡠䡠䡠
Hypercholesterolemia
21 (67.7%)
䡠䡠䡠
Coronary heart disease
5 (16.1%)
䡠䡠䡠
Peripheral arterial disease
4 (12.9%)
䡠䡠䡠
Parameter
Sanofi-Synthelabo Inc. Intake of study drugs was controlled by
counting of delivered tablets.
For the control group, we investigated 21 presumably healthy
subjects who had none of the above exclusion criteria and no
vascular risk factor or arterial vascular disease. None of the control
subjects took aspirin, clopidogrel, or other platelet-inhibiting drugs.
Control subjects were matched to patients for age and sex
distribution.
Assessment of Platelet Function
Venipuncture of forearm veins was performed under minimal stasis,
with the first few milliliters rejected. Flow cytometry was done
according to the consensus protocol for flow cytometric characterization of platelet function as reported recently.8,9 Briefly, peripheral
venous blood was collected into an aldehyde-based fixation solution
that blocks metabolic processes within milliseconds and preserves
CD62p and CD63 for at least 24 hours at room temperature. The
samples were incubated with saturating concentrations of FITClabeled antibodies (Beckman Coulter) against CD62p (clone
CLBThromb6) or CD63 (Clone CLBGran12) and with PE-labeled
antibodies against CD41a (clone P2) for 15 minutes at room
temperature in the dark. As control experiments, platelets were
incubated with FITC-coupled unspecific mouse IgG1 (Beckman
Coulter) with the same fluorochrome-to-protein ratio and concentration as the specific IgG. These controls yielded the same fluorescence as platelets whose binding of FITC-labeled specific antibodies
was blocked by an excess of unlabeled antibodies of the same clone.
After immunolabeling, the samples were analyzed by FACScan
(Becton Dickinson). Forward light scatter and expression of CD41a
were used for platelet identification. Platelet-bound anti-CD62p or
anti-CD63 antibodies were then determined by analyzing 5000
platelets for FITC fluorescence. Results were expressed as a percentage of antibody-positive platelets, defined as those with a
fluorescence intensity exceeding that of 98% to 99% of the control
platelets. The intra-test variability was low (mean coefficient of
variation, 0.023 for CD62p; 0.039 for CD63).
For PFA-100 studies, blood was collected into tubes containing
3.2% buffered citrate (Sarstedt), kept at room temperature, and
analyzed within 3 hours after venipuncture. The PFA-100 simulates
an injured blood vessel by aspirating blood through disposable test
cartridges coated with collagen and either ADP (50 ␮g) or epinephrine (10 ␮g). Whole blood (900 ␮L) flows under constant high shear
rates (5000 to 6000/s) through a capillary and a microscopic aperture
(147 ␮m) cut into the coated membrane. When blood comes into
contact with the membrane, platelets adhere, aggregate, and form a
plug that occludes the aperture with consecutive cessation of blood
flow. The time required to occlude the aperture is automatically
reported as the CT. Measurements are terminated after a maximum
of 300 seconds. Aspirin usually prolongs the CEPI-CT but does not
extend the CADP-CT. CADP cartridges detect platelet dysfunction
not mediated by aspirin.10,11 On the basis of a recent review10 and our
own control group, CEPI-CT ⬎193 seconds and CADP-CT ⬎133
seconds were considered prolonged. Measurements were performed
in duplicate. The intratest variability was 0.050 for CEPI and 0.085
Grau et al
Antiplatelet Drugs and Platelet Function
851
TABLE 2. Platelet Activation Parameters Under Different Treatment Regimens in
Patients With a History of Ischemic Stroke
Treatment Regimens
Parameter
CD62p-positive platelets (%)
CD63-positive platelets (%)
Collagen-epinephrine closure time(s)
Collagen-ADP closure time(s)
Aspirin
Clopidogrel Aspirin⫹Clopidogrel
2.3
2.5
3.3
(1.1–3.9)
(1.3–4.0)
(1.5–3.9)
2.8
2.5
1.8
(0.9–4.5)
(0.9–4.0)
(0.9–3.9)
⬎300
120
⬎300
(270–⬎300)
(106–148)
(⬎300–⬎300)
86
89
100
(72–94)
(76–105)
(88–196)
P Value*
0.043
0.87
⬍0.0001
⬍0.0001
Data are given as median and 25 to 75 percentile.
*P values by Friedmann test; in order to adjust for multiple tests, P values ⬍0.0125 were
considered as significant.
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for CADP cartridges. CRP was determined by immunoturbidimetric
assay (Aeroset; threshold of detectability: 3 mg/L). Fibrinogen was
measured by functional coagulation testing (derived fibrinogen) with
Recombiplastin (Instrumentation Laboratory) as reagent. Leukocyte
counts were assessed by Coulter counter analysis.
Data are presented as mean and SD or median and percentiles as
appropriate. We used the Friedman test for intraindividual comparisons between the 3 treatment regimens. For comparisons of the 4
platelet function parameters between treatment groups, differences
were considered significant at Pⱕ0.0125. The Wilcoxon signed-rank
test was applied for further analyses of treatment regimens if the
Friedman test yielded significant results. The Mann-Whitney U test
was used to compare patients and control subjects; the Spearman
rank correlation coefficient was used to correlate parameters; and
Fisher’s exact test was used for analyses of categorical variables.
Analysis of variance (ANOVA) was applied to analyze the influence
of various factors on a parameter. Variables without normal distribution received logarithmic transformation before ANOVA. We
used the software package SAS (version 8.02) for the analyses.
Results
None of the 31 stroke patients had symptoms attributable to
cerebral ischemia or developed any cerebral or systemic
bleeding during the study. One patient withdrew from the
study during therapy with aspirin plus clopidogrel because of
sudden severe shoulder pain. Bleeding into the shoulder joint
was excluded by MRI. One patient was excluded shortly after
initiation of the combination therapy because of a first
epileptic seizure. Therefore, 29 patients completed the entire
study.
Under the 3 treatment regimens, the expression of CD62p
and CD63 was not significantly different (Table 2). In
contrast, CEPI-CT and CADP-CT showed significant differences between treatments (P⬍0.0001, respectively; Table 2,
Figures 1 and 2). Regarding CEPI-CT, differences were due
to prolonged clot formation under aspirin (P⬍0.0001) and
aspirin plus clopidogrel (P⬍0.0001) compared with clopidogrel monotherapy. The CEPI-CT was maximally inhibited
(⬎300 seconds) in 23 of 31 patients (74.2%) under aspirin
and in 26 of 29 patients (89.7%) under both aspirin and
clopidogrel (P⫽0.18). Normal values (ⱕ193 seconds) were
found in 5 of 31 patients (16.1%) despite medication with
aspirin and in 1 of 29 patients under the combination therapy
(3.4%; P⫽0.20). The CADP-CT was prolonged under the
combination therapy compared with monotherapy with either
aspirin (P⫽0.0009) or clopidogrel (P⫽0.0074). Results under
aspirin and clopidogrel were not different (P⫽0.51). The
CADP-CT was outside the normal range (⬎133 seconds) in 2
patients (6.5%) under clopidogrel but in 8 patients (27.6%)
under the combination therapy (P⫽0.039). Complete inhibi-
Figure 1. CEPI-CT assessed with the
PFA-100 under treatment with aspirin,
clopidogrel, or both.
852
Stroke
April 2003
Figure 2. CADP-CT assessed with the
PFA-100 under treatment with aspirin,
clopidogrel, or both.
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tion of CADP-CT (⬎300 seconds) occurred in 7 patients
under the combination therapy but in 0 of the patients under
clopidogrel (P⫽0.004). CEPI-CT and CADP-CT were correlated with each other under clopidogrel (R⫽0.32, P⫽0.006)
and the combination therapy (R⫽0.54, P⬍0.001) but not
under aspirin monotherapy (R⫽0.17, P⫽0.15). CD62p and
CD63 expression showed significant correlations under all
medications (R⬎0.53, P⬍0.001). CEPI-CT and CADP-CT
on one side and CD62p and CD63 expression on the other
side were not significantly correlated with each other.
Platelet activation parameters did not differ between patients who took 300 mg of aspirin and those who took a
smaller doses (P⬎0.30) or between patients with lacunar
stroke and patients with stroke resulting from large-artery
atherosclerosis (P⬎0.25). Smoking, hypertension, hypercholesterolemia, diabetes mellitus, and the presence of coronary
heart disease and/or peripheral arterial disease did neither
influence CEPI-CT, CADP-CT, or CD63 expression. Diabetes mellitus (P⫽0.003) and hypercholesterolemia (P⫽0.010)
were associated with higher CD62p expression (ANOVA).
Leukocyte counts, platelet counts, and CRP and fibrinogen
levels were not different under the 3 treatment regimens
(P⬎0.10).
Control subjects had lower CD63 expression (median,
1.0%; 25th and 75th quartiles, 0.2% and 2.8%) than patients
under all 3 therapeutic regimens (P⬍0.05). In contrast, there
was no difference regarding CD62p expression (control
subjects: median, 2.3%; 25th and 75th quartiles, 0.7% and
3.6%). The CEPI-CT was similar in patients under treatment
with clopidogrel and control subjects (median, 117 seconds;
25th and 75th quartiles, 108 and 127 seconds). The
CADP-CT (median, 83; 25th and 75th quartiles, 81 and 93
seconds) was not different from patients treated with aspirin
or clopidogrel; however, it was shorter than in patients under
aspirin plus clopidogrel (P⫽0.001). Leukocyte counts were
5.17⫾1.40⫻109/L compared with 6.77⫾1.70x109/L (P⫽0.003),
and fibrinogen levels tended to be lower in control subjects
than in patients under therapy with aspirin (2.97⫾0.65 versus
3.50⫾1.06 g/L; P⫽0.069), whereas no difference existed
regarding platelet counts and CRP levels.
Discussion
Combination therapy with aspirin and clopidogrel was well
tolerated and safe in our patients during the short treatment
period of 4 weeks. Both serious adverse events that occurred
were not causally related to the study medication. The main
hypothesis of the present investigation was that in patients
after stroke, aspirin plus clopidogrel may reduce platelet
activation to below the level reached with each single agent
alone. This study shows that the CADP-CT assessed with the
PFA-100 was prolonged under combination therapy, whereas
the 3 other platelet activation markers were not significantly
changed.
We chose flow cytometry and the PFA-100 because both
methods allow platelet function analysis in whole blood.12
Aggregometry with platelet-rich plasma and measurement of
platelet release products have the problem of potential artifactual platelet activation during blood sampling, and release
products may lack sensitivity because of dilution effects in
plasma. Flow cytometric analysis of platelets is a relatively
new and powerful technique that has been developing into a
useful clinical tool.12 It has the advantage of little artifactual
in vitro activation, particularly because blood fixation was
used in our study. Previous studies showed that CD62p and
CD63 expression by platelets is increased in acute ischemic
stroke.9,13 CD62p (p-selectin) is a glycoprotein localized on
the ␣-granule membrane that is rapidly translocated to the
cell surface after stimulation and mediates the adhesion of
platelets to leukocytes. P-selectin upregulates tissue factor in
monocytes, promotes fibrin deposition, and leads to leukocyte accumulation in areas of vascular injury associated with
thrombosis and inflammation.14,15 CD63 is expressed on the
platelet surface after the release of lysosomes. It may protect
the plasma membrane against degradation by lysosomal
proteins; however, its biological function is not sufficiently
understood.16 In a baboon model, degranulated platelets shed
CD62p from the cell surface but continued to circulate and
function.17 Such shedding after cell activation may explain
why CD62p expression was not increased in our patients in
the chronic stage after stroke. In contrast, CD63, for which
shedding is not known to occur, was expressed more strongly
in patients than control subjects.
Grau et al
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In our study, neither clopidogrel nor the combination of
aspirin and clopidogrel led to lower CD63 expression than
therapy with aspirin. These results indicate that currently
available antiplatelet drugs, even if combined, do not inhibit
the release of platelet lysosomes in vivo, a mechanism that
requires strong platelet activation.18 In patients with previous
myocardial infarction, clopidogrel with and without aspirin
suppressed CD62p and CD63 expression after stimulation
with ADP or thrombin.19 Assessment of drug effects on
platelet reactivity in vitro was not performed in our study but
may be a meritorious focus of future research.
The PFA-100 assesses platelet-related function, in particular adhesion and aggregation, under shear stress. Its advantages are ease of operation, rapidity, and comparability
between different laboratories. It was shown to reliably
monitor platelet function under treatment with aspirin and
GPIIb/IIIa receptor antagonists and to be useful in screening
for congenital and acquired platelet dysfunction.10 –12,20 –23 It
is not sensitive to heparin or defects in coagulation proteins,
eg, hemophilia A.12,21 To the best of our knowledge, the
PFA-100 has not been applied in clinical stroke. As expected,
the CEPI-CT was strongly prolonged under treatment with
aspirin compared with clopidogrel or the control group.
Despite intake of aspirin, 16.1% of our stroke patients had
normal CEPI-CT values. In a PFA-100 – based study using
the same cutoff value, the rate of “aspirin resistance” was
9.5% in patients with stable cardiovascular disease receiving
325 mg/d aspirin.11 Measuring platelet aggregation in platelet-rich plasma, Helgason et al24 detected aspirin resistance in
20.6% of their stroke patients taking ⱕ325 mg/d aspirin.
Aspirin resistance defined by in vivo thromboxane biosynthesis was recently shown to predict ischemic events.25
Patients with normal CEPI-CT values despite aspirin medication may be at increased risk of ischemic events; however,
prospective data based on the PFA-100 are not yet available.
Clopidogrel acts via inhibition of ADP-induced platelet
activation. In accordance with previous studies,9 treatment
with clopidogrel was not reflected by prolonged CADP-CT.
This shows that the current setup of the PFA-100, mainly the
high ADP concentration in CADP cartridges, is not suitable
for monitoring therapy with clopidogrel or ticlopidine. Most
interestingly, CADP-CT was longer under combined aspirin
and clopidogrel than under either monotherapy. In patients
undergoing percutaneous transluminal coronary angioplasty,
the combination of ticlopidine and aspirin led to a nonsignificant increase in CADP-CT compared with aspirin alone21 or
was associated with normal CADP-CT23; however, ticlopidine medication was shorter than in our study. The combination therapy was associated with a heterogenous response
among our patients. About one fourth of the subjects showed
a very strong inhibitory effect, whereas in the other patients,
the difference from both monotherapies was not significant.
In the subjects who showed a strong inhibition, the combination therapy could be associated with good protection
against ischemic events but also with an increased bleeding
risk. It is unknown why some individuals react with a strong
response to combined aspirin and clopidogrel and others do
not. It was recently shown that closure times in the PFA-100
are highly dependent on the von Willebrand factor.26 Differ-
Antiplatelet Drugs and Platelet Function
853
ences in von Willebrand factor levels could possibly explain
variations between patients. It is a limitation of our study that
the von Willebrand factor was not assessed. Furthermore, the
nonrandomized, non–placebo-controlled sequential treatment
design could be regarded as a limitation, but it is unlikely that
it affects the data interpretation. Similarly, it is unlikely that
effects seen under the combination therapy represent late
treatment effects of clopidogrel rather than effects of the
combination therapy.
Aspirin but not clopidogrel possesses an anti-inflammatory
mode of action; however, the 3 inflammatory parameters
assessed were not different under the medication with or
without aspirin. In accordance with previous studies,27 leukocyte counts and fibrinogen levels were higher after stroke
than in healthy control subjects, although the difference was
not significant for fibrinogen. We did not use a highsensitivity test for CRP assessment and may therefore have
overlooked small differences in CRP.
From the results of this study, it appears meritorious to
investigate in larger studies whether the CADP-CT is a
predictor of ischemic events or bleeding complications under
treatment with combined aspirin and clopidogrel and whether
normal CEPI-CT predicts an increased rate of ischemic
events under monotherapy with aspirin. CD62p and CD63
expression may not be suitable for monitoring therapy with
aspirin, clopidogrel, or both after stroke. Although not influenced by the antiplatelet agents studied here, CD63 expression could still be a predictor of the risk of ischemic events,
an issue that requires investigation in a larger prospective
study.
Acknowledgments
This study was supported by a grant from Sanofi-Synthelabo Inc.
Dade-Behring Inc financially supported our measurements with the
PFA-100. We wish to thank Dagmar Oehmichen for her valuable
technical assistance.
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Clevers HC. CD63 antigen, a novel lysosomal membrane glycoprotein,
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19. Mosfegh K, Redondo M, Julmy F, Wuillemin WA, Gebauer MU,
Haeberli A, Meyer BJ. Antiplatelet effects of clopidogrel compared with
aspirin after myocardial infarction: enhanced inhibitory effects of combination therapy. J Am Coll Cardiol. 2000;36:699 –705.
20. Mammen EF, Comp PC, Gosselin R, Greenberg C, Hoots WK, Kessler
CM, Larkin EC, Liles D, Nugent DJ. PFA-100 system: a new method for
assessment of platelet dysfunction. Semin Thromb Hemost. 1998;24:
195–202.
21. Kottke-Marchant K, Powers JB, Brooks L, Kundu S, Christie DJ. The
effect of antiplatelet drugs, heparin, and preanalytical variables on platelet
function detected by the platelet function analyzer (PFA 100). Clin Appl
Thromb Hemost. 1999;5:122–130.
22. Homoncik M, Jilma B, Hergovich N, Stohlawetz P, Panzer S, Speiser W.
Monitoring of aspirin (ASA) pharmacodynamics with the platelet
function analyzer PFA-100. Thromb Haemost. 2000;83:316 –321.
23. Hézard N, Metz D, Nazeyrollas P, Droulle C, Elaerts J, Potron G, Nguyen
P. Use of the PFA-100 apparatus to assess platelet function in patients
undergoing PTCA during and after infusion of c7E3 Fab in the presence
of other antiplatelet agents. Thromb Haemost. 2000;83:540 –544.
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McClelland TJ, Brace LD. Aspirin response and failure in cerebral
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26. Homoncik M, Blann AD, Hollenstein U, Pernerstorfer T, Eichler HG,
Jilma B. Systemic inflammation increases shear stress-induced platelet
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Editorial Comment
Synergistic Antiplatelet Effects of Clopidogrel and Aspirin Detected With
the PFA-100 in Stroke Patients
Currently, it is neither standard nor clinical practice to
measure platelet function under treatment with antiplatelet
drugs. This practice follows a “one size fits all” principle,
which is not likely to be correct. Historically, platelet function tests, mainly aggregometry, have been cumbersome and
time consuming and may have lacked reproducibility if they
were not carried out by highly trained personnel. Aggregometric measurements have another major disadvantage: they
evaluate platelet function at low shear rates, which is in stark
contrast to the high shear rates prevailing at sites of arterial
stenosis. Thus, their relevance for predicting platelet behavior
in stenotic arteries remains to be determined.
A number of point-of-care platelet function tests have
recently become available.1 One of these novel test systems is
the PFA-100 platelet function analyzer, which is designed to
overcome many of the drawbacks of aggregometry. In particular, the PFA-100 is a high-shear system2 that has successfully been applied to detect and monitor the effects of various
antiplatelet drugs,3–5 most importantly aspirin.
Until recently, the PFA-100 was considered insensitive to
the effects of thienopyridines such as clopidogrel,2,6 whereas
the in vivo bleeding time could detect clopidogrel effects.7
In this issue of Stroke, Grau et al report a nonrandomized,
crossover trial in which 31 patients with previous stroke
received aspirin, clopidogrel, and both in a sequential manner. Consistent with previous reports, aspirin maximally
prolonged collagen/epinephrine-induced closure times
(CEPI-CT ⬎300 seconds) in 74% of patients, while one
fourth of patients were more or less resistant to aspirin
treatment. However, the study is underpowered to allow any
conclusions on dose-effect relationships because most patients received 300 mg aspirin. Although the authors conclude
that the CEPI-CT was insensitive to clopidogrel effects, a
subgroup of 4 to 6 patients appeared to have prolonged
CEPI-CT values under clopidogrel. Along similar lines,
combination of both drugs enhanced “aspirin” responder rates
to 90%.
Neither aspirin nor clopidogrel alone prolonged collagen/
ADP-induced closure time (CADP-CT), whereas the combination of both significantly prolonged CADP-CT in 25% of
patients; most patients displayed even maximal CADP-CT
values (⬎300 seconds). This degree of platelet dysfunction is
typically observed only in patients with severe thrombocytopenia,8 von Willebrand disease,6 or a congenital deficiency in
Grau et al
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platelet glycoprotein GPIb or GPIIb/IIIa9 or in those under
treatment with infusable GPIIb/IIIa antagonists.10 Thus,
CADP-CT values ⬎300 seconds can be regarded as clinically
relevant, and it is conceivable that this degree of platelet
inhibition translates into greater clinical benefit in terms of
reduction of vascular accidents but also poses a greater risk
for bleeding complications. However, response patterns were
not continuous over the range of possible CADP-CT values
but rather appeared to follow an “all or none” principle
(normal or ⬎300 seconds). Hence, it is tempting to speculate
that 1 single hereditary or environmental factor could largely
determine the response to combined treatment with aspirin
and clopidogrel in this test system.
Although provocative, the small sample size and nonrandomized design are evident limitations of the present trial.
Thus, confirmation of these interesting findings in larger
randomized trials and other patient populations is desirable.
Such trials should ideally try to identify the predictive value
of the PFA-100 for either vascular or bleeding complications
in these patients and provide some mechanistic insight into or
explanations for the variability in response.
Bernd Jilma, MD, Guest Editor
Department of Clinical Pharmacology
Vienna University
Vienna, Austria
Antiplatelet Drugs and Platelet Function
855
References
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Platelet Function Under Aspirin, Clopidogrel, and Both After Ischemic Stroke: A
Case-Crossover Study
Armin J. Grau, Sven Reiners, Christoph Lichy, Florian Buggle and Andreas Ruf
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Stroke. 2003;34:849-854; originally published online March 13, 2003;
doi: 10.1161/01.STR.0000064326.65899.AC
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