□ ORIGINAL ARTICLE □

Dual Antiplatelet Therapy Clopidogrel with Low-dose

Cilostazol Intensifies Platelet Inhibition in Patients
with Ischemic Stroke

Hajime Maruyama, Takuya Fukuoka, Ichiro Deguchi, Yasuko Ohe, Harumitsu Nagoya,
Yuji Kato, Yohsuke Horiuchi, Takeshi Hayashi and Norio Tanahashi

Abstract
Objective We previously reported that the antiplatelet action is intensified with combined use of clopido-
grel and cilostazol in ischemic stroke patients using the VerifyNow P2Y12 Assay. In this study, the relation-
ship between the cilostazol dose and the platelet function achieved with combination therapy was investi-
gated.
Methods The subjects included 231 patients with noncardiogenic ischemic stroke treated at our hospital (18
patients treated with a combination of clopidogrel (75 mg) and cilostazol (100 mg), 52 patients treated with a
combination of clopidogrel (75 mg) and cilostazol (200 mg), 126 patients treated with clopidogrel (75 mg)
alone and 35 patients treated with cilostazol (200 mg) alone). The platelet function achieved with 20 μM of
adenosine diphosphate was measured using the VerifyNow P2Y12 Assay. Clopidogrel resistance was defined
as P2Y12 Reaction Units (PRU) >230 and/or % inhibition <20%.
Results The PRU was >230 in 32 patients (25.4%) receiving clopidogrel alone, one patient (5.6%) receiv-
ing combination therapy with cilostazol (100 mg) and one patient (1.9%) receiving combination therapy with
cilostazol (200 mg). The rate of PRU >230 was significantly lower in both of the cilostazol combination
groups than in the clopidogrel alone group. The percent inhibition was <20% in 41 patients (32.5%) receiv-
ing clopidogrel alone, one patient (5.6%) receiving a combination with cilostazol (100 mg) and one patient
(1.9%) receiving a combination with cilostazol (200 mg). The rate of % inhibition <20% was significantly
lower in both of the cilostazol combination groups than in the clopidogrel alone group.
Conclusion Clopidogrel resistance was clearly decreased with combination clopidogrel (75 mg) and low-
dose (100 mg) cilostazol therapy. The use of combination therapy with clopidogrel and low-dose cilostazol
may be one means of overcoming clopidogrel resistance.

Key words: clopidogrel resistance, cilostazol, ischemic stroke, VerifyNow P2Y12 Assay, dual antiplatelet
therapy

(Intern Med 52: 1043-1047, 2013)

(DOI: 10.2169/internalmedicine.52.9550)

(Accumetrics Inc., San Diego, CA, USA) and reported that

Introduction
There are individual differences in the response to clopi-
dogrel, which is affected by the presence or absence of drug
metabolizing enzyme CYP2C19 gene polymorphism (1-5).
We previously measured the platelet function ability of
ischemic stroke patients using the VerifyNow P2Y12 Assay
clopidogrel resistance was present in 29% of the pa-
tients (6). We also reported that the antiplatelet action is
strengthened with combined use of clopidogrel and cilosta-
zol (6). However, we did not investigate the dose of cilosta-
zol administered in combination therapy with clopidogrel
and cilostazol. In this study, a greater number of patients re-
ceiving combination therapy with clopidogrel and cilostazol

Department of Neurology and Cerebrovascular Medicine, Saitama Medical University International Medical Center, Japan
Received for publication December 18, 2012; Accepted for publication January 22, 2013
Correspondence to Dr. Hajime Maruyama, hmaruyam@saitama-med.ac.jp

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 Intern Med 52: 1043-1047, 2013
Table. Clinical Stroke Type and Risk Factors
Clopidogrel 75mg
Cilostazol 100mg
(n=18)
Clinical stroke type
Atherothrombotic infarction, n (%) 14 (77.8)
Lacunar infarction, n (%) 2 (11.1)
Transient ischemic attack, n (%) 2 (11.1)
Risk factors
Diabetes mellitus, n (%) 7 (38.9)
Hypertension, n (%) 13 (72.2)
Dyslipidemia, n (%) 15 (83.3)
Smoking, n (%) 8 (44.4)
was included and the relationship between the dose of
cilostazol used in combination therapy and the platelet func-
tion was therefore investigated.
Materials and Methods
The subjects consisted of 231 noncardiogenic ischemic
stroke patients treated at our hospital between October 2009
and September 2012 (174 men, 57 women, mean age 67.0±
9.5 years, 161 patients with atherothrombotic infarction, 51
patients with lacunar infarction and 19 patients with tran-
sient ischemic attack). The treatment included combined
clopidogrel (75 mg) and cilostazol (100 mg) in 18 patients
(16 men, two women, mean age 69.1±8.2 years), combined
clopidogrel (75 mg) and cilostazol (200 mg) in 52 patients
(38 men, 14 women, mean age 66.9±7.8 years), clopidogrel
(75 mg) alone in 126 patients (95 men, 31 women, mean
age 66.8±9.6 years) and cilostazol (200 mg) alone in 35 pa-
tients (25 men, 10 women, mean age 66.6±11.9 years). The
recommended dose of cilostazol in Japan is 200 mg. The
reason for selecting clopidogrel at a dose of 75 mg and
cilostazol at a dose of 100 mg was that, in seven of 18 pa-
tients, headaches and tachycardia occurred with the admini-
stration of 200 mg of cilostazol. The dose was therefore re-
duced to 100 mg. In the 11 other patients, a dose of 100 mg
was selected by the primary physician out of concern for
headaches or tachycardia. All patients received an antiplate-
let agent in a given oral dose for seven days or more, and
the number of days from onset until measurement of the
platelet function was !14 days. The clinical stroke type and
risk factors in each patient group are shown in the Table.
The cilostazol combination groups exhibited a higher fre-
quency of atherothrombotic infarction and a lower frequency
of lacunar infarction than the clopidogrel alone group or the
cilostazol alone group. The frequencies of risk factors were
not clearly different between the two groups receiving com-
bination cilostazol. The frequencies of diabetes, hypertension
and dyslipidemia were lower in the cilostazol alone group
than in the other three groups.
Blood (1.8 mL) was collected into a vacuum blood col-
lection tube containing 0.2 mL of 3.2% sodium citrate using
a 21-G blood collection needle. The platelet function was
measured with 20 μM adenosine diphosphate (ADP) using
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DOI: 10.2169/internalmedicine.52.9550
Clopidogrel 75mg
Clopidogrel 75mg Cilostazol 200mg
Cilostazol 200mg (n=126) (n=35)
(n=52)
46 (88.5) 83 (65.9) 18 (51.4)
4 (7.7) 33 (26.2) 12 (34.3)
2 (3.8) 10 (7.9) 5 (14.3)
21 (40.4) 51 (40.5) 8 (22.9)
38 (73.1) 84 (66.7) 17 (48.6)
42 (80.8) 90 (71.4) 16 (45.7)
29 (55.8) 60 (47.6) 15 (42.9)
the VerifyNow P2Y12 Assay between 10 minutes and four
hours after collection. The platelet function was then com-
pared among the clopidogrel (75 mg) and cilostazol (100
mg) combination group, the clopidogrel (75 mg) and cilosta-
zol (200 mg) combination group, the clopidogrel (75 mg)
alone group and the cilostazol (200 mg) alone group. Next,
the rate of clopidogrel resistance was compared among the
clopidogrel (75 mg) and cilostazol (100 mg) combination
group, the clopidogrel (75 mg) and cilostazol (200 mg)
combination group and the clopidogrel (75 mg) alone group.
With reference to previous reports, clopidogrel resistance
was defined in this study as P2Y12 Reaction Units (PRU) >
230 (7-9) and/or % inhibition <20% (10-12).
VerifyNow P2Y12 Assay
The VerifyNow P2Y12 Assay is a system to measure P2Y
12 receptor inhibition of platelets with whole blood speci-
mens. This system measures the platelet function based on
the fibrinogen binding capacity of activated platelets. There
are reaction chambers with ADP 20 μM + prostaglandin E1
22 nM and isothrombin receptor activating peptide (iso-
TRAP) as platelet activating substances in the measurement
cartridge. Fibrinogen is placed into each chamber. The fi-
brinogen aggregates in whole blood are measured in propor-
tion to the number of glycoprotein (GP) IIb/IIIa receptors on
activated platelets. Changes in platelet activation are de-
tected by monitoring changes in the light transmittance pro-
duced by aggregate formation. The extent of aggregation is
expressed by the PRU and % inhibition. The PRU is the
amount of aggregation due specifically to ADP in platelet
P2Y12 receptors, calculated from the speed and extent of
platelet aggregation in the reaction chamber containing ADP.
The percent inhibition is the percent change from the base-
line aggregation ability, calculated from the PRU results and
baseline results (BASE). BASE is an independently meas-
ured value based on the speed and extent of platelet aggre-
gation due to thrombin receptors, especially protease-
activated receptor-1, 4 (PAR-1, 4) receptors. To activate
platelets, iso-TRAP and PAR-4 activating peptide (PAR-4
AP) are incorporated into the reaction chamber for BASE
measurement. The percent inhibition is obtained from the
following formula: % inhibition=100×(BASE-PRU)/BASE.
 Intern Med 52: 1043-1047, 2013 DOI: 10.2169/internalmedicine.52.9550

* * * Ɖ <0.001
400

P2Y12 ReacƟŽn Units (PRU)

300

230
200

100

0
ClŽƉiĚŽgrel 75mg ClŽƉiĚŽgrel 75mg ClŽƉiĚŽgrel 75mg CiůŽstazŽl 200mg
CiůŽstazŽl 100mg CiůŽstazŽl 200mg

Figure 1. P2Y12 Reaction Units (PRU) in each treatment group. The PRU was significantly lower
in the three groups that received clopidogrel than in the cilostazol alone group (p<0.001). In addi-
tion, the PRU was significantly lower in the clopidogrel (75 mg) and cilostazol (200 mg) combination
group than in the clopidogrel (75 mg) alone group (p<0.001); however, there were no significant dif-
ferences compared with that observed in the clopidogrel (75 mg) and cilostazol (100 mg) combina-
tion group.

tion was 49.2%±21.6% in the clopidogrel (75 mg) and

Statistical analysis
The IBM SPSS Statistics 20 software program (IBM
SPSS Inc., Chicago, IL, USA) was used for the statistical
analysis. A one-way analysis of variance and Tukey’s hon-
estly significant difference test were used in the comparisons
of PRU and % inhibition. The results are expressed as the
mean ± SD. A one-tailed Fisher’s exact test was used to
compare the rate of clopidogrel resistance. p<0.05 was taken
to be significant.
Ethical approval
This study was approved by the ethics committee of Sai-
tama Medical University.
Results
Fig. 1 shows the PRU results. The PRU was 141±62 in
the clopidogrel (75 mg) and cilostazol (100 mg) combina-
tion group, 102±74 in the clopidogrel (75 mg) and cilostazol
(200 mg) combination group, 174±89 in the clopidogrel (75
mg) alone group and 249±57 in the cilostazol (200 mg)
alone group. The PRU was significantly lower in the three
groups receiving clopidogrel than in the cilostazol alone
group (p<0.001). The PRU was also significantly lower in
the clopidogrel (75 mg) and cilostazol (200 mg) combina-
tion group than in the clopidogrel (75 mg) alone group (p<
0.001); however, it was not significantly different from that
observed in the clopidogrel (75 mg) and cilostazol (100 mg)
combination group.
Fig. 2 shows the % inhibition results. The percent inhibi-
cilostazol (100 mg) combination group, 63.9%±25.2% in the
clopidogrel (75 mg) and cilostazol (200 mg) combination
group, 37.4%±28.0% in the clopidogrel (75 mg) alone
group and 12.7%±11.8% in the cilostazol (200 mg) alone
group. The percent inhibition was significantly higher in the
three groups that received clopidogrel than in the cilostazol
alone group (p<0.001). The percent inhibition was also sig-
nificantly higher in the clopidogrel (75 mg) and cilostazol
(200 mg) combination group than in the clopidogrel (75
mg) alone group (p<0.001); however, it was not signifi-
cantly different from that observed in the clopidogrel (75
mg) and cilostazol (100 mg) combination group.
Fig. 3 shows the rate of PRU >230 in the three groups
that received clopidogrel. The PRU was >230 in 1/18 pa-
tients (5.6%) in the clopidogrel (75 mg) and cilostazol (100
mg) combination group, 1/52 patients (1.9%) in the clopido-
grel (75 mg) and cilostazol (200 mg) combination group and
32/126 patients (25.4%) in the clopidogrel (75 mg) alone
group. The rate of PRU >230 was significantly lower in
both groups with combination cilostazol than in the clopido-
grel alone group.
Fig. 4 shows the rate of % inhibition <20% in the three
groups that received clopidogrel. The rate was 1/18 patients
(5.6%) in the clopidogrel (75 mg) and cilostazol (100 mg)
combination group, 1/52 patients (1.9%) in the clopidogrel
(75 mg) and cilostazol (200 mg) combination group and 41/
126 patients (32.5%) in the clopidogrel (75 mg) alone
group. The rate of % inhibition <20% was significantly
lower in both groups with combination cilostazol than in the
clopidogrel alone group.

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 Intern Med 52: 1043-1047, 2013 DOI: 10.2169/internalmedicine.52.9550

* * * Ɖ <0.001
100

80
% inhibiƟŽŶ (%)

60

40

20

0
ClŽƉiĚŽgrel 75mg ClŽƉiĚŽgrel 75mg ClŽƉiĚŽgrel 75mg CiůŽstazŽl 200mg
CiůŽstazŽl 100mg CiůŽstazŽl 200mg

Figure 2. The percent inhibition in each treatment group. The percent inhibition was significantly
higher in the three groups that received clopidogrel than in the cilostazol alone group (p<0.001). The
percent inhibition was also significantly higher in the clopidogrel (75 mg) and cilostazol (200 mg)
combination group than in the clopidogrel (75 mg) alone group (p<0.001); however, there were no
significant differences compared with that observed in the clopidogrel (75 mg) and cilostazol (100
mg) combination group.

50
* p<0.05 vs. Clopidogrel 75mg doses of 200 mg of cilostazol in combination treatment;
** p<0.001 vs. Clopidogrel 75mg
none investigated the effects of 100 mg of cilostazol in
40
combination treatment.
Rate of PRU >230 (%)

There are very few reports on the use of combined clopi-

30
20
10 * ured the platelet function in patients who had undergone en-
5.6
0 carotid artery stenting) and reported that the antiplatelet ac-
Clopidogrel 75mg
Cilostazol 100mg
Figure 3. Rate of P2Y12 Reaction Units (PRU) >230 in each
treatment group. The rate of clopidogrel resistance was signif-
icantly lower in both the cilostazol combination groups than in
the clopidogrel alone group.
Discussion
In this study, the inhibition of ADP aggregation with a
low cilostazol dose of 100 mg in combined use with clopi-
dogrel and cilostazol was almost the same as that observed
with 200 mg, and clopidogrel resistance was clearly de-
creased.
In the past, it has been reported that, when combination
cilostazol is given to patients receiving clopidogrel after
coronary artery stent placement for myocardial infarction, a
good antiplatelet action is obtained in platelet aggregation
tests with ADP (10-18). However, all of these reports used
25.4 dogrel and cilostazol therapy in patients with cerebral ische-
mia. We presented the first such report, with ischemic stroke
patients as subjects (6). Next, Haraguchi et al. (19) meas-
** dovascular treatment (coil embolization for brain aneurysm,
1.9
Clopidogrel 75mg Clopidogrel 75mg tion was strengthened with combined clopidogrel and
Cilostazol 200mg
cilostazol. They found no significant differences between the
cilostazol 100 mg and 200 mg combination groups. Yama-
gami et al. (20) reported decreased incidences of in-stent
stenosis and new stenosis within treated vessels with combi-
nation cilostazol in patients who underwent carotid artery
stenting. Therefore, the combined use of clopidogrel and
cilostazol has also attracted attention in recent years from a
clinical viewpoint.
The mechanism underlying the strengthened antiplatelet
action observed with the concurrent use of clopidogrel and
cilostazol, as described in our previous report, is thought to
involve an increase in cyclic adenosine monophosphate
(cAMP) within platelets as a result of inhibition of P2Y12
receptors by clopidogrel and inhibition of phosphodiesterase
3 (PDE III) by cilostazol (6).
This study is associated with several limitations. First, the
cutoff value to define clopidogrel resistance using the Veri-
fyNow P2Y12 Assay has yet to be determined in Japanese
patients. Second, this study was not a randomized trial.
Third, the number of subjects was small. In the future, large

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 Intern Med 52: 1043-1047, 2013
50
Rate of % inhibiƟon <20% (%)
40
30
20
10 * a time-dependent analysis of the Gauging Responsiveness with a
5.6 **
1.9
0
Clopidogrel 75mg Clopidogrel 75mg
Cilostazol 100mg Cilostazol 200mg
Figure 4. Rate of % inhibition <20% in each treatment
group. The rate of clopidogrel resistance was significantly
lower in both the cilostazol combination groups than in the
clopidogrel alone group.
clinical trials are needed to confirm the effects of combina-
tion therapy with clopidogrel and low-dose cilostazol in the
prevention of ischemic stroke events. Finally, CYP2C19
gene polymorphism has been shown to contribute to clopi-
dogrel resistance; however, gene polymorphism was not in-
vestigated in the present study. Nevertheless, the present re-
sults showed that clopidogrel resistance is clearly reduced
with the combined use of clopidogrel and cilostazol, and
even a low dose of 100 mg of cilostazol seems to be suffi-
cient for use in combination therapy. Since low-dose cilosta-
zol leads to a decrease in adverse effects such as headaches
and tachycardia, the use of combination therapy with clopi-
dogrel and low-dose cilostazol may be one means of over-
coming clopidogrel resistance in the future.
The authors state that they have no Conflict of Interest (COI).
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