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□ 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 clopidogrel resistance was present in 29% of the pa-
tients (6). We also reported that the antiplatelet action is
There are individual differences in the response to clopi- strengthened with combined use of clopidogrel and cilosta-
dogrel, which is affected by the presence or absence of drug zol (6). However, we did not investigate the dose of cilosta-
metabolizing enzyme CYP2C19 gene polymorphism (1-5). zol administered in combination therapy with clopidogrel
We previously measured the platelet function ability of and cilostazol. In this study, a greater number of patients re-
ischemic stroke patients using the VerifyNow P2Y12 Assay 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 DOI: 10.2169/internalmedicine.52.9550

Table. Clinical Stroke Type and Risk Factors

Clopidogrel 75mg Clopidogrel 75mg


Clopidogrel 75mg Cilostazol 200mg
Cilostazol 100mg Cilostazol 200mg (n=126) (n=35)
(n=18) (n=52)
Clinical stroke type
Atherothrombotic infarction, n (%) 14 (77.8) 46 (88.5) 83 (65.9) 18 (51.4)
Lacunar infarction, n (%) 2 (11.1) 4 (7.7) 33 (26.2) 12 (34.3)
Transient ischemic attack, n (%) 2 (11.1) 2 (3.8) 10 (7.9) 5 (14.3)
Risk factors
Diabetes mellitus, n (%) 7 (38.9) 21 (40.4) 51 (40.5) 8 (22.9)
Hypertension, n (%) 13 (72.2) 38 (73.1) 84 (66.7) 17 (48.6)
Dyslipidemia, n (%) 15 (83.3) 42 (80.8) 90 (71.4) 16 (45.7)
Smoking, n (%) 8 (44.4) 29 (55.8) 60 (47.6) 15 (42.9)

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

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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
cilostazol (100 mg) combination group, 63.9%±25.2% in the
The IBM SPSS Statistics 20 software program (IBM clopidogrel (75 mg) and cilostazol (200 mg) combination
SPSS Inc., Chicago, IL, USA) was used for the statistical group, 37.4%±28.0% in the clopidogrel (75 mg) alone
analysis. A one-way analysis of variance and Tukey’s hon- group and 12.7%±11.8% in the cilostazol (200 mg) alone
estly significant difference test were used in the comparisons group. The percent inhibition was significantly higher in the
of PRU and % inhibition. The results are expressed as the three groups that received clopidogrel than in the cilostazol
mean ± SD. A one-tailed Fisher’s exact test was used to alone group (p<0.001). The percent inhibition was also sig-
compare the rate of clopidogrel resistance. p<0.05 was taken nificantly higher in the clopidogrel (75 mg) and cilostazol
to be significant. (200 mg) combination group than in the clopidogrel (75
mg) alone group (p<0.001); however, it was not signifi-
Ethical approval
cantly different from that observed in the clopidogrel (75
This study was approved by the ethics committee of Sai- mg) and cilostazol (100 mg) combination group.
tama Medical University. Fig. 3 shows the rate of PRU >230 in the three groups
that received clopidogrel. The PRU was >230 in 1/18 pa-
Results tients (5.6%) in the clopidogrel (75 mg) and cilostazol (100
mg) combination group, 1/52 patients (1.9%) in the clopido-
Fig. 1 shows the PRU results. The PRU was 141±62 in grel (75 mg) and cilostazol (200 mg) combination group and
the clopidogrel (75 mg) and cilostazol (100 mg) combina- 32/126 patients (25.4%) in the clopidogrel (75 mg) alone
tion group, 102±74 in the clopidogrel (75 mg) and cilostazol group. The rate of PRU >230 was significantly lower in
(200 mg) combination group, 174±89 in the clopidogrel (75 both groups with combination cilostazol than in the clopido-
mg) alone group and 249±57 in the cilostazol (200 mg) grel alone group.
alone group. The PRU was significantly lower in the three Fig. 4 shows the rate of % inhibition <20% in the three
groups receiving clopidogrel than in the cilostazol alone groups that received clopidogrel. The rate was 1/18 patients
group (p<0.001). The PRU was also significantly lower in (5.6%) in the clopidogrel (75 mg) and cilostazol (100 mg)
the clopidogrel (75 mg) and cilostazol (200 mg) combina- combination group, 1/52 patients (1.9%) in the clopidogrel
tion group than in the clopidogrel (75 mg) alone group (p< (75 mg) and cilostazol (200 mg) combination group and 41/
0.001); however, it was not significantly different from that 126 patients (32.5%) in the clopidogrel (75 mg) alone
observed in the clopidogrel (75 mg) and cilostazol (100 mg) group. The rate of % inhibition <20% was significantly
combination group. lower in both groups with combination cilostazol than in the
Fig. 2 shows the % inhibition results. The percent inhibi- 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
25.4 dogrel and cilostazol therapy in patients with cerebral ische-
mia. We presented the first such report, with ischemic stroke
20
patients as subjects (6). Next, Haraguchi et al. (19) meas-
10 * ured the platelet function in patients who had undergone en-
5.6 ** dovascular treatment (coil embolization for brain aneurysm,
1.9
0 carotid artery stenting) and reported that the antiplatelet ac-
Clopidogrel 75mg Clopidogrel 75mg Clopidogrel 75mg tion was strengthened with combined clopidogrel and
Cilostazol 100mg Cilostazol 200mg
cilostazol. They found no significant differences between the
Figure 3. Rate of P2Y12 Reaction Units (PRU) >230 in each cilostazol 100 mg and 200 mg combination groups. Yama-
treatment group. The rate of clopidogrel resistance was signif- gami et al. (20) reported decreased incidences of in-stent
icantly lower in both the cilostazol combination groups than in stenosis and new stenosis within treated vessels with combi-
the clopidogrel alone group. 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.
Discussion The mechanism underlying the strengthened antiplatelet
action observed with the concurrent use of clopidogrel and
In this study, the inhibition of ADP aggregation with a cilostazol, as described in our previous report, is thought to
low cilostazol dose of 100 mg in combined use with clopi- involve an increase in cyclic adenosine monophosphate
dogrel and cilostazol was almost the same as that observed (cAMP) within platelets as a result of inhibition of P2Y12
with 200 mg, and clopidogrel resistance was clearly de- receptors by clopidogrel and inhibition of phosphodiesterase
creased. 3 (PDE III) by cilostazol (6).
In the past, it has been reported that, when combination This study is associated with several limitations. First, the
cilostazol is given to patients receiving clopidogrel after cutoff value to define clopidogrel resistance using the Veri-
coronary artery stent placement for myocardial infarction, a fyNow P2Y12 Assay has yet to be determined in Japanese
good antiplatelet action is obtained in platelet aggregation patients. Second, this study was not a randomized trial.
tests with ADP (10-18). However, all of these reports used Third, the number of subjects was small. In the future, large

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

50 * p<0.05 vs. Clopidogrel 75mg say. Intern Med 50: 695-698, 2011.
** p<0.001 vs. Clopidogrel 75mg
7. Brar SS, ten Berg J, Marcucci R, et al. Impact of platelet reactiv-
Rate of % inhibiƟon <20% (%)

40
ity on clinical outcomes after percutaneous coronary intervention.
32.5
A collaborative meta-analysis of individual participant data. J Am
30
Coll Cardiol 58: 1945-1954, 2011.
8. Price MJ, Angiolillo DJ, Teirstein PS, et al. Platelet reactivity and
20
cardiovascular outcomes after percutaneous coronary intervention:
10 * a time-dependent analysis of the Gauging Responsiveness with a
5.6 ** VerifyNow P2Y12 assay: Impact on Thrombosis and Safety
1.9
0
(GRAVITAS) trial. Circulation 124: 1132-1137, 2011.
Clopidogrel 75mg Clopidogrel 75mg Clopidogrel 75mg 9. Ueno M, Ferreiro JL, Tomasello SD, et al. Impact of pentoxifyl-
Cilostazol 100mg Cilostazol 200mg
line on platelet function profiles in patients with type 2 diabetes
Figure 4. Rate of % inhibition <20% in each treatment mellitus and coronary artery disease on dual antiplatelet therapy
with aspirin and clopidogrel. J Am Coll Cardiol Intv 4: 905-912,
group. The rate of clopidogrel resistance was significantly
2011.
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clopidogrel alone group. responsiveness to clopidogrel in patients undergoing primary per-
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gene polymorphism has been shown to contribute to clopi- platelet inhibition in patients undergoing percutaneous coronary
dogrel resistance; however, gene polymorphism was not in- intervention. Int Heart J 53: 1-4, 2012.
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sults showed that clopidogrel resistance is clearly reduced tiplatelet therapy achieves greater platelet inhibition than high
maintenance dose clopidogrel in patients with acute myocardial
with the combined use of clopidogrel and cilostazol, and infarction: result of the adjunctive cilostazol versus high mainte-
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patients with high post-treatment platelet reactivity: result of the
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