Circ J 2008; 72: 1844–1851

Evaluation of the Antiplatelet Effects of , a 3 Inhibitor, by VASP Phosphorylation and Aggregation

Hiromi Yamamoto, MD; Kanako Takahashi, MT; Haruyo Watanabe, MT; Yuka Yoshikawa, MT; Ryutaro Shirakawa, PhD; Tomohito Higashi, PhD; Mitsunori Kawato, MD; Tomoyuki Ikeda, MD; Arata Tabuchi, MD**; Takeshi Morimoto, MD*; Toru Kita, MD; Hisanori Horiuchi, MD

Background Cilostazol, a inhibitor, is an antiplatelet that is widely used for prevent- ing cardiovascular events, although, to date, there are few methods for evaluating its effects. Methods and Results samples were taken at baseline and at 3 and 12h in 10 healthy male subjects after 100mg cilostazol intake. Each sample was examined by Western blot for phosphorylation levels of vasodilator- stimulated phosphoprotein (VASP), an abundant cAMP-dependent kinase substrate in , and by the optical aggregometer for ADP- and collagen-induced aggregation, before and after 8nmol/L prostaglandin E1 (PGE1) treatment. Cilostazol intake did not affect VASP phosphorylation levels or the maximal aggregation rates without PGE1 treatment. However, cilostazol intake apparently enhanced PGE1-induced VASP phosphorylation and PGE1- mediated reduction of ADP- and collagen-induced maximal aggregation rates. Levels of VASP phosphorylated at Ser157 were correlated and the maximal aggregation rates induced by ADP were inversely correlated with cilostazol concentrations in the plasma. Conclusion The antiplatelet effects of cilostazol intake could be evaluated by measuring VASP phosphorylation levels and maximal aggregation rates in platelets by ex vivo treatment with a low concentration of PGE1.(Circ J 2008; 72: 1844–1851) Key Words: Antiplatelet; Cilostazol; Platelets; Prostaglandins; Signal transduction

ecause of its antiplatelet and vasodilating effects,1,2 dantly expressed in platelets16 and plays an important role in cilostazol is widely used in clinical practice for pre- their aggregation.17 VASP is phosphorylated at serine 157 B vention of stent and stent restenosis.3–6 (Ser157) and serine 239 (Ser239) by both cAMP-dependent It also reduces the risk of by approximately 40%,7 and protein kinase (A-kinase) and cGMP-dependent protein prevents recurrence of cerebral infarction.8 Furthermore, kinase (G-kinase). Therefore, VASP phosphorylation levels cilostazol has been recommended in the international guide- are considered to reflect levels of cAMP and cGMP in plate- line, TASCII,9,10 as a first-line therapy for peripheral arterial lets.18,19 Recently, VASP phosphorylation levels were used disease because it improves the symptoms.11 to monitor the effects of Gαi-coupled ADP Some agonists, including , a well established inhibitors (ie, derivatives such as antiplatelet agent,12 and adenosine13 increase cAMP via Gαs- and )20 because these inhibit the reduction coupled receptors. A synthetic compound, prostaglandin E1 of cAMP levels induced by ADP in platelets. (PGE1) also increases cAMP levels in platelets via the Although the effectiveness of antiplatelet drugs such as prostacyclin receptor14 and is used in the clinical setting for and thienopyridine have been evaluated, evaluation its antiplatelet and vasodilating effects.15 Thus, increased of cilostazol’s effectiveness is limited to a few reports,21,22 cAMP levels inhibit platelet activation. Cilostazol, a phos- because there are few methods available for monitoring its phodiesterase 3 inhibitor, blocks cAMP degradation and is antiplatelet effect. In this report, we show that the effects of thus expected to have an antiplatelet function. cilostazol intake can be monitored by analyzing the VASP Vasodilator-stimulated phosphoprotein (VASP), which phosphorylation levels and the ADP- and collagen-induced is a regulator of actin-cytoskeletal reorganization, is abun- maximal aggregation rates (MARs) of platelets under PGE1 treatment. Because VASP phosphorylation can not be de- tected without PGE1 stimulation, our data suggest that the (Received March 19, 2008; revised manuscript received June 17, 2008; accepted July 2, 2008; released online October 3, 2008) function of cilostazol is to enhance the platelet-inactivating Department of Cardiovascular Medicine, *Center for Medical Educa- cAMP signals downstream of Gαs-coupled receptors stim- tion, Graduate School of Medicine, Kyoto University, Kyoto, Japan and ulated by agonists such as prostacyclin and . **Institute of Physiology, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Mailing address: Hisanori Horiuchi, MD, Department of Cardio- Methods vascular Medicine, Graduate School of Medicine, Kyoto University, Yoshida, Honmachi, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: Cilostazol Intake and Platelet-Rich Plasma (PRP) [email protected] Preparation All rights are reserved to the Japanese Circulation Society. For per- This study was approved by the Ethics Committee, Fac- missions, please e-mail: [email protected] ulty of Medicine, Kyoto University. Ten healthy male

Circulation Journal Vol.72, November 2008 Evaluation of Cilostazol Antiplatelet Effect 1845

Fig1. Study protocols for (A) blood sampling after cilostazol intake and (B) analysis of platelet aggregation and vaso- dilator-stimulated phosphoprotein (VASP) phosphorylation after blood sampling. At the 12-h time point post-cilostazol intake, only VASP phosphorylation was examined. PGE1, prostaglandin E1; PRP, platelet-rich plasma.

Fig2. Effects of cilostazol intake on vasodilator-stimulated phosphoprotein (VASP) phosphorylation of platelets under prostaglandin E1 (PGE1) treatment. (A) VASP phosphorylation in platelets at baseline, and at 3 and 12 h after cilostazol intake, were examined under PGE1 treatment for 0.5 and 3h by immunoblotting with anti-pan-VASP and anti-VASP-P239 antibodies as described in the Methods. (B–D) Quantification of phosphorylated VASP shown in (A) (n=10) using Image J 1.33u software (NIH). The signals in the “a”, “b”, “c” and “d” bands in (A) are VASP-P157/239, VASP-P239, VASP-P157, and non-phosphorylated VASP, respectively, as described in the Methods. (B) VASP-P157 levels expressed as c/(c+d)× 100% are shown. (C) The level of phosphorylation of Ser239 was quantified by measuring a+b. The signal of a+b (* in Fig2A) was defined as the standard (1.0) and other signals were expressed as ratios of the standard. (D) The level of VASP-P157/239 was quantified by measuring “a”. The signal of “a” (* in Fig2A) was defined as the standard (1.0) and other signals were expressed as ratios of the standard. †p<0.05 and ††p<0.01 vs before cilostazol. The data are mean±SE (n=10). physicians, aged 32–48 years old, from Kyoto University tant for 7 days prior to and during the study Hospital, who understood the significance of this investiga- period. Each subject took a single dose of 100mg cilostazol tion and possible adverse side-effects of cilostazol, volun- (Otsuka Pharmaceutical Co Ltd, Tokushima, Japan) after tarily participated in this study, and gave written informed breakfast. A blood sample, containing 1.79–2.53×105 (2.31± consent. Subjects were not permitted to take any concomi- 0.23×105, mean±SE) platelets/μl, was collected at baseline

Circulation Journal Vol.72, November 2008 1846 YAMAMOTO H et al.

Table 1 Clinical and Biochemical Characteristics of the Study quantified by measuring a+b. The a+b level at 3h post- Subjects cilostazol intake with PGE1 treatment for 3h (* in Fig2A) was defined as the standard (1.0) and other signals were Age, years 36.8±6.12 RBC×1012 /L 5.04±0.18 expressed as ratios of the standard. The level of VASP- Hemoglobin×102 g/L 1.55±0.04 P157/239 was quantified by measuring “a”. The “a” level WBC×109 /L 5.84±0.78 at 3h post-cilostazol intake, with PGE1 treatment for 3h Platelets×1011 /L 2.31±0.27 (* in Fig2A), was defined as the standard (1.0) and other Fibrinogen, g/L 2.29±0.22 signals were expressed as ratios of the standard. PT (INR) 1.05±0.06 aPTT, s 32.2±3.11 Fasting glucose, g/L 0.94±0.07 Analysis of Aggregation of PRP HbA1c, % 4.74±0.24 The PRPs, with or without 8nmol/L PGE1 treatment for AST, IU/L 19.7±2.5 1h at 30°C, prepared from blood samples taken at baseline ALT, IU/L 20.2±5.26 and at 3h after 100mg cilostazol intake were individually Gamma GTP, IU/L 33.1±24.7 stimulated by 5 and 20μmol/L ADP (Sigma) and 1μg/ml Total cholesterol, g/L 2.04±0.21 collagen (Horm, Germany) at 30°C, and the aggregations HDL-cholesterol, g/L 0.51±0.09 Triglyceride, g/L 1.18±0.63 were analyzed under stirring using a light transmission CK, IU/L 112±32 aggregometer, MCM HEMA TRACER 313 (MC Medical, UA, mg/L 58.4±8.11 Tokyo, Japan), where the degree of light transmission of BUN, mg/L 131±25.3 PRP was defined as 0% of the aggregation rate, and the cog- Creatinine, mg/L 7.92±0.69 nitive platelet-poor plasma as 100%.25 The MAR of platelets Na, mmol/L 140±2.12 and the rate of inhibition of platelet aggregation (IPA) were K, mmol/L 4.2±0.14 used for data analysis. IPA was calculated using the fol- Data are mean±SE; n=10 males. lowing formula: IPA (%)=[(MAR0–MAR3)/MAR0]×100, RBC, erythrocyte; WBC, leukocyte; PT (INR), prothrombin time (international where MAR0 is the MAR at baseline and MAR3 is the normalized ratio); aPTT, activated partial thromboplastin time; HbA1c, hemo- MAR at 3h after cilostazol intake. globin A1c; AST, aspartate aminotransferase; ALT, alanine aminotransferase; gamma GTP, gamma glutamyl transpeptidase; HDL, high-density lipoprotein; CK, creatine kinase; UA, ; BUN, blood urea nitrogen. Laboratory Testing and Analysis of the Concentration of Cilostazol General serum values were measured by the SRL Labo- and at 3 and 12h after cilostazol intake (Fig1), using a 21G ratory (Tokyo, Japan). Plasma concentrations of cilostazol needle with tourniquet, into a glass tube containing sodium at baseline and at 3 and 12h after 100mg cilostazol intake citrate at a final concentration of 0.313%. PRP was prepared were measured by BML, Inc (Tokyo, Japan). by centrifugation of the blood sample at 200g at 25°C for 10min, and platelet-poor plasma was prepared by centrifu- Statistical Analyses gation at 2,000g at 25°C for 10min. Data are expressed as mean±SE (n=10). Paired t-test was used for comparison of continuous variables over time. Analysis of VASP Phosphorylation Correlation between cilostazol plasma concentrations and The PRPs prepared from blood samples taken at base- IPA (%) was assessed by Spearman’s rank test. Correlation line, and at 3 and 12h after 100mg cilostazol intake, were between cilostazol plasma concentrations and VASP-P157 treated with or without PGE1 (Sigma, St Louis, MO, USA) levels was assessed by longitudinal analysis methods be- at 8nmol/L for 0.5 and 3h at 30°C. Platelets were collected cause the cilostazol concentrations changed over time. We by centrifugation at 700g at 4°C for 5min and added to used the PROC MIXED command of SAS, with serine 157 sodium dodecylsulfate (SDS)-containing Laemmli buffer,23 phosphorylation of VASP of platelets as a dependent varia- followed by incubation at 95°C for 10min. The samples ble, and cilostazol concentration and time as independent were subjected to SDS-polyacrylamide gel electrophoresis variables. Correlation between cilostazol plasma concen- (PAGE) and immunoblotted with anti-pan-VASP (M4, trations and MARs was assessed in the same way. As for Alexis, San Diego, CA, USA) and anti-phospho (239)- VASP-P157, the linearity of time was evaluated by the Log VASP (16C2, Nano Tools Antikörpertecnik, Tiningen, likelihood method. If the linearity of time was appropriate, Germany) mouse monoclonal antibodies as primary anti- then longitudinal analysis was conducted with the time as bodies and horseradish peroxidase-conjugated anti-mouse linear. Otherwise, we conducted it using the profile method. IgG polyclonal sheep antibody (GE Healthcare) as the sec- We used SAS software version 9.1 (SAS Institute Inc, Cary, ondary antibody, followed by visualization with ECL NC, USA); p values less than 0.05 were defined as signifi- chemiluminescent material (GE Healthcare). A typical cant. result is shown in Fig2A. It has been reported that VASP phosphorylated at Ser157 (VASP-P157) migrates to a 50 kD position in SDS-PAGE analysis, whereas VASP Results phosphorylated at Ser239 (VASP-P239) migrates to a Effects of Cilostazol Intake on Platelet Aggregation and 46kD position similar to the non-phosphorylated form.24 VASP Phosphorylation Therefore, the “a”, “b”, “c” and “d” bands shown in Fig2A Baseline laboratory parameter values were within normal were VASP phosphorylated at both Ser157 and Ser239 limits for all subjects (Table1). It has been shown that, fol- (VASP-P157/239), VASP-P239, VASP-P157 irrespective lowing administration, the maximum plasma concentration of phosphorylation at Ser239, and non-phosphorylated of cilostazol is reached in approximately 3h, and that it has VASP, respectively. These bands were quantified using a half-life of approximately 12h.26 Therefore, we analyzed Image J 1.33u software (NIH). VASP-P157 levels were platelet functions at baseline and at 3 and 12h after 100mg expressed as c/(c+d)×100%. The level of VASP-P239 was cilostazol intake. Compared with the baseline data, no reduc-

Circulation Journal Vol.72, November 2008 Evaluation of Cilostazol Antiplatelet Effect 1847

Fig3. Effects of cilostazol intake on aggre- gation of platelets without prostaglandin E1 treatment. (A–D) Platelets at baseline and at 3h after cilostazol intake were examined for 5μmol/L ADP-induced (A,B) and 1μg/ml collagen-induced (C,D) aggregation. Repre- sentative results of the aggregation curve (A,C), and the maximal aggregation rates expressed as mean ± SE (n=10) (B,D) are shown.

Fig 4. Effects of cilostazol intake on ADP- induced platelet aggregation under prostaglan- din E1 (PGE1) treatment. (A) Representative curves of 5μmol/L ADP-induced aggrega- tion of platelets at baseline and at 3h after cilostazol intake, with or without 8nmol/L PGE1 treatment for 1h. (B) Quantification of the maximal aggregation rates of the data shown in (A). At baseline (▲) and at 3h after cilostazol (○). The data are mean±SE (n= 10). ††p<0.01 vs before cilostazol. tion was detected in ADP- or collagen-induced aggregation bility induced by collagen or ADP. of platelets after cilostazol intake (Figs3A–D). Because cilostazol is an inhibitor of phosphodiesterase 3, which de- Cilostazol Enhanced VASP Phosphorylation Levels Induced grades cAMP, it would be expected to cause an increase in by PGE1 the cAMP level in platelets, and also to raise phosphorylated Although cilostazol intake did not induce VASP phospho- VASP levels through the activation of A-kinase. As shown rylation, we considered that it might have an effect on VASP in Fig2A, however, phosphorylated VASP was not detected phosphorylation, if cAMP was produced in response to in the platelets prepared from samples collected at baseline, stimuli. Therefore, VASP phosphorylation in isolated plate- or at 3 or 12h post-cilostazol intake. Thus, cilostazol failed lets under treatment of 8nmol/L PGE1 was analyzed by to increase cAMP to a level adequate enough to increase Western blot with anti-pan-VASP and anti-phospho (239)- VASP phosphorylation levels or to reduce platelet aggrega- VASP antibodies. We evaluated VASP, VASP-P157, VASP-

Circulation Journal Vol.72, November 2008 1848 YAMAMOTO H et al.

Fig5. Effects of cilostazol intake on colla- gen-induced platelet aggregation under pros- taglandin E1 (PGE1) treatment. (A) Repre- sentative curves of 1μg/ml collagen-induced aggregation of platelets at baseline and at 3h after cilostazol intake, with or without 8nmol/L PGE1 treatment for 1h. (B) Quanti- fication of the maximal aggregation rates of the data shown in (A). At baseline (▲) and at 3h after cilostazol (○). The data are mean± SE (n=10). ††p<0.01 vs before cilostazol.

P239 and VASP-P157/239 individually (see Methods). significantly decreased the 5μmol/L ADP-induced MAR Treatment with PGE1 induced VASP phosphorylation at (52.4% vs 22.3%, p<0.001) (Fig4B). When the stimulus was Ser157 and Ser239 in a time-dependent manner (Fig2). Al- 20μmol/L ADP (data not shown), without cilostazol intake though VASP-P157 per total VASP was 29.2±3.4% before the MAR without PGE1 treatment was 81.7±2.3% and that cilostazol intake at 0.5h after PGE1 treatment, the ratio was at 1h after 8nmol/L PGE1 treatment was 69.8±3.59% (p= increased at 3h after cilostazol intake to 44.7±3.17% (p= 0.004), whereas for platelets at 3h after cilostazol intake, 0.001) (Fig2B). At 0.5h after PGE1 treatment, VASP-P239 the MAR without PGE1 treatment was 82.2±1.61% and before cilostazol intake was 0.19±0.02 (arbitrary units), that at 1h after 8nmol/L PGE1 treatment was 48.7±5.58% while at 3h after cilostazol intake it was 0.82±0.22 (p< (p<0.001). As with 5μmol/L ADP, cilostazol intake signifi- 0.001) (Fig2C). Similarly, at 0.5h after PGE1 treatment, cantly decreased the 20μmol/L ADP-induced MAR under VASP-P157/239 before cilostazol intake was 0.05±0.03 PGE1 treatment (69.8% vs 48.7%, p=0.001). Similar results (arbitrary units), while at 3h after cilostazol intake it was were obtained with 1μg/ml collagen as the stimulus, where 0.93±0.19 (p<0.001) (Fig2D). The effect of cilostazol intake without cilostazol intake the MAR without PGE1 treatment on VASP phosphorylation under PGE1 treatment lasted for was 83.0±1.35% and that at 1h after 8nmol/L PGE1 treat- 12h (Figs2B–D). Although VASP-P157/239 under 8nmol/L ment was 57.4±8.22% (p=0.009) (Fig5A), whereas the MAR PGE1 for 0.5h was barely detectable (0.05±0.01 arbitrary without PGE1 treatment was 82.4±1.16% and that at 1h units) without cilostazol intake (Figs2A,D), at 3 and 12h after 8nmol/L PGE1 treatment was 13.7±2.41% (p<0.001) after cilostazol intake it increased 18.6-fold (0.93±0.19, for platelets 3h after cilostazol intake (Fig5A). Under PGE1 p=0.001) and 15.4-fold (0.77±0.24, p=0.016), respectively treatment, cilostazol intake significantly decreased the (Fig 2D). Therefore, the effects of cilostazol were most MAR (57.4% vs 13.7%, p<0.001) (Fig5B). Thus, we could clearly detected in the evaluation of VASP phosphorylated clearly detect the effect of cilostazol intake by analyzing at both Ser157 and Ser239, so the effect of cilostazol intake ADP- and collagen-induced platelet aggregation under could be clearly detected by analyzing VASP phosphoryla- PGE1 treatment. tion under PGE1 treatment. Correlation of Cilostazol Levels in the Plasma With VASP Cilostazol Intake Reduced Aggregability of PGE1-Treated Phosphorylation and Aggregation of Platelets Platelets The plasma concentrations of cilostazol in all subjects We next examined platelet aggregability under PGE1 were below the limit of detection (<20ng/ml) at baseline, but treatment. Without cilostazol intake, the 5μmol/L ADP- were 732±89.9ng/ml and 192±22.7ng/ml at 3h and 12h, induced MAR without PGE1 treatment was 76.7±2.12% respectively, after 100mg cilostazol intake (Fig6A); these and at 1h after 8nmol/L PGE1 treatment was 52.4±5.56% findings were similar to those reported previously.26 (p<0.001) (Fig4A). On the other hand, for platelets at 3h Individual plots of cilostazol levels and VASP-P157 after cilostazol intake, the 5μmol/L ADP-induced MAR levels are shown in Fig6B. Assessed by longitudinal analy- without PGE1 treatment was 76.9±1.13% and that at 1h sis, considering change of cilostazol plasma concentration after 8nmol/L PGE1 treatment was drastically decreased to over time, VASP-P157 levels induced with 8nmol/L PGE1 22.0±2.60% (p<0.001) (Fig4A). Without PGE1 treatment, for 3h strongly correlated with the plasma concentration of cilostazol intake did not decrease the MAR (76.7% vs 76.9%, cilostazol (p=0.001) (Fig6A). Because the values related to p=0.932). Under PGE1 treatment, however, cilostazol intake VASP-P239 and VASP-P157/239 were expressed as rela-

Circulation Journal Vol.72, November 2008 Evaluation of Cilostazol Antiplatelet Effect 1849

Fig6. Relationship of plasma cilostazol concentrations with vasodilator-stimulated phosphoprotein (VASP)-P157 levels under prostaglandin E1 (PGE1) treatment. (A) The mean plasma concentration of cilostazol and the mean VASP-P157 levels under PGE1 treatment for 3h over time are shown. The data are mean±SE (n=10). (B) Individual plots of cilostazol plasma concentrations and VASP-P157 levels under PGE1 treatment for 3h are shown.

Fig7. Relationship of plasma cilostazol concentrations with platelet aggregation under prostaglandin E1 (PGE1) treat- ment. (A) Individual plots of cilostazol plasma concentrations and the 5μmol/L ADP, 20μmol/L ADP and 1μg/ml colla- gen-induced maximal aggregation rates under PGE1 treatment are shown. (B) Correlation of plasma concentrations of cilostazol with the rate of inhibition of platelet aggregation induced by 20μmol/L ADP is shown. This correlation was assessed by Spearman’s rank test.

tive values using a standard, which was VASP-P239 and of platelets treated with 8nmol/L PGE1 for 1h (Figs4,5) at VASP-P157/239 at 3h after 100mg cilostazol intake treated 3h after cilostazol intake, as assessed by longitudinal analy- with 8nmol/L PGE1 for 3h, the correlations were not ob- sis; there was a significant inverse correlation between the tained. plasma concentration of cilostazol and the 5μmol/L and Similarly, cilostazol intake apparently inhibited the MAR 20μmol/L ADP-induced MARs of platelets treated with

Circulation Journal Vol.72, November 2008 1850 YAMAMOTO H et al.

8nmol/L PGE1 for 1h (p=0.038 and p=0.004, respectively) VASP-P157/239 might be the best way of monitoring the (data not shown). On the other hand, we did not detect effect of cilostazol. a correlation of cilostazol plasma concentrations with the Although we could not detect a cilostazol effect on ADP- 1μg/ml collagen-induced MAR of platelets under PGE1 or collagen-induced platelet aggregation without PGE1 treatment (p=0.255) (data not shown). Individual plots of treatment, we could clearly detect its effect on both agonist- cilostazol levels and MARs are shown in Fig7A. induced aggregations under PGE1 treatment. Furthermore, We used Spearman’s rank test to analyze the correlation the 20μmol/L ADP-induced MARs of platelets treated with between cilostazol plasma concentrations and the rates of PGE1 inversely correlated with the plasma concentration of IPA (%) under PGE1 treatment induced by 20μmol/L ADP; cilostazol, although the inverse-correlation between colla- we found a strong correlation between them (r=0.903, gen-induced aggregation and the plasma concentration of p<0.001) (Fig7B). On the other hand, we did not detect a cilostazol was not significant, probably because the agonist correlation of plasma cilostazol level with 5μmol/L ADP- concentrations were inappropriately weak, as these ag- induced IPA (%) (r=0.413, p=0.235) and 1μg/ml collagen- gregations were strongly inhibited in the presence of even induced IPA (%) (r=0.503, p=0.138) (data not shown), prob- low concentrations of cilostazol. Thus, the 20μmol/L ADP- ably because of inappropriately weak stimulation where induced platelet aggregation under PGE1 treatment could aggregation was strongly suppressed even at low concentra- reflect the level of cilostazol in the plasma. Based on these tions of cilostazol (Fig7A). findings, 20μmol/L ADP stimulation might be a better Thus, VASP phosphorylation levels and platelet aggre- choice for monitoring the effect of cilostazol on platelet gation under PGE1 treatment could reflect the levels of aggregation under PGE1 treatment. cilostazol in the plasma. Although analysis of the levels of VASP phosphorylation by the Western blotting method would more directly reflect the concentration of cAMP and activity of cAMP-dependent Discussion protein kinase, it takes more time than the analysis of aggre- We have demonstrated that the antiplatelet effect of gation, because a much complicated process is involved. cilostazol can be monitored by analyzing intracellular VASP Given that we could clearly detect the effect of cilostazol in phosphorylation levels and platelet aggregation induced by the evaluation of 20μmol/L ADP-induced platelet aggrega- collagen and ADP under PGE1 treatment. Although a pre- tion under ex vivo PGE1 treatment, it would be the more vious report showed an antiplatelet effect of cilostazol feasible method for use in the clinical setting. intake using the optical aggregometer and ADP or collagen Evaluation of a drug’s efficacy sometimes provides in- as stimuli,21 another report showed that cilostazol intake did sight into its functional mechanism; this is the case for not affect ADP- or collagen-induced platelet aggregation.22 antiplatelet drugs. In this study, under PGE1 treatment, we In the present study, we were also unable to detect an anti- detected apparent enhancement of VASP phosphorylation platelet effect of cilostazol by optical aggregometer using and antiplatelet effect on the aggregation induced by ADP the same agonists without PGE1 treatment. and collagen. Although PGE1 is a synthetic compound, VASP is an abundant substrate of A-kinase and G-kinase which reduces platelet reactivity through the prostacyclin in platelets. Cilostazol is a phosphodiesterase 3 inhibitor and receptor,14 adenosine and prostacyclin are conceivable in- inhibits the degradation of cAMP.1,2 Theoretically, phospho- trinsic agonists that increase cAMP.13 Therefore, the func- diesterase 3 inhibitor could also inhibit the degradation of tion of cilostazol in pathophysiological conditions would be cGMP. Because cilostazol can affect the cAMP and cGMP to enhance and prolong the effects of these cAMP-producing levels in platelets, we used analysis of phosphorylated VASP factors in platelets, which could at least partially contribute levels to evaluate cilostazol’s effect on platelets. However, to their cardiovascular protective effects. we could not detect VASP phosphorylation without PGE1 PGE1 increases cAMP via its Gαs-coupled receptor. On treatment even after cilostazol intake. Thus, it might be the other hand, it has been demonstrated that increased difficult to detect the effect of cilostazol intake not only by cAMP subsequently induces the endothelial type of nitric platelet aggregation but also by VASP phosphorylation, oxide synthase, thereby increasing cGMP in platelets.27 Be- probably because of low concentrations of cAMP and cGMP cause not only A-kinase, but also G-kinase, has been dem- in isolated platelets. However, in situations where cAMP onstrated to phosphorylate VASP at serines 157 and 237,16 concentrations increase, cilostazol might achieve antiplatelet the effect of cilostazol, which theoretically inhibits degra- effects. Because the expected function of cilostazol is to dation of both cAMP and cGMP,28 might be partly medi- maintain increased cAMP levels in platelets, but not to pro- ated by inhibition of cGMP degradation. The half-lives of duce cyclic , it is understandable that we were prostacyclin and nitric oxide are 6–7 min29 and several unable to detect an antiplatelet effect by analyzing levels of seconds,30 respectively. The reason why cilostazol did not VASP phosphorylation or platelet aggregation without achieve antiplatelet effects without PGE1 treatment might PGE1 treatment, even after cilostazol intake. be loss of cyclic producing factors, because it In the present study, a low concentration of PGE1 took at least 20min to prepare PRP after blood sampling. (8nmol/L) induced VASP phosphorylation at Ser157 and It was recently reported that some people are poor re- Ser239 to a certain extent, both of which were not fully phos- sponders to antiplatelet drugs such as aspirin31,32 and clopi- phorylated. Under these conditions, we observed apparent dogrel,33,34 and that cardiovascular risk is elevated in such enhancement of the phosphorylation of each residue by patients. As for cilostazol, it remains to be seen whether cilostazol. The level of VASP phosphorylated at both the poor responders exist, because its efficacy has not been Ser157 and Ser239 residues exhibited the most striking evaluated extensively because of the lack of suitable moni- change following cilostazol intake, because VASP-P157/239 toring methods. Given that the cilostazol-effect monitoring was not detected even after treatment with PGE1 without methods have been established by this study, it should now cilostazol intake (Figs2A,D). Therefore, to evaluate the be possible, and important, to evaluate the antiplatelet level of VASP phosphorylation, measuring the levels of effects of cilostazol in a number of patients groups. Values

Circulation Journal Vol.72, November 2008 Evaluation of Cilostazol Antiplatelet Effect 1851 of any parameter that reflect a patient’s clinical status can Drugs Ther 1997; 11: 441–448. acquire even greater importance when they are linked to 16. Reinhard M, Jarchau T, Walter U. Actin-based motility: Stop and go with Ena/VASP proteins. Trends Biochem Sci 2001; 26: 243–249. clinical outcomes. Therefore, further examination, especial- 17. Massberg S, Gruner S, Konrad I, Garcia Arguinzonis MI, Eigenthaler ly through a prospective clinical study, is required in order M, Hemler K, et al. Enhanced in vivo platelet adhesion in vasodilator- to determine which value might be best for the evaluation stimulated phosphoprotein (VASP)-deficient mice. Blood 2004; 103: of the cilostazol-effect in the prevention of cardiovascular 136–142. 18. Aszodi A, Pfeifer A, Ahmad M, Glauner M, Zhou XH, Ny L, et al. events. The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet Acknowledgments aggregation, but is dispensable for smooth muscle function. EMBO J 1999; 18: 37–48. This work was supported by a Health and Labor Sciences Research 19. Hauser W, Knobeloch KP, Eigenthaler M, Gambaryan S, Krenn V, Grant for Cardiovascular Research and a Grant from the Japan Cardio- Geiger J, et al. Megakaryocyte hyperplasia and enhanced agonist- vascular Research Foundation. induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice. Proc Natl Acad Sci USA 1999; 96: 8120–8125. References 20. Schwarz UR, Geiger J, Walter U, Eigenthaler M. Flow cytometry analysis of intracellular VASP phosphorylation for the assessment of 1. Goto S. Cilostazol: Potential mechanism of action for activating and inhibitory signal transduction pathways in human effects accompanied by a low rate of bleeding. Atheroscler Suppl platelets: Definition and detection of ticlopidine/clopidogrel effects. 2005; 6: 3–11. Thromb Haemost 1999; 82: 1145–1152. 2. Horiuchi H. Recent advance in antiplatelet therapy: The mechanisms, 21. Yasunaga K, Mase K. Antiaggregatory effect of oral cilostazol and evidence and approach to the problems. Ann Med 2006; 38: 162– recovery of platelet aggregability in patients with cerebrovascular 172. disease. Arzneimittelforschung 1985; 35: 1189–1192. 3. Schleinitz MD, Olkin I, Heidenreich PA. Cilostazol, clopidogrel or 22. Tanemoto K, Kanaoka Y, Kuinose M. Assessment of antithrombotic ticlopidine to prevent sub-acute stent thrombosis: A meta-analysis of agents using the platelet aggregation test. Curr Ther Res 2000; 61: randomized trials. Am J 2004; 148: 990–997. 798–806. 4. Sekiguchi M, Hoshizaki H, Adachi H, Ohshima S, Taniguchi K, 23. Laemmli UK. Cleavage of structural proteins during the assembly of Kurabayashi M. Effects of antiplatelet agents on subacute thrombosis the head of bacteriophage T4. Nature 1970; 227: 680–685. and restenosis after successful coronary stenting: A randomized com- 24. Smolenski A, Bachmann C, Reinhard K, Honig-Liedl P, Jarchau T, parison of ticlopidine and cilostazol. Circ J 2004; 68: 610–614. Hoschuetzky H, et al. Analysis and regulation of vasodilator-stimu- 5. Ahn Y, Jeong MH, Jeong JW, Kim KH, Ahn TH, Kang WC, et al. lated phosphoprotein serine 239 phosphorylation in vitro and in intact Randomized comparison of cilostazol vs clopidogrel after drug- cells using a phosphospecific monoclonal antibody. J Biol Chem eluting stenting in diabetic patients: Cilostazol for diabetic patients in 1998; 273: 20029–20035. drug-eluting stent (CIDES) trial. Circ J 2008; 72: 35–39. 25. Tabuchi A, Taniguchi R, Takahashi K, Kondo H, Kawato M, 6. Douglas JS Jr, Holmes DR Jr, Kereiakes DJ, Grines CL, Block E, Morimoto T, et al. Action of aspirin on whole blood-aggregation Ghazzal ZM, et al. restenosis in patients treated with evaluated by the screen filtration pressure method. Circ J 2008; 72: cilostazol. Circulation 2005; 112: 2826–2832. 420–426. 7. Gotoh F, Tohgi H, Hirai S, Terashi A, Fukuuchi Y, Otomo E, et al. 26. Bramer SL, Forbes WP, Mallikaarjun S. Cilostazol Cilostazol stroke prevention study: A -controlled double- after single and multiple oral doses in healthy males and patients with blind trial for secondary prevention of cerebral infarction. J Stroke intermittent claudication resulting from peripheral arterial disease. Cerebrovasc Dis 2000; 9: 147–157. Clin Pharmacokinet 1999; 37(Suppl 2): 1–11. 8. Inoue T, Kobayashi M, Uetsuka Y, Uchiyama S. Pharmacoeconomic 27. Russo I, Doronzo G, Mattiello L, De Salve A, Trovati M, Anfossi G. analysis of cilostazol for the secondary prevention of cerebral infarc- The activity of constitutive nitric oxide synthase is increased by the tion. Circ J 2006; 70: 453–458. pathway cAMP/cAMP-activated protein kinase in human platelets: 9. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes New insights into the antiaggregating effects of cAMP-elevating FG, et al; TASC II Working Group. Inter-society consensus for the agents. Thromb Res 2004; 114: 265–273. management of peripheral arterial disease. Int Angiol 2007; 26: 81– 28. Conti M, Beavo J. Biochemistry and physiology of cyclic nucleotide 157. : Essential components in cyclic nucleotide sig- 10. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes naling. Annu Rev Biochem 2007; 76: 481–511. FG; TASC II Working Group. Inter-Society Consensus for the Man- 29. Lucas FV, Skrinska VA, Chisolm GM, Hesse BL. Stability of prosta- agement of Peripheral Arterial Disease (TASC II). J Vasc Surg 2007; cyclin in human and rabbit whole blood and plasma. Thromb Res 45(Suppl S): S5–S67. 1986; 43: 379–387. 11. Hiatt WR, Money SR, Brass EP. Long-term safety of cilostazol in pa- 30. Ignarro LJ. Nitric oxide: A novel signal transduction mechanism for tients with peripheral disease: The CASTLE study (Cilostazol: transcellular communication. Hypertension 1990; 16: 477–483. A Study in Long-term Effects). J Vasc Surg 2008; 47: 330–336. 31. Gum PA, Kottke-Marchant K, Poggio ED, Gurm H, Welsh PA, 12. Tanaka Y, Yamaki F, Koike K, Toro L. New insights into the intra- Brooks L, et al. Profile and prevalence of aspirin resistance in patients cellular mechanisms by which PGI2 analogues elicit vascular relaxa- with cardiovascular disease. Am J Cardiol 2001; 88: 230–235. tion: Cyclic AMP-independent, Gs-protein mediated-activation of 32. Zimmermann N, Wenk A, Kim U, Kienzle P, Weber AA, Gams E, et MaxiK channel. Curr Med Chem Cardiovasc Hematol Agents 2004; al. Functional and biochemical evaluation of platelet aspirin resis- 2: 257–265. tance after coronary artery bypass surgery. Circulation 2003; 108: 13. Nakamura T, Uchiyama S, Yamazaki M, Iwata M. Synergistic effect 542–547. of cilostazol and mediated by adenosine on shear- 33. Matetzky S, Shenkman B, Guetta V, Shechter M, Bienart R, induced platelet aggregation. Thromb Res 2007; 119: 511–516. Goldenberg I, et al. Clopidogrel resistance is associated with increased 14. Dutta-Roy AK, Kahn NN, Sinha AK. Prostaglandin E1: The endoge- risk of recurrent atherothrombotic events in patients with acute myo- nous physiological regulator of platelet mediated blood . cardial infarction. Circulation 2004; 109: 3171–3175. Prostaglandins Leukot Essent Fatty Acids 1989; 35: 189–195. 34. Buonamici P, Marcucci R, Migliorini A, Gensini GF, Santini A, 15. Makita S, Nakamura M, Ohhira A, Itoh S, Hiramori K. Effects of Paniccia R, et al. Impact of platelet reactivity after clopidogrel ad- prostaglandin E1 infusion on limb hemodynamics and vasodilatory ministration on drug-eluting stent thrombosis. J Am Coll Cardiol response in patients with arteriosclerosis obliterans. Cardiovasc 2007; 49: 2312–2317.

Circulation Journal Vol.72, November 2008