Cilostazol, 6-[4-(1-cyclohexy 1-IH-tetrazol-5yl) butoxy]-3,4-dihydro-2 (1H)-quinolinone, is an anti-platelet/anti-thrombotic agent that has been used in several Asian countries for the treatment of chronic peripheral arterial occlusive diseases (PAOD) since 1988. In 1999, the United States Food and Drug Administration approved cilostazol for the treatment of intermittent claudication, the most common debilitating symptom of PAOD. Cilostazol not only inhibits platelet activation but also induces vasodilation (1,2). Although it is generally believed that one of the mechanisms of these effects is mediated by inhibition of cyclic nucleotide phosphodiesterase (PDE) 3 to elevate intracellular cAMP, the exact mechanism of action is not fully understood (3). Recently we detected a secondary function of cilostazol during a comparison study with another selective PDE3 inhibitor, milrinone, on inhibition of collagen-induced platelet aggregation and cardiac function, even though cilostazol and milrinone have similar potency on PDE3 inhibition (4). Cilostazol and milrinone were equally effective in inhibiting collagen-induced platelet aggregation, corresponding with similar potency in increasing cAMP levels in rabbit and human platelets. In isolated rabbit ventricular myocytes, however, cilostazol elevated the cAMP level to a significantly lesser extent, compared with milrinone. Furthermore, experiments involving Langendorff rabbit heart preparations showed milrinone to be a very potent cardiotonic agent, whereas cilostazol was much less effective in increasing left ventricular developed pressure and contractility. But both compounds increased coronary flow equally. Subsequent studies demonstrated that cilostazol inhibited adenosine uptake by isolated rabbit ventricular myocytes, vascular smooth muscle cells, and endothelial cells in culture. Thus there was an increase in cardiac interstitial adenosine levels (5). Adenosine acts as an anti-platelet aggregating agent both in vitro and in vivo (6,7). It increases intracellular cAMP levels by stimulating adenylyl cyclase activity through Gs protein–coupled A2A receptor in the platelet membrane (8). Adenosine and other PDE inhibitors at low concentrations have been shown to exert a synergistic effect on platelet aggregation and intraplatelet cAMP levels (9–12). Without addition of exogenous adenosine, we have previously shown that cilostazol and milrinone have equal effects on collagen-induced platelet aggregation and cAMP elevation (4). In the present studies, we demonstrate that cilostazol and adenosine synergistically inhibit collagen-induced washed human platelet aggregation with a greater effect in comparison with milrinone. Inhibition of adenosine uptake by platelets and erythrocytes by cilostazol contributes considerably to its overall anti-platelet effect in whole blood.
Peripheral blood samples were collected from 10 healthy volunteers (medication-free for at least 10 days) by a two-syringe technique using a 19G butterfly needle. The procedure to draw blood was approved by institutional review committee according to the Helsinki convention. Nine volumes of blood were directly collected into a syringe containing 1 volume of trisodium citrate (3.8%). Platelet-rich plasma (PRP) was collected following centrifugation at 150 g for 15 min at room temperature. Washed platelet suspension was prepared from citrated PRP by citrate wash method as described previously (4). Platelets were finally resuspended in Tyrode HEPES (N-2-hydroxyethylpiperazine-N ′-2-ethanesulfonic acid) buffer (136.7 m M NaCl, 5.5 m M dextrose, 2.6 m M KCl, 13.8 m M NaHCO3, 1 m M MgCl2, 0.36 m M NaH2PO4, and 10 m M HEPES; pH 7.4). Platelet concentration was adjusted to 3.8 × 108 platelets/ml.
Washed platelet aggregation
Aggregation was quantified by the change in light transmission using an AG-10 Aggregation Analyzer (Kowa, Japan) (4). Washed platelets were maintained at room temperature and the study was performed within 3 h of blood collection. The platelet suspension (400 μl) was pipetted into an aggregation cuvette and allowed to incubate with stirring at 1,000 rpm at 37°C for 1 min. Drug or vehicle (dimethyl sulfoxide) was then added (0.4 μl) and incubated for another 3 min. When testing the adenosine synergism, 1 μM of adenosine was added 1 min following the addition of drug. Then the suspension was stimulated with collagen (Chrono-Log Corp., Havertown, PA, U.S.A.). Maximal light transmission values after collagen stimulation are presented as the percentage of control aggregation.
Whole blood platelet aggregation
Blood samples were diluted 1:1 with physiologic saline and tests were performed using a Chrono-Log Whole Blood Aggregometer (Chrono-Log Corp.) with a stirring rate of 1,000 rpm. Diluted whole blood (1 ml) was added into a plastic cuvette and allowed to incubate at 37°C. Test agents (1 μl) were incubated for 3 min before collagen was added. The collagen concentration chosen was the one that allowed cilostazol or milrinone to inhibit 30–50% of platelet aggregation when compared with control (dimethyl sulfoxide). ZM241385 (100 n M, Tocris Cookson, Ballwin, MO, U.S.A.) or CGS15943 (Sigma RBI, St. Louis, MO, U.S.A.) was added 1 min after the addition of the drugs. Administration of adenosine deaminase (2 U/ml, Boehringer Mannheim Corp., Indianapolis, IN, U.S.A.) was added immediately before the addition of cilostazol or milrinone. Adenosine deaminase alone at this concentration was tested without any effect on the platelet aggregation. The maximal amplitude in impedance after collagen stimulation was used and the data were expressed as the percentage of the controls.
cAMP measurement in washed platelets
Test agents alone or in combinations were mixed with 200 μl of washed platelets in triplicates by brief vortex, followed by incubation at 37°C for 3 min. The reaction was terminated by adding 50 μl of ice-cold perchloric acid (1.25 N). After freezing and thawing once, the mixture was neutralized with 50 μl of KHCO3 (1.25 N) and centrifuged at 20,000 ×g for 15 min at 4°C. The resulting supernatants were collected and diluted with acetate buffer provided with the kit. The cAMP concentration was measured in duplicates using a cAMP radioimmunoassay kit (NEK-033, NEN Life Science, Boston, MA, U.S.A.).
Adenosine uptake into washed platelets and erythrocytes
Washed erythrocytes were prepared as follows: After initial centrifugation and removal of PRP and buffy coat, 100 μl of the red pellet portion was diluted into 12 ml of phosphate-buffered saline (PBS) containing calcium and magnesium. Erythrocytes were spun at 150 g for 5 min. After one more wash with PBS, the pellet was resuspended in PBS to 1 × 108 erythrocytes/ml. Adenosine uptake experiments were performed according to the method described previously (5). Washed platelets, 100 μl, or washed erythrocytes were incubated with 50 μl of cilostazol or milrinone at 37°C for 5 min. Then 50 μl of 1 μCi of [3H]-adenosine (Amersham Pharmacia, Piscataway, NJ, U.S.A.), 1 μM adenosine, and 25 μM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA, final concentration, Sigma Chemical) were added, followed by 200 μl oil (dibutyl phthalate: dioctyl phthalate = 1:1, Aldrich) and then incubated for 1 min. The cells were separated from free adenosine in the water phase by centrifugation at 16,000 g for 2 min. After removing the oil and water phases, the radioactivity of the cell pellet was measured using a β-liquid scintillation counter (1209 Rackbeta, LKB, Turku, Finland).
Establishment of Chinese hamster ovary cells expressing human adenosine A2A receptor
Total RNA was extracted from fresh human platelets and 5 μg was reverse transcribed into cDNA and used as a template for the polymerase chain reaction. Specific primers with a Kozak sequence (CCCACC) for A2A adenosine receptor were designed (forward primer: 5´-CCCACCATGCCCATCATGGGCT-3´, reverse primer: 5´-TCAGGACACTCCTGCTCC-3´) and synthesized by Life Technologies (Rockville, MD, U.S.A.). Using these primers, full coding regions were amplified by polymerase chain reaction and further recombined into the cloning vector, pCR2.1 (Invitrogen, Carlsbad, CA, U.S.A.). The DNA sequence of the insert was confirmed before being inserted into the mammalian expression vector, pcDNA3.1+ (Invitrogen). An expression vector (pCRE-Luc) containing a cAMP-response element in the promoter region, which drives the expression of luciferase, was purchased from Stratagene (La Jolla, CA, U.S.A.). The level of luciferase expression therefore reflects the concentration of intracellular cAMP. It is known that adenosine A2A receptor is coupled to Gs proteins (8). Therefore, the activation of the receptors would be reflected by luciferase expression, where the expression level can be measured by the luciferase activity assay. Co-transfection of the luciferase reporting vector with the vectors containing A2A receptors was carried out by calcium phosphate precipitation into Chinese hamster ovary (CHO) cells. Stable transfectants were selected with 1.0 mg/ml G418 (Life Technologies) for 12 days. The cell clones overexpressing functional A2A receptors were determined by luciferase expression under the stimulation of adenosine.
To test the synergistic effect of cilostazol with adenosine on cAMP elevation, the cells were subcultured into a white-wall 96-well plate with clear bottom (Corning Costar Co., Cambridge, MA, U.S.A.) at near-confluence. The next day, the cells were washed once with F12K medium supplemented with 0.5% fetal bovine serum and then incubated with 100 μl of the medium only (basal), plus forskolin (0.03 μM, a direct adenylyl cyclase activator used as positive control), or plus test agents for 4 h at 37°C. After equilibrating to room temperature, 100 μl of detection substrate (Bright-Gl luciferase assay system, Promega, Madison, WI, U.S.A.) was added to each well. The luciferase activity was measured after 5 min using a Mediators PhL luminescence plate reader (ImmTech, New Windsor, MD, U.S.A.). The value of luminescence (arbitrary unit) detected during half a second was taken as luciferase activity.
Measurement of adenosine concentration in plasma
Blood was drawn and mixed with recombinant human hirudin (100 units/ml). PRP and washed platelets were prepared as described previously. The number of platelets in both the PRP and washed platelets suspension was the same. The same procedure for platelet aggregation was used to stimulate these platelets with either collagen (2 μg/ml) or add ATP (100 μM). After a 5-min incubation, 500 μl of whole blood, PRP, or washed platelets was mixed quickly with 500 μl of ice-cold saline. The cells were spun at 20,000 ×g for 4 min at 4°C. Supernatant (600 μl) was first mixed with 300 μl perchloric acid (2.5 N) and then followed by 300 μl of KHCO3 (2.5 M). Finally, the mixture was centrifuged at 20,000 ×g for 15 min at 4°C. The adenosine concentration in the supernatants was measured using reverse-phase high-performance liquid chromatography (Waters, Watford, U.K., Alliance 2690) with a Hypersil 3μ C18 column (150 mm × 4.6 mm) and a gradient from 5–20% methanol in 20 m M of KH2PO4. Adenosine was detected using a diode array detector (Water 996) with an absorbance change at 258 nm and quantified by comparison of retention times and peak height with those of a known external standard. Quantifications were performed using Waters Millennium 32 Client/Server software (5). Detection limit for adenosine is 30 n M.
Radioligand binding assays for A2A receptor
Membrane preparations of CHO cells overexpressing human A2A adenosine receptor and [carboxyethyl-3H-(N)]CGS 21680 ([3H]CGS 21680) were both purchased from NEN Life Sciences. Triplicate samples of [3H]CGS 21680 (20 and 30 n M; 30 ci/mmol) were incubated with membrane suspension (14 μg protein/tube) in tromethamine hydrochloride buffer (50 m M tromethamine hydrochloride, 10 m M MgCl2, and 1 m M ethylenediamine tetra-acetic acid; pH 7.4) at ambient temperature (19–25°C) for 1 h and in the absence (total) or presence (nonspecific) of 10 μM unlabeled CGS 21680 (Sigma RBI). To determine any effects of cilostazol on the binding, 10 μM of cilostazol was included. Specific binding was obtained by subtracting nonspecific from total binding. Incubations were terminated by the addition of 4 ml of ice-cold buffer (50 m M tromethamine hydrochloride, 0.9% NaCl, pH 7.4), followed by the collection of membranes onto Reeves angel 934AH filters (Environmental Express, Mt. Pleasant, SC, U.S.A.) (Brandel, presoaked in 0.5% polyethylenimine) by vacuum filtration. Filters were washed three times with 3 ml ice-cold tromethamine hydrochloride buffer to remove unbound ligand. Filter disks containing trapped membrane protein and radioligand were placed in 2.5 ml of Ultra Gold XR (Packard, Meriden, CT, U.S.A.) and the radioactivity was quantified using a liquid scintillation counter (1209 Rackbeta, LKB). Dissociation experiments were performed by adding CGS 21680 (300 n M) alone or with cilostazol (10 μM), following a 1-h incubation of [3H]CGS 21680 (20 n M) with the membrane. Displacement of ligand binding was terminated every 5 min up to 25 min.
All data are expressed as the mean ± SD. Differences between groups were statistically analyzed by means of analysis of variance with the post hoc Newman-Keuls test or Student t test. Values of p < 0.05 were considered statistically significant.
To study the synergistic effect of adenosine and cilostazol on washed platelet aggregation, the amount of collagen was titrated for each individual donor in the presence of 1 μM of adenosine. The minimum concentration of collagen (1–5 μg/ml) in which 1 μM of adenosine showed no effect on aggregation was used. Cilostazol (1 μM) or adenosine (1 μM) by itself had little effect on collagen-induced platelet aggregation (Fig. 1A: a,b,c). However, combining both completely inhibited platelet aggregation (Fig. 1A: d). The concentration-dependent inhibition of collagen-induced aggregation of washed platelets by cilostazol and milrinone was compared in the absence and presence of adenosine. As shown in Fig. 1B, both cilostazol and milrinone dose-dependently inhibited platelet aggregation. Addition of adenosine (1 μM) shifted the inhibitory curve to the left for both cilostazol and milrinone. The calculated median inhibitory concentration (IC50) was reduced from 2.66 ± 0.41 μM (n = 6) to 0.38 ± 0.05 μM (n = 5, p < 0.001) for cilostazol and from 2.07 ± 0.22 μM (n = 6) to 0.47 ± 0.07 μM (n = 7, p < 0.001) for milrinone. Without adenosine, cilostazol was slightly less potent than milrinone. However, cilostazol became more potent when combined with adenosine. The range of shifting by adenosine when combined with cilostazol is significantly larger than that with milrinone (p < 0.01).
We next measured the intracellular cAMP concentration in washed platelets. Adenosine (1 μM) in combination with cilostazol (0.5 μM) significantly increased cAMP levels (p < 0.05 versus cilostazol alone, n = 3, Fig. 2), whereas adenosine (1 μM), cilostazol (0.5 μM), or milrinone (0.5 μM) alone elevated cAMP levels to a lesser extent. Higher concentrations of cilostazol or milrinone (10 μM) significantly increased cAMP levels (p < 0.05 versus basal). Further increment was achieved by the addition of adenosine (1 μM, p < 0.05 versus cilostazol or milrinone alone). The increase of cAMP by the combination of cilostazol (10 μM) with adenosine was much greater (p < 0.01) than that by the combination of milrinone and adenosine, though cilostazol and milrinone individually increased cAMP equally. A similar trend was also seen with a combination of 0.5 μM cilostazol or milrinone with adenosine but did not reach statistical significance. These differences may be due to the effect of cilostazol on adenosine uptake, as demonstrated previously in other cells (5).
[3H]-adenosine uptake experiments were performed with washed platelets and washed erythrocytes and the results are shown in Fig. 3. Cilostazol inhibited adenosine uptake in both platelets and erythrocytes with an IC50 of about 7 μM (n = 3). The potency of cilostazol on the uptake inhibition is similar to the values reported previously on rabbit cardiac myocytes, human vascular smooth muscle, and endothelial cells (5–10 μM) (5). In contrast, milrinone had virtually no effect on adenosine uptake by platelets or erythrocytes. To further confirm the role of cilostazol in inhibiting the adenosine uptake, we established a CHO cell line overexpressing functional human A2A receptors. Cilostazol inhibited [3H]-adenosine uptake into CHO cells with similar potency to platelets and erythrocytes, whereas milrinone had no effect (data not shown). Cilostazol (up to 50 μM) inhibited cAMP PDE activity in CHO cells by < 10%. Milrinone is two- to threefold more potent than cilostazol on inhibiting cAMP metabolism in these cells (unpublished observation), probably due to its PDE-inhibitory effect (e.g., on PDE4). Intracellular cAMP levels were measured by luciferase assay (see “Methods”). In mock-transfected cells, milrinone slightly increased luciferase expression after a 4-h incubation whereas cilostazol had no effect (Fig. 4A). Addition of 0.03 μM of forskolin also elevated luciferase expression, and a further increase was observed when combined with milrinone (p < 0.05 versus basal). In A2A receptor–expressing CHO cells, cilostazol increased luciferase activity (p < 0.05 versus basal) to a much greater extent than milrinone alone, or when they were combined with forskolin (0.03 μM, p < 0.01 versus forskolin alone) or with adenosine (0.3 μM, p < 0.01 versus cilostazol or adenosine alone, Fig. 4B). Significant difference was seen between the combination of cilostazol and milrinone with adenosine (p < 0.01). The enhancement of luciferase activity was also demonstrated by the addition of a potent adenosine uptake inhibitor, dipyridamole (1 μM) to adenosine (0.3 μM, p < 0.01 versus dipyridamole alone) and to adenosine (0.3 μM) plus milrinone (50 μM, p < 0.01). The increase of luciferase expression induced by adenosine was reversed by 100 n M of ZM241385, an A2A receptor–selective antagonist (13). These observations strongly support the notion that the inhibitory effect of cilostazol on adenosine uptake contributed to the enhanced elevation of intracellular cAMP levels.
Cardiac myocytes have been shown to be a main source of adenosine during cardiac ischemia (5,14). Though activated platelets may be another important source for adenosine generation, no measurement of adenosine levels was performed (15). Using high-performance liquid chromatography, we measured adenosine concentrations in the extracellular medium of whole blood, PRP, and washed platelets 5 min after stimulating with 2 μg/ml collagen. As shown in Fig. 5, a large amount of adenosine (in the μM range) was generated in whole blood and PRP after collagen stimulation; however, a very small amount was detected in the buffer of activated washed platelets. These results suggest that the majority of adenosine came from degradation of released ATP and ADP in the plasma rather than the direct release of adenosine from activated platelets. This was supported by the following experiments. AMPCP (100 μM, Sigma RBI), a 5´-nucleotidase inhibitor, inhibited adenosine generation in such a case (Table 1). Furthermore, addition of ATP (100 μM) to platelet-rich and platelet-poor plasma, but not to washed platelets, increased adenosine concentration equivalent to collagen stimulation (Table 1). Neither cilostazol nor milrinone was shown with any effect on adenosine generation under similar conditions. As cilostazol and milrinone inhibit platelet aggregation, we were unable to compare the adenosine concentration in the plasma after collagen stimulation. When cilostazol (50 μM) was incubated with 100 μM of ATP in whole blood at 37°C for 5 min, plasma adenosine concentration increased to 582 ± 72 n M, in comparison with controls (410 ± 57 n M) and milrinone (50 μM, 383 ± 40 n M, n = 3), though no statistically significant difference was found. The newly generated adenosine during platelet activation may play an important role in preventing subsequent thrombus formation.
To establish whether the adenosine uptake effect of cilostazol plays a role in inhibiting platelet activation, we next studied collagen (0.1–2 μg/ml)-induced platelet aggregation in whole blood without adding any exogenous adenosine, because we already knew from these experiments that a large amount of adenosine can be generated in whole blood during platelet activation by collagen stimulation. As shown in Fig. 5, ZM241385 (100 n M) reversed the inhibitory effect of cilostazol from 47.6 ± 3.4% (3 μM) of control to 72.6 ± 3.1% and from 33.3 ± 7.0% (100 μM) of control to 56.9 ± 5.7% (both p < 0.05), without affecting the inhibitory effect of milrinone. Another structurally different A2A receptor antagonist, CGS15943 (1 μM), was shown to have similar effect with cilostazol (data not shown). Furthermore, adenosine deaminase (2 U/ml), which degrades adenosine in the plasma, was also shown to reverse the inhibitory effect of cilostazol (100 μM) significantly (to 54.3 ± 3.8%), but without any effect on that of milrinone (Fig. 5).
Some compounds (e.g., PD 81,723) have been shown to have an allosteric effect on adenosine receptors (16). We investigated any possible direct effect of cilostazol on A2A receptor with radioligand membrane binding studies. Cilostazol (0.3, 1, 3, and 10 μM) had no effect on either saturation or time-dependent dissociation of [3H]CGS15943 binding to the membrane of CHO cells expressing A2A receptor (data not shown). These results ruled out any possible allosteric effect of cilostazol on the A2A receptor in the platelets.
In the present studies, we have demonstrated that cilostazol and adenosine synergistically increase intracellular accumulation of cAMP levels, resulting in enhanced inhibitory effect on platelet aggregation. By comparing with another PDE3-selective inhibitor, milrinone, an additional inhibitory effect of cilostazol on adenosine uptake in platelets and erythrocytes was revealed. In combination with increased generation of adenosine during platelet activation, this effect of cilostazol was shown to play an additional role during platelet activation in whole blood. The mechanism by which cilostazol inhibits adenosine uptake remains to be determined.
Adenosine has been shown to regulate platelet function. Many investigators have demonstrated the anti-platelet activity of adenosine by adding exogenous adenosine. In addition, it has been shown that lowering plasma adenosine levels decreased platelet cAMP concentrations and the platelet response to various agonists was greatly increased (17). In contrast, nucleoside transport inhibitor such as dipyridamole, which causes an increase in plasma adenosine levels, was shown to suppress agonist-induced platelet activation (10). Rat plasma contains much higher adenosine levels (586 ± 124 n M) as compared with human plasma (230 ± 90 n M) (17). Accordingly, a five- to 10-fold greater amount of collagen was required to induce platelet aggregation in rats as compared with humans. This is perhaps due to a higher concentration of plasma adenosine (9). The anti-platelet actions of adenosine were previously shown to be potentiated by direct activation of adenylyl cyclase with forskolin (18) or with the inhibitors of cAMP PDE (e.g., HL-725, EG626, RA 233, IBMX, milrinone) (9–12), both of which elevate intraplatelet cAMP levels. Similarly the enhancement of the inhibitory effect of cilostazol in the presence of low adenosine concentration was observed on collagen-induced platelet aggregation in the present studies. It has been shown previously that endothelium-derived prostacyclin enhanced the anti-platelet aggregatory effect of cilostazol (19).
Both vascular endothelium and platelets contribute significantly to the plasma adenosine levels. As shown in the present studies, adenosine can be generated from large amounts of adenine nucleotides released from platelets by ecto-ATPase in the plasma (or on the surface of endothelial cells in vivo) and by 5´-nucleotidase (20,21). The concentration of locally generated adenosine may be high enough to prevent further platelet recruitment, thus inhibiting large thrombus growth, similar to that proposed in the case of newly released nitric oxide from activated platelets (22). Human plasma normally contains low levels of adenosine (100–300 n M) because of rapid uptake and metabolism by erythrocytes (10). The disappearance of adenosine can be reduced or retarded by adenosine uptake inhibitors. Experiments have shown that, in human whole blood, [14C]-adenosine had a half-life (t1/2) of < 15 s in the plasma. In the presence of dipyridamole, the t1/2 increased to about 5 min (23). Also, exogenous adenosine (IC50, 2 μM) was strongly inhibitory to platelet aggregation in the presence of dipyridamole, compared with no significant effect at 10–50 μM in its absence (24). Cilostazol is slightly weaker than milrinone in inhibiting the activity of PDE3A from human platelets (4), consistent with the present platelet aggregation study. Although the dose-dependent inhibition curves for both cilostazol and milrinone were shifted to the left in the presence of 1 μM adenosine, the shift for cilostazol was significantly larger than that of milrinone. We have ruled out any direct allosteric effect of cilostazol on adenosine receptor by radioligand binding studies. The release of ATP and adenosine from cultured CHO cells seemed not affected by cilostazol. Using luciferase assay and high-performance liquid chromatography to detect released ATP and adenosine in the cell culture medium, we did not observe any increase in the release of ATP or adenosine from the CHO cells treated with cilostazol. Experiments on [3H]-adenosine uptake by CHO cells were performed in the presence of forskolin (1 μM) to increase intracellular cAMP. Increased cAMP concentrations did not affect adenosine uptake (data not shown). The degradation of ATP in plasma was not altered by cilostazol, in agreement with previous observations that cilostazol did not affect 5´-nucleotidase activity (5). The fact that cilostazol inhibited adenosine uptake by platelets and erythrocytes, as shown in Fig. 3, suggests that the relatively higher potency of cilostazol than milrinone in inhibiting platelet aggregation is partially due to adenosine uptake inhibition. This is further supported by the whole blood platelet aggregation studies, in which the effect of cilostazol was reversed partially by adenosine receptor antagonists (ZM241385 and CGS15943) and by adenosine deaminase. However, ZM241385 and adenosine deaminase had no effect on milrinone-mediated platelet inhibition (Fig. 5).
Cilostazol is currently used to treat intermittent claudication. The therapeutic effect of cilostazol was generally believed to be mediated by platelet inhibition and vasodilation due to PDE3 inhibition (1,2). The additional effect of cilostazol on adenosine uptake, as demonstrated in the present and previous (5) studies, may also contribute to its overall anti-platelet aggregation and vasodilation effect in PAOD patients. Intermittent claudication is a condition caused by reduced blood flow to the legs, resulting in a characteristic pain on walking. It is known that ischemia can cause adenosine concentration increase by several-fold. Energy-demanding activities, such as exercise, may also increase adenosine in tissues considerably. Two clinical studies have demonstrated beneficial effect of inhibiting adenosine uptake by dipyridamole in patients with intermittent claudication (25,26). Furthermore, adenosine is recognized as an important mediator in cardiac preconditioning, which protects subsequent ischemic cardiac tissue from infarction (14). In patients with intermittent claudication, it was recently suggested by Capecchi et al. (27) that, based on an experimental model, adenosine may also be involved in short-term exercise-induced preconditioning in limb skeletal muscle. By inhibiting adenosine uptake, cilostazol has been previously shown to increase the accumulation of interstitial adenosine and to augment preconditioning in ischemic tissues (5). Together with the present studies of cilostazol on platelet aggregation with adenosine, our findings suggest that combination of PDE with adenosine uptake inhibition may provide additional therapeutic efficacy in intermittent claudication.
We have demonstrated a new mechanism of action for cilostazol on adenosine uptake inhibition, which may enhance the synergistic effect between Gs protein–coupled receptor activation (e.g., by adenosine or prostaglandin I2) and PDE3 inhibition in inhibiting platelet activation, thrombus formation, and vasodilation. The inhibitory effects of cilostazol on both adenosine uptake and PDE3 may account for the superior therapeutic effect of cilostazol in the treatment of intermittent claudication.
The authors would like to thank Jess Li for expression vector construction and DNA sequencing, James Hensley and Dr. Yasmin Shakur for the determination of PDE activity in the CHO cells, and Marina Samuel for adenosine measurement.
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