Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro

Abstract

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.

Figures

Figure 1

Representative original tracings demonstrating the effects of clopidogrel (0.3–30 μM) on ADP (6 μM)-induced aggregation and shape change of washed human platelets.

Figure 2

Effects of clopidogrel (0.3–30 μM) on ADP (6 μM)-, collagen (2.5 μg ml−1), U46619 (1 μM) or thrombin (0.1 u ml−1)-induced aggregation of washed human platelets. The aggregation was assessed by measuring the maximum change of light transmission (A) and the change of light transmission 4 min after addition of the stimulus (B). Data are given as % of aggregation response induced by the respective agonists without clopidogrel (control=100%). (means±s.e.mean of n=5–6 independent experiments, *P<0.05 vs control, Student's t-test for paired data).

Figure 3

Original tracings demonstrating the effects clopidogrel (30 μM) on ADP (0.2–20 μM)-induced platelet shape change. The experiments were carried out in a Ca2+- and fibrinogen-free medium supplemented with 2 mM EGTA. Shown is one representative experiment of n=3 with similar results.

Figure 4

Effects of clopidogrel (30 μM) on collagen (2.5 μg ml−1) (A) and U46619 (1 μM) (B)-induced aggregation of acetylsalicylic acid (10 μM)-treated washed human platelets. Experiments were carried out in the absence and presence of apyrase (1 u ml−1). The aggregation was assessed by measuring the maximum change of light transmission. Data are given as % of aggregation response induced by the respective agonists without clopidogrel (control=100%). (means±s.e.mean of n=4 independent experiments, *P<0.05 vs control, Student's t-test for paired data).

Figure 5

ADP (0.2–600 μM)-induced platelet aggregation in control and in clopidogrel (30 μM)-treated washed platelets. Data are given as % of maximum aggregation response (control=100%). (means ±s.e.mean of n=3 independent experiments).

Figure 6

Original tracings demonstrating the time course of the inhibitory actions of clopidogrel (30 μM) on ADP (6 μM)-induced platelet aggregation. Shown is one representative experiment of n=4 with similar results.

Figure 7

Effects of clopidogrel (3–30 μM) on the inhibitory effects of ADP (6 μM) on prostaglandin E1 (PGE1, 1 μM)-stimulated platelet adenylate cyclase. (means±s.e.mean of n=4 independent experiments, *P<0.05 vs control, #P<0.05 vs PGE1, Student's t-test for unpaired data).