BINDING OF [H-3] 2-METHYLTHIO ADP TO RAT PLATELETS - EFFECT OF CLOPIDOGREL AND TICLOPIDINE

Thienopyridine compounds, including ticlopidine and clopidogrel, have been found to selectively inhibit adenosine 5' diphosphate (ADP)-induc ed platelet aggregation and adenylyl cyclase ex vivo, but the mechanis m of their antiplatelet action remains to be determined. This study wa s aimed at investigating the effect of clopidogrel and ticlopidine on the binding of [H-3]-2-methylthio-adenosine-5'-diphosphate (2-MeS-ADP) to rat platelets. Binding of [H-3]-2-MeS-ADP to rat platelets was tim e-dependent and saturable. Scatchard analysis of the saturation bindin g data indicated that [H-3]-2-MeS-ADP bound to one population of speci fic binding sites with high affinity (K-D = 0.78 +/- 0.05 nM; B-max = 156.3 +/- 4.8 fmole/10(8) cells) (n = 3). Unlabeled 2-MeS-ADP and ADP competitively and selectively inhibited the specific binding of [H-3]- 2-MeS-ADP with IC50 values of 11.3 +/- 1.2 nM and 11.3 +/- 0.7 mu M, r espectively (n = 3). Other nucleotide analogs such as ADP-beta S, ATP and ATP-alpha S also antagonized [H-3]-2MeS-ADP binding. When administ ered orally at doses ranging from 1 to 25 mg/kg, clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation as well as ADP or 2-Me S-ADP-induced inhibition of intraplatelet adenylyl cyclase. When measu red in parallel, clopidogrel reduced in a dose-dependent manner the bi nding of [H-3]-2-MeS-ADP to rat platelets ex vivo. Clopidogrel adminis tration resulted in the decrease of [H-3]-2-MeS-ADP binding sites on p latelets without any significant change in the affinity; this indicate s noncompetitive binding. Ticlopidine (200 mg/kg a day for 3 days) beh aved in the same way. These effects closely correlated with the compou nds' respective antiaggregating activity, indicating that they were ac ting via a direct, irreversible inhibition of ADP binding to its adeny lyl cyclase-coupled receptor sites on platelets.