Effect of clopidogrel treatment on ADP-induced phosphorylations in rat platelets

Phosphorylations induced by 2-MeS-ADP, a potent agonist of platelet ADP receptors, have been studied in rat platelets, and the effect of clopidogrel, a compound which inhibits platelet aggregation by selectively reducing the binding of ADP to its low affinity receptors on platelets, has been determined, 2-MeS-ADP induced platelet activation (shape change and aggregation) simultaneously with the phosphorylation of myosin light chain (P-20) and plekstrin (P-47). Phosphorylation of P-20 and P-47 was transient, a maximum being observed 10s after addition of the agonist when shape change reached its maximum. P-20 and P-47 phosphorylations were not strongly affected by clopidogrel treatment. Following stimulation of platelets with 2-MeS-ADP, several proteins were phosphorylated at tyrosine residues. Clopidogrel treatment inhibited the increase in phosphorylation of P-140, P-100, P-80/85, P-66 and P-55 concomitantly with the inhibition of platelet aggregation. However, clopidogrel did not interfere with the early phosphorylation of the P-80/85 kD doublet which occurs at the time of the shape change. P-80/85, identified by immunodetection as cortactin, could be involved in the reorganization of the cytoskeleton necessary for morphological changes. Thus, by using clopidogrel-treated rat platelets, we were able to determine some of the phosphorylations coupled either to clopidogrel-resistant high-affinity ADP receptors leading to shape change or to clopidogrel sensitive low-affinity ADP receptors coupled to the aggregation process.