Evidence for the existence of two different ADP-binding sites on rat platelets

Abstract

[ 3H]-2-Methylthio-ADP ( [ 3H]-2-MeS-ADP ), a stable analogue of ADP bound to one type of specific binding sites on rat platelets ( K D = 0.77 ± 0.07 nM, Bmax = 160 ± 11 fmol/10 8 cells ). 2-MeS-ADP and ADP antagonized [ 3H]-2-MeS-ADP binding, showing respective Ki values of 1.4 ± 0.1 nM and 486 ± 78 nM. Clopidogrel, a potent and specific inhibitor of ADP-induced platelet aggregation partially inhibited ( ∼70% inhibition ) the binding of [ 3H]-2-MeS-ADP at the same time it abrogated 2-MeS-ADP-and ADP-induced adenylyl cyclase inhibition and aggregation. A population of clopidogrel-resistant [ 3H]-2-MeS-ADP binding sites was detected on platelets from treated animals. These receptor sites ( K D = 0.9 ± 0.2 nM, Bmax = 47 ± 5 fmol/10 8 platelets ) which showed high affinity for both ADP and 2-MeS-ADP (Ki values in the nanomolar range) might be involved in the ADP-induced shape change, a clopidogrel-resistant ADP-induced event. Using clopidogrel which acts via a direct and irreversible inhibition of ADP binding to its adenylyl cyclase-coupled receptor sites on platelets, we were able to discriminate between two types of ADP receptor sites. The former which was clopidogrel-sensitive represented about 70% of the total [ 3H]-2-MeS-ADP receptors and was responsible for ADP-induced platelet aggregation and adenylyl cyclase inhibition. The latter which was not affected by clopidogrel might be involved in ADP-induced shape-change.