Evaluation of the Binding to Fixed Platelets of Agonists and Antagonists of ADP-Induced Aggregation

Abstract

SummarySteady state binding of eleven different ADP analogues to formaldehyde-fixed platelets has been determined in a competitive binding assay using 3H-ADP. The compounds tested were the inactive analogues L-ADP and L-ATP; the agonists 2-chloroadenosine 5’-diphosphate, adenosine 5’-O-(2-thiodiphosphate)and the diastereoisomeric pair Sp-adenosine 5’-(1-thiodiphosphate) (Sp-ADP-α-S) and Rp-adenosine 5’-(1-thiodiphosphate) (Rp-ADP-α-S); and the antagonists adenosine 5’-O-thiomonophosphate, 2-chloroadenosine 5’-O-thiomonophosphate, 2-chloroadenosine 5’-triphoshate, and the diastereoisomeric pair 5’-(1-thiotriphosphate) (Sp-ATP-α-S) and Rp-adenosine 5’-(1-thiotriphosphate) (Rp-ATP-α-S). All compounds tested competed at the high affinity binding sites for ADP previously identified (Blood 1988; 71: 110-6) but in some cases competition could not be demonstrated at the low affinity sites because of the high nucleotide concentrations required. As a group, C2-substituted analogues bound less strongly (Ki >2 μM) than did the analogues without substituents in the purine ring (Ki <0.7 μM). With the pair of diastereoisomeric agonists Sp-ADP-α-S and Rp-ADP-α-S the Ki values at the high affinity site (210 nM and 560 nM) were of the same relative magnitude and in the same direction as their reported potencies as agonists (Ki 4 μM and 20 μM). With the diastereoisomeric antagonists Sp-ATP-α-S and Rp-ATP-α-S a similar relationship was seen between affinity (17 nM and 156 nM) and inhibitory potency (Ki 4 μM and 20 μM). These results may help to differentiate possible mechanisms in the interaction of ADP with its receptors.