Identification of High-Affinity (Kd 0.35 μmol/L) and Low-Affinity (Kd 7.9 μmol/L) Platelet Binding Sites for ADP and Competition by ADP Analogues

Abstract

AbstractSteady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2-3H]ADP bound to two sites with a small amount (-5% of total) of nonspecific binding: 410,000 ± 40,000 sites of low affinity (Kd 7.9 ± 2.0 μmol/L) and 160,000 ± 20,000 sites of high affinity (Kd 0.35 ± 0.04 μmol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1-0.5 μmol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3-aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 μmol/L and 200 μmol/L, respectively. The partial agonist 2‘ x2018;,3’- dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 μmol/L and 49 μmol/L. respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 μmol/L) = ATP (Kd 0.45 μmol /L) » AMP (Kd 360 μmol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuri-benzene sulfonate (Ki 250 μmol/L) but only very weakly by 5’-p-f1uorosulfonylbenzoyladenosine (K, 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 μmol/L), 2-MeS ADP (200 μmol/L) and 2‘ x2018;,3’-dialdehyde ADP (270 μmol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 μmol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50, -100 μmol/L) but not by collagen, thrombin, or ionophore A23187. These studies (a) provide accurate parameters for the binding of ADP to fixed platelets in the absence of complications due to metabolism and secretion, (b) demonstrate that both high-affinity (Kd 0.35 μmol/L) and low-affinity (Kd 7.9 μmol/L) binding sites are present and that both are accessible to ADP and C-2-substituted ADP analogues; and (c) suggest that the high-affinity binding site corresponds to the receptor modulating platelet activation by ADP. These results help to define possible platelet receptors for ADP and may provide a useful indicator system for evaluating the interaction of agonists and antagonists with platelets.