Characterization of platelet thromboxane A 2/prostaglandin H 2 receptor by a novel thromboxane receptor antagonist, [ 3H]S-145

Abstract

The specific binding sites for S-145, a novel thromboxane A 2/prostaglandin H 2 (TXA 2/PGH 2) receptor antagonist with weak partial agonistic activity, were studied in human platelet membranes. [ 3H]S-145 displayed high affinity and specificity, as well as saturable and displaceable binding, to a single class of recognition sites with the same maximum number of sites (2100 fmol/mg protein) as the other two TXA 2/PGH 2 receptor antagonists, [ 3H]SQ29,548 and [ 3H]ONO3708. Binding of S-145 to the platelet membranes was enhanced by divalent cations (Mg 2+ and Ca 2+), and the binding affinity in the presence of 20 mM MgCl 2 was 0.75 nM, a value which was smaller than those of SQ29,548 (8.7nM) and ONO3708 (3.7 nM). The rank order of potency ( K i) for a series of TXA 2/PGH 2 receptor antagonists to displace [ 3H]S-145 binding to the membranes was correlated with those determined from [ 3H]SQ29,548 or [ 3H]ONO3708 binding to the same preparations. Kinetic analysis for the binding of the above radiolabeled antagonist to the crude platelet membranes revealed that the dissociation rate constant ( K −1) for S-145 was much smaller than that for other ligands in human, rat and rabbit platelets. The extremely slow dissociation of S-145 from the receptors may explain the long-lasting characteristic of this compound in vivo as well as the abolishment of partial agonistic activity.