Characterization of U46619 binding in unactivated, intact human platelets and determination of binding site affinities of four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548)

The binding of U46619 and the inhibition of this binding by four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548) were studied in unactivated, intact human platelets. Washed platelets were equilibrated with [3H]-U46619 (5 nM) and the time course of binding determined. The receptor-specific binding reached equilibrium within 2-4 minutes, and could be displaced by addition of excess unlabelled ligand. Saturation of this binding was achieved at 750 nM. Scatchard transformation of the saturation binding curve yielded a single class of binding site with a Kd of 108 nM and Bmax of 360 fmole/10(8) platelets. When [3H]-U46619 (4 nM) was incubated with platelets in the presence of increasing concentrations of the antagonists, binding of U46619 was inhibited in a dose dependent manner. The potency series for inhibition of U46619 binding was: SQ 29,548 (IC50 = 7.9 nM) greater than ONO 3708 (IC50 = 38 nM) greater than BM 13.177 (IC50 = 0.91 microM) greater than 13-APA (IC50 = 6.2 microM). These findings are consistent with the notion that these compounds all act as competitive antagonists at the level of the platelet TXA2/PGH2 receptor.