Binding characteristics of the new thromboxane A 2/prostaglandin H 2 receptor antagonist [ 3H]BAY U 3405 to washed human platelets and platelet membranes

Abstract

The new thromboxane A 2 antagonist [ 3H]BAY U 3405 was characterized for its binding to washed human platelets and platelet membranes. In washed platelets the specific binding was reversible, selective and stereospecific, but not saturable. The dissociation constant ( K d) was 6 ± 2.5 nM, the number of specific binding sites 1177 ± 306 per platelet. Three structurally different thromboxane A 2 (TXA 2)/prostaglandin H 2 (prostaglandin endoperoxide) (PGH 2) receptor ligands completely inhibited the specific binding of [ 3H]BAY U 3405 in a concentration-dependent manner, indicating that the observed high affinity binding site is the TXA 2/PGH 2 receptor. In platelet membranes, however, specific [ 3H]BAY U 3405 binding showed saturability in addition to reversibility, selectivity, and stereospecifity. The K d of the binding was 9.6 ± 2.3 nM in kinetic studies and 8.7 ± 3.7 nM in saturation studies, the inhibition constant ( K i) was 10 ± 1.1 nM in displacement studies. The TXA 2/PGH 2 receptor agonists U 46619 and CTA 2, and the antagonists Daltroban (BM 13505), I-PTA-OH and SQ 29548 all completely inhibited the specific binding of [ 3H]BAY U 3405 thus defining the observed binding site as the TXA 2/ PGH 2 receptor. In conclusion, the data suggest that the previously reported TXA 2 antagonism of BAY U 3405 is mediated by binding to a specific high affinity binding site of human platelets and platelet membranes that represents the TXA 2/PGH 2 receptor.