Binding of a radioiodinated 13‐azapinane thromboxane antagonist to platelets: correlation with antiaggregatory activity in different species

Binding of a 125I-labelled derivative of the 13-azapinane thromboxane antagonist (ONO-11120), 125I-9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyp hen yl)-13, 14-dihydro-13-aza-15-beta-omega-tetranor-thromboxane A2 (125I-PTA-OH), to washed platelets of human, dog and rabbit was studied. Results were compared with the in vitro inhibitory potency of ONO-11120 on platelet aggregation induced by arachidonate and a thromboxane agonist, 9,11-epithio-11,12-methano-thromboxane A2 (STA2). 125I-PTA-OH bound to washed human platelets in a reversible, saturable and temperature-dependent manner, and specific binding displaced by 20 microM ONO-11120 constituted about 40% of the total binding. Scatchard analyses revealed a single class of specific binding and the equilibrium dissociation constant (KD) and maximal concentration of binding sites (Bmax) were 22 nM and 390 fmol per 10(8) platelets (about 2,300 sites per platelet), respectively. In addition to ONO-11120, STA2 and another thromboxane receptor agonist, (15S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid (U-46619), effectively displaced the binding with IC50 values of 44 and 125 nM respectively. Prostaglandin D2 (PGD2) partially displaced the binding only at a concentration above 1 microM. PGE1 and thromboxane B2 (TXB2) were without effect up to 100 microM. Similar binding of 125I-PTA-OH was observed on dog platelets. The KD and Bmax were 12 nM and 110 fmol per 10(8) platelets (about 680 sites per platelet), respectively, and these values did not change significantly after adrenaline treatment which potentiated arachidonate-induced aggregation of platelets in this species. On the other hand, no specific binding of '251-PTA-OH was found on rabbit platelets. 4 Consistent with the results from binding studies, ONO-11120, 0.5 microM, completely suppressed arachidonate-induced aggregation of human platelets, whereas, at concentrations up to 5 microM, this agent did not significantly inhibit aggregation of rabbit platelets induced by the same stimulus. STA2- induced aggregation of rabbit platelets also showed less sensitivity to ONO-I 1120. When a similar extent ofirreversible aggregation was induced by STA2 and the inhibitory potency ofONO-1 1120 was compared in human and rabbit platelets, about one hundred times greater concentration of ONO- 11120 was required to suppress aggregation of rabbit platelets than that of human platelets. 5 These results suggest that 1251-PTA-OH binds to a platelet thromboxane receptor, and that the structure of the binding site(s) on the receptor may vary between species.