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

Abstract

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‐hydroxyphenyl)‐13,14‐dihydro‐13‐aza‐15‐β‐ω‐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‐methanothromboxane A2 (STA2). [125I]‐PTA‐OH bound to washed human platelets in a reversible, saturable and temperature‐dependent manner, and specific binding displaced by 20 μM 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 108 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 μM. PGE1 and thromboxane B2 (TXB2) were without effect up to 100 μM. Similar binding of [125I]‐PTA‐OH was observed on dog platelets. The KD and Bmax were 12 nM and 110 fmol per 108 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 [125I]‐PTA‐OH was found on rabbit platelets. Consistent with the results from binding studies, ONO‐11120, 0.5 μM, completely suppressed arachidonate‐induced aggregation of human platelets, whereas, at concentrations up to 5 μM, 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‐11120. When a similar extent of irreversible aggregation was induced by STA2 and the inhibitory potency of ONO‐11120 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. These results suggest that [125I]‐PTA‐OH binds to a platelet thromboxane receptor, and that the structure of the binding site(s) on the receptor may vary between species.

Thromboxane A2, released by the anti‐ tumour drug irinotecan, is a novel stimulator of Cl− secretion in isolated rat colon

“…STA2 is a highly potent agonist of TXA2 receptors in smooth muscle; it contracted the canine and human saphenous veins with EC50 values of 0·2-0·4 nÒ (Mais et al 1985) and the canine cerebral, coronary, renal and mesenteric arteries with EC50 values of 0·9-1·1 nÒ (Toda, Nakajima, Okamura & Miyazaki, 1986). In contrast, a higher concentration of STA2 was required to produce platelet aggregation; the EC50 values for human (Mais et al 1985;Narumiya, Okuma & Ushikubi, 1986) and rabbit (Narumiya et al 1986) plateletrich plasma were 1·1-1·8 ìÒ and 8·2 ìÒ, respectively. In the present study, STA2 acted as an intermediary potent agonist, causing Cl¦ secretion in the rat colon with an EC50 value of more than 30 nÒ (Fig.…”

Heterogeneity of thromboxane A2 (TP‐) receptors: evidence from antagonist but not agonist potency measurements

“…Its aggregatory potency appears to be lower: EC50 = 0.4flM (Katsura et al, 1983) and 1.1UM (Mais et al, 1985) for human PRP, 0.68juM (Mais et al, 1985) for dog PRP and 8jM for rabbit PRP (Narumiya et al, 1986).…”

“…In a preliminary report Anderson & MacIntyre (1982) also showed that EP 045 and several other TP-receptor antagonists were less potent as inhibitors of U-46619-induced aggregation in rabbit platelet-rich plasma (PRP) compared with human PRP. More extensive studies (Narumiya et al, 1986) have shown that the TP antagonist, ONO 11120 (Katsura et al, 1983) (Figure 1), is a less effective inhibitor of aggregation induced by STA2 (a stable TXA2 analogue, Figure 1) in rabbit PRP than in human PRP. In addition, radiolabelled I-PTA-OH, a close relative of ONO 11120, was found to bind with high affinity to TP-receptors on human and dog washed platelets but not rabbit washed platelets.…”

Distinct platelet thromboxane A2/prostaglandin H2 receptor subtypes. A radioligand binding study of human platelets.

“…Detailed studies of the human platelet TXA2/PGH2 receptor have also been performed with ['25I]PTA-OH, a stable radioiodinated TXA2 antagonist. Two different groups have re-ported that [125I]PTA-OH binds to a single site with a capacity of -2,000 sites per platelet (9,10). Though [1251]PTA-OH has a higher specific activity and greater affinity for the receptor than does [3H]U46619, its properties as an antagonist may make it less than ideal for discriminating between different TXA2/PGH2 agonist binding sites (11)(12)(13).…”