Inhibition of ligand binding to thromboxane A2/prostaglandin H2 receptors by diethylpyrocarbonate. Protection by receptor ligands and reversal by hydroxylamine.

The potential of histidines to modulate the binding of agonists and antagonists to human platelet thromboxane A2 (TXA2) receptors was investigated. TXA2 receptors were purified from crude platelet membranes via affinity and wheat germ lectin chromatography. Radioligand binding studies were conducted using the TXA2, mimetic [125I]BOP (I-BOP (I-BOP = [1S-(1 alpha,2 beta(5Z),3 alpha(1E, 3R*),4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)7-oxabicyclo- [2.2.1]heptan-2-yl]-5-heptenoic acid) and the TXA2 receptor antagonist [125I]SAP (I-SAP = 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-iodobenzene- sulfonylamino)-bicyclo-[3.1.1]hept-2-yl]-(5Z)-heptenoic acid). The histidine modifying reagent diethyl-pyrocarbonate (DEPC) produced a concentration (30-100 microM) dependent inhibition of binding of both [125I]BOP and [125I]SAP. DEPC treatment significantly (P < 0.05, N = 6) decreased the affinity of the receptor for [125I]SAP (Kd = 2.4 +/- 0.4 and 5.4 +/- 0.4 nM, control and DEPC, respectively) without significantly decreasing the Bmax. The effects of DEPC were reversed by hydroxylamine. The inhibition of [125I]BOP and [125I]SAP binding produced by DEPC was reduced significantly by prior incubation of the purified receptors with the TXA2 receptor agonist U-46619 or the TXA2 receptor antagonist SQ 29548. The results strongly support the notion that one or more histidines reside in a domain that can modulate ligand binding to the TXA2 receptor.