Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets

P.R Mayeux,T.A Morinelli,T.C Williams,E.S Hazard,D.E Mais,J.E Oatis,D.A Baron,P.V Halushka

doi:10.1016/s0021-9258(18)92764-x

Abstract

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.

Highlights

From the Departmentof Cell and Molecular Pharmacology, and Experimental Therapeutics, Medical Universityof South Carolina, Charleston, South Carolina29425
The effects of changes in pH on the binding of ago- PGHJ' receptors which when activated by the nativeligands nists and antagonists to the human platelet thromboxT- XA2 and PGHo,r by stable mimeticsp, roduce shape change, ane Az/prostaglandin H, (TXA2/PGH2)receptor were aggregation, secretion, and several other events [1,2,3,4,5]
This study was designed to determine if changes in pH coulddifferentiallyinfluence thebinding of TXA2/PGH2 agonists and antagonists to the human platelet TXA2/PGH2 receptor

Summary

Introduction

From the Departmentof Cell and Molecular Pharmacology, and Experimental Therapeutics, Medical Universityof South Carolina, Charleston, South Carolina29425. Binding assays were performed at pH 7.4 in the presence of 0.01 unit/ml thrombin to determine if the K d or TABLEI Effect of pH on equilibrium bindingof ['Z51]BOP to washed human platelets N = 5; all values are means f S.E. B,,, values for ['251]BOPbinding to platelets in a state of changed shape was different than in thaebsence of thrombin.

Results

Conclusion