title: Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.
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.
authors.affiliation: Department of Cell and Molecular Pharmacology, and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425.
authors.forename: P R
authors.initials: PR
authors.lastname: Mayeux
authors.forename: T A
authors.initials: TA
authors.lastname: Morinelli
authors.forename: T C
authors.initials: TC
authors.lastname: Williams
authors.forename: E S
authors.initials: ES
authors.lastname: Hazard
authors.forename: D E
authors.initials: DE
authors.lastname: Mais
authors.forename: J E
authors.initials: JE
authors.lastname: Oatis
authors.forename: D A
authors.initials: DA
authors.lastname: Baron
authors.forename: P V
authors.initials: PV
authors.lastname: Halushka
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001792:Blood Platelets / Q000378:metabolism*; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001974:Bromosuccinimide / Q000737:chemistry; D002227:Carbazoles / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006834:Hydrazines / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D015539:Platelet Activation; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D011485:Protein Binding; D011982:Receptors, Prostaglandin / Q000378:metabolism*; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D012995:Solubility; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002227:Carbazoles; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D008024:Ligands; D011450:Prostaglandin Endoperoxides, Synthetic; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; C069994:L 657925; C061750:7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid; D006639:Histidine; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; C037866:STA 2; C045749:SQ 29548; C046957:I-PTA-OH; D001974:Bromosuccinimide; D004047:Diethyl Pyrocarbonate