Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

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Abstract

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (B(max)) from 1.51 ± 0.11 pmol/mg to 0.51 ± 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (K(d) = 647 ± 64 pM (untreated), 363 ± 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the B(max) to 0.23 ± 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (K(d) = 334 ± 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 ± 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20°C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 ± 0.12 pmol/mg to 2.70 ± 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 ± 106 μM and occurred in less than 1 min at 37°C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP B(max) in platelet membranes to 0.85 ± 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 ± 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20°C. Collectively, these data indicate that the human platelet TXA2/PGH2 receptor possesses disulfide bond(s) and sulfhydryl group(s) located at or near the ligand binding site. The abilities of oxidizing and reducing agents to respectively up and down regulate the density of receptors strongly suggest that the membrane receptor exists in variable redox states and that cyclic disulfide-sulfhydryl interchange reactions are a possible mechanism for acute reversible heterospecific regulation of TXA2/PGH2 receptors in human platelets.

Original languageEnglish
Pages (from-to)4240-4246
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number8
StatePublished - 1990

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