Cyclic Oxidation-Reduction Reactions Regulate Thromboxane A2/Prostaglandin Hz Receptor Number and Affinity in Human Platelet Membranes*

The radiolabeled thromboxane As/prostaglandin HZ (TXAZ/PGHZ) agonist “‘1-BOP bound to the TXAs/ PGHZ 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 “‘IBOP binding (B,,,*J from 1.51 2 0.11 pmol/mg to 0.61 2 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 2 64 pM (untreated), 363 2 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the B,,,** to 0.23 2 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 2 117 pM, p = 0.035 from untreated). Kinetic analysis of 12’I-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 12’I-BOP binding were dose-dependent with an EC&,, of 8.1 2 0.2 mM. DTT inactivation of TXA2/PGHZ receptors was time-dependent with a second order rate constant (&J of 0.123 Mm1 s-l at 20 ‘C. The platelet membrane 12’I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGHZ 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 “‘1-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 12’I-BOP binding sites from 1.65 2 0.12 pmol/mg to 2.70 2 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 12’I-BOP binding were concentration-dependent with an ECbO of 668 2 106 PM and occurred in less than 1 min at 37 ‘C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced “‘1-BOP Bmax in platelet membranes to 0.85 2 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining