Solubilization of a thromboxane A2/prostaglandin H2 antagonist binding site from human platelets.

A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-hydroxyph eny l)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, was solubilized into the 200,000 X g supernatant from human platelet membranes by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Binding to the solubilized site was saturable, displaceable, and reversible. Displaceable binding was not affected by sodium, potassium, or phosphate concentrations up to 50 mM or by magnesium to 5 mM but was increased 14% (P less than 0.05) by 5 mM calcium. A pH optimum for displaceable binding occurred between pH 7.0 and 7.5. Scatchard analysis of [125I]-PTA-OH binding to the solubilized binding site revealed a single class of sites, having a dissociation constant (Kd) of 66 +/- 16 nM (n = 3) and a Bmax of 750 +/- 80 fmol/mg of protein. The Kd for the membranes prior to solubilization was 47 +/- 11 nM (n = 3) and the Bmax was 700 +/- 90 fmol sites per mg of protein. The association rate constant, k1, was 1.57 X 10(7) M-1 X min-1 and the dissociation rate constant, k-1, was 0.61 +/- 0.04 min-1 (n = 4), yielding a Kd (k-1/k1) of 39 nM. Several thromboxane A2/prostaglandin H2 agonists and antagonists displaced bound [125I]-PTA-OH at concentrations similar to those at which they affect platelet aggregation. Collectively, these observations suggest that the solubilized protein is the thromboxane A2/prostaglandin H2 binding site that mediates platelet aggregation.