Subcellular localization of a thromboxane A2/prostaglandin H2 receptor antagonist binding site in human platelets.

The subcellular localization of a binding site for the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4- hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 ([125I]-PTA-OH), was determined. Subcellular fractions of platelets were prepared by glycerol lysis or nitrogen cavitation, and were characterized by the use of enzymatic markers specific for plasma membranes, endoplasmic reticulum (dense tubular system), mitochondria, granules, and cytosolic constituents. The Kd and density of binding sites in the subcellular fractions were determined by Scatchard analysis of equilibrium binding data. The Kd and Bmax for [125I]-PTA-OH determined in the lysates were 49 +/- 11 nM and 4.1 +/- 1.7 pmol/mg protein respectively (N = 6). The Kd values were not significantly different in any of the fractions assayed. The binding sites were coenriched (4.5 +/- 0.66 fold) with the enzymatic markers for plasma membranes (3.7 +/- 0.5 fold) and dense tubular system (2.4 +/- 0.4 fold). The binding sites were not coenriched with markers for cytoplasmic constituents, mitochondria, or granules. The ability of the TXA2/PGH2 mimetic U46619 to compete with [125I]-PTA-OH for the binding site was also determined for the various subcellular fractions. The IC50 for U46619 was 5.4 +/- 1.2 microM in the lysate, and was not significantly different in the subcellular fractions. These data suggest that the binding site is the TXA2/PGH2 receptor described previously. These data are consistent with the notion that the putative TXA2/PGH2 receptor is localized in the plasma membranes and/or the dense tubular system.