Separate receptors for prostacyclin and prostaglandin E₂ on human gel-filtered platelets

Human gel-filtered platelets (GFP) and radiolabeled prostacyclin (PGI₂), prostaglandin (PG) E₂ and PGE₁, were used to ascertain whether PGI₂ and PGs of the E series share a common receptor or have their own specific receptors on platelets. Attention was given to ensuring the proper experimental conditions to compensate for the rapid half-life of PGI₂ at physiologic pH. Specific [³H]PGI₂ binding to GFP was maximal at 5 min and pH 7.45. Scatchard analysis indicated a single class of binding sites with an apparent K(D) of 4.52 x 10^-8 M and 1130 sites per platelet. Approximately 90% of specifically bound [³H]PGI₂ could be dissociated by excess unlabeled PGI₂ by 5 min. The IC₅₀ for PGI₂ was 66 nM. By 5 min, PGE₁ and PGE₂ were only 7.17 and 0.03%, respectively, as potent inhibitors of binding. Maximal specific binding of either [³H]PGE₂ or [³H]PGE₁ to GFP occurred by 60 min. During 60-min incubations with [³H]PGE₂, the IC₅₀ values for PGE₂ and PGE₁ were 3 and 6 nM, respectively. When [³H]PGE₁ was used, the IC₅₀ values for PGE₁ and PGE₂ were 30 and 10 nM, respectively. To examine PGI₂ competition for [³H]PGE₂ and [³H]PGE₁ binding sites, 5-min incubation periods were used. PGI₂ was only 0.38% as potent an inhibitor of [³H]PGE₂ compared to PGE₂ and only 30% as potent an inhibitor of [³H]PGE₁ compared to PGE₁. Scatchard analysis of the 60-min competition experiments using [³H]PGE₂ and [³H]PGE₁ and the homologous unlabeled ligand yielded curvilinear plots in both instances. However, when GFP were preincubated with a saturating concentration of PGE₂, only low affinity/high density binding sites were available for subsequent [³H]PGE₁ binding. In contrast, this maneuver did not affect subsequent [³H]PGI₂ binding. Consequently, high affinity binding [³H]PGE₁ appears to be fully accounted for by binding of this ligand to PGE₂ sites. The authors interpret these data to indicate that there are separate and distinguishable receptors for PGI₂ and PGE₂ on human GFP because 1) PGI₂ competes rather poorly for either [³H]PGE₂ or [³H]PGE₁ binding; 2) neither PGE₂ nor PGE₁ compete well for [³H]PGI₂ binding; 3) preincubation with a saturating concentration of PGE₂ before [³H]PGI₂ binding does not influence [³H]PGI₂ binding; and 4) the concentration of PGE₂ required to complete for [³H]PGI₂ binding is approximately 1000 times greater than the concentration required to saturate fully all binding sites occupied by [³H]PGE₂. There is no evidence for a high affinity PGE₁-specific receptor because all PGE₁ binding can be accounted for by interactions with the PGE₂ sites.