1 The interaction of the specific thromboxane (TP-) receptor blocking drug, [H-3]-GR32191 with human intact platelets and platelet membranes has been investigated in vitro. 2 On intact platelets association of specific [H-3]-GR32191 binding at 37-degrees-C was biphasic, with an initial rapid component and a slower secondary phase. Dissociation experiments indicated displacement from two sites with t1/2 values of 8.1 and 65.6 minutes. K(d) values derived from the kinetic rate constants for the rapid onset/offset and slow onset/offset phases were 0.4 and 0.5 nM respectively. 3 Competition binding of [H-3]-GR32191 and GR32191 on intact platelets gave an IC50 of 2.3 nM. Scatchard analysis indicated a single class of binding site with a K(d) of 2.2 nM. Further analysis of the data yielded a Hill slope of - 1.0 again indicating an interaction at a single binding site. Saturation binding experiments gave a similar estimate of the K(d) value for [H-3]-GR32191 to that obtained from competition binding experiments. A possible explanation for the biphasic interaction of the GR32191 in intact platelets may lie in restriction of its access to and egress from a population of TP-receptors. 4 In platelet membranes at 37-degrees-C, specific [H-3]-GR32191 binding was complete within 5 min with a calculated association rate constant of 3.2 x 10(8) M-1 min-1. Dissociation of [H-3]-GR32191 was relatively slow, with measurable specific binding persisting for > 40 min. Analysis of these data yielded a t1/2 of 17.7 min and a dissociation rate constant of 0.04 min-1 and indicated dissociation from a single site. The t1/2 for dissociation appeared to be related to the contact time of platelet membranes with [H-3]-GR32191. Derivation of a K(d) from the kinetic rate constants gave a value of 0.13 nM. 5 Competition binding of [H-3]-GR32191 and GR32191 to platelet membranes gave an IC50 value of 3.5 nM. Scatchard analysis of these data indicated a single binding site with a K(d) of 2.1 nM. Saturation binding experiments with [H-3]-GR32191 yielded similar IC50 and K(d) values to those from competition experiments. 6 In further competition binding experiments, the TP-receptor agonists U-46619, STA2, EP171 and 9,11-azo PGH2 and antagonists SQ29,548, BM 13.177 and EP092 all competed with specific [H-3]-GR32191 binding on intact platelets and, where determined, on platelet membranes. All compounds fully displaced specific [H-3]-GR32191 binding. However, where tested, the IC50 values for a particular compound were always greater when [H-3]-GR32191 was the radioligand than when [H-3]-SQ29,548 was used. At the concentrations used in these studies (2 and 5 nM respectively), platelets appeared to bind approximately twice as much [H-3]-GR32191 as [H-3]-SQ29,548. 7 In conclusion, the interaction of [H-3]-GR32191 with human intact platelets was complex but the data were consistent with an action at a single class of binding site; from competition experiments this appears to be the functional TP-receptor. The interaction of the drug with this binding site is, however, characterized by a slow dissociation. This characteristic was confirmed in studies with platelet membranes and does not therefore appear to be an artefact of diffusion. Estimates of the K(d) of the drug differed depending on the method of determination. Because of the slow dissociation of [H-3]-GR32191, those relying upon equilibrium of the radioligand with competing agent may be unreliable. The rate of dissociation also appeared to be related to the contact time of drug with receptor. An explanation for this phenomenon may lie in the ability of GR32191 to induce a change in the conformational state or location of the human platelet TP-receptor.
