CHARACTERISTICS OF THE BINDING OF [H-3] GR32191 TO THE THROMBOXANE (TP-) RECEPTOR OF HUMAN PLATELETS

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 i nitial rapid component and a slower secondary phase. Dissociation expe riments 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 indica ted a single class of binding site with a K(d) of 2.2 nM. Further anal ysis of the data yielded a Hill slope of - 1.0 again indicating an int eraction at a single binding site. Saturation binding experiments gave a similar estimate of the K(d) value for [H-3]-GR32191 to that obtain ed from competition binding experiments. A possible explanation for th e biphasic interaction of the GR32191 in intact platelets may lie in r estriction of its access to and egress from a population of TP-recepto rs. 4 In platelet membranes at 37-degrees-C, specific [H-3]-GR32191 bi nding was complete within 5 min with a calculated association rate con stant of 3.2 x 10(8) M-1 min-1. Dissociation of [H-3]-GR32191 was rela tively 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 s ite. The t1/2 for dissociation appeared to be related to the contact t ime of platelet membranes with [H-3]-GR32191. Derivation of a K(d) fro m the kinetic rate constants gave a value of 0.13 nM. 5 Competition bi nding 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 b inding 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 comp etition experiments. 6 In further competition binding experiments, the TP-receptor agonists U-46619, STA2, EP171 and 9,11-azo PGH2 and antag onists 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 bindi ng. However, where tested, the IC50 values for a particular compound w ere 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 interac tion 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-rec eptor. The interaction of the drug with this binding site is, however, characterized by a slow dissociation. This characteristic was confirm ed in studies with platelet membranes and does not therefore appear to be an artefact of diffusion. Estimates of the K(d) of the drug differ ed depending on the method of determination. Because of the slow disso ciation of [H-3]-GR32191, those relying upon equilibrium of the radiol igand 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 hum an platelet TP-receptor.