Characterization of the inhibition of U46619-mediated human platelet activation by the trimetoquinol isomers: Evidence for endoperoxide/thromboxane A 2 receptor blockade

Abstract

Sites of inhibition for the trimetoquinol (TMQ) isomers on 15S-hydroxy-11α,9α-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619)-, 12- O-tetradecanoylphorbol 13-acetate (TPA)- and A23187-induced human platelet activation were investigated. Experiments using washed human platelets were designed to characterize relationships among functional (aggregation, secretion) and biochemical (protein phosphorylation, metabolism of inositol phospholipids and radioligand displacement analysis) processes of platelet activation by U46619 and the specificity of inhibition by the TMQ isomers. Thromboxane A 2 receptor stimulation by U46619 in human platelets resulted in a time- and concentration-dependent breakdown of inositol phospholipids [phosphatidylinositol 4,5-bisphosphate (PIP 2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI)], phosphatidic acid (PA) accumulation, phosphorylation of 20 and 45 kD proteins, aggregation and serotonin secretion. The TMQ isomers stereoselectively inhibited all U46619-mediated platelet activation processes. R(+)TMQ was 40- and 22-fold more potent than S(−)-TMQ as an inhibitor of U46619-induced platelet aggregation and serotonin secretion respectively. In addition, R(+)-TMQ blocked U46619-induced 20 kD protein phosphorylation, 45 kD protein phosphorylation, PIP 2, PIP and PI breakdown, and PA accumulation with a potency which was 8-, 13-, 45-, 37-, 33- and 33-fold greater than the S(−)-isomer respectively. In contrast to S(−)-TMQ, R(+)-TMQ produced a concentration-dependent inhibition of specific [ 3H]U46619 binding to endoperoxide/thromboxane A 2 receptor sites in washed platelets. In other experiments, S(−)-TMQ was more potent than R(+)-TMQ as an inhibitor of TPA- and A23187-induced platelet aggregation and serotonin secretion, and of TPA-induced phosphorylation of 45 and 20 kD proteins. The inhibitory potencies of S(−)-TMQ against TPA- or A23187-induced responses were similar to those needed for antagonism of U46619-mediated platelet activation. In contrast, much higher concentrations of R(+)-TMQ were required for blockade of TPA or A23187 versus U46619-mediated responses in human platelets. Taken collectively, the data show that the TMQ isomers interfered with the endoperoxide/thromboxane A 2 receptor-mediated phospholipase C-signal cascade of inositol phospholipid hydrolysis, calcium mobilization, and protein phosphorylation leading to platelet aggregation and secretion. R(+)-TMQ acted as a pharmacologically selective and highly stereospecific [ R(+)-TMQ S(−)-TMQ] antagonist of endoperoxide/thromboxane A 2 receptor sites in platelets. Additionally, S(−)-TMQ was more potent than R(+)-TMQ as an antagonist of platelet activation by inducers of protein kinase C activity and calcium mobilization. Thus, the isomers of TMQ may also interfere by a yet undefined mechanism with calcium-regulated cytosolic processes in platelets.