Receptor-mediated effects of a PGH2 analogue (U 46619) on human platelets

The specific effects of U 46619 (9,11-dideoxy,9 alpha-11 alpha-methanoepoxyprostaglandin F2 alpha), thromboxane A2-prostaglandin H2 (TxA2/PGH2) analogue, on human platelet shape change, myosin light-chain phosphorylation, serotonin release, fibrinogen receptor exposure, and platelet aggregation were measured and compared with binding of [3H]U 46619 to platelets. Shape change and myosin light-chain phosphorylation were found to be saturable and dose dependent, having effective concentration producing 50% of the maximum response (EC50) values of 0.035 +/- 0.005 and 0.057 +/- 0.021 microM, respectively (mean +/- SE). These two effects were competitively inhibited by specific antagonists of TxA2/PGH2 receptors (BM 13177, PTA-OH, and 1.PTA-OH) indicating that they are receptor mediated. Binding of [3H]U 46619 showed two components. Occupancy of high-affinity binding sites [dissociation constant (Kd) = 0.041 +/- 0.009 microM, maximum binding site (Bmax) = 19.4 +/- 5.3 fmol/10(7) platelets, with 1,166 +/- 310 sites/platelet; n = 12] correlated with platelet shape change and myosin light-chain phosphorylation. We propose that a second component with an apparent Kd of 1.46 +/- 0.47 microM (n = 12) represents a second, low-affinity site. Mean EC50 values for U 46619-induced serotonin release, platelet aggregation, and fibrinogen receptor exposure were 0.54 +/- 0.13. 1.31 +/- 0.34 and 0.53 +/- 0.21 microM, respectively. Therefore, the platelet release reaction was not directly correlated with occupancy of high-affinity receptors but could be related to the second binding component of U 46619. Fibrinogen receptor exposure and platelet aggregation caused by U 46619 appeared to be events mediated by the release of adenosine diphosphate from platelet-dense granules.