The relationships of human platelet thromboxane A2-prostaglandin H2 (TxA2/PGH2) receptor occupation as assessed by equilibrium binding of the TxA2/PGH2 agonist [125I]BOP to the functional responses of I-BOP-induced platelet shape change and aggregation were determined before and after specific, irreversible inactivation of platelet TxA2/PGH2 receptors with the photolyzable TxA2/PGH2 antagonist I-PTA-PON3. I-BOP stimulated platelet shape change and aggregation with concentrations producing a half-maximal response of 173 +/- 39 pM (n = 4) and 1.8 +/- 0.4 nM (n = 6), respectively (means +/- SE). Covalent inactivation of TxA2/PGH2 receptors with I-PTA-PON3 caused rightward shifts of I-BOP shape change and aggregation dose-response curves and resulted in pharmacological dissociation constants (Kd) of 134 pM and 1.95 nM, respectively. Isotherms of [125I] BOP binding to intact platelets (n = 6) were shallow with Hill coefficients of -0.68 +/- 0.03 and were best described by a two-site model with 222 +/- 58 sites/platelet of high affinity (Kd = 270 +/- 60 pM) and 818 +/- 90 sites/platelet of lower affinity (Kd = 3.9 +/- 1.2 nM). The relationship of I-BOP occupancy of high-affinity receptors to shape-change responses was linear (r = 0.97, P less than 0.001), but the occupancy-response relationship for the lower affinity receptor and platelet aggregation was hyperbolic with half-maximal aggregation occurring after occupation of 25% of the receptors. After covalent inactivation of a variable proportion of the receptors with I-PTA-PON3, the occupancy-response relationship for platelet aggregation resulted in a similar hyperbola indicating an excess of low-affinity receptors coupled to aggregation (spare receptors).(ABSTRACT TRUNCATED AT 250 WORDS)