Distinct platelet thromboxane A₂/prostaglandin H₂ receptor subtypes. A radioligand binding study of human platelets

Thromboxane A₂ (TXA₂) and prostaglandin H₂ (PGH₂) may aggregate platelets via a common membrane receptor(s). To further characterize this receptor, binding of the radiolabeled TXA₂/PGH₂ mimetic [¹²⁵I]BOP to washed human platelets (WP) was investigated. [¹²⁵I]BOP was competitively displaced from its platelet binding site by stable TXA₂/PGH₂ analogues. Competition curves were shallow with Hill coefficients of -0.73 ± 0.05 (P < 0.001 different from unity) (90 ± 1% specific binding). Scatchard plots were curvilinear and most consistent with two binding sites; a high-affinity site with K(d) of 234 ± 103 pM, B(max) of 0.7 ± 0.3 pM/mg protein (180 ± 87 sites/WP), and a lower affinity site with K(d) of 2.31 ± 0.86 nM, B(max) of 2.2 ± 0.3 pM/mg protein (666 ± 65 sites/WP). [¹²⁵I]BOP association and dissociation kinetics gave a K(d) of 157 pM without evidence of negative cooperativity. The EC₅₀ for I-BOP-induced initial Ca²⁺ increase was 209 ± 24 pM, shape change was 263 ± 65 pM, and aggregation was 4.4 ± 0.5 nM. Parallel binding studies using the TXA₂/PGH₂ receptor antagonist [¹²⁵I]PTA-OH showed a single binding site. The rank order for TXA₂/PGH₂ analogues to displace [¹²⁵I]PTA-OH was identical to that for [¹²⁵I]BOP. These studies indicate that [¹²⁵I]BOP binds to two distinct sites on human platelets that may represent platelet TXA₂/PGH₂ receptor subtypes. The close correlation of IC₅₀ values for I-BOP-induced platelet shape change and aggregation with the two K(d)s for [¹²⁵I]BOP binding suggests that these platelet responses may be independently mediated by the two putative receptors.