Photoaffinity receptor antagonist for human platelet thromboxane A₂/prostaglandin H₂ receptors

9,11-Dimethylmethano -11,12-methano-16-(3-azido-4-iodophenoxy)-13, 14-dihydro-13-aza-15αβ-ω-tetranor TXA₂ (I-PTA-PON₃) was synthesized and evaluated as a potential photoaffinity probe of the human platelet thromboxane A₂/prostaglandin H₂ ( TXA₂ PGH₂) receptor. I-PTA-PON₃ inhibited the aggregation of washed human platelets induced by the TXA₂ mimetic U46619 [(15S)-hydroxy-11α,9α-(epoxymethano)prosta-5Z,13E-dienoic acid]. Schild analysis of the data revealed a K_d of 9.5 nM and a slope not significantly different from -1. Equilibrium binding studies using [¹²⁵I]PTAOH, a TXA₂ PGH₂ receptor antagonist, showed that I-PTA-PON₃ plus photolysis resulted in a 52% reduction in the number of binding sites (1252 ± 202/platelet) compared to the nonphotolyzed group (2557 ± 293/platelet) (N = 5, P < 0.05) with no significant change in the K_d. Repetition of the incubation with I-PTA-PON₃ and photolysis a second time resulted in a further 77% (578 ± 163 binding sites/ platelet) reduction in the number of binding sites. Incubation of washed human platelets with I-PTAPON₃ (163 nM) followed by photolysis and removal of the non-covalently bound I-PTA-PON₃ resulted in no change in the 6050 value for the TXA₂ mimetic, U46619, when compared to controls that were either exposed to I-PTA-PON₃ and not photolyzed or exposed only to photolysis. The second photolysis of I-PTA-PON₃ resulted in a significant 42% increase in the ec₅₀ value of U46619-induced aggregation compared to the non-photolyzed group (N = 4, P < 0.05). These results suggest that I-PTA-PON₃ is a useful probe for the study of TXA₂ PGH₂ receptors and that spare TXA₂ PGH₂ receptors may exist in the platelet.