IRREVERSIBLE BLOCKADE OF THE THROMBOXANE A2/PROSTAGLANDIN H2 RECEPTOR OF HUMAN PLATELETS BY AZIDO-BSP
Azido-BSP (4-[2-(4-azido-benzenesulphonylamino)-ethyl]phen-oxyacetic acid) is a photolabile derivative of the competitive thromboxane A2 /prostaglandin H2 (TXA2/PGH2) receptor antagonist sulotroban (=BM 13.177). If protected from short wave light, azido-BSP reversibly inhibited the platelet shape change induced by the PGH2 analogue U 46619 but notthe shape change induced by ADP or PAF. Schild analysis revealed an apparent KD=0.2 μM with washed platelets. The irreversible inhibition requiredirradiation of the platelet suspensionwith UVlight (254 nm) for 5 minutes in the presenceof azido-BSP. After this treatment,the platelets were washed twice and used forplatelet function tests. Treatment with 0.5 μM of azido-BSP suppressed the U 46619(10 μM)-induced (3H)serotonin release and 1 μM of azido-BSP was necessary to block the U 46619(2 μM)-inducedaggregation.The platelet shape change induced by U 46619 (0.01μM) was only partially inhibited, even at very high concentrations (50μM) of the antagonist.This suggests that a small portion of the TXA2/PGH2 receptors could not be blocked bythe photoaffinity treatment with azido-BSP. After treatment with 1 μM azido-BSP, the shape change stimulated by ADP or PAF was not reduced. This indicates a high specificity of thephotoaffinity ligand for the TXA2/PGH2 receptor. It is concluded that UV irradiation of azido-BSP generates anitrene intermediate that covalently links to the majority of the TXA2/PGH2 receptors. Azido-BSP provides a specific tool for tagging and subsequent purification of the TXA2/PGH2 receptor of platelets.(Supported by the Deutsche Forschungsgemeinschaft, Grant Pa263).