title: Irreversible inhibition of the TXA2/PGH2 receptor of human platelets by a photoaffinity ligand.
abstract: In order to tag the TXA2/PGH2 receptor of human platelets, we synthesized azido-BSP (= 4-[2-(4-azido-benzenesulfonylamino)-ethyl]phenoxyacetic acid), a photolabile derivative of the specific TXA2/PGH2 receptor antagonist sulotroban (= BM 13.177). If protected from UV light, azido-BSP competitively inhibited the shape change of human washed platelets stimulated by the TXA2 mimetic U 46619. Schild analysis revealed a pA2 = 6.7 (apparent KD = 0.2 mumol/l). Irreversible inhibition of the U 46619-induced platelet activation was achieved by irradiating for 5 min with UV light of 254 nm a platelet suspension containing azido-BSP. After subsequent washing, the platelets were stimulated with U 46619, ADP or PAF. Under these conditions azido-BSP inhibited the shape change, aggregation and [3H]serotonin release induced by U 46619 but not the shape change induced by ADP or PAF. The concentrations of azido-BSP which blocked the U 46619-induced [3H]serotonin release and the aggregation were 0.5 mumol/l and 1.0 mumol/l, respectively, whereas even 50.0 mumol/l of azido-BSP only partially inhibited the U 46619-stimulated shape change. Obviously, increasing numbers of thromboxane receptors have to be blocked in order to inhibit the [3H]serotonin release, the aggregation and the shape change. Even at an azido-BSP concentration equal to 250 times the apparent dissociation constant, enough receptor sites remained active to allow U 46619 to induce the shape change. In sulotroban was added prior to irradiation, the blocking effect of azido-BSP decreased with increasing concentrations of sulotroban. These results show that azido-BSP is a specific and high affinity ligand of the TXA2/PGH2 receptor and that it covalently links to the receptor under irradiation. Azido-BSP is a new tool to identify and characterize the TXA2/PGH2 receptor.
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000345:Affinity Labels / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D001792:Blood Platelets / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011449:Prostaglandin Endoperoxides / Q000378:metabolism*; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism* / Q000494:pharmacology; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000378:metabolism*; D011982:Receptors, Prostaglandin / Q000187:drug effects / Q000378:metabolism*; D017482:Receptors, Thromboxane; D013449:Sulfonamides / Q000494:pharmacology; D013928:Thromboxane A2 / Q000097:blood*
chemical_list: D000345:Affinity Labels; D001386:Azides; D005343:Fibrinolytic Agents; D011449:Prostaglandin Endoperoxides; D011450:Prostaglandin Endoperoxides, Synthetic; D011463:Prostaglandins H; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D013449:Sulfonamides; D044262:Prostaglandin H2; D013928:Thromboxane A2; C040624:sulotroban; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid