Zehender H, Witte E C, Wolff H P, Patscheke H
Institute for Clinical Chemistry, Klinikum Mannheim, University of Heidelberg, Federal Republic of Germany.
Biochem Pharmacol. 1988 Feb 1;37(3):491-5. doi: 10.1016/0006-2952(88)90219-5.
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.
为标记人血小板的血栓素A2/前列环素H2（TXA2/PGH2）受体，我们合成了叠氮基-苯磺酰基苯丙酸（azido-BSP，即4-[2-(4-叠氮基-苯磺酰氨基)-乙基]苯氧基乙酸），它是特异性TXA2/PGH2受体拮抗剂舒洛地尔（sulotroban，即BM 13.177）的光不稳定衍生物。若避免紫外线照射，叠氮基-苯磺酰基苯丙酸能竞争性抑制由TXA2模拟物U 46619刺激引起的人洗涤血小板的形态变化。希尔德分析显示pA2 = 6.7（表观解离常数KD = 0.2 μmol/L）。通过用254 nm紫外线照射含有叠氮基-苯磺酰基苯丙酸的血小板悬液5分钟，可实现对U 46619诱导的血小板活化的不可逆抑制。随后洗涤后，用U 46619、二磷酸腺苷（ADP）或血小板活化因子（PAF）刺激血小板。在这些条件下，叠氮基-苯磺酰基苯丙酸抑制U 46619诱导的形态变化、聚集和[3H]5-羟色胺释放，但不抑制ADP或PAF诱导的形态变化。阻断U 46619诱导的[3H]5-羟色胺释放和聚集的叠氮基-苯磺酰基苯丙酸浓度分别为0.5 μmol/L和1.0 μmol/L，而即使50.0 μmol/L的叠氮基-苯磺酰基苯丙酸也只能部分抑制U 46619刺激的形态变化。显然，为抑制[3H]5-羟色胺释放、聚集和形态变化，必须阻断越来越多的血栓素受体。即使在叠氮基-苯磺酰基苯丙酸浓度等于表观解离常数250倍时，仍有足够的受体位点保持活性，使U 46619能诱导形态变化。若在照射前加入舒洛地尔，叠氮基-苯磺酰基苯丙酸的阻断作用会随舒洛地尔浓度增加而降低。这些结果表明，叠氮基-苯磺酰基苯丙酸是TXA2/PGH2受体的特异性高亲和力配体，且在照射下能与该受体共价连接。叠氮基-苯磺酰基苯丙酸是鉴定和表征TXA2/PGH2受体的新工具。
