Puri R N, Colman R F, Colman R W
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Eur J Biochem. 1996 Mar 15;236(3):862-70. doi: 10.1111/j.1432-1033.1996.00862.x.
Platelet responses induced by ADP are mediated by a unique P21-purinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP-binding site(s), the identity of the receptor protein has not been firmly established. 2-(4-Bromo-2,3-dioxobutylthio)- ADP [2-BrCH2(CO)2CH2-S-ADP], a well-characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP-requiring enzymes [Kapetanovic, E., Bailey, J.B. & Colman, R.F. (1985) Biochemistry 24, 7586-7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non-stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP-induced shape change, aggregation, and the ability to ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP-induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H], showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)2CH2S-ADP and NaB[3H]4-8-BrCH2(CO)2CH2-S-ADP was previously shown by us to irreversibly antagonize ADP-induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2-BrCH2(CO)2CH2S-ADP completely blocked labeling of aggregin in platelets by 8-BrCH2(CO)2CH2S-[32P]ADP. These results show that 2-BrCH2(CO)2CH2S-ADP initially interacts reversibly with aggregin (100kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)2CH2S-ADP was found to be a weak and reversible inhibitor of ADP-induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8-BrCH2(CO)2CH2S-[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.
ADP诱导的血小板反应由一种独特的P21-嘌呤能受体介导。尽管多种在C2位被取代的ADP类似物已被用于描述ADP结合位点的药理学特性，但受体蛋白的身份尚未得到确凿证实。2-(4-溴-2,3-二氧代丁基硫代)-ADP [2-BrCH2(CO)2CH2-S-ADP]，一种特性明确的ADP类似物，此前已被用作亲和标记物来研究需要ADP的酶的结构/功能关系[卡佩塔诺维奇，E.，贝利，J.B.和科尔曼，R.F.（1985年）《生物化学》24，7586 - 7593]。我们发现它能诱导血小板形状改变、聚集、纤维蛋白原结合位点暴露、分泌以及细胞内钙的动员，但效力比ADP弱。在非搅拌条件下，用这种类似物长时间孵育血小板会阻断ADP诱导的形状改变、聚集以及ADP拮抗依洛前列素（一种前列腺素I2类似物）诱导的细胞内cAMP水平升高的能力。在所检测的多种激动剂中，只有ADP诱导的聚集在被该类似物不可逆修饰的血小板中几乎完全被抑制。通过SDS/PAGE对经ADP类似物修饰的溶解血小板进行凝胶电泳，然后用NaB[3H]还原二氧代基团，得到的放射自显影片显示在100 kDa处有一条单一的放射性标记蛋白带。先用ADP、ATP或2-甲硫基-ADP孵育的血小板不会被2-BrCH2(CO)2CH2S-ADP和NaB[3H]标记，并且我们之前已表明4-8-BrCH2(CO)2CH2-S-ADP通过选择性修饰聚集蛋白不可逆地拮抗ADP诱导的血小板反应。用2-BrCH2(CO)2CH2S-ADP孵育血小板完全阻断了8-BrCH2(CO)2CH2S-[32P]ADP对血小板中聚集蛋白的标记。这些结果表明，2-BrCH2(CO)2CH2S-ADP最初与一种假定的ADP受体聚集蛋白（100 kDa）可逆性相互作用，并诱导血小板形状改变和聚集，且在较长孵育时间下会不可逆地反应以阻断ADP拮抗刺激的腺苷酸环化酶活性的能力。相比之下，发现6-BrCH2(CO)2CH2S-ADP是ADP诱导的血小板聚集的一种弱且可逆的抑制剂。先用后一种类似物孵育血小板会减少8-BrCH2(CO)2CH2S-[32P]ADP对聚集蛋白的标记。综上所述，结果进一步表明，在C2和C8位被BrCH2(CO)2CH2基团取代是可以耐受的，而C6位存在游离氨基功能对其与聚集蛋白的相互作用至关重要。
