Puri R N, Colman R F, Colman R W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Arch Biochem Biophys. 1997 Jul 1;343(1):140-5. doi: 10.1006/abbi.1997.0160.
The 2-substituted ADP derivatives are known to activate human blood platelets with varying degrees of potency. For example, 2-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate [2-BDB-TADP], an ADP-affinity analog, was previously shown by us to be 50% as potent as ADP in inducing human blood platelet responses [Puri, R. N., Colman, R. F., and Colman, R. W. (1996) Eur. J. Biochem. 236, 862-870]. 2-Methylthio-ADP (2-MeS-ADP) has been known to be a far more potent agonist than ADP. However, the molecular basis for defining the rank order of potency of the 2-substituted ADP derivatives as agonists of platelet responses have been incompletely understood. We now report that 2-BOP-TADP (a one carbon atom lower homolog of 2-BDB-TADP) at equimolar concentration is as potent as ADP in inducing platelet responses. Prolonged incubation of platelets with 2-BOP-TADP abolished its ability to elicit cellular responses. An autoradiogram of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets labeled by incubating the platelets with 2-BOP-TADP for 1 h followed by reduction by NaB[3H]4 showed the presence of a single covalently radiolabeled protein band at 100 kDa. Preincubation of platelets with either ADP or ATP reduced the intensity of the band corresponding to the 100-kDa protein radiolabeled by 2-BOP-TADP and NaB[3H]4. The results show that (i) 2-BOP-TADP modulates ADP-induced platelet responses by interacting with aggregin and (ii) 2-BOP-TADP was twice as potent as 2-BDB-TADP, and (iii) the chain length of the substituent in a homologous series has an important bearing on the potency of a 2-substituted ADP analog.
已知2-取代的ADP衍生物能不同程度地激活人血小板。例如，2-(4-溴-2,3-二氧代丁基硫基)腺苷-5'-二磷酸[2-BDB-TADP]，一种ADP亲和力类似物，我们之前已表明其在诱导人血小板反应方面的效力是ADP的50%[普里，R.N.，科尔曼，R.F.，和科尔曼，R.W.(1996)《欧洲生物化学杂志》236，862 - 870]。已知2-甲硫基-ADP(2-MeS-ADP)是比ADP更强效的激动剂。然而，确定2-取代的ADP衍生物作为血小板反应激动剂效力等级顺序的分子基础尚未完全明了。我们现在报告，等摩尔浓度的2-BOP-TADP(2-BDB-TADP的一个碳原子更低的同系物)在诱导血小板反应方面与ADP效力相当。血小板与2-BOP-TADP长时间孵育消除了其引发细胞反应的能力。通过将血小板与2-BOP-TADP孵育1小时，然后用NaB[3H]4还原，对溶解的血小板进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳得到的凝胶放射自显影片显示，在100 kDa处有一条单一的共价放射性标记蛋白带。用ADP或ATP预孵育血小板会降低与2-BOP-TADP和NaB[3H]4放射性标记的100 kDa蛋白相对应的条带强度。结果表明：(i)2-BOP-TADP通过与聚集蛋白相互作用调节ADP诱导的血小板反应；(ii)2-BOP-TADP的效力是2-BDB-TADP的两倍；(iii)同系物系列中取代基的链长对2-取代的ADP类似物的效力有重要影响。
