Puri R N, Colman R W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
J Cell Biochem. 1996 Apr;61(1):97-108. doi: 10.1002/(sici)1097-4644(19960401)61:1<97::aid-jcb11>3.0.co;2-e.
ADP-induced platelet responses play an important role in the maintenance of hemostasis. There has been disagreement concerning the identity of an ADP receptor on the platelet surface. The chemical structure of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-CI) shows considerable resemblance to that of the adenine moiety of adenine-based nucleotides. The reagent has been previously used by other investigators as an affinity label for adenine nucleotide-requiring enzymes, such as mitochondrial ATPase and the catalytic subunit of cAMP-dependent protein kinase. Since ADP-induced platelet responses depend on the binding of ADP to its receptor, we investigated the effect on ADP-induced platelet responses and the nature of ADP-binding protein modified by NBD-CI. NBD-CI inhibited ADP-induced shape change and aggregation of platelets in platelet-rich plasma in a concentration- and time-dependent manner. NBD-CI also inhibited ADP-induced shape change, aggregation, exposure of fibrinogen binding sites, secretion, and calcium mobilization in washed platelets. NBD-CI did not act as an agonist for platelet shape change and aggregation. Covalent modification of platelets by NBD-CI blocked the ability of ADP to antagonize the increase in intracellular levels of cAMP mediated by iloprost (a stable analogue of prostaglandin I2). NBD-CI was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), that completely or partially depend on the binding of ADP to its receptor. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platelets modified by [14C]-NBD-CI showed the presence of a predominant radiolabeled protein band at 100 kDa corresponding to aggregin, a putative ADP receptor. The intensity of this band was considerably decreased when platelets were either preincubated with ADP and ATP or covalently modified by a sulfhydryl group modifying reagent before modification by [14C]-NBD-CI. These results (1) indicate that covalent modification of aggregin by NBD-CI contributed to loss of the ADP-induced platelet responses, and (2) suggest that there is a sulfhydryl group in the ADP-binding domain of aggregin.
ADP诱导的血小板反应在维持止血过程中起重要作用。关于血小板表面ADP受体的身份一直存在分歧。7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂茂（NBD-CI）的化学结构与腺嘌呤核苷酸的腺嘌呤部分有很大相似性。该试剂先前已被其他研究人员用作腺嘌呤核苷酸依赖性酶的亲和标记，如线粒体ATP酶和cAMP依赖性蛋白激酶的催化亚基。由于ADP诱导的血小板反应依赖于ADP与其受体的结合，我们研究了NBD-CI对ADP诱导的血小板反应的影响以及被NBD-CI修饰的ADP结合蛋白的性质。NBD-CI以浓度和时间依赖性方式抑制富含血小板血浆中ADP诱导的血小板形状改变和聚集。NBD-CI还抑制洗涤血小板中ADP诱导的形状改变、聚集以及纤维蛋白原结合位点的暴露、分泌和钙动员。NBD-CI不作为血小板形状改变和聚集的激动剂。NBD-CI对血小板的共价修饰阻断了ADP拮抗由伊洛前列素（前列腺素I2的稳定类似物）介导的细胞内cAMP水平升高的能力。NBD-CI在抑制那些完全或部分依赖于ADP与其受体结合的激动剂（如ADP、胶原和U44619（一种血栓素模拟物））诱导的血小板聚集方面非常具有特异性。通过对用[14C]-NBD-CI修饰的溶解血小板进行SDS-PAGE获得的凝胶放射自显影片显示，在100 kDa处存在一条主要的放射性标记蛋白带，对应于聚集素（一种假定的ADP受体）。当血小板在用[14C]-NBD-CI修饰之前先用ADP和ATP预孵育或用巯基修饰试剂进行共价修饰时，这条带的强度显著降低。这些结果（1）表明NBD-CI对聚集素的共价修饰导致ADP诱导的血小板反应丧失，（2）提示聚集素的ADP结合结构域中存在一个巯基。
