INHIBITION OF ADP-INDUCED PLATELET ACTIVATION BY 7-CHLORO-4-NITROBENZ-2-OXA-1,3-DIAZOLE - COVALENT MODIFICATION OF AGGREGIN, A PUTATIVE ADPRECEPTOR

ADP-induced platelet responses play an important role in the maintenan ce 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 res emblance to that of the adenine moiety of adenine-based nucleotides. T he reagent has been previously used by other investigators as an affin ity label for adenine nucleotide-requiring enzymes, such as mitochondr ial 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 respo nses and the nature of ADP-binding protein modified by NBD-CI. NBD-CI inhibited ADP-induced shape change and aggregation of platelets in pla telet-rich plasma in a concentration- and time-dependent manner. NBD-C I also inhibited ADP-induced shape change, aggregation, exposure of fi brinogen 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 I-2). NBD-CI was quite specific in inhibiting platelet aggregation by those agonists, e.g., ADP, collagen, and U44619 (a thromboxane mimetic), th at completely or partially depend on the binding of ADP to its recepto r. Autoradiogram of the gel obtained by SDS-PAGE of solubilized platel ets modified by [C-14]-NBD-CI showed the presence of a predominant rad iolabeled protein band at 100 kDa corresponding to aggregin, a putativ e 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 [C-14]-NBD-CI. These results (1) indicate that covalent modificatio n of aggregin by NBD-CI contributed to loss of the ADP-induced platele t responses, and (2) suggest that there is a sulfhydryl group in the A DP-binding domain of aggregin. (C) 1996 Wiley-Liss, Inc.