PLATELET ACTIVATION BY 2-(4-BROMO-2,3-DIOXOBUTYLTHIO)ADENOSINE 5'-DIPHOSPHATE IS MEDIATED BY ITS BINDING TO A PUTATIVE ADP RECEPTOR, AGGREGIN

Platelet responses induced by ADP are mediated by a unique P-2T-purine rgic receptor. Although a variety of ADP analogs, substituted al C2, h ave been used to delineate pharmacological properties of the ADP-bindi ng site(s), the identity of the receptor protein has not been firmly e stablished. o-2,3-dioxobutylthio)-ADP[2-BrCH2(CO)(2)CH2-S-ADP] a well- characterized ADP analog, has been previously used as an affinity labe l to examine the structure/function relationship of ADP-requiring enzy mes [Kapetanovic, E., Bailey, J. B. & Colman, R. F. (1985) Biochemistr y 24, 7586-7593]. We found that it induced platelet shape change, aggr egation, exposure of fibrinogen binding sites, secretion and mobilizat ion of intracellular calcium, but was less potent than ADP. Under non- stirring conditions, incubation of platelets with this analog for long er time periods blocked ADP-induced shape change, aggregation, and the ability of ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I-2 analog). Of a variety of ago nists examined, only ADP-induced aggregation was almost completely inh ibited in platelets irreversibly modified by the analog. An autoradiog ram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[H-3] (4) showed the presence of a single radiolabeled protein band at 100 k Da. Platelets incubated first with either ADP ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)(2)CH2S-ADP and NaB[H-3](4). 8-BrCH2(C O)(2)CH2-S-ADP was previously shown by us to irreversibly antagonize A DP-induced platelet responses by selectively modifying aggregin. Incub ation of platelets with 2-BrCH2(CO)(2)CH2S-ADP completely blocked labe ling of aggregin in platelets by 8-BrCH2(CO)(2)CH2S-[P-32]ADP. These r esults show that 2-BrCH2(CO)(2)CH2S-ADP initially interacts reversibly with aggregin (100 kDa), a putative ADP receptor, and induces platele t shape change and aggregation, and at longer periods of incubation re acts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)(2)CH2S-ADP was f ound to be a weak and reversible inhibitor of ADP-induced platelet agg re gation. Prior incubation of platelets with the latter analog reduce d labeling of aggregin by 8-BrCH2(CO)(2)CH2S-[P-32]ADP. Taken together , the results further show that substitution by the BrCH2(CO)(2)CH2 gr oup at the C2 and C8 positions is tolerated, while the presence of a f ree amino function at the C6 position is essential for its interaction with aggregin.