MODULATION OF PLATELET RESPONSES BY 3-(BROMO-2-OXOPROPYLTHIO)]ADENOSINE-5'-DIPHOSPHATE INVOLVES ITS BINDING TO AS WELL AS COVALENT MODIFICATION OF AN ADP-RECEPTOR, AGGREGIN

The 2-substituted ADP derivatives are known to activate human blood pl atelets with varying degrees of potency, For example, bromo-2,3-dioxob utylthio)-adenosine-5'-diphosphate [2-BDB-TADP], an ADP-affinity analo g, was previously shown by us to be 50% as potent as ADP in inducing h uman 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-s ubstituted 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 pot ent as ADP in inducing platelet responses, Prolonged incubation of pla telets with 2-BOP-TADP abolished its ability to elicit cellular respon ses, An autoradiogram of the gel obtained by sodium dodecyl sulfate po lyacrylamide gel electrophoresis of solubilized platelets labeled by i ncubating the platelets with 2-BOP-TADP for 1 h followed by reduction by NaB[H-3](4) showed the presence of a single covalently radiolabeled protein band at 100 kDa, Preincubation of platelets with either ADP o r ATP reduced the intensity of the band corresponding to the 100-kDa p rotein radiolabeled by 2-BOP-TADP and NaB[H-3](4), The results show th at (i) 2-BOP-TADP modulates ADP-induced platelet responses by interact ing with aggregin and (ii) 2-BOP-TADP was twice as potent as 2-BDB-TAD P, and (iii) the chain length of the substituent in a homologous serie s has an important bearing on the potency of a 2-substituted ADP analo g. (C) 1997 Academic Press