title: Modulation of platelet responses by 2-[3-(bromo-2-oxopropylthio)]adenosine-5'-diphosphate involves its binding to as well as covalent modification of an ADP-receptor, aggregin.
abstract: 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.
authors.affiliation: Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: R N
authors.initials: RN
authors.lastname: Puri
authors.forename: R F
authors.initials: RF
authors.lastname: Colman
authors.forename: R W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*
chemical_list: C106826:2-(3-(bromo-2-oxopropylthio))adenosine 5'-diphosphate; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D018048:Receptors, Purinergic P2; C055422:aggregin; D000244:Adenosine Diphosphate