IMMUNOAFFINITY METHOD TO IDENTIFY AGGREGIN, A PUTATIVE ADP-RECEPTOR IN HUMAN BLOOD-PLATELETS

The ADP-receptor on the surface of human platelets and cells of megaka ryocytic lineage has been classified as P2T purinergic receptor for wh ich ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has ne ither been purified nor cloned, We have developed an immunoaffinity me thod for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal ant ibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-flu orosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutama te dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sul fate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled pl atelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated wit h ADP and ATP or covalently modified by the chemically reactive ADP-af finity analogs, 2- and -bromo-2,3-dioxobutylthio)adenosine-5'-diphosph ate (2- and 8-BDB-TADP) and 2-(3-bromo-2-oxopropylthio) adenosine-5'-d iphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibi t ADP-induced platelet responses by selectively and covalently modifyi ng aggregin (100 kDa), an ADP-receptor in intact human blood platelets . The results show that polyclonal antibody to FSBA-labeled GDH is cap able of recognizing FSBA-labeled aggregin on platelets and, thus, coul d be used to purify aggregin by immunoaffinity column chromatography, The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses. (C) 1997 Academic Press.