PHARMACOLOGICAL AND BIOCHEMICAL-CHARACTERIZATION OF PURIFIED A(2A) ADENOSINE RECEPTORS IN HUMAN PLATELET MEMBRANES BY [H-3] CGS-21680 BINDING

1 The binding properties of human platelet A(2a) adenosine receptors, assayed with the A(2a)-selective agonist, [H-3]-2-[p-(2-carboxyethyl)- phenethylamino]-5'-N- ethylcarboxamidoadenosine ([H-3]-CGS 21680), are masked by a non-receptorial component, the adenotin site. In order to separate A(2a) receptors from adenotin sites, human platelet membrane s were solubilized with 1% olamidopropyl)dimethylammonio]-1-propanesul phonate (CHAPS). The soluble platelet extract was precipitated with po lyethylene glycol (PEG) and the fraction enriched in adenosine recepto rs was isolated from the precipitate by differential centrifugation. 2 The present paper describes the binding characteristics of the select ive A(2a) agonist, [H-3]-CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several ad enosine agonists and antagonists were determined in binding and adenyl yl cyclase studies. 3 Saturation experiments revealed a single class o f binding site with K-d and B-max values of 285 nM and 2.07 pmol mg(-1 ) of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [H-3]-CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A(2a) adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to th at observed in binding experiments. 4 Thermodynamic data indicated tha t [H-3]-CGS 21680 binding to the purified receptor is totally entropy- driven in agreement with results obtained in rat striatal A(2a) adenos ine receptors. 5 It is concluded that in the purified platelet membran es there is a CGS 21680 binding site showing the characteristic proper ties of the A(2a) receptor. This makes it possible to use this compoun d for reliable radioligand binding studies on the A(2a) adenosine rece ptor of human platelets.