Specific binding of the thromboxane A2 antagonist 13-azaprostanoic acid to human platelet membranes.
In the present study we characterized the interaction between the thromboxane A2/prostaglandin H2 antagonist, trans-13-azaprostanoic acid (13-APA), and isolated human platelet membranes. In these studies, we developed a binding assay using trans [3H] 13-APA as the ligand. It was found that trans [3H] 13-APA specific binding was rapid, reversible, saturable and temperature dependent. Scatchard analysis of the binding data yielded a curvilinear plot which indicated the existence of two classes of binding sites: a high-affinity binding site with an estimated dissociation constant (Kd) of 100 nM; and a low-affinity binding site with an estimated Kd of 3.5 microM. At saturation, approximately 1 pmol/mg protein of [3H] 13-APA was bound to the high affinity site. In order to further characterize the nature of the [3H] 13-APA binding site, we evaluated competitive binding by cis 13-APA, cis 15-APA, prostaglandin F2 alpha, U46619, 6-ketoprostaglandin F1 alpha and thromboxane B2. It was found that the [3H] 13-APA binding site was stereospecific and structurally specific. Thus, the cis isomer of 13-APA exhibited substantially reduced affinity for binding. Furthermore, the prostaglandin derivatives, thromboxane B2 and 6-ketoprostaglandin F1 alpha, which do not possess biological activity, also did not compete for [3H] 13-APA binding. On the other hand, U46619 which acts as a thromboxane A2/prostaglandin H2 mimetic, and prostaglandin F2 alpha which acts as a thromboxane A2/prostaglandin H2 antagonist, both effectively competed for [3H] 13-APA binding. These findings indicate that trans 13-APA binds to a specific site on the platelet membrane which presumably represents the thromboxane A2/prostaglandin H2 receptor.
D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000378:metabolism; D011466:Prostanoic Acids / Q000378:metabolism* / Q000494:pharmacology; D011982:Receptors, Prostaglandin / Q000378:metabolism; D017482:Receptors, Thromboxane; D013928:Thromboxane A2 / Q000037:antagonists & inhibitors*; D013931:Thromboxanes / Q000037:antagonists & inhibitors*
D005227:Fatty Acids; D011450:Prostaglandin Endoperoxides, Synthetic; D011463:Prostaglandins H; D011466:Prostanoic Acids; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D013931:Thromboxanes; D044262:Prostaglandin H2; D013928:Thromboxane A2; C022574:13-azaprostanoic acid