EFFECTS OF FREE AND MACROMOLECULAR-BOUND TXA(2)-RECEPTOR ANTAGONIST BM-13.505 ON U46619-INDUCED PLATELET-AGGREGATION

The goal of this study was to synthesize a macromolecular probe of the TXA2 receptor antagonist BM13.505 which is unable to penetrate the pl atelet membrane for localization and characterization of the TXA2 rece ptor. The active NHS-ester of BM13.505 was synthesized and purified. I t was used for covalent coupling of BM13.505 to bovine serum albumin, a macromolecular carrier. Inhibitory effects of free and macromolecula r bound BM13.505 on aggregatory properties of U46619-stimulated platel ets were measured and compared to TXA2 generation in platelets, as det ermined by TXB2 radioimmuno assay. No inhibitory effects of free and m acromolecular-bound BM13.505 on ADP- or thrombin-induced platelet aggr egation were observed. Equimolar concentrations of free or macromolecu lar bound BM13.505 inhibited U46619-induced platelet aggregation and T XA2 generation with equal potency. IC50-values for platelet aggregatio n inhibition by free and macromolecular bound BM13.505 were 64 nM and 96 nM respectively. It appears that the TXA2 receptor ligand binding s ite is located close to the outer membrane surface of platelets. Inter action of macromolecular bound BM13.505 with the platelet thromboxane receptor does not depend on the availability of the free carboxyl resi due in BM13.505. The method for coupling a TXA2 receptor antagonist to a macromolecule will aid in constructing probes for the localization and characterization of the TXA2 receptor.