Effects of free and macromolecular-bound TXA2 receptor antagonist BM 1..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 platelet membrane for localization and characterization of the TXA₂ receptor. The active NHS-ester of BM13.505 was synthesized and purified. It was used for covalent coupling of BM13.505 to bovine serum albumin, a macromolecular carrier. Inhibitory effects of free and macromolecular bound BM13.505 on aggregatory properties of U46619-stimulated platelets were measured and compared to TXA₂ generation in platelets, as determined by TXB₂ radioimmuno assay. No inhibitory effects of free and macromolecular-bound BM13.505 on ADP- or thrombin-induced platelet aggregation were observed. Equimolar concentrations of free or macromolecular bound BM13.505 inhibited U46619-induced platelet aggregation and TXA₂ generation with equal potency. IC₅₀-values for platelet aggregation inhibition by free and macromolecular bound BM13.505 were 64 nM and 96 nM respectively. It appears that the TXA₂ receptor ligand binding site is located close to the outer membrane surface of platelets. Interaction of macromolecular bound BM13.505 with the platelet thromboxane receptor does not depend on the availability of the free carboxyl residue 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.