Vol. 285, Issue 3, 1317-1326, June 1998

Identification of Low Molecular Weight GP IIb/IIIa Antagonists That Bind Preferentially to Activated Platelets

Rodney A. Bednar, S. Lee Gaul, Terence G. Hamill, Melissa S. Egbertson, Jules A. Shafer, George D. Hartman, Robert J. Gould and Bohumil Bednar

Department of Pharmacology (R.A.B., S.L.G., T.G.H., R.J.G., B.B.), Biological Chemistry (J.A.S.) and Medicinal Chemistry (M.S.E., G.D.H.), Merck Research Laboratories, West Point, Pennsylvania

A critical function of fibrinogen in hemostasis and thrombosis is to mediate platelet aggregation by binding selectively to an activated form of glycoprotein (GP) IIb/IIIa. Although numerous peptide and nonpeptide fibrinogen receptor antagonists have been described, their binding selectivity for resting and activated platelets has not been explored. Therefore, dissociation constants of GP IIb/IIIa antagonists for two biochemically separated forms of purified GP IIb/IIIa and for resting and activated platelets were determined by competitive displacement of the dansyl fluorophore containing GP IIb/IIIa antagonist L-736,622. Also, coating either form of the purified GP IIb/IIIa onto yttrium silicate scintillation proximity assay fluomicrospheres produced an activated form of the receptor, whose binding affinity for GP IIb/IIIa antagonists was measured conveniently by competition with the arginine-glycine-aspartic acid (RGD) containing heptapeptide [¹²⁵I]L-692,884. In addition, direct binding measurements with radiolabeled GP IIb/IIIa antagonists also were performed on resting and activated platelets. We identified two classes of compounds. One class binds to both forms of GP IIb/IIIa, as well as resting and activated platelets, with similar K_d values (e.g., L-736,622 and Echistatin). The other class of compounds binds with much higher affinity to the activated form of GP IIb/IIIa (purified or on platelets) as compared with the resting form (e.g., L-734,217, MK-852, tirofiban and L-692,884). Selective antagonists, like L-734,217 (K_d^Activated = 5 nM and K_d^Resting = 620 nM), can effectively inhibit ex vivo platelet aggregation at concentrations of drug that produce low levels of occupancy of the circulating platelet receptors. The potential clinical advantages of selective versus nonselective GP IIb/IIIa antagonists remain to be explored in clinical trials.