IDENTIFICATION OF LOW-MOLECULAR-WEIGHT GP IIB IIIA ANTAGONISTS THAT BIND PREFERENTIALLY TO ACTIVATED PLATELETS/

A critical function of fibrinogen in hemostasis and thrombosis is to m ediate platelet aggregation by binding selectively to an activated for m of glycoprotein (GP) IIb/IIIa. Although numerous peptide and nonpept ide 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 o f the dansyl fluorophore containing GP IIb/IIIa antagonist L-736,622. Also, coating either form of the purified GP IIb/IIIa onto yttrium sil icate scintillation proximity assay fluomicrospheres produced an activ ated form of the receptor, whose binding affinity for GP IIb/IIIa anta gonists was measured conveniently by competition with the arginine-gly cine-aspartic acid (RGD) containing heptapeptide [I-125]L-692,884. In addition, direct binding measurements with radiolabeled GP IIb/IIIa an tagonists also were performed on resting and activated platelets. We i dentified two classes of compounds. One class binds to both forms of G P 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 compoun ds binds with much higher affinity to the activated form of GP IIb/III a (purified or on platelets) as compared with the resting form (e.g., L-734,217, MK-852, tirofiban and L-692,884). Selective antagonists, li ke L-734,217 (K-d(Activated) = 5 nM and K-d(Resting) = 620 nM), can ef fectively inhibit ex vivo platelet aggregation at concentrations of dr ug that produce low levels of occupancy of the circulating platelet re ceptors. The potential clinical advantages of selective versus nonsele ctive GP IIb/IIIa antagonists remain to be explored in clinical trials .