AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY TO IDENTIFY INHIBITORS OF ACTIVATION OF PLATELET INTEGRIN ALPHA(IIB)BETA(3)

The affinity of integrin alpha(IIb)beta(3) for adhesive ligands is tig htly regulated by the platelet such that fibrinogen binding is observe d only after platelet activation. Ligand binding is necessary for plat elet aggregation, which contributes to vascular occlusion in pathologi cal states. Therefore, we have developed an ELISA assay to screen for compounds that inhibit alpha(IIb)beta(3) activation. Washed platelets were incubated in microtitre wells with potential inhibitory compounds and stimulated with an agonist to activate alpha(IIb)beta(3). After t he addition of biotin-PAC1, a fibrinogen-mimetic monoclonal antibody, the activation state of alpha(IIb)beta(3) was measured by sedimenting the platelets and quantitating the residual biotin-PAC1 in the cell-fr ee supernatant in a streptavidin-based ELISA. This assay detected (1) specific PAC1 binding to activated platelets in response to a variety of agonists, and (2) dose-dependent inhibition of PAC1 binding by func tion-blocking anti-alpha(IIb)beta(3) monoclonal antibodies, by the tet rapeptide, RGDS, and by an alpha(IIb)beta(3)-selective RGD peptidomime tic, Furthermore, the assay detected inhibition of PAC1 binding by int racellular inhibitors of platelet activation, including bisindolylmale imide, a selective protein kinase C antagonist, and wortmannin, an inh ibitor of phosphatidylinositol 3-kinase. These studies demonstrate tha t this integrin activation ELISA can detect pharmacological blockade o f platelet alpha(IIb)beta(3) by extracellular and intracellular inhibi tors. Its use may facilitate the search for clinically useful anti-pla telet drugs.