This investigation addressed the paradox that disintegrins and small RGD-ligands readily bind to the resting alphaIIbbeta3 integrin, while macromolecules with similar integrin recognition motifs require an activated, or primed, receptor. Three structurally similar pharmaceutical integrin antagonists (eptifibatide, tirofiban, and roxifiban) were each incubated with resting alphaIIbbeta3; after drug wash-out, the receptor's ability to recognize PAC-1, an activation-dependent IgM with an RYD integrin-targeting site was measured. Their promotion of PAC-1:alphaIIbbeta3 binding (solid phase assay), eptifibatide > tirofiban > roxifiban, correlated with their ability to shift the receptor to an open conformer, as measured by analytical ultracentrifugation. Surface plasmon resonance (SPR) demonstrated that PAC-1 bound rapidly (k(on) approximately 5 x 10(5) l/mol-s, 25 degrees C) and tightly (Kd approximately 1 nM) to eptifibatide-primed integrins, captured on a biosensor using an IgG specific for alphaIIb's cytoplasmic domain. Varying the interval between integrin capture and antagonist dissociation indicated that transiently primed alphaIIbbeta3 retains the ability to rapidly bind PAC-1 from 2-90 min, although the dissociation rate increased at later times, indicative of a weakening of the complex. Fluorescence anisotropy (fluorophore-tagged analogue exchange assay) demonstrated that eptifibatide dissociates rapidly from alphaIIbbeta3 (half-time <2 min), consistent with the priming window determined by SPR. van't Hoff analysis of alphaIIbbeta3:PAC-1's temperature-dependent Kd indicated entropy/enthalpy compensation, similar to (resting) integrin binding to the disintegrin echistatin. Eyring analysis of k(on) yielded DeltaG degrees approximately 10 kcal/mol for PAC-1 binding to primed alphaIIbbeta3, 3 kcal/mol lower than that of echistatin. These observations suggest that priming lowers the transition-state energy barrier, enabling rapid macromolecular ligand binding to activated integrins. Recognizing the limitations in extrapolating from laboratory to pathophysiological conditions, we propose that similar priming mechanisms may contribute to the unexpected platelet-activating effects of pharmaceutical integrin antagonists.