Integrin Priming Dynamics: Mechanisms of Integrin Antagonist-Promoted αIIbβ3:PAC-1 Molecular Recognition

This investigation addressed the paradox that disintegrins and small RGD-ligands readily bind to the resting alpha IIb beta 3 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 alpha IIb beta 3; 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:alpha IIb beta 3 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) similar to 5 x 10(5) l/mol-s, 25 degrees C) and tightly (K(d) similar to 1 nM) to eptifibatide-primed integrins, captured on a biosensor using an IgG specific for alpha IIb's cytoplasmic domain. Varying the interval between integrin capture and antagonist dissociation indicated that transiently primed alpha IIb beta 3 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 alpha IIb beta 3 (half-time < 2 min), consistent with the priming Window determined by SPR. van't Hoff analysis of alpha IIb beta 3:PAC-1's temperature-dependent K(d) indicated entropy/enthalpy compensation, similar to (resting) integrin binding to the disintegrin echistatin. Eyring analysis of k(on) yielded Delta G degrees double dagger similar to 10 kcal/mol for PAC-1 binding to primed alpha IIb beta 3, 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.