PROBING alpha IIb beta 3: LIGAND INTERACTIONS BY DYNAMIC FORCE SPECTROSCOPY AND SURFACE PLASMON RESONANCE

The interaction between platelet integrin alpha IIb beta 3 and fibrin(ogen) plays a key role in blood clot formation and stability. Integrin antagonists, a class of pharmaceuticals used to prevent and treat cardiovascular disease, are designed to competitively interfere with this process. However, the energetics of the integrin - drug binding are not fully understood, potentially hampering further development of this class of pharmaceuticals. We integrated dynamic force spectroscopy (DFS) and surface plasmon resonance (SPR) to probe the energetics of complex formation between alpha IIb beta 3 and cHarGD, a cyclic peptide integrin antagonist. Analysis of alpha IIb beta 3: cHarGD DFS rupture force data at pulling rates of 14 000 pN/s, 42 000 pN/s and 70 000 pN/s yielded k(off) = 0.02 = 0.09s(-1), a dissociation energy barrier Delta G(d)(double dagger) = 22 - 29 kJ/mol, and a potential well width x(-1) = 0.5-0.8 nm. SPR kinetic data yielded an association rate constant k(on) = 7 x 103 L/mol-s and a dissociation rate constant k(off) = 10(-2)s(-1), followed by a slower stabilization step (T similar to 400s). Both DFS and SPR detected minimal interactions between alpha IIb beta 3 and cHarGA demonstrating a key role for electrostatic interactions between the ligand aspartate and the integrin metal ion-dependent adhesion site (MIDAS). Our work provides new insights into the energy landscape of alpha IIb beta 3's interactions with pharmacological and physiological ligands.