Integrating Dynamic Force Spectroscopy and Surface Plasmon Resonance to Define the Energy Landscape for Integrin:Ligand Binding

Abstract

Background: Blood clots, aggregates of platelets trapped in a mesh of fibrin fibers, can impede normal blood flow, causing heart attacks and strokes. Therapeutic interventions use drugs with Arg-Gly-Asp (RGD) sequences to disrupt interactions between platelet αIIbβ3 integrins and the fibrin network's subunits. Determination of the αIIbβ3-ligand energy landscape will elucidate the successes and limitations of integrin antagonists.Objectives: Integrating surface plasmon resonance (SPR) and dynamic force spectroscopy (DFS), we studied the energetics of αIIbβ3: ligand interactions. We focused on cHArGD, a cyclic peptide structurally similar to eptifibatide, a cardiovascular disease drug, as well as to fibrinogen's KQAGDV integrin-recognition sequence.Methods: DFS determined single bond rupture forces, the dissociation constant koff, and the rupture distance x-1 for αIIbβ3: cHArGD interactions. SPR determined the kinetic and thermodynamic parameters for αIIbβ3: cHArGD binding.Results: DFS performed at three different pulling rates (14000, 42000, and 70000) pN/s) yielded rupture forces of 77, 86 and 88 pN; Bell model analysis yielded a dissociation constant, koff ∼ 0.03 sec−1 and rupture distance x-1∼ 0.6 nm. Excess cHArGD in solution dramatically reduced the rupture forces, confirming specificity. SPR yielded kon ∼ 7000 L/mol-sec and koff ∼ 0.01 sec−1 at 25 °C. SPR equilibrium and transition state thermodynamic data, obtained at 15 - 37 °C, show that αIIbβ3:cHArGD interactions must overcome an entropy-unfavorable activation energy barrier (ΔGao‡ 12 kcal/mol) before gaining a favorable ΔH and ΔS for binding (ΔG° - 8 kcal/mol).Conclusions: SPR and DFS gave comparable dissociation rates for αIIbβ3:cHArGD interactions and a critical rupture distance that agrees with the dimensions of the complementary electrostatic contacts shared by all integrin:RGD complexes. Our energy landscape adds a nanoscale to the mechanisms that regulate αIIbβ3's interactions with pharmacological and physiological ligands.