PROBING αIIbβ3: LIGAND INTERACTIONS BY DYNAMIC FORCE SPECTROSCOPY AND SURFACE PLASMON RESONANCE

Abstract

The interaction between platelet integrin αIIbβ3 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 αIIbβ3 and cHarGD, a cyclic peptide integrin antagonist. Analysis of αIIbβ3:cHarGD DFS rupture force data at pulling rates of 14 000 pN/s, 42 000 pN/s and 70 000 pN/s yielded koff = 0.02–0.09 s−1, a dissociation energy barrier ΔGd‡ = 22–29 kJ/mol, and a potential well width x−1 = 0.5–0.8 nm. SPR kinetic data yielded an association rate constant kon = 7 × 103 L/mol-s and a dissociation rate constant koff = 10−2 s−1, followed by a slower stabilization step (τ ~ 400 s). Both DFS and SPR detected minimal interactions between αIIbβ3 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 αIIbβ3’s interactions with pharmacological and physiological ligands.

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

“…16,48,49 Moreover, the calculated values of the rupture forces reported here are also qualitatively consistent with our previous studies in which we found that cRGD- α IIb β 3 interactions had a greater binding strength than that of α IIb β 3 with γ C-12. 16 Further, the two-pathway unbinding of cRGDFK in the MD simulations agrees with a bimodal distribution of rupture forces observed experimentally.…”

“…16 Similar to our results, Lee and Marchant, using atomic force microscopy and live platelets, found that the kinetic off-rate ( k off ) was significantly greater for γ C-12 than for RGD-containing peptides, indicating that the γ C-12– α IIb β 3 complex dissociates faster under tension than does the cRGDFK- α IIb β 3 complex. 48 Lastly, based on a theoretical model, Dutta et al 49 found that the dissociation energy of a cyclic RGD peptide cHarGD [cyclo ( S,S )-L-lysyl-L-tyrosyl-glycyl-L-cystinyl-L-homoarginyl-glycyl-L-aspartyl-L-trytopanyl-L-prolyl-L-cystine] was ~7–9 kcal/mol, less than the value we obtained for cRGDFK (~16 kcal/mol), but within the same order of magnitude.…”

Preliminary Therapy Evaluation of 225Ac-DOTA-c(RGDyK) Demonstrates that Cerenkov Radiation Derived from 225Ac Daughter Decay Can Be Detected by Optical Imaging for In Vivo Tumor Visualization

“…Samples of the lyophilized peptide were dissolved in water (5 mg/mL) and then diluted in sodium acetate buffer (50 µg/mL; pH 5.0) for coupling to biosensor chips. CM-5 biosensor chips (Biacore, Inc., Piscataway, NJ) were activated for amine coupling by reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in a Biacore T100 instrument 24 , 25 . c(RGDyK) (50 µg/mL) in 0.1 M sodium acetate buffer (pH 5.0) was delivered to the sample chamber, yielding immobilization densities of 222 response units (RU) for the α v β 3 :c(RGDyK) binding series and 196 RU for the La-DOTA-c(RGDyK) competition series.…”

Probing of miniPEGγ-PNA–DNA Hybrid Duplex Stability with AFM Force Spectroscopy

“…This technique has been extensively used to study several systems including folding/unfolding of biomolecules 17-21 , peptide-peptide interactions 22-24 , ligand-receptor interactions 25-27 , and other molecular interactions 28 . In the SMFS experiment, the molecule of interest and its target are functionalized on the probe and surface.…”