Abstract

Binding of soluble fibrinogen to the activated open conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. Paradoxically, however, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3. Moreover, the Aα chain RGD motifs make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. The interaction of αIIbβ3 with a variety of RGD- and γC-12 peptides has been studied extensively, but the characteristics of their binding to the active open and inactive closed conformations of αIIbβ3 are largely unknown. Here, we have used both experimental and computational approaches to compare the two-dimensional kinetics, thermodynamics and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. First, we employed optical trap-based single-molecule nanomechanical measurements to determine the probability of peptide binding to αIIbβ3 as a function of the time the peptides interact with αIIbβ3. In the optical trap-based experimental system, a microscopic bead coated with either cRGDFK or γC-12 is trapped in a fluid chamber by a focused laser beam and moved in an oscillatory manner to touch a stationary pedestal coated with αIIbβ3. When the peptide on the bead interacts with αIIbβ3 on the pedestal, tension is generated when the bead is displaced from the laser focus until the αIIbβ3-peptide bond ruptures. The percentage of binding/unbinding events at a particular time of contact duration enabled us to plot the force-free binding probability as a function of contact duration. From these curves, we extracted first-order binding rates: 0.61×10-14 cm2/s for γC-12 and 0.71×10-14 cm2/s for cRGDFK and unbinding rates: 2.05/s and 1.53/s for the γC-12 and cRGDFK, respectively. Corresponding binding affinity constants were 0.3×10-14 cm2 and 0.46×10-14 cm2 for the γC-12 and cRGDFK, respectively, indicating that cRGDFK binds to αIIbβ3 somewhat tighter than γC-12. These measurements were then followed by computational modeling using NMR-determined 3D solution structures of cRGDFK and γC-12, as well as the crystallographically resolved αIIbβ3 conformers of the αIIbβ3 headpiece (PDB entries: 3ZDX and 3ZE2). Docking analysis confirmed that both cRGDFK and γC-12 bound to the αIIbβ3 conformers at a site located in the interface between the α subunit β-propeller domain and the β subunit βI domain. Estimated docking energies revealed that the closed αIIbβ3 conformation is almost as reactive towards the peptides as the open conformation. Next, we probed the strength of the αIIbβ3-peptide interactions and determined their comparative binding energies and the dynamics of interatomic contacts by performing dynamic force-ramp unbinding in silico using all-atom Molecular Dynamics simulations in implicit solvent on Graphics Processing Units. Force was increased linearly at a pulling rate of 2×104 μm/s and applied to specified Cα-atom of the peptides. Analysis of the molecular force profiles and the number of ligand-receptor binding contacts revealed that both peptides interact strongly with αIIbβ3, forming stable bimolecular complexes that dissociate in the 60-120 pN range through several competing unbinding pathways that display multi-step kinetics and distinct bond lifetimes. We profiled the Gibbs free energy (ΔG) of the αIIbβ3-peptide complexes as a function of the separation distance between the open form of αIIbβ3 headpiece and the peptide using the Umbrella Sampling technique. The results showed that the energies of ligand binding to the αIIbβ3 headpiece are ΔG~16 kcal/mol for cRGDFK and ΔG~13 kcal/mol for γC-12, meaning that the overall thermodynamic stability of the αIIbβ3-peptide complex is higher for cRGDFK than for γC-12. Thus, these results provide a kinetic and thermodynamic explanation for previous observations that the affinity of RGD for αIIbβ3 is greater than AGDV and support our hypothesis that the RGD motifs preferentially support the interaction of αIIbβ3 with immobilized fibrinogen and fibrin.

Disclosures

Weisel:Bayer: Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.