ABSTRACT
Binding of soluble fibrinogen to the activated conformation of the integrin αIIbß3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbß3 and make substantial contributions to αIIbß3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbß3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbß3 conformers at the interface between the αIIb ß-propeller domain and the ß3 ßI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbß3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide-αIIbß3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbß3 that dissociate in the 60-120 pN range. The Gibbs free energy profiles of the αIIbß3-peptide complexes revealed that the overall stability of the αIIbß3-cRGDFK complex was comparable with that of the αIIbß3-γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbß3-fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of αIIbß3 with immobilized fibrinogen and fibrin.
