Time-Dependent Single-Molecule Interactions of the Platelet Integrin αIIbβ3 with Cyclic Arg-Gly-Asp and the Fibrin(ogen) γC-Dodecapeptide
Authors: Litvinov Rustem I. , Billings Paul C. , Streu Craig N. , Weisel John W. , Bennett Joel S. 
Published: October 2019

Abstract
Abstract 2103
Both fibrinogen and fibrin bind to the platelet integrin αIIbβ3 (GPIIb-IIIa). There are at least two motifs in human fibrin(ogen) that bind to αIIbβ3: HHLGGAKQAGDV located at position 400–411 at the C-terminus of the γ chain and RGD located at position 572–574 in the Aα (or α) chain. A second RGD motif located at position 95–97 in the Aα (or α) chain may also play a role in αIIbβ3-mediated platelet adhesion on fibrin(ogen). Recent crystal structures of αIIbβ3 containing either a γ chain peptide corresponding to residues 404–411 or linear RGD-containing peptide (Springer et al., J. Cell Biol. 182:791-800, 2008) revealed that although the RGD and γ chain binding sites completely overlap, the γ chain interface with αIIbβ3 is much more extensive, implying that αIIbβ3 should bind more tightly to the γ chain peptide. To directly test this possibility, we applied single-molecule optical trap-based rupture force spectroscopy to study the interactions of RGD and HHLGGAKQAGDV with purified αIIbβ3. Experiments were performed with the cyclo(RGDFK) peptide (cRGD) and the HHLGGAKQAGDV peptide (H12) covalently attached to dextran-functionalized latex beads via their primary amines, simulating more closely the physiological situation and leaving their αIIbβ3-binding groups exposed. The cyclo(RADFK) peptide (cRAD), as well as weakly reacting surfaces containing dextran alone or albumin, were used as negative controls. We tested whether the strength and probability of formation of the αIIbβ3-ligand complex depend on the precisely controlled duration of contact between the interacting surfaces coated with the receptor and the ligand. A peptide-coated microscopic (1.9 μm) bead was trapped by the focused laser beam and repeatedly brought into contact with an αIIbβ3-coated silica pedestal (5 μm) attached to the bottom of a chamber. The repeated touching and separation of bead and pedestal were allowed to occur with a compressive force of 10–20 pN and a bead pulling speed of 0.8 μm/s. The flexible parameter was the stopping time of the oscillating bead, which corresponds to the duration of contact between the interacting surfaces. Varying the time of contact between the surface-bound peptides and αIIbβ3 from 0.01s to 2s revealed that the average binding strength for cRGD and H12 with αIIbβ3 increased to 40–50 pN until it reached a plateau at ~0.5s, indicating that the interactions were time-dependent and occurred on the time scale of tens to hundreds of milliseconds. The strength of the cRGD-αIIbβ3 binding was consistently greater than that of H12-αIIbβ3 by 15–20% at the corresponding contact times. The probability of the peptide-αIIbβ3 interaction was also time-dependent. Within an interval of contact duration of 0.01–2s, the cumulative binding probability for cRGD-αIIbβ3 bonds increased from zero up to ~35%, whereas the binding probability for H12-αIIbβ3 bonds was 21% at the same surface density of the peptides and integrin. Both values were reached at a contact duration time of ~1.0s. Importantly, because a relatively small number of interface contacts resulted in the establishment of αIIbβ3-peptide bonds during the applied range of contact time, it is likely that the majority of the measured bond rupture events resulted from the dissociation of single bonds. Thus, these data demonstrate that cRGD has a higher binding preference to αIIbβ3 compared to H12. Further, they support a hypothesis that the RGD motifs, exposed either when fibrinogen is immobilized on a surface or following its conversion to fibrin, may account for the remarkable platelet reactivity with these adhesive substrates.

Disclosures:
No relevant conflicts of interest to declare.