Interaction of the Integrin αIIbβ3 with Monomeric Fibrin at the Single-Molecule Level.

Abstract 4018 Poster Board III-954 Although platelets aggregated with fibrin constitute major components of in vivo blood clots and thrombi, heretofore most research has focused on fibrinogen's role in platelet aggregation. The integrin αIIbβ3 has been shown to primarily mediate platelet-fibrin interactions, but the mechanism of αIIbβ3 binding to fibrin is largely unknown and may be significantly different from binding to fibrinogen. To elucidate mechanisms of platelet interactions with fibrin, we have compared the overall reactivity, binding strength, and specificity towards αIIbβ3 of human fibrinogen vs. monomeric fibrin, using single-molecule optical trap-based rupture force spectroscopy of the surface-bound proteins. In this system, a ligand-coated bead is trapped by the laser and repeatedly brought into contact with a receptor-coated surface so that the forces required to separate the two can be measured and displayed as rupture force histograms. We have applied this technique to the interaction of fibrin(ogen) and αIIbβ3 by measuring the force required to separate a laser-trapped bead coated with either fibrinogen or monomeric fibrin from an immobilized pedestal coated with purified αIIbβ3. Experiments were performed with plasma-purified or recombinant homodimeric γA/γA and γ′/γ′ fibrin(ogen)s. The latter protein represents a splicing variant in which the γ chain has a substitution in the C-terminal four amino acids in 400-411 dodecapeptide, the major αIIbβ3-binding site in fibrinogen. Surface-bound monomeric fibrin was obtained by treating fibrinogen-coated surfaces with thrombin. Control integrin-fibrinogen interactions manifested as a characteristic bimodal rupture force histogram with rupture forces ranging from 20 pN to 140 pN, similar to what we had observed previously (PNAS, 2002, 99, 7426). The interactions were highly sensitive to inhibitory effects of abciximab and eptifibatide, specific αIIbβ3 antagonists, and to Mn2+-induced activation, indicating that they were mediated by the functional integrin. Monomeric fibrin γA/γA was at least as reactive towards αIIbβ3 as the parental fibrinogen γA/γA, sometimes exhibiting even higher binding probabilities and slightly stronger rupture forces. Fibrin-integrin interactions were less sensitive to the inhibitory effect of abciximab compared to fibrinogen, suggesting that some additional structures in αIIbβ3, not completely blocked by this Fab fragment, might be involved in binding to fibrin. Similar to fibrinogen, fibrin-integrin interactions were partially sensitive to eptifibatide, cRGD peptide and γC-dodecapeptide, indicating that their specificity is akin to fibrinogen but may not be identical. Fibrinogen γ′/γ′, lacking the established binding site for the integrin, was reactive with αIIbβ3, but the binding strength was somewhat smaller. The effect of replacing the γA with the γ′ chains in fibrinogen was qualitatively similar to competitive inhibition by the γC-dodecapeptide and resulted in complete cutoff of the larger force peak as well as in partial reduction of weaker interactions. That fibrinogen γ′/γ′ maintained its ability to bind αIIbβ3 suggests that the γ400-411 motif is not the only structure involved in the binding of immobilized fibrinogen to the integrin. Fibrin γ′/γ′ was as reactive with αIIbβ3 as fibrinogen γ′/γ′. Both of them interacted with αIIbβ3 in an RGD-sensitive manner. The data show that both surface-bound fibrinogen and monomeric fibrin are highly reactive with the integrin αIIbβ3. Fibrin is somewhat more reactive than fibrinogen in terms of binding probability and strength and is less sensitive to a specific inhibitor, abciximab, suggesting that αIIbβ3-fibrin interactions have distinct specificity. Susceptibility to competitive inhibitors, such as cRGD and γC-dodecapeptide, along with maintenance of integrin-binding activity of fibrinogen γ′/γ′ suggest that the αIIbβ3-binding sites in fibrin(ogen) are complex and include, but are not confined to, the γC-terminal 400-411 motif. Disclosures: No relevant conflicts of interest to declare.