Abstract

Fibrinogen binding to the integrin αIIbβ3 can mediate platelet aggregation and platelet spreading on fibrinogen-coated surfaces. However, in vivo, where blood clotting is catalyzed by thrombin, αIIbβ3 activation and fibrinogen conversion to fibrin occur simultaneously, although the relative contributions of fibrinogen versus monomeric and polymerized fibrin to αIIbβ3-mediated platelet aggregation and adhesion are unknown. The interaction of αIIbβ3 with fibrin is responsible for clot retraction. Nonetheless, differences in the efficacy of αIIbβ3 antagonists with regard to clot retraction versus platelet aggregation suggest that the interaction of αIIbβ3 with fibrinogen and fibrin is different. Here, we have compared nanomechanical measurements of the interaction of αIIbβ3 with fibrin and fibrinogen to explain these differential effects. Briefly, a microscopic bead covalently coated with purified αIIbβ3 was captured by a focused laser beamand repeatedly brought into contact with surface-attached fibrinogen, monomeric fibrin produced by treating the immobilized fibrinogen with thrombin, or a naturally-formed hydrated fibrin fiber at the edge of a plasma clot. When an αIIbβ3-ligand complex was detected, the force required to dissociate the complex was measured at piconewton resolution. By analyzing the distribution of these forces, we determined the overall reactivity and the binding strength of the interacting protein pairs. Besides native fibrinogen, experiments were performed with recombinant fibrinogen variants. Rupture force histograms for αIIbβ3-fibrinogen interactions displayed forces ranging from 20 pN to 140 pN and could be segregated into moderate (20-60 pN) and strong (>60 pN) interactions. The inhibitory effects of EDTA and αIIbβ3antagonists such as abciximab and eptifibatide confirmed that fully functional αIIbβ3 was required to obtain these force signals. Monomeric fibrin displayed a higher cumulative probability of interacting with αIIbβ3 and a greater binding strength. αIIbβ3-fibrin interactions were also less sensitive to inhibition by abciximab and eptifibatide, suggesting that they had a different binding specificity. Both fibrinogen- and fibrin- interactions with αIIbβ3 were partially inhibited by RGD-containing peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants αD97E or αD574E whose mutated RGD motifs were less reactive with αIIbβ3 than those of wild type fibrin. Monomeric fibrin made from a homodimeric fibrinogen γ chain splice variant lacking the γC αIIbβ3 binding motif was more reactive with αIIbβ3 than the parent fibrinogen, suggesting that this binding motif is less important in fibrin. Polymeric fibrin displayed a rupture force profile similar to fibrinogen and monomeric fibrin with moderate (20-60 pN) and strong (>60 pN) forces that peaked at 70-80 pN. The αIIbβ3 antagonist eptifibatide, the αVβ3-specific cyclic RGD-containing peptide XJ735, a non-selective cyclic RGD-containing peptide, and a dodecapeptide corresponding to the C-terminus of the fibrinogen γ chain each produced a sharp drop in the medium-to-high force range. Interaction forces >60 pN were more effectively reduced by EDTA than by any of the inhibitory peptides, indicating their dependence on the structural integrity and functionality of αIIbβ3. The free γC dodecapeptide was also less efficient than the cRGD peptides or eptifibatide in preventing the stronger interactions. Taken together, these results demonstrate that surface-bound fibrinogen and monomeric, as well as polymeric fibrin, are highly reactive with the αIIbβ3. Fibrin is more reactive than fibrinogen in terms of its binding probability and it has greater binding strength. Fibrin binding to αIIbβ3 is also less sensitive to αIIbβ3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding requirements. In particular, the maintenance of αIIbβ3-binding activity when the C-terminus of the fibrinogen γ chain and the α chain RGD sequences are mutated suggests that the αIIbβ3-binding sites in fibrin are not confined to its known γ chain and RGD motifs.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.