Measurement of the Lifetime of Bonds Between αIIbβ3 and Fibrinogen Using Constant Unbinding Forces Generated by Optical Tweezers
Authors: Litvinov Rustem I. , Shuman Henry , Weisel John , Bennett Joel S. 
Published: April 2020

We have shown that the distribution of rupture forces between individual αIIbβ3 and fibrinogen molecules displays at least two components that differ in kinetics, loading rate dependence, and susceptibility to activation and inhibition of the integrin. This suggests that the binding and unbinding of αIIbβ3 and fibrinogen is a complex multistep process that depends on the conformational state of both αIIbβ3 and fibrinogen, the duration of their interaction, and environmental factors such as externally-applied shear force. To directly test these possibilities, we quantified the lifetime of bonds stabilizing individual αIIbβ3-fibrinogen complexes with a novel nanoscale laser tweezers-based system that uses an optical trap to apply a constant unbinding force to single-molecule protein-protein interactions. When a ligand-coated bead is brought into repeated contact with a receptor-coated silica pedestal using an optical trap, the signal parameters that are measured correspond to both compressive and rupture forces. To measure bond lifetimes, the amplitude of the generated tensile force signal must remain constant throughout the lifetime of the bond. This can be accomplished by incorporating an analog feedback circuit within the optical system. The system also enables us to control the time of contact between interacting surfaces, the magnitude of compressive force during contact, the magnitude of the tensile force, and the time of protein-protein separation when binding occurs. To quantify the forced unbinding of fibrinogen molecules covalently bound to latex beads and αIIbβ3 molecules covalently attached to silica microspheres, we measured the distribution of bond lifetimes obtained under constant tensile force, mimicking the effect of hydrodynamic blood flow on an adherent platelet. We found that the separation times of the αIIbβ3- and fibrinogen-coated surfaces varied, indicating that the interactions occurring at the interface had heterogeneous kinetic and thermodynamic properties. Discrimination of specific αIIbβ3-fibrinogen binding events versus non-specific interactions was based on comparison of bond lifetime distributions in the absence and presence of abciximab or eptifibatide, specific inhibitors of fibrinogen binding to activated αIIbβ3. We found that the separation times of the αIIbβ3- and fibrinogen-coated surfaces were bimodal, with specific integrin-fibrinogen interactions lasting more than 2s under a constant tensile force of 50 pN. Varying the time of contact between αIIbβ3 and fibrinogen from 0.1s to 2.0s at the same unbinding force revealed that the bond lifetimes increased as the duration of contact between that interacting surfaces increased, suggesting that stability of αIIbβ3-fibrinogen interactions is time-dependent. Because these measurements mimic the binding/unbinding parameters and the time course of the αIIbβ3-fibrinogen interactions under conditions of shear, they are relevant to physiological processes of fibrinogen-mediated platelet adhesion and platelet aggregation. Taken together, our data suggest a model for fibrinogen binding to αIIbβ3 in which the initial interaction is followed by reorganization of the binding interface, thereby enhancing the strength and stability of fibrinogen binding.