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Wednesday, August 22, 2012

Resolving two-dimensional kinetics of the integrin αIIbβ3-fibrinogen interactions using Binding-Unbinding Correlation Spectroscopy

Rustem I. Litvinov, Andrey Mekler, Henry Shuman, Joel S. Bennett,Valeri Barsegov and John W. Weisel

Using a combined experimental and theoretical approach named Binding-Unbinding Correlation Spectroscopy (BUCS), we describe the 2D kinetics of interactions between fibrinogen and the integrin αIIbβ3, the ligand-receptor pair essential for platelet function during hemostasis and thrombosis. The methodology uses the optical trap to probe force-free association of individual surface-attached fibrinogen and αIIbβ3 molecules and forced dissociation of an αIIbβ3-fibrinogen complex. This novel approach combines force-clamp measurements of bond lifetimes with the binding mode to quantify the dependence of the binding probability on the interaction time. We found that fibrinogen-reactive αIIbβ3 pre-exists in at least two states that differ in their zero-force on-rates (kon1=1.4x10-4 and kon2=2.3x10-4 μm2/s), off-rates (koff1=2.42 and koff2=0.60 s-1) and dissociation constants (Kd1=1.7x104 andKd2=2.6x103 1/μm2). The integrin activator Mn2+ changed the on-rates and affinities (Kd1=5x104 and Kd2=0.3x103 1/μm2) but did not affect the off-rates. The strength of αIIbβ3-fibrinogen interactions was time-dependent due to progressive increase in the fraction of the high-affinity state of the αIIbβ3-fibrinogen complex characterized by a faster on-rate. Upon Mn2+-induced integrin activation, the force-dependent off-rates decrease while the complex undergoes a conformational transition from a lower- to higher-affinity state. The results obtained provide quantitative estimates of the 2D kinetic rates for the low- and high-affinity αIIbβ3 and fibrinogen interactions at the single-molecule level, and offer direct evidence for the time- and force-dependent changes in αIIbβ3 conformation and ligand-binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.

DOI

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