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Resolving two-dimensional kinetics of the integrin ?IIb?3-fibrinogen interactions using binding-unbinding correlation spectroscopy.

ABSTRACT: Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional kinetics of interactions between fibrinogen and the integrin ?IIb?3, 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 ?IIb?3 molecules and forced dissociation of an ?IIb?3-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 ?IIb?3 pre-exists in at least two states that differ in their zero force on-rates (k(on1) = 1.4 × 10(-4) and k(on2) = 2.3 × 10(-4) ?m(2)/s), off-rates (k(off1) = 2.42 and k(off2) = 0.60 s(-1)), and dissociation constants (K(d)(1) = 1.7 × 10(4) and K(d)(2) = 2.6 × 10(3) ?m(-2)). The integrin activator Mn(2+) changed the on-rates and affinities (K(d)(1) = 5 × 10(4) and K(d)(2) = 0.3 × 10(3) ?m(-2)) but did not affect the off-rates. The strength of ?IIb?3-fibrinogen interactions was time-dependent due to a progressive increase in the fraction of the high affinity state of the ?IIb?3-fibrinogen complex characterized by a faster on-rate. Upon Mn(2+)-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 two-dimensional kinetic rates for the low and high affinity ?IIb?3 and fibrinogen interactions at the single molecule level and offer direct evidence for the time- and force-dependent changes in ?IIb?3 conformation and ligand binding activity, underlying the dynamics of fibrinogen-mediated platelet adhesion and aggregation.

SUBMITTER: Litvinov RI

PROVIDER: S-EPMC3471694 | biostudies-other | 2012 Oct

REPOSITORIES: biostudies-other

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Resolving two-dimensional kinetics of the integrin αIIbβ3-fibrinogen interactions using binding-unbinding correlation spectroscopy.

Litvinov Rustem I RI Mekler Andrey A Shuman Henry H Bennett Joel S JS Barsegov Valeri V Weisel John W JW

The Journal of biological chemistry 20120814 42

Using a combined experimental and theoretical approach named binding-unbinding correlation spectroscopy (BUCS), we describe the two-dimensional 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 n ...[more]

PMID: 22893701

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Project description:By following single fluorescent molecules in a microscope, single-particle tracking (SPT) can measure diffusion and binding on the nanometer and millisecond scales. Still, although SPT can at its limits characterize the fastest biomolecules as they interact with subcellular environments, this measurement may require advanced illumination techniques such as stroboscopic illumination. Here, we address the challenge of measuring fast subcellular motion by instead analyzing single-molecule data with spatiotemporal image correlation spectroscopy (STICS) with a focus on measurements of confined motion. Our SPT and STICS analysis of simulations of the fast diffusion of confined molecules shows that image blur affects both STICS and SPT, and we find biased diffusion rate measurements for STICS analysis in the limits of fast diffusion and tight confinement due to fitting STICS correlation functions to a Gaussian approximation. However, we determine that with STICS, it is possible to correctly interpret the motion that blurs single-molecule images without advanced illumination techniques or fast cameras. In particular, we present a method to overcome the bias due to image blur by properly estimating the width of the correlation function by directly calculating the correlation function variance instead of using the typical Gaussian fitting procedure. Our simulation results are validated by applying the STICS method to experimental measurements of fast, confined motion: we measure the diffusion of cytosolic mMaple3 in living Escherichia coli cells at 25 frames/s under continuous illumination to illustrate the utility of STICS in an experimental parameter regime for which in-frame motion prevents SPT and tight confinement of fast diffusion precludes stroboscopic illumination. Overall, our application of STICS to freely diffusing cytosolic protein in small cells extends the utility of single-molecule experiments to the regime of fast confined diffusion without requiring advanced microscopy techniques.

Dataset Information

Resolving two-dimensional kinetics of the integrin ?IIb?3-fibrinogen interactions using binding-unbinding correlation spectroscopy.

Publications

Resolving two-dimensional kinetics of the integrin αIIbβ3-fibrinogen interactions using binding-unbinding correlation spectroscopy.

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