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Biophys. J. BioFAST: First Published July 22, 2005. doi:10.1529/biophysj.105.061887
© 2005 by the Biophysical Society.

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CELL BIOPHYSICS

Multi-Step Fibrinogen Binding to the Integrin {alpha}IIb{beta}3 Detected Using Force Spectroscopy

Rustem I. Litvinov 1, Joel S. Bennett 1, John W. Weisel 1 and Henry Shuman 1*

1 University of Pennsylvania School of Medicine

* To whom correspondence should be addressed. E-mail: shuman{at}mail.med.upenn.edu.

Submitted on February 24, 2005
Revised on April 4, 2005
Accepted on 20 June 2005


  Abstract
The regulated ability of integrin {alpha}IIb{beta}3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. We have developed a model system based on laser tweezers, enabling us to measure specific rupture forces needed to separate single receptor-ligand complexes. First of all, we performed a thorough and statistically representative analysis of non-specific protein-protein binding versus specific {alpha}IIb{beta}3-fibrinogen interactions in combination with experimental proof for single-molecule measurements. The rupture force distribution of purified {alpha}IIb{beta}3 and fibrinogen, covalently attached to underlying surfaces, ranged from about 20 pN to 150 pN. This distribution could be fit with a sum of an exponential curve for weak to moderate (20-60 pN) forces, and a Gaussian curve for strong (>60 pN) rupture forces that peaked at 80-90 pN. The interactions corresponding to these rupture force regimes, differed in their susceptibility to {alpha}IIb{beta}3 antagonists or Mn2+, an {alpha}IIb{beta}3 activator. Varying the surface density of fibrinogen changed the total binding probability linearly more than 3.5-fold but did not affect the shape of the rupture force distribution indicating that the measurements represent single molecule binding. The yield strength of {alpha}IIb{beta}3-fibrinogen interactions was independent of loading rate (160-16,000 pN/s) while their probability markedly correlated with the duration of contact. The aggregate of data provides evidence for complex multi-step binding/unbinding pathways of {alpha}IIb{beta}3 and fibrinogen revealed at the single molecule level.

Key Words: binding kinetics, receptor-ligand interactions, rupture forces




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R. I. Litvinov, O. V. Gorkun, S. F. Owen, H. Shuman, and J. W. Weisel
Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the single-molecule level
Blood, November 1, 2005; 106(9): 2944 - 2951.
[Abstract] [Full Text] [PDF]


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Copyright © 2005 by the Biophysical Society.