Journal Article

Multi-Step Fibrinogen Binding to the Integrin αIIb β3 Detected Using Force Spectroscopy

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

2005

Overview

The regulated ability of integrin αIIb β3 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 nonspecific protein-protein binding versus specific αIIb β3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified αIIb β3 and fibrinogen, covalently attached to underlying surfaces, ranged from ∼20 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 αIIb β3 antagonists or Mn 2+, an αIIb β3 activator. Varying the surface density of fibrinogen changed the total binding probability linearly >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 αIIb β3-fibrinogen interactions was independent of the loading rate (160–16,000 pN/s), whereas their binding probability markedly correlated with the duration of contact. The aggregate of data provides evidence for complex multi-step binding/unbinding pathways of αIIb β3 and fibrinogen revealed at the single-molecule level.

• Publisher: Elsevier Inc,Biophysical Society
• Subject: Cell Biophysics