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Multi-Step Fibrinogen Binding to the Integrin IIbß3 Detected Using Force Spectroscopy

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

^* Department of Cell and Developmental Biology, Hematology-Oncology Division of the Department of Medicine, and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Correspondence: Address reprint requests to Henry Shuman, Dept. of Physiology, University of Pennsylvania School of Medicine, 601 Goddard Labs, 3710 Hamilton Walk, Philadelphia, PA 19104-6085. Tel.: 215-573-2757; Fax: 215-898-2653; E-mail: shuman{at}mail.med.upenn.edu.

The regulated ability of integrin IIbß3 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 IIbß3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified IIbß3 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 IIbß3 antagonists or Mn²⁺, an IIbß3 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 IIbß3-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 IIbß3 and fibrinogen revealed at the single-molecule level.

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