title: Multi-step fibrinogen binding to the integrin (alpha)IIb(beta)3 detected using force spectroscopy.
abstract: The regulated ability of integrin alphaIIbbeta3 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 alphaIIbbeta3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified alphaIIbbeta3 and fibrinogen, covalently attached to underlying surfaces, ranged from approximately 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 alphaIIbbeta3 antagonists or Mn2+, an alphaIIbbeta3 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 alphaIIbbeta3-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 alphaIIbbeta3 and fibrinogen revealed at the single-molecule level.
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085, USA.
authors.forename: Rustem I
authors.initials: RI
authors.lastname: Litvinov
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bennett
authors.forename: John W
authors.initials: JW
authors.lastname: Weisel
authors.forename: Henry
authors.initials: H
authors.lastname: Shuman
mesh_terms: D001665:Binding Sites; D004548:Elasticity; D005340:Fibrinogen / Q000032:analysis* / Q000737:chemistry* / Q000648:ultrastructure; D007834:Lasers; D018625:Microscopy, Atomic Force / Q000379:methods*; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis* / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D013057:Spectrum Analysis / Q000379:methods*; D013314:Stress, Mechanical
chemical_list: D046912:Multiprotein Complexes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen