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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Litvinov RI,Bennett JS,Weisel JW,Shuman H

doi

10.1529/biophysj.105.061887

subject

Has Abstract

pub_date

2005-10-01 00:00:00

pages

2824-34

issue

4

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(05)72923-4

journal_volume

89

pub_type

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