期刊
BIOPHYSICAL JOURNAL
卷 89, 期 4, 页码 2824-2834出版社
CELL PRESS
DOI: 10.1529/biophysj.105.061887
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资金
- NHLBI NIH HHS [P01 HL040387, R01 HL030954, P01 HL062250, HL40387, HL62250, HL30954, HL57407] Funding Source: Medline
The regulated ability of integrin alpha llb 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 nonspecific protein-protein binding versus specific alpha llb beta 3-fibrinogen interactions in combination with experimental evidence for single-molecule measurements. The rupture force distribution of purified alpha llb beta 3 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 alpha llb beta 3 antagonists or Mn2+, an alpha llb beta 3 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 alpha llb beta 3-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 alpha llb beta 3 and fibrinogen revealed at the single-molecule level.
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