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We used laser tweezers-based force spectroscopy to measure the binding strength between fibrinogen molecules covalently bound to latex beads and either wild-type αIIbβ3 molecules or αIIbβ3 molecules containing the transmembrane domain mutations β3 G708N or αIIb G972N expressed on Chinese hamster ovary cells. As we demonstrated previously for αIIbβ3 on agonist-stimulated platelets and for purified αIIbβ3 molecules incubated with Mn2+, two regimes of rupture forces were present when wild-type αIIbβ3 was activated by the monoclonal antibody PT25-2: rupture forces of 20−60 pN with an exponentially decreasing probability of detection and rupture forces in the range of 60−150 pN with a maximum at ∼70−80 pN. Both rupture force regimes were specific for fibrinogen binding to the activated conformation of αIIbβ3 because they were inhibited by αIIbβ3-specific antagonists. Identical rupture force regimes were present constitutively when cells expressing the αIIb and β3 transmembrane domain mutants were studied, confirming that these mutations induced an active αIIbβ3 conformation. Moreover, there were no significant differences in the yield strength of the low-to-moderate and strong force regimes when αIIbβ3 was activated by PT25-2 or the transmembrane domain mutations, implying that there was no fundamental difference in the way these forms of activated αIIbβ3 interacted with fibrinogen. Thus, the two-step pathway of the interaction of αIIbβ3 with fibrinogen we have identified appears to be a fundamental property of the high-affinity state of αIIbβ3 and is identical regardless of whether this affinity state is achieved by intracellular, extracellular, or membrane-associated events.
Sprache
Englisch
Identifikatoren
ISSN: 0006-2960
eISSN: 1520-4995
DOI: 10.1021/bi0526581
Titel-ID: cdi_crossref_primary_10_1021_bi0526581
Format
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