Activation of individual αIIbβ3 integrin molecules by disruption of transmembrane domain interactions in the absence of clustering

We used laser tweezers-based force spectroscopy to measure the binding strength between fibrinogen molecules covalently bound to latex beads and either wild-type alpha IIb beta 3 molecules or alpha IIb beta 3 molecules containing the transmembrane domain mutations beta 3 G708N or alpha IIb G972N expressed on Chinese hamster ovary cells. As we demonstrated previously for alpha II beta 3 on agonist-stimulated platelets and for purified alpha IIb beta 3 molecules incubated with Mn2+, two regirnes of rupture forces were present when wild-type alpha IIb beta 3 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 similar to 70-80 pN. Both rupture force regimes were specific for fibrinogen binding to the activated conformation of alpha IIb beta 3 because they were inhibited by alpha IIb beta 3-specific antagonists. Identical rupture force regimes were present constitutively when cells expressing the alpha IIb and beta 3 transmembrane domain mutants were studied, confirming that these mutations induced an active alpha IIb beta 3 conformation. Moreover, there were no significant differences in the yield strength of the low-to-moderate and strong force regimes when alpha IIb beta 3 was activated by PT25-2 or the transmembrane domain mutations, implying that there was no fundamental difference in the way these forms of activated alpha IIb beta 3 interacted with fibrinogen. Thus, the two-step pathway of the interaction of alpha IIb beta 3 with fibrinogen we have identified appears to be a fundamental property of the high-affinity state of alpha IIb beta 3 and is identical regardless of whether this affinity state is achieved by intracellular, extracellular, or membrane-associated events.