Multi-Step Fibrinogen-αIIbβ3 Binding/Unbinding Revealed at the Single Molecule Level Using Laser Tweezers.

The regulated ability of the platelet integrin αIIbβ3 to bind fibrinogen plays a crucial role in platelet aggregation and primary hemostasis. We have developed a model system based on laser tweezers that enables us to measure directly the mechanical forces needed to separate single receptor-ligand complexes. In this system, a ligand-coated bead can be trapped by the laser and repeatedly brought into contact with a receptor-coated pedestal so that the forces required to separate the two can be measured and displayed as “rupture force” histograms. We have applied this system to the interaction of fibrinogen and αIIbβ3 by measuring the rupture force required to separate a laser-trapped bead coated with fibrinogen from an immobilized pedestal coated with purified αIIbβ3. These measurements revealed that bimolecular αIIbβ3-fibrinogen interactions are characterized by rupture forces ranging from 20 pN to 150 pN. We found that the yield strength of αIIbβ3-fibrinogen interactions was independent of the rate at which force was applied over a range of 160 to 16,000 pN/s, but the frequency of fibrinogen binding to αIIbβ3 during repeated contacts correlated strongly with the duration of contact between the αIIbβ3- and fibrinogen-coated surfaces. The observed rupture forces could be segregated into small (20–40 pN), intermediate (40–60 pN), and high (>60 pN) force regimes, each of which displayed unique kinetic behavior. Thus, the contact time needed to reach half maximal fibrinogen binding at a constant loading rate of 1,600 pN/s was ~ 1 ms for the 20–40 pN, 20 ms for the 40–60 pN, and 70 ms for the >60 pN force regimes. Increasing the surface density of immobilized fibrinogen changed the probability of αIIbβ3 binding to fibrinogen >3.5-fold, but did not affect the yield strength profile, implying that these measurements represent interactions between individual molecules. Each force regime also differed in its susceptibility to the inhibitory effect of αIIbβ3 antagonists and to the activating effect of Mn²⁺. Thus, the low molecular weight αIIbβ3 antagonists H12 peptide, tirofiban, and RGDS were most effective in inhibiting weak interactions, whereas the monoclonal antibodies A2A9 and abciximab were most effective in inhibiting the stronger ones. In the presence of Mn²⁺, the strongest component of the yield force distribution increased more than 2.5-fold while the moderate force component increased only 1.5-fold. In addition, rupture forces in the range of 60 to 150 pN disappeared gradually when αIIbβ3 preparations were stored at 4°C for 7 d, suggesting that the αIIbβ3 conformation corresponding to these rupture forces is labile. Taken together, these data suggest that fibrinogen binding to αIIbβ3 is a complex, time-dependent multi-step process during which the strength of the bond between αIIbβ3 and fibrinogen appears to progressively increase.