Figure 1.
Force dependence of binding of FVIII to VWF. (A) BFP brightfield scheme (left panel) and protein functionalization (right panel). A micropipette-aspirated RBC with a bead attached to the apex (probe, left) was aligned against a bead held by an apposing micropipette (target, right). The probe bead was covalently linked with purified human plasma VWF and streptavidin for attachment of the bead to biotinylated RBC (left). VWF is the focus for interaction with human recombinant full-length FVIII on the target bead (right). The FVIII was either covalently immobilized via a PEG3500 linker or by using NB11B2 antibody that binds to the A2 domain of FVIII on the opposite face that binds VWF. (B) Force vs time traces of 2 representative BFP touch cycles. The target was driven to impinge, contact, and retract the probe. In a “no bond” event, the cycle ended after retraction (black). In a “bond” event (blue), the target was held (marked by ∗) at clamp force (20 pN) until the bond between probe and target dissociated, signified by the force dropping back to 0, and the lifetime of the bond was measured. (C) Adhesion frequencies of binding of VWF to FVIII immobilized either covalently or using NB11B2 antibody. The negative controls were performed in the absence of either FVIII or VWF. The data points are from 20 touch cycles. Each probe–target pair was tested repeatedly for 200 approach-contact-retract cycles to estimate an adhesion frequency. Errors are mean ± SEM. ∗P < .05 assessed by unpaired, two-tailed Student t test. (D) Rupture force of the VWF-FVIII vs FVIII-NB11B2 bonds. The force applied to the bonds increased until dissociation was detected. The magnitudes of rupture forces were measured at the instant force traces dropped. N ≥ 3 probe–target pairs were tested to obtain mean ± SEM. ∗∗∗∗P < .001 assessed by unpaired, two-tailed Student t test. (E) Lifetime of VWF-FVIII bonds as a function of clamp force in BFP. The FVIII was immobilized either covalently or using NB11B2 antibody. Results represent mean ± SEM of >50 measurements per point. SEM, standard error of the mean.

Force dependence of binding of FVIII to VWF. (A) BFP brightfield scheme (left panel) and protein functionalization (right panel). A micropipette-aspirated RBC with a bead attached to the apex (probe, left) was aligned against a bead held by an apposing micropipette (target, right). The probe bead was covalently linked with purified human plasma VWF and streptavidin for attachment of the bead to biotinylated RBC (left). VWF is the focus for interaction with human recombinant full-length FVIII on the target bead (right). The FVIII was either covalently immobilized via a PEG3500 linker or by using NB11B2 antibody that binds to the A2 domain of FVIII on the opposite face that binds VWF. (B) Force vs time traces of 2 representative BFP touch cycles. The target was driven to impinge, contact, and retract the probe. In a “no bond” event, the cycle ended after retraction (black). In a “bond” event (blue), the target was held (marked by ∗) at clamp force (20 pN) until the bond between probe and target dissociated, signified by the force dropping back to 0, and the lifetime of the bond was measured. (C) Adhesion frequencies of binding of VWF to FVIII immobilized either covalently or using NB11B2 antibody. The negative controls were performed in the absence of either FVIII or VWF. The data points are from 20 touch cycles. Each probe–target pair was tested repeatedly for 200 approach-contact-retract cycles to estimate an adhesion frequency. Errors are mean ± SEM. ∗P < .05 assessed by unpaired, two-tailed Student t test. (D) Rupture force of the VWF-FVIII vs FVIII-NB11B2 bonds. The force applied to the bonds increased until dissociation was detected. The magnitudes of rupture forces were measured at the instant force traces dropped. N ≥ 3 probe–target pairs were tested to obtain mean ± SEM. ∗∗∗∗P < .001 assessed by unpaired, two-tailed Student t test. (E) Lifetime of VWF-FVIII bonds as a function of clamp force in BFP. The FVIII was immobilized either covalently or using NB11B2 antibody. Results represent mean ± SEM of >50 measurements per point. SEM, standard error of the mean.

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