Blocking disulfide formation in VWF prevents stiffening of the VWF-FVIII bond in response to force. VWF or VWF-IPA on the bead was the focus for interaction with human recombinant full-length FVIII on the target. The FVIII was immobilized using NB11B2 antibody that binds to the A2 domain of FVIII on the opposite face that binds VWF. (A) Adhesion frequencies of binding of VWF or VWF-IPA to FVIII. The negative control is performed in the absence of FVIII. The data points are from 20 or 29 touch cycles for VWF or VWF-IPA, respectively. Each probe–target pair was tested repeatedly for 200 approach-contact-retract cycles to estimate an adhesion frequency. Errors are mean ± SEM. (B) Lifetime of VWF-FVIII or VWF-IPA·FVIII bonds as a function of clamp force in BFP. Results represent mean ± SEM of >50 measurements per point. (C) The molecular spring constant (k_mol) of the VWF-FVIII and VWF-IPA·FVIII bonds during the retraction phase of the BFP cycle at 10, 20, and 30 pN clamp force levels. Data at each force level were obtained from >20 points. Errors are mean ± SEM. ∗∗∗∗P < .0001, assessed by an unpaired, two-tailed Student t test.