Kinetics of tether bond formation and dissociation. (A) Frequency of transient adhesive interactions between microspheres coated with recombinant VWF-A1 protein and surface-immobilized mouse platelets (WSRs of 10 s⁻¹ to 300 s⁻¹). (B-E) Representative graphs depicting the distribution of pause times for more than 30 individual transiently tethered VWF-A1–coated microspheres at the indicated wall shear stresses. The dissociation rate constant (k_off) is the negative slope of the linear regression through the experimental data. (F) Effect of force on the GPIbα–VWF-A1 tether bond as a function of k_off. Tethering frequency and estimation of k_off values for VWF-A1–coated microspheres transiently interacting with surface-immobilized platelets were determined by recording digital images with a Nikon ×10 plan or 60× DIC objective (oil immersion) at 30 fps or 235 fps, respectively (Speed Vision Technologies, San Diego, CA). Data represent the mean ± SD. pN, piconewtons.