Dependence of VWF self-association on free VWF concentration. (A) Free VWF binding kinetics (solid lines) after flow increase from 80 to 960 dyn cm⁻² at different concentrations of free VWF. For each tension bin, the curves were fitted using a homogeneous binding model assuming the free VWF molecules bind as individual monomers. Fitting to the following equation yielded the dissociation and association rate (k_off and k_on) for free VWF binding to regions of tethered VWF:⁶ $NfreeVWF=Ntotal⋅(1−exp (−(kon[C]+koff)Δt⋅i))⋅kon[C]/(kon[C]+koff)$ where N_total is the total number of binding sites in tethered VWF that belong to the tension bin, N_freeVWF is the monomer number of bound VWF in the same region, [C] is the concentration of free VWF monomers in solution, i is the frame number, and Δt is the time lag between consecutive frames. The predictions of the fitted model are shown in corresponding colors (dashed lines). Data for 1, 5, 10, and 50 μg mL⁻¹ free VWF are with 52, 69, 62, and 51 tethered VWF concatemers, respectively, which were long enough to include a 40-to-60 pN tension bin. Data for 10 μg mL⁻¹ free VWF are the same as in Figure 3. (B) Initial VWF binding rate per μm of tethered VWF in each tension bin at 960 dyn cm⁻² wall shear stress (circles). The data points corresponding to the tension bins are jittered along the x-axis to avoid overlap. Dashed lines show linear fit (inset formula) to the data points from 1 to 10 μg mL⁻¹ VWF, that is, with data at 50 μg mL⁻¹ VWF omitted from the fit (see “Results”). a(f) is a tension-dependent, numerical fitting parameter. (C) Tension dependence of the a(f) value determined in (B). Red lines connect points.