Association of VWF in flow to tethered VWF. (A) Schematic of total internal reflection fluorescence (TIRF) microscopy with air pressure-actuated flow. (B) Dual-color fluorescence images showing the extension and relaxation of a representative tethered Alexa Fluor 488–labeled VWF molecule and the binding and dissociation of Alexa Fluor 647–labeled VWF in flow at the indicated wall shear stresses. (C) Average time course of free VWF association to single tethered VWF concatemers. The y-axis is the mass ratio of bound VWF:tethered VWF. The red triangle marks the increase in flow from 80 dyn cm⁻² to the shear rate keyed by color, and black triangles mark resumption of flow at 80 dyn cm⁻². (D) Profile of final VWF binding density at relative locations along individual tethered VWF concatemers. N_freeVWF is the monomer number of bound VWF. (E) Dissociation rate at 80 dyn cm⁻² of VWF that had been bound at different wall shear stresses. Dissociation in (panel C) was fit to a double exponential function $RfreeVWF(t)=RfreeVWF,fast⋅exp(−koff,fast⋅t)+RfreeVWF,slow⋅exp(−koff,slow⋅t).$ In panels C-E, binding of free VWF (10 μg mL⁻¹) was measured on 93 tethered VWF molecules. The average end-to-end distance of these VWF molecules was 3.6 ± 1.1 μm at 960 dyn cm⁻², and they contained 149 ± 32 monomers based on integrated fluorescence intensity.