Fig. 3.

Tension-regulated VWF binding to GPIbα. a Representative time-lapse dual color fluorescence images of equilibrium VWF extension and reversible binding to GPIbα during cycling between stasis and a range of wall shear stresses. b Kinetics (57 fps) of VWF extension/relaxation and binding/dissociation of GPIbα following flow initiation/cessation at 1280 and 0 dyn cm⁻², respectively. Data are mean ± SD for 20 concatemers for three flow cycles each, with VWF length and GPIbα binding for each cycle normalized to its average, equilibrium value between 0.37 and 0.52 s after flow initiation. c Average fluorescence intensity of bound GPIbα per μm of VWF with increasing wall shear stress. VWF concatemers (N = 240) are binned by number of monomers per concatemer (N _VWF), estimated by total fluorescence intensity. Bins are keyed according to the color bar; see Supplementary Fig. 3 for details. d Percentage of activated VWF concatemers by gray scale from black (0%) to white (100%) as a function of N _VWF (same binning as c). Activation is defined as binding over background to GPIbα (Methods). Data in 150 and 10 mM NaCl are with N = 240 and 210 concatemers, respectively. Activation is plotted according to wall shear stress (upper) or maximal tension (lower). Red lines show the median (50%) activation in each bin (dotted) or fits to the equation shown to all bins (solid). Fits to shear stress in 150 and 10 mM NaCl gave b values of (6.9 ± 1.2) × 10⁴ and (3.0 ± 0.2) × 10⁴ dyn cm⁻², respectively. e Equilibrium binding of GPIbα per μm (n _GPIbα) in tension bins along the length of VWF concatemers extended at 1280 dyn cm⁻². Data are mean for 103 concatemers that were long enough to include all seven tension bins. Red line: two-state model fit (inset formula). a–e Show data with 100 nM GPIbα in 150 mM NaCl; additionally, d and e (right) show data with 20 nM GPIbα in 10 mM NaCl (Supplementary Fig. 3). a and c–e Average four to six 10 ms exposures taken every 300 ms. f Dependence on tension of k _on, k _off, and K _D values with 95% confidence interval. Lines show fit of the two-state model to k _on. Binding was too low in the 0–8 pN force bin to yield meaningful data. Measurements were with 78–141 concatemers at five concentrations of GPIbα in each of 10 and 150 mM NaCl, respectively, at six force bins with kinetics following shift from 0 to 1280 dyn cm⁻² (Supplementary Fig. 5). ΔG values in e and f are within error of one another; uncertainty in f, which lacks the data point in the 0–8 pN bin and has higher variance for each data point. In e, f, error bars are SD for tension and 95% confidence intervals for the other variables