Stability and dynamics of the D3 interface probed by MT force spectroscopy. (A) Extension-time traces of VWF dimers in MT. Three-state transitions shift with decreasing force toward the bottom state. The 3 states (top, middle, and bottom) are indicated by dashed lines. Histograms of the extensions are shown on the right. Black lines show triple Gaussian fits. (B) Analysis of the relative population of the different states as a function of force. Each circle corresponds to experimental data from a 5-minute force plateau measurement. Data are well described by a 3-state model with 2 independent transitions (solid lines; Table 1, Equations 1-3). Fit parameters are the mid-force F_1/2 (here F_1/2 = 7.85 pN) and the distance between the states Δz (here Δz = 6.5 nm). (C) Pseudo-dwell time distributions for the open and closed domains calculated from dwell times in the top, middle, and bottom state. For each plateau, pseudo-dwell times are determined and fit with an exponential to determine the mean dwell times for each force. Mean dwell times in the open (open circles) and the closed state (filled circles) depend exponentially on the applied force (solid lines are exponential fits; Table 1, Equations 6 and 7). (D) Histogram of mean midpoint forces determined from fitting the 2- or 3-state model (B; supplemental Figure 4B). The solid line is a Gaussian fit with mean ± standard deviation of 8.3 ± 1.1 pN. (E) Histogram of Δz determined from fitting the 2- or 3-state model (B; supplemental Figure 4B). The solid line is a Gaussian fit with mean ± standard deviation of 7.6 ± 1.7 nm. Histograms in panels D and E show distributions of 33 molecules.