Conformational change in the D’D3 domain is unaffected by other domains. (A) Three-state transitions occur independent of the unfolding of the 2 A2 domains. Schematic of wild-type (wt) VWF dimer and extension-time trace at 8.3 pN. Three-state transitions are observable before and after A2 unfolding. Extension histogram of the segment marked in dark orange reveals 3 distinct states that can be fitted with a 3-term Gaussian (solid black line). (B) Three-state transitions occur independent of a deletion of the D4 domain. Schematic of VWF dimer with deletion of both D4 domains, and extension-time trace at 9.0 pN. Extension histogram reveals 3 distinct states that can be fitted with a 3-term Gaussian (solid black line). (C) Three-state transitions occur independent of a deletion of the A1 domain. Schematic of VWF dimer with deletion of both A1 domains, and extension-time trace at 7.5 pN. Extension histogram reveals 3 distinct states that can be fitted with a 3-term Gaussian (solid black line). (D-E) Neither midpoint forces (D) nor Δz (E) (see main text for a discussion of the model) of the deletion constructs are significantly different from the wt. In the boxplots in panels D and E, each data point corresponds to an individual molecule. The line in the boxes indicates the median of all data points, the box the 25th and 75th percentile, and the whiskers the furthest data point outside the box, but within 1.5 times the box width. Number of molecules included in panels D and E: wt, 33; delD4, 13; delA1, 15.