Force-extension assay of recombinant VWF forms using AFM. (A) The force-extension curve shows a saw-tooth pattern⁵⁴  for the WT form. (B) The heterozygous in cis mutant shows a much flatter profile. The microscope probe consisted of an ultrasharp silicon nitride cantilever of nominal force constant k = 0.1 N/m, with a tip radius of <10 nm (MikroMasch NSC16; NanoWorld, Neuchâtel, Switzerland). Image analysis was performed using Gwyddion software (Czech Metrology Institute, Brno, Czech Republic). (B) The force-extension curve concerning the in cis heterozygous form shows constant force plateaus, which are the expression of the mechanical unzipping of linear interactions between multiple linear polymer chains.⁵⁵  (C) Statistical analysis of the experimental data showed that the WT form has a median unfolding force of ∼17 pN in good agreement with the average VWF unfolding force value previously reported.²¹  (D) The in cis heterozygous form has an average unfolding force of ∼7 pN. These unfolding force profiles agree with the morphological counterparts (Figure 5).