Multimer analysis of wildtype and mutant VWF by AFM-based imaging of individual molecules.

(A, B) Representative AFM images of individual native (A) and non-native (B) VWF molecules. Numbers in images indicate the multimer size i (i = 1 corresponds to a dimer). White arrowheads mark paired, red arrowheads unpaired CK domains. For more details on the identification of dimeric and monomeric building blocks within VWF molecules, see Materials and Methods and S1 Fig. Scale bars represent 20 nm, range of color scale is 2.4 nm. (C) Size distributions of wtVWF (i) and mutant p.Cys1099Tyr (C1099Y, ii), and schematic of step-growth multimerization (iii). Insets in subpanels i and ii show linear fits to the data represented in logarithmic space, yielding values for the extent p of multimerization of 0.43 and 0.21, respectively. (D) Size distributions of VWF mutants p.Cys2771Arg (C2771A, i) and p.Cys2773Arg (C2773R, ii), and schematic of the underlying multimerization process (iii). Native and non-native molecules are depicted in blue and red, respectively. Non-native molecules are characterized by ending on a C-terminal CK domain (small, closed circle) at one or both termini, while native molecules end on N-terminal D’D3 assemblies (open circle). Monomers are “non-native” because they are never secreted after expression of wtVWF. From the observed size distributions, values for the dimerization abolishment of 87% and 73% were determined for p.Cys2771Arg and p.Cys2773Arg, respectively. (Ei) Size distribution of VWF mutant p.Cys2811Ala, for which non-native molecules had been hypothesized to result from reopening of disulfide-linked CK domains. The overall ratio of non-native molecules was found to be 51%. The (apparent) reopening probability was determined to be 23%. (Eii) Size distribution (shown for i ≤ 5) obtained from a simulation that assumed multimers to initially follow an exponential size distribution–with p = 0.43 as observed for wtVWF–and to afterwards reopen partially at their CK domains with the experimentally determined probability for p.Cys2811Ala. Simulations yielded, similarly to p.Cys2811Ala, a ratio of 53% non-native molecules, and very low fractions of even-numbered non-native molecules. (Eiii) Schematic representation of the hypothesized scenario of initial wildtype-like step-growth multimerization and subsequent reopening of C-terminal disulfide bonds within constituent dimers. The data presented in this figure are listed in S1 Table.