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Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging

By Achim Löf (6208778), Gesa König (6208781), Sonja Schneppenheim (6208784), Reinhard Schneppenheim (832567), Martin Benoit (385575), Ulrich Budde (6208787), Jochen P. Müller (6208790) and Maria A. Brehm (6208793)


<div><p>The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773’, Cys2773-Cys2771’, and Cys2811-Cys2811’. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants’ different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found ‘even-numbered’ non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.</p></div

Topics: Biophysics, Biochemistry, Medicine, Microbiology, Cell Biology, Hematology, Chemical Sciences not elsewhere classified, Physical Sciences not elsewhere classified, single-molecule level, monomer, C-terminal disulfide bonds Cys 2771-Cys Cys 2773-Cys, disorder von Willebrand Disease, von Willebrand factor, impairs N-terminal multimerization, Cys 2811-Cys Mutations, multimer analysis, mutants p.Cys 2771Arg, VWD, multimer size distribution, Cys 2811, dimerization, electrophoretic multimer analysis, C-terminally dimerized, VWF processing defects, form alternative C-terminal disulfides, mutants p.Cys 2771Arg p.Cys 2773Arg, multimeric glycoprotein von Willebrand factor, single-molecule AFM imaging
Year: 2019
DOI identifier: 10.1371/journal.pone.0210963
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Provided by: FigShare
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