Abstract
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.
Highlights
The plasma glycoprotein von Willebrand factor (VWF) plays a crucial role in primary hemostasis [1,2,3]
Investigating several mutants that exhibit disturbed multimerization, we further demonstrate that atomic force microscopy (AFM) imaging can provide detailed insights into VWF processing and pathological defects at the single-molecule level that cannot be gained from electrophoretic multimer analysis alone
Cys2811 in contrast appears to play a secondary role in dimerization, as loss of Cys2811 still allows for significant formation of native dimers and larger multimers
Summary
The plasma glycoprotein von Willebrand factor (VWF) plays a crucial role in primary hemostasis [1,2,3]. Mutations that alter the normal size distribution of VWF and result in reduction of large multimers can lead to the subtype 2A of von Willebrand Disease (VWD), the most common hereditary bleeding disorder [5,7]. In a recent study [11], which employed quantitative electrophoretic multimer analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy-based photobleaching, it was reported that the size distribution of VWF is exponential and may well result from a simple step-growth polymerization mechanism, where the number N of multimers of size i (i = 1 representing a dimer), is given by the expression N(i) = N1 × p(i-1). For mutant p.Cys1099Tyr, which impairs N-terminal linkage of dimers, an exponential size distribution was still observed, albeit with a lower extent of multimerization
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