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

Date Published: January 15, 2019

Publisher: Public Library of Science

Author(s): Achim Löf, Gesa König, Sonja Schneppenheim, Reinhard Schneppenheim, Martin Benoit, Ulrich Budde, Jochen P. Müller, Maria A. Brehm, Toshiyuki Miyata.

http://doi.org/10.1371/journal.pone.0210963

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.

Partial Text

The plasma glycoprotein von Willebrand factor (VWF) plays a crucial role in primary hemostasis [1–3]. Activated by increased hydrodynamic forces at sites of vascular injury [4], it recruits platelets to exposed subendothelial collagen and promotes the formation of platelet plugs. The hemostatically most active forms of VWF are its ultra-large multimers (UL-VWF), as they experience the highest forces and are capable of multivalent binding [1,5–7]. Mutations that alter the normal size distribution of VWF and result in reduction of large multimers can hence lead to the subtype 2A of von Willebrand Disease (VWD), the most common hereditary bleeding disorder [5,7]. Linear multimers are assembled in the trans-Golgi exclusively from dimers–VWF’s smallest repeating subunits–which are linked via the disulfide bonds Cys1099-Cys1099’ and Cys1142-Cys1142’ between their N-terminal D’D3 assemblies [8] (Fig 1). Multimers can partially be secreted constitutively or stored in Weibel Palade Bodies (WPBs) until release is stimulated by agonists [9,10]. 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). Here, N1 is a constant fitting parameter representing the number of dimers after multimerization, and p describes the extent of multimerization. Larger values of p are indicative of samples in which large multimers are more abundant. 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.

In this study, we introduced the use of single-molecule AFM imaging as a research tool to characterize the size distribution of VWF multimers. We used recombinant VWF expressed in HEK293 cells, which do not intrinsically possess WPBs, but form pseudo-WPBs induced by expression of VWF. Although WPBs and pseudo-WPBs most likely are not identical in their composition, HEK293 cells have been shown to produce multimer size distributions that very closely match those found in plasma samples [18], and therefore are a commonly used cell line for recombinant VWF production (e.g.: [19–21]). The usage of recombinant protein has a couple of advantages: 1) VWF is secreted into the serum-free medium of choice, making complex purification protocols unnecessary. 2) Absence of ADAMTS13 provides uncleaved VWF samples. 3) Patients are not burdened with voluminous blood draws, which would be necessary for complex purification protocols to yield plasma VWF of high enough purity for imaging. 4) The expression in HEK293 cells reproduces the homozygous state. Although it is clinically much less common than the heterozygous state, it can be analyzed more straight-forwardly and helped us to gain deeper insights into how the different mutations disturb VWF processing. 5) If necessary, reproduction of the heterozygous state is also possible by co-expression of wildtype and variant VWF, and good agreement between the size distribution of heterozygous recombinant multimers and respective patient plasma samples has been reported [18].

Put succinctly, our data on selected VWF mutants are fully in line with the recently proposed picture of VWF dimerization: Cys2771 and Cys2773 are the essential starting points of dimerization and bonds Cys2771-Cys2773’ and Cys2773-Cys2771’ are the structural bonds that connect CK domains of monomers. A loss of Cys2771 has a more severe effect than a loss of Cys2773, indicating that an alternative bond Cys2773-Cys2773’ is formed less readily than Cys2771-Cys2771’. Cys2811 in contrast appears to play a secondary role in dimerization, as loss of Cys2811 still allows for significant formation of native dimers and consequently larger multimers. From a more methodological point of view, we showed that AFM imaging is a powerful approach to assess the size distribution of VWF, which can help to gain a quantitative understanding of the processes involved in VWF’s multimerization and in multimerization defects at the single-molecule level. In particular, direct visualization of individual molecules by AFM imaging enables detection of structurally anomalous molecules, even if they are indistinguishable from native molecules in electrophoretic multimer analysis. In the future, AFM imaging may therefore aid electrophoretic multimer analysis as a complementary method to better comprehend the pathological mechanisms of elusive VWF variants, especially in cases where subtle structural differences are expected to play a role.

 

Source:

http://doi.org/10.1371/journal.pone.0210963

 

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