Figure Legends
Figure 1. Impact of the VWF propeptide mutations on secretion,
function and multimer structures of the VWF protein. (A) HEK293T cells
were transiently transfected with rVWF-wt, p.Gly55Glu, p.Val86Glu,
p.Trp191Arg, p.Asn211Asp, p.Gly334Glu and p.Cys608Trp (left side); the
mutant constructs were co-transfected with VWF-wt at a 1:1 ratio (right
side). Recombinant VWF antigen levels (VWF:Ag) in medium (dark gray
bars) and lysate (light gray bars) were measured and expressed as a
percentage relative to the VWF-wt. Each bar represents the average value
and \(\pm\) SEM of three independent experiments in triplicate.(B) The VWF binding activities, VWF:GPIb and VWF:CB. The
functional assessments are presented as mean and \(\pm\) SEM of ratios
VWF:GPIb and VWF:CB to VWF:Ag from three independent experiments in
triplicate. The n/a indicates where the amount of mutated VWF antigen in
media was not adequate for reliable quantification evaluations.(C) The multimer pattern of rVWF-wt and mutants in the medium
of transfected HEK293T cells (both single and co-transfected cells)
analyzed by electrophoresis on 1.6% SDS-agarose gel.
Lines 1 and 2 show normal pool plasma
and rVWF-wt, respectively. Lines 3, 5, 7, 9, 11 and 13 represent
single-expressed VWF mutants, whereas lines 4, 6, 8, 10, 12 and 14
illustrate co-expressed mutants with VWF-wt in a ratio of 1:1.
wt, wild type; VWF, von Willebrand factor;rVWF , recombinant VWF; VWF:Ag, VWF antigen;VWF:GPIb, VWF binding to platelet GPIb; VWF:CB, VWF
binding to collagen type I.
Figure 2. Intracellular localization and storage of
recombinant VWF (rVWF) variants in transfected HEK293 cells. (A
and B) rVWF-wt and pathogenic VWF variants were expressed in HEK293,
subsequently, intracellular trafficking and storage of VWF were
investigated. Cells were fixed and immunostained to visualize VWF
(green, left channels) and endoplasmic reticulum (ER) using the PDI
marker (red, middle channels). Right channels illustrate the merge of
green and red staining, showing co-localization of the two proteins VWF
and PDI. The punctate staining (green) represents a storage of VWF in
pseudo-WPBs, whereas the diffuse green staining indicates VWF retained
in ER. Scale bar = 10µm. (A) HEK293 cells were transiently
transfected with rVWF-wt or the pathogenic VWF variants p.Gly55Glu,
p.Val86Glu, p.Trp191Arg, and p.Cys608Trp. (B) HEK293 cells were
co-transfected with VWF mutant and wt in a 1:1 ratio to imitate
heterozygous expression. (C ) Degree of co-localization of VWF
mutants with ER. This bar graph represents the mean of Pearson’s
coefficient for rVWF-wt and pathologic variants (single and
co-expressed) analyzed in this study. Error bars represent the SEM. The
Pearson’s coefficient has been calculated from at least 100 cells for
each construct on the Zeiss ZEN 2 (blue edition) software.
Figure 3. Immunostaining and quantitative morphological analysis
of WPBs-like vesicles in co-transfected HEK293 cells. (A)Immunofluorescence images show a 3D model of formation of the WPBs-like
granules in transiently transfected HEK293 cells with recombinant VWF wt
as well as transiently co-transfected wt with variants p.Gly55Glu,
p.Val86Glu, p.Trp191Arg, and p.Cys608Trp in a 1:1 ratio imitating
heterozygous expression. Scale bar = 10µm. Boxes represent close-up
views of the processed 3D models of the WPBs-like granules surrounding
with oriented bounds which are generated by arivis Vision4D 3.2
software. The size measurements (length, depth, and width) of some wt
and mutant pseudo-WPBs are provided jus as examples; the sizes are
presented as µm (B) The fraction of cells forming WPBs-like
granules are presented as % of the cells detected with green signal
(indicating expressing VWF) after inspecting at least 100 cells for each
HEK293 cells transfected with wt VWF and HEK293 cells co-transfected
with wt and VWF variants. Bars represent means ± SEM from a minimum of
eight fields of view. (C) The relative average number of
WPBs-like organelles per cell in transfected HEK293 cells, calculated by
dividing the global count of the WPBs in each acquired image arena by
the cells encompassing at least one WPB (D) The mean intensity
signal value (green, VWF) for every single WPB-like organelle was
calculated. Bars display average ± SEM from the whole population of
WPBs-like granules (hundreds of WPBs). (E, F, and G) panels
demonstrate the average value of the longest (length), middle (depth),
and shortest sides (width) from the whole population of WPBs-like
granules (hundreds of WPBs), respectively. Each bar represents the
average value and \(\pm\) SEM. (H) The Sphericity factor (Ψ),
describing the roundness of the 3D granules were computed. It is
represented as a value between 0 and 1, where 1 is an ideal sphere. Each
value implies the average rate ± SEM of the whole population of
WPBs-like granules.
ns: non-significant; *: P<0.05; **: P<0.002;
***: P<0.0001 by Student t-test.
Figure 4. The generated merged model of the D1-D2-D´-D3 domains
and its docking with ARF1 protein and Sec24. (A) The structural model
represents the joined models of the D1-D2-D´-D3 domains. D1 and D2
domains (VWFpp) models (orange and green, respectively) were generated
individually on the ITASSER threading server, and the crystal structure
of the D´-D3 assembly (cyan and yellow, respectively) was downloaded
from the structure database (PDB ID: 6N29; Resolution: 2.5 Å).
Subsequently, the joined model of the VWFpp and the D´-D3 assembly was
created. All propeptide cysteines are represented in blue colored stick
format. The mutated residues have been depicted with their molecular
surface, and are colored red. (B) The 3-dimensional model
illustrates an orientational depiction of the joined model of the
propeptide and the D´-D3 domains. The model itself is depicted by its
molecular surface area. The molecular surface area of the linearly
contiguous D1,D2,D’ and D3 domains are colored in a darker shade of grey
while the D3 domain of an adjacent dimer and part of the complete model
is colored in a lighter shade of grey. (C) The schematic
illustration of the multimeric arrangement and tubular packing of the
N-terminal of the VWF. In the ER, VWF monomers dimerized through
disulfide bonds formation between C-terminal cysteine-knot (CK) domains,
later in trans-Golgi each pro-VWF dimer zip up into a dimeric bouquet
(transparent light blue boxes display the arrangement of the D1 (orange)
D2 (green) D´ (cyan) D3 (yellow) domains in each dimeric bouquet).
Forming the dimeric bouquets are crucial for accurate VWF dimer
incorporation into growing tubules inside WPBs. Homotypic interactions
between the D1 and D2 domains (non-covalent dimerization of N-terminal
ends within proVWF dimeric bouquet, shown with broken arrow) places one
D´D3 domain within a dimer apart from one another, and brings one D´D3
nearby to one D´D3 domain from another assembled dimer, facilitating
N-terminal disulfide bond between dimers . (D) panel shows the
ten best docking poses resulting from docking the crystal structure of
ARF1 on our propeptide model. The backbone of both participating
proteins is depicted in ribbon format with ARF1 colored magenta and VWF
colored grey. The residues on which causative mutations have been
reported in our study have been depicted with their molecular surface
are colored red. (E) panel shows a close-up view of the four
mutated residues reported in our study on the model, especially for
their position vis-à-vis the ARF1 interaction interfaces (residues
Gly55, Val86, Trp191) and also free unpaired Cysteines (residue Cys608).
All protein structures are depicted in ribbon format, with VWF colored
grey and ARF-1 colored magenta. The regions on ARF-1 which undergoes a
conformational change during its transition from an inactive to an
active state (upon GTP binding) are colored yellow. The mutated wild
type residues are depicted in their stick formats. The wild type
residues of Gly55, Val86, and Trp191 are colored red. The Cysteines
forming disulfide bonds are also depicted in blue colored stick format,
while the unpaired cysteines are depicted in green colored stick
formats. (F) panel shows the ten best docking poses resulting
from docking the crystal structure of Sec24 on our propeptide model. The
backbone of both participating proteins is depicted in ribbon format
with Sec24 colored magenta and VWF colored grey. The residues on which
causative mutations have been reported in our study have been depicted
with their molecular surface are colored red. (G) panel shows a
close-up view of the four mutated residues reported in our study on the
model, especially for their position vis-à-vis the Sec24 interaction
interfaces (residues Gly55, Val86, Trp191 and Cys608).