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THROMBOSIS AND HEMOSTASIS
Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol
in the C2 domain
Tim Ganderton,1 Jason W. H. Wong,1 Christina Schroeder,1 and Philip J. Hogg1
1Adult
Cancer Program, Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, Australia
VWF is a plasma protein that binds platelets to an injured vascular wall during
thrombosis. When exposed to the shear
forces found in flowing blood, VWF molecules undergo lateral self-association
that results in a meshwork of VWF fibers.
Fiber formation has been shown to involve thiol/disulfide exchange between
VWF molecules. A C-terminal fragment of
VWF was expressed in mammalian cells
and examined for unpaired cysteine thiols using tandem mass spectrometry
(MS). The VWF C2 domain Cys2431Cys2453 disulfide bond was shown to be
reduced in approximately 75% of the molecules. Fragments containing all 3 C domains or just the C2 domain formed monomers, dimers, and higher-order oligomers
when expressed in mammalian cells. Mutagenesis studies showed that both the
Cys2431-Cys2453 and nearby Cys2451Cys2468 disulfide bonds were involved in
oligomer formation. Our present findings
imply that lateral VWF dimers form when
a Cys2431 thiolate anion attacks the
Cys2431 sulfur atom of the Cys2431Cys2453 disulfide bond of another VWF
molecule, whereas the Cys2451-Cys2468
disulfide/dithiol mediates formation of
trimers and higher-order oligomers. These
observations provide the basis for exploring defects in lateral VWF association in
patients with unexplained hemorrhage
or thrombosis. (Blood. 2011;118(19):
5312-5318)
Introduction
VWF is a large, multimeric glycoprotein responsible for chaperoning the blood coagulation cofactor factor VIII and tethering
platelets to the site of vascular endothelial injury.1 It is
synthesized by vascular endothelial cells and megakaryocytes
and circulates as a series of multimers containing variable
numbers of 500-kDa dimeric units. Circulating multimers range
between 500 and 20 000 kDa in size, and although any size
multimer is able to chaperone factor VIII,2 it is the only the
largest sizes that are able to effectively tether platelets. Each
subunit contains binding sites for collagen and for platelet
glycoproteins Ib and ␣IIb␤3.1
Multimer size is regulated in the circulation. An excess of
high-molecular-weight multimers can cause unwanted thrombosis
and is associated with thrombotic thrombocytopenic purpura,3
whereas a paucity of large multimers is associated with the
bleeding disorder VWD.1 Under normal conditions, the size of
VWF multimers is controlled by ADAMTS13 (a disintegrin and
metalloproteinase with thrombospondin type-1 motifs) proteolysis
of the Tyr1605-Met1606 peptide bond in the A2 domain.4
The tertiary and quaternary structure of VWF is influenced by
the shear forces typically found in flowing blood. VWF undergoes
a shear-dependent conformational change,5 and individual molecules can self-associate under both static6 and shear conditions.7-9
VWF reversibly transitions from a loosely coiled ball to an
elongated structure in response to shear.5,10,11 Self-association of
VWF has been reported in different systems. Soluble VWF
perfused over a VWF-coated surface undergoes “homotypic”
self-association that facilitates platelet adhesion.9 In addition, VWF
self-associates into a flexible clump at a critical shear rate and,
when perfused over a collagen-coated surface, forms a web of
fibers that binds platelets.7,10 VWF fibers retain the shape acquired
under shear when the shear is removed.
Endothelial cell–derived VWF also forms fibers when exposed
to shear. VWF secreted by sheared endothelial cells forms “strings”
that are several cell diameters in length and also bind platelets.8,12,13
The mechanism of formation of the VWF strings involves cysteine
thiol/disulfide exchange, because the formation of the strings is
inhibited by thiol alkylation12 and the string structure is disrupted
by the small thiol N-acetylcysteine.14 Mature VWF, both purified
from plasma and made recombinantly, contains unpaired cysteine
thiols12,15-17 that have been localized to the N-terminal D3 and
C-terminal C domains.12,13,17 Presumably, one or more of these
cysteine thiols is involved in VWF self-association.
In the present study, we explored the thiol/disulfide mechanism
of self-association of VWF. The location and abundance of the
unpaired cysteines in the C-terminal part of VWF was elucidated
using mass spectrometry (MS), and their involvement in lateral
association was examined by mutating the cysteine residues. The
Cys2431-Cys2453 and possibly Cys2451-Cys2468 thiols/disulfides in
the C2 domain were found to mediate lateral association.
Submitted June 9, 2011; accepted August 29, 2011. Prepublished online as Blood
First Edition paper, September 12, 2011; DOI 10.1182/blood-2011-06-360297.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
5312
Methods
VWF expression constructs
C-terminal fragments of human VWF cDNA (OriGene) were cloned into
the pCS2⫹ expression plasmid, which contained a chordin signal peptide to
direct secretion followed by a c-Myc tag. C1-CK (residues 2255-2813),
C1-C3 (residues 2255-2648), and C2 (residues 2429-2495) constructs were
made. The Cys2431, Cys2453, Cys2451, and Cys2468 residues in the
BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
MECHANISM OF VWF FIBER FORMATION
5313
Figure 1. UniProt domain structure of mature VWF and the C-domain
fragments analyzed in this study. The mature N-terminus begins with a
protease inhibitor I8 domain (I8, residues 772-828), followed by a
D domain (residues 856-1074), a cysteine-rich domain (CR, residues
1053-1127), another I8 domain (residues 1140-1196), 3 type A domains
(residues 1275-1458, 1496-1669 and 1689-1871), another D (residues
1938-2153) and I8 (residues 2199-2255) domain, 3 type C domains
(residues 2255-2328, 2429-2495 and 2580-2645), and finishes with the
C-terminal knot (CK, residues 2724-2812). Fragments encompassing the
C1-CK (residues 2255-2813), C1-C3 (residues 2255-2648), and
C2 (residues 2429-2495) domains were expressed in mammalian
HEK cells and purified from the conditioned medium. The residue
numbering is that for the preproprotein.
C2 expression construct were mutated to alanine, individually or in pairs,
using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). All
primers were from Sigma-Aldrich.
Expression and analysis of recombinant VWF fragments
The human embryonic kidney cell line HEK 293 was cultured in DMEM
(Invitrogen) supplemented with 10% FCS, 2mM L-glutamine, 10 units/mL of
penicillin, and 10 ␮g/mL of streptomycin. T75 flasks of cells at ⬃ 70%
confluence were transfected with 25 ng of vector DNA using poly(ethyleneimine) (Sigma-Aldrich)18 and incubated for 5 days at 37°C and 5% CO2. The
conditioned medium was collected and centrifuged to remove cellular debris.
Samples were resolved on NuPAGE Novex 4%-12% Bis-Tris Gel (Invitrogen)
with MOPS running buffer under nonreducing and reducing conditions, and
transferred to polyvinylidene difluoride membrane (Millipore). Proteins were
detected by Western blot using 1:1500 dilutions of either peroxidase-conjugated
anti–c-Myc mAbs (Roche) or anti-VWF rabbit polyclonal Abs (Dako). Chemiluminescence films were analyzed using a GS-700 Imaging Densitometer and
Multi-Analyst software (Bio-Rad).
Purification and analysis of the C1-CK fragment
Conditioned medium (10 mL) was incubated with Red c-Myc Agarose
(Sigma-Aldrich; 0.5 mL of packed beads) on a rotating wheel overnight at
4°C. The beads were collected, washed 3 times with TBS, and incubated
with 50␮M 3-(N-maleimidylpropionyl)biocytin (MPB; Invitrogen) for
60 minutes at 25°C. The beads were washed 3 times with TBS and the
protein eluted from the beads with 0.05% SDS for 30 minutes at 70°C. The
C1-CK fragment (10-20 ␮g) was resolved on NuPAGE Novex 4%-12%
Bis-Tris Gel with MOPS running buffer under nonreducing conditions,
stained with colloidal Coomassie, or transferred to polyvinylidene difluoride membrane and blotted with 1:1500 dilutions of either anti-VWF rabbit
polyclonal Abs or streptavidin peroxidase (Dako) to detect the MPB label.
to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nL/min for 60 minutes.
Positive ions were generated by electrospray and analyzed in a LTQ FT
Ultra (Thermo Electron) mass spectrometer operated in data-dependent
MS/MS acquisition mode. MS data were searched using Mascot
(V2.2, Matrix Science) against the nonredundant database from the
National Center for Biotechnology Information. Search parameters
were: precursor tolerance 10 ppm and product ion tolerances ⫾ 0.4 Da.
Cys-carboxyamidomethyl and Cys-methyldisulfide were selected as
variable modifications with full tryptic cleavage of up to 5 missed
cleavages. To determine the extent of alkylated cysteine residues in the
C1-CK fragment, the relative ion abundance of peptides containing
Cys-carboxyamidomethyl and Cys-methyldisulfide was used. To calculate ion abundance of peptides, extracted ion chromatograms were
generated using the XCalibur Qual Browser software (Version 2.0.7,
Thermo). The area was calculated using the automated peak detection
function built into the software.
Structure prediction
The UniProt amino acid sequence of the type C2 domain of human VWF
(accession number P04275, residues 2429-2495) was submitted to the
I-TASSER protein structure prediction tool.19
Results
The domain structure of mature VWF and the recombinant
C-domain fragments produced and analyzed in this study are
described in Figure 1A.
Identification of the unpaired cysteine residues in the VWF
C1-CK fragment
MS
Conditioned medium was incubated with Red c-Myc Agarose on a rotating
wheel overnight at 4°C. The beads were collected, washed 3 times with
TBS, and incubated with 50mM iodoacetamide for 30 minutes at 25°C. The
beads were washed 3 times with TBS and the fragment eluted from the
beads with 0.05% SDS for 30 minutes at 70°C. The C1-CK fragment was
resolved on NuPAGE Novex 4%-12% Bis-Tris Gel under nonreducing
conditions and stained with colloidal Coomassie. The protein band was
excised from the gel, destained, dried, and incubated with 100mM DTT in
25mM NH4CO2 for 1 hour at 25°C. The gel slice was washed with
NH4CO2, dried, and incubated with 50mM methyl methanethiolsulfonate
(Sigma-Aldrich) in 25mM NH4CO2. The slices were washed and dried
before digestion of the C1-CK fragment with 20 ng/␮L of trypsin
(Promega) and 50 ng/␮L of Glu-C endopeptidase (Roche) in 25mM
NH4CO2 overnight at 25°C.
Peptides were eluted from the slices with 5% formic acid, 50% acetonitrile for 30 minutes at 25°C and separated by nano-LC on a Ultimate
3000 HPLC (Dionex) using a fritless nano column (75 ␮ ⫻ ⬃ 10 cm)
containing C18 medium (5 ␮, 200 Å Magic; Michrom). Peptides were
resolved using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid)
To elucidate the mechanism of lateral association of VWF, we first
chose to analyze the redox status of the cysteine residues in the
VWF C1-CK fragment. Myc-tagged C1-CK fragment was expressed in mammalian HEK cells and secretion was directed by a
chordin signal sequence. The fragment was purified from the
conditioned medium by c-Myc affinity chromatography. The
protein was homogeneous by staining with colloidal Coomassie
and was recognized by anti-VWF polyclonal Abs (Figure 2A). The
C1-CK fragment has a molecular weight of ⬃ 120 kDa, which is
consistent with a dimer linked by disulfide bonds through the
C-terminal knot.20 The fragment also labeled with a biotin-linked
maleimide, indicating that it contains unpaired cysteine thiols
(Figure 2A). This is in accordance with several reports of labeling
of native plasma and recombinant VWF with thiol alkylators.12,15-17
The C1-CK fragment was digested with trypsin and Glu-C
endopeptidase and the peptides were analyzed using tandem MS.
Seventy percent of the amino acids and 68% of the cysteine
residues of the fragment were covered in the analysis (Figure 2B).
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5314
BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
GANDERTON et al
cysteine in the population that is in the reduced state. Peptides with
ratios ⬎ 0.1 are listed in Table 1. The remaining cysteinecontaining peptides with ratios ⬍ 0.1 are listed in supplemental
Table 1 (available on the Blood Web site; see the Supplemental
Materials link at the top of the online article). Cys2453 in the
C2 domain is clearly the most abundant unpaired cysteine of those
analyzed in the C1-CK protein. The ratio of labeling for this
cysteine was 3.00, compared with the next highest of 0.25 for
Cys2574. Further results and discussion of this analysis is found in
the supplemental materials.
Although this analysis covered only 68% of the cysteine
residues in the C1-CK fragment, the relevant cysteines with respect
to lateral association of VWF are likely all accounted for based on
the findings of Choi et al.12 These investigators used a thiol-reactive
matrix (thiopropyl-Sepharose) to capture the VWF C-terminal
fragment that contains unpaired cysteine(s). As shown by MS
analysis, the fragment consisted of residues 2435-2464, 24792493, and 2516-2535 and contained 7 cysteines, all of which were
covered in our analysis (Figure 2B). Choi et al concluded that 1, 2,
or possibly all 7 cysteine residues in the fragment were unpaired.12
Our analysis indicated that Cys2453 was the unpaired thiol that
reacted with thiopropyl-Sepharose in their study.
Homology of the VWF C domains and joiner regions
Figure 2. Identification of the unpaired cysteine residues in the VWF C1-CK
fragment. (A) The C1-CK fragment contains unpaired cysteine thiols. The fragment
was expressed in mammalian HEK cells, purified from the conditioned medium,
labeled with a biotin-linked maleimide (MPB), and resolved on SDS-PAGE. The
fragment was visualized by staining with colloidal Coomassie or blotted with either
anti-VWF Abs or with streptavidin-peroxidase to detect the MPB label. (B) Amino acid
sequence and peptide coverage (underlined) of the VWF C1-CK fragment by tandem
MS analysis. Seventy percent of the residues and 68% of the cysteines were covered
in the analysis. (C) Tandem mass spectrum of the GCDVCTCTDME peptide showing
Cys2453 (underlined) labeled with carboxyamidomethyl (free cysteine) and Cys2448
and Cys2451 labeled with methyldisulfide (paired cysteines). The accurate mass
spectrum of the peptide is also shown in the inset (observed [M ⫹ 2H]2⫹ ⫽ 663.1872
m/z; expected [M ⫹ 2H]2⫹ ⫽ 663.1859 m/z).
Both the unpaired cysteines and the disulfide-bonded cysteines
were identified in the analysis. The unpaired cysteines were
alkylated with iodoacetamide and the disulfide-bonded cysteine
thiols with methyl methanethiolsulfonate after reduction with DTT.
A tandem mass spectrum of the GCDVCTCTDME peptide
(residues 2447-2457) is shown in Figure 2C. Based on the peptide
fragmentation pattern, it is evident that Cys2453 in this peptide is
labeled with carboxyamidomethyl (the iodoacetamide adduct). The
ratio of carboxyamidomethyl to methyldisulfide (the methyl methanethiolsulfonate adduct) labeling represents the fraction of the
The C domain is 60-80 amino acids in length and is defined by a
conserved spacing of 10 cysteine residues.21 In many cases, the
domains have low sequence homology. Interestingly, we noticed
that the C-domain joiner regions shared 8 of the 10 conserved
C-domain cysteine residues (Figure 3A). All 3 C-joining regions in
human VWF are missing Cys3 and Cys5, a pattern shared with
several hemolectin (fruit fly) and hemocytin (silk worm) C domains (Figure 3A). Hemolectin and hemocytin are insect hemocytespecific proteins that are believed to be important in the maintenance of invertebrate hemostasis and the immune system.22 A family
of single C-domain proteins (SVC) that is also missing Cys3 and
Cys5 has been described in the fruit fly.23 Based on X-ray crystal
and predicted structures of the C domain, Cys3 and Cys5 form a
disulfide pair (see next section). Whereas it is not possible to
determine whether the joining regions and other hemolectin/
hemocytin domains missing Cys3-Cys5 truly form the C fold
without structural evidence, it is a reasonable assumption that they
adopt a C fold. By analyzing the conservation of disulfide bonds in
eukaryotes, we have recently shown that disulfide-bonded cysteines are highly conserved and that, once these cysteines are
gained, they are rarely lost in protein evolution.24 It may be that the
C domains missing the Cys3-Cys5 disulfide bond represent a
less-evolved domain.
Table 1. Identification and quantification of the unpaired cysteine residues in the C1-CK fragment
Abundance in sample
Peptide
GCDVCTCTDME
Free cysteine residue
Carboxyamidomethyl
Methyldisulfide
Ratio of carboxyamidomethyl
to methyldisulfide
2453
521 147
173 452
3.00
2468/2473
36 701
284 780
0.13
TSACCPSCR
2574
161 978
656 725
0.25
TVMIDVCTTCR
2600
43 408
180 645
0.24
EENNTGECCGR
2640
95 707
561 872
0.17
2715/2716
194 631
914 022
0.21
VAQCSQKPCEDSCR
IPGTCCDTCEEPE
The ratio of carboxyamidomethyl (free cysteines) to methyldisulfide (paired cysteines) labeling represents the fraction of the cysteine (underlined) in the population that is in
the reduced state. Peptides with ratios ⬎ 0.1 are listed. The remaining cysteine containing peptides detected by mass spectrometry have ratios ⬍ 0.1 and are listed in
supplemental Table 1.
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
MECHANISM OF VWF FIBER FORMATION
5315
allosteric disulfides.27 For example, the allosteric disulfides in the
immune coreceptor CD428 and the HIV envelope protein gp12029
are ⫺RHStaple bonds. A feature of ⫺RHStaple bonds is the close
proximity of the ␣-carbon atoms of the 2 cysteine residues that can
impart strain on the bond.27,30 The distance between the ␣-carbons
of the Cys3-Cys5 bond is 4.01 Å (Table 2, average of the
2 structures), compared with a mean of 5.62 Å for all disulfides in a
nonredundant set of X-ray structures.31
The crossveinless 2 and procollagen 2A C domain structures
were used to predict the structure of the VWF C2 domain using the
I-TASSER protein structure prediction tool.19 It is apparent that the
structures have a similar architecture and the pairing of the cysteine
residues is the same (Figure 3C). Cys2431 and Cys2453 are
predicted to be unpaired in this structure, but would form the
Cys1-Cys4 disulfide bond. Cys2451 and Cys2468 form the
Cys3-Cys5 disulfide bond. The structure prediction was not
compatible with any other cysteine pairings. The finding that
Cys2453 is unpaired in the C1-CK fragment (Figure 2) implies that
the Cys2431-Cys2453 disulfide bond is redox active. Peptides
containing Cys2431 were not resolved in the MS analysis, but this
residue is predicted to be unpaired based on this structural analysis.
Oligomerization of the VWF C1-C3 and C2 fragments
Figure 3. Analysis of the VWF C domains and joiner regions and predicted
structure of the C2 domain of VWF. (A) Alignment of human VWF C domains and
joining regions with C domains from hemocytin (silk worm) and hemolectin (fruit fly) using
the Clustal 2.1 multiple sequence alignment tool. Insect C domains are derived and
numbered as described previously.41 The 10 disulfide-forming cysteines in C domains are
labeled above the alignment. (B) Ribbon structure of the C domain of crossveinless
2 (PDB ID 3BK3) showing the 5 disulfide bonds in yellow. The cysteine residues that form
the Cys1-Cys4 and Cys3-Cys5 disulfide bonds are indicated. The X-ray structure has a
resolution of 2.7 Å.25 (C) Predicted structure of the C2 domain of VWF. The structure was
derived using the crossveinless 2 and procollagen 2A C-domain structures and the
I-TASSER protein structure prediction tool.19 The C-score is ⫺1.05. The cysteine residues
are indicated in yellow. Cys2451 and Cys2468 form a disulfide bond in this structure,
whereas Cys2431 and Cys2453 are unpaired.
Myc-tagged VWF C1-C3 and C2 fragments (Figure 1) were
expressed in mammalian HEK cells, purified from the conditioned
medium by c-Myc affinity chromatography, resolved on SDSPAGE, and examined by Western blot (Figure 4). The C1-C3
fragment was recognized by anti-VWF polyclonal Abs under
nonreducing conditions but not when reduced with DTT The
C2 fragment was not recognized by anti-VWF Abs, but was
detected using Abs against the c-Myc tag.
The C1-C3 fragment was present as molecular weight species
of ⬃ 55 kDa, ⬃ 110 kDa, and ⬃ 160 kDa (Figure 4A). These sizes
are consistent with monomers, homodimers, and homotrimers of
the fragment, respectively. The C2 domain was present as molecular weight species of ⬃ 10 kDa, ⬃ 20 kDa, ⬃ 30 kDa, ⬃ 40 kDa,
and larger oligomers (Figure 4B). These sizes are consistent with
monomers, dimers, trimers, tetramers, and higher-order oligomers,
respectively. All of the C2 species were disulfide linked, because
they resolved to monomers on reduction with DTT (Figure 4B).
Predicted structure of the C2 domain of VWF
Oligomerization of the VWF C2 domain fragment is mediated by
the Cys2431-Cys2453 and Cys2451-Cys2468 disulfides/dithiols
The structures of 2 C domains have been determined: the C domain
of crossveinless 2 (X-ray structure, PDB ID 3BK3)25 and procollagen 2A (NMR structure, PDB ID 1U5M).26 The structure of the
crossveinless 2 C domain is shown in Figure 3B. The 10 cysteine
residues of the domain form 5 disulfide bonds. The disulfide
connectivity is Cys1-Cys4, Cys2-Cys8, Cys3-Cys5, Cys6-Cys9,
and Cys7-Cys10. An analysis of the disulfide bonds in this structure
is shown in Table 2.
The configuration of a disulfide bond can be described by the
5 bond angles of the cystine.27 There are 20 possible disulfide bond
configurations using this criterion. The Cys1-Cys4 bond links a
␤-strand (Cys4) to a loop structure (Cys1) and has a ⫹/⫺RHSpiral
configuration. Cys1 is exposed to solvent. The Cys3-Cys5 disulfide
is nearby (⬃ 10 Å) the Cys1-Cys4 bond and has an interesting
structure. It has a ⫺RHStaple configuration and the cysteines link
adjacent ␤-strands of a small antiparallel ␤-sheet. Both Cys3 and
Cys5 are exposed to solvent. This type of disulfide bond has been
associated with controlling mature protein function, the so-called
Mutants of the Cys2431-Cys2453 and Cys2451-Cys2468 disulfide
bonds of the C2 fragment were analyzed for differential states of
oligomerization. The Cys2431-Cys2453 bond was targeted because it was found to be reduced in the majority of the C1-CK
molecules (Figure 2). We also targeted the Cys2451-Cys2468
disulfide bond for the following reasons: (1) this bond is uniquely
missing in the C joiner regions and in some C domains in other
organisms, which suggests a functional relevance; (2) the bond has
all of the features of other functional (or allosteric) disulfides; and
(3) the bond is very close to the Cys2431-Cys2453 bond in the
tertiary structure, which suggests a relationship between the 2.
The C2431A and C2431A,C2453A C2 mutants were monomers
only, the Cys2451A mutant monomers and dimers, and the
C2453A and C2468A mutants demonstrated enhanced oligomerization compared with wild-type protein (Figure 4C). The
C2451A,C2468A double mutant did not appear in the conditioned
medium of the HEK cells, suggesting that the protein was
recognized as misfolded and targeted for degradation.
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GANDERTON et al
Table 2. Properties of the disulfide bonds of the C domain of crossveinless 225*
Chain
Cys1 residue†
1
2
Cys1 solvent‡
Cys2 residue
Cys2 solvent‡
C␣-C␣, ŧ
Configuration
9 (1)
17
31 (4)
0
6.04
⫹/⫺RHSpiral
26 (2)
26
60 (8)
4
4.89
⫹/⫺RHHook
29 (3)
4
38 (5)
16
3.98
⫺RHStaple
43 (6)
46
61 (9)
35
5.39
⫺LHSpiral
50 (7)
37
64 (10)
48
6.03
⫺LHHook
9 (1)
21
31 (4)
0
5.96
⫹/⫺RHSpiral
26 (2)
31
60 (8)
12
5.08
⫹/⫺RHHook
29 (3)
9
38 (5)
12
4.04
⫺RHStaple
43 (6)
54
61 (9)
34
5.51
⫺LHSpiral
50 (7)
36
64 (10)
57
6.12
⫺RHSpiral
*Calculated using the disulfide bond analysis tool.40 The protein crystallized as a dimer and the analysis of both chains (1 and 2) is shown.
†The number in the bracket is the order of the cysteine in the primary sequence from 1-10.
‡Solvent accessibility is the area of the cysteine residue exposed to solvent in approximately Å2.
§Distance between the ␣ carbon atoms of the 2 cysteine residues.
Discussion
There is emerging evidence that lateral self-association of VWF is
important for the hemostatic function of the protein. This interaction can result in large fibers of VWF (also called strings or ropes)
that form a meshwork. The formation of collagen-tethered VWF
meshes at sites of vascular injury is expected to be better able to
capture platelets in flowing blood than individual molecules. The
mechanism by which VWF molecules self-associate was explored
in this study. Self-association has been reported to be covalent and
involve unpaired cysteine residues.12,14 Considering the cluster of
potential free thiols in and around the C2 domain,12,13 we focused
on identifying the particular cysteines involved in this region and
understanding how they exchange between VWF molecules during
lateral self-association.
The following observations can be made from our results with
the C-terminal VWF fragments and modeling of the C2 domain
structure. The redox state of the Cys2431-Cys2453 disulfide bond
in the C2 domain is ⬃ 75% reduced and ⬃ 25% oxidized. This
suggests that the bond is reduced during or after secretion by a
reductase. Ablating Cys2431 and both Cys2431 and Cys2453 in the
C2 fragment by mutagenesis resulted in production of monomers
only. This implies that the C2 dimers are at least held together by
interchain disulfide bonds between Cys2431 residues. Ablating
Cys2451 resulted in production of monomers and dimers only. This
observation implies that trimers and higher-order oligomers of the
C2 domain are held together by interchain disulfide bonds between
Cys2451 and possibly Cys2431 residues. Ablating Cys2453 and
Cys2468 resulted in enhanced multimerization of the C2 fragment
compared with the wild-type protein. This suggests that the
rate-limiting step in multimerization is the reduction of the
Cys2431-Cys2453 and Cys2451-Cys2468 disulfide bonds; that is,
cleaving the bonds by mutating the cysteine residues not involved
in interchain disulfides removes the energy burden of reducing the
bonds. These conclusions are brought together in the proposed
mechanism of lateral association of VWF shown in Figure 5.
The Cys2431-Cys2453 disulfide bond in one molecule of VWF
is reduced by a reductase, and the resulting Cys2431 thiolate
attacks the same disulfide bond in another VWF molecule, forming
the dimer. A third VWF molecule can be added to the dimer when
the Cys2451 thiolate of one of the molecules in the dimer attacks
the Cys2431-Cys2453 disulfide bond of the third molecule. Other
molecules can then added by the same reaction. This is the simplest
mechanism that can explain the data and is consistent with the
findings that have been published to date. Considering that native
VWF consists of variable numbers of homodimers linked through
the N-terminal CR domain (Figure 1), the Cys2451-Cys2468
disulfide bond does not have to be involved to form trimers and
higher-order oligomers of this molecule. The Cys2431-Cys2453
Figure 4. Oligomerization of the VWF C2 domain is mediated by the Cys2431Cys2453 and Cys2451-Cys2468 disulfides/dithiols. (A) The VWF C1-C3 fragment was
expressed in mammalian HEK cells and purified from the conditioned medium. The
fragment was resolved on SDS-PAGE and blotted with anti-VWF polyclonal Abs. The
fragment was present as monomers, dimers, and trimers. (B) The Myc-tagged VWF C2
fragment was expressed in mammalian HEK cells and purified from the conditioned
medium. The fragments were resolved on SDS-PAGE and blotted with anti-Myc Abs. The
C2 domain was present as monomers, dimers, trimers, tetramers, and higher-order
oligomers. The C2 domain multimers were disulfide-linked, because they resolved to
monomers on incubation with DTT. (C) Wild-type or mutants of the Cys2431-Cys2453 and
Cys2451-Cys2468 disulfide bonds of the Myc-tagged C2 fragment were expressed in
mammalian HEK cells, purified from the conditioned medium, resolved on SDS-PAGE, and
blotted with anti-Myc Abs. The wild-type C2 fragment was present as monomers, dimers,
trimers, and higher-order oligomers. The C2431A and C2431A,C2453A C2 mutants were
only present as monomers, the Cys2451A mutant only as monomers and dimers,
whereas the C2453A and C2468A mutants demonstrated enhanced oligomerization.
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BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
MECHANISM OF VWF FIBER FORMATION
5317
Figure 5. Proposed mechanism of lateral association of VWF. The
Cys2431-Cys2453 disulfide bond in the C2 domain (shown in red) is
reduced by a reductase in blood or in the injured vascular wall. The
Cys2431 thiolate anion (present as a certain percentage of all thiols by
action of the buffer) attacks the Cys2431 sulfur atom of the Cys2431Cys2453 disulfide bond of another VWF molecule resulting in a disulfidelinked VWF lateral dimer. A VWF trimer may be formed when the Cys2451
thiolate anion (shown in green) of one of the VWF molecules in the dimer
attacks the Cys2431 sulfur atom of the Cys2431-Cys2453 disulfide bond
of a third VWF molecule. Higher-order oligomers of VWF may be formed
by the addition of other molecules by the same reaction.
disulfide/dithiol of different monomeric units of the multimer can
simply exchange with other monomeric units of another multimer.
Studies of lateralization of cysteine mutants of the full-length
protein secreted by cells under shear will answer this question.
Our future studies will focus on the identity of the reductase that
cleaves the Cys2431-Cys2453 and Cys2451-Cys2468 disulfide
bonds. Two candidates are thioredoxin and protein disulfide
isomerase (PDI). Thioredoxin has been shown to cleave the
Cys288-Cys326 disulfide bond in ␤2-glycoprotein I in plasma,32-34
as well as in 2 other disulfide bonds with the same ⫺RHStaple
configuration as the VWF Cys2451-Cys2468 bond.28,29 Conversely, PDI35-37 is on the surface of platelets38 and platelet
microparticles39 and so will be in the vicinity of VWF during
thrombus formation. Moreover, PDI ligands inhibit thrombus
formation in vivo.36,37 Identifying the reductase is important
because its action appears to be the rate-limiting step in the lateral
association of VWF.
The redox chemistry of VWF lateral association is predicted to
be influenced by compounds that react with the C2 domain
disulfides/dithiols. Two plasma proteins that may play a role in this
chemistry are ␤2-glycoprotein I and ADAMTS13. ␤2-Glycoprotein
I reduced with thioredoxin forms disulfide-linked complexes with
VWF.33 This suggests that the Cys288 and/or Cys326 thiols of
reduced ␤2-glycoprotein I can attack the Cys2431-Cys2453
(or Cys2451-Cys2468) disulfide of VWF. Similarly, ADAMTS13
contains unpaired cysteine thiols in the C-terminal region that react
with the C2 domain of VWF.17 Another layer of control of VWF
lateral association may be redox active small molecules generated
during inflammation. Activation of inflammatory cells (neutrophils
and macrophages) results in the production of large amounts of
myeloperoxidase-generated HOCl, which rapidly reacts with cysteine thiols. By influencing thiol/disulfide–exchange events in
VWF, HOCl may promote or inhibit VWF fiber formation.
The importance of lateral association of VWF for its hemostatic
role in humans has not been measured. Defects of lateralization
would not be reflected in abnormal results in standard tests of VWF
antigen, activity, or multimer patterns. It is possible, therefore, that
impaired VWF lateralization is responsible for hemostatic defects
in some patients with apparently normal VWF and platelet
function. From the results presented herein, point mutations in
cysteine residues 2431 and 2451 result in impaired lateral association that may present as bleeding, whereas mutations in cysteine
residues 2453 and 2468 result in enhanced lateral association that
may present as thrombosis. An assay for VWF lateralization that
could be performed in whole blood or plasma would be required to
identify these patients.
Acknowledgments
The authors thank Bruno Reversade for the pCS2⫹ expression
plasmid containing the chordin signal peptide to direct secretion
and Vivien Chen for helpful discussions.
This study was supported by grants from the National Health
and Medical Research Council of Australia, the Cancer Council
New South Wales, and the Cancer Institute New South Wales.
Authorship
Contribution: T.G. performed the experiments and contributed to
the experimental design; J.W.H.W. performed the homology analysis and contributed to the analysis of the data; C.S. contributed to
the analysis of the data; and P.J.H. conceived the study and wrote
the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Prof Philip Hogg, Director, Lowy Cancer
Research Centre, Level 2, Corner High and Botany Streets,
University of New South Wales, Sydney, NSW 2052, Australia;
e-mail: [email protected].
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
5318
GANDERTON et al
BLOOD, 10 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 19
References
1. Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem. 1998;67:395424.
2. Vlot AJ, Koppelman SJ, Meijers JC, et al. Kinetics
of factor VIII-von Willebrand factor association.
Blood. 1996;87(5):1809-1816.
3. Moake JL, Rudy CK, Troll JH, et al. Unusually
large plasma factor VIII:von Willebrand factor
multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med. 1982;
307(23):1432-1435.
4. Furlan M, Robles R, Lammle B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to
fragments produced by in vivo proteolysis. Blood.
1996;87(10):4223-4234.
5. Schneider SW, Nuschele S, Wixforth A, et al.
Shear-induced unfolding triggers adhesion of von
Willebrand factor fibers. Proc Natl Acad Sci
U S A. 2007;104(19):7899-7903.
6. Ulrichts H, Vanhoorelbeke K, Girma JP, Lenting PJ,
Vauterin S, Deckmyn H. The von Willebrand factor self-association is modulated by a multiple
domain interaction. J Thromb Haemost. 2005;
3(3):552-561.
7. Barg A, Ossig R, Goerge T, et al. Soluble plasmaderived von Willebrand factor assembles to a
haemostatically active filamentous network.
Thromb Haemost. 2007;97(4):514-526.
8. Bernardo A, Ball C, Nolasco L, Choi H, Moake JL,
Dong JF. Platelets adhered to endothelial cellbound ultra-large von Willebrand factor strings
support leukocyte tethering and rolling under high
shear stress. J Thromb Haemost. 2005;3(3):562570.
9. Savage B, Sixma JJ, Ruggeri ZM. Functional
self-association of von Willebrand factor during
platelet adhesion under flow. Proc Natl Acad Sci
U S A. 2002;99(1):425-430.
10. Steppich DM, Angerer JI, Sritharan K, et al. Relaxation of ultralarge VWF bundles in a microfluidic-AFM hybrid reactor. Biochem Biophys Res
Commun. 2008;369(2):507-512.
11. Siedlecki CA, Lestini BJ, Kottke-Marchant KK,
Eppell SJ, Wilson DL, Marchant RE. Sheardependent changes in the three-dimensional
structure of human von Willebrand factor. Blood.
1996;88(8):2939-2950.
12. Choi H, Aboulfatova K, Pownall HJ, Cook R,
Dong JF. Shear-induced disulfide bond formation
regulates adhesion activity of von Willebrand factor. J Biol Chem. 2007;282(49):35604-35611.
13. Li Y, Choi H, Zhou Z, et al. Covalent regulation of
ULVWF string formation and elongation on endothelial cells under flow conditions. J Thromb Haemost. 2008;6(7):1135-1143.
14. Chen J, Reheman A, Gushiken FC, et al. Nacetylcysteine reduces the size and activity of
von Willebrand factor in human plasma and mice.
J Clin Invest. 2011;121(2):593-603.
15. Ganderton T, Berndt MC, Chesterman CN, Hogg PJ.
Hypothesis for control of von Willebrand factor
multimer size by intra-molecular thiol-disulphide
exchange. J Thromb Haemost. 2007;5(1):204206.
16. McKinnon TA, Goode EC, Birdsey GM, et al. Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor. Blood.
2010;116(4):640-648.
17. Yeh HC, Zhou Z, Choi H, et al. Disulfide bond reduction of von Willebrand factor by ADAMTS-13.
J Thromb Haemost. 2010;8(12):2778-2788.
18. Aricescu AR, Lu W, Jones EY. A time- and costefficient system for high-level protein production
in mammalian cells. Acta Crystallogr D Biol Crystallogr. 2006;62(pt 10):1243-1250.
19. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and
function prediction. Nat Protoc. 2010;5(4):725738.
20. Katsumi A, Tuley EA, Bodo I, Sadler JE. Localization of disulfide bonds in the cystine knot domain
of human von Willebrand factor. J Biol Chem.
2000;275(33):25585-25594.
21. Zhang JL, Huang Y, Qiu LY, Nickel J, Sebald W.
von Willebrand factor type C domain-containing
proteins regulate bone morphogenetic protein
signaling through different recognition mechanisms. J Biol Chem. 2007;282(27):20002-20014.
22. Lesch C, Goto A, Lindgren M, Bidla G, Dushay MS,
Theopold U. A role for Hemolectin in coagulation
and immunity in Drosophila melanogaster. Dev
Comp Immunol. 2007;31(12):1255-1263.
23. Sheldon TJ, Miguel-Aliaga I, Gould AP, Taylor WR,
Conklin D. A novel family of single VWC-domain
proteins in invertebrates. FEBS Lett. 2007;
581(27):5268-5274.
24. Wong JW, Ho SY, Hogg PJ. Disulfide bond acquisition through eukaryotic protein evolution. Mol
Biol Evol. 2011;28(1):327-334.
25. Zhang JL, Qiu LY, Kotzsch A, et al. Crystal structure analysis reveals how the Chordin family
member crossveinless 2 blocks BMP-2 receptor
binding. Dev Cell. 2008;14(5):739-750.
26. O’Leary JM, Hamilton JM, Deane CM, Valeyev NV,
Sandell LJ, Downing AK. Solution structure and
dynamics of a prototypical chordin-like cysteinerich repeat (von Willebrand Factor Type C Module) from Collagen 2A. J Biol Chem. 2004;279:
53857-53866.
27. Schmidt B, Ho L, Hogg PJ. Allosteric disulfide
bonds. Biochemistry. 2006;45(24):7429-7433.
28. Matthias LJ, Azimi I, Tabrett CA, Hogg PJ. Reduced monomeric CD4 is the preferred receptor
for HIV. J Biol Chem. 2010;285(52):40793-40799.
29. Azimi I, Matthias LJ, Center RJ, Wong JW, Hogg PJ.
Disulfide bond that constrains the HIV-1 gp120
V3 domain is cleaved by thioredoxin. J Biol
Chem. 2010;285(51):40072-40080.
30. Wouters MA, Lau KK, Hogg PJ. Cross-strand disulphides in cell entry proteins: poised to act.
Bioessays. 2004;26(1):73-79.
31. Schmidt B, Hogg PJ. Search for allosteric disulfide bonds in NMR structures. BMC Struct Biol.
2007;7:49.
32. Passam FH, Rahgozar S, Qi M, et al. Beta 2 glycoprotein I is a substrate of thiol oxidoreductases.
Blood. 2010;116(11):1995-1997.
33. Passam FH, Rahgozar S, Qi M, et al. Redox control of ␤2GPI-von Willebrand factor interaction by
thioredoxin-1. J Thromb Haemost. 2010;8(8):
1754-1762.
34. Ioannou Y, Zhang J-Y, Passam FH, et al. Naturally occurring free thiols within ␤2-glycoprotein I
in vivo: nitrosylation, redox modification by endothelial cells and regulation of oxidative stress induced cell injury. Blood. 2010;116(11):1961-1970.
35. Ahamed J, Versteeg HH, Kerver M, et al. Disulfide isomerization switches tissue factor from coagulation to cell signaling. Proc Natl Acad Sci
U S A. 2006;103(38):13932-13937.
36. Cho J, Furie BC, Coughlin SR, Furie B. A critical
role for extracellular protein disulfide isomerase
during thrombus formation in mice. J Clin Invest.
2008;118(3):1123-1131.
37. Reinhardt C, von Bruhl ML, Manukyan D, et al.
Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via
tissue factor activation. J Clin Invest. 2008;
118(3):1110-1122.
38. Essex DW, Chen K, Swiatkowska M. Localization
of protein disulfide isomerase to the external surface of the platelet plasma membrane. Blood.
1995;86(6):2168-2173.
39. Raturi A, Miersch S, Hudson JW, Mutus B. Platelet microparticle-associated protein disulfide
isomerase promotes platelet aggregation and
inactivates insulin. Biochim Biophys Acta. 2008;
1778(12):2790-2796.
40. Wong JW, Hogg PJ. Analysis of disulfide bonds in
protein structures. J Thromb Haemost. 2010;8:
2345.
41. Goto A, Kumagai T, Kumagai C, et al. A Drosophila haemocyte-specific protein, hemolectin, similar to human von Willebrand factor. Biochem J.
2001;359(pt 1):99-108.
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2011 118: 5312-5318
doi:10.1182/blood-2011-06-360297 originally published
online September 12, 2011
Lateral self-association of VWF involves the Cys2431-Cys2453
disulfide/dithiol in the C2 domain
Tim Ganderton, Jason W. H. Wong, Christina Schroeder and Philip J. Hogg
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