From the Thrombosis and Haemostasis Laboratory,
Department of Haematology, University Medical Center and Institute of
Biomembranes, HP G03.647, P. O. Box 85500, 3508 GA Utrecht, The
Netherlands and § Bijvoet Center for Biomolecular Research,
Department of Crystal and Structural Chemistry, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
Received for publication, September 3, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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The multimeric glycoprotein von
Willebrand factor (VWF) mediates platelet adhesion to collagen at sites
of vascular damage. The binding site for collagen types I and III is
located in the VWF-A3 domain. Recently, we showed that
His1023, located near the edge between the
"front" and "bottom" faces of A3, is critical for collagen
binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon,
J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985-9991). To map the binding site in detail,
we introduced 22 point mutations in the front and bottom faces of A3.
The mutants were expressed as multimeric VWF, and binding to
collagen type III was evaluated in a solid-state binding assay and by
surface plasmon resonance. Mutation of residues Asp979,
Ser1020, and His1023 nearly abolished collagen
binding, whereas mutation of residues Ile975,
Thr977, Val997, and Glu1001 reduced
binding affinity about 10-fold. Together, these residues define a flat
and rather hydrophobic collagen-binding site located at the front face
of the A3 domain. The collagen-binding site of VWF-A3 is distinctly
different from that of the homologous integrin Under conditions of high shear stress, platelet adhesion to
collagen at sites of vascular injury is initiated by the interaction of
platelet receptor glycoprotein
(Gp)1 Ib-IX-V with
collagen-bound von Willebrand factor (VWF) (1). Transient interactions
between VWF and GpIb-IX-V mediate platelet rolling, which slows down
the platelet and allows other platelet receptors such as integrin
VWF is a multimeric glycoprotein consisting of ~270-kDa monomers that
are linked by disulfide bonds (5). The affinity of VWF for collagen
depends on multimer size (6). The binding site for fibrillar collagens
type I and III is located in the VWF-A3 domain (7), whereas collagen
type VI has been shown to bind to the VWF-A1 domain (8, 9). The latter
domain also contains the binding site for GpIb VWF A-type domains and homologous integrin I domains adopt a so-called
dinucleotide-binding fold, or Rossman fold, composed of a central
In this study, we identify the collagen-binding site in the VWF-A3
domain in detail by means of site-directed mutagenesis. We constructed
22 point mutants, expressed these as multimeric VWF, and evaluated
their collagen binding characteristics.
Selection and Construction of VWF Point Mutants--
Selection
of amino acid residues for mutagenesis was based on the approximate
location of the collagen-binding site as identified in our previous
study (23) and the crystal structure of the VWF-A3 domain (12, 13).
Selected residues are solvent-exposed in the isolated A3 domain.
Residues Gln966, Ser974, Ile975,
Thr977, Asp979, Val980,
Pro981, Asn983, Val984,
Val985, Ser993, Val997,
Gln999, Glu1001, Gln1006,
Asp1009, Ser1020, Glu1021, and
Met1022 were mutated to alanine. In addition,
Pro962, Pro981, and Pro1027 were
mutated to histidine to inhibit collagen binding by steric hindrance.
Backbone conformations of these proline residues suggested that the
histidine side chain would protrude from the protein surface. Point
mutations were introduced in the VWF-A3 domain using the QuikChange
method (Stratagene, La Jolla, CA) as described previously
(23).
Expression, Purification, and Characterization of VWF--
VWF
was stably expressed in baby hamster kidney cells overexpressing furin
required for proper removal of VWF propeptide (7). Cells were cultured
in serum-free medium. VWF was purified by immuno-affinity
chromatography using monoclonal antibody RU8, which is directed against
the D4 domain (7), and stored in 50 mM Hepes, 100 mM NaCl (pH 7.4) at
VWF concentration was determined by a sandwich enzyme-linked
immunosorbent assay using polyclonal
The multimeric structure of VWF was analyzed by agarose gel
electrophoresis followed by Western blotting as described by
Lawrie et al. (24).
Binding of monoclonal antibody RU5 (22) to VWF was analyzed as
described previously (23), with some modifications. Microtiter plate
wells (Costar, Cambridge, MA) were coated with a polyclonal antibody directed against the D' and D3 domains of VWF (22) diluted to
2.5 µg/ml with 50 mM carbonate buffer (pH 9.6). Coating was carried out for 3 h at 37 °C. Wells were washed with PBS-T (phosphate-buffered saline containing 0.1% Tween 20) and blocked with
3% bovine serum albumin in PBS-T for 1 h at 37 °C. Expression medium containing VWF was diluted with mock medium to a final concentration of 1 µg/ml. Wells were incubated with diluted medium for 1 h at 37 °C. After washing, 2 µg/ml RU5 in PBS-T
containing 3% bovine serum albumin was added for 1 h at 37 °C.
Wells were washed again and incubated for 1 h at 37 °C with
horseradish peroxidase-conjugated rabbit anti-mouse antibodies (DAKO)
diluted 1:2500 in PBS-T containing 3% bovine serum albumin.
O-Phenylenediamine was used as substrate for detection.
Static Collagen Binding Assay--
Binding of VWF in expression
medium to fibrillar human placenta collagen type III (catalogue no.
C-4407; Sigma) was studied in a solid-state binding assay at a VWF
concentration of 0.1 µg/ml as described previously (23).
Surface Plasmon Resonance Collagen Binding Assay--
Surface
plasmon resonance (SPR) binding studies were performed using a
Biacore2000 system (Biacore AB, Uppsala, Sweden). Fibrillar collagen
cannot be used in a Biacore system because it blocks the flow channels.
Therefore, we used acid-soluble collagen type III. Previously, we have
shown that binding of wt-VWF to acid-soluble collagen is similar to the
binding of wt-VWF to fibrillar collagen in enzyme-linked immunosorbent
assay (22). Human placenta collagen type III was dissolved in 50 mM acetic acid at a final concentration of 1 mg/ml (16 h,
4 °C) and immobilized on a CM5 biosensor chip using the amine
coupling kit as instructed by the supplier. Approximately 3000 response
units of collagen type III, which corresponds to 30 ng/mm2,
were immobilized. A reference channel was coated with a similar amount
of human placenta collagen type IV (catalogue no. C-7521; Sigma) that
does not interact with VWF (25). Analysis was performed in Biacore
standard buffer containing 25 mM Hepes, 125 mM
NaCl, 2.5 mM CaCl2, and 0.005% (v/v) Tween 20 (pH 7.4) at 25 °C at a flow rate of 10 µl/min. Binding of VWF to
the collagen type III-coated channel was corrected for nonspecific
binding to the control channel (between 2% and 5%). VWF monomer
concentrations were calculated on the basis of a monomer mass of 270 kDa. Collagen binding at equilibrium was determined at different VWF
concentrations. To this end, increasing concentrations of VWF were
injected. Each injection was continued for 30 min. The delay between
injections was 7 min, during which time the biosensor chip was flushed
with Biacore standard buffer. After measuring all concentrations of a
VWF variant, the biosensor chip was regenerated by injection of 1 mM EDTA, 1 M NaCl, 0.1 M sodium
citrate (pH 5.0) (1 min, 10 µl/min) and 10 mM
taurodeoxycholic acid, 100 mM Tris (pH 9.0) (1 min, 10 µl/min), and 0.1 M H3PO4 (1 min,
10 µl/min).
Dissociation constants (KD) and the number of
binding sites expressed as the response at infinite VWF
concentration (Req,max) were calculated as
follows. First, the response at equilibrium (Req) was calculated for each association curve
by fitting the data points with a 1:1 Langmuir interaction model
(Biaeveluation software version 3.0.1). This interaction model fitted
the experimental data well, despite the multivalent nature of the
VWF-collagen interaction, with Characterization of VWF Mutants--
The conformation of the A3
domain and the multimeric size of VWF determine its reactivity toward
collagen. Multimer distributions of VWF mutants and recombinant wt-VWF
were indistinguishable (data not shown). The conformation of the A3
domain was evaluated with conformation-dependent monoclonal
antibody RU5. Binding of RU5 to 18 of the 22 point mutants was similar
to binding to wt-VWF. As expected, RU5 did not bind to Collagen Binding Affinity of VWF Mutants--
In a first screen,
the effect of mutations on binding to collagen type III was assessed by
a solid-state collagen binding assay (Fig. 1B). Immobilized
collagen was incubated with expression medium containing 0.1 µg/ml
VWF. Binding of
To further investigate the effect of the seven mutations, these mutants
and mutant H1023A from our previous study (23) were purified by
immuno-affinity chromatography, and collagen binding was analyzed by
SPR. We also purified and analyzed wt-VWF and mutants P981A and E1021A
that bound normally to collagen in the solid-state collagen binding
assay. The multimer distribution of the VWF variants after purification
was similar (Fig. 2).
Addition of the RU5 Fab fragment to wt-VWF almost completely inhibited
collagen binding, confirming that the A3 domain contains the binding
site for collagen type III (Fig. 3).
Mutants P981A and E1021A, which bound normally to collagen in the
solid-state collagen binding assay, also had similar binding
affinities and a similar number of binding sites as wt-VWF in the
SPR-based collagen binding assay (Fig. 3). Mutants that exhibited
strongly reduced collagen binding in the solid-state assay divided in
two populations in the SPR collagen binding assay. Mutations I975A,
T977A, V997A, and E1001A reduced the affinity of VWF for collagen
5-10-fold, whereas at saturation, the number of binding sites was at
least 60% compared with wt-VWF (Table
I). Mutants D979A, P981H, S1020A, and
H1023A had a residual binding at saturation of less than 20% compared
with wt-VWF.
As shown in Fig. 4A, mutations
that reduce collagen binding are located at the front face of the
domain and define a rather flat collagen-binding site. Mutations at the
bottom face did not have an effect. Surface properties of the
collagen-binding site are shown in Fig. 4, B and
C. The upper part of the collagen-binding site contains a
small negatively charged patch. In addition, the collagen-binding site
contains one large hydrophobic patch and two smaller hydrophobic
patches.
Docking of a Collagen Triple Helix on A3--
The amino acid
sequence of collagen that is recognized by the VWF-A3 domain is not
known. Under these circumstances, the use of automated docking
procedures, such as FTdock (26) and AutoDock (27), that use scoring
functions based on shape complementarity and interaction energies is
not meaningful. Therefore, we used the interactive molecular graphics
program O (28) to obtain an impression of possible collagen-binding
modes compatible with structural and mutagenesis data. Because the
amino acids of A3 that are involved in collagen binding define an
extended and rather flat surface at the front face of the domain, a
bound collagen triple helix must lie nearly parallel to the front face.
A model of a triple helix restricted to lie parallel to the front face of A3 was rotated and translated with respect to A3, and two criteria were evaluated to select possible binding modes. Firstly, the collagen
triple helix should contact (d < 4 Å) all residues of A3
involved in collagen binding. Secondly, monoclonal antibody RU5 bound
to A3, as observed in the crystal structure of the A3·RU5 complex
(23), should block (d < 2 Å) binding of collagen by sterical
hindrance. For the evaluation of distances, we assumed a fairly large
radius for the collagen triple helix of 9 Å, which corresponds to the
approximate distance from the tip of an extended lysine side chain to
the center of the triple helix. Using a smaller radius would have
reduced the range of possible binding modes, but in the absence of
knowledge about the collagen residues actually involved, this did not
seem justified.
A range of orientations of the collagen triple helix fulfilled the
criteria (Fig. 4D). In these potential binding modes, the collagen triple helix lies at an angle of about 60° to 90° to strand Von Willebrand factor A-type domains are found in many proteins
including collagens, complement proteins, and integrins, where they are
named I domains. These proteins are involved in several biological
functions such as cell-cell interaction and ligand-receptor binding. In
integrins, these interactions involve a divalent cation present in the
MIDAS motif located at the top of the domain.
VWF contains three A-type domains, of which the A3 domain binds to
collagen. The VWF-A3 domain does not contain a functional MIDAS motif,
and collagen binding is cation-independent (20, 21). Previously, we
excluded the top face of A3 from being involved in collagen
binding (22) and showed that His1023, located close
to the edge of the front and bottom face, is critical for binding of
VWF to collagen (23). Based on these results, we constructed a panel of
22 point mutants in which solvent-exposed residues were mutated to
either alanine or histidine. These mutations were introduced in
multimeric VWF, and the binding of these VWF mutants to collagen type
III was evaluated.
In a solid-state collagen binding assay, 7 of the 22 mutants, namely,
I975A, T977A, D979A, P981H, V997A, E1001A, and S1020A, displayed
reduced collagen type III binding. None of these mutations are located
at the bottom face of A3, excluding its involvement in collagen
binding. We further characterized these seven mutants and mutant
H1023A, which also displays strongly reduced collagen binding (23), by
SPR. In contrast to the solid-state binding assay, SPR analysis
measures collagen binding under equilibrium conditions and is not
affected by washing steps. The apparent dissociation constant for
binding of wt-VWF to collagen type III as determined by SPR was 3.3 nM, which is similar to values previously determined by us
and others (22, 29). Interestingly, mutants that were qualified as
"strongly reduced" in the solid-state assay separated in two groups
in the SPR analysis. Mutants D979A, S1020A, and H1023A displayed a
large reduction in affinity and in the number of binding sites, showing
that these residues are critical for collagen type III binding. In
contrast, mutants I975A, T977A, V997A, and E1001A were characterized by
a 5-10-fold reduced affinity but had a near normal number of binding
sites, indicating that these residues contribute to collagen binding
but are not essential. Residues essential for collagen binding are
located in strand Mutation of Pro981 to alanine did not affect collagen
binding, whereas mutation to histidine markedly decreased VWF binding
to collagen. Apparently, Pro981 is not required for binding
to collagen type III, but the introduction of a bulky histidine side
chain at the lower half of the front face of the domain interferes with
collagen binding via sterical hindrance. This observation further
supports our conclusion that the collagen-binding site is located at
the front face of A3.
Docking of a collagen triple helix on the front face of the A3 domain
suggested a range of possible engagements (Fig. 4D) and
predicts that at most eight consecutive residues in a collagen molecule
interact with A3. Based on the surface characteristics of the
collagen-binding site (Fig. 4, B and C), we
propose that collagen sequences recognized by A3 contain positively
charged and hydrophobic residues.
Despite their similarity in fold and ligand, VWF-A3 and
collagen-binding integrin I domains have distinctly different binding sites. In A3, a rather hydrophobic and flat binding site is located at
the front face, whereas integrin I domains bind collagen in a
predominantly hydrophilic groove at their top face and require a
functional MIDAS motif (18, 30). Integrin I domains undergo a large
conformational change upon collagen binding that makes them
particularly suited for signal transduction and modulation of ligand
binding affinity (18). In contrast, the VWF-A3 domain appears to
function as an independent structural unit, and there is no evidence
for modulation of its collagen binding affinity, nor does binding of A3
to collagen appear to affect the affinity of the VWF-A1 domain for
platelet receptor GpIb Recently, we determined the crystal structure of the VWF-A1 domain in
complex with an amino-terminal fragment of platelet receptor GpIb Genetic screening has identified four mutations, S968T, Q971H, I978T,
and Q999R, that affect binding of VWF to collagen (32, 33). These
mutations are located at or just below the front surface of the domain,
and their effect on collagen binding is consistent with our mutagenesis
data. Surprisingly, these mutations caused no (32) or only moderate
(33) bleeding symptoms, which questioned the relevance of the A3 domain
for immobilization of VWF to the vascular matrix (32). However, Wu
et al. (34) recently showed that collagen binding by the A3
domain is relevant because an antibody blocking the VWF-A3-collagen
interaction prevented the formation of platelet-rich thrombi and
prolonged the skin bleeding time at high doses. Further investigations
are required to reconcile these conflicting observations and to
establish the physiological importance of VWF-A3 in platelet adhesion.
After submission of this manuscript, Nishida et al. (35)
reported mapping of the collagen-binding site of the VWF-A3 domain using a novel NMR technique. Their findings with regard to the location
of the binding site and the orientation of a bound collagen triple
helix are in complete agreement with our results.
In summary, the binding site for collagen type III is located at the
front face of the VWF-A3 domain. Residues in the lower half of the
collagen-binding site are essential for collagen binding, whereas
residues in the upper half contribute to binding but are not essential.
We suggest that a collagen triple helix that interacts with A3 contains
hydrophobic and positively charged residues. Further understanding of
the VWF-collagen interaction requires the identification of specific
collagen sequences involved in VWF binding.
2 I
domain, which has a hydrophilic binding site located at the top face of
the domain. Based on the surface characteristics of the
collagen-binding site of A3, we propose that it interacts with collagen
sequences containing positively charged and hydrophobic residues.
Docking of a collagen triple helix on the binding site suggests
a range of possible engagements and predicts that at most eight
consecutive residues in a collagen triple helix interact with A3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 (2) and GpVI to bind to collagen (2-4). These interactions result in firm adhesion and activation of
platelets at the site of vascular injury.
of the GpIb-IX-V
complex (10, 11).
-sheet flanked on both sides by amphipathic
-helices (12-15).
Binding of the I domains of integrins
1
1,
2
1,
10
1, and
11
1 to collagen involves a divalent
cation (16, 17) located in the metal ion-dependent adhesion
site (MIDAS) motif, and amino acid residues at the top face of the
domain (18, 19). Binding of the I domain of integrin
2
1 to collagen induces a major
displacement of its carboxyl-terminal
-helix that is thought to be
critical for integrin signaling (18). The A3 domain of VWF does not
contain a functional MIDAS motif, and binding of A3 to collagen is
cation-independent (20, 21). The involvement of the top face of A3 in
collagen binding has been excluded by mutagenesis studies (13, 22).
Recently, we showed that His1023, located close to the edge
of the front and bottom face of A3, is critical for binding of VWF
to collagen (23).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
-VWF and horseradish
peroxidase-conjugated polyclonal
-VWF (DAKO, Glostrup, Denmark) for
immobilization and detection, respectively (7). Normal pooled plasma
from 40 healthy donors was used as a reference.
2 values being typically
lower than 1.5. Use of more complex binding models did not improve the
fit significantly. Next, KD and
Req,max were determined from the binding
isotherms (Req plotted against VWF
concentration) by fitting equation Req = Req,max*[VWF]/(KD + [VWF]), which describes a 1:1 interaction. The fit was calculated using computer program GraphPad Prism (GraphPad Prism version 3.00 for
Windows; GraphPad Software, San Diego, CA). Because interaction between
VWF and collagen is multivalent and we used a 1:1 interaction model,
calculated dissociation constants must be regarded as apparent values.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A3-VWF, a
deletion mutant lacking the A3 domain (7) (Fig.
1A). Four of the point
mutants, P962H, P981A, V984A, and V985A, showed a significantly reduced
RU5 binding, ranging from 60% to 80% compared with wt-VWF. However,
collagen binding of these mutants was normal (see below). Reduced RU5
binding can be explained by direct interactions of the mutated residues with RU5, as observed in the crystal structure of the A3·RU5 complex (23). Overall, these data suggest that the A3 domains of all VWF
variants are correctly folded.
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Fig. 1.
Characterization of VWF-A3 mutants.
A, binding of conformation-dependent antibody RU5.
VWF was immobilized in microtiter plate wells via polyclonal
antibody D'D3. Immobilized VWF was incubated with RU5, and bound RU5
was detected as described under "Experimental Procedures."
Wild-type VWF and
A3-VWF, which lacks the A3 domain (7), were used
as a positive and negative control, respectively. Binding of RU5 to VWF
mutants is expressed as a percentage of wt-VWF binding. Each data
point represents the mean ± S.D. of three experiments
performed in duplicate. B, binding of VWF point mutants to
fibrillar collagen type III in a solid-state collagen binding assay.
Human placenta collagen type III was coated in microtiter plate wells.
Wells were incubated with VWF at a concentration of 0.1 µg/ml. Bound
VWF was detected as described under "Experimental Procedures."
A3-VWF (7) was used as a negative control. Bound VWF is expressed as
a percentage of wt-VWF binding. Each data point represents
the mean ± S.D. of three independent experiments performed in
duplicate.
A3-VWF to collagen was only 8% compared with
wt-VWF. Mutations I975A, T977A, D979A, P981H, V997A, E1001A, and S1020A
strongly reduced collagen binding, showing a residual binding of less
than 25%.
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Fig. 2.
Multimer distributions of purified VWF
variants. The multimeric structure of purified VWF variants used
in the SPR collagen binding assay was analyzed by agarose gel
electrophoresis followed by Western blotting. The multimeric
distributions of VWF mutants are similar to that of recombinant
wt-VWF.
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Fig. 3.
Binding isotherms of VWF mutants to collagen
type III in an SPR-based assay. Human placenta collagen types III
and IV were immobilized on a CM5 biosensor chip for a detection and
reference channel, respectively. Increasing concentrations of VWF were
injected, and binding at equilibrium was determined. Wild-type VWF and
wt-VWF preincubated with a 5-fold molar excess of RU5 Fab-fragment were
used as a positive and negative control, respectively. Each variant was
measured once, except for wt-VWF (×) and mutant I975A ( ), which
were measured four times using two independently purified batches of
VWF and two sensor chips prepared with different batches of collagen.
The error bars represent the S.D. calculated from
these four measurements. Coefficients of variation determined from
these multiple measurements are 10% and 25% for the dissociation
constants and 1.9% and 4.3% for the maximum binding of wt-VWF and
mutant I975A, respectively. Binding constants derived from the binding
isotherms are listed in Table I.
Apparent dissociation constants and maximum binding for the
VWF-collagen interaction
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Fig. 4.
Characteristics of the collagen-binding site
on the VWF-A3 domain. A, stereoview of the A3 domain
that shows the effect of mutations on collagen binding by color.
Mutation of residues shown in magenta strongly
reduces collagen binding. Mutation of residues shown in
green partially reduces collagen binding, whereas mutation
of residues shown in gray has no effect. Pro981
is shown in red. Mutation P981A has no effect on collagen
binding, whereas mutation P981H strongly reduces binding. The effect of
five mutations investigated in our previous study (23), R963A, E987A,
H990A, R1016A, and H1023A, are included in the figure. B,
solvent-accessible surface with electrostatic potential contoured from
red ( 260 mV) to blue (+260 mV). C,
solvent-accessible surface with hydrophobic and hydrophilic regions in
green and white, respectively. D,
space-filling representation of the A3 domain with two collagen triple
helices that indicate the putative range of orientations for the
A3-collagen interaction. Residues of A3 are color-coded as described in
A, except that residues that show no effect on collagen
binding are colored yellow, and residues for which no data
are available are shown in gray. Figures were generated with
MOLSCRIPT (36), RASTER3D (37), and GRASP (38). The atomic coordinates
for the crystal structure of A3 were taken from Protein Data Bank entry
1ATZ (12).
3 (located at the front face of the domain) and interacts with the A3 domain via six to eight consecutive residues.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 and loop
3
4 in the lower half of the front
face of A3, whereas nonessential residues are located in the upper half
of the front face in loops
2
3,
2
3, helix
3, and strand
3 (Fig. 3A).
. Thus, the collagen-binding site of A3 merely
performs an adhesive function, whereas binding sites of I domains are
more sophisticated and also play a regulatory role (31).
(11). Like A3, the A1 domain does not contain a MIDAS motif. GpIb
binds to two distinct sites on A1. The larger of the two binding sites
is located at the front face of A1 and consists of residues from strand
3, helix
3, and loop
3
4. Interestingly, the same structural
elements contribute residues to the collagen-binding site of A3,
suggesting that ligand-binding sites in A-type domains that lack a
MIDAS motive may all be located in a similar position at the front face
of the domain.
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FOOTNOTES |
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* This work was supported by the Council of Medical Science Program 902.26.193 from the Netherlands Organization for Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Laboratory of Medical Oncology, University Medical Center, HP F02.126, P. O. Box 85500, 3508 GA Utrecht, The Netherlands.
To whom correspondence should be addressed. Tel.:
31-30-2533502; Fax: 31-30-2533940; E-mail:
E.G.Huizinga@chem.uu.nl.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M208977200
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ABBREVIATIONS |
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The abbreviations used are: Gp, glycoprotein; MIDAS, metal ion-dependent adhesion site; SPR, surface plasmon resonance; VWF, von Willebrand factor; wt, wild-type.
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REFERENCES |
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---|
1. | Savage, B., Saldívar, E., and Ruggeri, Z. M. (1996) Cell 84, 289-297[Medline] [Order article via Infotrieve] |
2. | Savage, B., Almus-Jacobs, F., and Ruggeri, Z. M. (1998) Cell 94, 657-666[Medline] [Order article via Infotrieve] |
3. |
Goto, S.,
Tamura, N.,
Handa, S.,
Arai, M.,
Kodama, K.,
and Takayama, H.
(2002)
Circulation
106,
266-272 |
4. |
Kehrel, B.,
Wierwille, S.,
Clemetson, K. J.,
Anders, O.,
Steiner, M.,
Knight, C. G.,
Farndale, R. W.,
Okuma, M.,
and Barnes, M. J.
(1998)
Blood
91,
491-499 |
5. | Sadler, J. E. (1998) Annu. Rev. Biochem. 67, 395-424[CrossRef][Medline] [Order article via Infotrieve] |
6. | Fischer, B. E., Kramer, G., Mitterer, A., Grillberger, L., Reiter, M., Mundt, W., Dorner, F., and Eibl, J. (1996) Thromb. Res. 84, 55-66[CrossRef][Medline] [Order article via Infotrieve] |
7. | Lankhof, H., van Hoeij, M., Schiphorst, M. E., Bracke, M., Wu, Y. P., Ijsseldijk, M. J., Vink, T., de Groot, P. G., and Sixma, J. J. (1996) Thromb. Haemostasis 75, 950-958[Medline] [Order article via Infotrieve] |
8. | Hoylaerts, M. F., Yamamoto, H., Nuyts, K., Vreys, I., Deckmyn, H., and Vermylen, J. (1997) Biochem. J. 324, 185-191[Medline] [Order article via Infotrieve] |
9. | Denis, C., Baruch, D., Kielty, C. M., Ajzenberg, N., Christophe, O., and Meyer, D. (1993) Arterioscler. Thromb. 13, 398-406[Abstract] |
10. |
Lankhof, H.,
Wu, Y. P.,
Vink, T.,
Schiphorst, M. E.,
Zerwes, H. G.,
de Groot, P. G.,
and Sixma, J. J.
(1995)
Blood
86,
1035-1042 |
11. |
Huizinga, E. G.,
Tsuji, S.,
Romijn, R. A. P.,
Schiphorst, M. E.,
De Groot, Ph. G.,
Sixma, J. J.,
and Gros, P.
(2002)
Science
297,
1176-1179 |
12. | Huizinga, E. G., Van der Plas, R. M., Kroon, J., Sixma, J. J., and Gros, P. (1997) Structure 5, 1147-1156[Medline] [Order article via Infotrieve] |
13. |
Bienkowska, J.,
Cruz, M. A.,
Atiemo, A.,
Handin, R. I.,
and Liddington, R.
(1997)
J. Biol. Chem.
272,
25162-25167 |
14. | Colombatti, A., Bonaldo, P., and Doliana, R. (1993) Matrix 13, 297-306[Medline] [Order article via Infotrieve] |
15. |
Emsley, J.,
King, S. L.,
Bergelson, J. M.,
and Liddington, R. C.
(1997)
J. Biol. Chem.
272,
28512-28517 |
16. |
Tuckwell, D. S.,
Calderwood, D. A.,
Green, L. J.,
and Humphries, M. J.
(1995)
J. Cell Sci.
108,
1629-1637 |
17. |
Dickeson, S. K.,
Walsh, J. J.,
and Santoro, S. A.
(1997)
J. Biol. Chem.
272,
7661-7668 |
18. | Emsley, J., Knight, C. G., Farndale, R. W., Barnes, M. J., and Liddington, R. C. (2000) Cell 100, 47-56 |
19. |
Tulla, M.,
Pentikainen, O. T.,
Viitasalo, T.,
Kapyla, J.,
Impola, U.,
Nykvist, P.,
Nissinen, L.,
Johnson, M. S.,
and Heino, J.
(2001)
J. Biol. Chem.
276,
48206-48212 |
20. | Pietu, G., Fressinaud, E., Girma, J. P., Nieuwenhuis, H. K., Rothschild, C., and Meyer, D. (1987) J. Lab. Clin. Med. 109, 637-646[Medline] [Order article via Infotrieve] |
21. | Bockenstedt, P. L., McDonagh, J., and Handin, R. I. (1986) J. Clin. Invest. 78, 551-556[Medline] [Order article via Infotrieve] |
22. | Van der Plas, R. M., Gomes, L., Marquart, J. A., Vink, T., Meijers, J. C. M., de Groot, Ph. G., Sixma, J. J., and Huizinga, E. G. (2000) Thromb. Haemostasis 84, 1005-1011[Medline] [Order article via Infotrieve] |
23. |
Romijn, R. A.,
Bouma, B.,
Wuyster, W.,
Gros, P.,
Kroon, J.,
Sixma, J. J.,
and Huizinga, E. G.
(2001)
J. Biol. Chem.
276,
9985-9991 |
24. | Lawrie, A. S., Horser, M. J., and Savidge, G. F. (1990) Thromb. Haemostasis 59, 369-373 |
25. | Sixma, J. J., van Zanten, G. H., Huizinga, E. G., Van der Plas, R. M., Verkley, M., Wu, Y. P., Gros, P., and de Groot, P. G. (1997) Thromb. Haemostasis 78, 434-438[Medline] [Order article via Infotrieve] |
26. | Gabb, H. A., Jackson, R. M., and Sternberg, M. J. E. (1997) J. Mol. Biol. 272, 106-120[CrossRef][Medline] [Order article via Infotrieve] |
27. | Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R. K., and Olson, A. J. (1998) J. Comput. Chem. 19, 1639-1662[CrossRef] |
28. | Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
29. | Saenko, E., Kannicht, C., Loster, K., Sarafanov, A., Khrenov, A., Kouiavskaia, D., Shima, M., Ananyeva, N., Schwinn, H., Gruber, G., and Josic, D. (2002) Anal. Biochem. 302, 252-262[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Smith, C.,
Estavillo, D.,
Emsley, J.,
Bankston, L. A.,
Liddington, R. C.,
and Cruz, M. A.
(2000)
J. Biol. Chem.
275,
4205-4209 |
31. |
Dickeson, S. K.,
Mathis, N. L.,
Rahman, M.,
Bergelson, J. M.,
and Santoro, S. A.
(1999)
J. Biol. Chem.
274,
32182-32191 |
32. | Schneppenheim, R., Obser, T., Drewke, E., Grosse-Wieltsch, U., Oyen, F., Sutor, A. H., Wermes, C., and Budde, U. (2001) Blood 98 (suppl.), 41a |
33. | Ribba, A. S., Loisel, I., Lavergne, J. M., Juhan-Vague, I., Obert, B., Cherel, G., Meyer, D., and Girma, J. P. (2001) Thromb Haemostasis 86, 848-854[Medline] [Order article via Infotrieve] |
34. |
Wu, D.,
Vanhoorelbeke, K.,
Cauwenberghs, N.,
Meiring, M.,
Depraetere, H.,
Kotze, H. F.,
and Deckmyn, H.
(2002)
Blood
99,
3623-3628 |
35. | Nishida, N., Sumikawa, H., Sakakura, M., Shimba, N., Takahashi, H., Terasawa, H., Suzuki, E. I., and Shimada, I. (2003) Nat. Struct. Biol. 10, 53-58[CrossRef][Medline] [Order article via Infotrieve] |
36. | Kraulis, P. (1991) J. Appl. Crystallogr. 25, 649-950[CrossRef] |
37. | Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524 |
38. | Nicholls, A., Sharp, K. A., and Honig, B. (1993) Biophys. J. 64, 166-170 |