From the
The von Willebrand factor (vWF) mediates platelet adhesion to
the vascular subendothelium by binding to collagen, other matrix
constituents, and the platelet receptor glycoproteins Ib/IX and
IIb/IIIa. Although substantial progress has been made in defining vWF
structure-function relationships, there are conflicting data regarding
the location of its collagen-binding site(s). Possible collagen-binding
sites have been localized in the A1 and A3 domains of vWF. To study the
proposed binding sites, we have expressed cDNA sequences encoding the
A1 and A3 domains of vWF in Escherichia coli and purified the
resulting proteins from bacterial inclusion bodies. In addition, a
chimeric molecule containing residues 465-598 of the vWF A1
domain polypeptide (vWF-A1) fused in frame to residues 1018-1114
of the vWF A3 domain polypeptide (vWF-A3) was also expressed. Each of
the three recombinant proteins purified as a monomer and contained a
single disulfide bond. As previously reported (Cruz, M. A., Handin, R.
I., and Wise, R. J. (1993) J. Biol. Chem. 268,
21238-21245), recombinant vWF-A1 inhibited ristocetin-induced
platelet agglutination, but did not compete with vWF multimers for
collagen binding. In contrast, vWF-A3 inhibited the binding of
multimeric vWF to immobilized collagen, but did not inhibit
ristocetin-induced platelet agglutination. Metabolically labeled vWF-A3
bound to immobilized collagen in a saturable and reversible manner with
a K
The von Willebrand factor (vWF)
Analysis of vWF cDNA and its predicted amino acid sequence shows an
interesting pattern of homologous repeats
(20, 21, 22) . There is strong evidence that the
vWF-A1 repeat, which encodes amino acids 479-717, contains
binding sites for GPIb/IX, glycosaminoglycans, sulfatides, and collagen
(23, 24, 25, 26, 27) . The
vWF-A3 repeat, which encodes amino acids 910-1111
(28) , is also
reported to have a collagen-binding site
(15, 17, 18, 29, 30) . Thus, two
of the triplicated A repeats contain sequences that have been
implicated in collagen binding. In addition to their sequence
similarity, the A1 and A3 repeats each contain a single intrachain
disulfide bond. The A1 disulfide bond links Cys-509 and Cys-695, and
the A3 disulfide links Cys-923 and Cys-1109, forming a 185-amino acid
``loop'' structure in each domain
(19, 31) .
Given the similarities in primary and secondary structure, the A1 and
A3 domains could easily have overlapping functions.
Despite the data
obtained with tryptic fragments of vWF
(13, 15, 17, 18, 19) , there is
still some uncertainty regarding the existence of a collagen-binding
site in the A1 domain. One early study, utilizing unpurified bacterial
lysate, reported an interaction between recombinant vWF-A1 protein in
the bacterial lysate and collagen
(32) . Other investigators who
have subsequently expressed and studied the function of purified vWF-A1
protein have reported conflicting results
(33, 34, 35) . For example, our laboratory
recently reported that a highly purified monomeric vWF-A1 protein bound
to platelet GPIb/IX and heparin, but did not bind to collagen
(36) . In addition, it has been reported that recombinant vWF
from which the entire A1 domain has been deleted, which forms the
normal spectrum of multimers, no longer binds to platelet GPIb/IX, but
still binds to collagen
(37) .
To resolve these conflicting
results and to learn more about the collagen-vWF interaction, we have
cloned and expressed vWF A1 and A3 domain polypeptides and compared
their biochemical properties with a chimeric vWF A1 domain polypeptide
containing sequences derived from the vWF A1 and A3 domains
(vWF-A1/A3). The studies reported here clearly demonstrate that vWF-A3
protein and the vWF-A1/A3 chimera both bind to type I collagen. Results
obtained with vWF-A1/A3 help to localize the collagen-binding sequence
in the A3 domain to a sequence between amino acids 1018 and 1114. We
have concluded that the vWF-A3 binding site can account for all of the
interactions between multimeric vWF and type I collagen and that this
site probably represents the physiologically relevant collagen-binding
site in vWF.
For
purification of vWF-A3 protein, the washed pellet was solubilized by
the addition of 7.5 M urea in 25 mM Tris-HCl, pH 8.8,
and the solubilized proteins were dialyzed against 25 mM
Tris-HCl, pH 8.2. The solubilized proteins were passed over a
Q-Sepharose column (Pharmacia Biotech Inc.) equilibrated with 25
mM Tris-HCl, pH 8.2. vWF-A3 eluted from the column with sodium
chloride. The fractions containing vWF-A3 were pooled, concentrated by
ultrafiltration, and dialyzed against Tris-buffered saline (TBS; 25
mM Tris-HCl, 150 mM NaCl, pH 7.4)
Recombinant
vWF-A1 or chimeric vWF-A1/A3 protein was expressed in E. coli as described above. They were both purified as described
previously
(36) . Briefly, after induction with IPTG and
collection of inclusion bodies from lysed E. coli cells, the
washed inclusion bodies were solubilized in 6 M guanidine
hydrochloride, dialyzed into 6 M urea, and purified by fast
protein liquid chromatography on an S-Sepharose column (Pharmacia
Biotech Inc.). Urea was removed by slow dialysis, and the recombinant
proteins were dialyzed against TBS
(36) . Each of the purified
recombinant proteins was then concentrated by adsorption to and elution
from a heparin column (Bio-Rad) and/or ultrafiltration and dialyzed
against TBS.
As shown in Fig. 4,
The interaction between vWF and collagen has been studied
most extensively with proteolytic fragments derived from purified
plasma vWF. These reports have identified peptides that inhibit
collagen binding and that contain either A1 or A3 domain sequences
(13, 15, 17, 18, 19, 23, 29) .
Despite these data, when highly purified recombinant vWF A1 domain
polypeptides were studied in several laboratories, collagen binding
could not be demonstrated
(33, 34, 35, 36) . This raised the
interesting possibility that the major collagen-binding site might be
located in the A3 domain of vWF. To study this, we expressed a vWF cDNA
encoding Ser-908-Gly-1111 and compared its properties with those
of recombinant vWF-A1 and a vWF-A1/A3 chimera. All three proteins were
readily purified from E. coli inclusion bodies in quantities
sufficient for biochemical studies. Based on our comparative studies,
we have concluded that vWF-A3 1) binds to immobilized type I collagen,
2) blocks the binding of multimeric vWF to type I collagen, and 3) does
not inhibit ristocetin-dependent platelet agglutination by multimeric
vWF. In contrast, vWF-A1 does not compete for collagen-binding sites.
Finally, a chimeric molecule, vWF-A1/A3, which contains the
amino-terminal half of vWF-A1 fused in frame to the carboxyl-terminal
half of vWF-A3, also inhibits vWF binding to collagen. Although not the
major focus of this study, the chimeric molecule is bifunctional and
also inhibits vWF binding to the platelet GPIb/IX receptor site.
These results extend five previous reports that predicted a
collagen-binding site in the vWF A3 domain. Roth et al.(17) first reported that a tryptic fragment of vWF containing
residues 948-998 inhibited the binding of vWF to immobilized type
III collagen. Three subsequent studies of tryptic fragments spanning
residues 730-1114 and/or 911-1365 have demonstrated binding to collagen
types I, III, and VI
(15, 18, 29) . Jorieux
et al.(30) then expressed a recombinant protein
containing residues 914-1364 and demonstrated binding to fibrillar
collagen types I and III using as ligand a radiolabeled partially
purified bacterial extract. Our study extends and clarifies these
previous studies by using a highly purified monomeric recombinant
protein rather than a proteolytic fragment of plasma vWF or a crude
bacterial extract. In addition, our recombinant vWF-A3 protein is
considerably smaller than that of Jorieux et al. (206
versus 450 residues). This is of some importance as the
recombinant vWF-A3 protein used by Jorieux et al. also
contains a portion of the D4 domain, making localization of the
collagen-binding site exclusively within the A3 domain less certain.
While the recombinant vWF A3 domain polypeptide used in this study
bound saturably and reversibly to immobilized type I collagen, its
affinity ( K
The failure of
monomeric recombinant vWF-A1 to compete with vWF for binding to type I
collagen remains a puzzle. The most likely explanation, and the one
that is supported by our data, is that the vWF A1 domain does not, in
fact, contain a collagen-binding site. This is supported by the
observation that a recombinant form of multimeric vWF from which the A1
domain sequence has been deleted still binds to collagen normally
(37) . There is also a recent preliminary report confirming that
vWF from which the A1 domain has been deleted, which no longer binds to
GPIb/IX, still binds to collagen
(43) . This study also showed
that deletion of the A3 domain preserves the GPIb/IX interaction, but
abolishes collagen binding. It is widely appreciated that deletion
mutagenesis experiments must be interpreted cautiously since deletion
of large portions of the vWF subunit could induce major conformational
changes in the remaining portion of the molecule; however, the
selectivity of the two deletions suggests that this is not the cause
for the observed losses of function. Finally, our data show that
recombinant vWF-A3 can completely inhibit vWF multimer binding to
collagen (Fig. 6). If both the A1 and A3 domains of vWF were
binding to collagen, blockade of one domain should either have no
effect on binding or cause only partial inhibition. The data we have
obtained show that preventing binding via the vWF A3 domain completely
abolishes vWF binding to type I collagen.
Further proof for the role
of the vWF A3 domain in collagen binding comes from the
characterization of a vWF-A1/A3 chimera containing the amino-terminal
half of the A1 domain (amino acids 475-598) and the
carboxyl-terminal half of the A3 domain (amino acids 1018-1114).
As expected, the vWF-A1/A3 chimera inhibited ristocetin-induced
platelet agglutination with an IC
One
limitation of our study is that the analysis was limited to a single
collagen subtype. There is, in fact, evidence that vWF interacts with
multiple collagen subtypes and may also bind to noncollagenous
components of the extracellular matrix. For example, Rand et al.(14) identified type VI collagen as one potential binding
protein for vWF in the vascular subendothelium. Denis et al.(15) recently analyzed the binding of proteolytic fragments of
vWF to the extracellular matrix and to type VI collagen. Their study
documents that vWF binds to the endothelial matrix via sequences in the
A1 domain and that both the A1 and A3 domains contain a binding site
for type VI collagen
(15) .
Thus, while there is little doubt
that the vWF A3 domain is essential for the binding of vWF to type I
collagen, it is possible that other regions of vWF, including sequences
within the A1 domain, may mediate vWF interactions with other collagen
subtypes or other matrix components. The techniques described here for
the expression, purification, and biochemical analysis of recombinant A
domain polypeptides can be used in future studies to clarify which vWF
domains interact with other collagen subtypes. They may also provide
interesting models for designing agents that can selectively inhibit
flow-dependent platelet adhesion, the initial event in normal
hemostasis as well as in arterial thrombosis.
of 1.8
10
M. The vWF-A1/A3 chimera was bifunctional. It inhibited
vWF binding to platelet glycoprotein Ib/IX with an IC
of
0.6
10
M and inhibited vWF binding
to collagen with an IC
of 0.5-1.0
10
M. These results, taken together,
provide firm evidence that the major collagen-binding site in vWF
resides in the A3 domain.
(
)
is a
multimeric plasma glycoprotein that plays an important role in primary
hemostasis
(1, 2) . vWF mediates the adhesion of
platelets to exposed subendothelium by forming a bridge between
collagen, heparin-like glycosaminoglycans and other components of the
subendothelium, and platelet receptor sites on glycoproteins Ib/IX and
IIb/IIIa. vWF stabilizes adherent platelets under conditions of high
flow and shear stress
(3, 4, 5, 6) .
Although the sites on vWF that bind to platelet GPIb/IX and GPIIb/IIIa
have been well characterized, the collagen-binding site(s) within vWF
is not well defined. Results vary with the source of vWF, the type of
collagen, and the nature of the binding assay. Previous investigators
have reported that vWF binds to collagen types I, III, and VI
(7, 8, 9, 10, 11, 12, 13, 14, 15) .
Studies with proteolytic fragments of vWF have defined three potential
collagen-binding sites in vWF. One is localized in the propeptide,
which is cleaved during the assembly of vWF multimers and is unlikely
to play a major role in platelet adhesion
(16) . The other two
sites have been localized to amino acids 542-622 and
948-998 of the mature vWF subunit polypeptide
(17) .
Construction of vWF Expression Vectors
A map for
each of the three vWF cDNAs expressed is shown in Fig. 1. For
expression of the vWF A3 domain in Escherichia coli, a cDNA
fragment encoding amino acids 908-1111 of mature vWF was constructed by
mutagenesis of vWF cDNA in M13 with two oligonucleotides.
Oligonucleotide 1 spanned codons 1666-1674
(28) and
introduced a BamHI restriction site (encoding Gly and Ser) at
codons 1670 and 1671. Oligonucleotide 2 spanned codons 1870-1882
and introduced a termination codon and a HindIII restriction
site. Following sequence confirmation, the vWF-A3 cDNA fragment was
isolated by digestion with BamHI and HindIII and
inserted into the expression vector pQE9 (QIAGEN Inc.). Insertion of
the vWF-A3 fragment in pQE9 produces an amino-terminal fusion protein
containing the vWF sequence fused in frame to 10 amino acids (6
histidines) contributed by the vector. vWF-A1 cDNA was prepared as
described previously and used to transform pQE9 as well
(36) .
Figure 1:
Maps of the recombinant vWF-A1, vWF-A3,
and vWF-A1/A3 constructs used for bacterial expression studies.
A, the vWF-A1 sequence (amino acids 475-709) is shown in
black. B, the vWF-A3 sequence (amino acids 908-1111)
is shown in white. C, the vWF-A1/A3 chimera (amino
acids 475-598/1018-1114) is shown with respective A1 and A3
domain contributions in black and white. The native
vWF-A1 disulfide bond (Cys-509-Cys-695), the native vWF-A3
disulfide bond (Cys-923-Cys-1109), and the proposed disulfide
bond in the vWF-A1/A3 chimera (Cys-509-Cys-1109) are also
depicted.
Chimeric vWF-A1/A3 cDNA was constructed with a polymerase chain
reaction-based mutagenesis strategy. In the first round of
amplification, the amino-terminal half of the A1 domain (residues
475-598) and the carboxyl-terminal half of the A3 domain
(residues 1018-1114) were amplified. The 3`-primer for amplifying
the A1 domain was designed to contain 12 extra bases of A3 domain
sequence (5`-TCGCACAGCAAACAAGACCTCGCTGGTGGA-3` (vWF-A1 sequence is
underlined)). Thus, the amplified A1 cDNA contained a short A3 sequence
at its 3`-end. Similarly, A3 cDNA contained a short A1 sequence at its
5`-end (5`-AGCGAGGTCTTGGCTGTGCGATACTTG-3` (vWF-A1 sequence is
underlined)). The two chimeric cDNA fragments were then incubated
together so that they were annealed via their overlapping sequences.
The annealed material was used as the template for the second round of
polymerase chain reaction. The chimeric DNA fragment was amplified
using 5`-A1 (5`-CCTCACCTGTGAAGGATCCCAGGAGCCGGGAG-3`) and 3`-A3
(5`-CATTCCAAGCTTGAATTCATCAAGATCTAACAAATCCAGAGC-3`) primers designed to
introduce BamHI and HindIII restriction sites,
respectively, for cloning as described previously above. The chimeric
vWF-A1/A3 cDNA fragment was isolated by digestion with BamHI
and HindIII and used to transform pQE9.
Purification of Recombinant Proteins
E. coli M15(pREP4) cells (QIAGEN Inc.) containing pQE9-vWF-A3,
pQE9-vWF-A1, or pQE9-vWF-A1/A3 were cultured overnight at 37 °C in
8 liters of 25 g/liter Tryptone, 15 g/liter yeast extract, 5 g/liter
NaCl, pH 7.3, containing 100 µg/ml ampicillin and 25 µg/ml
kanamycin. The overnight culture was diluted 1:20 and grown to
A = 0.7. The culture was adjusted to 1.5
mM IPTG and incubated for 5 h at 37 °C. The cells were
then harvested, resuspended in 125 ml of lysis buffer (50 mM
Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 8.0) containing
lysozyme at a final concentration of 250 µg/ml, and allowed to
stand for 1 h at 4 °C. The bacterial cells were lysed in the
presence of 1.25 mg/ml deoxycholic acid and 7 µg/ml DNase I. The
lysate was centrifuged at 12,000
g for 15 min, and the
resulting pellet was washed with lysis buffer containing 0.5% Triton
X-100 and 10 mM EDTA, followed by recentrifugation.
Radiolabeling of vWF-A3 Protein
E. coli cells were grown overnight and diluted in medium described above.
When grown to A = 0.7, the cells (500 ml)
were pelleted by centrifugation, washed with M9 minimal medium (6 g of
Na
HPO
/liter, 3 g of
KH
PO
/liter, 0.5 g of NaCl/liter, 1 g of
NH
Cl/liter, 1 mM MgCl, 0.1 mM
CaCl
, and 0.2% glucose), and pelleted again. The pellet was
resuspended in 500 ml of M9 minimal medium supplemented with a 0.02%
concentration of 18 amino acids except Met and Cys. Bacteria were grown
for 45 min; IPTG was added; and the medium was supplemented with 1.5
µCi/ml [
S]H
SO
(DuPont NEN) overnight at 37 °C. Labeled vWF-A3 was purified
as described above. Specific activity of
S-labeled vWF-A3
was 3.6
10
cpm/µg.
Production and Purification of Multimeric Recombinant
vWF
Recombinant vWF was purified from the conditioned medium of
Chinese hamster ovary cells that had been stably transformed with
full-length vWF cDNA as described previously
(21, 39, 40) . Confluent cells were rinsed and
incubated for 24-48 h in -minimal essential medium
containing 0.05% bovine serum albumin, insulin/transferrin/sodium
selenite supplement (Sigma), and 1% (v/v) aprotinin (Sigma, A-6279).
The conditioned medium was collected and centrifuged to remove cellular
debris, and Na
EDTA and phenylmethanesulfonyl fluoride were
added to final concentrations of 5 and 2 mM, respectively. To
isolate vWF wild-type protein, Chinese hamster ovary cell-conditioned
medium was concentrated in dialysis tubing by using Aquacide II
(Calbiochem) and passed over a Sepharose CL-4B gel filtration
chromatography column (Pharmacia Biotech Inc.; 5
100 cm).
Fractions containing vWF protein were pooled, concentrated by Aquacide
II, and dialyzed against TBS. For radiolabeling of vWF produced in
Chinese hamster ovary cells, Cys- and Met-deficient serum-free medium
was supplemented with 10 µCi/ml [
S]Met and
[
S]Cys
(39, 40) . Radiolabeled
vWF was purified as described above. Specific activity of
S-labeled vWF was 2.0
10
cpm/µg.
Collagen Binding Assay
A final concentration of
1.8 mg/ml acid-soluble bovine type I collagen (Collaborative
Biomedical, Boston) was added to microtiter wells in 20 mM
sodium citrate buffer, pH 6.0, for 90 min. An acid-soluble calfskin
type I collagen (Sigma) was also used in some experiments. After
washing three times with TBS to remove nonadsorbed collagen, wells were
blocked with 1% bovine serum albumin in TBS for 30 min. Increasing
concentrations of S-labeled vWF proteins were added to the
wells and incubated for 60 min at room temperature. For competition
assays, a constant concentration (1-4 µg/ml) of
S-labeled vWF was added to the wells with increasing
concentrations of the unlabeled ligands. Wells were washed with TBS,
and bound radioactivity was removed for scintillation counting by
overnight incubation in 1% SDS/TBS. Nonspecific binding was determined
in the presence of a 40-fold excess of nonradioactive vWF-A3 or a
50-fold excess of nonradioactive vWF. In each case, nonspecific binding
was always <5% of total binding. The amount of collagen bound to
wells was measured in duplicate as described previously
(12) .
Bound collagen was removed with 1% SDS and submitted to protein
quantitation. At the concentration used in the assays, 6 ± 1.0
µg of collagen bound to each well.
Platelet Agglutination Assay
Ristocetin-induced
platelet agglutination was carried out in siliconized glass cuvettes at
37 °C with constant stirring at 1200 rpm in a four-channel
aggregometer (Bio/Data Corp.). A suspension of 2
10
/ml formaldehyde-fixed platelets containing 8 µg/ml
purified vWF
(41) and increasing concentrations of the
recombinant proteins was prepared. After 5 min of incubation at 37
°C, agglutination was initiated by the addition of ristocetin
(Sigma) to a final concentration of 1 mg/ml.
Protein Quantitation
Protein concentrations were
determined by the bicinchoninic acid method (Pierce). Purity was
assessed by Coomassie Blue staining of SDS-polyacrylamide gels
(42) . Radiolabeled protein was visualized by autoradiography
utilizing ENHANCE (DuPont NEN). Gel filtration analysis was carried out
in a Sephacryl 300-HR column (Sigma; 0.8 30 cm) using a Waters
650E-APPS apparatus
(36) .
Production, Purification, and Collagen-binding Activity
of vWF-A3
After induction with IPTG, transformed bacteria
expressing vWF-A3 cDNA, pQE9-vWF-A3, were lysed, and their inclusion
bodies were collected. As shown in Fig. 2 A, when
analyzed by SDS-PAGE under reducing conditions, the washed inclusion
bodies contained a prominent protein band of 27,000 Da. The inclusion
bodies could be solubilized in 7.5 M urea, and all of the
protein remained in solution after dialysis against 20 mM
Tris, pH 8.2. The soluble proteins were then fractionated by fast
protein liquid chromatography using a Q-Sepharose ion-exchange column
and a linear NaCl gradient. As shown in Fig. 3, vWF-A3 eluted in
a sharp peak at 160 mM NaCl. This peak represented 50% of
the total vWF-A3 protein expressed. The remaining 50%, which formed
high molecular mass aggregates, eluted in the flow-through volume of
the column. The final yield of purified monomeric protein was 8
mg/liter of bacterial culture. To assess the purity of vWF-A3,
radiolabeled protein was produced by incubating E. coli containing pQE9-vWF-A3 in medium containing
SO
and IPTG. Radiolabeled vWF-A3 was then purified by the procedure
described above. A single radiolabeled band was seen after purification
by SDS-PAGE and autoradiography (Fig. 2 B, lane4).
Figure 2:
SDS-PAGE analysis of recombinant vWF-A3
protein. Bacterial inclusion bodies and purified bacterial vWF-A3
protein were analyzed by SDS-PAGE (12.5%). A, the gel,
analyzed under reducing conditions, shows molecular mass markers
( lane1) and washed bacterial inclusion bodies
( lane2) from E. coli transformed with
pQE-vWF-A3 and induced with IPTG. B, lanes 1,
2, and 4 were analyzed under reducing conditions.
Lane1 shows molecular mass markers. Lane 2 shows vWF-A3 eluted from a Q-Sepharose column. Lane 3 shows vWF-A3 analyzed under nonreducing conditions. Lane 4 shows an autoradiograph of vWF-A3 metabolically labeled with
SO
.
Figure 3:
Ion-exchange chromatography of vWF-A3.
Following solubilization of inclusion bodies, vWF-A3 was purified using
a Q-Sepharose column equilibrated with 20 mM Tris-HCl, pH 8.2.
The bound protein was eluted with a gradient of NaCl. The eluted
protein was concentrated by ultrafiltration and dialyzed against TBS.
Abs, absorbance.
The calculated molecular mass for the sequence
between Ser-908 and Gly-1111 is 21,538 Da. The 10 additional amino
acids from the vector sequence add another 1254 Da, bringing the
estimated molecular mass to 22,792 Da. This is in good agreement with
the estimated molecular mass of the purified material of 27,000 Da
(Fig. 2 B, lane2). In addition,
purified vWF-A3 eluted from a Sephacryl 300 column with the
K of a globular monomeric 24-kDa protein (data
not shown). When purified vWF-A3 was analyzed by SDS-PAGE under
nonreducing conditions (Fig. 2 B, lane3), it migrated slightly faster than the reduced form,
suggesting a compact globular structure that is extended following
reduction of the single disulfide bond between Cys-923 and Cys-1109.
S-labeled vWF-A3 bound to
immobilized soluble type I collagen derived from bovine Achilles tendon
or calfskin collagen in a saturable and reversible manner. At the
highest concentration of added
S-labeled vWF-A3,
nonspecific binding accounted for <5% of total bound radioactivity.
The relevant binding parameters for vWF-A3 were derived by Scatchard
analysis of binding isotherms (Fig. 4, inset). The
Scatchard plot demonstrated a single class of binding sites with a
K
of 1.8 ± 0.3 µM. At
saturation, there were 300 fmol of vWF-A3 bound per µg of
immobilized collagen.
Figure 4:
Binding of S-labeled
recombinant vWF-A3 protein to collagen-coated microtiter wells. A final
concentration of 1.8 mg/ml acid-soluble bovine type I collagen was
added to microtiter wells in 20 mM sodium citrate buffer, pH
6.0, for 90 min at 37 °C. After washing with TBS to remove
nonadsorbed collagen, wells were blocked by the addition of 1% bovine
serum albumin for 60 min at room temperature. Increasing concentrations
of
S-labeled recombinant vWF-A3 were added to the wells
and incubated for 30 min at 37 °C. Wells were washed with TBS, and
bound radioactivity was removed with 1% SDS/TBS and counted.
Nonspecific binding, measured in parallel wells with the addition of a
40-fold excess of unlabeled vWF-A3 protein, was subtracted from each
point. The values shown represent specific binding from five separate
experiments. B, bound; F,
free.
Characterization of Chimeric vWF-A1/A3 Protein
The
purified vWF-A1/A3 chimera, when analyzed by SDS-PAGE under reducing
conditions, had a band of 32,000 Da. This is in good agreement with the
predicted molecular mass of 27,000 Da. The anticipated shift in
mobility between reduced and unreduced protein was also demonstrated,
suggesting formation of a disulfide bond between Cys-509 and Cys-1109
(data not shown). As shown in Fig. 5, both vWF-A1 and vWF-A1/A3
proteins inhibited ristocetin-induced platelet agglutination in a
dose-dependent manner. The IC for both proteins was
between 200 and 600 nM. This finding provides additional
evidence for proper folding of the chimeric protein and helps to
localize the GPIb/IX-binding domain to the first 98 residues of the vWF
A1 domain. In contrast, at concentrations up to 2 µM,
vWF-A3 did not inhibit ristocetin-induced platelet agglutination.
Figure 5:
Inhibition of ristocetin-induced platelet
agglutination by vWF-A1 and vWF-A1/A3. Increasing concentrations of
vWF-A1, vWF-A1/A3, and vWF-A3 proteins were incubated with 2
10
/ml Formalin-fixed platelets in the presence of 8
µg/ml purified vWF for 5 min at 37 °C. Agglutination was
initiated by the addition of 1 mg/ml ristocetin. Platelets were stirred
continuously at 1200 rpm at 37 °C, and the change in light
transmission was recorded. 100% is defined relative to the
agglutination of platelets without any recombinant proteins (control).
The IC
for both vWF-A1 and vWF-A1/A3 was similar
(200-600 nM). vWF-A3 did not inhibit ristocetin-induced
platelet agglutination at any concentration
tested.
Inhibition of
The ability of the three
recombinant proteins to block the binding of multimeric vWF to type I
collagen was then examined. Inhibition of S-Labeled Recombinant vWF
Binding to Type I Collagen
S-labeled vWF
multimer binding to immobilized type I collagen by unlabeled vWF,
vWF-A1, vWF-A3, and vWF-A1/A3 proteins is shown in Fig. 6.
Multimeric vWF, vWF-A3, and the vWF-A1/A3 chimera compete with
S-labeled vWF for binding to collagen, with varying
affinities. The IC
for multimeric vWF was 8 ± 2
nM using the molecular mass of its 275-kDa subunit to
calculate molarity. The IC
for vWF-A3 was 1.0 ± 0.5
µM, which is comparable to the previously determined
K
of 1.8 µM established by
direct binding assays. The IC
for vWF-A1/A3 was 0.5
± 0.3 µM, which is very similar to the IC
for vWF-A3. In contrast, we were unable to inhibit vWF binding to
collagen with up to 9 µM vWF-A1.
Figure 6:
Inhibition of S-labeled vWF
binding to type I collagen. The binding of
S-labeled vWF
(1-4 µg/ml) to immobilized acid-soluble type I collagen was
measured in the presence of purified unlabeled recombinant vWF, vWF-A3,
vWF-A1/A3, and vWF-A1 at various concentrations as indicated or the
same volume of TBS in the control mixture. 100% is defined as the
fraction of added
S-labeled vWF bound with no competing
ligand. The values shown represent four separate
experiments.
= 1.8 µM)
is substantially lower than the estimated K
for multimeric vWF
(12, 13, 17, 18) . This suggests that
other structural features within multimeric vWF may enhance binding and
that the sequences present in vWF-A3, while necessary, are not
sufficient for optimal binding. It is also possible that the presence
of multiple collagen-binding sites in each vWF multimer enhances the
overall affinity of multimeric vWF for collagen.
that was identical to
that of vWF-A1 (0.2-0.6 µM). This observation
confirms the importance of residues 474-488
(25) and
514-542
(38) for binding to GPIb/IX. The binding of the
chimera to collagen, however, conflicts with previous studies of Roth
et al.(17) , who reported that collagen-binding
sequences in vWF lie between amino acids 542 and 622 (vWF-A1) and
between amino acids 948 and 998 (vWF-A3). Although the chimera contains
a portion of the reported vWF-A1 collagen-binding sequence (amino acids
542-598), its relevance to collagen binding is unclear since the
native vWF A1 domain does not bind to type I collagen. In contrast, the
collagen-binding sequence in vWF-A3 originally proposed by Roth et
al. (residues 948-998) is not present in the chimera.
However, the observation that the fusion of residues 1018-1114
from vWF-A3 to a vWF-A1 sequence that previously did not interact with
collagen reconstitutes collagen-binding activity is fairly strong
evidence that this sequence contains the collagenbinding site.
-D-thiogalactopyranoside; TBS, Tris-buffered
saline; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.