From the
At sites of vascular injury, von Willebrand factor (VWF)
mediates platelet adhesion through binding to platelet glycoprotein Ib
(GPIb). The VWF-GPIb interaction was investigated by clustered
charged-to-alanine scanning mutagenesis of VWF domain A1 between
His-473 and Gly-716. Recombinant variants of VWF were assayed for
binding to conformation-dependent monoclonal antibody NMC-4, for
ristocetin-induced and botrocetin-induced binding to platelets, and for
direct binding to botrocetin. Substitutions at 32 amino acids had no
effect on VWF function. The epitope of NMC-4 depended on charged
residues between Asp-514 and Arg-632 and not on segments previously
implicated by peptide inhibition studies, Cys-474-Pro-488 and
Leu-694-Pro-708. Substitutions at Glu-626 and in the segment
Asp-520-Lys-534 abolished ristocetin-induced binding of VWF to
GPIb but did not affect botrocetin-induced binding, suggesting that
these regions are required for modulation by ristocetin but not for
binding of VWF to GPIb. Mutations at Glu-596 and Lys-599 decreased
binding of VWF to GPIb without affecting its binding to botrocetin,
suggesting that this segment interacts directly with GPIb. Alanine
substitutions at Arg-545 and in the segments Glu-497-Arg-511 and
Arg-687-Glu-689 caused increased binding of VWF to GPIb. These
results, and the locations of von Willebrand disease type 2B mutations,
suggest that two acidic regions containing the Cys-509-Cys-695
disulfide (Glu-497-Arg-511, Arg-687-Val-698) and one
predominantly basic region (Met-540-Arg-578) cooperate to inhibit
a distinct GPIb binding site in the VWF A1 domain. This inhibition is
relieved by specific mutations, by the modulators ristocetin and
botrocetin, or by binding to subendothelial connective tissue.
von Willebrand factor (VWF)
Binding of VWF to GPIb in vitro can be induced by the antibiotic ristocetin or by the snake venom
protein botrocetin. Ristocetin apparently can bind both to platelets
and to VWF
(4) , whereas botrocetin binds to VWF but not to
GPIb
(5) . The precise mechanism by which these agents promote
VWF-GPIb interaction is not known.
The binding site on VWF for
platelet GPIb corresponds approximately to the first of three repeated
A domains in the VWF subunit. Domain A1 extends from Glu-497 to Gly-716
and contains an intrachain disulfide loop that is defined by the
disulfide bond Cys-509-Cys-695
(6, 7) . A
proteolytic fragment of VWF that contains residues Leu-480/Val-481 to
Gly-718
(7) binds directly to platelet GPIb. This binding is
stimulated by ristocetin or botrocetin
(8) , suggesting that at
least some structures required to regulate the VWF-GPIb binding
interaction are contained in this proteolytic fragment.
The VWF
domain that binds to GPIb has been studied by using peptide inhibitors
of VWF function
(9, 10, 11) , by mapping the
epitopes of inhibitory monoclonal antibodies
(9, 12) ,
and by deletion mutagenesis (13). The results suggest that several
discontinuous segments of VWF domain A1 interact with GPIb, ristocetin,
and botrocetin. However, none of these approaches has sufficient
resolution to identify single amino acid residues that are necessary
for VWF function. The low potency of most synthetic peptide inhibitors
also raises concerns about their specificity and mechanism of action.
Scanning mutagenesis has proved to be a powerful method for the
accurate high resolution mapping of protein interaction
sites
(14, 15) . In this study, we have employed
charged-to-alanine scanning mutagenesis to define functional amino acid
residues within the A1 domain of human VWF. The results implicate
residues of the A1 domain not previously known to participate in the
positive and negative regulation of VWF binding to platelet GPIb.
Botrocetin-induced binding of VWF to platelets was assayed
essentially as previously described
(19) . Each reaction mixture
(25 µl) contained 570 ng/ml rVWF, 0.2% BSA and various
concentrations (0-20 µg/ml) of botrocetin in TBS. After
incubation for 30 min at room temperature, the reaction mixtures were
centrifuged, and VWF in the supernatant was measured by ELISA. Control
assays were performed in the absence of platelets.
With two
exceptions, the mutant rVWF proteins did not bind spontaneously to
platelets, and the results of platelet binding assays are expressed as
the percentage of unbound VWF antigen relative to the value obtained
with platelets but without ristocetin or botrocetin. In the case of the
variant R545A in both assays and the variant (687-689)3A in the
botrocetin-induced platelet binding assay, spontaneous binding to
platelets occurred, and the values obtained in the presence of
modulators were compared with those obtained without platelets or
modulators.
To summarize binding data in graphical form, values
obtained for each mutant at specific concentrations of ristocetin or
botrocetin were normalized to the corresponding values obtained for
wild type rVWF as follows.
On-line formulae not verified for accuracy
The binding of botrocetin to rVWF variants was determined at a fixed
concentration of 3 µg/ml
The contact sites between proteins can be shown directly by
structural methods such as x-ray crystallography, although structure
alone cannot indicate the functional importance of a specific contact.
At this time, no three-dimensional structures are available for VWF or
GPIb. Mutagenesis provides an indirect approach to define contacts and
also can define the energetic contribution of individual amino acids to
protein-protein interactions
(31) .
A clustered
charged-to-alanine scanning strategy
(14, 15) was chosen
for the production and analysis of mutant VWF. The rationale underlying
this approach has been reviewed
(15) . In particular, alanine was
selected as the replacement residue because it is the most common amino
acid in proteins, is compatible with all types of secondary structures,
and does not impose new structural effects related to hydrogen bonding,
unusual hydrophobicity, or steric bulk
(15) . Charged amino acids
usually are at or near the protein surface. For this reason,
charged-to-alanine substitution generally does not interfere with the
packing of buried residues and disrupt the structural integrity or
expression of the protein
(15) . Each of these critical points
has been confirmed during the successful application of this strategy
to study the binding interactions of many proteins
(15) .
Recombinant human VWF proved to be quite tolerant to mutations
within domain A1. Among 34 different constructs covering 68 charged
residues, only one was not secreted. Deletion of the entire A1 domain
also is compatible with the secretion of VWF multimers that retain the
ability to bind collagen and factor VIII
(32) . These results
suggest that domain A1, containing the GPIb binding site, is
structurally independent of the remainder of the VWF subunit.
Peptide inhibition studies appear to implicate segments
Cys-474-Pro-488 and Leu-694-Pro-708 of VWF in the
interaction with antibody NMC-4
(9) , but recent deletion
mutagenesis experiments do not support this proposal. A rVWF fragment
that lacked all residues amino-terminal to Tyr-508 still reacted
strongly with NMC-4; another fragment that lacked all residues
carboxyl-terminal to Glu-700 also retained strong reactivity with
NMC-4
(13) . These results suggest that the NMC-4 epitope does
not contain most of the residues in the synthetic peptides
Cys-474-Pro-488 and Lys-694-Pro-708. Further study will be
required to define the mechanism by which these peptides inhibit NMC-4
binding to VWF.
Since GPIb and modulators
(ristocetin or botrocetin) can bind simultaneously to VWF, a strong
correlation between loss of binding to GPIb and to modulators was not
expected. These results suggest several explanations. Mutations that
cause nonselective loss-of-function could grossly disrupt the folding
of domain A1 and impair the binding of all of these macromolecules.
However, reduced and alkylated fragments of VWF will bind normally to
GPIb in the presence of botrocetin at concentrations (5 µg/ml) that
were employed in these studies
(13, 30) , indicating that
little secondary structure is required. Alternatively, botrocetin
binding and GPIb binding may be conformationally linked, as suggested
by quantitative binding studies (30). In that case, mutations at either
the GPIb or botrocetin binding site could impair binding to both
ligands. More than one residue was altered in five of these
loss-of-function constructs. Therefore, further mutagenesis will be
required to determine which amino acids are necessary for normal
function and whether modification of single amino acids can dissociate
the effects on botrocetin binding and GPIb binding.
Peptide inhibition studies have suggested that
botrocetin binds to VWF through discontinuous sites that are
represented in three synthetic peptides: D539-V553, K569-Q583, and
R629-K643
(10) . The results of scanning mutagenesis are
consistent with the participation of residues in the segments
Asp-539-Val-553 and Arg-629-Lys-643 but do not support a
role for the segment Lys-569-Gln-583.
The strongest support
for the participation of specific residues in botrocetin binding is
provided by the two constructs, R636A and (663-667)3A, that
exhibited normal ristocetin-induced binding but defective
botrocetin-induced binding to GPIb. The loss of botrocetin-induced GPIb
binding correlated with decreased direct binding to botrocetin. The
preservation of ristocetin-induced binding indicates that the GPIb
binding site is intact. Therefore, these mutations identify amino acid
residues that are required specifically for botrocetin binding and may
contribute to the binding site.
Peptide inhibition experiments suggested that segments of domain A1
outside the Cys-509-Cys-695 loop interact directly with
GPIb
(9) , but subsequent studies do not support this
interpretation. Synthetic peptides corresponding to VWF residues
Cys-474-Pro-488 and Leu-694-Pro-708 blocked
ristocetin-induced binding of VWF to platelets; they also inhibited the
binding of asialo-VWF to platelets in the absence of
modulators
(9) . These peptides did not inhibit
botrocetin-induced binding of VWF to GPIb
(12, 35) ,
however, suggesting that they do not interact with GPIb but may instead
interact with ristocetin. Proteins that bind ristocetin often are rich
in proline, and a proline-containing motif in the Cys-474-Pro-488
peptide was proposed to be a ristocetin binding site
(4) . This
conclusion was supported by ultraviolet difference spectroscopy;
peptides Cys-474-Pro-488 and Leu-694-Pro-708 and other
unrelated proline-rich peptides were shown to bind directly to
ristocetin
(11) . A role for proline residues in the binding of
ristocetin also is supported by mutagenesis of VWF. Substitution of
proline residues 702-704 either by aspartate or by arginine
abolished ristocetin-induced binding to GPIb but had no significant
effect on the spontaneous binding of the reduced recombinant
proteins
(36) . Finally, recombinant VWF fragments that lack the
Cys-474-Pro-488 and Leu-694-Pro-708 peptide segments bind
to GPIb with high affinity
(13) . Thus, the VWF sequences
represented in peptides Cys-474-Pro-488 and Leu-694-Pro-708
are dispensable for binding to platelet GPIb but may associate with
ristocetin.
A similar peptide inhibition study suggests that VWF
residues Asp-514-Glu-542 contribute to the binding site for
GPIb
(11) . A synthetic peptide with this sequence inhibited
ristocetin-induced and botrocetin-induced binding of VWF to GPIb, and
it inhibited the agglutination of platelets by asialo-human VWF or
bovine VWF. However, this peptide also inhibited the direct binding of
botrocetin to VWF (11); whether it binds to botrocetin, VWF, or GPIb or
to more than one site has not been determined.
The results of scanning mutagenesis reinforce this
concept and identify additional residues that inhibit the binding of
VWF to GPIb. Constructs (497-501)3A,(505-506)2A, R511A,
R545A, and(687-689)3A had increased affinity for GPIb. Three of
these constructs affect residues within segments that were identified
previously by naturally occurring mutations in VWD type 2B, but
constructs R511A and(687-689)3A mark sites in which no type 2B
mutations have been observed. These sites are within the
Cys-509-Cys-695 disulfide loop and adjacent to either cysteine
residue.
These data also suggest a speculative but
testable model for the regulation of the GPIb-VWF interaction
(Fig. 8). As defined by mutagenesis and the location of natural
VWD type 2B mutations, residues within three VWF segments cooperate to
inhibit the binding of VWF to GPIb: Glu-497-Arg-511,
Arg-687-Val-698, and Met-540-Arg-578. The first two
segments are acidic, whereas the last contains many basic residues. The
GPIb binding site itself may be located in a second region that
contains amino acids from several discontinuous segments, including
Glu-596-Arg-616, Arg-629-Arg-632, and
Lys-642-Lys-645; these segments contain many basic amino acid
residues. Inhibition of binding is relieved by certain mutations, by
the modulators ristocetin and botrocetin, or by binding to
subendothelial connective tissue. The striking distribution of positive
and negative charges in distinct regions suggests that intramolecular
electrostatic interactions among these sites play a major role in the
regulation of VWF binding to GPIb. An acidic segment
Asp-252-Asp-287 of the GPIb
We thank Lisa Westfield for preparing
oligonucleotides, Dr. David Ginsburg (University of Michigan) for
providing 293T cells, Dr. Claudine Mazurier (CRTS, Lille, France) for
providing monoclonal antibody 33E12, Dr. Barry Coller (Mt. Sinai
Medical Center, NY) for providing monoclonal antibody 6D1, Dr. Midori
Shima (Nara Medical College, Japan) for generously providing monoclonal
antibody NMC-4, and Dr. Yoshihiro Fujimura (Nara Medical College,
Japan) for his gift of highly purified botrocetin. We also thank Dr.
Zhengyu Dong for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a
multimeric glycoprotein that plays an important role in primary
hemostasis. It circulates in the blood as disulfide-linked multimers
that are assembled from subunits of
250 kDa. The VWF multimers
range in size from dimers of
500 kDa to >10,000 kDa. VWF does
not bind spontaneously to platelets in blood but promotes thrombus
formation by mediating platelet adhesion at sites of vascular injury.
This activation of adhesive properties is induced in vivo upon
the binding of VWF to subendothelial connective tissue, particularly
under the conditions of high shear stress that occur in the
microcirculation. Activated VWF binds to the
chain of platelet
glycoprotein Ib (GPIb)
(1, 2, 3) . This
interaction results in platelet adhesion, followed by platelet
activation and aggregation.
Materials
Restriction enzymes were
obtained from New England BioLabs (Beverly, MA). Taq DNA
polymerase was from Perkin Elmer Corp. Highly purified two-chain
botrocetin was provided by Dr. Yoshihiro Fujimura (Nara Medical
College, Japan). Monoclonal antibody 6D1 against human platelet GPIb
(16) was provided by Dr. Barry Coller (Mt. Sinai Medical Center,
NY). Monoclonal antibody NMC-4 against the GPIb binding domain of human
VWF
(9, 12, 17) was provided as ascites fluid by
Dr. Midori Shima (Nara Medical College, Japan). Anti-VWF monoclonal
antibody 33E12
(18) was provided by Dr. Claudine Mazurier (CRTS,
Lille, France).
Plasmid Constructs
The strategy for
mutagenesis was similar to that previously described
(19) .
Expression plasmid pSVHVWF1
(20, 21) contains a unique
NgoMI site at nucleotide 3608 and two KpnI sites at
nucleotides 298 and 4748 (relative to the initiation codon) in the
full-length coding sequence of human VWF. The first KpnI site
was mutated by changing the codon for G100 from GGT to GGA, generating
the plasmid pSVHVWF1.1. Plasmid pGEM-4ZNK
(19) contains the
1140-base pair NgoMI-KpnI human VWF cDNA fragment
that encodes amino acid residues 442-821 of the mature subunit;
this fragment was cloned into the NgoMI and KpnI
sites of plasmid pGEM-4ZNae
(19) (NgoMI is an
isoschizomer of NaeI but leaves a 3`-overhang). DNA fragments
containing each of 33 mutations were produced by
oligonucleotide-directed mutagenesis using a polymerase chain reaction
method essentially as previously described (19) except for the choice
of mutagenic primers. Mutated NgoMI-KpnI fragments
were recloned into pGEM-4ZNae, and the entire sequence was confirmed by
dideoxy sequencing (Sequenase 2.0, U. S. Biochemical Corp.). The
mutated NgoMI-KpnI fragments then were cloned into
pSVHVWF1.1.
Expression and Characterization of Recombinant
VWF
Human 293T cells
(22) were kindly provided by
Dr. D. Ginsburg (University of Michigan, Ann Arbor, MI) and were grown
in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum (Life Technologies,
Inc.). Cells were transfected by a calcium-phosphate
method
(23) . In mock transfections, vector pSV7D DNA
(24) was used. 24 h after transfection, cells were washed once
with phosphated-buffered saline (PBS) and then incubated with
serum-free medium (Optimem-1, Life Technologies, Inc.). Recombinant VWF
(rVWF) secreted in the medium was harvested 48 h later and concentrated
using Centriprep-30 and Centricon-100 devices (Amicon, Beverly, MA).
The VWF antigen was measured by an ELISA assay using polyclonal rabbit
anti-human VWF antibody 082 and peroxidase-conjugated rabbit anti-human
VWF antibody P226 (DAKO, Carpinteria, CA)
(25) . Multimer
analysis was performed as described
(26) . Briefly, samples
(<40 µl) of concentrated medium containing 70 ng of VWF were
subjected to SDS-1.5% agarose gel electrophoresis and blotted onto
polyvinylidene difluoride membranes (Millipore). The VWF was detected
with polyclonal rabbit anti-human VWF 082 (DAKO) and the Vectastain ABC
kit (Vector Laboratories, Burlingame, CA).
Epitope Mapping of Antibody
NMC-4
Monoclonal antibody NMC-4 was purified from ascites
fluid by chromatography on recombinant protein A-agarose (RepliGen,
Cambridge, MA)
(27) . The binding of NMC-4 to rVWF was assayed by
ELISA as described above
(25) except that microtiter ELISA
plates with U-shaped bottoms (Coster, Cambridge, MA) were coated for 24
h at 4 °C with 25 µl of NMC-4, 7.5 µg/ml, in 0.1 M
sodium carbonate, pH 9.6. The wells were washed with PBS containing
0.1% Tween 20 and then incubated for 105 min at room temperature with
15 µl of various concentrations of wild type or mutant rVWF diluted
in PBS containing 3% BSA. The wells were washed again and incubated for
105 min at room temperature with 20 µl of peroxidase-conjugated
rabbit polyclonal anti-human VWF P226 (DAKO) diluted 1:5000 in PBS
containing 3% BSA. The plates were washed and incubated for 7 min with
100 µl of o-phenylenediamine solution (Sigma). The
reaction was stopped by adding 3 M sulfuric acid, and
absorbance at 490 nm was determined. Control assays performed with
concentrated conditioned media from mock-transfected 293T cells gave
absorbance values of zero.
Platelet Binding
Assays
Ristocetin-induced binding of VWF to platelets was
assayed as described
(20) . Briefly, the total reaction volume
(100 µl) contained 500 ng/ml rVWF, 2 10
/ml
human lyophilized platelets (Biodata, Hatboro, PA), 4% BSA (Sigma), and
various concentrations (0-1.5 mg/ml) of ristocetin (Helena
Laboratories, Beaumont, TX) in Tris-buffered saline (TBS, 50
mM Tris-HCl, pH 7.5, 150 mM NaCl). The reaction
mixtures were incubated for 30 min at room temperature and centrifuged
for 10 min at 10,000
g. The VWF antigen present in the
supernatant was measured by ELISA. Reaction mixtures without platelets
were tested simultaneously to verify the absence of nonspecific VWF
flocculation and sedimentation in the presence of ristocetin.
Radiolabeled Botrocetin Binding
Assay
Botrocetin binding to VWF was assayed according to
Fujimura et al.(28) with some modifications.
Botrocetin (60 µg) was radioiodinated with I using a
chloramine-T method
(29) to a specific radioactivity of
2.5
10
cpm/µg, and the concentration of botrocetin
was adjusted to 3 µg/ml in TBS containing 1% BSA. Anti-VWF
monoclonal antibody 33E12
(18) binds to the carboxyl-terminal
region of the VWF subunit and has no effect on VWF binding to platelets
in the presence of either ristocetin or botrocetin. The concentration
of each mutant rVWF was determined by ELISA, with antibody 33E12 as the
first antibody, and adjusted to 5 µg/ml with TBS. Polystyrene
microtiter plates with eight strips (Coster) were coated for 16 h at 4
°C with 100 µl of antibody 33E12 (10 µg/ml). After coating,
the wells were washed three times with TBS, blocked with 3% BSA in TBS
for 2 h at room temperature, and incubated with 25 µl of rVWF (5
µg/ml) for 3 h at room temperature. The wells were washed four
times with TBS containing 0.1% Tween 20 (TBS-Tween), and radiolabeled
botrocetin solution (5 µl,
35,000 cpm) was added for 30 min at
room temperature. The wells were washed rapidly five times with
TBS-Tween; the total time of washing wells was <1 min. After drying,
each well was cut out, and the bound radioactivity was measured by
spectroscopy. Nonspecific binding was obtained by testing
concentrated supernatant from mock-transfected cells in the same assay
system and was
1,400 cpm. Binding to wild type rVWF was
7,500
cpm. Specific binding for each mutant rVWF was calculated by
subtracting nonspecific from total binding and normalized to the value
obtained for wild type rVWF.
Design, Construction, and Expression of Human
Recombinant VWF Variants
The segment of VWF that was
targeted for mutagenesis consists of 254 amino acid residues
between His-463 and Gly-716. This segment contains domain A1 and part
of VWF domain D3 (Fig. 1) and encompasses a fragment produced by
digestion of VWF with dispase (Leu-480/Val-481-Gly-718); this
fragment binds GPIb in a ristocetin-dependent or botrocetin-dependent
manner
(7, 8) . To simplify the production and analysis
of mutant constructs, a clustered charged-to-alanine scanning strategy
was chosen
(14, 15) . All 68 charged amino acids
including arginine, lysine, aspartate, glutamate, and histidine were
changed singly or in small clusters to alanine. The 68 charged residues
in the target sequence were covered in a total of 33 constructs
(Fig. 1). For convenience the variant proteins were named
according to the residue number of the mutated amino acid in the mature
VWF subunit. If more than one charged amino acid was mutated, the range
of residue numbers and the number of alanine substitutions is
indicated. For example, in construct R545A, one arginine at position
545 was changed to alanine; in construct(557-563)4A, the four
residues Glu-557, His-559, Asp-560, and His-563 were changed to
alanine. To facilitate the mutagenesis procedure, the expression
construct pSVHVWF1 was modified by eliminating the KpnI site
at codon 99 of the VWF cDNA sequence. The resultant plasmid pSVHVWF1.1
has unique KpnI and NgoMI sites that facilitated the
mutagenesis of amino acids 442-821 by a cassette replacement
method.
Figure 1:
Amino acid residues of human VWF
targeted for clustered charged-to-alanine scanning mutagenesis. The
target VWF segment contains a part of domain D3 (463-496) and
entire A1 domain (497-716). Segments that contain mutations in
each variant protein are indicated by shading, and the mutated
charged residues are shown in boldcharacters. Note
that the segment mutated in variant (527-531)3A is divided
between two lines. The positions of two single residue mutant
constructs, E527A and R632A, are indicated by underlinesbelowshadedsegments. Above the sequence, solidarrows indicate the
two segments (474-488 and 694-708) reported to mediate
ristocetin-induced platelet binding (9) and three segments
(539-553, 569-583, 629-643) reported to bind
botrocetin (10). Below the sequence, a dashedarrow marks another segment (514-542) proposed to
interact with GPIb (11), and mutations in VWD type 2B are shown. The
mutation V551F was identified originally as a VWD type 2A mutation, but
a recent study indicates that it causes a type 2B phenotype
(42).
Human kidney 293T cells were transfected with each
construct, and serum-free media were analyzed for the expression of
mutant human rVWF. Initially, a construct containing the two mutations
R511A and D514A was not detectably secreted; therefore, the independent
mutants R511A and D514A were constructed and analyzed instead. All
other mutant constructs were expressed and secreted efficiently. By
ELISA, the level of expression of the variant rVWF proteins was 66
± 23% (S.D.) of wild type rVWF. The rVWF was concentrated and
subjected to multimer analysis using SDS-agarose gel electrophoresis
without protein reduction. The multimer distribution of all the mutant
proteins was similar to that of wild type rVWF and plasma VWF
(Fig. 2). In every case, at least 12 multimer bands could be
detected.
Figure 2:
Multimer analysis of secreted recombinant
VWF variants. Samples (70 ng) of rVWF were analyzed by SDS, 1.5%
agarose gel electrophoresis as described under ``Experimental
Procedures.'' The abbreviated name of each variant is indicated
above the corresponding
lane.
The functional properties of the mutant VWF proteins were
studied in four assays that depend on the GPIb binding domain. Antibody
NMC-4 recognizes a conformation-dependent epitope within the A1 domain
of VWF
(9, 12, 17) . Reactivity with NMC-4
therefore was determined to provide an index of the structural
integrity of the A1 domain of rVWF, and decreases in reactivity were
used to localize the epitope of NMC-4. The ability to bind to platelet
GPIb was assessed in the presence of ristocetin or botrocetin. In
addition, the direct binding of radiolabeled botrocetin to each mutant
rVWF protein was determined.
Epitope Mapping of Monoclonal Antibody
NMC-4
Plasma VWF and wild type rVWF bound to NMC-4 with
similar concentration dependence (Fig. 3A). Wild type
rVWF appeared to bind slightly more avidly than plasma VWF; the cause
of this apparent minor difference is not known but might be due to
subtle differences in glycosylation or multimer structure or to
proteolytic degradation of plasma VWF. The binding data for the mutant
rVWF proteins are summarized in Fig. 3B. Compared to
wild type rVWF, 26 mutant rVWF proteins bound normally to NMC-4. Seven
mutant rVWF proteins exhibited 50% of normal binding to NMC-4:
D514A,(520-524)2A,(549-552)2A,(608-611)3A,(613-616)2A,
(629-632)2A, and R632A. These results suggest that amino acids in
at least three discontinuous segments of domain A1 contribute directly
to the epitope for NMC-4 or are required indirectly for its
conformation.
Figure 3:
Binding of VWF to anti-human VWF
monoclonal antibody NMC-4. Antibody NMC-4 was immobilized in microtiter
plates, and binding of VWF variants was determined as described under
``Experimental Procedures.'' Panel A, dose response
to plasma VWF (open circles) and wild type rVWF (closed
circles). Nonspecific binding (open squares) was
determined with concentrated conditioned medium from mock-transfected
293T cells and was undetectable. Panel B, binding of rVWF
variants to NMC-4 was determined at a fixed concentration of rVWF (500
ng/ml) and normalized to the value obtained for wild type
rVWF.
Ristocetin-induced Binding of rVWF to
Platelets
The conditions for ristocetin-induced binding of
rVWF to platelets were optimized for wild type rVWF. In the absence of
platelets, addition of ristocetin (1.5 mg/ml) directly to wild type
rVWF (500 ng/ml) in conditioned medium was associated with
precipitation and sedimentation of the VWF during centrifugation. This
nonspecific aggregation was prevented by addition of 4% BSA (data not
shown). Under these conditions, the binding of wild type rVWF to
formalin-fixed human platelets increased as a function of ristocetin
concentration, reaching a maximum of 40% binding at 1.5 mg/ml
ristocetin (Fig. 4). Binding was blocked by monoclonal antibody
6D1 to platelet GPIb
(16) (data not shown) as previously
reported
(20) .
Figure 4:
Binding
of VWF to platelets with increasing concentrations of ristocetin. Each
rVWF variant (rVWF) was incubated at a concentration of 500 ng/ml with
2 10
/ml human lyophilized platelets and ristocetin
as described under ``Experimental Procedures.'' After 30 min
at room temperature, the samples were centrifuged, and VWF antigen
present in the supernatant was measured by ELISA. Platelet binding is
expressed as the percentage of unbound VWF antigen compared with the
values obtained with no ristocetin (A-C)or
with neither ristocetin nor platelets (D). In each
panel, VWF binding to platelets is shown for one mutant rVWF
(closed circles) and for wild type rVWF (open
circles) assayed at the same time. The mutant proteins are
(671-673)2A (panel A), (629-632)2A (panel
B), (687-689)3A (panel C), and R545A (panel
D). Each data point represents the mean ± S.D. of values
obtained in at least two independent sets of duplicate
assays.
Ristocetin-induced binding to platelets was
determined for each mutant rVWF at several concentrations of
ristocetin.(
)
The dose response for wild type
rVWF was determined simultaneously to control for variations among
platelet preparations. Normal and loss-of-function phenotypes were
distinguished clearly at the higher ristocetin concentrations. A total
of 16 mutant constructs, representing 36 charged amino acid residues,
had essentially normal ristocetin-induced binding to platelets. For
another 12 constructs, containing mutations at 23 charged residues,
little or no ristocetin-induced binding was observed. Representative
results for several mutant rVWF proteins with these phenotypes are
shown in Fig. 4B. The results for variants with normal
and decreased binding can be summarized satisfactorily by histograms of
the values obtained at 1.0 and 1.5 mg/ml ristocetin, normalized to wild
type rVWF (Fig. 5).
Figure 5:
Histogram of ristocetin-induced binding of
VWF to platelets. The binding assay was performed as described in the
legend to Fig. 4. The value for each mutant rVWF is expressed relative
to that for wild type rVWF performed at the same time. Binding to
platelets was determined in the presence of 1.5 mg/ml ristocetin
(closed column) and 1.0 mg/ml ristocetin (hatched
column). Each column represents the mean ± S.D. of
values obtained in at least two independent sets of duplicate
assays.
For 5 constructs, representing 10 charged
residues, a gain-of-function phenotype was demonstrated most clearly at
a low ristocetin concentration of 0.5 mg/ml (Fig. 4, C and D). These constructs
were(497-501)3A,(505-506)2A, R511A, R545A, and
(687-689)3A. One variant, R545A, bound spontaneously to platelets
in the absence of ristocetin (Fig. 4D). For this group
of mutant proteins, the increase in binding also was evident at higher
ristocetin concentrations, although it was less dramatic
(Fig. 5).
Botrocetin-induced Binding of rVWF to
Platelets
The conditions selected for botrocetin-induced
binding of rVWF to platelets are similar to those for
ristocetin-induced binding, except that high concentrations of BSA were
not required to prevent platelet-independent precipitation of VWF.
Representative dose responses to botrocetin are shown in
Fig. 6
for wild type rVWF and for the mutant rVWF proteins that
were assayed for ristocetin-induced binding in Fig. 4. Two
variants exhibited spontaneous binding. Approximately 30% of mutant
R545A sedimented with platelets in the absence of botrocetin
(Fig. 6D); this variant also exhibited spontaneous
binding to platelets in the ristocetin-dependent assay
(Fig. 4D). Approximately 27% of mutant(687-689)3A
bound spontaneously to platelets under the botrocetin-dependent assay
conditions, but none bound spontaneously under the ristocetin-dependent
assay conditions. This difference appears to depend on the BSA
concentration in the respective assays (data not shown).
Figure 6:
Binding of VWF to platelets with
increasing concentrations of botrocetin. Highly purified two-chain
botrocetin was incubated with 570 ng/ml rVWF and 2
10
/ml lyophilized human platelets as described under
``Experimental Procedures.'' After 30 min at room
temperature, the samples were centrifuged, and VWF antigen in the
supernatant was measured by ELISA. Binding is expressed as the
percentage of unbound VWF compared with the values obtained with no
botrocetin (A, B) or with neither botrocetin nor platelets
(C, D). In each panel, VWF binding to platelets is
shown for one mutant rVWF (closed circles) and for wild type
rVWF (open circles) assayed at the same time. The mutant
proteins are (671-673)2A (panel A), (629-632)2A
(panel B), (687-689)3A (panel C), and R545A
(panel D). Each data point represents the mean ± S.D.
of values obtained in at least two independent sets of duplicate
assays.
Half
maximal binding of wild type rVWF occurred at 3 µg/ml
botrocetin (Fig. 6). The binding of the mutant proteins at this
fixed concentration of botrocetin is summarized in Fig. 7. With
few exceptions, mutant rVWF proteins behaved similarly in both the
ristocetin-dependent and botrocetin-dependent binding assays. A total
of 18 constructs, encompassing 39 charged residues, exhibited normal
botrocetin-induced binding. Interestingly, four constructs had normal
botrocetin-induced binding but no ristocetin-induced
binding:(520-524)2A,(527-531)3A, K534A, and E626A. Ten
constructs, including 20 amino acids, exhibited decreased
botrocetin-induced binding. Only two of these variants with decreased
botrocetin-induced binding had normal ristocetin-induced binding: R636A
and(663-667)3A. Two constructs with normal botrocetin-induced
binding exhibited increased ristocetin-induced binding to platelets:
(505-506)2A and R511A.
Figure 7:
Histogram of botrocetin-induced binding of
VWF to platelets and direct binding of radiolabeled botrocetin to VWF.
Botrocetin-induced platelet binding (closed columns) was
measured at 3 µg/ml botrocetin as described in the legend to Fig.
6. Direct binding of radiolabeled botrocetin to rVWF (hatched
columns) was measured as described under ``Experimental
Procedures.'' Each column represents the mean ±
S.D. of values obtained in at least two independent duplicated
assays.
Binding of Botrocetin to rVWF
Decreased
botrocetin-induced platelet binding could be the result of mutations
that affect the interaction of VWF with either botrocetin or GPIb;
these two binding properties also could be linked conformationally
(30). To address this point, the binding of radiolabeled botrocetin to
rVWF mutants was measured directly as described by Fujimura et
al.(28) with minor modifications. In this assay,
I-botrocetin (two chain) bound to wild type rVWF with an
EC
of
3.0 µg/ml (data not shown). For comparison,
an EC
of
1.7 µg/ml was determined for botrocetin
binding to purified human plasma VWF in a similar assay
(28) .
I-botrocetin (Fig. 7).
With one exception, decreased binding of botrocetin to rVWF correlated
with decreased botrocetin-induced binding of rVWF to platelets. For
mutant (596-599)2A, direct botrocetin binding was normal, but
botrocetin-induced binding to platelets was decreased.
Localization of the NMC-4 Epitope
Recognition
of VWF by monoclonal antibody NMC-4 is decreased markedly by reduction
and alkylation of the Cys-509-Cys-695 disulfide bond or by
proteolytic excision of the sequences between Leu-512 and
Lys-674
(9, 12, 13, 33) , so that normal
reactivity with NMC-4 indicates the normal folding of a complex epitope
in VWF domain A1. In the present study, all of the rVWF proteins with
decreased binding to NMC-4 had mutations between Leu-512 and Lys-674.
Three mutant rVWF proteins had 60% of normal NMC-4 binding; another
two had
35% of normal NMC-4 binding (Fig. 3). These results
suggest that the functional epitope of NMC-4 contains amino acid
residues in the segments Asp-514-Arg-524, Lys-549-Arg-552,
Lys-608-Arg-616, and Arg-629-Arg-632. Several of these
segments are widely separated in the primary sequence of VWF and may be
held in proximity by the Cys-509-Cys-695 disulfide bond; if so,
this would explain the effect of reduction on the recognition of VWF by
NMC-4.
Normal and Nonselective Loss-of-Function
Phenotypes
Mutations of 32 different charged amino acid
residues did not significantly change the properties of VWF in any of
the assays tested, suggesting that the corresponding amino acid side
chains do not participate in the regulation of VWF binding to
platelets. Mutations of an additional 14 residues markedly reduced
modulator-induced binding to platelets and direct binding to
botrocetin. Five loss-of-function constructs contain mutations between
Lys-608 and Lys-645; two other constructs, D514A and(549-552)2A,
also exhibited this phenotype. These results implicate three
discontinuous segments of domain A1 in binding to botrocetin and in
ristocetin-induced binding to GPIb.
Selective Loss of Modulation by Ristocetin or
Botrocetin
Four constructs with mutations at a total of
seven charged residues exhibited a selective loss of ristocetin-induced
binding to platelets:(520-524)2A,(527-531)3A, K534A, and
E626A. The single mutant E527A had normal ristocetin-induced binding,
so the abnormal phenotype of(527-531)3A may be due to mutation of
Glu-529 or Glu-531, or both. Botrocetin-induced binding to GPIb was
normal for all four of these constructs, so the mutated amino acid
residues probably are not part of the GPIb binding site. Instead, these
mutations may identify a specific ristocetin modulator site. This
phenotype is similar to that of the naturally occurring VWF mutation
G561S, which was identified in a patient with VWD and severe
bleeding
(34) .
GPIb Binding
Sites
Construct(596-599)2A exhibited normal binding
to botrocetin but defective modulator-induced binding to GPIb. Thus,
Glu-596 or Lys-599 may bind directly to GPIb. Mutations at nearby
residues also impaired VWF binding to GPIb (Fig. 5), suggesting
that the GPIb binding site may require several amino acid side chains
in this predominantly basic segment of domain A1. All mutations that
decreased the binding of VWF to GPIb are located within the
Cys-509-Cys-695 disulfide loop, and they overlap with the sets of
mutations that decreased binding to botrocetin or to antibody NMC-4.
Inhibition of Binding to GPIb
Type 2B VWD
is a relatively rare subtype of VWD that is characterized by increased
affinity of the mutant VWF for platelet GPIb
(37) . The natural
mutations that cause this phenotype all are in three major clusters
within domain A1: amino-terminal to Cys-509 (e.g. P503L,
H505D), between Met-540 and Arg-578, and carboxyl-terminal to Cys-695
(e.g. L697V, A698V)
(38, 39) . These
observations suggest that the segments affected by VWD type 2B
mutations normally inhibit the binding of VWF to GPIb; type 2B
mutations relieve this inhibition, causing VWF to bind GPIb
constitutively.
Model for Binding of VWF to GPIb
The
mutagenesis results and the interactions that have been reported among
antibody NMC-4, botrocetin, VWF, and GPIb suggest a model for the
location of ligand binding sites on VWF. Both botrocetin and GPIb can
bind simultaneously to VWF
(7) , whereas NMC-4 binding is not
compatible with either botrocetin
(40) or GPIb
binding
(9) . The scanning mutagenesis data suggest that the
binding sites of NMC-4, GPIb, and botrocetin overlap but are not
identical. In particular, construct(596-599)2A binds normally to
botrocetin and NMC-4 but not to GPIb; constructs R636A
and(663-667)3A bind normally to NMC-4 and GPIb (with ristocetin)
but not to botrocetin; construct(520-524)2A binds normally to
botrocetin and GPIb (with botrocetin) but has decreased binding to
NMC-4; construct(642-645)4A binds normally to NMC-4 but not to
GPIb or botrocetin. Seven other constructs are defective in binding to
all three ligands. These data suggest that botrocetin and GPIb bind to
adjacent sites and that the epitope of NMC-4 may overlap with both the
botrocetin and GPIb sites.
chain was identified as a
binding site for VWF
(2) , and botrocetin is notable for a
striking preponderance of acidic residues, especially in the
chain
(41) . Thus, botrocetin and GPIb both may interact
electrostatically with positively charged sites on VWF.
Figure 8:
Model
for the regulation of VWF binding to platelet GPIb. Panel A shows a schematic diagram of the VWF A1 domain. A disulfide bond
links Cys-509 to Cys-695. Functionally important VWF segments are
indicated that were identified by mutagenesis or VWD type 2B mutations.
Mutations within three segments (grayshaded;
497-511, 540-578, and 687-798) cause mainly
gain-of-function phenotypes and are labeled Inhibitor. Mutations within two acidic regions (hatched) abolish
only ristocetin-induced binding to platelets; these are labeled
Activator because the defect is bypassed by botrocetin. Three
predominantly basic regions (black-filled;
596-616, 629-632, and 642-645) are labeled GPIb
binding. Participation of segments 629-632 and 642-645
in GPIb binding is uncertain because mutations in them cause
nonselective loss of function, as do mutations at Asp-514 and
Lys-549-Arg-552. Segments that are required specifically for
botrocetin binding are labeled (Arg-636 and Arg-663-Lys-667).
Panel B shows a model that is discussed in the text for
interactions within domain A1. Positive regulation is denoted by
[b|]m+ and negative regulation by .
Inhibitor segments prevent the spontaneous binding of VWF to
GPIb, and this repression is relieved by ristocetin. Inhibitor segments may act directly on GPIb binding segments or
indirectly, possibly through Activator segments that also are
required for ristocetin-induced binding to GPIb. Botrocetin bypasses
the putative regulatory segments.
Modulation
of VWF binding by ristocetin requires a third region that may contain
the discontinuous acidic segments Asp-520-Lys-534 and Glu-626.
The requirement for these segments is bypassed by botrocetin.
Therefore, this region might act as an endogenous
``botrocetin-like'' activator that stimulates binding to GPIb
but that is regulated by the inhibitor regions (Fig. 8). This
model will be tested and refined by further mutagenesis and by
structural studies of VWF domain A1.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.