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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 164-172
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Structure and function of the von Willebrand factor A1 domain:
analysis with monoclonal antibodies reveals distinct binding sites
involved in recognition of the platelet membrane glycoprotein
Ib-IX-V complex and ristocetin-dependent activation
Mariagrazia De Luca,
David A. Facey,
Emmanuel J. Favaloro,
Mark S. Hertzberg,
James C. Whisstock,
Tracy McNally,
Robert K. Andrews, and
Michael
C. Berndt
From the Hazel and Pip Appel Vascular Biology Laboratory, Baker
Medical Research Institute, Melbourne; the Department of Hematology,
Westmead Hospital, Westmead, New South Wales; and the Department of
Biochemistry and Molecular Biology, Monash University, Clayton,
Australia.
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Abstract |
Binding of the adhesive glycoprotein, von Willebrand factor (vWf),
to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates
platelet adhesion and aggregation at high shear stress in hemostasis
and thrombosis. In this study, the GP Ib-IX-V binding site within the
vWf A1 domain was analyzed using a panel of murine monoclonal
antibodies raised against a 39/34-kd vWf fragment
(Leu-480/Val-481-Gly-718) encompassing the A1 domain. One antibody,
6G1, strongly inhibited ristocetin-dependent vWf binding to platelets,
but had no effect on botrocetin- or jaracetin-dependent binding, or
asialo-vWf-dependent platelet aggregation. The 6G1 epitope was mapped
to Glu-700-Asp-709, confirming the importance of this region for
modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf
A1 domain, because it enhanced binding of the 39/34-kd
fragment to platelets. In contrast, 5D2 and CR1 completely inhibited
asialo-vWf-induced platelet aggregation and ristocetin-induced vWf
binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and
jaracetin-induced vWf binding to platelets and binding of vWf to
botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were
conformationally dependent, but not congruent. Other antibodies mapped
to epitopes within the A1 domain (CR2 and CR15, Leu-494-Leu-512; CR2,
Phe-536-Ala-554; CR3, Arg-578-Glu-596; CR11 and CR15,
Ala-564-Ser-582) were not functional, identifying regions of the vWf
A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The
combined results provide evidence that the proline-rich sequence
Glu-700-Asp-709 constitutes a regulatory site for ristocetin, and that
ristocetin and botrocetin induce, at least in part, separate
receptor-recognition sites on vWf. (Blood. 2000;95:164-172)
© 2000 by The American Society of Hematology.
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Introduction |
Platelet adhesion to the damaged vasculature is not
only the initial event in the hemostatic response to injury but is also an important factor in thrombosis underlying cardiovascular disease and
stroke. Thrombus formation at high shear stress in hemostasis and
thrombosis depends on the adhesive multimeric glycoprotein von
Willebrand factor (vWf) binding to its specific platelet membrane receptor, the glycoprotein (GP) Ib-IX-V complex.1-3 This
interaction requires activation of either vWf or the receptor or both.
Association of vWf with the subendothelial extracellular matrix appears
to activate vWf to bind to GP Ib-IX-V constitutively expressed on platelets.1,4 Alternatively, binding of plasma vWf to
platelet GP Ib-IX-V is induced by pathologic shear stress by a
mechanism that may involve activation of receptor.2
vWf, derived from a complex gene (~180 kb, 52 exons), is synthesized
as pre-pro-vWf, including a 22-residue signal peptide and a
741-residue propeptide, and undergoes extensive posttranslational processing, glycosylation, and assembly in the endoplasmic reticulum, Golgi and post-Golgi.5 vWf consists of 270-kd subunits and forms disulfide-linked multimers of ~1000 to >10 000 kd.
Each vWf subunit contains 2050 amino acids made up of
conserved modular domains in the order
D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2.6 The GP
Ib-IX-V-binding site on vWf is contained within the A1
domain.7,8 A 39/34-kd proteolytic fragment of vWf
(Leu-480/Val-481-Gly-718) encompassing the A1 domain binds to GP
Ib-IX-V on platelets.8 This fragment contains an
intramolecular disulfide bond, Cys-509-Cys-695, defining a
predominantly positively charged sequence within the loop, and 2 discontinuous anionic flanking sequences containing sialylated
glycosylation sites at residues 468, 485, 492, 493, 500, 705, and
714. The sequence Asp-514-Glu-542 within the disulfide loop has been
identified as a potential site involved in binding GP
Ib-IX-V.9 Other sequences within the loop have been
identified as potential binding sites for sulfatide
(Gln-628-Val-646),10 Type III collagen
(Glu-542-Met-622),11 and heparin
(Tyr-565-Ala-587).12 The vWf-activating compounds, the
snake venom proteins botrocetin and jaracetin, and the bacterial
glycopeptide ristocetin, also interact with the A1 domain. Botrocetin
binds to 1 or more sequences within the disulfide loop,9,13
whereas there is evidence that ristocetin binds to proline-rich
sequences (Cys-474-Pro-488 and Leu-694-Pro-708) contained in the
anionic flanking sequences of the A1 domain.9,14,15 These
findings suggested an electrostatic mechanism for activation of the A1
domain.9
The aim of this study was to further investigate
structure-activity relationships of the vWf A1 domain. We therefore
generated a panel of monoclonal antibodies raised against the 39/34-kd
vWf fragment. These antibodies were functionally characterized in terms
of their ability to inhibit (1) the interaction of vWf with platelet GP
Ib-IX-V in the presence of ristocetin, botrocetin, or jaracetin; (2)
asialo-vWf- and bovine vWf-induced platelet aggregation; and (3)
binding of vWf directly to the modulators, botrocetin and jaracetin.
Specific antibodies discriminated between vWf binding to platelets
depending on how the interaction was induced. In addition, epitopes for
several functional and nonfunctional antibodies were mapped by
immunoblotting the 39/34-kd vWf fragment (Leu-480-Gly-718) and
overlapping synthetic peptides, based on the vWf A1 domain sequence and
by cross blocking between different antibodies. Overall, the combined
results identified sites that are potentially involved in binding the
platelet membrane GP Ib-IX-V complex in the presence of ristocetin
compared with botrocetin, and support the role of the C-terminal
proline-rich sequence Glu-700-Asp-709 in ristocetin-dependent
activation of the vWf A1 domain.
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Materials and methods |
Materials
Bovine serum albumin (BSA), Tween 20, pristane, and Freund's
adjuvant were purchased from Sigma, St Louis, MO. Chloramine T was
obtained from Riedel-de Häen, Selze, Germany. Neuraminidase was
purchased from Hoechst-Behring (Marburg, Germany). DMEM and HAT media
were from Trace Scientific (Castle Hill, Australia). Ristocetin sulfate
was from Paesel and Lorei (Frankfurt, Germany). Synthetic peptides
based on the vWf A1 domain amino acid sequence,6 containing
an N-terminal cysteine residue to facilitate coupling, were purified by
reverse-phase high-performance liquid chromatography (HPLC) and
characterized by mass spectroscopy (Chiron, Clayton, Australia or
Auspep, Parkville, Australia). Botrocetin and jaracetin were purified
from Bothrops jararaca viper venom (Sigma) as previously described.16,17 Human Factor VIII concentrate was a gift of the Commonwealth Serum Laboratories, Melbourne, Australia.
Purification of vWf, asialo-vWf, and the 39/34-kd vWf
fragment
vWf was purified from Factor VIII concentrates as described
elsewhere.16 Asialo-vWf was prepared from native vWf by
neuraminidase treatment as previously described.9 The
39/34-kd dispase fragment of vWf, Leu-480/Val-481-Gly-718, was
prepared and isolated by heparin-affinity chromatography as described
elsewhere.8 The reduced (2-mercaptoethanol) and alkylated
(2-iodoacetamide) 39/34-kd vWf fragment was also prepared as previously
described.8 Where appropriate, vWf and the 39/34-kd vWf
fragment were radio-iodinated with sodium [125I]iodide
(Australian Radioisotopes, Lucas Heights, Australia) using the
chloramine T method18 and separated from excess label by
gel filtration on a Sephadex G25 PD10 column (Pharmacia, Uppsala, Sweden) equilibrated with TS buffer (0.01 mol/L Tris, 0.15 mol/L sodium
chloride, pH 7.4).
Antibodies
Murine monoclonal antibodies, AK2 and WM23, directed against the
extracytoplasmic domain of GP Ib were purified and characterized as
described in detail elsewhere.16,18-21 Anti-vWf monoclonal antibodies were produced in BALB/C mice immunized against the 39/34-kd
vWf fragment by 3 to 4 intraperitoneal injections (140 µg 39/34-kd
fragment/0.2 mL adjuvant/injection) over 8 weeks. A booster injection
was given 3 to 4 days before fusion of spleen cells with the NS-1 cell
line. Briefly, the spleen was macerated in serum-free DMEM cell culture
medium. Both spleen cells and NS-1 cells were washed in serum-free
DMEM, equal numbers of both cell types (~26 × 106
cells) were mixed by inversion, centrifuged at 200g for 5 minutes, and resuspended in 1 mL of 40% polyethylene glycol in DMEM.
After 5 minutes at 37°C, the solution was diluted to 10 mL with
serum-free DMEM, incubated for 1 hour at 37°C with 5% carbon
dioxide, and HAT media added to a final volume to 15 mL before plating
onto macrophage feeder plates. Positive clones as assessed by
immunoblotting vWf (see below) were subcloned to homogeneity by
limiting dilution. Ascites was produced from clonal hybridomas by
washing cells twice in serum-free DMEM, diluting to
1.5 × 106 cells/mL, and injecting 0.5 mL
intraperitoneally into BALB/C mice primed with pristane for 1 to 2 weeks. Ascites was collected after 10 to 30 days from carbon dioxide
asphyxiated mice. Anti-vWf monoclonal antibodies were purified on
protein-G Sepharose 4B (Pharmacia) and subtyped using a commercial
isotyping kit (Pierce, Rockford, IL) according to the manufacturer's
instructions. All purified antibodies were radio-iodinated where
appropriate using the chloramine T method18 and separated
from free label by gel filtration on Sephadex G25.
Immunoblotting
For Western blotting, protein was electrophoresed on
SDS-polyacrylamide gels under reducing or nonreducing conditions,
electrotransferred to nitrocellulose, and immunoblotted by standard
methods.18-21 For dot blotting of synthetic peptides with
antibodies, peptides were coupled through an N-terminal Cys residue to
BSA (1 mg peptide/10 mg BSA) with
m-maleimidobenzoyl-N-hydroxysuccinimide (Pierce) essentially as
previously described for coupling peptides to hemocyanin.22 Peptide-BSA conjugates (1 µg), native or asialo-vWf (1 µg), the 39/34-kd vWf fragment (0.2 µg), or reduced and alkylated 39/34-kd fragment (0.2 µg), all in TS buffer, were dotted onto nitrocellulose strips (1 µL). After air drying, membranes were blocked with 5% (w/v) skim milk powder in TS buffer for 1 hour and incubated with anti-vWf monoclonal antibodies (1 µg/mL, final concentration) in
0.5% (w/v) skim milk powder in TS buffer for a further 2 hours. The
nitrocellulose strips were washed extensively with TS buffer, and
incubated with a 1:500 dilution of peroxidase-labeled sheep antimouse
IgG (Silenus, Melbourne, Australia) for 90 minutes in 0.5% (w/v) skim
milk powder in TS buffer, washed thoroughly with TS buffer, and
visualized using the ECL detection system (Amersham, UK).
Binding of 125I-labeled monoclonal antibodies to
immobilized vWf or 39/34-kd vWf fragment
Detachable microtiter wells (Immulon I; Dynatech, Chantilly, VA)
were coated with either vWf (10 µg/mL) or the 39/34-kd fragment (~7
µg/mL) in TS buffer for 4 hours at 22°C. Wells were aspirated and
replaced with 125I-labeled monoclonal antibody (1 µg/mL)
in 0.1% (w/v) BSA in TS buffer. Nonspecific binding was assessed using
a 100-fold excess of the unlabeled antibody in a parallel assay. To
test the ability of other anti-vWf monoclonal antibodies to cross
block, some assays also included a 100-fold excess of these individual
antibodies. Alternatively, other assays included synthetic peptides at
a final concentration of 100 µM. After 30 minutes at 22°C, the
wells were aspirated and washed twice with 100-µL aliquots of 0.1%
(w/v) BSA in TS buffer. Radioactivity bound to the vWf-coated wells was
measured in a  counter. Assays were performed in triplicate or
quadruplicate, with standard deviation from the mean typically  5%.
Platelet aggregometry
Platelet aggregation was performed at 37°C in a Lumiaggregometer
(Chronolog, Havertown, PA) using citrated platelet-rich plasma stirred
at 900 rpm as previously described.9,21,23 The effect of
anti-GP Ib or anti-vWf monoclonal antibodies (final concentration, 20 µg/mL) was determined by preincubating the antibodies with platelets or vWf, respectively, for 3 minutes at 37°C before the addition of ristocetin (final concentration, 1.5 mg/mL), botrocetin (final concentration, 25 µg/mL), asialo-vWf (final concentration, 70 µg/mL), or 10% (v/v) bovine plasma (as a source of bovine vWf).
Binding of 125I-labeled vWf or 39/34-kd vWf fragment
to platelets
The effect of monoclonal antibodies on the binding of
125I-labeled vWf to platelets in the presence of 1 mg/mL
ristocetin or 25 µg/mL botrocetin or jaracetin as vWf modulators was
measured using a previously described assay.16,17,19
Monoclonal antibodies (final concentration, 50 µg/mL) were incubated
with 125I-labeled vWf (1 µg/mL) for 5 minutes at
22°C, before the addition of washed platelets (final concentration,
5 × 108/mL) in TS buffer containing 0.1% (w/v)
BSA. After 30 minutes, samples were centrifuged at 8750g for 2 minutes, and label associated with the pellet was measured in a
-counter after aspiration of the supernatant. Specific binding was
calculated from total binding by subtracting nonspecific binding
measured in a parallel assay containing a 100-fold excess of unlabeled
vWf or 20 µg/mL of the blocking monoclonal antibody,
AK2.16,19 Binding of 125I-labeled 39/34-kd vWf
fragment to platelets in the presence of 25 µg/mL botrocetin or 10 µg/mL of monoclonal antibody was measured using the same
method.8 Nonspecific binding was determined in the absence
of botrocetin or antibody in a parallel assay, or by including 20 µg/mL of AK2.
Binding of vWf to botrocetin- or jaracetin-coated beads
Purified botrocetin or jaracetin was covalently coupled to the
surface of polyacrylamide Immunobeads (Bio-Rad, Richmond, CA) according
to the manufacturer's instructions. Routine assays incorporated 25%
(v/v) beads and 0.5 µg/mL 125I-labeled vWf in a final
volume of 0.1 mL TS buffer containing 0.1% (w/v) BSA. After 30 minutes
at 22°C, samples were centrifuged at 8750g for 2 minutes,
and the radioactivity associated with the pellet was measured in a counter. Nonspecific binding was determined in a parallel assay by
including a 100-fold excess of unlabeled vWf. To assess the effect of
monoclonal antibodies on binding of 125I-labeled vWf to the
beads, some assays included 50 µg/mL anti-vWf or control IgG.
Standard assays were performed in triplicate or quadruplicate, with
standard deviation from the mean typically 5%.
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Results |
The A1 internal repeat domain of vWf is encompassed by a 39/34-kd
dispase fragment of vWf (Leu-480/Val-481-Gly-718) that includes the
binding site for the platelet membrane vWf receptor, the GP Ib-IX-V
complex.8 Unlike native vWf, this fragment has the capacity
to spontaneously bind receptor in the absence of shear stress or vWf
modulators. As part of our analysis of structure-activity relationships
of the vWf A1 domain, we used the 39/34-kd fragment to generate a
panel of monoclonal antibodies to identify receptor-binding and
regulatory sites involved in vWf-dependent platelet adhesion and
activation. A total of 9 murine monoclonal antibodies were produced
from 6 separate fusions using purified 39/34-kd vWf fragment as
immunogen. Four of the monoclonal antibodies, 5D2, 6G1, CR1, and CR3,
were of the IgG1 subtype, whereas the other 5, CR2, CR4, CR7, CR11, and
CR15, were of the IgG2b subtype. The physicochemical and functional
characteristics of these antibodies were determined, along with the
previously described murine monoclonal antibodies, 2C9, raised against
native vWf23 and AK2 and WM23, directed against platelet GP
Ib.19,21
Characterization of anti-vWf monoclonal antibodies by
immunoblotting
The anti-vWf monoclonal antibodies were initially characterized by
immunoblotting native vWf, asialo-vWf, 39/34-kd vWf fragment and
reduced and alkylated 39/34-kd vWf fragment
(Table). First, all 9 of the antibodies
raised against the 39/34-kd vWf fragment, but not 2C9, Western blotted
the 39/34-kd fragment following SDS-polyacrylamide gel electrophoresis
under nonreducing or reducing conditions (Table), although CR4
and CR7 reacted only weakly. Both the 39-kd and the 34-kd species have
an identical amino acid sequence (Leu-480/Val-481-Gly-718) and differ
in apparent molecular mass due to variable glycosylation.8 None of the antibodies immunoreacted preferentially with either the
39-kd or 34-kd species under reducing or nonreducing conditions (data
not shown).
Second, in immunodot assays, all the antibodies, including 2C9, had
similar reactivity toward native and asialo-vWf spotted onto
nitrocellulose, with 6G1, CR1, CR3, and CR11 being most reactive (Table). This result confirmed that all the antibodies raised against
the proteolytic fragment of vWf had epitopes that were conserved in the
native protein. As expected, all 9 antibodies raised against the
39/34-kd vWf fragment reacted with the native, purified 39/34-kd
fragment. However, CR2, CR4, CR7, and CR15 were only weakly reactive
with the immobilized protein. 2C9 failed to recognize the 39/34-kd vWf
fragment suggesting its epitope did not involve the sequence
Leu-480-Gly-718. 5D2, CR1, and CR3 showed markedly less reactivity
with reduced and alkylated 39/34-kd fragment compared with
native fragment suggesting that these epitopes were sensitive to
disruption of the Cys-509-Cys-695 disulfide bond. Interestingly,
this observation was in contrast to the Western blotting results, where
5D2, CR1, and CR3 were all essentially equally reactive under
nonreducing and reducing conditions (Table). This suggests
that the epitopes for these 3 antibodies may in part encompass the
Cys-509-Cys-695 region and that the effect seen in the immunodot
assays may primarily be a consequence of disruption of the epitope by
alkylation rather than by reduction.
To further characterize the epitopes for these antibodies, overlapping
19-mer peptides together comprising the entire Leu-480-Gly-718 sequence were coupled to bovine serum albumin and their reactivity with
the antibodies assessed by immunodot analysis (Table). Four of the
antibodies 5D2, CR1, CR4, and CR7 did not react with any of the
peptide-albumin conjugates suggesting that these antibodies may be
recognizing conformational epitopes. The 6G1 reacted intensely with 2 peptide sequences, Ile-690-Pro-708 and Glu-700-Gly-718, suggesting
that the overlap sequence contained the epitope for 6G1. To further map
the epitope of 6G1, a series of peptides were evaluated for their
ability to inhibit binding of 125I-labeled 6G1 to
immobilized vWf (Figure 1). These results
showed that the sequence Glu-700-Asp-709 represented a minimal 6G1
epitope. CR2 showed weak binding to 2 noncontiguous sequences,
Leu-494-Leu-512 and Phe-536-Ala-554 (Table). CR3 reacted with the
sequence Arg-578-Glu-596, whereas CR11 and CR15 both reacted with the
peptide Ala-564-Ser-582. CR15 also reacted with Leu-494-Leu-512. The
control monoclonal antibodies, AK2 and WM23, were unreactive in
all the immunoblotting experiments (data not shown). The location of
the epitopes for these antibodies is shown in Figure
2. Thus, while CR2 and CR15 reacted with
non-contiguous peptide sequences, the sequences recognized by these 2 antibodies were proximal in the 3-dimensional structure.
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| Fig 1.
Mapping the epitope for the anti-vWf monoclonal antibody,
6G1.
Inhibition of binding of 125I-labeled monoclonal antibody
6G1 (final concentration, 1 µg/mL) to immobilized vWf by synthetic
peptides (final concentration, 100 µM) in 30 minutes at 22°C.
Inhibition is expressed relative to maximal specific binding in the
absence of peptide. Results are the mean of quadruplicate
determinations, with standard deviation from the mean of 5%.
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| Fig 2.
Epitopes for anti-vWf monoclonal antibodies.
Structure of the von Willebrand factor A1 domain based on the x-ray
crystal coordinates25 with highlighted sequences
representing epitopes for the anti-39/34-kd vWf fragment monoclonal
antibodies determined using synthetic peptides.
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Cross blocking of anti-vWf monoclonal antibodies
To further characterize the topographic association of the
antibodies within the vWf A1 domain and its flanking sequences, cross-blocking studies were performed using immobilized vWf or the
39/34-kd vWf fragment (Figure 3). Although
all the antibodies bound to vWf or the 39/34-kd vWf fragment
immobilized on nitrocellulose (see above), only 4 antibodies, 2C9, 5D2,
6G1, and CR1, bound to native vWf immobilized on plastic and only 2 antibodies, 6G1 and CR11, recognized plastic-bound 39/34-kd vWf
fragment. This suggests that either the immobilization of vWf or
fragment is not random (that is, a specific face of the A1 domain binds
to plastic), that binding of vWf or fragment to plastic results in conformational changes not recognized by some of the antibodies, or a
combination of these possibilities. The simplest interpretation of
cross-blocking studies, that is, if antibody A blocks binding of
antibody B and the converse is true, then the epitopes for A and B are
either identical or sterically proximal, was not the case for the
anti-vWf antibodies. This suggested that the assays were affected by
conformational effects within the vWf A1 domain. First, binding of
radiolabeled 2C9 to immobilized vWf was only inhibited by unlabeled 2C9
(Figure 3A), and 2C9 did not block binding of the other antibodies.
However, while binding of 5D2 to vWf was only inhibited by excess cold
5D2 (Figure 3B), unlabeled 5D2 blocked the binding of 6G1 and CR1
(Figures 3C and 3D, respectively). This suggests the epitope for 5D2
was distinct from those of CR1 and 6G1, and that binding of 5D2 induced
conformational changes that masked the CR1 and 6G1 epitopes. 6G1 and
CR1 cross blocked each other (Figures 3C and 3D), suggesting their
epitopes were proximal, even though 6G1 recognized the linear sequence
Glu-700-Asp-709; whereas the epitope for CR1 was apparently dependent
on conformation and markedly affected by reduction and alkylation of
the Cys-509 to Cys-695 disulfide bond (see above). Of the 2 antibodies
that bound to the 39/34-kd vWf fragment on plastic, 6G1 binding was only inhibited by itself (Figure 3E), but binding of CR11 was inhibited
by both 6G1 and CR11 (Figure 3F), suggesting that 6G1 induces a
conformational change that causes the CR11 epitope to become cryptic.
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| Fig 3.
Cross blocking of anti-vWf monoclonal antibodies.
Binding of 125I-labeled monoclonal antibodies (final
concentration, 1 µg/mL) to immobilized native vWf (A-D) or 39/34-kd
vWf fragment (E-F) in 30 minutes at 22°C. Unlabeled antibodies were
at a final concentration of 100 µg/mL. Specific binding in the
presence of blocking antibodies was expressed as a percentage of
maximal specific binding measured in the absence of blocking antibody.
Panel, monoclonal antibody: A, 2C9; B, 5D2; C, 6G1; D, CR1; E, 6G1; F,
CR11. Results are the mean of triplicate determinations, with standard
deviation from the mean of 5%.
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Effect of anti-vWf antibodies on vWf binding to GP Ib-IX-V on
platelets
In initial studies, the anti-vWf monoclonal antibodies at 50 µg/mL
(final concentration) were tested for the ability to inhibit aggregation of platelet-rich plasma induced by either asialo-vWf or
bovine-vWf. Of the 10 antibodies tested, 5D2 and CR1 inhibited asialo-vWf-induced platelet aggregation, whereas 2C9 and the other anti-vWf antibodies were without effect (data not shown). CR1 was the
only monoclonal antibody to inhibit bovine vWf-induced platelet
agglutination (not shown), although whether the other antibodies
recognized bovine vWf was not determined. Anti-vWf antibodies were then
tested for their ability to inhibit botrocetin-, jaracetin-, and
ristocetin-induced vWf binding to washed platelets. 5D2 was the only
antibody that significantly inhibited botrocetin- and jaracetin-induced
vWf binding to washed platelets (Figures 4A
and 4B, respectively). In both cases, binding of vWf to platelets was
inhibited by ~90%. Three antibodies, 5D2, CR1, and 6G1, blocked ristocetin-induced vWf binding to platelets (Figure 4C). Consistent with previously reported results,16,19,21 the anti-GP Ib antibody, AK2, completely blocked vWf binding to platelets in all the
assay systems used, whereas WM23 against GP Ib (data not shown) and
2C9 against vWf (Figure 4) were without effect in any of the assays.
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| Fig 4.
Effect of anti-vWf monoclonal antibodies on vWf binding
to platelets.
Antibodies (final concentration, 50 µg/mL) were included in assays
measuring binding of 125I-labeled vWf (1 µg/mL) to GP
Ib-IX-V on washed platelets induced by (A) botrocetin (25 µg/mL), (B)
jaracetin (25 µg/mL), or (C) ristocetin (1 mg/mL). Antibodies were
preincubated with vWf for 5 minutes at 22°C before the addition of
washed platelets (final concentration, 5 × 108/mL).
Specific binding was expressed as a percentage of maximal specific
binding measured in the absence of antibody. Nonspecific binding was
assessed in the presence of 50 µg/mL of the inhibitory anti-GP Ib
monoclonal antibody, AK2. Results are the mean of quadruplicate
determinations, with standard deviation from the mean of 5%.
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Effect of anti-vWf antibodies on binding of vWf to botrocetin-
and jaracetin-coated beads
Previously established assays measuring the interaction of vWf with
the modulators botrocetin or jaracetin immobilized on beads16,17,24 were used to determine whether any of the
anti-vWf monoclonal antibodies blocked access to regulatory sites
within the A1 domain. Binding of 125I-labeled vWf to
botrocetin- or jaracetin-coated beads was only completely inhibited by
5D2 (Figures 5A and 5B, respectively). 6G1
partially inhibited binding of vWf to botrocetin and jaracetin under
these conditions, by ~30% and ~60%, respectively.
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| Fig 5.
Effect of anti-vWf monoclonal antibodies on vWf binding
to botrocetin or jaracetin.
Antibodies (final concentration, 50 µg/mL) were included in assays
measuring binding of 125I-labeled vWf (1 µg/mL) to (A)
botrocetin-coated beads or (B) jaracetin-coated beads for 30 minutes at
22°C. Specific binding was expressed as a percentage of maximal
specific binding measured in the absence of antibody. Nonspecific
binding was assessed in the presence of a 100-fold excess of unlabeled
vWf. Results are the mean of triplicate determinations, with standard
deviation from the mean of 5%.
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Effect of 6G1 on binding of the 39/34-kd vWf fragment to washed
platelets
Because the anti-vWf monoclonal antibody 6G1 mapped into a
proline-rich sequence at the C-terminal flank of the vWf A1 domain (Glu-700-Asp-709) previously identified as a binding site for the
modulator, ristocetin,9,14,15 and because it induced conformational changes in the vWf A1 domain as suggested by
cross-blocking studies with CR11, we tested whether 6G1 was itself able
to activate vWf to bind GP Ib-IX-V on platelets. In preliminary
experiments, 6G1 did not induce binding of 125I-labeled vWf
to washed platelets (data not shown). However, saturable binding of
125I-labeled 39/34-kd vWf fragment to platelets was induced
by increasing concentrations of 6G1, with maximal binding at
concentrations above 10 µg/mL (Figure
6A). Binding induced by 6G1 was
specifically inhibited by the blocking anti-GP Ib monoclonal
antibody, AK2, by ~80% (data not shown), indicating that 6G1 induced
a specific interaction between the 39/34-kd vWf fragment and GP
Ib-IX-V. To examine the relationship between binding of the 39/34-kd
vWf fragment to platelets as induced by 6G1 and botrocetin,
botrocetin-dependent binding of the 39/34-kd vWf fragment to platelets
was evaluated over the botrocetin concentration range, 1 to 50 µg/mL,
in the absence and presence of 50 µg/mL of 6G1. The modulatory
effects of botrocetin and 6G1 on vWf binding to platelets were additive consistent with these modulators activating vWf by distinct mechanisms (Figure 6B). In previous studies, it was not technically feasible to
directly demonstrate modulator-independent binding of the 39/34-kd vWf
fragment to the GP Ib-IX-V complex. Direct binding was inferred by its
capacity to inhibit asialo-vWf-induced platelet aggregation and by
cross-linking analysis.8 Consistent with this, there was no
measurable specific binding of 125I-labeled 39/34-kd vWf
fragment to platelets in the absence of 6G1 or other modulator (Figure
6B), and the level of nonspecific binding was unaffected by the
presence of AK2 (not shown).
View larger version (16K):
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| Fig 6.
Effect of the anti-vWf monoclonal antibody 6G1 on binding
of the 39/34-kd vWf fragment to platelets.
(A) Specific binding of 125I-labeled 39/34-kd vWf fragment
(final concentration, 1 µg/mL) to washed platelets (final
concentration, 5 × 108/mL) in the presence of 6G1
in 30 minutes at 22°C. Nonspecific binding was determined in the
absence of 6G1 (see Materials and Methods). (B) Specific binding of
125I-labeled 39/34-kd vWf fragment (final concentration, 1 µg/mL) to platelets in the presence of botrocetin and either the
absence (squares) or presence (circles) of 6G1 (final concentration, 50 µg/mL).
|
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| |
Discussion |
Binding of von Willebrand factor (vWf) to its adhesive
receptor on the platelet surface, the glycoprotein (GP) Ib-IX-V
complex, initiates platelet adhesion and activation at high shear
stress. In the normal circulation, plasma vWf does not bind to
platelets, but becomes platelet adhesive when bound to the
subendothelial matrix, or when platelets and vWf are exposed to
pathologic hydrodynamic shear stress.1-4 The interaction of
vWf and GP Ib-IX-V is induced by activation of the ligand, the receptor
or both. The GP Ib-IX-V-binding domain on vWf has been localized to
the internal A1 domain,7 encompassed by a 39/34-kd
proteolytic fragment of vWf (Leu-480/Val-481-Gly-718), including the
disulfide bond between Cys-509 and Cys-695.8 The crystal
structure of the vWf A1 domain has recently been solved at 0.23 nm,25 revealing a central core of 5 parallel
and 1 short antiparallel  -sheets surrounded by 7 -helices and
loop regions (Figure 2). A sequence within the A1 domain
(Asp-514-Glu-542) identified as a GP Ib-IX-V binding site9
overlaps a 1 sheet-loop-1 helix structure. NMR analysis of a
synthetic peptide based on the Asp-514-Glu-542 sequence shows it
adopts a solution structure consistent with the crystal
structure.26 In this study, characterization of a panel of
monoclonal antibodies directed against the vWf A1 domain provides
further evidence on functionally important regions of the domain, and
how binding to the GP Ib-IX-V complex may be regulated.
First, a monoclonal antibody, 6G1, was identified which blocked
ristocetin-dependent binding of vWf to platelets but had no effect on
botrocetin-dependent binding or on asialo-vWf-induced platelet
agglutination. These results were consistent with 6G1 competing for a
ristocetin-binding site rather than a site that recognized receptor.
6G1 mapped into 2 linear overlapping peptides corresponding to residues
Ile-690-Pro-708 and Glu-700-Gly-718 (Table), and further analysis
using synthetic peptides based on these sequences localized the 6G1
epitope to Glu-700-Asp-709. Previous evidence has suggested that
proline-rich sequences Cys-474-Pro-488 and Leu-694-Pro-708 flanking
the vWf A1 domain constituted binding sites for ristocetin. Synthetic
peptides, based on these sequences, specifically inhibited
ristocetin-dependent vWf binding and directly bound
ristocetin,9 while mutagenesis of a triple proline
sequence, 702-704, in native vWf specifically abolished ristocetin
modulation of vWf binding to platelets.15 6G1 also enhanced
binding of the 39/34-kd vWf fragment to platelets, consistent with the
sequence Glu-700-Asp-709 acting as a regulatory site, binding to which can trigger conformational activation of the vWf A1 domain.
Interestingly, 6G1 did not induce binding to receptor of intact vWf.
This difference may reflect that the 39/34-kd vWf fragment, as opposed
to intact vWf, has the capacity to weakly bind receptor in the absence
of modulators8 and therefore may be more susceptible to a
trigger of conformational change.
The functional effects of 6G1 combined with the mapping of its epitope
to the C-terminal proline-rich sequence Glu-700-Asp-709 strongly
support this region being a ristocetin recognition site, and suggest it
is more important than the N-terminal flank. Consistent with this
supposition, CR2 and CR15 that map into Leu-494-Leu-512 on the
N-terminal side of the disulfide bond (Table; Figure 2) did not inhibit
ristocetin-dependent vWf binding. In the 3-dimensional structure of the
domain (Figure 2), Glu-700-Asp-709 is proximal to the putative GP
Ib-IX-V-binding sequence Asp-514-Glu-542. The finding that 6G1
induced binding of the 39/34-kd vWf fragment to platelets additive to
that induced by botrocetin also suggests that 6G1 activates the vWf A1
domain by a different mechanism to that of botrocetin. This is
consistent with previous evidence that ristocetin (that uses the 6G1
binding site) and botrocetin modulate vWf by distinct mechanisms. Three
noncontiguous peptide sequences within the vWf A1 domain,
Asp-539- Val-553, Lys-569-Gln-583, and
Arg-629-Lys-64313, in addition to
Asp-514-Glu-5429 have been proposed as potential
botrocetin binding sites mediating vWf binding to the GP Ib-IX-V
complex. In the present study, 4 monoclonal antibodies, CR2, CR3, CR11,
and CR15, were identified whose epitopes overlapped the 539-553 or
569-583 sequences (Table; Figure 2). None of these antibodies affected
botrocetin-dependent binding of vWf to platelets or vWf binding to
immobilized botrocetin or jaracetin. This is consistent with the
observation that although the 539-553 and 569-583 sequences block
botrocetin-dependent vWf binding to platelets, they do not inhibit
binding of vWf to botrocetin,13 suggesting their effects on
receptor-ligand interaction are either nonspecific or occur by a
different mechanism than interference with modulator. In this study,
binding of vWf to botrocetin and jaracetin was only blocked by the
monoclonal antibody, 5D2, and partially by 6G1, suggesting that the
binding site for botrocetin may be proximal to the Glu-700-Asp-709
sequence constituting the 6G1 epitope. The epitope for 5D2 has not been
determined and appears dependent on conformation.
Binding of ristocetin to the negatively charged Glu-700-Asp-709
sequence (this study9,14,15) or botrocetin to the
predominantly positively charged Asp-514-Glu-542/Arg-629-Lys-643
sequences9,13 conceivably exposes a GP Ib-IX-V binding
site within Asp-514-Glu-542 or elsewhere by altering electrostatic
interactions within this region. Previous evidence supports an
electrostatic mechanism for vWf A1 domain activation. First, vWf
from type 2B von Willebrand's disease patients, where vWf
spontaneously binds GP Ib-IX-V, contains point mutations within the A1
domain.29 A number of these gain-of-function mutations are
clustered in the region immediately C-terminal to the GP
Ib-IX-V-binding sequence (Asp-514-Glu-542). Three major loci are
Arg-543/Trp or Gln, Arg-545/Cys or Pro, and Arg-578/Gln or Leu,
mutations that involve loss of a positively charged arginine residue.
Second, mutagenesis of charged residues within or proximal to the
sulfatide-binding region (Gln-628-Val-646) inhibits binding of vWf to
platelets.27 Third, a number of sulfated glycans and polyanionic compounds block ristocetin- and botrocetin-dependent binding of vWf to platelets,10,24,30-32 implying that
positive charge in the vWf A1 domain is important in mediating receptor binding. The heparin-binding sequence in the A1 domain,
Tyr-565-Ala-587, is unlikely to be directly involved in binding GP
Ib-IX-V, because antibodies that mapped into this region (CR3, CR11,
and CR15) had no effect on vWf-GP Ib-IX-V interaction. However,
heparin and some analogous compounds that inhibit both ristocetin- and botrocetin-dependent vWf binding to platelets do not inhibit vWf binding to sulfatides.10,24,30-32 A possible explanation
for these observations is that heparin binding to Tyr-565-Ala-587 prevents formation of an active GP Ib-IX-V-binding state, heparin sterically inhibits vWf binding, or heparin interacts with other positively charged residues within the A1 domain.
Unlike 6G1 that specifically blocked ristocetin-dependent binding, 2 monoclonal antibodies, 5D2 and CR1, had profound effects on the vWf-GP
Ib-IX-V complex interaction. Cross-blocking studies, while complicated
by apparent comformational effects, indicated that the 2 antibodies
recognized distinct epitopes, neither of which could be identified by
immunoblotting linear peptides. CR1 inhibited ristocetin-dependent
binding of vWf to platelets as well as asialo-vWf- and
bovine-vWf-induced platelet agglutination, but had no effect on
botrocetin-dependent binding of vWf to platelets. In this regard, CR1
appears similar to the previously described anti-vWf monoclonal
antibody, 3F8.16,19 (The clone for 3F8 has been lost,
precluding further characterization.) In contrast, 5D2 inhibited both
ristocetin- and botrocetin-dependent binding of vWf to platelets as
well as asialo-vWf-induced platelet aggregation. This difference
therefore cannot be explained on the basis that ristocetin and
botrocetin bind to different modulation sites, because both CR1 and 5D2
inhibited asialo-vWf-dependent aggregation. The data for these
antibodies strongly suggest that different regions of vWf, possibly
overlapping, interact with receptor depending on whether vWf is
activated by ristocetin or botrocetin. This proposition is consistent
with analysis of recombinant vWf fragments by scanning mutagenesis
within the A1 domain. Specific amino acid substitutions at Glu-626 or
within Asp-520-Lys-534 preferentially inhibited ristocetin-dependent
vWf binding as opposed to botrocetin-dependent binding.27
These studies, however, do not exclude the possibility that mutations
are inducing conformational changes in the A1 domain that preclude
activation by ristocetin, rather than directly affecting a
receptor-binding sequence.
A model in which different forms of vWf have the capacity to bind
receptor depending on the mechanism of vWf activation is supported by
complementary structure-activity studies on the GP Ib-IX-V complex. vWf
binds to the N-terminal domain of the GP Ib chain (His-1-Glu-282),
consisting of 7 leucine-rich repeats, their conserved N- and C-terminal
flanking sequences, and an anionic sequence, Tyr-276-Glu-282,
containing 3 sulfated tyrosines.1-3 The sulfated tyrosine
sequence is more important for binding vWf activated by botrocetin
compared with ristocetin, as demonstrated using proteolytic fragments
of native vWf lacking the sulfated tyrosine domain21 or
recombinant GP Ib where sulfation is deficient.33,34 In
addition, 2 anti-GP Ib monoclonal antibodies that map to this region, SZ2 and ES85, blocked botrocetin-induced binding of vWf to
platelets and asialo-vWf-dependent platelet aggregation, but not
ristocetin-dependent binding.21 In contrast, several
antibodies against the N-terminal region of GP Ib (His-1-Leu-275)
blocked ristocetin-dependent, botrocetin-dependent, and asialo-vWf
binding.16,19,21 Finally, consistent with separate
ristocetin- and botrocetin-dependent receptor-binding sites on vWf, an
anti-GP Ib monoclonal antibody, OP-F1, has been described that
abolishes ristocetin-induced, but not botrocetin-induced vWf binding to
platelets.35
In conclusion, characterization of a panel of anti-39/34-kd vWf
fragment monoclonal antibodies, and epitope analysis of inhibitory and
noninhibitory antibodies, has provided further insight into the
structure and function of the vWf A1 domain. In particular, this study
provides strong additional support for the involvement of the
proline-rich A domain C-terminal flanking sequence in binding the
modulator, ristocetin, and supports a model in which separate sites
within the A1 domain are critical for binding GP Ib-IX-V, depending on
how vWf is activated to recognize receptor.
| |
Acknowledgments |
We are grateful to Ms Carmen Llerena for outstanding technical
assistance. We also thank the National Health and Medical Research Council of Australia for financial support.
| |
Footnotes |
Submitted May 10, 1999; accepted August 27, 1999.
M.D.L. and D.A.F. are co-first authors.
Reprints: Michael C. Berndt, Baker Medical Research Institute,
PO Box 6492, St Kilda Road Central, Melbourne, VIC, Australia, 8008;
Robert K. Andrews, Baker Medical Research Institute, PO Box 6492, St
Kilda Road Central, Melbourne, VIC, Australia, 8008.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction