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INTRODUCTION |
von Willebrand factor
(vWf)1 is a large multimeric
glycoprotein that functions both as an adhesive protein and as a
carrier for factor VIII (antihemophilic factor) in plasma. vWf is
secreted both from the apical surface of cells into plasma and from the basolateral surface of cells, where it binds to type VI collagen and is
incorporated into the vascular matrix (1). As a carrier for factor
VIII, plasma vWf functions to prevent factor VIII from binding to
phosphatidylserine-containing membranes, where it may be degraded by
anticoagulant proteases such as activated protein C (2-4). As an
adhesive protein, plasma vWf forms the primary link between exposed
extravascular collagen at a site of vascular rupture and platelets,
which are sequestered from flowing blood by collagen-bound vWf (5, 6).
vWf secreted from the basolateral surface of endothelial cells also
functions as an adhesive protein. When the vascular matrix is exposed
to flowing blood, the vWf-bound to type VI collagen supports adhesion
of platelets and development of a thrombus (7). Factor VIII is released
from vWf following proteolytic activation by thrombin (8) and is then
free to bind to phosphatidylserine-containing membranes, where it may assemble with the serine protease, factor IXa, to form an efficient procoagulant enzyme complex (9) or be degraded by activated protein C
(4). Only suspended, plasma vWf is known to bind factor VIII. Thus, we
were motivated to determine the consequences that may occur when
circulating vWf with bound factor VIII binds to collagen at a site of
vascular injury.
vWf, which is synthesized in both endothelial cells and megakaryocytes
(1, 10), consists of disulfide-bonded monomeric subunits of 260,000 Da
with multimers ranging from dimers to 80-mers with masses of up to
20 × 106 Da (11, 12). The primary amino acid sequence
of the mature subunit entails homologous domains arranged in the order
D'-D3-A1-A2-A3-D4-C1-C2 (13-15). The subunits are linked by C-terminal
disulfide bonds prior to transport from the endoplasmic reticulum to
the Golgi apparatus (16, 17). Within the Golgi apparatus, the dimers are assembled into high molecular mass multimers under the influence of
a large propeptide (18, 19). Each vWf subunit contains a factor
VIII-binding motif with functional residues clustered in the D' domain
(20, 21), whereas the primary collagen-binding motif of vWf is
localized in the A3 domain (22). The D domains of vWf are remarkable
for a high cysteine content (10), with the vast majority of these
residues forming intradomain disulfide bonds. We have recently observed
that formation of the intersubunit disulfide bonds between D3 domains
of dimers, linking them to form multimers, increases the affinity of
vWf subunits for factor VIII 6-fold (23). This suggests that high
affinity factor VIII-binding sites reside on the internal subunits of
vWf multimers rather than on the two terminating subunits and that the
D' domain is subject to conformational change despite its
eight-intradomain disulfide bond restraints (17).
Factor VIII is a trace plasma glycoprotein with a molecular mass of 280 kDa. It functions as cofactor for factor IXa in the highly efficient
factor X-activating enzyme complex (9). Within this complex, factor
VIII binds both to a platelet-binding site (24, 25) and to the enzyme
factor IXa (26). The enzymatic product of this complex, activated
factor X, activates prothrombin to thrombin (27). Hemophilia, an
X-linked bleeding disorder in which factor VIII or IX levels are
decreased in plasma, illustrates the importance of factor VIII for
normal hemostasis. Because von Willebrand factor protects factor VIII
in plasma, factor VIII levels are decreased in patients with reduced or
absent circulating vWf and in patients with mutant vWf with impaired
factor VIII binding (28).
In plasma, vWf multimers carry only one factor VIII molecule/50 vWf
subunits. Binding studies conducted in vitro have yielded conflicting data for the number of available factor VIII-binding sites
on plasma vWf multimers. A recent investigation from this laboratory
(23) demonstrated a 1:1 stoichiometry of binding sites/vWf subunit,
which is in agreement with the data of Lollar and Parker (29) and Vlot
et al. (30). In contrast, other studies reported different
factor VIII/vWf ratios, such as 1:4, 1:10, 1:50, and 1:70 (30-33).
Several investigators reported that factor VIII forms a high affinity
complex with vWf, with dissociation constants range from 0.2 to 0.94 nM (30, 32-35). A study by Leyte et al. (33)
suggested that the high affinity interaction is restricted to 1-2% of
the available sites. We were motivated to determine whether any
proteins in plasma reduce the affinity or availability of factor
VIII-binding sites on vWf multimers, possibly explaining the discrepancies.
In vitro, plasma vWf does not bind to its platelet receptor,
glycoprotein Ib, in the absence of nonphysiologic cofactors such as
ristocetin. However, after vWf binds to collagen or is adsorbed to a
plastic surface, platelets bind to the immobilized vWf via the platelet
glycoprotein Ib-IX receptor (6). The altered affinity of vWf for
platelet glycoprotein Ib-IX is presumed due to a collagen-induced conformational change. We hypothesized that the apparent reduction in
factor VIII-binding sites on vWf bound to microtiter wells may reflect
a conformational change, induced by binding to collagen, that affects
the affinity for factor VIII as well as for platelet glycoprotein
Ib-IX.
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EXPERIMENTAL PROCEDURES |
Materials--
Fluorescein-5-maleimide, fluorescein
isothiocyanate, and Alexa-conjugated streptavidin were purchased from
Molecular Probes, Inc. (Eugene, OR). 20-µm polystyrene microspheres
(density = 1.05 g/liters) were from Duke Scientific Corp. (Palo
Alto, CA). Optiprep (1.3 g/liter) was from Life Technologies, Inc.
Bovine serum albumin (BSA), benzamidine,
-aminocaproic acid, and
Histopaque (1.077 g/liter) were purchased from Sigma. 34-µm Superose
12 microspheres were purchased from Amersham Pharmacia Biotech.
Cryoprecipitate was obtained from the American Red Cross. Recombinant
factor VIII, anti-factor VIII monoclonal antibody F8, and anti-vWf
antibodies 13.7.9 and 16.9.2 were generously provided by D. Pittman
(Genetics Institute Inc., Cambridge, MA). mAb W5-6A was a kind gift of
Dr. D. N. Fass. Calf skin collagen was from Worthington. Heparin
was from Becton and Dickinson.
Proteins--
vWf was purified from cryoprecipitate and
quantified with an enzyme-linked immunosorbent assay as described
previously (23). Recombinant vWf lacking the propeptide, therefore
present only as C-terminal dimers, was expressed and purified as
described previously (19). Recombinant human factor VIII was labeled
with fluorescein-5-maleimide as described previously (3). Anti-von Willebrand factor monoclonal antibody 16.9.2 was labeled with fluorescein isothiocyanate as described (23).
Preparation of Microspheres--
Anti-vWf mAb 13.7.9 was linked
to cyanogen bromide-activated Superose 12 microspheres as described
previously (23). For calf skin collagen immobilization, cross-linked
agarose microspheres 34 µm in diameter (Superose 12; 500 µl) were
equilibrated with 1 M
CO3Na2/HNaCO3 buffer, pH 11. CNBr
(500 µg) dissolved in 500 µl of acetonitrile was added to the
microspheres and incubated for 10 min on an ice bath while swirling. If
necessary, 4 N NaOH was added to the mixture to maintain
the pH between 10.5 and 11. The microspheres were extensively washed
with distilled H2O and 100 mM
carbonate/bicarbonate buffer, pH 9.2. Subsequently, they were washed
with cold 0.1 M sodium citrate, pH 6.5. The slurry of
activated microspheres was added to a tube containing 12 ml of calf
skin collagen (1-1.5 mg/ml) diluted in 0.1 M sodium
citrate and incubated at 4 °C for 48-72 h on an inverting mixer.
The microspheres were then washed with cold 0.1 M sodium
citrate, pH 6.5. Nonspecific active sites were blocked by incubating
the coated microspheres with 10 volumes of 100 mM Tris, pH
8, for 4 h to overnight at 4 °C with gentle mixing.
Microspheres were kept at 4 °C until used, but no longer than 10 days. The microspheres were counted using a Coulter ZM counter.
Collagen deposition was confirmed by indirect immunofluorescence
microscopy; 10 µl of microspheres were incubated with anti-type I
collagen mAb (Sigma) in a final volume of 500 µl on a inverting mixer. After 15 min of incubation at room temperature, the microspheres were washed with Trizma (Tris base)-buffered saline (TBS; 150 mM NaCl and 20 mM Trizma, pH 7.8), and the
fluorescein-labeled anti-mouse antibody (Dako Corp.) was added and
allowed to incubate for 15 min. Control microspheres were
gelatin-coated microspheres (Sigma) and CNBr-activated, uncoated
Superose 12 microspheres. Collagen-positive microspheres had a
distinctive, bright fluorescent rim as seen by fluorescence microscopy,
whereas this property was not observed either on the gelatin
microspheres or on the uncoated ones.
Calf skin collagen was adsorbed to 20-µm diameter polystyrene
microspheres after extensive washing to remove surfactant. Microspheres (1 ml) were sedimented at 3000 rpm × 1 min using a tabletop
microcentrifuge. The pellet was washed with H2O (1 ml), and
the procedure was repeated six times. The microspheres were resuspended
and incubated with 1 ml of 600 µg/ml collagen, pH 6.5, in 50 mM Tris and 150 mM NaCl overnight at 4 °C
while rocking. Unbound collagen was removed by washing once with 10 volumes of TBS. Nonspecific sites were blocked with whole milk
following a 1-h incubation at room temperature. Microspheres were
counted with a Coulter ZM counter. They were flash-frozen in liquid
nitrogen and kept at
80 °C until used.
Type VI collagen purified from human placenta (80 µg/ml) was dialyzed
into 0.5 M
Na2CO3/NaHCO3 with the pH adjusted
to 9.5. 1 ml of CNBr-activated Superose 12 microspheres was added to a 1-ml suspension of type VI collagen, and the reaction was allowed to
proceed for 16 h at 4 °C with gentle rocking. Unreacted active sites were blocked with 0.1 M
-aminocaproic acid, pH 7. Deposition of type VI collagen was confirmed by staining with mAb VI-I,
a secondary biotinylated goat anti-mouse polyclonal antibody, followed by staining with Alexa-conjugated streptavidin with fluorescence detection of microspheres containing and lacking type VI collagen by
flow cytometry. For factor VIII binding experiments, nonspecific binding of factor VIII to type VI collagen was reduced by incubating the microspheres with skim milk for 60 min at room temperature, followed by washing with TBS.
Endothelial cell-conditioned microspheres were prepared as follows.
Following activation with CNBr as described above, Superose microspheres were incubated with 2% (w/v) gelatin in 50 mM
Na2HPO4, pH 7.8, for 16 h at 4 °C.
Gelatin-coated microspheres were stored in 20% ethanol at 4 °C to
minimize the likelihood of microbial contamination. Prior to incubation
with human umbilical vein endothelial cells, prepared by standard
techniques, microspheres were washed twice with endothelial cell growth
medium. Human umbilical vein endothelial cells were grown to confluence
on 100-mm gelatin-coated tissue culture dishes. Cells from one dish
(~2 × 106 cells) were removed by trypsin treatment,
sedimented by gentle centrifugation, and resuspended in 5 ml of growth
medium. The resuspended cells were combined with a 1-ml suspension of
~2.5 × 106 gelatin-conjugated Superose 12 microspheres in endothelial cell growth medium and placed on a 100-mm
bacteriological Petri dish on a rocker at 37 °C. Following 3 days of
growth, the microspheres were harvested and washed by centrifugal
sedimentation with Hanks' buffered saline. The endothelial cells were
removed by treatment with 0.1 M NH4OH as
described previously (36). Briefly, microspheres were resuspended in 5 ml of 0.1 M NH4OH, incubated for 15 min at room
temperature, sedimented, and resuspended in 0.1 M
NH4OH for 15 min. Microspheres were then washed three times
with Hanks' buffered saline and finally resuspended in TBS. The
conditioned microspheres were stored at 4 °C. All experiments were
performed within 2 weeks of endothelial cell stripping.
Equilibrium Binding Assay--
Binding of fluorescein-labeled
factor VIII to vWf immobilized on mAb 13.7.9 or collagen-Superose 12 was measured as described previously (23). The same assay was employed
to detect binding of fluorescein-labeled mAb W5-6A to vWf.
Factor VIII binding to collagen-bound vWf on polystyrene
microspheres was conducted as following. vWf (6.6 µg) was incubated with 3 × 106 collagen-coated polystyrene microspheres
in a final volume of 720 µl in TBS containing 0.01% Tween 80 and BSA
(TBST-BSA) for 45 min at room temperature while shaking in a Vortex
mixer. Free vWf was separated from collagen-bound vWf on microspheres
by sedimentation onto a density gradient. vWf/collagen-coated
microspheres were layered onto 5.5 ml of TBST-BSA/Histopaque (1.015 g/liter) over a sublayer of 1 ml of TBST-BSA/Optiprep (1.221 g/liter).
The preparation was centrifuged for 30 min at 400 × g
before removal of microspheres from the interface between the two
density layers. Microspheres were washed with 1 ml of TBST-BSA, and
aliquots containing 30,000 microspheres were incubated with increasing
concentrations of fluorescein-labeled factor VIII or mAb 16.9.2, as
described above. After 15 min, vWf-bound fluorescein-labeled factor
VIII or fluorescein-labeled mAb 13.7.9 was measured by flow cytometry
as described previously (23).
Kinetic Binding Assays--
vWf (117 ng) was immobilized onto
200,000 mAb- or collagen-coated Superose 12 microspheres by incubating
for 45 min at room temperature with continuous mixing on a Vortex
mixer. The association of fluorescein-labeled factor VIII with vWf was
monitored after the addition of either 1 or 10 nM
fluorescein-labeled factor VIII to suspended vWf-coated mAb
13.7.9-Superose microspheres or 5 or 10 nM
fluorescein-labeled factor VIII to vWf-coated collagen-Superose microspheres. At timed intervals, 50-µl aliquots were diluted to 500 µl, and bound factor VIII was measured immediately using flow
cytometry. The average elapsed time between sample dilution and
completion of data acquisition was ~20 s.
For dissociation experiments, 10 nM fluorescein-labeled
factor VIII was allowed to equilibrate for 15 min with vWf bound to collagen-Superose or mAb 13.7.9-Superose. The dissociation of factor
VIII from bound vWf was measured following the addition of 100 nM unlabeled factor VIII. At timed intervals, 50-µl
aliquots were taken, and the fluorescence/microsphere was monitored as described above.
Data Analysis--
Control experiments were performed to measure
fluorescence due to nonspecific binding of fluorescein-labeled factor
VIII to microspheres lacking vWf. This background fluorescence, ~20%
of total fluorescence at 1 nM factor VIII and 50% of total
fluorescence at 16 nM factor VIII, was subtracted from the
total fluorescence to obtain the specific fluorescence. The figures
display specific fluorescence, and Table I reports values derived from
specific fluorescence. Equilibrium binding data were analyzed using the equation F = F0 + Fmax(VIIIf/(VIIIf + KD)), where F is the measured
fluorescence/Superose bead, F0 is the
fluorescence/Superose bead in the absence of fluorescein-labeled factor
VIII, Fmax is the maximum fluorescence with
saturating concentrations of factor VIII, KD is the
dissociation constant for factor VIII binding to vWf, and
VIIIf is the molar concentration of fluorescein-labeled factor VIII. We assumed that the fraction of total factor VIII bound
could be neglected (the nominal concentration of factor VIII-binding
sites was 0.1 nM, assuming one binding site/vWf subunit). The variables KD and Fmax
were determined using nonlinear least-squares analysis with FitAll
Version 4.0 software (MTR Software, Toronto). The stoichiometry between
factor VIII-binding sites and vWf subunits was determined using
fluorescein-labeled mAb 13.7.9 as described under "Results."
Kinetic association data were fitted according to first-order binding
kinetics using nonlinear least-squares software (FitAll Version 4.0),
assuming that the concentration of free fluorescein-labeled factor VIII
did not change significantly in the course of the reaction (see above).
Kinetic dissociation data were fitted according to exponential decay
with a non-zero plateau using FitAll Version 4.0.
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RESULTS |
Plasma Proteins and Factor VIII-vWf Binding--
Purified
suspended vWf displays one high affinity binding site for factor VIII
on each vWf subunit; yet in plasma, only 1-2% of these sites are
actually occupied by factor VIII. We asked whether plasma contains
protein(s) that inhibit binding of factor VIII to some fraction of vWf
subunits. To address this question, we evaluated binding of factor VIII
to immobilized vWf in the presence and absence of plasma. Purified vWf
was immobilized on the non-inhibitory mAb 13.7.9 linked to 34-µm
diameter cross-linked agarose microspheres (Superose). We have recently
shown that vWf immobilized in this way displays one high affinity
binding site/subunit, equivalent to suspended vWf (23). Varying
concentrations of fluorescein-labeled factor VIII were incubated with
vWf microspheres and either buffer or a vWf-deficient plasma/buffer
mixture at a 1:1 ratio. Fluorescein-labeled factor VIII bound saturably
and with high affinity to immobilized vWf in the presence or absence of
plasma (Fig. 1). The equivalent shapes
and plateaus of the two binding isotherms indicated that plasma does
not contain proteins that substantially inhibit binding of factor VIII
to vWf.

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Fig. 1.
Effect of plasma on factor VIII binding to
von Willebrand factor. vWf was immobilized on mAb 13.7.9 coupled
to Superose microspheres as described under "Experimental
Procedures." Increasing amounts of fluorescein-labeled factor VIII
were incubated with the immobilized vWf for 15 min in buffer ( ) or
50% plasma ( ) prior to evaluation by flow cytometry. Plasma and the
buffer control each contained 10 mM sodium citrate and 12.5 mM CaCl2 with 1 unit/ml heparin, 5 mM benzamidine, and 5 mM -aminocaproic acid
to inhibit plasma proteases. The results displayed are from a single
experiment representative of two such experiments.
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Calf Skin Collagen and Factor VIII-vWf Binding--
We
hypothesized that vWf has decreased affinity for factor VIII when bound
to collagen. To test this hypothesis, calf skin collagen was adsorbed
to 20-µm diameter polystyrene microspheres, and the microspheres were
incubated with suspended vWf. Saturable binding of vWf to collagen was
detected by flow cytometry with fluorescein-labeled mAb 13.7.9. Half-maximal binding occurred at a vWf concentration of 1 µg/ml. We
performed kinetic association and dissociation experiments to determine
whether collagen-bound vWf would remain stably bound over the time
period necessary for factor VIII binding. The results indicated that
~40% of the bound material dissociated over 20 min, but that
following the initial dissociation, at least 80% of bound vWf remained
stably bound for >2 h. Thus, all equilibrium and kinetic factor VIII
binding experiments were initiated at least 20 min after washing free vWf from the microspheres and completed within 2 h.
Increasing concentrations of fluorescein-labeled factor VIII were added
to the vWf/collagen-coated microspheres, and binding of factor VIII was
measured by flow cytometry. The affinity of collagen-bound vWf for
factor VIII (KD = 5 nM) was decreased compared with the affinity of antibody-bound vWf (KD = 0.9 nM) (Table I). To
quantify the number of vWf subunits on the collagen-coated
microspheres, binding of fluorescein-labeled anti-vWf mAb 16.9.2 to
microspheres was measured (data not shown). Comparison of the maximum
fluorescence/microsphere from vWf-bound mAb 16.9.2 with the maximum
fluorescence from vWf-bound factor VIII allowed calculation of the
apparent stoichiometry between bound factor VIII and vWf subunits. The
results indicated that one vWf subunit bound one factor VIII molecule
whether the vWf was bound to mAb 13.7.9 or to collagen (Table I).
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Table I
Measured parameters for the binding of factor VIII to vWf on collagen
versus anti-vWf monoclonal antibody
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We were concerned that the decreased affinity of collagen-bound vWf for
factor VIII may not be due solely to an effect of collagen on vWf, but
may also reflect an effect of the polystyrene matrix to which collagen
was adsorbed. Therefore, we prepared cyanogen bromide-activated
Superose microspheres and allowed them to couple to calf skin collagen,
which contains type I and III collagens. Deposition of collagen on the
Superose microspheres was confirmed by indirect immunofluorescence
microscopy using an anti-type I collagen mAb. Collagen attachment was
also evident by inspection as the Superose microspheres sedimented to a
less dense pellet than control microspheres and had a fuzzy-appearing margin when observed by phase-contrast microscopy. vWf bound to collagen-Superose microspheres was detected by fluorescein-labeled mAb
16.9.2. Binding of fluorescein-labeled factor VIII to vWf on
collagen-Superose microspheres was evaluated in comparison with vWf on
mAb 13.7.9-Superose microspheres (Fig.
2). The results confirmed that the
affinity of collagen-bound vWf for factor VIII was decreased ~4-fold
compared with that of mAb-immobilized vWf (Fig. 2 and Table I). The
stoichiometry of factor VIII to vWf subunits was 1:1 on
collagen-Superose and mAb 13.7.9-Superose microspheres.

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Fig. 2.
Decreased affinity of collagen-bound vWf for
factor VIII. vWf was allowed to adhere to collagen-Superose and
mAb 13.7.9-Superose microspheres as described under "Experimental
Procedures." Increasing concentrations of fluorescein-labeled factor
VIII were incubated for 45 min with vWf bound to mAb 13.7.9 ( ) or
collagen ( ). The quantity of bound factor VIII/bead was measured by
flow cytometry. The fitted curves correspond to KD
values of 4.3 and 1.3 nM for collagen-bound and mAb
13.7.9-bound vWf, respectively.
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Subendothelial Matrix and Type VI Collagen--
The experiments
described above provide a model of plasma vWf that adheres to collagen
at a site of vascular injury. In vivo vWf is also secreted
from the basolateral surface of endothelial cells and is then found
associated with type VI collagen fibrils in the subendothelial matrix.
We wished to know if vWf deposited in the subendothelial matrix also
bound factor VIII with reduced affinity. Accordingly, we cultured
endothelial cells on gelatin-coated Superose microspheres. After
72 h, the endothelial cells were removed, and microspheres were
stained with fluorescein-labeled mAb 16.9.2. The results indicated that
vWf had been deposited. Factor VIII bound saturably to vWf, with a
KD of 4.1 nM (Fig.
3A and Table I), comparable to
vWf bound to calf skin collagen. There were 3.5-fold more factor
VIII-binding sites accessible than mAb 16.9.2 epitopes, indicating that
access to the mAb 16.9.2 epitope is hindered by matrix proteins to a
greater degree than access to factor VIII-binding sites. These results
indicate that vWf in the subendothelial matrix has decreased affinity
for factor VIII.

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Fig. 3.
Decreased affinity of subendothelial matrix
vWf and type VI collagen-bound vWf for factor VIII. A,
vWf and other matrix proteins were deposited by human umbilical vein
endothelial cells on Superose microspheres as described under
"Experimental Procedures." Increasing concentrations of
fluorescein-labeled factor VIII were incubated 45 min with endothelial
cell-conditioned Superose microspheres, and the quantity of bound
factor VIII/bead was measured by flow cytometry. The fitted curve
corresponds to a KD of 5.1 nM for this
representative experiment. B, varying concentrations of
fluorescein-labeled factor VIII were incubated with vWf bound to
purified type VI collagen coupled to Superose microspheres. After 15 min, bound factor VIII was evaluated by flow cytometry. The fitted
curve corresponds to a KD of 6.4 nM for
this representative experiment.
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Smooth muscle cells efficiently produce type VI collagen, which is
incorporated into the vascular matrix, whereas endothelial cells are
inefficient producers of type VI collagen (37). Therefore, it is likely
that the results in Fig. 3A do not reflect the affinity of
factor VIII for vWf bound to type VI collagen. To explicitly examine
the factor VIII-binding properties of vWf bound only to type VI
collagen, we linked purified human type VI collagen to Superose
microspheres. vWf was incubated with these microspheres, and deposition
of vWf was confirmed with fluorescein-labeled mAb 16.9.2. Factor VIII
bound saturably to type VI collagen-bound vWf, with an average
KD of 8.3 nM (Fig. 3B and
Table I). This dissociation constant indicates that vWf has reduced affinity for factor VIII when it is bound to type VI collagen, comparable to the reduced affinity when bound to calf skin collagen. In
contrast to calf skin collagen-bound vWf, type VI collagen-bound vWf
displayed only one factor VIII-binding site for every three epitopes of
mAb 16.9.2. Thus, binding of vWf to type VI collagen differs from
binding to calf skin collagen in that some factor VIII-binding sites
are made inaccessible by the interaction between vWf and collagen.
Collagen and Factor VIII-vWf Binding Kinetics--
To determine
whether the decreased affinity of factor VIII for collagen-bound vWf
was due to a decreased association rate or to an increased dissociation
rate, we performed kinetic binding experiments. vWf was immobilized on
mAb 13.7.9- or collagen-Superose microspheres. Fluorescent factor VIII
was added to mAb 13.7.9-immobilized or collagen-immobilized vWf. At
timed intervals, 50-µl aliquots were removed from the reaction
mixture, and bound factor VIII was measured by flow cytometry (Fig.
4A). The averaged association rates were 0.07 nM
1 min
1 for
mAb-immobilized vWf and 0.05 nM
1
min
1 for collagen-immobilized vWf (Table I), indicating
that the rate of association of factor VIII with vWf is not
significantly affected after vWf binds to collagen. The dissociation of
factor VIII from collagen-bound and mAb 13.7.9-bound vWf was monitored after the addition of 100 nM unlabeled factor VIII to the
fluorescent factor VIII-vWf equilibrium complex (Fig. 4B).
The dissociation of factor VIII from mAb 13.7.9-immobilized vWf was
relatively slow, comparable to the previously reported value (34). By
comparison, the dissociation from collagen-bound vWf was 7-fold faster.
The averaged separation constants (koff) were
0.07 and 0.77 min
1 for mAb 13.7.9-immobilized and
collagen-immobilized vWf, respectively (Fig. 4B). The
precision of experiments in which the dissociation of factor VIII from
collagen-bound vWf was measured is limited because the 30-s increments
at which bound factor VIII was measured allowed most of the
dissociation to occur within only the first two readings. However, the
data are sufficient to indicate that the lower affinity of factor VIII
for collagen-bound vWf relates primarily to a more rapid dissociation
rate.

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Fig. 4.
Accelerated dissociation of factor VIII from
collagen-bound vWf. A, representative experiments
comparing the association of fluorescein-labeled factor VIII with vWf
on collagen-Superose ( ) versus mAb 13.7.9-Superose ( ).
10 nM fluorescein-labeled factor VIII was added to
suspended vWf-coated microspheres. At timed intervals, 50-µl aliquots
were removed, and the quantity of bound factor VIII/microsphere was
measured immediately by flow cytometry. The fitted curves correspond to
kon values of 0.084 and 0.044 nM 1 min 1 for collagen-Superose
and mAb 13.7.9-Superose, respectively. B, representative
experiments comparing the dissociation of factor VIII from vWf on
collagen-Superose ( ) versus mAb 13.7.9-Superose ( )
after the addition of excess unlabeled factor VIII. After 10 nM fluorescein-labeled factor VIII was allowed to
equilibrate with immobilized vWf for 15 min, 100 nM
unlabeled factor VIII was added, and the residual bound vWf was
measured at timed intervals. The fitted curves displayed correspond to
koff values of 1.3 and 0.1 min 1
for collagen-Superose and mAb 13.7.9-Superose, respectively.
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Collagen and Affinity of vWf for a Monoclonal Antibody--
The
decreased affinity of collagen-bound vWf for factor VIII is most
readily explained by a collagen-induced conformational change in vWf.
If the conformation of the factor VIII-binding motif is altered, then
the affinity of vWf for other molecules that bind this motif may be
changed. We asked whether the affinity of mAb W5-6A, which competes
with factor VIII for its binding site on vWf (21), may also be
sensitive to conformational changes that occur within the vWf site.
Binding of fluorescein-labeled mAb W5-6A to vWf was measured on
collagen-Superose microspheres versus mAb 13.7.9-Superose
microspheres (Fig. 5). The results indicated that the affinity of mAb W5-6A was decreased ~6-fold when
vWf was bound to collagen. This reduction in affinity parallels the
reduced affinity of factor VIII for collagen-bound vWf (Fig. 2),
suggesting that the same change in vWf alters binding affinity for both
proteins. We also measured the affinity of mAb W5-6A for vWf deposited
on Superose microspheres by endothelial cells and for vWf bound to
purified type VI collagen (data not shown). In both cases, the affinity
was reduced at least 6-fold, with a KD of ~35
nM, comparable to the affinity for vWf bound to calf skin
collagen.

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Fig. 5.
Anti-vWf mAb W5-6A, with an epitope in the
factor VIII-binding site, has reduced affinity for collagen-bound
vWf. Collagen-Superose-immobilized vWf ( ) and mAb
13.7.9-Superose-immobilized vWf ( ) were prepared as described under
"Experimental Procedures." Increasing concentrations of
fluorescein-labeled mAb W5-6A were incubated with vWf for 15 min, and
binding was monitored by flow cytometry. The dissociation constants
corresponding to the fitted curve were 27 nM for
collagen-bound vWf and 4.7 nM for mAb 13.7.9-bound vWf,
respectively. The fluorescence data for the two experiments shown are
normalized to the asymptotic saturation point of the fitted curves for
visual comparison. Data are representative of three experiments for
collagen-bound vWf and six experiments for mAb 13.7.9-bound vWf.
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Since binding of mAb W5-6A is apparently sensitive to the
collagen-induced change in the factor VIII-binding motif of vWf, we
asked whether it may also be sensitive to another conformational change. We have recently observed that dimeric vWf, lacking N-terminal intersubunit disulfide bonds, has a 6-fold decreased affinity for
factor VIII (23). We have hypothesized that the gain in affinity
exhibited by dimeric vWf upon formation of the N-terminal intersubunit
disulfide bond reflects a conformational change in the factor
VIII-binding motif. We therefore compared binding of fluorescein-labeled mAb W5-6A to plasma vWf versus
recombinant dimeric vWf lacking N-terminal intersubunit disulfide bonds
(Fig. 6). Both forms of vWf were
immobilized on mAb 13.7.9-Superose microspheres and incubated with
fluorescein-labeled mAb W5-6A. The results indicated that mAb W5-6A
recognized plasma vWf and dimeric vWf with affinities that were within
2-fold of each other. We interpret these results as indicating that mAb
W5-6A is not sensitive to the conformational change in vWf that
results from formation of N-terminal intersubunit disulfide bonds.
These data suggest that the N-terminal portion of vWf subunits is
susceptible to two conformational changes. One change is induced by
formation of intersubunit disulfide bonds, increasing affinity for
factor VIII; and the other is induced by collagen binding, decreasing affinity for factor VIII.

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Fig. 6.
mAb W5-6A binds to dimeric vWf and
multimeric plasma vWf with the same affinity. Various
concentrations of fluorescein-labeled mAb W5-6A were incubated with
vWf ( ) or a mutant vWf dimer lacking N-terminal intersubunit
disulfide bonds ( ) immobilized on mAb 13.7.9-Superose microspheres.
Binding was measured by flow cytometry. The dissociation constants
corresponding to the best fit curves were 4.4 and 3.4 nM
for vWf and dimeric vWf, respectively. The data displayed are
representative of six experiments (multimeric vWf) and two experiments
(dimeric vWf).
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DISCUSSION |
The results in this report indicate that plasma proteins do not
alter the affinity of factor VIII for vWf or the stoichiometry with
which factor VIII binds to vWf subunits. Therefore, the ~1:50 ratio
between factor VIII and vWf subunits in circulating blood (31, 38)
cannot be explained by plasma proteins that modify the availability of
vWf subunits for factor VIII binding or the affinity of those sites for
factor VIII. However, our results indicate that when vWf adheres to
collagen, the affinity of vWf subunits for factor VIII is reduced. The
decreased affinity of vWf is due to an enhanced off-rate of factor
VIII, which is increased ~10-fold. The dissociation half-time is 1 min for collagen-bound vWf versus 10 min for suspended vWf.
The more rapid release suggests the possibility that, when vWf binds to
collagen at the site of vascular injury, factor VIII released by this
mechanism may contribute to initiation of the coagulation cascade. It
also suggests that collagen-bound vWf, which is a ubiquitous
constituent of the subendothelial vascular matrix, has a reduced
capacity to compete for factor VIII compared with plasma vWf.
We have found that the number of factor VIII-binding sites/vWf subunit
is 1:1. These findings agree with those of Lollar and Parker (29), who
studied factor VIII-vWf association by velocity sedimentation; with
those of Vlot et al. (30), who studied VIII/vWf stoichiometry either on colloidal gold or using gel-filtration chromatography on Sepharose; and with those of Saenko and Scandella (35), who measured factor VIII binding to suspended vWf in a competition assay. In contrast, other studies have concluded that the
number of factor VIII-binding sites/vWf subunit is <1:1. From binding
competition experiments, Nesheim et al. (32) inferred that
the factor VIII/vWf stoichiometry is ~1:10 at saturation. In studies
in which vWf was adsorbed to polystyrene microtiter wells, the factor
VIII/vWf subunit stoichiometries were 1:40 (39), 1:50 (30), and 1:70
(33). The detected factor VIII binding on microtiter plates is greatly
diminished if the rate of dissociation of factor VIII from vWf is
increased so that bound factor VIII is removed in the wash steps.
Therefore, it remains possible that interaction of vWf with microtiter
plate plastic causes a reduced affinity of most vWf subunits for factor
VIII, analogous to the effect of collagen. Binding to the subunits with
reduced affinity may not have been detected because of the experimental
design. In our preliminary experiments in which vWf was immobilized on collagen adsorbed to polystyrene microspheres, we detected reduced affinity of vWf subunits comparable to the reduced affinity on collagen-Superose microspheres, suggesting that a polystyrene matrix
does not necessarily inhibit the affinity of vWf subunits for factor
VIII to a greater degree than collagen alone.
The results in this report indicate that the affinity of vWf subunits
for factor VIII is reduced 4-6-fold when vWf is bound to collagen. The
reduced affinity results from an enhanced dissociation rate with an
essentially unchanged association rate. By comparing the effects of
collagen adsorbed to solid polystyrene microspheres versus
collagen covalently coupled to porous agarose microspheres, we have
confirmed that the decreased affinity is related to an effect of
collagen rather than an effect of the matrix to which the collagen was
attached. In these experiments, we have measured an average affinity of
vWf subunits for factor VIII. Our results do not indicate what fraction
of vWf subunits actually bind to collagen versus reside as
constituents of vWf multimers tethered to the collagen surface.
Therefore, it is possible that vWf subunits that bind to collagen have
a lower affinity for factor VIII and that non-interacting subunits
retain higher affinity. Alternatively, all vWf subunits may have
affinity reduced to the same degree if a conformational change induced
by collagen propagates through intersubunit disulfide bonds from
collagen-binding subunits to non-binding subunits.
We believe that our results may be best explained by a conformational
change in the factor VIII-binding D' domain of vWf. We argue that the
alternative explanation, steric hindrance of the factor VIII-binding
site by collagen, is less likely, first because the collagen-binding
motif in the A3 domain of vWf is 922 amino acids distant from the D'
domain in linear sequence, and second because steric hindrance would
probably ablate factor VIII-binding sites rather than reduce affinity
4-6-fold. The probability of a collagen-induced conformational change
is further increased by the effect of collagen on the affinity of vWf
subunits for mAb W5-6A, which recognizes part of the factor
VIII-binding motif in the D' domain of vWf (21). The affinity of vWf
for mAb W5-6A was reduced ~6-fold, suggesting that the epitope of
mAb W5-6A, including amino acid residues 78-96 within the D' domain
of vWf, is a local region within the factor VIII-binding motif that is subject to conformational change.
We have recently observed that the terminal subunits of vWf multimers,
which lack N-terminal intersubunit disulfide bonds, have 6-fold reduced
affinity for factor VIII (23). The similarity of this reduction in
affinity to the effect produced by collagen led us to ask whether
collagen induces a relaxed conformation of the factor VIII-binding
motif equivalent to non-disulfide-linked vWf N termini. To address this
question, we asked whether the affinity of mAb W5-6A is altered to the
same degree on vWf dimers lacking N-terminal intersubunit disulfide
bonds as on collagen-bound vWf. Since the affinity of mAb W5-6A is not
reduced on dimeric vWf, but is reduced 6-fold on collagen-bound vWf, we
speculate that the factor VIII-binding motif, located primarily in the
D' domain of vWf subunits, has three conformations. In this scheme, the
conformation is relaxed, with a low affinity for factor VIII, prior to
formation of intersubunit disulfide bonds. Following formation of
N-terminal intersubunit disulfide bonds (multimer formation), the
conformation of the D' domain is strained into a high affinity factor
VIII-binding conformation. This high affinity conformation is altered
when vWf binds to collagen. Our results do not elucidate the mechanism
through which binding of vWf to collagen via the A3 domain may transmit
a conformational change to the D' domain, nor do they indicate whether
a conformational change may be propagated from one collagen-binding vWf
subunit to another that may not bind vWf.
The affinity of vWf for platelet glycoprotein Ib-IX is increased when
vWf is exposed to shear stress (40, 41) or binds to polycations such as
ristocetin and polymerizing fibrin (42) or collagen (6). The most
likely explanation for the increased affinity upon collagen binding is
a conformational change in the platelet glycoprotein Ib-IX-binding A1
domain (43). The relationship of this conformational change to the
affinity of vWf subunits for factor VIII is currently unknown. We are
attracted to the possibility that a single conformational change
increases the affinity for platelet glycoprotein Ib-IX and decreases
the affinity for factor VIII.
Because vWf lines the entire vascular tree, our results have
implications for the possible partitioning of factor VIII between plasma vWf and subendothelial matrix vWf. Our results indicate that
factor VIII should preferentially partition to plasma vWf (as it
apparently does) because of higher affinity for suspended vWf subunits.
However, they leave open the possibility of subendothelial matrix vWf
serving as a lower affinity reservoir for factor VIII, which rapidly
equilibrates with the plasma factor VIII compartment. In support of
this possibility, our experiments with endothelial cell-deposited vWf
indicate an affinity for factor VIII equivalent to the affinity of
collagen-bound vWf for factor VIII. However, the ability of factor VIII
to traverse the endothelial cell barrier between plasma and the
subendothelial matrix is a critical unknown factor that may limit the
capacity of matrix vWf to serve as a factor VIII reservoir.