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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 529-538
Binding of Factor VIII to von Willebrand Factor Is Enabled by
Cleavage of the von Willebrand Factor Propeptide and Enhanced by
Formation of Disulfide-Linked Multimers
By
Ana Victoria Bendetowicz,
Jill A. Morris,
Robert J. Wise,
Gary E. Gilbert, and
Randal J. Kaufman
From the Brockton/West Roxbury VAMC, West Roxbury, MA; the Brigham
and Women's Hospital, Boston, MA; the Harvard University Medical
School, Boston, MA; the Department of Biological Chemistry and the
Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI.
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ABSTRACT |
von Willebrand factor (vWF) is a multimeric adhesive glycoprotein
with one factor VIII binding site/subunit. Prior reports suggest that
posttranslational modifications of vWF, including formation of
N-terminal intersubunit disulfide bonds and subsequent cleavage of the
propeptide, influence availability and/or affinity of factor
VIII binding sites. We found that deletion of the vWF propeptide
produced a dimeric vWF molecule lacking N-terminal intersubunit
disulfide bonds. This molecule bound fluorescein-labeled factor VIII
with sixfold lower affinity than multimeric vWF in an equilibrium flow
cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide
in trans yielded multimeric vWF that displayed increased affinity for
factor VIII. Insertion of an alanine residue at the N-terminus of the
mature vWF subunit destroyed binding to factor VIII, indicating that
the native mature N-terminus is required for factor VIII binding. The
requirement for vWF propeptide cleavage was shown by (1) a point
mutation of the vWF propeptide cleavage site yielding pro-vWF that was
defective in factor VIII binding and (2) correlation between efficiency
of intracellular propeptide cleavage and factor VIII binding.
Furthermore, in a cell-free system, addition of the propeptide-cleaving
enzyme PACE/furin enabled factor VIII binding in parallel with
propeptide cleavage. Our results indicate that high-affinity factor
VIII binding sites are located on N-terminal disulfide-linked vWF
subunits from which the propeptide has been cleaved.
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INTRODUCTION |
FACTOR VIII FUNCTIONS in the intrinsic
pathway of blood coagulation as a cofactor to accelerate the activation
of factor X by factor IXa, a reaction that occurs on phosphatidylserine
(PS)-containing membranes in the presence of calcium ions.1
Deficiency in factor VIII is the cause of the X-chromosome
linked bleeding disorder hemophilia A.2 Factor VIII
circulates in plasma bound to von Willebrand factor (vWF) in a
noncovalent protein complex. While bound to vWF in plasma, factor VIII
is prevented from binding to PS-containing membranes, an interaction
that inhibits both binding to factor IXa and degradation by activated
protein C.3,4 In addition, the presence of vWF in the
conditioned medium can promote stable accumulation of factor VIII
secreted from transfected cells in culture.5,6
vWF is a large adhesive glycoprotein that is synthesized in
megakaryocytes and endothelial cells. The primary amino acid sequence of vWF identified homologous domains arranged in the order
D1-D2-D -D3-A1-A2-A3-D4.7-9 vWF is synthesized as a
precursor with a large propeptide, encompassing domains D1-D2, that is
cleaved upon transport through the secretory apparatus at the Arg-Ser
bond at residue 763-764. Fully processed vWF is a multimeric protein
consisting of disulfide-bonded monomeric subunits that range from
dimers to multimers extending up to 20 × 106
Daltons.10,11 Initial dimerization mediated through
disulfide linkages within the carboxyl (C)-terminal region of
the protein occurs within the endoplasmic reticulum.12-14
The vWF propeptide is required to promote polymerization of vWF dimers
in the trans-Golgi compartment through disulfide bonds between
D3 domains of opposing subunits.15,16 Identified factor
VIII binding site determinants lie within the D domain at the
amino (N)-terminus of mature vWF subunits.17-20
In endothelial cells, multimeric vWF assembly proceeds to a high degree
before propeptide cleavage.21 Furthermore, mutagenesis studies have shown that propeptide cleavage is not required for vWF
multimerization.15,16 Constitutively secreted vWF from endothelial cells as well as vWF from recombinant expression systems is
not completely processed, with some subunits retaining the propeptide.9,13,22 Coexpression of PACE/furin, an
intracellular propeptide processing enzyme, with vWF enhances the
secretion of completely processed vWF.23,24 Deletion of the
vWF propeptide produces efficiently secreted dimeric vWF ( Pro-vWF)
that is not multimerized, indicating that the propeptide is necessary
to promote the formation of N-terminal intersubunit disulfide
bonds.15,16 However, coexpression of the propeptide with
Pro-vWF leads to limited multimerization via N-terminal disulfide
bond formation, indicating that the propeptide does not have to be
covalently linked to vWF to promote multimerization.15
Although each subunit of a vWF multimer contains one factor VIII
binding site, the ratio of circulating factor VIII to vWF observed in
vivo is approximately 1:50.25-28 In vitro binding studies have yielded conflicting data for the number of available factor VIII
binding sites/vWF subunit, with reported ratios from 1:1 to as low as
1:70.29-33 Although the source for the
differences remains uncertain, these reports raise the possibility that
either not all factor VIII binding sites are accessible or that
affinity of sites is altered by other factors. Because each circulating vWF molecule contains 2 terminal non-disulfide-linked N-termini and
2-80 internal disulfide-linked N-termini, altered binding due to
disulfide bond formation may influence the location of factor VIII on
vWF molecules in vivo. Determining the relationship between the
maturation events of disulfide bond formation and propeptide cleavage
and formation of high affinity factor VIII binding sites is a chief
motivation of the following studies.
The influence of vWF processing events on factor VIII binding was
previously studied by expression of site-directed mutants of vWF and
yielded conflicting results.34,35 The propeptide cleavage
site mutation R763K at the P1 residue yielded pro-vWF that did not bind
or stabilize factor VIII expressed from Chinese hamster ovary (CHO)
cells.34 Deletion of the propeptide yielded dimeric vWF
that was capable of binding and stabilizing factor VIII expressed from
CHO cells.34 Although these studies were not designed to
quantitate binding affinity, they suggested that propeptide removal was
required to expose a factor VIII binding site. In contrast, studies
using a different expression system and a different method of analysis
with a similar propeptide deletion mutant that contained an extra
alanine at the junction of the signal peptide and the mature vWF
sequences (vWFdelpro),35 designated Pro+A herein, did
not detectably bind factor VIII. To elucidate the structural
requirements for vWF binding to factor VIII, we have examined the
binding of factor VIII to vWF containing and lacking the propeptide,
vWF containing and lacking N-terminal intersubunit disulfide bonds, and
dimeric vWF containing and lacking the N-terminal alanine residue. Our
studies demonstrate that formation of N-terminal disulfide bonds
between vWF dimers is necessary for formation of the highest affinity
factor VIII binding sites and that these sites remain inaccessible
until the propeptide has been cleaved.
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MATERIALS AND METHODS |
Materials
Fetal bovine serum (FBS) and Dulbecco's Modified Essential Medium
(DMEM) were obtained from Mediatech (Herndon, VA). OptiMEM medium and
goat antirabbit secondary antibody were purchased from GIBCO-BRL
(Gaithersburg, MD). Aprotinin and phenylmethyl sulfonyl fluoride (PMSF)
were from Boehringer Mannheim (Chicago, IL). Bovine brain PS,
phosphatidyl ethanolamine synthesized by transphosphatidylation of egg
phosphatidyl-choline, and egg phosphatidyl-choline were from Avanti
Polar Lipids Inc (Alabaster, AL). Cholesterol was from Calbiochem (San
Diego, CA). Soybean trypsin inhibitor, bovine serum
albumin (BSA), and o-phenylenediamine dihydrochloride (OPD) tablets
were from Sigma Corp (St Louis, MO). Unless otherwise noted, anti-vWF
polyclonal antibody and anti-vWF horseradish peroxidase (HRP)-conjugated antibody were from Dako Corp (Carpinteria, CA). Fluorescein-5-maleimide and fluorescein isothiocyanate (FITC) were
purchased from Molecular Probes, Inc (Eugene, OR). Superose-12 beads
(34 µm) were purchased from Pharmacia Biotech (Piscataway, NJ). Centricon and Microcon concentrators were purchased
from Amicon Corp (Danvers, MA). Immulon 2 membranes and microtiter plates were from Dynatech (Chantilly, VA). vWF normal reference plasma
was from Bio/Data Corp (Horsham, PA). The enhanced chemiluminescence system (ECL) was from Amersham Corp (Arlington Heights, IL).
Cryoprecipitate was obtained from the American Red Cross. Recombinant
factor VIII, monoclonal antibody (MoAb) to factor VIII,
F8,36 and anti-vWF MoAbs vW/13.7.9 and vW/16.9.2 were
generously provided by D. Pittman (Genetics Institute Inc, Cambridge,
MA).
Fluorescein Labeling
Factor VIII was labeled with fluorescein-5-maleimide, as described
previously.3,37 Protein concentration of factor VIII was
determined using a micro-bicinchoninic acid assay (Pierce, Rockford,
IL) using BSA as a standard. Anti-vWF MoAb vW/16.9.2 was
labeled with FITC as described.38 Protein concentration was
measured by absorbance at 280 nm with correction for fluorescein absorbance at 280 nm (A280  0.21 × A490). Efficiency of labeling was determined by comparing
absorbance at 490 nm to corrected absorbance at 280 nm.
Expression Vectors
pMT2-vWF,15 pMT2-KRX (the vWF
propeptide alone), pMT2-Pro-vWF, and Pro+A were
previously described.15,35 The expression vectors encoding
wild-type PACE/furin (pMT2-PACE) and mutant
PACE/furin (pMT2-PACE Sol and pMT2-PACE
SA) and vWF propeptide cleavage site mutant molecules
(pMT2vWF-R760K and pMT2-vWF-RXK/KXD, herein
described as R760K/K762D) were previously
described.9,15,23,39 Escherichia coli DH5 was
used to prepare plasmid DNA that was purified by standard cesium
chloride-ethidium bromide gradient equilibrium centrifugation.
vWF Expression in COS-1 and CHO Cells
COS-1 monkey cells were transfected with plasmid DNA by the
diethylaminoethyl dextran procedure.40 Cells were
maintained in 10% FBS in DMEM. At 48 hours posttransfection,
transfected cells were washed with phosphate-buffered saline (PBS) to
remove residual serum and incubated in OptiMEM medium containing 0.2 mg/mL aprotinin. After 24 hours, conditioned medium was harvested and
fresh protease inhibitors were added (0.2 mg/mL aprotinin, 1 mg/mL
soybean trypsin inhibitor, and 1 mmol/L PMSF). The concentration of vWF
in the conditioned medium was measured by enzyme-linked immunosorbent
assay (ELISA). The stably transfected CHO cell lines vWF-wt and
vWF-Pro were previously described.34
vWF Preparation and Evaluation
Human plasma-derived vWF was purified from cryoprecipitate on
sepharose CL4-b (Pharmacia Biotech) equilibrated with 20 mmol/L imidazole, 20 mmol/L  -n-caproic acid, 150 mmol/L sodium chloride, 10 mmol/L sodium citrate, and 0.02% sodium azide, pH 6.5. Its concentration was measured by ELISA, using the polyclonal anti-vWF (and
HRP-conjugated anti-vWF) antibodies cited above. vWF multimers were
analyzed by gel electrophoresis on discontinuous sodium dodecyl sulfate
(SDS)-agarose gels based on methods described
previously.11 Where indicated, vWF was purified from
conditioned medium of transfected cells by immunoaffinity
chromatography as described.34
Preparation of MoAb vW/13.7.9-Coated Superose-12 Microspheres
Superose-12 microspheres (500 µL) were first equilibrated with 1 mol/L CO3Na2/HNaCO3 buffer, pH 11. CNBr (500 µg) dissolved in 500 µL acetonitrile was added to the
microspheres and incubated for 10 minutes on an ice bath while
swirling. If necessary, 4 N NaOH was added to the mix to maintain the
pH between 10.5 and 11. The microspheres were extensively washed with
distilled H2O, 95% acetone, and 200 mmol/L
Na2CO3/HNaCO3 buffer, pH 9.75. Anti-vWF MoAb vW/13.7.9 (500 µL, 0.75 mg/mL) was added to the
microspheres slur and incubated overnight at room temperature on a
rocker. The beads were washed twice with 0.2 mol/L
Na2CO3/HNaCO3, pH 9.75, and once
with 1 mol/L NaCl, 0.05 mol/L
Na2CO3/HNaCO3. Nonspecific active
sites were blocked by incubating the coated beads with 10 vol of 100 mmol/L Tris-HCl 4 hours to overnight at room temperature with gentle
mixing. Beads were kept at 4°C until used. The microsphere concentration was counted using a ZM Coulter-counter (Coulter Instruments, Hialeah, FL).
Factor VIII-vWF Binding Assays
VIII-vWF Binding Assays on Microtiter Plates
Factor VIII-vWF binding assays on microtiter plates were conducted
using two methods. First, Immulon 2 microtiter plates were coated with
5 µg/mL anti-vWF polyclonal antibody in 50 mmol/L Na2CO3/HNaCO3, pH 9.6. Plates were
subsequently washed after each step with TBST buffer (50 mmol/L Tris
HCl, pH 7.6, 150 mmol/L NaCl, and 0.05% Tween 20). Plates were blocked
for 1 hour at 37°C in TBST buffer with 3% BSA. Increasing amounts
of recombinant factor VIII were preincubated with COS-1 cell
conditioned medium containing vWF (100 ng/mL of conditioned medium),
and this was added to the plates. After incubation, the amount of
factor VIII bound to immobilized vWF was measured by factor VIII
Coatest endpoint assay (Kabi Pharmacia, Uppsala, Sweden). The optical
densities at 490 nm were read using an EL340 microtiter plate Bio
Kinetics Reader (Bio-Tek Instruments Inc, Winooski, VT). In the second method, the buffers and washes were the same as in the first method. Microtiter plates were coated with anti-factor VIII MoAb F8 at 2 µg/mL in carbonate buffer. Conditioned medium from transfected COS-1
cells was incubated with increasing concentrations of recombinant factor VIII in TBST buffer containing 10 mmol/L CaCl2 and
3% BSA in polypropylene tubes at 37°C for 2 hours. After
incubation, the conditioned medium/factor VIII samples were added to
coated and blocked microtiter plates for 2 hours at 37°C. The
amount of vWF bound to immobilized factor VIII was measured by adding anti-vWF HRP-conjugated antibody to the plate at 10 µg/mL. After the
addition of OPD, the reaction was stopped by the addition of 2.5 mol/L
H2SO4. The optical density was read and
analyzed as described above.
Flow Cytometry/Equilibrium Binding Assays
Factor VIII binding to antibody immobilized vWF.
vWF (117 ng) was added to 200,000 MoAb vW/13.7.9-Superose microspheres
in a final volume of 50 µL with TBS containing 0.01% Tween 80 and
0.1% BSA, pH 7.85 (TBST-BSA), and incubated at room temperature while
shaking in a vortex mixer. After 45 minutes, the microsphere suspension
was diluted with 50 µL TBST-BSA. At time zero, aliquots containing
6,500 vWF-microspheres were incubated with increasing concentrations of
fluorescein-labeled factor VIII (in buffer containing 0.5 mmol/L
CaCl2) or FITC-MoAb vW/16.9.2 in a final volume of 100 µL
for 15 minutes. The samples were diluted to 500 µL before reading the
fluorescence/microsphere by flow cytometry within 30 seconds of
dilution. Equilibrium binding data were analyzed using the equation
![F = F<SUB>0</SUB> + F<SUB>max</SUB>(VIII<SUB>f</SUB>/[K<SUB>D</SUB> + VIII<SUB>f</SUB>]) + VIII<SUB>f</SUB> × k<SUB>f</SUB>](/content/vol92/issue2/fulltext/529/img001.gif)
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(1)
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where
F is the measured fluorescence/Superose bead, F0 is the
fluorescence for 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. The term kf is the measured fluorescence per
concentration unit of free factor VIII due to unbound factor VIII and
any nonspecific binding of fluorescein-labeled factor VIII to control
Superose beads lacking vWF. We assumed that the fraction of total
factor VIII bound could be neglected (the nominal concentration of
factor VIII binding sites was 0.1 nmol/L, assuming 1 binding site/vWF
subunit). The variables, KD and Fmax, were
determined using nonlinear least squares analysis with the software,
FitAll, or alternatively were fitted by eye using Microsoft Excel
(Redman, WA) v. 5.0 on a Macintosh computer (Cupertino,
CA). The stoichiometry between factor VIII binding sites and vWF
subunits was determined using fluorescein-labeled MoAb vW/16.9.2 as
described under the Results.
For binding experiments with Pro-vWF, which had formed
disulfide-linked oligomers under the influence of the cotransfected vWF
propeptide, binding of factor VIII was not optimally fitted by equation
1. However, binding was well fitted by a modification of equation 1, which took into account two classes of binding sites with differing
affinities for factor VIII:
![F = F<SUB>0</SUB> + F<SUB>1max</SUB>(VIII<SUB>f</SUB>/[K<SUB>D1</SUB> + VIII<SUB>f</SUB>]) + F<SUB>2max</SUB>(VIII<SUB>f</SUB>/[K<SUB>D2</SUB> + VIII<SUB>f</SUB>]) + VIII<SUB>f</SUB> × k<SUB>f</SUB>](/content/vol92/issue2/fulltext/529/img002.gif)
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(2)
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where F1max and F2max refer to the
fluorescence from saturating the higher and lower affinity binding
sites with fluorescein-labeled factor VIII. The dissociation constants,
KD1 and KD2, pertain to the higher and lower
affinities of the two types of sites. Curve fitting of this data was
performed by eye using Microsoft Excel v. 5.0 on a Macintosh computer.
Factor VIII binding to vWF in solution.
Fluorescein-labeled factor VIII (2 nmol/L) was incubated with
increasing concentrations of vWF for 15 minutes at room temperature. Phospholipid bilayers supported by 2-µm diameter glass microspheres were added and, after 10 minutes, phospholipid-bound factor VIII was
measured using flow cytometry. vWF-bound factor VIII was calculated as
fraction of fluorescence competed from liposphere binding. Curve
fitting was performed by eye using Microsoft Excel on a Macintosh
computer. The equation used is in the initial description of this
assay.3 The composition of the phospholipid bilayers was
PS: phosphatidyl-choline: phosphatidyl-ethanolamine: cholesterol (4:70:26:10).
Western Blot Analysis
After measuring the concentration of vWF in the conditioned medium by
ELISA, equal amounts of vWF in conditioned medium were electrophoresed
on reducing SDS-polyacrylamide gels. Transfer of proteins to
nitrocellulose filters was performed in 0.192 mol/L glycine and 0.025 mol/L Tris HCl as described.41 Nitrocellulose filters were
blocked in PBS containing 2% Blotto42 and with 0.1% Tween
20. Anti-vWF antibody was added at 5 µg/mL in a solution of PBS with
Blotto and 0.1% Tween 20. After several washes of the blot in PBS with
0.1%Tween 20, goat antirabbit secondary antibody was added at 0.15 µg/mL. After multiple washings, the filters were developed by ECL,
and band intensities were quantified using the ImageQuant program and
scanning hardware (Molecular Dynamics, Sunnyvale, CA).
Protein Sequence Analysis
COS-1 cells were transfected with the expression vector encoding
Pro+A vWF. At 60 hours posttransfection, conditioned medium was
harvested. Protein was immunoprecipitated with polyclonal anti-vWF
antibody (The Binding Site, Birmingham, UK) and subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was
electroblotted onto ProBlott membrane using CAPS buffer (10 mmol/L
3-[cyclohexylamino]-1-propanesulfonic acid, pH 11, 10% methanol) at
20 V overnight. The protein was detected with Coomassie Blue staining
and excised from the membrane. Protein was sequenced by the University
of Michigan protein sequencing core. The sequential recovery of
residues for Pro+A-vWF was A (4 pmol/L), S (1 pmol/L), L (5 pmol/L), S (1 pmol/L), A (2 pmol/L), R (1 pmol/L), P (2 pmol/L), P (2 pmol/L), M (1 pmol/L), V (2 pmol/L), K (2 pmol/L), and
L (4 pmol/L).
In Vitro vWF Propeptide Cleavage Analysis
Conditioned medium from PACE-Sol and mock-transfected cells were
harvested using OptiMEM medium without protease inhibitors. This
conditioned medium was then concentrated by centrifugation in Centricon
10 filters and dialyzed against 0.15 mol/L
CO3Na2/HNaCO3, pH 6.5. Microtiter
plates were coated with anti-vWF antibody (The Binding Site), as
described above. After blocking the plates, equal amounts of vWF in
conditioned medium were added to the plates and incubated at 37°C
for 2 hours. The vWF-containing conditioned medium was removed and the
plates were washed four times with 0.15 mol/L sodium acetate, pH 6.5. Equal volumes of PACE-Sol or mock-transfected cell conditioned medium
in sodium acetate buffer with 5 mmol/L CaCl2 were added to
the plates and incubated for 2 hours at 37°C. After washing plates
in TBST buffer, purified recombinant factor VIII was added to the wells
and incubated for an additional 2 hours. After washing, factor VIII
binding was determined by factor VIII Coatest assay. A parallel plate
was washed and SDS-PAGE sample buffer was added for subsequent SDS-PAGE and Western immunoblot analysis.
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RESULTS |
Multimeric vWF Has Higher Affinity for Factor VIII Than Dimeric vWF
Deletion of the vWF propeptide yields dimeric vWF, the two subunits
linked by C-terminal disulfide bonds. In the absence of the propeptide,
the cysteine residues near the N-terminus of vWF subunits that
ordinarily link dimers to form high molecular weight multimers are
presumably not oxidized. To characterize the importance of intersubunit
disulfide bonds that contribute to multimer formation, we compared
binding of factor VIII to dimeric vWF (Pro-vWF) versus wild-type
multimeric vWF. Recombinant vWF in conditioned medium from transfected
COS-1 cells was quantitated by vWF ELISA and characterized by Western
blot analysis. This analysis demonstrated that each construct expressed
vWF at similar levels and that the mature vWF from the wild-type vWF
expression vector comigrated with Pro-vWF
(Fig 1, lanes 1 and 3). To measure factor
VIII binding, equal concentrations of vWF in conditioned medium were
incubated with increasing concentrations of recombinant factor VIII.
The incubation mixtures were immobilized on microtiter plates with an
anti-factor VIII heavy chain antibody (data not shown) or with anti-vWF
antibody. Binding of factor VIII to Pro-vWF was substantially reduced by comparison to wild-type vWF, but significantly greater than
that obtained from conditioned medium from mock-transfected cells
(Fig 2). Similar results were obtained when
complexes were captured with the factor VIII MoAb.
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| Fig 1.
Western immunoblot analysis of vWF propeptide processing.
Equal aliquots of conditioned medium from transiently transfected COS-1
cells were analyzed by SDS-PAGE under reducing conditions. Proteins
were transferred to nitrocellulose and probed with anti-vWF antibody as
described in the Materials and Methods. Where cotransfection was
performed, equal amounts of the two plasmid DNAs were used in the
transfection. The ratio of pro-vWF to mature was quantified by scanning
as described in the Materials and Methods. The amount of pro-vWF in
this analysis is underrepresented due to the poorer transfer efficiency
of the high molecular weight pro-vWF. ( ) Pro-vWF; ( ) mature
vWF.
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| Fig 2.
Factor VIII binding to vWF. Based on ELISA, equal amounts
of vWF protein in the conditioned medium from transfected cells (100 ng/mL) were incubated with increasing concentrations of
recombinant-derived factor VIII. The complexes were immobilized by
capture using microtiter plates coated with anti-vWF polyclonal
antibody. Bound proteins were quantified by factor VIII activity as
described in the Materials and Methods. () Wild-type vWF; ( )
Pro-vWF; ( ) Pro+A-vWF; () conditioned medium from
mock-transfected cells. Although these results represent single values
from a single experiment in which protein was analyzed in parallel by
Western blot analysis (Fig 1), these binding curves were performed at
least 5 times from different transfection experiments and yielded data
qualitatively similar to that reported here.
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To investigate whether the reduced binding of factor VIII to Pro-vWF
was due to a reduced affinity or loss of some binding sites, we
developed a quantitative equilibrium binding assay. Our system was
based on the recent observation that vWF captured by anti-vWF MoAbs on
agarose beads binds factor VIII equivalently to vWF in
solution.43 We activated cross-linked agarose beads of
uniform size (Superose) with cyanogen bromide and coupled the noninhibitory anti-vWF MoAb vW/13.7.9 to the beads. In preliminary experiments, we found that Pro-vWF labeled with
fluorescein-maleimide bound to the MoAb vW/13.7.9-Superose beads and
that bound Pro-vWF did not dissociate more than 10% over 30 minutes
(data not shown). Therefore, we concluded that Pro-vWF and vWF
immobilized in this way were suitable for quantitative studies of
factor VIII binding. Purified vWF was captured by the suspended MoAb
vW/13.7.9-Superose beads, the beads were washed, and binding of
fluorescein-conjugated factor VIII was investigated by flow cytometry.
We found that factor VIII bound saturably to plasma vWF with a
KD of 0.94 nmol/L (Fig 3A). To
estimate the fraction of vWF binding sites available to factor VIII, we
estimated the number of bound vWF subunits using fluorescein-labeled
anti-vWF MoAb vW/16.9.2. By comparing the ratio of maximum fluorescence
from bound MoAb vW/16.9.2 to fluorescence from bound
fluorescein-labeled factor VIII, correcting from the
fluorescein/protein ratio, we were able to compare the ratio of factor
VIII binding sites with the number of vWF subunits recognized by MoAb
vW/16.9.2. The results indicated a ratio of 0.99 (n = 2). Thus, the
factor VIII binding sites are accessible to the same degree as an
unrelated epitope.
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| Fig 3.
Equilibrium binding of factor VIII to plasma vWF and to
recombinant vWF. (A) Binding of fluorescein-labeled factor VIII to immobilized plasma vWF. Purified vWF from human plasma was immobilized onto MoAb vW/13.7.9-coated Superose-12 beads. After 1 hour of incubation at room temperature, increasing amounts of
fluorescein-labeled factor VIII were added and incubated for 15 minutes. Fluorescence per bead was monitored by flow cytometry, as
described under the Materials and Methods. The best fit curve
corresponds to a KD of 0.8 nmol/L. (B) Displacement of
factor VIII binding to PS-containing lipospheres by plasma vWF and
recombinant vWF purified from CHO cells. ( ) Plasma vWF; ()
recombinant vWF. In this assay, displacement of factor VIII from
lipospheres does not reach zero, apparently because vWF binds with low
affinity to lipospheres (data not shown). Therefore, the portion of the
curve fitted correlates to displacement of 60% of factor VIII. The
residual fluorescence, apparently not subject to displacement under
these conditions, was excluded from quantitative analysis.
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To confirm that immobilization of vWF on MoAb vW/13.7.9-Superose beads
did not influence binding of factor VIII and to verify that the
production of vWF in cell culture (wild-type) does not affect its
binding to factor VIII, binding of factor VIII to plasma vWF and
wild-type vWF from CHO cells in solution was measured in competition
binding assay. Because vWF and PS-containing membranes compete for the
same factor VIII binding motif, we were able to detect binding of
factor VIII to vWF in solution as decreased binding to PS-containing
membranes supported by glass microspheres, as previously
described.3 In this system, increasing vWF concentrations decreased membrane binding of factor VIII by 80% to 90% (Fig
3B). The observed displacement for both vWF types was analyzed by
fitting the data points to the equation cited in the Materials and
Methods. The dissociation constants obtained under these conditions
were 0.68 nmol/L and 1.05 nmol/L for plasma vWF and wild-type
recombinant vWF, respectively (Table 1).
These results indicate that binding of factor VIII to vWF is not
influenced by posttranslational processing differences between
endothelial cells and CHO cells. They also indicated that
immobilization of vWF on MoAb vW/13.7.9 linked to Superose does not
alter binding of factor VIII.
We next evaluated the importance of N-terminal intersubunit vWF
disulfide bonds by immobilizing Pro-vWF onto MoAb vW/13.7.9 and
compared the affinity of factor VIII for Pro-vWF versus plasma vWF.
The results indicate that the affinity of factor VIII for Pro-vWF is
lower than wild-type vWF. The best fit curves indicate that factor VIII
binds to plasma vWF with a KD of 0.94 nmol/L versus 5 nmol/L for Pro-vWF (Figs 3A and
4A and Table 1). In these experiments, the
number of vWF subunits immobilized on the microspheres was measured
with fluorescein-labeled anti-vWF MoAb vW/16.9.2. The ratio between the
number of factor VIII binding sites and the number of MoAb vW/16.9.2
epitopes detected was 0.99 for vWF and 1.37 for Pro-vWF (Table 1).
Therefore, the intersubunit disulfide bonds predominately affect the
affinity of factor VIII for vWF but not the stoichiometry between
factor VIII and subunit binding sites.
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| Fig 4.
Multimerization increases the affinity of vWF subunits
for factor VIII. vWF purified from human plasma or dimeric mutant
Pro-vWF, incapable of multimerizing and purified from CHO cells,
were immobilized on MoAb vW/13.7.9-Superose-12 beads. Increasing
concentrations of fluorescein-conjugated factor VIII were added and
incubated for 15 minutes to allow equilibrium. Fluorescence per
particle was monitored by flow cytometry and shown in (A). Parameters
fitted were KD = 0.94 and 5 nmol/L for vWF and
Pro-vWF, respectively. Binding of factor VIII to vWF in solution was
measured in a competition binding assay and is shown in (B). Purified
vWF or Pro-vWF, at various concentrations, was incubated with 2 nmol/L fluorescein-labeled factor VIII for 15 minutes at room
temperature. Lipospheres were added and incubated for an additional 10 minutes and liposphere-bound fluorescein-labeled factor VIII was
measured by flow cytometry. vWF-bound factor VIII was interpreted as
the fraction not available for binding to lipospheres. The best-fit
curves corresponds to the following parameter values: KD
= 0.68 ± 0.28 (n = 3) and 6.4 ± 0.64 (n = 3) nmol/L and a
stoichiometry of 0.9 and 0.8 factor VIII molecules per vWF subunit, for
plasma vWF and pro-vWF, respectively. () vWF; () pro-vWF.
|
|
Results obtained with immobilized vWF and Pro-vWF were confirmed
using the competition experiment described above in Fig 3B. The
dissociation constant for plasma vWF was 0.68 nmol/L and for Pro-vWF
was 6.4 nmol/L (Fig 4B), whereas the binding stoichiometry between
factor VIII and vWF subunits was 0.9:1 for both plasma vWF and
Pro-vWF.
vWF Propeptide Coexpression Increases Factor VIII Binding to
Pro-vWF
To further probe the possibility that multimer formation increases
affinity of vWF subunits for factor VIII, we coexpressed the vWF
propeptide with Pro-vWF. COS-1 cells were cotransfected with
Pro-vWF and the vWF propeptide expression construct
pMT2-KRX.15 By means of gel electrophoresis
under nonreducing conditions, we confirmed that coexpression of the vWF
propeptide with Pro-vWF yields a multimeric vWF
(Fig 5A), as we have previously
reported.15 We then immobilized the partially multimerized
Pro-vWF to MoAb vW/13.7.9-Superose beads and added saturable amounts
of fluorescein-labeled factor VIII, as described above. We observed
saturable binding of factor VIII with an apparent affinity intermediate
between plasma vWF and Pro-vWF (Fig 5B). These results confirm that
formation of multimers increases the affinity of vWF subunits for
factor VIII.
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| Fig 5.
Coexpression of the vWF propeptide increases binding
affinity of Pro-vWF for factor VIII. COS-1 cells were transfected
with Pro-vWF in the presence or absence of the vWF propeptide
expression vector pMT2-KRX. The resulting protein was
evaluated by agarose gel electrophoresis under nonreducing conditions
and transferred to nitrocellulose for detection as described in the
Materials and Methods and shown in (A). Lane 1, plasma-derived vWF;
lane 2, Pro-vWF coexpressed with the propeptide; lane 3, Pro-vWF without the propeptide. The bracket on the left indicates the distribution of vWF multimers in plasma. The arrow indicates the migration of the dimeric form of vWF. (B) Factor VIII binding as the
fluorescence per Superose microsphere monitored by flow cytometry,
measured as described for Fig 4. The solid fitted curve corresponds to
a model with two classes of binding sites corresponding to lower
affinity sites on non-disulfide-linked N-termini and higher affinity
sites on disulfide-linked N-termini. The corresponding KDs
were 0.77 and 6.5 nmol/L. The dashed curve corresponds to the same
maximum fluorescence/microsphere but a single class of binding sites
with intermediate affinity, KD of 3 nmol/L.
|
|
Inspection of the nonreducing gel indicated that, although the
propeptide had induced vWF multimer formation, a significant fraction
of vWF remained as dimers (Fig 5A, lane 2). The dimers are predicted to
bind factor VIII with lower affinity. In addition, each higher
molecular weight multimer is predicted to have two terminal vWF
subunits with low affinity for factor VIII in addition to the internal
subunits with high affinity for factor VIII. We used this reasoning,
together with the measured quantity of vWF of various multimer sizes
determined by laser densitometry, to predict that the partially
multimerized vWF in lane 2 of Fig 5A would contain 42% high-affinity
sites and 58% low-affinity sites. The solid curve depicted in Fig 5B
was fit to the data using a model with two classes of binding sites.
The KDs corresponding to the curve were 0.77 and 6.5 nmol/L. The proportion of low-affinity sites was 64%, with 36%
higher-affinity binding sites. Although the data may also be fitted for
a single class of binding sites of intermediate affinity
(KD = 3 nmol/L, dashed curve, Fig 5B), the fit is less good
and we lack a rationale for predicting formation of actual
intermediate-affinity binding sites. Thus, these data are consistent
with the interpretation that formation of N-terminal disulfide-linked
multimer bonds increases the affinity of factor VIII binding sites.
vWF Propeptide Cleavage and a Native Mature N-Terminus Is Required
for Factor VIII Binding
The results in this report indicate that each vWF subunit in a
Pro-vWF dimer contains one factor VIII binding site with
approximately sixfold lower affinity than the internal factor VIII
binding sites on vWF multimers. In contrast, a prior report suggested
that binding of factor VIII to vWF was abolished on vWF dimers secreted
in the absence of the propeptide.35 We hypothesized that
this apparent discrepancy was due to one extra Ala residue at the
junction of the signal peptide and the mature vWF polypeptide in the
propeptide-deletion construct prepared in the latter study. To evaluate
the importance of the addition of this Ala for factor VIII binding, we
expressed that construct, Pro+A-vWF, in parallel with Pro-vWF and
compared binding of factor VIII. Under conditions in which binding to
Pro-vWF was detected, binding to Pro+A-vWF was not detected (Fig
2). We also evaluated binding of Pro+A-vWF using the equilibrium binding assay. Successful capture of Pro+A-vWF was demonstrated with
fluorescein-labeled MoAb vW/16.9.2. However, no binding of factor VIII
was detected (data not shown). The difference in the ability for
Pro-vWF and Pro+A-vWF to bind factor VIII supported our
hypothesis and indicated that minor perturbations at the vWF N-terminus
may decrease affinity for factor VIII by more than 20-fold.
Pro-vWF and Pro+A-vWF differ by the presence of a single alanine
residue at the junction of the signal peptide sequence and the mature
sequence in Pro+A-vWF. Because the addition of this residue could
alter the site of signal peptide processing, we determined the
N-terminal sequence of mature secreted Pro+A-vWF to identify the
site of signal peptide cleavage. Conditioned medium was harvested from
Pro+A-vWF-transfected cells and immunoprecipitated for SDS-PAGE
after reduction. The polypeptide migrating at the position of monomeric
vWF was isolated. The sequence was determined to contain an N-terminal
Ala immediately upstream from the N-terminal 10 residues of mature vWF
(see the Materials and Methods).
We previously reported that a mutant form of vWF in which the
propeptide was not cleaved during intracellular processing did not bind
factor VIII.34 In that construct, the P1 position with respect to the vWF propeptide cleavage site, Arg763, was mutated to
Lys. Because factor VIII binding may be perturbed by small alterations
in this region, such as the presence of an extra alanine at the
N-terminus of mature vWF, we were concerned that the failure of the
R763K mutant to bind factor VIII resulted from the amino acid
substitution at residue 763 rather than the attached
propeptide.34 To further evaluate the importance of the vWF
propeptide cleavage for factor VIII binding, we studied the factor VIII
binding of two different pro-vWF propeptide cleavage site mutants.
Western blot analysis of the single mutant R760K or the double mutant R760K/K762D demonstrated that the propeptides of these mutants were not
detectably processed by the endogenous COS-1 processing enzyme (Fig 1,
lanes 6 and 7). Analysis of factor VIII binding indicated that both
mutants were defective in binding factor VIII (Fig 6). The defective factor VIII binding
observed for these noncleavage mutants supports the conclusion that
propeptide cleavage is required for factor VIII binding.
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| Fig 6.
The presence of the vWF propeptide inhibits factor VIII
binding. COS-1 cells were transfected with wild-type and propeptide cleavage site mutant (R760K and R760K/K762D) vWF, as well as with the
expression vectors encoding wild-type vWF in the absence or presence of
PACE-Sol or a catalytically inactive mutant PACE-SA. Conditioned medium
was harvested for factor VIII binding assays and Western immunoblot
analysis of vWF (Fig 1). The reduced amount of vWF detected upon
PACE-SA cotransfection was reproducible and was corrected for in the
factor VIII binding assay by using increased amounts of conditioned
medium for this sample. The concentration of vWF in the conditioned
medium was determined by ELISA and the binding assays were performed
using equal amounts of vWF and were measured by factor VIII activity
assay as described in the Materials and Methods. () Wild-type vWF;
() R760K vWF; ( ) R760K/K762D vWF; () vWF+PACE-Sol; ()
vWF+PACE-SA; () mock.
|
|
To further demonstrate the requirement for vWF propeptide cleavage
within wild-type pro-vWF in factor VIII binding, we modulated cleavage
of wild-type pro-vWF that occurred by coexpression of PACE-Sol (a
soluble secreted form of PACE/furin) that was previously shown
to improve vWF processing39 or by coexpression of a
catalytically inactive serine to alanine active site mutant of
PACE/furin (PACE-SA) that was previously shown to inhibit
pro-vWF processing.39 Analysis of vWF processing by Western
blot analysis of conditioned medium indicated that processing of
wild-type pro-vWF expressed alone was incomplete, whereas one third of
the secreted vWF was in the precursor form (Fig 1, lane 3), similar to
previous observations.23,24 Upon cotransfection with
PACE-Sol, all the secreted vWF was in the mature form (Fig 1, lane 4).
In contrast, coexpression with the PACE-SA mutant yielded at least 60%
of the vWF precursor form as pro-vWF (Fig 1, lane 5). Factor VIII
binding assays performed on the vWF conditioned medium demonstrated
that cotransfection of PACE-Sol increased factor VIII binding to
wild-type vWF (Fig 6). In contrast, cotransfection with mutant PACE-SA
reduced the binding to factor VIII (Fig 6). As a control,
cotransfection of PACE-Sol with Pro-vWF did not affect the binding
of Pro-vWF to factor VIII (data not shown). These results correlate
the degree of vWF propeptide processing with the degree of factor VIII
binding to wild-type vWF.
In Vitro Propeptide Cleavage Increases Factor VIII Binding
The ability for PACE-Sol to cleave the vWF propeptide in vitro was
tested by immobilizing wild-type vWF from COS-1 transfected cells onto
microtiter plates. Subsequently, conditioned medium from
PACE-Sol-transfected or mock-transfected COS-1 cells was incubated with the samples and cleavage was monitored by Western immunoblotting analysis using anti-vWF antibody. The results
demonstrated that incubation with PACE-Sol increased propeptide
cleavage to 100% in this cell-free system
(Fig 7A, lanes 1 and 2). Incubation of
PACE-Sol with either Pro-vWF or the propeptide cleavage site mutant
R760K/K762D had no effect (Fig 7A, lanes 3 through 6). These results
support that cleavage occurred at the authentic propeptide cleavage
site. These results show that PACE-Sol can mediate propeptide
processing of pro-vWF in a cell-free system.
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| Fig 7.
In vitro cleavage of pro-vWF by PACE/furin
increases binding affinity to factor VIII. COS-1 cells were transfected
with wild-type vWF, Pro-vWF, or cleavage site mutant R760K/K762D vWF
and conditioned medium was harvested. vWF from the conditioned medium
was immobilized on anti-vWF polyclonal antibody-coated microtiter
plates. The immobilized vWF was then treated with concentrated
conditioned medium from PACE-Sol-transfected or mock-transfected COS-1
cells as described in the Materials and Methods. In (A), proteins were recovered from the wells and analyzed by SDS-PAGE under reducing conditions and Western blot analysis using polyclonal anti-vWF antibody. () Pro-vWF; () mature vWF. (B) Factor VIII binding (1.25 nmol/L) to vWF preparations (wild-type, Pro-vWF and
R760K/K762D vWF) that were immobilized on anti-vWF polyclonal
antibody-coated microtiter plates and then treated with conditioned
medium from mock-transfected or PACE-Sol-transfected (+ PACE) COS-1
cells. Although the results depicted here are from a single experiment with a single concentration of factor VIII in which Western blot analysis was performed in parallel (A), this experiment was performed several times with varying concentrations of factor VIII and gave consistent results.
|
|
We next asked whether in vitro cleavage of the vWF propeptide affects
factor VIII binding. Under these in vitro conditions, it is unlikely
that disulfide bond rearrangements occur; thus, any change in factor
VIII binding affinity could be attributed only to cleavage of the
propeptide. After in vitro cleavage of the propeptide, wild-type vWF
binding to factor VIII increased approximately 2.5-fold (Fig 7B). For
control, PACE-Sol conditioned medium was incubated with Pro-vWF or
with the vWF cleavage site mutant R760K/K762D. PACE-Sol did not
increase the binding of factor VIII to either of these vWF mutants (Fig
7B). These results show that in vitro cleavage of the propeptide
increases vWF binding to factor VIII.
| |
DISCUSSION |
The data in this report elucidate the importance of two vWF processing
events that occur late in the biosynthetic pathway, propeptide cleavage
and N-terminal disulfide bond formation. Removal of the vWF propeptide
is necessary for factor VIII to interact with its binding site in the
D domain of vWF. Because the addition of a single alanine to the
mature N-terminus, like the presence of the large propeptide, abolished
binding of factor VIII, the native N-terminus of vWF is necessary for
factor VIII binding. These results are consistent either with direct
interaction of the N-terminal Ser residue of vWF with factor VIII or
with the native N-terminal Ser residue being required for correct
folding of the N-terminus. The affinity of factor VIII for vWF binding sites is enhanced approximately sixfold upon vWF multimer formation via
disulfide bonds, a change likely reflecting a conformational change of
the D domain. These data imply that, in plasma, factor VIII is
not bound to vWF subunits with attached propeptides and that factor
VIII is far more likely to be found on internal vWF subunits than those
that terminate the high molecular weight multimers.
Our studies have now resolved the previous conflicting results
concerning the role of the vWF propeptide in binding to factor VIII.34,35 The results demonstrated that Pro-vWF,
previously described by Wise et al,34 bound factor VIII but
at a sixfold lower affinity than wild-type vWF. In contrast, Pro+A
vWF, described by Leyte et al,35 did not bind factor VIII
at all concentrations tested. Previous protein sequence analysis
confirmed the correct N-terminus for the Pro-vWF deletion
molecule.15 We have now confirmed that the secreted
Pro+A contains an additional Ala at the N-terminus of the secreted
vWF molecule and believe that inhibition of binding by the additional
alanine is the only likely explanation for failure of factor VIII to
bind to the dimeric vWF.
Previous studies demonstrated that the vWF propeptide can also act in
trans to mediate multimerization of Pro-vWF.15 We have
shown by cotransfection of a vWF propeptide expression vector with a
Pro-vWF expression vector that the resulting multimers possess
increased factor VIII binding. The propeptide sequence contains vicinal
cysteine residues (2 cysteine residues separated by 2 amino acids) that
are similar to the catalytic active sites of protein disulfide
isomerase,44 which facilitates disulfide bond formation and
exchange in the endoplasmic reticulum. In vivo, the vWF propeptide
promotes disulfide bond formation between N-terminal ends and
subsequently the propeptide is cleaved to generate the mature vWF
N-terminus.21 In the present study, we conducted equilibrium binding studies by flow cytometry to demonstrate multimeric vWF prepared by coexpression of propeptide-deleted vWF with the propeptide displays increased binding affinity to factor VIII. When the
binding data are corrected for the fraction of sites on
disulfide-linked terminal vWF subunits, these multimers have the same
affinity for factor VIII as plasma vWF (Fig 5B). There are two
mechanisms by which the propeptide could increase the factor VIII
binding affinity. First, the process of multimer formation may
juxtapose two factor VIII binding sites that may increase affinity
through proximity so that a single factor VIII molecule might interact
with two adjacent vWF subunits. Second, multimer formation may cause a
conformational change in the factor VIII binding motif of each vWF
subunit. We prefer the second explanation, noting that the relatively
large factor VIII binding motif spanning vWF amino acid residues 1-102 and the corresponding vWF binding motifs of factor VIII, separated by
600 amino acids in linear sequence,45-47 suggest a
relatively large interacting surface where modest geometric changes
could alter affinity. Alternatively, the propeptide may mediate
disulfide bond exchange reactions within the N-terminal end
of the mature vWF subunit to change the conformation of vWF to create a
high-affinity factor VIII binding site. However, we think this latter
possibility is unlikely because proteolytic fragments of plasma-derived
vWF exhibit similar affinities to factor VIII, as observed for
Pro-vWF,48 suggesting that disulfide exchanges may not
be responsible for the difference between high-affinity and
low-affinity sites.
In summary, these results indicate that propeptide-induced formation of
N-terminal disulfide bonds between vWF dimers is necessary for
formation of the highest affinity factor VIII binding sites. However,
these sites remain cryptic until the propeptide is cleaved, eliminating
steric hindrance or allowing correct folding of the vWF N-terminus.
| |
FOOTNOTES |
Submitted December 5, 1997;
accepted March 19, 1998.
Supported in part by National Institutes of Health Grants No. HL42443,
HL53777, and HL52173; by the Medical Research Service of the Veterans
Administration; and by a Grant in Aid from the American Heart
Association (to R.J.W.). A.V.B. was supported by individual NRSA No.
HL09506 and G.E.G. was a recipient of AHA Established Investigator
Award No. 96001720.
Address reprint requests to Randal J. Kaufman, PhD, Howard Hughes
Medical Institute, MSRBII, 1150 W Medical Center Dr, University of
Michigan, Ann Arbor, MI 48109; e-mail: kaufmanr{at}umich.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr J. van Mourik for kindly providing
the vWFdelpro expression plasmid for these studies, K.C. Kye for
assistance in developing the microtiter plate binding assays used in
this study, and Dr Steven Pipe for critical review of this report.
| |
REFERENCES |
1.
van Dieijen G,
Tans G,
Rosing J,
Hemker H:
The role of phospholipid and factor VIIIa in the activation of bovine factor X.
J Biol Chem
256:3433,
1981[Abstract/Free Full Text]
2.
Patek A,
Taylor F:
Hemophilia. II. Some properties of a substance obtained from normal human plasma effective in accelerating the clotting of hemophilic blood.
J Clin Invest
16:113,
1937
3.
Gilbert GE,
Drinkwater D,
Barter S,
Clouse SB:
Specificity of phosphatidylserine-containing membrane binding sites for factor VIII: Studies with model membranes supported by glass microspheres (Lipospheres).
J Biol Chem
267:15861,
1992[Abstract/Free Full Text]
4.
Kaufman RJ,
Wasley LC,
Dorner AJ:
Synthesis, processing and secretion of recombinant human factor VIII expressed in mammalian cells.
J Biol Chem
263:6352,
1988[Abstract/Free Full Text]
5.
Kaufman RJ,
Wasley LC,
Davies MV,
Wise RJ,
Israel DI,
Dorner AJ:
The effect of von Willlebrand factor co-expression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells.
Mol Cell Biol
9:1233,
1989[Abstract/Free Full Text]
6.
Rick ME,
Esmon NL,
Krized DM:
Factor IXa and von Willebrand factor modify the inactivation of factor VIII by activated protein C.
J Lab Clin Med
115:415,
1990[Medline]
[Order article via Infotrieve]
7.
Sadler JE,
Shelton-Inloes BB,
Sorace JM,
Harlan JM,
Titani K,
Davie EW:
Cloning and characterization of two cDNAs coding for human von Willebrand factor.
Proc Natl Acad Sci USA
82:6394,
1985[Abstract/Free Full Text]
8.
Verweij CL,
Diergaarde PMH,
Pannekoek H:
Full-length von Willebrand factor (vWF) cDNA encodes a highly repetitive protein, considerably larger than the mature vWF subunit.
EMBO J
5:1839,
1986[Medline]
[Order article via Infotrieve]
9.
Bonthron DT,
Handin RI,
Kaufman RJ,
Wasley LC,
Orr EC,
Mitsock LM,
Ewenstein B,
Loscalzo J,
Ginsburg D,
Orkin SH:
Structure of pre-pro von Willebrand factor and its expression in heterologous cells.
Nature
324:270,
1986[Medline]
[Order article via Infotrieve]
10.
Hoyer LW,
Shainoff JR:
Factor VIII-related protein circulates in normal human plasma as high molecular weight multimers.
Blood
55:1056,
1980[Abstract/Free Full Text]
11.
Ruggeri ZM,
Zimmerman TS:
The complex multimeric composition of factor VIII/von Willebrand factor.
Blood
57:1140,
1981[Abstract/Free Full Text]
12.
Marti T,
Rosselet SJ,
Titani R,
Walsh KA:
Identification of disulfide-bridged structures within human von Willebrand factor.
Biochemistry
26:8099,
1987[Medline]
[Order article via Infotrieve]
13.
Wagner DD,
Marder VJ:
Biosynthesis of von Willebrand protein by human endothelial cells: Processing steps and their intracellular localization.
J Cell Biol
99:2123,
1984[Abstract/Free Full Text]
14.
Sadler JE:
von Willebrand factor.
J Biol Chem
266:22777,
1991[Free Full Text]
15.
Wise RJ,
Pittman DD,
Handin RI,
Kaufman RJ,
Orkin SH:
The propeptide of vWF is required for the assembly of von Willebrand subunits into disulfide-linked multimers.
Cell
52:229,
1988[Medline]
[Order article via Infotrieve]
16.
Verweij CL,
Hart M,
Pannekoek H:
Expression of a variant von Willebrand factor vWF cDNA in heterologous cells: Requirement of the propolypeptide in vWF multimer assembly.
EMBO J
6:2885,
1987[Medline]
[Order article via Infotrieve]
17. Koedam JA: Interaction Between Factor VIII and vWF. PhD Thesis,
University of Utrecht, Utrecht, The Netherlands, 1989
18.
Foster PA,
Fulcher CA,
Marti T,
Titani K,
Zimmerman TS:
A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor.
J Biol Chem
262:8443,
1987[Abstract/Free Full Text]
19.
Bahou WF,
Ginsburg D,
Sikkink R,
Litwiller R,
Fass DN:
A monoclonal antibody to von Willebrand factor (vWF) inhibits factor VIII binding.
J Clin Invest
84:56,
1989
20.
Takahashi Y,
Kalafatis M,
Girma J-P,
Sewerin K,
Andersson L-O,
Meyer D:
Localization of factor VIII binding domain on a 34 kilodalton fragment of the N-terminal portion of von Willebrand factor.
Blood
70:1987,
1987
21.
Vischer UM,
Wagner DD:
von Willebrand factor proteolytic processing and multimerization precede the formation of Weibel-Palade bodies.
Blood
83:3536,
1994[Abstract/Free Full Text]
22.
Lynch DC,
Williams R,
Zimmerman TS,
Kirby EP,
Livingston DM:
Biosynthesis of the subunits of factor VIIIR by bovine aortic endothelial cells.
Proc Natl Acad Sci USA
80:2738,
1983[Abstract/Free Full Text]
23.
Wise RJ,
Barr PJ,
Wong PA,
Kiefer MC,
Brake AJ,
Kaufman RJ:
Expression of a human proprotein processing enzyme: Correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.
Proc Natl Acad Sci USA
87:9378,
1990[Abstract/Free Full Text]
24.
Van der Ven WJM,
Voorberg J,
Fontijn R,
Pannekoek H,
Van den Ouweland AMW,
Van Duynhoven HLF,
Roebroek AJM,
Siezen RJ:
Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes.
Mol Biol Rep
14:15,
1990
25.
Tuddenham E,
Lane R,
Rotblat F,
Johnson A,
Snape T,
Middleton S,
Kernoff P:
Response to infusions of polyelectrolyte fractionated human factor VIII concentrate in human hemophilia A and von Willebrand's disease.
Br J Haematol
52:259,
1982[Medline]
[Order article via Infotrieve]
26.
Over J,
Sixma J,
Bruine M,
Trieschnigg M,
Vlooswijk R,
Beeser-Visser N,
Bouma B:
Survival of 125iodine-labeled factor VIII in normals and patients with classic hemophilia.
J Clin Invest
62:223,
1978
27. Weiss H, Sussman I, Hoyer L: Stabilization of factor VIII in
plasma by the von Willebrand factor. Studies on posttransfusion and
dissociated factor VIII and in patients with von Willebrand's disease.
J Clin Invest 60, 1977
28.
Mannucci PM,
Tenconi PA,
Castman G,
Rodegheiro F:
Comparison of four virus-inactivated plasma concentrates for treatment of severe von Willebrand disease. A cross-over randomized trial.
Blood
79:3130,
1992[Abstract/Free Full Text]
29.
Lollar P,
Parker C:
Stoichiometry of the porcine factor VIII-von Willebrand factor association.
J Biol Chem
262:17572,
1987[Abstract/Free Full Text]
30.
Zucker MB,
Soberano ME,
Johnson AJ,
Fulton AJ,
Kowalski S:
The in vitro association of antihemophilic factor and von Willebrand factor.
Thromb Haemost
49:37,
1983[Medline]
[Order article via Infotrieve]
31.
Hamer R,
Koedam J,
Beeser-Visser N,
Bertina R,
Van Mourik J,
Sixma JJ:
Factor VIII binds to von Willebrand factor via its Mr-80,000 light chain.
Eur J Biochem
166:37,
1987[Medline]
[Order article via Infotrieve]
32.
Leyte A,
Verbeet M,
Brodniewicz-Proba T,
van Mourik J,
Mertens K:
The interaction between human blood-coagulation factor VIII and von Willebrand factor: Characterization of a high-affinity binding site on factor VIII.
Biochem J
257:679,
1989[Medline]
[Order article via Infotrieve]
33.
Nesheim ME,
Pittman DD,
Giles AR,
Fass DN,
Wang J,
Slonosky D,
Kaufman RJ:
The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets.
J Biol Chem
266:17815,
1991[Abstract/Free Full Text]
34.
Wise RJ,
Dorner AD,
Krane M,
Pittman DD,
Kaufman RJ:
The role of von Willebrand factor multimerization and propeptide cleavage in the binding and stabilization of factor VIII.
J Biol Chem
266:21948,
1991[Abstract/Free Full Text]
35.
Leyte A,
Voorberg J,
van Schijndel H,
Duim B,
Pannekoek H,
van Mourik J:
The pro-polypeptide of von Willebrand factor is required for the formation of a functional factor VIII-binding site on mature von Willebrand factor.
Biochem J
274:257,
1991
36.
Pittman DD,
Millenson M,
Marquette K,
Bauer KA,
Kaufman RJ:
A2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage.
Blood
79:389,
1992[Abstract/Free Full Text]
37.
Bardelle C,
Furie B,
Furie BC,
Gilbert GE:
Membrane binding kinetics of factor VIII indicate a complex binding process.
J Biol Chem
268:8815,
1993[Abstract/Free Full Text]
38. Harlow E, Lane D: Antibodies: A Laboratory Manual. Cold Spring
Harbor, NY, Cold Spring Harbor Laboratory, 1988
39.
Rehemetulla A,
Kaufman RJ:
Preferred sequence requirements for cleavage of pro-vWF by propeptide processing enzymes.
Blood
9:2349,
1992
40.
Kaufman RJ:
Vectors used for expression in mammalian cells.
Methods Enzymol
185:487,
1990[Medline]
[Order article via Infotrieve]
41.
Faassem AE,
Schrager JA,
Klein DJ,
Oegema TR,
Couchman JR,
McCarthy JB:
A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion.
J Cell Biol
116:521,
1992[Abstract/Free Full Text]
42.
Towbin H,
Staehelin T,
Gordon J:
Electrophorestic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.
Proc Natl Acad Sci USA
76:4350,
1979[Abstract/Free Full Text]
43.
Vlot AJ,
Koppelman SJ,
van den Berg MH,
Bouma BN,
Sixma JJ:
The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor.
Blood
85:3150,
1995[Abstract/Free Full Text]
44.
Mayadas TN,
Wagner DD:
Vicinal cysteines in the prosequence play a role in von Willebrand factor multimer assembly.
Proc Natl Acad Sci USA
89:3531,
1992[Abstract/Free Full Text]
45.
Saenko EL,
Scandella D:
The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor.
J Biol Chem
272:18007,
1997[Abstract/Free Full Text]
46.
Shima M,
Scandella D,
Yoshioka A,
Nakai H,
Tanaka I,
Kamisue S,
Terada S,
Fukui H:
A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine.
Thromb Haemost
69:240,
1993[Medline]
[Order article via Infotrieve]
47.
Saenko EL,
Shima M,
Rajalakshmi KJ,
Scandella D:
A role for the C2 domain of factor VIII in binding to von Willebrand factor.
J Biol Chem
269:11601,
1994[Abstract/Free Full Text]
48.
Koppelman SJ,
van Hoeij M,
Vink T,
Lankhof H,
Schiphorts ME,
Damas C,
Vlot AJ,
Wise RJ,
Bouma BN,
Sixma JJ:
Requirements of von Willebrand factor to protect factor VIII from inactivation by activated protein C.
Blood
87:2292,
1996[Abstract/Free Full Text]
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Abstract
Introduction
Abstract