From the
von Willebrand factor (vWf) acts as a carrier for blood
coagulation factor VIII (fVIII) in the circulation. The amino-terminal
272 residues of mature vWf contain a high affinity fVIII binding site.
Upon thrombin activation, fVIII is released from vWf, thereby allowing
its binding to phospholipid which is required for its procoagulant
activity. Although phospholipid and vWf compete for fVIII binding, it
was previously suggested that their binding sites are not closely
juxtaposed within the fVIII protein because only amino-terminal vWf
proteolytic fragments larger than SPIII-T4(1-272) were able to
block the binding of fVIII to phospholipid. We have demonstrated,
however, that SPIII-T4 is able to inhibit fVIII binding to
phosphatidylserine (PS) in a dose-dependent fashion, but only at
concentrations higher than those used in previous experiments. Our
demonstration that the K
Inhibition (>95%) of
SPIII-T4 binding to fVIII by a purified recombinant fVIII C2 domain
polypeptide demonstrated that SPIII-T4 binds directly to C2, as we had
previously shown for vWf. The similarity of the C2 binding sites for
vWf and SPIII-T4 was further confirmed by the identical inhibitory
effects of synthetic peptides and monoclonal antibodies (mAbs) on
vWf
Factor VIII (fVIII)
fVIII internal protein
sequence homology has led to the designation of six domains arranged in
the order A1-A2-B-A3-C1-C2
(3) , Fig. 1. The light chain of
fVIII corresponding to the A3-C1-C2 sequence contains sites for binding
of vWf
(4, 5) , activated protein C
(6) , factor
IXa (7), and phosphatidylserine (PS)
(8) . A PS binding site was
localized to fVIII amino acid residues 2303-2332 within the C2
domain based on the ability of overlapping peptides from this region to
inhibit the binding of fVIII to immobilized PS
(9) . Observations
that the fVIII
Proteolytic fragments of mature vWf amino acids
1-272 (SPIII-T4)
(18) or 1-298 (P34)
(19) ,
but not fragments lacking these regions, bind to fVIII. Monoclonal
antibodies with epitopes localized to vWf residues 78-96
(20) and 51-60
(21) prevent binding of vWf or
SPIII-T4 to fVIII, and naturally occurring human point mutations of vWf
(residues 19, 28, 53, 54, and 91)
(22) abolish fVIII binding.
Thus, a major high affinity fVIII binding domain is believed to
correspond to amino acid residues 1-272 of the mature vWf
protein. A binding site for vWf was localized to the C2 domain of fVIII
(residues 2173-2332) based on the ability of a fusion protein,
glutathione S-transferase-C2, to bind to vWf. It was shown
that this site has some overlap with a PS binding site (residues
2303-2332) since a peptide corresponding to this sequence and a
monoclonal antibody with an epitope within amino acid residues
2170-2327 prevented fVIII binding to vWf
(23) . Two
antibodies with C2 epitopes 2170-2218 or 2248-2285, which
do not overlap the PS binding site, did not have any inhibitory
effect
(23) . These data suggest that the antagonistic binding of
vWf and phospholipid to fVIII is due to the involvement of some C2
domain amino acids in both processes.
It was previously shown that
the binding of fVIII to phospholipid was inhibited by vWf but not by
SPIII-T4 when each was used at a 10-fold molar excess over
fVIII
(11) , suggesting that the effect of vWf may be due to
steric hindrance. If SPIII-T4 is believed to contain the major fVIII
binding site, it would be expected to interfere with fVIII
Expression of C2
was measured by enzyme-linked immunosorbent assay, as follows. Immulon
I plates were coated with 5 µg/ml mAb ESH4, blocked with 200
µl/well TBS, 1% bovine serum albumin, and recombinant fVIII
(41-524 ng/ml), or dilutions of a C2 sample with unknown
concentration were added. All incubation steps were at 37 °C for 2
h with shaking, and wells were washed three times with 200 µl/well
TBS, 0.05% Tween 20 (Bio-Rad). Binding of C2 and fVIII was detected
with biotinylated mAb ESH8 followed by streptavidin-alkaline
phosphatase. The fVIII standard curve was analyzed by linear
regression, and it was used for calculation of the C2 concentration.
Molecular masses of 18.5 kDa and 265 kDa were used for C2 and fVIII,
respectively, to determine their molar concentration. Wells coated with
ESH4 and containing all components except fVIII were used as negative
controls. The average values of all the negative controls were
Fragments SPII
and SPIII were obtained by limited proteolysis of vWf with
Staphylococcus aureus V8 protease (ICN Biomedicals Inc.) and
purified by fast protein liquid chromatography (FPLC) using a Resource
Q column (Pharmacia) as described
(34) . The SPIII-T4 fragment
was prepared by digestion of SPIII with
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
bovine pancreatic trypsin (Sigma)
(29) . The digested mixture was
treated with the serine protease inhibitor Pefabloc (Boehringer
Mannheim) at 1 mM and subjected to FPLC on a Superose 12HR
(Pharmacia) column under denaturing conditions (8 M urea, 0.1
M Tris, pH 7.2) as described
(35) . Fractions containing
SPIII-T4 were identified by SDS-PAGE and staining with silver nitrate.
SPIII-T4 was further purified by FPLC on a Superdex G-75 (Pharmacia)
column in TBS and by affinity chromatography on a column of 1.9 mg of
purified plasma fVIII (36) immobilized on Affi-Prep 10 gel (Bio-Rad) at
1.4 mg/ml. Eighty µg of SPIII-T4 was loaded in TBS, and the bound
material was eluted with a linear, 20-ml gradient of CaCl
To study the
effect of C2 on vWf or SPIII-T4 binding to immobilized fVIII,
On-line formulae not verified for accuracy
Herein,
On-line formulae not verified for accuracy where dissociation constant K
Purified, reduced vWf migrated on an
SDS-polyacrylamide gel as four vWf-specific bands of 270, 215, 140, and
120 kDa, Fig. 2, lane 1, identified by immunoblotting
with a polyclonal monospecific anti-vWf antibody, which were present at
80%, 2%, 4%, and 6.3% of the total protein, respectively. There was
also a faint unidentified band at 80 kDa (<3% total loaded protein),
as observed in other studies
(23, 27) , and two faint
bands at 40-50 kDa (<4.5%), which may be
fibrinogen
(27) . Purified, reduced SPIII and SPII fragments are
shown in Fig. 2(lanes 2 and 3) where they
migrate as major bands containing >85% of total protein at 170 kDa
and 110 kDa, respectively, as previously
reported
(27, 28) . The identity of the fragments was
additionally confirmed by determination of the amino-terminal amino
acid sequences, which were identical with those reported
(28) .
It was previously proposed that the binding sites in fVIII
for phospholipid and vWf are not closely juxtaposed, because only
amino-terminal vWf fragments larger than SPIII-T4(1-272), which
is known to contain a major fVIII binding site
(18) , are able to
inhibit binding of phospholipid to fVIII
(11, 19) ,
possibly by steric hindrance. We have, however, demonstrated that
SPIII-T4 is able to block fVIII access to PS, but the molar
concentration of SPIII-T4 required for effective inhibition is almost
100 times greater than that for vWf or SPIII. In our experiments, the
K
Since vWf was previously shown to bind to fVIII residues
2303-2332, a PS binding site, we tested SPIII-T4 for binding to
the same region. SPIII-T4 binding to fVIII was inhibited by a
recombinant C2 domain polypeptide and by synthetic peptide
2303-2332. The epitope of mAb NMC-VIII/5 overlaps amino acids
2303-2332, but those of mAbs ESH8 and 37 do not. Only mAb
NMC-VIII/5 inhibited SPIII-T4 binding to fVIII. Since the peptide and
mAb competition results for SPIII-T4 were identical with those we
reported for vWf
(23) , we conclude that SPIII-T4 and vWf bind to
the same, or very closely spaced, sites within amino acid residues
2303-2332 of the C2 domain. Thus, we propose that the ability of
SPIII-T4 to inhibit fVIII
The diminished
fVIII binding capacity of SPIII-T4 compared to SPIII and vWf is similar
to that previously observed for the P34 fragment of vWf (residues
1-298)
(19) , and it may be due to the presence of the
three internal proteolytic cleavages in all SPIII-T4
molecules
(29) . Our experiments, however, do not completely
exclude the possibility that the low affinity of SPIII-T4(1-272)
for fVIII binding may be due to the presence of another fVIII binding
region within residues 272-1365 of vWf, both of which are required for
high affinity binding to fVIII. We believe that this is unlikely since
we have demonstrated that the properties of the sites for SPIII-T4 and
vWf binding to fVIII are similar or identical. In addition, it was
previously shown that proteolytic fragments SPIII-T2 (heterodimer of
273-511 and 674-728), SPI (monomer of 911-1365), and 39/34
(monomer of 480-718) did not bind fVIII
(18, 19) .
The presence of PS in membranes is required for mediation of fVIII
binding
(41) . An fVIII PS binding site was localized to C2
domain residues 2303-2332
(9) , and a recombinant
glutathione S-transferase-C2 domain fusion protein binds to
100% immobilized PS
(23) . Deletion analysis of coagulation
factor V (fV), an fVIII homolog, demonstrated that removal of the C2
domain resulted in a complete loss of PS binding by fV
(42) ,
whereas both fV proteolytic fragments containing A3 and C1-C2 bound to
pure phosphatidylcholine (PC) vesicles but only C1-C2 bound to 100%
PS
(43) . A model for fV interaction with phospholipid was
proposed to consist of a first step mediated by C2 interaction with PS.
This would be followed by fV penetration into a PS/PC membrane,
mediated by A3
(43, 44) . Since fV does not bind to pure
PC
(43, 45) both steps would be required for fV high
affinity interaction with PS/PC membrane. A multistep character of
fVIII interaction with PS/PC membranes
(46) and possible
parallels to fV membrane binding suggest that regions of fVIII other
than the C2 domain sequence 2303-2332 may also be involved in
membrane binding. The 100% PS binding assay we have used would
represent the first step in fVIII binding to phospholipid.
Our
finding that SPIII-T4 binding to fVIII is inhibited by mAb NMC-VIII/10
(epitope within residues 1675-1684) is consistent with the
suggestion from previous studies that the acidic region of the light
chain(1649-1689) either directly or indirectly participates in
the binding of fVIII to vWf. If both the acidic region of the fVIII
light chain and the carboxyl-terminal region of C2 are in close
proximity and form one high affinity binding site for vWf, removal of
either region may reduce binding to vWf. This could explain why
thrombin cleavage at residue 1689, which removes the acidic region of
the light chain, leads to dissociation of the fVIII
The numbers in parentheses indicate
picomoles of amino acid at the given cycle; - means that the
expected C residue cannot be determined by the method of sequencing
used.
We thank Dr. Midori Shima for the generous gift of
mAbs NMC-VIII/5 and NMC-VIII/10. We also thank Drs. Yury Matsuka and
Sergey Litvinovich for their helpful advice regarding purification of
proteolytic fragments of von Willebrand factor and Dr. Dudley
Strickland for advice on the data analysis. We also thank Matthew Felch
for his help in the preparation of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
values for vWf
and SPIII-T4 for fVIII are 0.52 nM and 48 nM,
respectively, explain this discrepancy.
fVIII or SPIII-T4
fVIII binding. In both cases, binding
was inhibited by synthetic peptide 2303-2332, containing a PS
binding site, and by mAb NMC-VIII/5 Fab` (epitope within C2 residues
2170-2327). We propose that vWf, via residues 1-272, and PS
compete for fVIII binding because they recognize overlapping sites
within fVIII C2 domain residues 2303-2332.
(
)
and von Willebrand
factor (vWf) are distinct glycoproteins that both have important but
different roles in hemostasis. fVIII functions as a cofactor for factor
IXa in the intrinsic activation of factor X on a membrane
surface
(1) , and vWf promotes the adhesion of platelets to the
injured vessel wall. fVIII and vWf circulate in plasma as a
noncovalently linked complex. Formation of a complex between fVIII and
vWf is crucial to the stability of fVIII, since patients with severe
von Willebrand's disease, who have a complete deletion of the vWf
gene or mutations which reduce binding between fVIII and vWf, have a
secondary deficiency of fVIII
(2) .
vWf complex is dissociated by phospholipid vesicles
(10) and that vWf prevents fVIII from binding to such
vesicles
(10, 11) and to platelets(12) demonstrated
antagonism between phospholipid and vWf for binding to fVIII. Thrombin
cleavage of the light chain of fVIII at amino acid residue 1689
separates an acidic region (amino acid residues 1649-1689) from
the rest of the light chain
(13) . This leads to release of fVIII
from vWf
(5) and allows its subsequent binding to phospholipid.
Figure 1:
Domain structure of fVIII and
binding sites for mAbs and PS. The domain structure of fVIII (top
line) was previously published (3). Epitopes of mAbs were
determined using immunoblotting assays (16, 25). The PS binding site
was determined based on the ability of overlapping fVIII synthetic
peptides to inhibit the binding of fVIII to immobilized PS
(9).
The importance of the fVIII light chain acidic region for binding to
vWf was also supported by the observation that several monoclonal
antibodies with epitopes within residues
1670-1689
(14, 15, 16) inhibit fVIII
binding to vWf. The presence of post-translationally sulfated
Tyr was shown to be essential for vWf
binding
(17) . However, synthetic peptide 1673-1689 failed
to inhibit fVIII binding to vWf, regardless of whether Tyr
was sulfated(17). It was suggested
(17) that residues
1673-1689 and sulfated Tyr
may be required
indirectly to maintain the necessary tertiary structure for high
affinity vWf binding to the light chain, but the exact function of this
region is not clear.
PS
binding as vWf does. Alternatively, the binding of vWf to the fVIII C2
domain may be mediated by a different region of vWf. In this study, we
have examined the interaction of vWf and various vWf proteolytic
fragments with the fVIII C2 domain in order to determine the mechanism
by which vWf prevents phospholipid binding of fVIII.
Materials
Purified IgG from monoclonal antibody (mAb) ESH8 was obtained
from American Diagnostica. Highly purified, human recombinant fVIII
(3809 units/mg) and gelatin-Sepharose were generously provided by
Baxter/Hyland (Glendale, CA) and Shelesa Brew (American Red Cross),
respectively.
Monoclonal Antibodies
mAbs 413 (epitope within fVIII A2 domain amino acid residues
373-606) and 37 were purified as described previously
(23) to 90-95%, as estimated by electrophoresis in 10%
polyacrylamide-SDS gels and staining with Fast Stain (Zoion Research
Inc.). The preparations of mAbs NMC-VIII/5 and NMC-VIII/10 IgG and
their Fab` or F(ab)` and biotinylation of mAb 413 were
described previously
(16, 24, 25) .
Quantification of Proteins
Concentrations of proteins were determined by the method of
Bradford
(26) . The molar concentrations of vWf, SPIII, and
SPIII-T4 were calculated using molecular masses of 270 kDa
(27) ,
170 kDa
(28) , and 34 kDa
(29) , respectively. The protein
concentration of recombinant fVIII was determined by absorbance at 280
nm, using the extinction coefficient previously reported for porcine
fVIII
(30) . The molar concentration of fVIII was calculated
using the molecular mass of the deduced fVIII amino acid sequence,
equal to 265 kDa. fVIII and vWf antigen concentrations were determined
using immunoradiometric
(31) assays with a normal plasma
standard.
C2 Expression
The C2 domain cDNA preceded by the prepro polypeptide cDNA of
tissue plasminogen activator was constructed as described
(32) .
It was subcloned into the baculovirus transfer vector pVL941, kindly
provided by Dr. Max Summers, by a polymerase chain reaction procedure
which introduced new restriction sites at the 5` and 3` ends. The
resulting plasmid, pDS188, and baculovirus Autographa californica DNA were used to cotransfect Spodoptera frugiperda Sf9
insect cells. A recombinant baculovirus encoding C2 was purified as
described (25, 33), and expression of C2 with the expected molecular
mass of 18.5 kDa was verified by immunoblotting with anti-C2 mAb ESH8.
The cDNA sequence of the entire C2 domain was verified by sequencing
using the dideoxy chain termination method. The C2 domain polypeptide
was produced in Sf9 cells as described
(25) .
10%
of the maximal signal. The color developed by alkaline phosphatase
cleavage of p-nitrophenyl phosphate was read at 410 nm on an
MR5000 microtiter plate reader (Dynatech Laboratories). All samples
were assayed in duplicate. The average values of negative controls were
subtracted from the average values for all other samples.
Purification of C2
To precipitate C2, ammonium sulfate was added to the growth
medium of the infected cells at a final concentration of 50%. The
precipitate containing C2 was dissolved in 20 mM Tris, 0.15
M NaCl, pH 7.4 (TBS) and subjected to FPLC on a Superose-12HR
(Pharmacia) gel filtration column (1.6 60 cm) equilibrated in
TBS. Pooled fractions containing C2 were further purified by FPLC using
a Superdex G-75 gel filtration column (Pharmacia) equilibrated in TBS.
The C2 peak (V
/V
= 0.54) was diluted 1:2 with 50 mM histidine, pH
6.0, 0.01% Tween 80, loaded on a Resource S column equilibrated with
the same buffer, and eluted with a linear gradient of NaCl (0-1.2
M). C2 was eluted as a single peak at 0.8 M NaCl.
Recovery of purified C2 from the initial extract was about 25% as
measured by enzyme-linked immunosorbent assay and the Quantigold
protein assay (Diversified Biotech). The two assays gave similar
results.
Purification and Characterization of vWf and Its
Proteolytic Fragments
vWf was prepared from cryoprecipitate (Cutter Biological) as
described
(5) with the following modifications: 1) after
precipitation with 1.55 M NaCl, the precipitate containing vWf
was dissolved in 20 ml of TBS, 0.3 M CaCl, and vWf
was further purified on a 2.6
100 cm gel filtration column of
Sepharose 4B-CL equilibrated in the same buffer; 2) fibronectin (
4%
of total protein) was removed from the resulting preparation by passage
of vWf in TBS over a gelatin-Sepharose column. The final vWf
preparation (1 mg/ml) contained <0.2 µg/ml fVIII, as determined
by the immunoradiometric fVIII antigen assay
(31) . The purity of
vWf was determined by SDS-7.5% polyacrylamide gel electrophoresis of
reduced protein and staining with Coomassie Blue. Protein distribution
among the bands was determined by laser densitometry.
from 0 to 0.5 M in TBS. SPIII-T4 was eluted at 0.2
M CaCl
as a single peak containing 85% of the
loaded protein, and no other protein peaks were eluted at higher
CaCl
concentrations.
Synthetic Peptides
Peptides were synthesized, purified, and characterized as
described
(23) . Peptide solutions of 0.7-0.9 mM
in 10% acetonitrile were diluted for use to 0.2 mM with 50
mM NHHCO
, and the pH was adjusted to
7.4 with 0.1 M NaOH. The final concentration of
NH
HCO
used had no effect on assays in which
peptides were used.
Protein Iodination
Ten micrograms of vWf, SPIII, or SPIII-T4 in 50 µl of 0.5
M Tris, pH 7.4, were radiolabeled by incubation with 0.5 mCi
of NaI (Amersham), 50 µl of Enzymobead reagent
(Bio-Rad), and 25 µl of 1%
-D-glucose for 15 min.
Free Na
I was removed on a PD10 column (Pharmacia).
Recombinant fVIII was iodinated with the Bolton-Hunter reagent
I kit (ICN Radiochemicals). fVIII (30 µg) in 60
µl of 100 mM sodium borate buffer, pH 8.5, 5 mM
CaCl
, was incubated with 1 mCi of the iodinating reagent
for 6 h at 4 °C. The reaction was terminated by addition of 100
µl of 0.5 M ethanolamine, 10% glycerol in sodium borate
buffer, pH 8.5. The specific radioactivity of the proteins labeled by
either method was 4-10 µCi/µg.
Solid Phase Radioimmunoassays
Anti-fVIII mAbs 413 or ESH8 (3.5 µg/ml) in 0.1 M
NaHCO, pH 9.6, were incubated in Immulon 1 (Dynatech)
microtiter plates for 16 h at 4 °C. Each well contained 100 µl,
except at the TBS, bovine serum albumin (BSA) blocking step (200
µl). All incubation steps were at 37 °C for 1 h with shaking,
and the plates were washed 4 times after each step with 200 µl/well
of TBS, 0.05% Tween 20 (Bio-Rad). After blocking with TBS, 1% bovine
serum albumin (Sigma, fatty acid-free), anti-fVIII antibodies were
saturated with fVIII by addition of 1.6 µg/ml fVIII, followed by
the addition of increasing concentrations of
I-vWf or
I-SPIII-T4. Negative controls were mAb 413 or mAb ESH8
coated wells containing all components except fVIII or uncoated blocked
wells with all other components, and these values were subtracted from
average values of duplicates for all other samples. The values of these
controls were similar to values of nonspecific binding of
I-labeled SPIII-T4, SPIII, or vWf in the presence of a
200-fold excess of unlabeled SPIII-T4 or vWf, respectively, i.e.
20% and
10% of the maximal signal for mAb 413 or ESH8
fVIII immobilization, respectively. In competition studies, fixed
concentrations below those achieving saturation of
I-vWf
or
I-SPIII-T4 were added together with increasing
concentrations of antibodies or synthetic peptides.
I-SPIII-T4 or
I-vWf were added to the wells
together with increasing concentrations of unlabeled purified C2 or
fVIII. All the determinations were done in triplicate, and inhibitory
data were analyzed as described below. After washing, radioactivity
bound to the wells was counted in a Pharmacia LKB 1274
counter.
Affinity Measurements
Step a: Binding of SPIII-T4, SPIII, and vWf to
fVIII
Binding affinities were determined by both homologous and
heterologous ligand displacement assays. I-labeled vWf,
SPIII, or SPIII-T4 were added together with varying concentrations of
the homologous unlabeled protein and incubated at 37 °C for 2 h in
microtiter wells containing fVIII immobilized on mAb ESH8, followed by
washing and determination of bound radioactivity. Preliminary kinetic
studies of the binding of
I-labeled ligands to
immobilized fVIII in the presence of unlabeled competitors demonstrated
that for all ligands binding approaches equilibrium within 2 h at 37
°C. Data from homologous ligand competitions were analyzed using
the computer program LIGAND
(37) . For heterologous ligand
displacement studies, in which the displacing ligand differs from the
labeled ligand, the data were analyzed assuming two distinct
equilibria, R + L
RL
and R + L
RL
, where R is immobilized
fVIII, L
is
I-SPIII-T4, and
L
is vWf or SPIII. These equilibria are described
by the equilibrium constants K
and
K
. Since there is insufficient
information in a single heterologous displacement curve to solve for
all of the unknown parameters (K
,
K
, and maximal number of binding sites,
B
), the values for K
and B
were determined in a parallel
homologous ligand displacement experiment measuring the binding of
I-SPIII-T4 to immobilized fVIII.
Step b: Binding of fVIII to Immobilized PS
PS was
immobilized on a microtiter plate, and wells were blocked as described
(23). To determine K and the maximal
number of fVIII binding sites (B
) for PS,
I-fVIII was homologously displaced by unlabeled fVIII and
the data were fitted to a model with a single class of binding sites as
in Step a. In competition studies, vWf, SPIII, SPIII-T4, or SPII were
added to the PS-coated, blocked wells together with 0.18 nM
I-fVIII. The binding step was at 37 °C for 2 h with
shaking, which is sufficient time to achieve equilibrium for fVIII
binding to PS (data not shown) and fVIII binding to vWf and its
fragments. Negative controls were uncoated, blocked wells containing
all other components. The values of the negative controls were <5%
of the positive signal, representing
I-fVIII binding to
PS in the absence of competitor. All samples were assayed in triplicate
for Steps a and b.
Analysis of Competition Data
Binding data obtained
in the presence of varying concentrations of competitor were analyzed
using the following equation
(7, 38) :
represents the ratio of bound ligand
(
I-fVIII,
I-SPIII-T4, or
I-vWf, respectively) in the presence and absence of
competitor, [L
] is the concentration of added
labeled ligand, and [I] is the concentration of competitor
added. K
, the apparent
inhibition constant, was derived from nonlinear regression analysis
fitting
versus [I] to Equation 1 using the
Sigmaplot 1.02 program (Jandel Scientific). Correcting the value of
K
for contribution of the
direct interaction between labeled ligand and immobilized PS or fVIII
led to determination of the K
as follows:
and
maximal number of binding sites B
are parameters
describing fVIII binding to immobilized PS or SPIII-T4 and vWf binding
to immobilized fVIII obtained from independent homologous displacement
experiments as described above.
Purification and Characterization of vWf and Its
Proteolytic Fragments
Our previous finding that vWf binds to the
fVIII C2 domain and that this binding is inhibited by peptide
2303-2332, a PS binding site (Fig. 1), indicated that vWf
and PS have overlapping fVIII binding sites. Since the major fVIII
binding site of mature vWf has been localized to residues 1-272,
this region should also prevent fVIIIPS binding. In order to
confirm this possibility, we prepared and characterized proteolytic vWf
fragment SPIII-T4, a monomer of 1-272, by limited proteolysis of
vWf with S. aureus V8 protease and trypsin. The larger vWf
fragments SPIII, a dimer of 1-1365 which binds to fVIII, and
SPII, a dimer of 1366-2050 which does not
(18) , were also
purified for use as controls.
Figure 2:
SDS-polyacrylamide gel electrophoresis of
vWf and its proteolytic fragments. Lane 1, 7.5% gel of 10
µg of purified, reduced vWf, 5 µg of SPIII (lane 2),
or SPII (lane 3) fragments stained with Coomassie Blue.
Left panel, 8-25% gel of SPIII-T4 fragment reduced
(lane 4) and unreduced (lane 5) stained with silver
nitrate. The positions of molecular mass markers in kilodaltons are
indicated to the left.
Since gel filtration of SPIII-T4 using buffer containing 8
M urea can lead to its partial denaturation, affinity
purification of SPIII-T4 on an immobilized fVIII column was the final
step of SPIII-T4 purification (see ``Experimental
Procedures''). Purified SPIII-T4 migrated on an 8-25%
SDS-polyacrylamide gel as a band containing >98% of the total
protein at approximately 31 kDa (reduced) and 34 kDa (unreduced),
Fig. 2
(lanes 4 and 5), as previously
reported
(29) . A faint unidentified band (1.1%) at 40 kDa is
seen on the unreduced gel (lane 5). During affinity
purification of SPIII-T4, the amount of the 40-kDa fragment decreased
by 8-fold, which indicates that it does not to bind to fVIII with high
affinity. SPIII-T4 was previously shown to contain 4 disulfide-linked
peptides, vWf residues 1-10, 11-19, 264-272, and
21-263, due to internal proteolytic cleavages between residues
10-11, 19-20, and 263-264
(29) . The data shown
in the demonstrate that our SPIII-T4 preparation contained
the 4 expected amino-terminal sequences in equal amounts. In sequencing
cycle numbers 11-15, only residues 30-34 of the von
Willebrand peptide 20-263 were detected, which indicates that
>90% of the SPIII-T4 molecules contained internal cleavages at
positions 10-11, 19-20, and 263-264. Since the
peptides with sequences 1-10, 11-19, and 264-272 are
attached to the 20-263 peptide through disulfide
bonds
(29) , upon reduction only the single band corresponding to
sequence 20-263, which migrates at the expected mass of 31 kDa,
is seen on the gel (Fig. 2, lane 4). The broader band of
reduced SPIII-T4 (lane 4) is likely to be due to the ability
of -mercaptoethanol to intensify silver stains, as
described
(39) , by reduction of the seven disulfide bonds within
SPIII-T4
(29) and generation of thiol groups. The higher
intensity of silver staining of reduced proteins could also explain
some additional minor contaminants visible on the reduced gel which are
not seen on the unreduced gel. The amino-terminal amino acid sequences
determined from affinity-purified SPIII-T4 () demonstrate
that it also contains these cleavages as does SPIII-T4 which is not
affinity-purified (not shown). These results rule out the possibility
that the original 34-kDa material contained a fraction of a different
structure which selectively binds to an immobilized fVIII column.
Inhibition of Human fVIII Binding to PS by
SPIII-T4
To test our hypothesis that SPIII-T4 should inhibit
fVIII binding to PS, the effect of SPIII-T4 on I-fVIII
binding to immobilized PS was investigated, and the results are shown
in Fig. 3. In order to preserve the post-translationally sulfated
Tyr
which is essential for fVIII binding to
vWf
(17) , radioactive iodine was introduced into fVIII lysine
residues using the Bolton-Hunter reagent
(40) . Binding of
I-fVIII to PS was inhibited by increasing concentrations
of vWf, SPIII, and SPIII-T4, but not by SPII. The concentration of
I-fVIII (0.18 nM) in this experiment was 6.5
times below K
for fVIII
PS binding.
The molar concentration of SPIII-T4 required to achieve an inhibitory
effect was about 100-fold higher than the molar concentration of vWf or
SPIII. The inhibition constants (K
) were
calculated using Equations 1 and 2 by nonlinear regression analysis as
described under ``Experimental Procedures.'' The
K
= 1.18 ± 0.21 nM
and B
= 0.155 ± 0.17 nM
for
I-fVIII binding to immobilized PS used in Equation 2
were determined from an independent homologous diplacement experiment
(data not shown). The calculated K
values
for vWf, SPIII, and SPIII-T4 were 0.76 ± 0.18 nM, 1.02
± 0.18 nM, and 68 ± 16 nM,
respectively.
Figure 3:
Effect of vWf and its proteolytic
fragments on fVIII binding to PS. I-fVIII (0.18
nM) with increasing concentrations of unlabeled vWf
(
--
) or vWf fragments SPIII
(
--
), SPIII-T4 (
--
), or
SPII (
- - -
) was added to wells with
immobilized PS (see ``Experimental Procedures''). fVIII
binding in the presence of competitor is expressed as the percentage of
fVIII binding when no competitor was added. Each point represents the
mean value ± S.D. of triplicates. The curves show a best fit of
the data to a model describing competitive inhibition (see
``Experimental Procedures,'' Equation
1).
Determination of SPIII-T4 Affinity for fVIII
The
greater molar concentration of SPIII-T4 required for inhibition of
fVIIIPS binding compared to SPIII or vWf may reflect a lower
affinity of SPIII-T4 for fVIII. Thus, quantitative measurements of the
affinities of SPIII-T4, SPIII, and vWf for fVIII were obtained in
homologous ligand displacement assays. fVIII was captured to microtiter
wells by mAb ESH8, and
I-labeled SPIII-T4, SPIII, or vWf
binding to fVIII was determined in the presence of increasing
concentrations of the corresponding unlabeled ligands. The data shown
in Fig. 4A were used to calculate the
K
by computer analysis from the best fit
of the data to a single class of binding sites by the LIGAND
program
(37) . The K
values derived
for the binding of vWf, SPIII, and SPIII-T4 to fVIII were 0.52 ±
0.11 nM, 0.82 ± 0.2 nM, and 48.5 ± 9.7
nM, respectively. These results indicate that the SPIII-T4
fragment binds to fVIII with 60 or 93 times lower affinity than SPIII
or vWf. The value of K
obtained for the
fVIII
vWf interaction is similar to the previously reported
K
values of 0.44 nM and 0.21
nM(12, 15) .
Figure 4:
Determination of affinity of vWf and its
fragments for fVIII. A, homologous displacement by unlabeled
analogs. Binding of I-labeled vWf (0.23 nM),
SPIII (0.33 nM), or SPIII-T4 (7.7 nM) to fVIII
immobilized on mAb ESH8 in the presence of increasing concentrations of
the corresponding unlabeled ligands is shown. Binding conditions are
described under ``Experimental Procedures.'' The data
represent mean values ± S.D. from three independent experiments.
The curves show a best fit of the data to a single class of sites using
the computer program LIGAND. B, heterologous displacement of
I-SPIII-T4 by vWf or its fragments.
I-SPIII-T4 (5.9 nM) and increasing
concentrations of competitors SPIII-T4, vWf, SPIII, or SPII were added
to wells with fVIII immobilized on mAb ESH8, as described under
``Experimental Procedures.'' Symbols used in A and
B are:
--
, SPIII-T4;
--
, vWf;
--
, SPIII; and
- - - - -
, SPII.
To further confirm that
SPIII-T4 affinity for fVIII is much lower than that of vWf or SPIII,
inhibition of SPIII-T4 binding to immobilized fVIII by vWf and SPIII
was studied (Fig. 4B). The concentration of
I-SPIII-T4 was 8.3 times below K
for SPIII-T4
fVIII binding. The inhibitory effects of vWf
and SPIII on
I-SPIII-T4 binding to immobilized fVIII were
similar, as would be predicted from their similar
K
for binding to fVIII. However, it
required a molar concentration of unlabeled SPIII-T4 almost 100 times
higher than that of vWf or SPIII to achieve the same inhibition of
I-SPIII-T4
fVIII binding. The SPII fragment was not
inhibitory in this assay. The inhibition constants for vWf and SPIII
calculated from heterologous displacement data using the LIGAND program
were 0.5 ± 0.15 nM and 1.03 ± 0.25 nM,
respectively. Values of the inhibition constants for both vWf and SPIII
are close to the corresponding K
calculated from homologous displacement data. Thus, both
homologous and heterologous displacement data demonstrate that the
affinity of SPIII-T4 for fVIII is substantially lower than that of vWf
and SPIII.
Localization of the SPIII-T4 Binding Site to the fVIII C2
Domain
In order to determine if inhibition of fVIII binding to
PS resulted from SPIII-T4 binding to the C2 domain of fVIII as reported
for vWf
(23) , the effect of a recombinant C2 domain polypeptide
on SPIII-T4 binding to fVIII was tested. The C2 domain was produced as
a soluble, secreted polypeptide by Sf9 insect cells infected with a
recombinant baculovirus encoding C2. The growth medium from a
baculovirus infection of Sf9 cells contained 8-10 µg/ml C2,
as measured by enzyme-linked immunosorbent assay using two different
anti-C2 antibodies, mAbs ESH8 and ESH4, which do not compete with each
other for binding to fVIII (data not shown). C2, purified as described
under ``Experimental Procedures,'' was characterized by
amino-terminal amino acid sequencing which revealed that cleavage of
the prepro polypeptide was complete in 60% of the C2 molecules. Twenty
percent of the expressed C2 contained 3 residues of the prepro sequence
and 20% contained 9 residues. SDS-PAGE of purified, reduced C2 is shown
in Fig. 5. Amino-terminal sequencing of each band of the doublet
was carried out. The bottom band contained predominantly C2 without
prepro residues, whereas the upper band contained C2 with 3 or 9
residues of the prepro sequence.
Figure 5:
SDS-polyacrylamide gel electrophoresis of
the purified recombinant C2 domain polypeptide. The reduced sample (2.5
µg) was analyzed by electrophoresis in a 12% gel stained with
Coomassie Blue. The positions of molecular mass markers in kilodaltons
are indicated to the left.
Inhibition of
I-SPIII-T4 binding to fVIII by C2 was tested in an assay
using fVIII immobilized in microtiter wells by mAb ESH8, which does not
interfere with vWf binding
(23) . A saturating concentration of
fVIII was used to prevent subsequent binding of fluid phase C2 or fVIII
to mAb ESH8. Dose-dependent inhibition of
I-SPIII-T4
binding to immobilized fVIII by fluid phase C2 or fVIII is shown in
Fig. 6A. Inhibition constants for C2 and fVIII were
calculated using the model describing competitive inhibition (see
``Experimental Procedures,'' Equations 1 and 2) were 960
± 203 nM and 47.7 ± 10.3 nM,
respectively. The C2 polypeptide and fVIII also inhibited
I-vWf (0.1 nM or 0.2
K
) binding to immobilized fVIII,
Fig. 6B. The calculated K
values for C2 and fVIII were 105.3 ± 17.3 nM and
0.50 ± 0.15 nM, respectively.
Figure 6:
Effect of recombinant C2 domain on
SPIII-T4 or vWf binding to immobilized fVIII. A,
I-SPIII-T4 (5.9 nM) and increasing
concentrations of unlabeled C2 or fVIII were added to the wells with
fVIII immobilized on mAb ESH8. Binding of
I-SPIII-T4 is
expressed as a percentage of its binding in the absence of competitor.
Each point represents the mean value ± S.D. of triplicates. The
curves show a best fit of the data to a model describing competitive
inhibition (Equation 1 under ``Experimental Procedures'').
B,
I-vWf (0.1 nM) and increasing
concentrations of C2 or fVIII were added to wells with immobilized
fVIII as in A. Symbols used in A and B:
--
, C2;
--
,
fVIII.
The binding site of
SPIII-T4 within C2 was further localized with an fVIII synthetic
peptide, 2303-2332, which contains PS and vWf binding sites
(Fig. 1). Peptide 2303-2332 but not its randomized version
(TLHQAEIWIRLGAMDPSYERQLTYHEVCVR) inhibited I-SPIII-T4
binding to fVIII in a dose-dependent fashion, Fig. 7. Peptides
with the amino acid sequences 2218-2233 from the C2 domain of
fVIII or 351-365 from the fVIII heavy chain were not inhibitory,
as was previously observed for fVIII binding to immobilized
vWf
(23) . The dose of peptide 2303-2332 that reduced the
binding of SPIII-T4 to fVIII to 50% was approximately 6
µM. In a control experiment, incubation of immobilized
fVIII with peptide 2303-2332 did not lead to dissociation of
fVIII from mAb ESH8 (not shown); therefore, peptide 2303-2332
directly interfered with SPIII-T4 or vWf binding to fVIII.
Figure 7:
Effect
of synthetic peptides on SPIII-T4 binding to fVIII. Increasing
concentrations of fVIII peptides were preincubated with
I-SPIII-T4 (6.7 nM) for 1 h at 37 °C, and
the mixture was added to wells containing fVIII immobilized on mAb
ESH8. Binding of
I-SPIII-T4 in the presence of peptide is
expressed as a percentage of fVIII binding when no peptide was added.
Symbols represent peptides with the following fVIII amino acid
sequences:
--
, 2303-2332;
--
, randomized version of 2303-2332;
--
, 2218-2233; and
--
, 351-365.
Inhibition of fVIIIvWf binding by mAbs with epitopes within
the C2 domain
(23, 24) and within the acidic region of
the fVIII light chain (1649-1689)
(14, 15, 16) (Fig. 1) suggested that both regions of fVIII are
critical for its binding to vWf. In order to determine if these two
regions are also important for binding of fVIII to SPIII-T4, the effect
of anti-C2 domain mAbs NMC-VIII/5, ESH8, 37, and NMC-VIII/10, with the
C2 epitopes shown in Fig. 1, was tested. Anti-A2 domain mAb 413
was used to capture fVIII onto microtiter wells. Each of the other mAbs
was added in increasing concentrations at the
I-SPIII-T4
binding step. As shown in Fig. 8A, binding was inhibited
in a dose-dependent fashion by mAb NMC-VIII/5 IgG and Fab`, but not by
IgG of mAbs ESH8 or 37. Similar concentrations of the mAb NMC-VIII/5
IgG and Fab` reduced binding by 50% (7.5 nM and 8 nM,
respectively), suggesting that whole IgG is not required for
inhibition. mAb NMC-VIII/10 IgG and F(ab)`
also
demonstrated dose-dependent inhibition of SPIII-T4
fVIII binding
(Fig. 8B), and the IgG and F(ab)`
concentrations for 50% inhibition were approximately 5
nM and 130 nM. Neither mAb NMC-VIII/5 nor mAb
NMC-VIII/10 led to dissociation of fVIII from mAb 413 (not shown);
therefore, they directly inhibited SPIII-T4
fVIII binding.
Figure 8:
Effect of monoclonal antibodies on the
binding of SPIII-T4 to fVIII. I-SPIII-T4 (5.9
nM) and increasing concentrations of antibodies with epitopes
in the C2 domain of fVIII (A) or with epitopes in the acidic
region of fVIII light chain (B) were added to wells with fVIII
immobilized on mAb 413, as described under ``Experimental
Procedures.'' The following symbols are used for antibodies in
A:
--
, NMC-VIII/5 IgG;
--
, NMC-VIII/5 Fab`;
--
,
mAb 37 IgG;
--
, mAb ESH8 IgG. Symbols used in
B:
--
, NMC-VIII/10 IgG;
--
, NMC-VIII/10
F(ab)
`.
values for the respective binding of
vWf, SPIII, and SPIII-T4 to fVIII demonstrated that the higher
concentration of SPIII-T4 than SPIII or vWf required for inhibition of
fVIII
PS binding is due to the lower affinity of SPIII-T4 for
fVIII binding. We propose that this explains why the inhibition of
fVIII binding to phospholipid vesicles by SPIII-T4 was not observed in
the previous study
(11) , in which equimolar concentrations of
SPIII-T4, vWf, and SPIII were used to compete for fVIII
PS
binding.
PS binding can also be explained by its
direct prevention of fVIII access to phospholipid.
vWf
complex
(5) . This hypothesis predicts that the affinities of the
C2 domain and thrombin-cleaved light chain for vWf are lower than that
of the uncleaved light chain. Since we showed that the
K
value (0.5 nM) for inhibition
of vWf binding to immobilized fVIII by fluid phase fVIII is similar to
the K
for fVIII
vWf interaction
(0.52 nM), the K
for C2 binding
to vWf is expected to be similar to the K
(105 nM), which we measured for inhibition of
vWf
fVIII binding by C2. This value is about 200 times lower than
that for fVIII. Similar affinities for mAb ESH8 binding to C2 and
fVIII, determined by homologous displacement of
I-ESH8 by
the unlabeled analog,
(
)
suggest that the
recombinant C2 is likely to be correctly folded. Further
characterization of the C2 structure and determination of its
affinities for binding to vWf and PS are in progress.
Table:
Amino-terminal sequencing of the affinity
purified SPIII-T4 fragment
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.