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Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 136-142
Some Human Inhibitor Antibodies Interfere With Factor VIII
Binding to Factor IX
By
Degang Zhong,
Evgueni L. Saenko,
Midori Shima,
Matthew Felch, and
Dorothea Scandella
From the American Red Cross, Holland Laboratory, Rockville, MD; and
Nara Medical College, Japan.
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ABSTRACT |
Factor VIII (fVIII) functions as a cofactor of factor IXa in the
intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit
its activity. We previously showed by inhibitor neutralization assays
that the fVIII A2 and C2 domain polypeptides contain common inhibitor
epitopes. Often hemophilic inhibitor plasmas were partially neutralized
by C2 and more completely neutralized by fVIII light chain (A3-C1-C2),
suggesting the presence of an additional major inhibitor epitope(s)
within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids
1811-1818 of the A3 domain comprise a binding site for factors IX and
IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII
light chain, and the binding of each IgG to light chain was competed by
A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa
complex in a chromogenic assay was prevented by these inhibitors.
Therefore, we propose that another important mechanism of fVIII
inactivation by human inhibitors is the prevention of fVIIIa/fIXa
association.
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INTRODUCTION |
COAGULATION FACTOR VIII (fVIII) functions
as a cofactor of the protease factor IXa (fIXa) in the intrinsic
pathway of blood coagulation. Activated fVIII (fVIIIa) forms a complex
with fIXa and factor X in the presence of CaCl2 on a cell
surface containing negatively charged phospholipid where it acts to
increase the Vmax for the conversion of factor X to
Xa.1
FVIII is a high-molecular-weight glycoprotein composed of three
different types of domains arranged in the order NH2
-A1-A2-B-A3-C1-C2-COOH, as deduced from the cDNA
sequence.2,3 It circulates in the plasma as a heterodimer
of a light chain (A3-C1-C2) and a heavy chain (A1-A2-B)4
linked by a metal ion bridge. Lack of or defective fVIII causes the
disease hemophilia A, and plasma-derived or recombinant fVIII is used
to correct this bleeding disorder. In eight studies, 23% (6.2% to
33%) of hemophiliacs developed antibodies that inhibit fVIII
activity,5 and they present a serious clinical
complication.
Epitopes of common inhibitor antibodies were previously localized to
the A2 and C2 domains by assays in which recombinant A2 and C2
polypeptides neutralized the inhibitor activity.6 Anti-A2
antibodies prevent the function of the fIXa/fVIIIa enzyme complex on a
phospholipid surface but not its formation,7 whereas anti-C2 antibodies prevent the interaction of fVIII with phospholipid and von Willebrand factor.8 The greater neutralization of
many inhibitor plasmas by fVIII light chain than by recombinant C2 suggested the presence of another common inhibitor epitope(s) within
the A3 and/or C1 domains.6
FVIII binding sites for fIXa were localized to amino acid residues
558-565 of the A2 domain9 and residues 1811-1818 of the A3
domain.10 Synthetic peptides corresponding to each region inhibited the factor Xase activity of fIXa, which suggested that both
sites are required for maximal affinity of fVIII for fIXa. The
monoclonal antibody (MoAb) CLB-CAg A with an epitope in A3 domain
residues 1804-1818 prevented fVIII light chain binding to fIXa and
inhibited fVIII cofactor activity.10 Because of the
characteristics of the MoAb, we investigated the possibility that the
anti-A3-C1 inhibitors suggested in our previous
experiments6 may also prevent fVIII/fIX binding.
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MATERIALS AND METHODS |
Antibodies.
MoAbs CLB-CAg A and NMC-VIII/5 were kindly provided by Drs Jan van
Mourik (Central Laboratory, Netherlands Red Cross) and Midori Shima
(Nara Medical College, Japan), respectively. The preparation of IgG from plasmas of severe hemophilia A patients with
inhibitors was previously described.6 Inhibitor titers were
determined in the Bethesda assay.11
Construction of recombinant A3-C1 and (His)5-C2
polypeptide expression vectors.
A cDNA construct encoding the signal peptide of interferon- in frame
with the A3 and C1 domains (amino acids 1690-2172) and two stop codons
was assembled by overlap extension12 and cloned into
plasmid pMT2,13 which also encodes dihydrofolate reductase (dhfr). The pMT2 A3-C1 construct was used to transfect
dhfr Chinese hamster ovary (CHO) cells, a stable
dhfr+ cell line expressing A3-C1 was isolated, and CHO
cells containing amplified copies of A3-C1, verified by Southern
blotting, were selected in increasing concentrations of
methotrexate13 to 0.32 µmol/L. The C2 cDNA including the
stop codon was attached to a 5 sequence encoding the prepro
polypeptide of tissue plasminogen activator,14 five
histidines, and a thrombin cleavage site by overlap
extension,12 and the resulting DNA was cloned into
baculovirus vector pVL1393 (Invitrogen, Carlsbad, CA). Using the Bac to
Bac expression system (Life Technologies, Gaithersburg, MD), the C2 cDNA was transferred to a baculovirus plasmid by in vivo site specific
transposition in Escherichia coli. The C2-baculovirus plasmid
was purified and used to transfect Sf9 insect cells for generation of
baculoviruses expressing C2. Secreted C2 was quantitated by
enzyme-linked immunosorbent assay (ELISA)14 as 27 µg/mL. All cDNA constructs were sequenced to verify the absence of DNA errors.
The amplified A3-C1 cell line, 2 × 106 in 100-mm
tissue culture dishes, was grown in Dulbecco's modified Eagle medium
(DMEM)  -medium (Life Technologies) with 10% fetal calf serum for 48 to 72 hours to 90% confluency. The cells were washed two times with
methionine-free DMEM and labeled in 4 mL of this medium
with 0.5 mCi Tran35S-Label (ICN Biomedicals, Costa Mesa,
CA) for 4 hours at 37°C. The culture medium was collected, and 1%
NP40 (Sigma, St Louis, MO) was added. Cell lysates were suspended in 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl (TBS), 1% NP40. The growth medium
and cellular fractions were centrifuged at 10,000 rpm for 10 minutes at
4°C. Aliquots of the supernatants were stored at  80°C.
Preparation and radiolabeling of fVIII fragments.
Plasma derived fVIII was purified from fVIII concentrate Method M
(American Red Cross),15 and heavy and light chains were further purified as described.15,16 The A1 and A2 domains
were purified from plasma fVIII after thrombin cleavage17,
and recombinant C2 polypeptide was purified from Sf9 insect cell growth
medium.14 Ammonium sulfate (38%) was added to the Sf9
medium containing 0.7 mg (His)5-C2, and the C2 precipitate was dialyzed into 50 mmol/L phosphate, 0.3 mol/L NaCl, 0.05% Tween 20, pH 8.0, and passed over a 2-mL Ni2+-NTA agarose column
(Qiagen, Valencia, CA). The column was washed with the above buffer
adjusted to pH 5.8 followed by elution of (His)5-C2 with a
pH gradient of 5.6 to 4.0 in the same buffer. Recovery of
(His)5-C2 was 65%. Recombinant fVIII and B domain were
generously provided by Baxter/Hyland (Glendale, CA).
Polypeptides were radiolabeled with 5 µL Na 125I (100 mCi/mL; Amersham, Arlington Heights, IL) by immobilized lactoperoxidase (Worthington, Freehold, NJ) as described.17 Specific
radioactivities ranged from 5 to 13 µCi/µg for all polypeptides.
They were aliquoted and stored at 80°C for up to 1 month.
Immunoprecipitation of radiolabeled polypeptides by inhibitor IgG.
Details were previously described.17 Briefly, each
125I-polypeptide (0.75 nmol/L) was incubated with inhibitor
IgG or plasma dilutions at 4°C overnight. Immune complexes were
precipitated with Protein G Sepharose (Pharmacia Biotech, Piscataway,
NJ) and washed to remove unbound 125I-polypeptide. The
negative control contained all components except antibody.
Dose-response binding curves were done for each IgG, and data points
from the linear portion of each curve were used to calculate
immunoprecipitation (IP) units per milliliter: [(Bound/Total 125I-fVIII Polypeptide Background) × Plasma
Dilution × 20 (to Convert to IP Units/mL)]. IP of
35S-labeled A3-C1 polypeptide in cell culture medium with
inhibitor was performed as described above, except that bound
35S-labeled A3-C1 was detected by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide)
and autoradiography.
To determine the concentration of A3-C1, aliquots of the CHO cell
culture medium containing A3-C1 were used to compete the binding of
125I-recombinant fVIII to MoAb CLB-CAg A IgG in competitive
IP assays, a modification of the above IP method. Recombinant fVIII was
used to generate a standard curve.
Depletion of anti-C2 antibodies from inhibitor IgG.
Purified recombinant (His)5-C2 was immobilized on
Ni+2-NTA agarose at 250 µg per mL of resin via the
(His)5 tag. Inhibitor IgGs were diluted to 2 mg/mL in HBS
(20 mmol/L HEPES, 0.15 mol/L NaCl, pH 7.4, 0.01% Tween 80), mixed with
resin at a 2:1 vol/vol ratio, and incubated for 18 hours at 4°C.
Upon removal of the resin by centrifugation, the supernatants were
tested for the presence of anti-C2 antibodies by IP assay using
125I-labeled C2 polypeptide.
Chromogenic factor Xase assay.
Details were previously described.16 Briefly, plasma fVIII
(2 nmol/L) was incubated with increasing concentrations of MoAb CLB-CAg
A or inhibitor IgG for 30 minutes at 37°C. Aliquots were diluted
10-fold into HBS (above) with 5 mmol/L CaCl2, 2 nmol/L fIXa
(Enzyme Research Laboratories, South Bend, IN), and 20 µmol/L phosphatidylserine/phosphatidylcholine vesicles (75/25, wt/wt). fVIII
was actived with 40 nmol/L thrombin for 30 seconds, and 300 nmol/L
factor X was added. Aliquots were withdrawn after 15, 30, 45, and 60 seconds, and factor X activation was stopped with 0.05 mol/L EDTA.
Factor Xa generation was measured by cleavage of synthetic substrate
S-2765, 0.3 mmol/L (Pharmacia Hepar, Franklin, OH) using a
Vmax microplate reader (Molecular Devices, Menlo Park, CA).
A purified factor Xa standard (Enzyme Research Laboratories) was used
to convert absorbance (410 nm) into factor Xa concentration. The molar
concentrations of fIXa, factor X, and factor Xa were calculated using
molecular masses of 45 kD, 57 kD, and 45.3 kD, respectively.
FVIII or light-chain binding to a ligand using biosensor technology.
The kinetics of light-chain binding to ligands was determined by
surface plasmon resonance using the IAsys biosensor (Fisons, Cambridge,
UK). Anti-C2 MoAb NMC-VIII/5 (50 µg/mL) in 10 mmol/L sodium acetate,
pH 5.0, was covalently coupled to an activated carboxymethyldextran
coated biosensor cuvette (Affinity Sensors, Paramus, NJ) via amino
groups using succinimide ester chemistry.18 fIXa was
inactivated for use in the binding studies by reaction with the active
site specific reagent DEGR-CK (Calbiochem, La Jolla, CA) as
described.19 All measurements of fIXa-DEGR binding to fVIII
light chain were performed in 200 µL HBS, 5 mmol/L CaCl2 at 37°C. Dissociation of bound fIXa-DEGR was initiated by
substitution with 200 µL buffer lacking fIXa-DEGR. Binding of MoAb or
inhibitor IgG to immobilized light chain in the presence or absence of
the synthetic peptide was performed under the above conditions. The cuvette was regenerated by addition of 0.1 mol/L glycine, pH 3.0, for 3 minutes, resulting in complete dissociation of light chain from MoAb.
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RESULTS |
Expression of recombinant A3-C1 polypeptide.
The previously observed greater neutralization of inhibitors by light
chain than by C26 suggested the presence of one or more
additional inhibitor epitopes in the A3-C1 domains of the light chain.
To determine if antibodies from inhibitor plasmas bind to this region,
we constructed an expression plasmid, pMT2,13 encoding the
A3 and C1 domains of the light chain but not its amino terminal acidic
region, residues 1649-1689. The interferon- (IFN-) signal
sequence cDNA was linked in frame to the 5 end of the A3-C1 cDNA
for potential secretion of the recombinant polypeptide. A stable CHO
cell line containing multiple copies of the A3-C1 cDNA was established
(Materials and Methods). Expression of recombinant A3-C1 was analyzed
by IP of the growth medium and the cellular fraction of the
35S-Met labeled CHO cell line
(Fig 1) with MoAb CLB-CAg A IgG (epitope A3
residues 1804-182010) followed by SDS-PAGE. Similar
concentrations of 35S-A3-C1 with the expected molecular
weight (~60 kD) were seen in the extracellular (lane 1) and the
intracellular (lane 2) fractions, demonstrating approximately 50%
secretion. Additional bands seen in the cell fraction were not fVIII
specific because they were also present when MoAb CLB-CAg A was omitted
(Fig 1, lane 3). The A3-C1 concentration in the growth medium was
determined to be 0.76 µg/mL in a competitive IP assay (Materials and
Methods).
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| Fig 1.
Expression and secretion of A3-C1 polypeptide.
Immunoprecipitation of 35 S-A3-C1 by MoAb CLB-CAg A IgG (30 µg/mL)
was analyzed by 10% SDS-PAGE and autoradiography. The cellular and
growth medium fractions were adjusted to equal volumes. Lane 1, culture
medium; lane 2, cell lysate; lane 3, cell lysate without CLB-CAg A. Molecular weight standards are shown in kilodaltons (kDa) at the
left.
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Binding of human antibodies to the A3-C1 polypeptide.
The binding of 35S-A3-C1 to decreasing concentrations of
IgG purified from each of 10 inhibitor plasmas was measured by IP
followed by SDS-PAGE (Materials and Methods), and representative
results are shown in Fig 2. The binding of
the positive control, anti-A3 MoAb CLB-CAg A IgG10 (Fig
2B), and human inhibitors RI (Fig 2A) and MU (Fig 2C) was dose
dependent, and controls without antibody were negative. As both complex
antibodies and impure A3-C1 were used, the results in Fig 2 cannot be
used for quantitative comparisons among the IgGs. The binding of the
eight other human IgGs was also dose dependent in this assay (not
shown). For a more extensive analysis of the frequency of A3-C1 binding
by IgG in inhibitor plasmas, eight were diluted 1:5 and tested in
single-point IP assays. Binding was positive in seven and negative in
one (not shown). A3-C1 binding by 17 of 18 human inhibitors from both
experiments demonstrated that specificity for A3-C1 is common.
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| Fig 2.
Immunoprecipitation of A3-C1 polypeptide by MoAb and
hemophilic inhibitor IgGs. Cell culture medium containing
35S-methionine-labeled rA3-C1 was incubated with
increasing concentrations of IgG from human inhibitors RI or MU and
from MoAb CLB-CAg A. Immunoprecipitated 35S-A3-C1 was
analyzed as in Fig 1. (A) RI, lanes 1 through 6: 20, 10, 5, 2.5, 1.25, and 0.625 µg/mL; lane 7: no antibody; lane 8: 3 µg/mL of CLB-CAg A
IgG. (B) MoAb CLB-CAg A, lanes 1 through 7: 2, 1, 0.5, 0.25, 0.12, 0.06, and 0.03 µg/mL; lane 8: no antibody. (C) MU, lanes 1 through 8:
20, 10, 5, 2.5, 1.25, 0.62, 0.31, and 0.15 µg/mL; lane 9: no
antibody; lane 10: 3 µg/mL of CLB-CAg A IgG.
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Characteristics of IgGs used in determination of inhibitor mechanism.
For further characterization of antibodies directed against A3-C1, we
selected three IgGs from hemophilia A patients with high Bethesda
titers. In previous inhibitor neutralization assays6 the
MU, RI, and MS IgGs were neutralized to a greater extent by light chain
than by C2 (Table 1), suggesting that they
contained 44%, 48%, and 79% anti-A3-C1 and 15%, 26%, and 30%
anti-C2 inhibitors, respectively. MU and RI were not detectably
neutralized by the A2 domain ( 10%) but MS was 27% neutralized. In
addition, binding of each IgG to the individual fVIII heavy-chain
domains, the intact light chain, and the isolated C2 domain by
semi-quantitative IP assays (Materials and Methods) was also tested.
Figure 3 contains representative binding
curves for RI binding to the isolated fVIII fragments, and the total
results are summarized in Table 1. The combined anti-heavy-chain
titers were 0.1 of the anti-light-chain titers. The percent
anti-A3-C1 antibodies (LCh-C2) was 61%, 37%, and 91% and anti-C2
antibodies was 39%, 64%, and 9% for MU, RI, and MS, respectively.
These percentages are not the same as those determined by the inhibitor
neutralization assay due to the presence in plasmas of both inhibitory
and noninhibitory antibodies.20
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| Fig 3.
Binding of inhibitor RI IgG to fVIII-derived
polypeptides. IP assays with radiolabeled fVIII domains A2 ( ), C2
( ), and light chain ( ) were performed as described in Materials
and Methods.
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Because the neutralization assays indicated that 15% to 30% of the
inhibitor titers were caused by anti-C2 antibodies, each IgG was
depleted of these antibodies (Materials and Methods) to measure only
the effects of anti-A3-C1 antibodies in the following experiments. IP
assays of 125I-C2 binding by each IgG before and after the
depletion showed the >300-fold removal of anti-C2 antibodies in all
three IgGs (Table 1).
Inhibition of fIXa binding to fVIII light chain by inhibitor IgG.
Although the fIXa binding site consists of amino acid residues within
the A29 and A3 domains,10 the light chain binds
to fIXa with moderate affinity (kd =14.8
nmol/L).21 Due to the fact that inhibitors RI,
MU, and MS had measurable levels of anti-A2 antibodies (Table 1) that
might complicate the interpretation of the results, we chose to examine
the effect of inhibitor IgG on fIXa binding to the light chain only.
For detection of protein-protein interactions we used a biosensor
technique based on the surface plasmon resonance phenomenon which
measures protein binding in real time. The change in the refractive
index due to association of a fluid-phase ligand with an immobilized
ligand is measured by generation of a signal of 200 Arc seconds per 1 ng of protein bound per mm2 of the biosensor cuvette. MoAb
NMC-VIII/5 (C2 epitope 2170-23278) was immobilized to the
cuvette, and the light chain (20 µg/mL) was maximally bound to 35 fmol/mm2.
To determine if all immobilized light-chain molecules were accessible
for fIXa binding, we measured their maximal binding capacity. To
prevent the possible fIXa-mediated cleavage of immobilized light chain
at Arg1721,22 we used an active-site modified fIXa (fIXa-DEGR), which lacks proteolytic activity19 but retains fVIII binding properties.23
Curve 1 in Fig 4 shows the resonance
response of the time course of fIXa-DEGR association with light chain
and dissociation of the complex upon replacement of fIXa-DEGR with
buffer (shown only for curve 1). No binding of fIXa-DEGR to the cuvette
was observed in the absence of light chain (data not shown). The
association rate constant (kon = 1.2 × 104 M1s1) and the
dissociation rate constant (koff = 3.4 × 104 s1) for fIXa-DEGR/light-chain
interaction were derived from the best fit of these kinetic data to a
model describing single-phase association and dissociation processes
using the Fast Fit 1.0b computer program (FISONS). Because the
concentration of fIXa-DEGR (1,000 nmol/L) is >25 times the
corresponding equilibrium dissociation constant (kd = 28 nmol/L,
calculated as koff/kon) for its interaction with light chain, the amount of fIXa-DEGR bound at equilibrium (36.6 fmol/mm2) represents the maximum binding capacity of
immobilized light chain. This result shows that the immobilized light
chain (35 fmol/mm2) was able to bind fIXa-DEGR with 1:1
stoichiometry. In addition, the similar values of the kd for
fIXa-DEGR/light chain determined in our assay (28 nmol/L) and that
determined for fIXa/fVIIIa interaction in solution (42 nmol/L)24 suggest that the light-chain binding properties
were not significantly altered by immobilization. The absence of
phospholipid is probably a major contributor to the relatively high kd
values for fVIII -fIX interaction obtained in both our (28 nmol/L) and
previous experiments (42 nmol/L).24 This interpretation is
consistent with reported kds of 2.3 and 46 nmol/L for fVIIIa/fIXa
association in the presence and absence of phospholipid,
respectively.24
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| Fig 4.
Effect of stoichiometric titration of the light chain
with inhibitors MU, RI, MS, and MoAb CLB-CAg A IgGs on fIXa binding. (A) Association of fIXa-DEGR (1,000 nmol/L) with light chain (35 fmol/mm2) in the presence of 0, 14, 24, 31, 38, 46, and 52 fmol/mm2 bound RI IgG is shown in curves 1 through 7, respectively. The resonance response curves recorded upon addition of
fIXa-DEGR were corrected by subtraction of the resonance signal
produced by bound IgG, and therefore the curves show the resonance
signals solely produced by binding of fIXa to immobilized light chain (curve 1) or to IgG/light chain complexes (curves 2 through 7). (B)
Binding of fIXa in the presence of MoAb CLB-CAg A IgG () or
inhibitor IgG from MU ( ), RI ( ), or MS ( ) prebound to
immobilized light chain at the indicated molar ratio was determined
from the equilibrium binding of fIXa-DEGR in the presence of each IgG
as in (A).
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Inhibitor or MoAb IgGs were bound to the light chain at varying
IgG/light-chain molar ratios calculated from the resonance signals
produced by bound IgG (not shown), which was followed by replacement of
the IgG solution with buffer containing 1,000 nmol/L fIXa-DEGR. The
binding of fIXa-DEGR to light chain in the presence or absence of bound
RI IgG approached equilibrium after 50 minutes, and the fIXa-DEGR
binding progressively decreased upon saturation of light chain with RI
IgG (Fig 4A). Similar results were obtained for IgG from MU, MS, and
MoAb CLB-CAg A (not shown). Because of the multiple binding components,
these experiments more closely represent a stoichiometric titration
rather than equilibrium binding. In a control experiment, dissociation
of bound IgG was less than 3% during 50 minutes, which showed that the
IgG/LCh ratio did not change significantly during fIXa-DEGR binding to
LCh.
FIXa-DEGR binding to light chain was inhibited more than 95% by MoAb
CLB-CAg A at a stoichiometric ratio (Fig 4B). As the MoAb binds to A3
residues 1804-1820, an fIX binding site, the binding of MoAb and fIXa
to light chain is mutually exclusive. MU, RI, and MS IgGs maximally
inhibited fIXa-DEGR binding by 95%, 91%, and 88%, respectively, at
IgG/light chain ratios of 1.5 (Fig 4B). The nonstoichiometric ratios
for the human inhibitors may be due to the additional presence of lower
concentrations of antibodies directed against regions of A3-C1 other
than the fIXa binding site.
Effect of synthetic A3 domain peptide 1804-1819 on inhibitor IgG
binding to light chain.
As the inhibitory effect of MoAb CLB-CAg A is caused by direct
competition with fIXa for fVIII binding,10 we tested the hypothesis that the human anti-A3-C1 antibodies have the same mechanism of inhibition. We used synthetic A3 peptide 1804-1819 to
compete for human IgG binding to light chain. MoAb CLB-CAg A (10 nmol/L) or inhibitor IgGs MU (50 nmol/L), RI (1,000 nmol/L), and MS
(1,000 nmol/L) were incubated with increasing concentrations of peptide
for 1 hour at 37°C before addition to the biosensor cuvette
containing light chain immobilized on MoAb NMC-VIII/5. The above
antibody concentrations were chosen as each inhibited the factor Xase
assay (see below) by approximately 80%. The concentration of
anti-fVIII antibodies was different in each IgG; therefore, the total
IgG required in our experiments also varied widely. Stoichiometric
titration, measured as above, was achieved within 30 minutes at all
peptide concentrations. The binding of human inhibitor MU, RI, and MS,
or MoAb CLB-CAg A IgG to light chain was inhibited  73% in a
dose-dependent mannner by the peptide (Fig
5). The maximal inhibition of binding was 94%, 92%, 76%, and 73%
for CLB-CAg A, MU, RI, and MS IgG, respectively. The less complete
inhibition of MS and RI binding by the peptide may be due to
insufficient peptide concentrations or to the presence of a minor
population of antibodies with an epitope(s) outside the 1804-1819 sequence. The millimolar concentrations of peptide required for
inhibition in each case suggests that the peptide-antibody affinities
are low.
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| Fig 5.
Effect of fVIII synthetic peptides on antibody binding to
immobilized light chain. Increasing concentrations of A3 peptide 1804-1819 were preincubated with IgG from MU (), RI (), MS (), or MoAb CLB-CAg A () for 1 hour at 37°C, and the mixture (200 µL) was added to the biosensor cuvette containing light chain immobilized as in Fig 4. In the control experiments, binding of MU in
the presence of peptide 417-428 (x), amino acids QRIGRKYKKVRF, or the
randomized version of 1804-1819 () was determined as above. Antibody
binding in the presence of peptide is expressed as the percentage of
antibody binding when no peptide was added.
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A randomized, soluble peptide (PVKETYKFNKKTVFNV) of the fVIII sequence
1804-1819 was used as a control for MU and CLB-CAgA (not shown) binding
to LCh. There was no inhibition up to 4 nmol/L peptide. Because peptide
1804-1819 contains four positively charged Lys residues and one
negatively charged Glu residue, its effect on antibody binding may be
caused by the overall positive charge. This possibility was excluded by
lack of inhibition of MU (Fig 5; RI and MS, negative but not shown) in
the above assay by an unrelated control peptide of A2 domain residues
417-428, containing six positively charged and two negatively charged
amino acids.
MU IgG inhibits fVIII activity in a chromogenic factor Xase assay.
To determine if anti-A3-C1 antibodies are able to inhibit fVIII
function in a purified system, we tested their effect in a chromogenic
factor Xase assay using purified components. This assay (Materials and
Methods) measures the ability of activated fVIII (fVIIIa) in the
presence of phosphatidylserine/phosphatidylcholine vesicles (PSPC) to
act as a cofactor for fIXa in the activation of factor X.16
Because fVIIIa activity cannot be directly measured in this assay, we
determined the initial rate of factor X activation using a chromogenic
peptide substrate, S-2765. Under our conditions, this rate was linearly
proportional to the fVIIIa activity.
Only inhibitor MU IgG was tested because it had the lowest ratio of
anti-A2 to anti-light-chain antibodies (2.5 × 105) and no other anti-heavy-chain antibodies by IP
assays. Due to further depletion of anti-C2 antibodies (Materials and
Methods), the ratio of anti-C2 to anti-light chain antibodies was
decreased to 6.7 × 105 (Table 1). The MU IgG
used in this experiment therefore contained more than 99.9%
anti-A3-C1 antibodies. MU and CLB-CAg A IgGs both inhibited the factor
Xase assay by more than 90% (Fig 6).
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| Fig 6.
Inhibition of fVIII activity in the factor Xase assay by
MoAb CLB CAg A and inhibitor MU. FVIII (2 nmol/L) was preincubated with
increasing concentrations of inhibitor MU () or MoAb CLB-CAg A ()
IgG for 30 minutes at 37°C followed by determination of fVIII
activity in the chromogenic factor Xase assay as described in Materials
and Methods.
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DISCUSSION |
The greater neutralization of many inhibitor plasmas by the fVIII light
chain than by C26 suggested that these plasmas may contain
inhibitor antibodies with epitopes in A3-C1. To examine this
possibility, we first demonstrated by an IP assay that 17 of 18 human
inhibitor IgGs bound to a recombinant 35S-A3-C1
polypeptide. The IgG from three such inhibitors with >10-fold higher
anti-light chain than anti-heavy chain antibody titers was further
depleted of anti-C2 domain antibodies to prevent their interference in
subsequent assays. One IgG (MU) with barely detectable anti-heavy
chain antibodies completely inhibited the intrinsic factor Xase assay;
therefore, some anti-A3-C1 antibodies are likely to have inhibitor
activity.
MoAb CLB-CAg, A which binds to the A3 domain peptide 1804-1818, a fIX
binding site, also inhibits fVIII activity.10 No other binding site for a physiological ligand of fVIII has been localized to
A3-C1. Therefore, we tested the possibility that some human anti-A3-C1
antibodies inhibit fVIII-fIX binding, which is required for assembly of
the factor Xase complex. In an assay of fIXa binding to light chain,
the three inhibitor IgGs bound to light chain inhibited subsequent fIXa
binding by 88%, compared with 97% by CLB-CAg A. The inhibition of
the human and MoAb CLB-CAg A IgG binding to light chain by synthetic
peptide 1804-1819 suggested that the epitopes of these antibodies
overlap the fIX binding site. These data are consistent with a model of
direct inhibition of fIX binding to fVIII by these antibodies.
We found that peptide 1804-1819 inhibited binding of inhibitor MU IgG
and MoAb CLB-CAg A to light chain by 88% to 90%. This result implies
that MU anti-A3-C1 IgG is mainly directed against the fIX binding
site, which is consistent with the observed 1.1:1.0 molar ratio of
bound MU IgG to light chain. In contrast, binding of the inhibitor IgGs
RI and MS to light chain was inhibited only 76% and 73%,
respectively, by the peptide. This may be due to insufficient
concentrations of peptide for complete inhibition of binding or to the
presence of a minor population of anti-A3-C1 antibodies that do not
bind to the fIXa site.
Although we tested only three anti-A3-C1 inhibitors, all of which
competed for fIX binding to fVIII, this may be a common inhibitory
mechanism as suggested by our earlier finding that 20 of 34 inhibitor
plasmas were more completely neutralized by light chain than by its C2
domain.6 This remains to be confirmed by characterization
of additional inhibitors.
| |
FOOTNOTES |
Submitted October 27, 1997;
accepted February 13, 1998.
D.Z. and E.L.S. made equal contributions to the experimental work.
Address reprint requests to Dorothea Scandella, PhD, American Red
Cross, 15601 Crabbs Branch Way, Rockville, MD 20855.
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 |
We thank Dr Jan van Mourik (Netherlands Red Cross, Amsterdam) for
providing the MoAb CLB-CAg A, which was crucial for our work.
| |
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Abstract
Introduction
Abstract