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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3701-3709
Residues Glu2181-Val2243 Contain a Major Determinant of the
Inhibitory Epitope in the C2 Domain of Human Factor VIII
By
John F. Healey,
Rachel T. Barrow,
Hiba M. Tamim,
Ira M. Lubin,
Midori Shima,
Dorothea Scandella, and
Pete Lollar
From Emory University, Atlanta, GA; Holland Laboratory, American Red
Cross, Rockville, MD; and the Department of Pediatrics, Nara Medical
College, Kashihara Nara, Japan.
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ABSTRACT |
The human blood coagulation factor VIII C2 domain (Ser2173-Tyr2332)
contains an epitope recognized by most polyclonal inhibitory anti-factor VIII alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and
porcine factor VIII and mapped a major determinant of the C2 epitope by
using a series of active recombinant hybrid human/porcine factor VIII
molecules. A series of five C2-specific human antibodies and a murine
anti-factor VIII monoclonal antibody, NMC-VIII/5, inhibited a hybrid
containing a substitution of porcine sequence for Glu2181-Val2243
significantly less than human factor VIII. In contrast, four of the
five patient antibodies and NMC-VIII/5 inhibited a hybrid containing a
substitution of porcine sequence for Thr2253-Tyr2332 equally well as
human factor VIII. Thus, a major factor VIII inhibitor epitope
determinant is bounded by Glu2181-Val2243 at the
NH2-terminal end of the C2 domain. Because C2 inhibitors
block the binding of factor VIII to phospholipid and von Willebrand
factor, for which binding sites have been localized to Thr2303-Tyr2332,
these results imply that the segment bounded by Glu2181-Val2243 also is
involved in these macromolecular interactions.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
INHIBITORY ANTIBODIES (inhibitors) to
factor VIII (FVIII) either develop as alloantibodies in approximately
25% of hemophilia A patients after FVIII infusions or as
autoantibodies in nonhemophiliacs.1 The characterization of
the epitopes recognized by these inhibitors has been the subject of
several investigations.2-10 The approximately 300-kD FVIII
molecule contains homologous A and C regions that define an
A1-a1-A2-B-a2-A3-C1-C2 domainal
sequence.11,12 The 36-residue a1 peptide is
proteolytically cleaved by factor IXa or activated protein C, whereas
the 41-residue a2 peptide is proteolytically cleaved by
thrombin or factor Xa. Although FVIII is synthesized as a single-chain
molecule, it circulates in heterodimeric form because of proteolysis
within the B domain. FVIII heavy and light chains are designated
A1-a1-A2-B and a2-A3-C1-C2, respectively.
Inhibitor neutralization assays indicate that antibodies to epitopes in
the A2, a2-A3-C1, and C2 domains are responsible for the
anticoagulant activity in most inhibitor plasmas.8 Patients can have antibodies that recognize any combination of these epitopes. Additionally, the epitope pattern can vary with time.13 The epitopes recognized by inhibitors from alloantibody and autoantibody patients appear to be identical, despite their different immunological backgrounds. However, the epitope pattern differs in that
autoantibodies are more likely to recognize single epitopes, whereas
alloantibodies usually recognize multiple epitopes.8
FVIII participates in several macromolecular interactions that are
potential targets for disruption by inhibitory antibodies. It binds to
and circulates with von Willebrand factor (vWF) as an inactive
precursor. It is activated by thrombin or factor Xa, which cleaves
FVIII at Arg372, between the A1 and A2 domains; at Arg740, between the
A2 and B domains; and at Arg1689, which releases the a2 peptide
from the light chain.14 FXa catalyzes additional cleavages
within the A1, A2, and A3 domains.14,15 Thrombin-activated
FVIII (FVIIIa) is an A1-a1/A2/A3-C1-C2
heterotrimer.14,16-19 It functions in the intrinsic pathway
of blood coagulation by binding to both factor IXa, phospholipid, and
possibly FX and promoting the activation of FX by FIXa.
The release of the a2 peptide is necessary for FVIII to
dissociate from vWF. This is a requisite step in blood coagulation, because the binding of FVIII to phospholipid membranes and to vWF are
mutually exclusive.20-22 vWF promotes release of FVIII into
the circulation and increases its circulatory
lifetime.23-26 However, it is not required for the
activation of FVIII or the function of FVIIIa. In principle, inhibitors
also could act by blocking the interaction of FVIII with vWF and
destabilize FVIII in vivo. However, this is not a common mechanism of
action.
A2 inhibitors bind to the FVIIIa/FIXa/phospholipid membrane complex and
inhibit FX activation noncompetitively.27 a2-A3-C1 inhibitors have not been as thoroughly characterized, but may inhibit
the binding of FVIIIa to FIXa.28 C2 inhibitors inhibit the
binding of FVIII to phospholipid,29 which is consistent with the finding that phospholipid protects FVIII from inactivation by
some inhibitors.29,30 Additionally, C2 inhibitors inhibit the binding of FVIII to vWF,31,32 suggesting that vWF and
phospholipid bind to common or overlapping sites on FVIII. However, the
a2 region is necessary for binding of FVIII to vWF, but not to
phospholipid.33-38 Thus, phospholipid and vWF binding
involve distinct sites in FVIII. The interplay between the binding of
FVIII to phospholipid, vWF, and C2 inhibitors implies a complexity to
C2 macromolecular interactions that is poorly understood.
In this study, we took advantage of the limited cross-reactivity of
human and porcine FVIII and constructed a series of active recombinant
hybrid human/porcine FVIII molecules to map C2 inhibitor epitopes by
homolog scanning mutagenesis. We previously used this method to map a
major determinant of the A2 inhibitor epitope to
Arg484-Ile508.10 Our results indicate that C2-specific
antibodies recognize a region bounded by Glu2181-Val2243 in the
NH2-terminal half of the C2 domain.
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MATERIALS AND METHODS |
Materials.
Citrated hemophilia A and normal pooled human plasmas were purchased
from George King Biomedical, Inc (Overland Park, KS). Heparin-Sepharose
was purchased from Sigma Chemical Co (St Louis, MO). Human
vWF was purified as described previously.37 Murine monoclonal anti-FVIII antibody, ESH5, was purchased from American Diagnostica (Greenwich, CT). Highly purified, albumin-free
human recombinant FVIII was a gift from Baxter Corp (Duarte,
CA) and was stored in aliquots at 0.45 mg/mL at
 80°C. Alkaline phosphatase-conjugated goat antimouse
antibody was purchased from Bio-Rad (Hercules, CA). Fetal
bovine serum, geneticin, penicillin, streptomycin, Dulbecco's modified
Eagle's medium-F12 (DMEM-F12) medium, and AIM-V medium were purchased from Life Technologies, Inc
(Gaithersberg, MD). Taq DNA polymerase was
purchased from Promega (Madison, WI). Vent DNA
polymerase was purchased from New England Biolabs (Beverley, MA). Pfu DNA polymerase and the
phagemid pBlueScript II KS were purchased from
Stratagene (La Jolla, CA). Synthetic
oligonucleotides were purchased from Life Technologies or
Cruachem, Inc (Sterling, VA). Restriction enzymes were
purchased from New England Biolabs or Promega. 5 -Phosphorylated
primers were used when polymerase chain reaction (PCR) products were
produced for cloning purposes. Nucleotide (nt) numbering of
oligonucleotides used as primers for PCR amplification of porcine FVIII
cDNA or genomic DNA uses the human FVIII cDNA as
reference.39 A FVIII expression vector, designated
HB/ReNeo, was a gift from Biogen, Inc
(Cambridge, MA). HB/ReNeo contains
ampicillin and geneticin resistance genes and a human FVIII cDNA that
lacks the entire B domain, defined as the Ser741-Arg1648 cleavage
fragment produced by thrombin. To simplify mutagenesis of FVIII C2
domain cDNA, which is at the 3 end of the FVIII insert in ReNeo,
a Not I site was introduced two bases 3 to the stop
codon of HB/ReNeo (Fig
1) by splicing-by-overlap extension (SOE) mutagenesis.40 This construct is designated HB/ReNeo/Not I.
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| Fig 1.
Recombinant FVIII constructs. Amino acid numbering refers
to mature, full-length human FVIII.39 The parent human
B-domainless FVIII construct (HB) lacks residues
741-1648. Porcine B-domain deficient FVIII (PSQ) contains a 14-residue
sequence (SQ) corresponding to Ser741-Asn745, Pro1640-Arg1648 in human
FVIII, instead of the B domain. Shaded regions correspond to areas of
porcine substitution. In constructs HP23-HP28, these boundaries are
defined by amino acid residues of human-porcine nonidentity at amino
acid residues 2181, 2199, 2207, 2243, 2253, 2311, and 2321.
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Total RNA was isolated by acid guanidinium
thiocyanate-phenol-chloroform extraction.41 cDNA was
synthesized from mRNA using Moloney murine leukemia virus reverse
transcriptase (RT) and random hexamers according to instructions
supplied by the manufacturer (First-Strand cDNA Synthesis Kit;
Pharmacia Biotech, Uppsala, Sweden). Plasmid DNA was
purified using a Qiagen Plasmid Maxi Kit (Qiagen, Inc, Valencia,
CA). PCR reactions were done using a Hybaid OmniGene
thermocycler using Taq, Vent, or Pfu DNA
polymerases, unless otherwise indicated. PCR products were
gel-purified, precipitated with ethanol, and ligated into plasmid DNA
using T4 DNA ligase (Rapid DNA ligation kit; Boehringer Mannheim,
Indianapolis, IN). Insert-containing plasmids were used
to transform Escherichia coli Epicurean XL1-Blue cells. All
novel FVIII DNA sequences generated by PCR were confirmed by dideoxy
sequencing using an Applied Biosystems 373a automated DNA sequencer and
the PRISM dye terminator kit (Applied Biosystems, Foster City,
CA).
FVIII assays.
The activity of recombinant FVIII proteins was measured by one-stage
clotting assay42 using either a manual tilt tube assay or
an ST4 BIO Coagulation Instrument (Diagnostica Stago,
Parsippany, NJ). One unit of FVIII is defined as the activity in 1 mL
of pooled normal citrated human plasma. FVIII inhibitor titers were
measured using the Bethesda assay.43 To measure the
inhibitor activity against recombinant FVIII, human B-domainless FVIII,
porcine B-domain-deficient FVIII, or hybrid human porcine B-domainless
FVIII were added to hemophilia A plasma to a final concentration of 0.8 to 1.2 U/mL. One Bethesda unit (BU) is defined as the amount of
inhibitory activity that produces 50% inhibition of FVIII activity in
the one-stage assay.
Construction of a hybrid FVIII expression vector, HP20, containing
the porcine C2 domain.
A porcine FVIII cDNA corresponding to the 3 end of the C1 domain
and all of the C2 domain was cloned into pBluescript by RT-PCR from
spleen total RNA using primers based on known porcine FVIII cDNA
sequence.44 This construct and HB/ReNeo
were used as templates to construct a human C1-porcine C2 fusion
product in pBlueScript by SOE mutagenesis. The C1-C2 fragment in this
plasmid was removed with Apa I and Not I and ligated
into Apa I/Not I-cut
HB/ReNeo/Not I to produce
HP20/ReNeo/Not I.
Construction of hybrid human/porcine FVIII molecules containing
porcine C2 domain substitutions.
HB/ReNeo/Not I and HP20/ReNeo/Not I
were used as templates for a series of SOE mutagenesis constructions
that produced hybrid human/porcine cDNAs HP23, HP24, HP25, HP26, HP27,
and HP28 in ReNeo/Not I (Fig 1). Because of sequence identity
between human and porcine FVIII, we define boundaries of porcine
substitutions by the first amino acids that differ between human and
porcine FVIII at the NH2-terminal and C-terminal ends of
the insertions. These boundaries in HP23, HP24, HP25, HP26, HP27, and
HP28 correspond to human FVIII residues Thr2253-Gln2311,
Glu2181-Val2243, Thr2253-Met2321, Glu2181-Met2199, Lys2207-Val2243, and
Lys2207-Met2321, respectively.
Construction of B-domain deleted hybrid human/porcine FVIII
containing the porcine light chain (HP18).
The human FVIII light chain consists of amino acid residues
Asp1649-Tyr2332. The corresponding residues in the porcine FVIII cDNA
were substituted for this region of HB to produce a
hybrid human/porcine FVIII molecule designated HP18. This was performed
by substituting a PCR product corresponding to porcine a2
region, the A3 domain, the C1 domain, and part of the C2 domain for the
corresponding region in HP20. To facilitate constructions, a synonymous
Avr II site was introduced into nt 2273 at the junction of the
A2 and A3 domains of HP20 by SOE mutagenesis.
Construction of B-domain-deleted hybrid human/porcine FVIII
containing the porcine signal peptide, A1 domain, and A2 domain (HP22).
The human FVIII signal peptide, A1 domain, and A2 domains consist of
amino acid residues Met(19)-Arg740. The corresponding residues
in the porcine FVIII cDNA were substituted for this region of
HB to produce a molecule designated HP22.
Additionally, a synonymous Avr II site was introduced into nt
2273 at the junction of the A2 and A3 domains of HP22 by SOE
mutagenesis. HP22 was constructed by fusion of a porcine signal
peptide-A1-partial A2 fragment in pBlueScript44 with a
B-domainless hybrid human/porcine FVIII containing the porcine A2
domain, designated HP1.9
Construction of porcine B-domain-deleted FVIII and a porcine
B-domain-deficient FVIII, PSQ.
A Spe I/Not I fragment of HP18/BS (+Avr II) was
digested with Avr II/Not I and ligated into Avr
II/Not I-digested HP22/BS (+Avr II) to produce a
construct PB/BS (+Avr II), which consists of
the porcine FVIII lacking the entire B domain. PB
was cloned into ReNeo by ligating an Xba I/Not I
fragment of PB/BS (+Avr II) into
HP22/ReNeo/Not I (+Avr II).
In human FVIII, the presence of a 14 amino acid segment,
SerPheSerGlnAsn-ProProValLeuLysArgHisGlnArg, corresponding to B-domain residues Ser741-Asn745, Pro1640-Arg1648, in place of the B domain produces a molecule designated r-VIII SQ.45 This molecule
reportedly is expressed more efficiently in mammalian cell culture
compared with B-domain-deleted FVIII.45 The cDNA coding
for this segment was inserted into the corresponding region of
PB/BS (+Avr II) by SOE mutagenesis and then
cloned into ReNeo, producing a product PSQ/ReNeo/Not I
(+Avr II).
Expression of recombinant FVIII molecules.
All transfected cell lines were maintained in DMEM-F12 containing 10%
fetal bovine serum, 50 U/mL penicillin, and 50 µg/mL streptomycin.
Fetal bovine serum was heat-inactivated at 56°C for 1 hour before
use. HB/ReNeo, PSQ/ReNeo/Not I (+Avr
II), and hybrid human/porcine FVIII cDNAs in ReNeo initially were
transfected into COS-7 cells to confirm that active protein could be
expressed. They were then stably transfected into BHK cells and
selected for geneticin resistance as described previously,9
except that expressing cells were maintained in growth medium
containing 600 µg/mL geneticin. Cells from Corning T-75 flasks
(Corning, Corning, NY) grown to confluence were
transferred to Nunc triple flasks (Nunc, Glostrup,
Denmark) in medium containing 600 µg/mL geneticin and
grown to confluence. The medium was removed and replaced with
serum-free, AIM-V medium without geneticin. FVIII expression was
monitored by coagulation assay and 100 to 150 mL of medium was
collected once daily for 4 to 5 days. Typical expression levels were
300 U (~60 µg) FVIII per 200 mL of medium per day. Pooled medium
was stored at 4°C in 0.05% (wt/vol) sodium azide.
Recombinant hybrid FVIII molecules and PSQ were partially purified and
concentrated from the growth medium by heparin-Sepharose chromatography. Heparin-Sepharose (10 mL) was equilibrated with 0.075 mol/L NaCl, 10 mmol/L HEPES, 2.5 mmol/L CaCl2, 0.005%
Tween-80, and 0.02% sodium azide, pH 7.40. Medium (100 to 200 mL) from
expressing cells was applied to the heparin-Sepharose, which then was
washed with 30 mL of equilibration buffer without sodium azide. FVIII was eluted with 0.65 mol/L NaCl, 20 mmol/L HEPES, 5 mmol/L
CaCl2, and 0.01% Tween-80, pH 7.40, and was stored at
80°C. The yield of FVIII coagulant activity was typically
50% to 75%.
PSQ also was purified to homogeneity from serum-free medium using W3-3
antiporcine FVIII immunoaffinity chromatography and MonoQ
high-performance liquid chromatography (HPLC) using
chromatography conditions as described previously for plasma-derived
porcine FVIII.46 The specific activity of PSQ was 18,300 U
per A280 based on the human FVIII standard curve.
Recombinant FVIII was quantitated using a sandwich enzyme-linked
immunosorbent assay (ELISA) in which human vWF was used to capture
FVIII and antihuman FVIII murine MoAb, ESH5, was used for detection.
ESH5 was selected because it binds the FVIII heavy chain (A1 or A2
domain) and is unlikely to be influenced by mutations in the C2 domain.
Wells in a Corning Immobilon microtiter plate were coated with 6 µg/mL vWF in 0.15 mol/L NaCl, 10 mmol/L sodium phosphate, and 0.05%
sodium azide, pH 7.4 (PBS-N) overnight at room temperature. The plate
was washed with H2O, blocked by submersion for 1 hour in
PBS-N, 0.05% Tween-20, and 0.05% nonfat dry milk, and then washed
again with H2O. Samples containing recombinant human FVIII standards (0 to 200 ng/mL) or test recombinant proteins were added and
incubated for 1 hour at 37°C. Samples were performed in duplicate and two dilutions on the standard curve were made for each test recombinant protein. After washing the wells with 0.15 mol/L NaCl, 20 mmol/L HEPES, 5 mmol/L CaCl2, and 0.01% Tween-80, pH 7.4 (HCT), 1 µg/mL ESH5 in HCT was added for 1 hour at 37°C. Wells
were washed with HCT and goat antimouse alkaline phosphatase-conjugated
antibody, diluted 1:500 in HCT, was added for 1 hour at 37°C. After
washing with HCT, the wells were developed by the addition of
p-nitrophenylphosphate (Bio-Rad) for 1 hour according to the
instructions supplied by the manufacturer. The reaction was stopped by
the addition of 0.4 mol/L NaOH and absorbances at 405 nm were read in a
microplate reader. The molecular masses of human B-domain-containing
and the B-domainless constructs differ (~250 kD v 160 kD,
respectively). ELISA readings were corrected accordingly to calculate
the concentration of test constructs in mass units.
The specific activity of recombinant FVIII was calculated from
coagulation assay and ELISA determinations and expressed in terms of
units per milligram. The following results were obtained: HB, 4,900; HP20, 8,000; HP23, 3,900; HP24, 7,400;
HP25, 7,100; HP26, 8,500; HP27, 7,000; and HP28, 15,000. The
uncertainty of each measurement, which is based on duplicate
determination in the coagulation assay and quadruplicate determination
in the ELISA assay, is approximately 20%. Thus, the apparent specific
activity of most of the hybrids is significantly higher than that of
HB. Whether this difference is true or represents a
small systematic error in the ELISA method, the results indicate that
C2 hybrids are at least as active as human FVIII and thus are suitable
for epitope mapping analysis.
Patient plasmas.
Citrated human plasmas from five inhibitor patients (HR, LK, AA, RvR,
and YA) were used either without further purification (HR, RvR, and YA)
or as IgG preparations (LK and AA). Inhibitor IgG was prepared as
described previously.5 The inhibitors in HR, LK, AA, and
RvR antibodies were specific for the C2 domain as judged by antibody
neutralization assays performed as described previously.8
Briefly, inhibitor plasmas were incubated with increasing
concentrations of recombinant human A2 domain, recombinant human C2
domain, or purified plasma-derived human FVIII light chain, which
contains the A3, C1, and C2 domains. The residual inhibitor was
measured using the Bethesda assay. In all plasmas, the inhibitor titer
was neutralized by less than 10% by A2 and by greater than 79% by C2
or FVIII light chain. There was no difference between neutralization of
HR, LK, AA, and RvR antibodies by C2 and FVIII light chain. In
contrast, YA plasma was neutralized by less than 10% by A2, by 62% by
C2, and by greater than 95% by light chain. This finding indicates
that most of the inhibitor activity of YA is directed toward the C2
domain, but there is additional activity directed toward the A3 or C1
domains.
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RESULTS |
We constructed a series of hybrid B-domainless human/porcine FVIII
cDNAs, human B-domainless FVIII, and a porcine B-domain-deficient FVIII (Fig 1), stably expressed them from BHK cells in serum-free medium, and partially purified them using heparin-Sepharose
chromatography. The hybrid molecules contained substitutions of the
porcine A3/C1/C2 domains, C2 domain only, or porcine substitutions
within the C2 domain. The specific coagulant activities of the hybrids
based on an ELISA were equal to or greater than that of recombinant human B-domainless FVIII, as described in Materials and Methods, indicating that they were suitable for epitope mapping studies.
The recombinant FVIII molecules were added to hemophilia A plasma, and
their inhibition by five anti-human C2 antibodies (HR, LK, AA, YA, and
RvR) and the murine MoAb NMC-VIII/5 (Table
1) was measured using the Bethesda assay. NMC-VIII/5 was selected for
study because it binds the C2 domain and, like human C2 inhibitors, inhibits the binding of FVIII to phospholipid and to vWF.47 The C2-specificity of HR, LK, AA, and YA was previously identified by
the observation that they were neutralized by greater than 78% by
soluble, recombinant human C2 domain.8 RvR is greater than
95% neutralized by recombinant C2.48
The Bethesda titer of the NMC-VIII/5 IgG preparation toward human
B-domainless FVIII, determined by the concentration at which it
produced 50% inhibition of its coagulant activity, was 260 BU/mL
(Fig 2). NMC-VIII/5 did not detectably
inhibit HP18 or HP20, confirming its C2 specificity. The Bethesda titer
of NMC-VIII/5 toward HP24 was less than 10 BU/mL. By comparison with
Fig 1, this indicates that residues Glu2181-Val2243 contain a major
determinant of the NMC-VIII/5 epitope. The Bethesda titer of NMC-VIII/5
toward HP27 and HP28 also was below the limits of detection, indicating that the regions bounded by residues Glu2181-Met2199 and
Lys2207-Val2243 each contribute to NMC-VIII/5 binding.
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| Fig 2.
Inhibition of hybrid human/porcine FVIII by murine MoAb
NMC-VIII/5. HB or hybrid FVIII was diluted into
hemophilia A plasma to a final concentration of 0.8 to 1.2 U/mL and
then incubated for 2 hours with the indicated dilutions of NMC-VIII/5
antibody. Residual FVIII activity was measured as described in
Materials and Methods. ( ) HB; ( ) HP20; ( ) HP23;
( ) HP24; ( ) HP25; ( ) HP26; ( ) HP27; ( ) HP28.
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In contrast, NMC-VIII/5 was a potent inhibitor of HP23 and HP25. The
Bethesda titers determined from the curves shown in Fig 2 were 530 BU/mL and 1,060 BU/mL for HP23 and HP25, respectively. This finding
suggests that residues Thr2253-Met2321 do not contribute significantly
to the binding of NMC-VIII/5 to FVIII. We cannot exclude the
possibility that amino acid residues that are identical between human
and porcine FVIII are present in this region that bind antibody.
However, the major reduction in inhibitor activity toward HP24 and the
lack of reduction in activity toward HP25 indicate that most of the
binding energy associated with NMC-VIII/5 is directed against the
region bounded by residues Glu2181-Val2243.
The Bethesda titers for HP23 and HP25 actually were significantly
higher than that for human B-domain-deleted FVIII. Although the
mechanism for this behavior is not clear, one possibility is that the
human FVIII segment bounded by residues Thr2253-Met2321 unfavorably
influences the NMC-VIII/5 binding. Thus, the unfavorable interaction
would be relieved by insertion of the homologous porcine sequence.
Inhibition studies using human antibodies were performed similarly
(Fig 3). In addition to the hybrid
human/porcine FVIII constructs described in Fig 1, the inhibition of a
porcine B-domain-deficient FVIII by human antibodies was measured. The
cross-reactivity levels of the antibodies, defined as the
ratio of the Bethesda titers of porcine and human FVIII, were 11%,
5%, 4%, less than 1%, and 34% for HR, LK, AA, YA, and RvR,
respectively (Table 2). Calculating the
difference of the Bethesda titers of HP18 and porcine
B-domain-deficient FVIII and dividing by the human B-domainless FVIII
titer is a measure of non-FVIII light chain, presumably anti-A2,
antibodies. These antibodies accounted for 5% of the AA titer and were
negligible in the other antibodies (Table 2). Calculating the
difference of the Bethesda titers of HP20 and HP18 and dividing by the
human B-domainless FVIII titer is a measure of antibodies to the
a2-A3-C1 region. LK and RvR a2-A3-C1 antibodies were
negligible. They accounted for 17%, 4%, and 16% of the activity of
HR, AA, and YA, respectively (Table 2). Thus, at least 80% of the
activity of all of the antibodies was directed against the C2 domain,
which is consistent with the antibody neutralization experiments.
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| Fig 3.
Inhibition of hybrid human/porcine FVIII by human
anti-FVIII antibodies. The inhibition of FVIII constructs by antibodies
from patients HR, LK, AA, YA, and RvR was measured using the Bethesda
assay as described in Materials and Methods.
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Similar to NMC-VIII/5, all five antibodies inhibited HP24 less than
human B-domainless FVIII. Calculating the difference of the Bethesda
titers of HP24 and HP20 and dividing by the human B-domainless FVIII
titer is a measure of C2 antibody activity outside the 2181-2243 region. It was 21%, 3%, 18%, 2%, and 22% for HR, LK, AA, YA, and
RvR, respectively. This indicates that, like NMC-VIII/5, the human
antibodies recognize residues Glu2181-Val2243 as a major determinant of
C2 inhibitor epitope.
The complementary mutant to HP24 is HP25. The inhibition of HP25 by HR,
LK, AA, and YA was similar to human B-domainless FVIII. Thus, C2
antibodies directed to a region COOH-terminal to residue 2253 were not
identified in these patients. In contrast, RvR showed significant
reactivity to this region, although less so than the region bounded by
residues Glu2181-Val2243. Curiously, LK and AA, but not HR, RvR, and
YA, inhibited HP23 less than HP25. A possible explanation for this is
that human amino acid residues in the region bounded by residues
Gln2311-Met2321 make an unfavorable interaction with LK and AA.
Inhibition of HP26, HP27, and HP28 by LK, AA, and RVR was reduced
compared with human B-domainless FVIII, indicating that, as with
NMC-VIII/5, the regions bounded by residues Glu 2181-Met2199 and
Lys2207-Val2243 both contribute to antibody binding. In contrast, HR
and YA were relatively potent inhibitors of HP26, HP27, and HP28. These
antibodies were significantly more cross-reactive with porcine FVIII. A
possible explanation of these results is that HR and YA have strong
interactions with amino acid residues that are identical between human
and porcine FVIII in the Ser2173-Thr2253 region in addition to
interactions that are human specific.
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DISCUSSION |
By using a series of active recombinant hybrid human/porcine FVIII
molecules, we have mapped a major determinant of an epitope recognized
by five human antibodies and by a murine MoAb, NMC-VIII/5, to the
NH2-terminal half of the C2 domain. The antibodies used in
this study were selected because most of their anticoagulant activity
is neutralized by recombinant C2 domain.8 The
C2-specificity of the antibodies was confirmed in the present study by
showing that they inhibit HP20, which contains a porcine C2 domain
substitution (Ser2173-Tyr2332), less than human FVIII. Additionally,
HP24, which contains substitution of a porcine sequence corresponding to residues Glu2181-Val2243, is inhibited less than human FVIII by all
of the antibodies. This establishes residues Glu2181-Val2243 as a major
determinant of a C2 epitope recognized by these antibodies (Fig 4).
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| Fig 4.
Sequence alignment of the C2 domains of human and porcine
FVIII. The region containing the C2 epitope identified in this study is
underlined.
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This result was unexpected, because several lines of evidence have
implicated the COOH-terminal end of the C2 domain as a target for FVIII
inhibitors. Inhibitory C2 antibodies block the binding of FVIII to
phospholipid.29 Synthetic peptides corresponding to mature
FVIII C2 domain residues Thr2303-Ile2317, Tyr2305-Tyr2332, Ile2308-Glu2322, Ala2318-Tyr2332,49
Thr2303-Tyr2332,32 and Thr2303-Leu232450
inhibit the binding of FVIII to phospholipid. This finding suggests
that the binding sites on FVIII for phospholipid and C2 antibodies
overlap and are at the COOH-terminal end of the C2 domain. Furthermore,
NMC-VIII/5, which also inhibits the binding of FVIII to
phospholipid,47 binds the Thr2303-Tyr2332 peptide,7 directly implying that at least part of its
epitope is at the COOH-terminal end of the C2 domain. The C2 peptides corresponding to this region bind phospholipid with micromolar affinity,32,49,50 which is three orders of magnitude lower than the interaction of native FVIII with phospholipid.51
This low affinity could be due to a conformational equilibrium of the peptides that favors an inactive state. However, it is also possible that additional regions in FVIII, such as the region identified in this
study, are necessary for high-affinity binding to phospholipid.
Several observations suggest that the C2 domain is involved in binding
vWF at a site that overlaps the phospholipid binding site. vWF and
phospholipid compete for binding to FVIII.20-22 A 34-kD,
monomeric, proteolytic fragment of vWF, which corresponds to amino acid
residues Ser1-Arg272 of the 2050 amino acid residue subunit of the
mature homopolymeric vWF multimer, also inhibits the binding of FVIII
to phospholipid.52 This makes it less likely that the
competition between vWF and phospholipid is merely due to steric
hindrance. Additionally, NMC-VIII-5 inhibits the binding of FVIII to
vWF as well as to phospholipid.47 Finally, the
Thr2303-Tyr2332 synthetic FVIII peptide inhibits the binding of FVIII
to vWF.32
The interaction of vWF with C2 has been studied directly by showing
that recombinant C2 binds vWF.38 The affinity of this interaction is approximately 150-fold lower than that of vWF binding to
FVIII light chain (a2-A3-C1-C2), which contains all of the binding energy for binding to vWF. Additionally, fragments of 14 kD
(Met1672-Glu1794) and 67 kD (Asp1795-Tyr2332) generated by
Staphylococcal aureus protease cleavage of the
FVIII light chain each bind vWF, albeit with the lower affinity
observed for C2. This is consistent with observations that have
indicated that the a2 region (Glu1649-Arg1689) is necessary for
high-affinity binding of FVIII to vWF33-37 but not to
phospholipid. However, when the 14-kD/67-kD complex is not dissociated,
its affinity for vWF is the same as that of intact FVIII light chain.
These results establish the presence of at least two distinct vWF
binding sites within the FVIII light chain, both of which are required for maximal binding.
The results of previous studies implicating the COOH-terminal end of
the C2 domain in the binding of FVIII to inhibitory antibodies, phospholipid, and vWF can be reconciled with the results of the present
study if the NH2-terminal and COOH-terminal sequences of
the C2 domain are close to one another and form a single epitope. This
may be the case, because residues Cys2174-Cys2326, which are at
opposite ends of the C2 domain, are disulfide bonded.53
Epitope mapping studies of human inhibitors that bind C2 and the
anti-C2 MoAb NMC-VIII/5 have been performed by immunoblotting analysis
of recombinant C2 domain deletion polypeptides expressed in E
coli.7,47 The parent polypeptide used in these studies corresponded to FVIII residues Ala1974-Tyr2332, which include the
COOH-terminal 47 residues of the A3 domain and all of the C1 and C2
domains. Nested NH2-terminal and COOH-terminal deletions of
the C2 domain of this polypeptide were made to determine their effect
on antibody binding. Epitopes within the C2 domain or near each
terminus could be identified, but not those that spanned both the
NH2-termini and COOH-termini, because the disulfide bonds were reduced before immunoblotting. The binding of NMC-VIII/5 to the C2
domain was abolished by all NH2-terminal deletions and by
all COOH-terminal deletions except deletion of
Ala2328-Tyr2332,47 suggesting that both regions were
directly or indirectly crucial for antibody binding and that the
epitope was localized to Asp2170-Glu2327.
The epitope of the murine anti-FVIII MoAb ESH8, which does not
interfere with either vWF or phospholipid binding of FVIII, was
similarly localized to Val2248-Gly2285.7 Therefore, this region of the C2 domain is probably not involved in binding to either
vWF or phospholipid. Six inhibitors that were at least partially
neutralized by soluble, recombinant C2 domain were also studied. One
epitope was identical to that of NMC-VIII/5. Four additional epitopes
were localized to regions Asp2170-Ser2312, Thr2303-Ser2312,
Ala2218-Ser2312 (two plasmas), and Val2248-Ser2312. The common epitope
endpoint of Ser2312 suggests that the residues NH2-terminal
to this region are either required for binding by antibodies present in
five of the six plasmas or its deletion indirectly prevents antibody
binding elsewhere. The requirements for antibody binding to residues
near the NH2-terminus of the C2 domain were more
heterogeneous. Two epitopes included residues beginning with Asp2170,
suggesting that the entire NH2-terminal region was
important for binding of less frequent antibodies. The other
NH2-termini included Trp2203, Ala2218 (two plasmas), and
Val2248.
The present study suggests that inhibitor antibodies that bind
COOH-terminal to Val2248 are infrequent compared with those that bind
NH2-terminal to this region because the former were present
in only one of five plasmas (RvR). In contrast, the earlier immunoblotting studies suggested that antibody binding is eliminated by
deletion of specific regions of either the NH2-terminus or COOH-terminus. An important consideration in reconciling these results
is that immunoblotting detects all antibodies, whereas hybrid
human/porcine FVIII experiments detect only inhibitors. It is known
that inhibitor plasmas contain both inhibitory and noninhibitory
antibodies.54 Because immunoblotting is performed with
denatured, reduced proteins, they may bind a different set of
antibodies than the human/porcine FVIII hybrids. An important advantage
of using hybrid human/porcine FVIII molecules to map inhibitor epitopes
is that they have FVIII activity, which assures that loss of antibody
binding is not due to protein denaturation. However, the method cannot
identify determinants of epitopes that are identical between human and
porcine FVIII. Thus, common amino acid residues that contribute to
antibody binding the COOH-terminal region of the C2 domain may not be
detected by this method.
Coagulation factor V is homologous to FVIII and contains a
A1-A2-B-A3-C1-C2 domain sequence. Ortel et al55 expressed a
series of hybrid factor V/FVIII proteins that contained substitutions of FVIII in the factor V C2 domain. Using these hybrids and an inhibitory patient autoantibody to factor V that blocks the binding of
factor V to phospholipid, they mapped the phospholipid binding epitope
to the NH2-terminal third of the C2 domain. This result is
consistent with the results of our study and suggests that the
NH2-terminal region of the C2 domain of both factors V and VIII contains an immunodominant epitope that is important for phospholipid binding.
| |
FOOTNOTES |
Submitted March 10, 1998;
accepted July 15, 1998.
Supported by National Institutes of Health Grants No. R01-HL46215
(P.L.) and P50-HL44336 (D.S.) and by a Hemophilia of Georgia research
fellowship (H.M.T.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Pete Lollar, MD, 1639 Pierce Dr, Room 1003 Woodruff Memorial Bldg, Emory University, Atlanta, GA 30322; e-mail:
jlollar{at}emory.edu.
| |
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E. N. van den Brink, E. A. M. Turenhout, J. Davies, N. Bovenschen, K. Fijnvandraat, W. H. Ouwehand, M. Peters, and J. Voorberg
Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes
Blood,
January 15, 2000;
95(2):
558 - 563.
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R. T. Barrow, J. F. Healey, D. Gailani, D. Scandella, and P. Lollar
Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules
Blood,
January 15, 2000;
95(2):
564 - 568.
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Abstract
Introduction