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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 564-568
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Reduction of the antigenicity of factor VIII toward complex
inhibitory antibody plasmas using multiply-substituted hybrid
human/porcine factor VIII molecules
Rachel T. Barrow,
John F. Healey,
David Gailani,
Dorothea Scandella, and
Pete Lollar
From Emory University, Atlanta, GA; the Division of
Hematology, Vanderbilt University, Nashville, TN; and the Holland
Laboratory, American Red Cross, Rockville, MD.
 |
Abstract |
Factor VIII (fVIII) circulates as a heavy chain/light chain
(A1-A2-B/ap-A3-C1-C2) heterodimer. The 41-residue light chain activation peptide, ap, is cleaved from fVIII during
proteolytic activation by thrombin or factor Xa. We constructed 7 active recombinant hybrid B-domainless human/porcine fVIII molecules
that contained combinations of porcine sequence replacements within the
A2, ap-A3, and C2 domains. The cross-reactivity of 23 high-titer inhibitory antibodies between human fVIII and the hybrids
was inversely related to the degree of porcine substitution. In all
plasmas, the substitution of all 3 regions yielded cross-reactivities
that were not significantly different from those of porcine fVIII. To
differentiate between inhibitor binding to the ap region and
the A3 domain, we constructed 2 additional hybrids that contained
porcine A2 and C2 domain substitutions and either porcine A3 or porcine
ap substitutions. The porcine ap segment was less
antigenic than the human ap segment in several plasmas that had
activity against the ap-A3 region. This indicates that some
inhibitor plasmas contain antibodies directed against the fVIII
ap segment in addition to A2, A3, and C2 domain epitopes identified in previous studies. Substitution of porcine sequences within the A2, A3, C2, and ap regions of human fVIII is
necessary and sufficient to achieve a maximal reduction in antigenicity relative to porcine fVIII with respect to most inhibitory antibody plasmas.
(Blood. 2000;95:564-568)
© 2000 by The American Society of Hematology.
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Introduction |
Inhibitory antibodies (inhibitors) to factor VIII
(fVIII) arise as alloantibodies in hemophiliacs who have undergone
transfusion and as autoantibodies in persons without
hemophilia.1-4 As a rule, they are polyclonal IgG
populations directed against multiple epitopes. FVIII contains a
sequence of domains designated
NH2-A1-A2-B-ap-A3-C1-C2-COOH.5 It is
cleaved intracellularly and circulates as an
A1-A2-B/ap-A3-C1-C2 heavy chain/light chain
heterodimer.6 During proteolytic activation by thrombin,
the B domain and the 41-residue light chain activation peptide,
ap, are released, and cleavage occurs between the A1 and A2
domains. The final product is a 160-kd A1/A2/A3-C1-C2 fVIIIa heterotrimer.7,8
Most domain-specific inhibitors interfere with fVIIIa function rather
than with the activation or the circulatory lifetime of fVIII. Anti-A2
inhibitors bind to an epitope bounded by amino acids
484-508.9 They are noncompetitive inhibitors of the
fVIIIa/factor IXa complex.10 Anti-C2 inhibitors bind to a
discontinuous epitope derived from the NH2-terminal and
COOH-terminal ends of the domain11,12 and block the binding
of fVIIIa to phospholipid.13 Anti-A3 inhibitors bind to an
epitope that includes amino acids 1811-1818 and block the binding of
fVIIIa to factor IXa.14,15 Inhibitor neutralization studies
indicate that the effects of anti-A2, anti-A3, and anti-C2 inhibitors
are additive.16 Thus, inhibitors to a given domain appear
to act independently of inhibitors to other domains. Despite the
different immunologic settings in which they arise, alloantibody and
autoantibody epitopes appear to be the same, though autoantibody plasmas are more likely to be specific for single
domains.16
Clinically significant inhibitors usually cross-react poorly with
porcine fVIII,17-19 forming the basis for the therapeutic use of porcine fVIII concentrate. We have used recombinant hybrid human/porcine fVIII molecules to map epitopes in the A2 and C2 domains.9,11 Epitopes are located by identifying porcine
amino acid substitutions that result in a decrease in antibody
cross-reactivity. These studies have taken advantage of exceptional
patient plasmas monospecific for the A2 or C2 domains. Antibodies that
are monospecific for domains outside A2 and C2 are rare,16
which makes analysis of epitope(s) in the ap-A3 region using
hybrid human/porcine fVIII molecules more difficult. To approach this
problem, we made a series of constructs that contained combinations of
porcine substitutions in the A2, ap, A3, and C2 domains, and we
studied the cross-reactivity of 23 high-titer, primarily polyspecific,
inhibitory antibody plasmas against them. Our results indicated that
multiply-substituted hybrid fVIII molecules are useful for identifying
inhibitor domain specificity. Using this method, we found that some
inhibitors have activity directed against the ap region, which
previously had not been recognized.
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Materials and methods |
Materials
Citrated hemophilia A and normal pooled human plasma were purchased
from George King Biomedical (Overland Park, KA).
pBlueScript II KS was purchased from Stratagene (La
Jolla, CA). Synthetic oligonucleotides were purchased from Life
Technologies (Gaithersburg, MD) or Cruachem (Sterling, VA). Restriction
enzymes were purchased from New England Biolabs (Beverly, MA) or
Promega (Madison, WI). Polymerase chain reaction (PCR) products and
restriction fragments were gel purified, precipitated with ethanol, and
ligated to plasmid DNA using T4 DNA ligase (Rapid DNA ligation kit;
Boehringer Mannheim). Insert-containing plasmids were used to transform
Escherichia coli Epicurean XL1-Blue cells (Stratagene). 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 (Perkin Elmer, Norwalk,
CT). Murine monoclonal antibody CLB-CAg A was a
generous gift of Dr Jan van Mourik (Central Laboratory, Netherlands Red Cross).
Construction and expression of hybrid human/porcine fVIII
molecules
The B-domainless hybrid fVIII molecules used in this study are shown
in Figure 1 along with B-domainless human
fVIII (HB ) and porcine fVIII (PB). These molecules lack
the entire B domain, defined as the thrombin cleavage fragment
corresponding to amino acids 741-1648. HB, PB, HP9, and
HP20 have been described previously.9,20
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| Fig 1.
Domain structure of recombinant fVIII constructs.
Amino acid numbering refers to mature, full-length human fVIII. The A3,
C1, and C2 domains are defined as amino acids 1690-2019, 2020-2172, and
2173-2332, respectively.36 The light chain activation
peptide, ap, corresponds to amino acids 1649-1689. The
constructs lack the B domain, which is defined as amino acids 741-1648. Stippled regions indicate areas of porcine substitution. Boundaries are
defined by amino acids in which porcine and human fVIII differ. Porcine
A2, ap, A3, and C2 substitutions correspond to amino acids
484-508, 1649-1687, 1694-2019, and 2181-2321, respectively.
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Initially, HP29, HP30, HP31, HP32, HP33, HP35, and HP41 were
constructed in pBluescript. HP29 was made by splicing-by-overlap extension (SOE) mutagenesis 21 using procedures described
previously.9,11 HB and HP18, a human heavy
chain/porcine light chain hybrid,11 were used as PCR
templates. The SOE product, a porcine ap-A3/human C1 fragment, was ligated to HincII/SphI-digested HP18 to produce
HP29 in pBluescript.
HP30 was made using SphI/NotI digestion and ligation,
with HP29 and HB as vector and insert, respectively. HP31 was
made using SpeI/BglII digestion and ligation, with HP30
and HP9 as vector and insert, respectively. HP32 was made using
SpeI/BglII digestion and ligation, with HP29 and HP9 as
vector and insert, respectively. HP33 was made using
SpeI/BglII digestion and ligation using HP20 and HP9 as
vector and insert, respectively. Similarly, HP35 and HP41, which are
complementary constructs, were made by SOE mutagenesis using HB
and HP30 as PCR templates.
The constructions were moved into ReNeo, a mammalian expression
vector,20 using SpeI/NotI digestions and
ligations. cDNA in ReNeo initially was transfected into COS-7 cells to
confirm that active protein could be expressed. Then it was stably
transfected into baby hamster kidney (BHK) cells,
partially purified, and concentrated as described
previously.11,20
FVIII assays
The activity of recombinant fVIII proteins was measured by one-stage
clotting assay1,22 using an ST4 BIO Coagulation Instrument (Diagnostica Stago). One unit of fVIII is defined as the activity in 1 mL pooled normal citrated human plasma.
FVIII inhibitor titers were measured by the Bethesda
assay.23 Inhibitor samples are identified by patient
initials. Inhibitors were measured either in dilutions of patient
plasma or IgG preparations (samples KB, MS, MU, RJ, and
SCN). To measure the inhibitor activity against fVIII,
constructs 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. The 50% inhibition point was identified by
interpolation23 using only data points falling within a
range of 40% to 60% inhibition. At least 3 data points were used for
each determination, from which the mean and sample SD were calculated.
The cross-reactivity of inhibitors with hybrid fVIII molecules or
porcine fVIII was defined as the percentage Bethesda titer relative to
human-B domainless fVIII.
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Results |
Initially, we constructed 7 B-domainless hybrid human/porcine fVIII
cDNA that contained combinations of substitutions of porcine sequence
for human sequences in the A2, ap-A3, and C2 domains (Figure
1). HP9 contains a substitution encoding porcine replacement of human
amino acids 484-508, a region that appears to contain most, if not all,
of the epitope recognized by anti-A2 human
inhibitors.9 HP30 and HP20 contain porcine replacement of
most of the ap-A3 and C2 regions of fVIII, respectively. HP31,
HP33, and HP29 contain the corresponding combinations of these mutants
in the A2/ap/A3, A2/C2, and ap/A3/C2 domains,
respectively. Finally, HP32 contains the quadruply-substituted
A2/ap/A3/C2 hybrid.
The hybrids were stably expressed in BHK cells and partially purified
as described in "Materials and Methods." The cross-reactivity of
23 human inhibitors between human B-domainless fVIII and the hybrids
was measured using the Bethesda assay and was compared to porcine
B-domainless fVIII. In Figure 2, the
hybrids are ranked from left to right in terms of increasing degree of
porcine substitution. Inhibitor plasmas are ranked from left to right
according to their cross-reactivity with porcine B-domainless fVIII.
The results show that the cross-reactivity of the hybrids decreased as
the degree of porcine substitution increased.
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| Fig 2.
Inhibitor cross-reactivity with human fVIII and hybrid
fVIII constructs or porcine fVIII.
Inhibitor activity was measured by the Bethesda assay as described in
"Materials and Methods." Cross-reactivity is expressed as the
percentage Bethesda titer relative to human B-domainless fVIII.
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Within the calculated coefficient of variation of
0.2, the cross-reactivity of HP32 was not
greater than porcine fVIII in any of the plasmas (Table 1). The
difference between HP32 and porcine fVIII was not statistically
significant by either the paired Student t test or the Wilcoxon
signed-rank test. Thus, the decrease in cross-reactivity toward porcine
fVIII appeared to be completely conferred by the porcine substitutions
in HP32, indicating that the epitopes recognized by the plasmas were
confined to A2 amino acids 484-508, ap amino acids
1649-1687, A3 amino acids 1694-2017, and C2 amino acids 2181-2321.
Some inhibitor plasmas contain antibodies whose specificity is
restricted to the A2 or C2 domains.16,24 These antibodies have been useful for mapping epitopes in the A2 and C2 domains using
hybrid human/porcine fVIII molecules.9,11,20,25 In the
current study, BN contained predominantly anti-A2 activity because
substitution of the porcine 484-508 sequence in the HP9 hybrid was
sufficient to decrease antibody cross-reactivity by >90%. We have
reported similar results with HP9 and patient plasmas SC, JM, NS, and
RC,9 which have been shown by
neutralization studies to be A2-specific.24 Similarly,
plasma from patient L was C2-specific because substitution of the
porcine C2 sequence in HP20 was sufficient to decrease antibody
cross-reactivity by >90%. This result was consistent with
neutralization of patient L's plasma with recombinant
C2.16 The agreement between results using HP20 and antibody
neutralization experiments for 5 other C2-specific inhibitors, HR, LK,
AA, YA, and RvR, has been reported.11
In contrast, plasmas with inhibitors specific for the ap-A3
region have been more difficult to identify. Table 1 shows that several
plasmas had a substantial amount of activity against ap-A3. Substitution of the porcine sequence within the ap-A3 region in HP30 decreased antibody cross-reactivity to <40% and produced at
least a 15% difference between HP32 and HP33 cross-reactivity in 7 of
the 23 plasmas (GK1832, GK1833, HG, KB, RI, RMA, and TM). The bulk of
the inhibitor activity of KB, GK1833, and TM appeared directed against
the ap-A3 region. This was consistent with antibody neutralization experiments on KB and GK1833, which required
ap-A3-C1-C2 to achieve most of the
neutralization.16
To determine whether antibodies to the ap-A3 region are
directed against the ap peptide, the A3 domain, or both, we
constructed 2 additional hybrids, HP35 and HP41 (Figure 1). HP35 was
identical to HP32 except that it contained the human ap peptide
instead of the corresponding porcine peptide. Similarly, HP41 differs from HP32 by containing the human A3 domain. The cross-reactivity of
HP41 and HB toward mAb CLB-CAg A, which binds the epitope recognized by human anti-A3 inhibitors,14,26 was not
significantly reduced (data not shown). This demonstrated that the A3
inhibitory epitope in HP41 was intact.
The cross-reactivity of GK1832, GK1833, HG, KB, RI, RMA, and TM
against HP32, HP33, HP35, and HP41 was compared (Figure
3). In 4 of the 7 plasmas (GK1833, RI, RMA,
and TM), HP35 was significantly more cross-reactive than HP32.
Conversely, HP41 was less cross-reactive than HP33 in all but 1 of the
plasmas (HG). These results are consistent with the presence of an
epitope in the human ap segment that is recognized by some
inhibitor plasmas.
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| Fig 3.
Identification of ap-directed inhibitory
activity.
Plasma from 7 patients with inhibitor activity against the ap
domain, the A3 domain, or both was defined by a decrease in HP30
cross-reactivity to <40% and at least a 15% difference in
cross-reactivity between HP32 and HP33 using the data in Table 1. Data
are expressed as mean cross-reactivity and SD, as described in
"Materials and Methods."
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Discussion |
In previous studies, we constructed novel hybrid human/porcine
fVIII molecules to map inhibitor epitopes.9,11,20 These studies were restricted to the use of hybrids with substitutions of
porcine sequence within single domains of fVIII. Thus, it has been
necessary to use monospecific inhibitor plasma to avoid the confounding
effects of activity against epitopes outside the region of porcine substitution.
In this study, we made combinations of substitutions of porcine
sequence in the A2, ap, A3, and C2 domains of fVIII (Figure 1).
The antigenicity of the quadruply-substituted (A2/ap/A3/C2) hybrid, HP32, was not significantly different from B-domainless porcine
fVIII (Figure 2, Table 1). This indicates that clinically significant
antibodies recognize epitopes restricted to A2 amino acids 484-508, ap amino acids 1649-1687, A3 amino acids 1694-2017, and C2
amino acids 2181-2321, and it specifically excludes significant effects
of anti-A1 and anti-C1 inhibitors.
There is considerable evidence for the presence of an inhibitor
epitope in the A3 domain. An anti-A3 inhibitor was identified in
a patient with hemophilia A by deletion mapping, which placed the
epitope within an A3 segment bounded by amino acids
1778-1823.14 The antibody also inhibited the binding of
factor IXa to the factor VIII light chain, which is necessary for
assembly of the intrinsic factor X activation complex.27
The fVIII light chain binding site for factor IXa has been localized to
amino acids 1811-1818 in the A3 domain.26 Consistent with
those observations, 3 inhibitor IgGs have been identified that
prevented the binding of factor IXa to the fVIII light
chain.15 In that study, the binding of these
inhibitors to the fVIII light chain was completed by a synthetic peptide corresponding to A3 amino acids 1804-1819 competed for the binding of those inhibitors to the fVIII light chain.
The results shown in Figure 2 and Table 1 are consistent with the
presence of an inhibitor epitope in the A3 domain, but they do not
exclude the possibility of an anti-ap inhibitor epitope. An anti-ap murine monoclonal antibody has been identified that inhibits fVIII in the Bethesda assay,28 but human
anti-ap inhibitory antibodies have not been clearly
demonstrated. To differentiate between inhibitor binding to the
ap region versus the A3 domain, we constructed 2 additional
triply-substituted hybrids, HP35 and HP41, that differed from HP32 by
containing the human ap or human A3 domain, respectively
(Figure 1). In most plasmas with antibodies against the ap-A3
region, the cross-reactivity of HP35 was greater than HP32 and the
cross-reactivity of HP41 was less than HP33 (Figure 3), indicating that
these inhibitors recognize the ap peptide, as well as the A3
domain epitope identified in earlier studies.
During the activation of fVIII by thrombin, the ap peptide is
released.29 This step is necessary for fVIII to dissociate from von Willebrand factor and to participate in blood
coagulation.30,31 Thus, antibodies to the ap
segment may block the activation of the fVIII-von Willebrand factor complex.
Porcine fVIII concentrate (Hyate:C) is useful in
inhibitor patients who do not have significant cross-reactivity with
porcine fVIII.32-34 After exposure to porcine fVIII,
anti-porcine fVIII antibodies develop in some of these patients. The
epitopes recognized by these antibodies have not been characterized in
detail. Western blotting has identified antiporcine antibodies specific
for the A1 domain,35 which is rarely targeted by antihuman
fVIII antibodies. Whether anti-A1 antibodies are inhibitory is unknown.
A hybrid human/porcine fVIII molecule could be superior to porcine
fVIII therapeutically if it lacks immunogenic porcine epitopes. The cross-reactivity of HP32 was as low as or slightly lower than porcine
B-domainless in the series of high-titer inhibitor plasmas in this
study (Table 1). HP32 or a more humanized derivative thereof that lacks
the potentially immunogenic porcine A1 domain is a potential
alternative to porcine fVIII.
| |
Footnotes |
Submitted May 10, 1999; accepted September 14, 1999.
Supported by grants R01-HL46215 and RO1-HL55273 from the National
Institutes of Health.
Reprints: Pete Lollar, Emory University, Room
1003 Woodruff Memorial Building, 1639 Pierce Dr, Atlanta, GA 30322;
e-mail: jlollar{at}emory.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Hoyer LW, Scandella D.
Factor VIII inhibitors: structure and function in autoantibody and hemophilia A patients.
Semin Hematol.
1994;31:1-5[Medline]
[Order article via Infotrieve].
2.
Scandella D, Kessler C, Esmon P, et al.
Epitope specificity and functional characterization of factor VIII inhibitors.
Adv Exp Med Biol.
1995;386:47-63[Medline]
[Order article via Infotrieve].
3.
Scandella D.
Human anti-factor VIII antibodies: epitope localization and inhibitory function.
Vox Sang.
1996;70(suppl 1):9-14.
4.
Lollar P.
Analysis of factor VIII inhibitors using hybrid human/porcine factor VIII.
Thromb Haemost.
1997;78:647-651[Medline]
[Order article via Infotrieve].
5.
Vehar GA, Keyt B, Eaton D, et al.
Structure of human factor VIII.
Nature.
1984;312:337-342[Medline]
[Order article via Infotrieve].
6.
Fass DN, Knutson GJ, Katzmann JA.
Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein.
Blood.
1982;59:594-600[Abstract/Free Full Text].
7.
Lollar P, Parker CG.
Subunit structure of thrombin-activated porcine factor VIII.
Biochemistry.
1989;28:666-674[Medline]
[Order article via Infotrieve].
8.
Curtis JE, Helgerson SL, Parker ET, Lollar P.
Isolation and characterization of thrombin-activated human factor VIII.
J Biol Chem.
1994;269:6246-6251[Abstract/Free Full Text].
9.
Healey JF, Lubin IM, Nakai H, et al.
Residues 484-508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII.
J Biol Chem.
1995;270:14,505-14,509[Abstract/Free Full Text].
10.
Lollar P, Parker ET, Curtis JE, et al.
Inhibition of human factor VIIIa by anti-A2 subunit antibodies.
J Clin Invest.
1994;93:2497-2504.
11.
Healey JF, Barrow RT, Tamim HM, et al.
Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII.
Blood.
1998;92:3701-3709[Abstract/Free Full Text].
12.
Scandella D, Gilbert GE, Shima M, et al.
Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248-2312 which overlap a phospholipid binding site.
Blood.
1995;86:1811-1819[Abstract/Free Full Text].
13.
Arai M, Scandella D, Hoyer LW.
Molecular basis of factor-VIII inhibition by human antibodies: antibodies that bind to the factor-VIII light chain prevent the interaction of factor-VIII with phospholipid.
J Clin Invest.
1989;83:1978-1984.
14.
Fijnvandraat K, Celie PHN, Turenhout EAM, et al.
A human alloantibody interferes with binding of factor IXa to the factor VIII light chain.
Blood.
1998;91:2347-2352[Abstract/Free Full Text].
15.
Zhong D, Saenko EL, Shima M, Felch M, Scandella D.
Some human inhibitor antibodies interfere with factor VIII binding to factor IX.
Blood.
1998;92:136-142[Abstract/Free Full Text].
16.
Prescott R, Nakai H, Saenko EL, et al.
The inhibitory antibody response is more complex in hemophilia A patients than in most nonhemophiliacs with fVIII autoantibodies.
Blood.
1997;89:3663-3671[Abstract/Free Full Text].
17.
Morrison AE, Ludlam CA, Kessler C.
Use of porcine factor VIII in the treatment of patients with acquired hemophilia.
Blood.
1993;81:1513-1520[Abstract/Free Full Text].
18.
Fiks-Sigaud M, Bendelac L, Parquet A, et al.
Comparison of anti-human and anti-porcine factor VIII inhibitor levels in 63 patients with severe hemophilia A.
Vox Sang.
1993;64:210-214[Medline]
[Order article via Infotrieve].
19.
Lloyd JV, Street AM, Berry E, et al.
Cross-reactivity to porcine factor VIII of factor VIII inhibitors in patients with haemophilia in Australia and New Zealand.
Aust N Z J Med.
1997;27:658-664[Medline]
[Order article via Infotrieve].
20.
Lubin IM, Healey JF, Scandella D, Runge MS, Lollar P.
Elimination of a major inhibitor epitope in factor VIII.
J Biol Chem.
1994;269:8639-8641[Abstract/Free Full Text].
21.
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR.
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
Gene.
1989;77:51-59[Medline]
[Order article via Infotrieve].
22.
Bowie EJW, Owen CA.
The clinical and laboratory diagnosis of hemorrhagic disorders. In:
Ratnoff OD,Forbes CD, eds.
Disorders of Hemostasis. Orlando: Grune & Stratton; 1984:43.
23.
Kasper CK, Aledort LM, Counts RB, et al.
A more uniform measurement of factor VIII inhibitors.
Thromb Diath Haemorrh.
1975;34:612[Medline]
[Order article via Infotrieve].
24.
Scandella D, Timmons L, Mattingly M, Trabold N, Hoyer LW.
A soluble recombinant factor VIII fragment containing the A2 domain binds to some human anti-factor VIII antibodies that are not detected by immunoblotting.
Thromb Haemost.
1992;67:665-671[Medline]
[Order article via Infotrieve].
25.
Lubin IM, Healey JF, Scandella D, Lollar P.
Analysis of the human factor VIII A2 inhibitor epitope by alanine scanning mutagenesis.
J Biol Chem.
1997;272:30,191-30,195[Abstract/Free Full Text].
26.
Lenting PJ, van de Loo JW, Donath MJ, van Mourik JA, Mertens K.
The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX.
J Biol Chem.
1996;271:1935-1940[Abstract/Free Full Text].
27.
Lenting PJ, Donath MSH, van Mourik JA, Mertens K.
Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.
J Biol Chem.
1994;269:7150-7155[Abstract/Free Full Text].
28.
Fulcher CA, Roberts JR, Holland LZ, Zimmerman TS.
Human factor VIII procoagulant protein: monoclonal antibodies define precursor-product relationships and functional epitopes.
J Clin Invest.
1985;76:117-124.
29.
Lollar P, Hill-Eubanks DC, Parker CG.
Association of the factor VIII light chain with von Willebrand factor.
J Biol Chem.
1988;263:10,451-10,455[Abstract/Free Full Text].
30.
Hill-Eubanks DC, Lollar P.
von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain.
J Biol Chem.
1990;265:17,854-17,858[Abstract/Free Full Text].
31.
Hill-Eubanks DC, Parker CG, Lollar P.
Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin.
Proc Natl Acad Sci U S A.
1989;86:6508-6512[Abstract/Free Full Text].
32.
Kernoff PBA.
Porcine factor VIII: preparation and use in treatment of inhibitor patients. In:
Hoyer LW, ed.
Factor VIII Inhibitors. New York: Alan R. Liss; 1984:207.
33.
Brettler DM.
Inhibitors in congenital haemophilia.
Baillieres Clin Haematol.
1996;9:319-329[Medline]
[Order article via Infotrieve].
34.
Cohen AJ, Kessler CM.
Acquired inhibitors.
Baillieres Clin Haematol.
1996;9:331-354[Medline]
[Order article via Infotrieve].
35.
Koshihara K, Qian J, Lollar P, Hoyer LW.
Immunoblot crossreactivity of factor VIII inhibitors with porcine factor VIII.
Blood.
1995;86:2183-2190[Abstract/Free Full Text].
36.
Wood WI, Capon DJ, Simonsen CC, et al.
Expression of active human factor VIII from recombinant DNA clones.
Nature.
1984;312:330337.
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M. E. Rick, C. E. Walsh, and N. S. Key
Congenital Bleeding Disorders
Hematology,
January 1, 2003;
2003(1):
559 - 574.
[Abstract]
[Full Text]
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C. B. Doering, J. F. Healey, E. T. Parker, R. T. Barrow, and P. Lollar
High Level Expression of Recombinant Porcine Coagulation Factor VIII
J. Biol. Chem.,
October 4, 2002;
277(41):
38345 - 38349.
[Abstract]
[Full Text]
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P. C. Spiegel Jr, M. Jacquemin, J.-M. R. Saint-Remy, B. L. Stoddard, and K. P. Pratt
Structure of a factor VIII C2 domain-immunoglobulin G4{kappa} Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII
Blood,
July 1, 2001;
98(1):
13 - 19.
[Abstract]
[Full Text]
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P. M. Mannucci and E. G.D. Tuddenham
The Hemophilias -- From Royal Genes to Gene Therapy
N. Engl. J. Med.,
June 7, 2001;
344(23):
1773 - 1779.
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E. N. van den Brink, E. A. M. Turenhout, N. Bovenschen, B. G. A. D. H. Heijnen, K. Mertens, M. Peters, and J. Voorberg
Multiple VH genes are used to assemble human antibodies directed toward the A3-C1 domains of factor VIII
Blood,
February 15, 2001;
97(4):
966 - 972.
[Abstract]
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M.-L. Liu, B. W. Shen, S. Nakaya, K. P. Pratt, K. Fujikawa, E. W. Davie, B. L. Stoddard, and A. R. Thompson
Hemophilic factor VIII C1- and C2-domain missense mutations and their modeling to the 1.5-angstrom human C2-domain crystal structure
Blood,
August 1, 2000;
96(3):
979 - 987.
[Abstract]
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E. N. van den Brink, E. A. M. Turenhout, C. M. C. Bank, K. Fijnvandraat, M. Peters, and J. Voorberg
Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain
Blood,
July 15, 2000;
96(2):
540 - 545.
[Abstract]
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Abstract
Introduction