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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Winship Cancer Institute, Emory University,
Atlanta, GA; and the Center for Molecular and Vascular Biology,
Katholieke Universiteit, Leuven, Belgium.
Most inhibitory antibodies to human factor VIII (fVIII) bind to
epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding
of fVIII to phospholipid. The x-ray structure of the human fVIII C2
domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and
Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of
a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged
phosphatidylserine. In this study, 8 active mutants of human fVIII
(Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu,
Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and
Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on
the basis of differences between human, porcine, murine, and canine
fVIII at proposed phospholipid binding sites, were expressed. The
antigenicity of the mutants toward 5 C2-specific polyclonal human
antibodies was measured by using the Bethesda assay. A human monoclonal
anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody,
NMC VIII-5, were also included in the analysis. In comparison with
wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252
single mutants had lower antigenicity toward most of the inhibitors. In
contrast, the Val2223Ala and Lys2227Glu mutants usually showed
increased antigenicity. These results suggest that C2 inhibitors
frequently target the Met2199/Phe2200 and Leu2251/Leu2252 Inhibitory antibodies (inhibitors) to factor VIII
(fVIII) develop either as alloantibodies in patients with hemophilia A
after infusions of fVIII or as autoantibodies in people without
hemophilia.1 Antibodies to epitopes in the A2,
ap-A3, and C2 domains in the A1-A2-B-ap-A3-C1-C2
fVIII molecule are responsible for all anticoagulant activity in most
inhibitor plasma samples.2,3 The 18-kd C2 domain, defined
as residues Ser2173 to Tyr2332 in single-chain human fVIII, contains a
phospholipid membrane-binding site that is necessary for the normal
procoagulant function of fVIII. Human C2-specific anti-fVIII antibodies
inhibit this interaction.4 Consistent with this activity,
phospholipid protects fVIII from inactivation by fVIII
inhibitors.4,5 The C2 domain also contains part of the von
Willebrand factor (vWf) binding site. Some inhibitors may act by
interfering with this interaction.8-10
Recently, a 1.5-Å x-ray structure of the human fVIII C2 domain was
described.11 Examination of this structure revealed 3 solvent-exposed hydrophobic "feet" consisting of Met2199/Phe2200, Val2223, and Leu2251/Leu2252. A ring of positively charged residues, including Lys2227, which is near Val2223, surrounds these residues. This motif suggested that membrane binding consists of insertion of the
hydrophobic feet into the membrane bilayer and is stabilized by
electrostatic interaction with negatively charged phospholipid.
Most fVIII inhibitors have been found to cross-react poorly with
porcine fVIII. This observation led to the mapping of a major determinant of the C2 epitope to residues Glu2181 to Val2243 by means
of a series of constructs that contained porcine substitutions in the
human fVIII C2 domain.12 In the current study, we used residues in porcine, murine, or canine fVIII that are homologous to
residues Met2199, Phe2200, Val2223, Lys2227, Leu2252, or a combination
of these in human fVIII to create a series of recombinant hybrid fVIII
molecules. A significant reduction in antigenicity was usually observed
in association with mutations at Met2199, Phe2200, and Leu2252,
indicating that these residues participate in binding of fVIII to
phospholipid membranes and often to inhibitory antibodies.
Materials
Albumin-free recombinant full-length fVIII was a gift from the
Hyland-Immuno Division of Baxter Healthcare, Duarte, CA.
Synthetic oligonucleotides were purchased from Life Technologies.
Restriction enzymes were purchased from New England Biolabs, Beverly,
MA, or Promega, Madison, WI. A cell line derived from baby
hamster kidney cells was a generous gift from Dr R.T.A.
Macgillivray.15 A B-domainless fVIII expression vector,
designated HB Plasmid DNA was purified by using a Qiagen Plasmid Maxi Kit (Qiagen,
Inc, Valencia, CA). Polymerase chain reactions (PCRs) were done
with an OmniGene thermocycler (Hybaid, Woodbridge, NJ) using
Pfu DNA polymerase. PCR products were gel purified,
precipitated with ethanol, and ligated into plasmid DNA by 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 (Foster City, CA) 373a automated DNA
sequencer and the PRISM dye terminator kit.
Construction of fVIII mutant complementary DNAs
HSQ/ReNeo was used as the template in the PCRs. The first PCR used the human C1 primer, 5'-GTG GAT TCA TCT GGG ATA AAA CAC-3', designated H3763+ and corresponding to nucleotides 3763 to 3786 in the HSQ sequence, as the sense primer. The following primers were used as antisense primers: Met2199Ile, 5'-AGG AGA CCA GGT GGC AAA GAT ATT GGT AAA GTA GGA TGA-3'; Phe2200Leu, 5'-TGA AGG AGA CCA GGT GGC CAA CAT ATT GGT AAA GTA GGA-3'; Val2223Ala, 5'-CCA CTC TTT TGG ATT ATT GGC CTG AGG TCT CCA GGC ATT-3'; Lys2227Glu, 5'-GTC CAC TTG CAG CCA CTC CTC TGG ATT ATT CAC CTG AGG-3'; Leu2252Phe, 5'-CTT CAC ATA CAT GCT GGT GAA CAG AGA TTT TAC TCC CTG-3'; Met2199Ile/Phe2200Leu, 5'-AGG AGA CCA GGT GGC CAA GAT ATT GGT AAA GTA GGA TGA-3'; and Val2223Ala/Lys2227Glu, 5'-CAC TTG CAG CCA CTC CTC TGG ATT ATT GGC CTG AGG TCT CCA GGC-3'. The second PCR used the ReNeo primer, 5'-AGT TTT TCT ACA ACA GAG GAA
GTG-3', designated RE1110 The SOE reaction used fragments from the PCRs as templates and H3763+ and RE1110 as primers. The SOE product and HSQ/RENeo ligation fragments were generated by using SwaI and NotI. Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu cDNA was constructed as
follows. Met2199Ile/Phe2200Leu cDNA was moved into pBluescript II KS Expression of recombinant fVIII molecules Transfected cell lines were maintained in DMEM-F12 containing 10% FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin. FCS was heat inactivated for 1 hour at 56°C before use. Mutant cDNAs in ReNeo were stably transfected into BHK cells, selected for geneticin resistance, switched to serum-free AIM-V medium for expression, and partly purified by heparin-Sepharose chromatography as described previously.12fVIII and fVIII inhibitor assays The activity of recombinant fVIII proteins was measured by a one-stage clotting assay.18 One unit of fVIII was defined as the activity in 1 mL normal citrated human plasma. fVIII inhibitor titers were measured by using the following modification of the Bethesda assay.19 Recombinant fVIII was added to plasma from patients with hemophilia A to a final concentration of 0.8 to 1.2 U/mL and incubated with various concentrations of inhibitor for 2 hours at 37°C. To determine the 50% inhibition point that defines the Bethesda unit, dilutions of inhibitor were made that produced residual activities that spanned at least the range of 35% to 65%. In some cases, replicate dilutions were made and the average value was used. An average of 10 dilutions was made to determine each Bethesda titer.Semilogarithmic plots of the percentage of residual activity compared
with the log of the reciprocal of the inhibitor dilution appeared to be
linear in all cases. The data were fitted by nonlinear regression using
the Marquardt algorithm (SigmaPlot 5.0; SPSS, Chicago, IL) to the
following equation: percentage of residual activity = m (log x The Bethesda titer equals x50 The mass concentration of fVIII in partly purified preparations was
determined by a sandwich enzyme-linked immunosorbent assay (ELISA)
using ESH4 as the capture antibody and biotinylated ESH8 as the
detection antibody as described previously.20 Full-length recombinant fVIII was used as the standard, and values were corrected for the difference in mass between full-length and B-domainless forms
of fVIII. Samples were assayed in quadruplicate. The average coefficient of variation was 9.0%. The specific activity of fVIII molecules was calculated by dividing the coagulant activity by the
concentration determined by ELISA. The following values were obtained:
HB
Hydrophobic residues Met2199, Phe2200, Val2223, Leu2251, and Leu2252 in the C2 domain of fVIII have been proposed to participate in phospholipid membrane binding.11 Additionally, 4 basic residues, Arg2215, Arg2220, Lys2227, and Lys2249, which lie underneath these residues, may stabilize binding to negatively charged phosphatidylserine (PS) head groups. We reasoned that replacement of putative phospholipid binding residues with nonidentical homologous residues from porcine, murine, or canine fVIII would likely produce active molecules that could be evaluated with respect to their antigenicity toward C2-specific inhibitors. Figure 1 shows the alignment of the
human, porcine, murine, and canine fVIII C2 domains. At 4 of the 5 proposed hydrophobic phospholipid binding residues, there was 1 species
that differs from human fVIII; these were Met2199 (Ile; porcine),
Phe2200 (Leu; canine), Val2223 (Ala; canine), and Leu2252 (Phe;
murine). There was a species difference in only 1 of the 4 proposed
basic residues, ie, Lys2227 (Glu; porcine). Accordingly, we made
Met2199Ile, Phe2200Leu, Val2223Ala, Leu2252Phe, and Lys2227Glu single
mutants in human B-domainless fVIII. Additionally, we made 2 double
mutants, Met2199Ile/Phe2200Leu (designated C2 D1) and
Val2223Ala/Leu2252Phe (C2 D2), and a quadruple mutant,
Met2199Ile/Phe2200Leu/Val2223Ala/Leu2252Phe (C2 Q). The locations of
the mutated residues in the x-ray structure of fVIII are shown in
Figure 2. Met2199/Phe2200 and
Leu2251/Leu2252 project from 2
The mutants were stably expressed in serum-free medium from a cell line
derived from baby hamster kidney and were then partly purified. The
specific coagulant activities of the hybrids on ELISA were equal to or
greater than the activity of HB Most fVIII inhibitors are polyclonal IgG populations directed against
epitopes both within and outside the C2 domain.2,21 However, some inhibitors are C2 specific and are useful for evaluating the effects of substituting nonhuman sequences in the C2
domain.12 We studied C2-specific polyclonal inhibitors
from 5 patients (AA, AJ, HR, LK, and RvR; Figure
3). A reduction in antigenicity due to
mutations at Met2199, Phe2200, Leu2252, or all 3 was always observed,
although individual inhibitors varied with respect to the residues they
recognized. Surprisingly, there was frequently a significant increase
in Bethesda titer, most notably with the Val2223Ala mutant. The double
mutant Met2199Ile/Phe2200Leu had low antigenicity toward all 5 antibodies, consistent with the fact that the antigenicity of
Met2199Ile or Phe2200Leu was always reduced. Paradoxically, the double
mutant Val2223Ala/Lys2227Glu showed a reduction in antigenicity toward
all 5 polyclonal antibodies, even though, in 3 cases (AA, AJ, and HR),
the corresponding individual mutants had unchanged or increased
antigenicity. The antigenicity of the quadruple mutant
Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu was equal to or lower than
that of the single or double mutants. The antigenicity of HP20 was the
lowest of all the mutants, a result consistent with the existence of
antigenic residues in addition to Met2199, Phe2200, and Leu2252 that
were not mutated in this study.
The Bethesda titers of antibodies BO2C1113 and NMC
VIII-522 toward HB
Although the x-ray structure of the human fVIII C2 domain was obtained in the absence of phospholipid, 3 hydrophobic feet, consisting of the side chains of Met2199/Phe2200, Val2223, and Leu2251/Leu2252, are present and were postulated to penetrate the membrane bilayer.11 Several positively charged residues, including Lys2227, which may stabilize membrane binding by means of electrostatic interactions with negatively charged PS head groups, surround these residues. Human C2-specific fVIII inhibitors exert their anticoagulant effect by blocking the binding of fVIII to PS.4 Therefore, epitope mapping of these inhibitors is an independent way to identify putative membrane-binding sites. We prepared a series of recombinant hybrid fVIII molecules in
which Met2199, Phe2200, Val2223, Lys2227, Leu2252, or a combination of
these were replaced by homologous residues in porcine, murine, or
canine fVIII. As anticipated, these conservative replacements did not
lead to loss of fVIII coagulant activity. This is important because
loss of antigenicity could otherwise be due to protein misfolding,
which would not necessarily indicate that mutated residues are part of
the antibody epitope. Mutations at Met2199, Phe2200, Leu2252, or a
combination of these were associated with a decrease in antigenicity in
most of the 7 antibodies tested (Table 1)
and the decrease was frequently pronounced (Figures 3-5). This finding
is consistent with the hypothesis that the Met2199/Phe2200 and
Leu2251/Leu2252 loops participate in membrane binding. Even though all
7 inhibitors recognized the M2199/Phe2200 loop, the effects of
mutations at M2199 and PHE2200 often differed. For example, Met2199Ile
showed decreased antigenicity and Phe2200Leu had increased antigenicity
toward antibody AJ, whereas the opposite was true for BO2C11. Thus, the
amino acid specificity of AJ and BO2C11 varied, although both
recognized the Met2199/Phe2200 loop.
Previously, we used a series of recombinant hybrid human/porcine fVIII molecules to map a major determinant of the C2 epitope or epitopes to a segment bounded by residues Glu2181 to Val2243.12 The Met2199/Phe2200 loop is in this region. The Leu2251/Leu2252 loop was neither included nor excluded by this analysis because porcine fVIII also contains leucines at residues 2251 and 2252. Substitution of the entire porcine C2 domain into human fVIII, which produces a molecule designated HP20, was associated with antigenicity lower than that with the more limited substitutions made in the current study (Figures 3-5). This indicates that there are residues outside the Met2199/Phe2200 and Leu2251/Leu2252 loops that contribute to binding by C2 inhibitors. Recently, x-ray structures of 2 conformations of the factor V (fV) C2 domain in the absence of phospholipid were described.23 The researchers proposed a model for phospholipid membrane binding that involves a loop containing tryptophans at positions 26 and 27 (human fV C2-domain numbering), which are homologous to Met2199 and Phe2200 in fVIII. A considerable amount of evidence supports the involvement of this loop in phospholipid membrane binding. An inhibitory monoclonal antibody, HV-1, which blocks the binding of fV to PS, maps to this loop.24-26 Substitution of alanine for residues equivalent to Trp26 and Trp27 in fVa is associated with decreased binding to PS and loss of coagulant activity.24 Additionally, a loop containing Leu79, which is homologous to Leu2251 in fVIII, and a loop containing residues Asn39 to Asn45 were also proposed to participate in phospholipid membrane penetration because of proximity to the Trp26/Trp27 loop.23 The fVIII segment that is homologous to the Asn39 to Asn45 loop, His211 to Asn217, has not been proposed as a hydrophobic phospholipid membrane-binding site.11 Conversely, the loop in fV that is homologous to the Val2223 loop in fVIII was not proposed to participate in phospholipid membrane penetration. In the current study, Val2223Ala and Lys2227Glu mutations were usually associated with an increase in antigenicity (Table 1). Thus, our results do not support the hypothesis that these residues participate in phospholipid membrane binding. However, it is possible that they bind phospholipid but are not often targeted by inhibitory antibodies. The 2 fV C2 structures have different conformations, designated "open" and "closed," which are associated with major movements of Trp26 and Trp27 at the phospholipid membrane-binding site.23 It was proposed that these states switch phospholipid membrane binding on and off, respectively.23 The reduction in antigenicity associated with Val2223 and Lys2227 may occur because these residues stabilize a similar closed conformational state in fVIII that is associated with low-affinity membrane and antibody binding. Relaxation of this state by the Val2223Ala and Lys2227Glu mutations would then lead to high-affinity antibody binding. Alternatively, Val2223 and Lys2227 may simply interfere with an antigen-antibody lock-and-key interaction that involves high-affinity contacts with other fVIII residues (eg, Met2199, Phe2200, and so forth). It is important to compare the human C2-specific monoclonal antibody, BO2C11, with polyclonal inhibitors because of the heterogeneity that may confound analysis of the inhibitors. The functional properties of BO2C11 are similar to those of the murine monoclonal antibody NMC VIII-5. Both antibodies inhibit the binding of fVIII to PS and vWf and promote dissociation of the fVIII-vWf complex.13,22 Our results indicate that Phe2200, but not Met2199, is an important part of the epitope recognized by both antibodies (Figures 4 and 5). However, NMC VIII-5 recognized Leu2252, whereas BO2C11 did not. Val2223 and Lys2227 reduced antigenicity with respect to both antibodies. Thus, BO2C11 and NMC VIII-5 appear to recognize overlapping but not identical epitopes. The RvR antibody was obtained from a patient with hemophilia A who was part of an inhibitor "epidemic" that resulted from exposure to a heat-pasteurized fVIII product, CPS-A.27 In 1990 and 1991, C2-specific antibodies developed promptly after exposure to this product in several previously treated patients without inhibitors in The Netherlands and Belgium. RvR antibodies block the binding of fVIII to both PS and vWf.27 The RvR epitope maps to the N-terminal, Glu2181 to Val2243 region of the fVIII C2 domain recognized by most C2 inhibitors.12 The high-resolution mapping in the current study indicated that RvR is a typical C2 inhibitor that primarily recognizes the Met2199/Phe2200 and Leu2251/Leu2252 loops. Thus, the immunogenicity associated with CPS-A appears to be due to enhanced immune recognition of a normal immunodominant epitope rather than to development of a neoepitope.
Submitted July 13, 2000; accepted September 14, 2000.
Supported by a grant from the National Institutes of Health (R01-HL46215) to P.L.
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.
Reprints: Pete Lollar, 1639 Pierce Dr, Rm 1003, Woodruff Memorial Bldg, Emory University, Atlanta, GA 30322; e-mail: jlollar{at}emory.edu.
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© 2001 by The American Society of Hematology.
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  T. Soeda, K. Nogami, K. Nishiya, M. Takeyama, K. Ogiwara, Y. Sakata, A. Yoshioka, and M. Shima The Factor VIIIa C2 Domain (Residues 2228-2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex J. Biol. Chem., February 6, 2009; 284(6): 3379 - 3388. [Abstract] [Full Text] [PDF]
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 S. L. Meeks, J. F. Healey, E. T. Parker, R. T. Barrow, and P. Lollar Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors Blood, August 15, 2008; 112(4): 1151 - 1153. [Abstract] [Full Text] [PDF]
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A. Vencesla, M. A. Corral-Rodriguez, M. Baena, M. Cornet, M. Domenech, M. Baiget, P. Fuentes-Prior, and E. F. Tizzano Identification of 31 novel mutations in the F8 gene in Spanish hemophilia A patients: structural analysis of 20 missense mutations suggests new intermolecular binding sites Blood, April 1, 2008; 111(7): 3468 - 3478. [Abstract] [Full Text] [PDF]
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S. L. Meeks, J. F. Healey, E. T. Parker, R. T. Barrow, and P. Lollar Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation Blood, December 15, 2007; 110(13): 4234 - 4242. [Abstract] [Full Text] [PDF]
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E. T. Parker, J. F. Healey, R. T. Barrow, H. N. Craddock, and P. Lollar Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope Blood, August 1, 2004; 104(3): 704 - 710. [Abstract] [Full Text] [PDF]
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C. B. Doering, J. F. Healey, E. T. Parker, R. T. Barrow, and P. Lollar Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion J. Biol. Chem., February 20, 2004; 279(8): 6546 - 6552. [Abstract] [Full Text] [PDF]
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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] [PDF]
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E. N. van den Brink, W. S. Bril, E. A. M. Turenhout, M. Zuurveld, N. Bovenschen, M. Peters, T. T. Yee, K. Mertens, D. A. Lewis, T. L. Ortel, et al. Two classes of germline genes both derived from the VH1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII Blood, April 15, 2002; 99(8): 2828 - 2834. [Abstract] [Full Text] [PDF]
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S. Stoilova-McPhie, B. O. Villoutreix, K. Mertens, G. Kemball-Cook, and A. Holzenburg 3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography Blood, February 15, 2002; 99(4): 1215 - 1223. [Abstract] [Full Text] [PDF]
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Y. Lin, L. Chang, A. Solovey, J. F. Healey, P. Lollar, and R. P. Hebbel Use of blood outgrowth endothelial cells for gene therapy for hemophilia A Blood, January 15, 2002; 99(2): 457 - 462. [Abstract] [Full Text] [PDF]
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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] [PDF]
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Top
Abstract
Introduction
-hairpins
and are consistent with the hypothesis that these residues participate
in binding to phospholipid membranes. In contrast, Val2223 and Lys2227
may oppose antibody binding sterically or through stabilization of a
low-affinity membrane-binding conformation of the C2 domain.
(Blood. 2001;97:169-174)
were purchased from Stratagene, La Jolla, CA.
Murine antihuman fVIII monoclonal antibodies ESH4 and ESH8 were
purchased from American Diagnostica, Greenwich, CT. The murine
fVIII C2-specific inhibitory monoclonal antibody NMC VIII-5 was a
generous gift from Dr Midori Shima, Nara Medical College, Japan. A
human fVIII C2-specific IgG4 monoclonal antibody BO2C11, which was
cloned from a transformed B-cell line from a patient with hemophilia, was prepared as described previously.
/ReNeo, which contains a NotI site 2 bases 3'
to the stop codon and ampicillin and geneticin resistance genes, was
prepared as described previously.
monoclonal antibody derived from transformed B cells from
a patient with hemophilia A and inhibitor. It is the only C2-specific
human antibody that has been cloned to date. BO2C11 and NMC VIII-5 both recognize the C2 domain of fVIII and inhibit the binding of fVIII to
vWF and phospholipid. NMC VIII-5 can compete for the binding of human
polyclonal inhibitors to fVIII. The results with BO2C11 and NMC VIII-5
were similar to those obtained with polyclonal antibody HR (Figure 
antibodies that bind to the factor-VIII light chain prevent the interaction of factor-VIII with phospholipid.
J Clin Invest.
1989;83:1978-1984.