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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 156-163
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Center for Molecular and Vascular Biology, Katholieke
Universiteit Leuven, Leuven, Belgium; Hôpital Bicêtre, le
Kremlin-Bicêtre, AP-HP, France; Institut für
Experimentelle Hämatologie und Transfusionsmedizin, Bonn,
Germany; Department of Hematology-Immunology, Vrije Universiteit
Brussels, Brussels, Belgium.
The occurrence of factor VIII (fVIII) inhibitory antibodies is a
rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the
C1 domain are, however, more frequently associated with inhibitor, for
reasons which remain unclear. To determine whether inhibitors could map
to the mutation site, we analyzed at the clonal level the immune
response of such a patient with an inhibitor to wild-type but not
self-fVIII and an Arg2150His substitution in the C1 domain.
Immortalization of the patient B lymphocytes provided a cell line
producing an anti-fVIII IgG4
Hemophilia A is an X-linked bleeding disorder
characterized by the absence or an insufficient amount of functional
factor VIII (fVIII). This deficiency affects 1 in 10 000 males and
results in bleeding in joints, muscles, and soft tissues. Patients
affected by the severe form of the disease (fVIII activity < 0.01 IU/mL) suffer from spontaneous bleedings, whereas in moderate or mild hemophilia A (fVIII activity 0.01 to 0.05 IU/mL and 0.06 to 0.4 IU/mL,
respectively), bleeding can occur after minor trauma or surgery. The
fVIII molecule is a 330 kD glycoprotein produced by the liver as a
single polypeptide chain of 2332 amino acids that subsequently
undergoes proteolytic processing.1 The circulating fVIII
molecule consists of 2 chains. The heavy chain is constituted of the A1
and A2 domains and variable lengths of the B domain; the light chain
contains the A3 domain and the C1 and C2 domains. In plasma, fVIII
circulates complexed to von Willebrand factor (vWf) which protects it
from rapid degradation.2 On cleavage by thrombin, activated
fVIII dissociates from vWf,3 binds to negatively charged
phospholipids, and participates as a cofactor to factor IXa in the
factor X activating (tenase) complex.
Replacement therapy relies on the use of fVIII concentrates prepared
from plasma or of recombinant fVIII (rfVIII). However, such a treatment
can elicit the production of specific antibodies neutralising fVIII
function, also called inhibitors.4 Several factors
contribute to the development of an immune response toward fVIII.
Recent evidence has shown that product-related factors can be involved
in induction of inhibitors.5,6 Host-related factors play a
major role in determining the incidence of inhibitors. Possible
association between inhibitor formation and HLA haplotypes has recently
been suggested.7,8 In severe hemophilia, there is a close
relationship between the fVIII gene defect and the production of
inhibitors: patients with severe gene defects such as nonsense
mutations, large deletions or gene inversions are more likely to
produce inhibitors than those with small deletions or missense
mutations.9-11
In agreement with the concept that the presence of self-fVIII, although
functionally altered or produced at reduced levels, contributes to
render the immune system tolerant to fVIII, the development of
inhibitor antibodies in mild hemophilia A is a rare
event.12 Usually, the inhibitor is of low titer and
transient.13 High-titer inhibitor associated with an
anamnestic humoral response after replacement therapy has, however,
been observed in some patients. Typically, in such cases, the inhibitor
causes a fall of plasma fVIII levels,13 which transforms
the bleeding phenotype of the patient into that of a severe
hemophiliac. An immune response restricted to wild-type fVIII has
recently been reported in mild hemophilia A patients with mutation
Arg593-> Cys14 and Arg2150-> His.15
Antibodies recognizing wild-type but not self-fVIII represent unique
opportunities to study the influence of B-cell epitope alteration on
the development of an immune response toward fVIII.
A detailed analysis of the specificity of fVIII inhibitors has proven
to be difficult because of the large diversity of the humoral response,
including antibodies which do not interfere with fVIII
activity.16 Moreover, anti-idiotypic antibodies have been
described that can neutralize fVIII inhibitors.17,18 To circumvent difficulties inherent to the use of polyclonal antibodies, we produced human monoclonal antibodies directed toward fVIII and
representative of patients' pathogenic antibodies. Preliminary experiments have established that memory B cells from hemophilia A
patients with inhibitor can be immortalized with EBV after CD40 cross-linking.19 We have applied the same strategy to
characterize at the clonal level the relationship between alteration of
fVIII B-cell epitopes and humoral response of a mild hemophilia A
patient with inhibitor who maintained significant endogenous fVIII
activity despite the presence of a high level of fVIII
inhibitor.15
The fVIII gene mutation carried by patient LE was located in the C1
domain, ie, a region in which mutations are associated with a higher
incidence of inhibitors in mild/moderate hemophilia A
patients.13 However, the reason for such an association is still unclear. The fact that LE polyclonal antibodies recognized normal
but not self-fVIII could be accounted for by elimination of a B cell
epitope(s) through conformational changes induced at the mutation site
in the C1 domain. Alternatively, other regions of the fVIII molecule
could be affected by the mutation, as recently demonstrated for the
substitution Arg2159Cys in the C1 domain, which altered an epitope
located in the C2 domain.20 Mutations in the C1 domain are
commonly identified in hemophilia A patients, yet the role of the
latter domain in fVIII function or stability is not
determined.21,22 Moreover, inhibitor antibodies recognizing the C1 domain have never been demonstrated. Clonal characterization of
patient LE antibodies offered, therefore, the potential of determining
whether epitopes recognized by inhibitor antibodies were located in the
C1 domain, but also provided an opportunity to analyze the
structure/function relationship of a fVIII domain whose function is
still completely unknown.
Reagents
Immortalization of human PBMC
Sequencing of immunoglobulin genes V region gene usage was determined by reverse transcriptase polymerase chain reaction (RT-PCR) amplification and sequence analyses using primers specific for the C region gene, and each of the V region gene families.24 The complete sequences of the VH and VL have been submitted to the EMBL Nucleotide Sequence Database under the accession numbers AJ009732 and AJ009733, respectively.Purification of human IgG Human monoclonal antibodies were purified by adsorption on immobilized Protein A (high-TRAP® Protein A; Pharmacia, Uppsala, Sweden). Fab fragments of human monoclonal antibody were prepared by papain digestion. One milligram of LE2E9 was diluted at 500 µg/mL in 100 mmol/L phosphate buffer, pH 7.0, containing 50 mmol/L L-cystein (Sigma Chemicals, St Louis, MO), 1 mmol/L EDTA (Merck, Darmstadt, Germany) and 10 µg papain (Sigma). The mixture was incubated for 3 hours at 37°C with continuous agitation. The reaction was stopped by addition of iodoacetamide to a final concentration of 75 mmol/L and a further incubation of 30 minutes at RT. The digested antibody was dialyzed against phosphate-buffered saline, pH 7.4. Undigested IgG and Fc fragments were then eliminated by passage over immobilized Protein A Sepharose. Fab fragments were further purified by gel filtration chromatography on a Superdex 200 (Pharmacia).Immunoassays Previously described methods were used for the detection of anti-fVIII IgG antibodies,19 the determination of IgG subclass,18 and the evaluation of inhibition of fVIII binding to vWf.19 For analysis of the inhibition of rfVIII binding to LE2E9 by Fab or native LE2E9, Maxisorb polystyrene plates (Nunc, Roskilde, Denmark) were incubated for 2 hours with 50 µL of LE2E9 diluted at 5 µg/mL in glycine-buffered saline, 20 mmol/L glycine, 34 mmol/L NaCl, pH 9.2, and washed. Biotin-labeled rfVIII (50 µL) diluted to 1 µg/mL in Tris-casein (10 mmol/L tris(hydroxymethyl)-aminoethane, pH 7.3, containing 150 mmol/L NaCl and 0.5% casein) were mixed for 1 hour at 37°C with 50 µL of human IgG or Fab fragments at various concentrations. A 50-µL aliquot of the mixture was added to the plates for an incubation of 2 hours at RT. After washing, the binding of biotin-labeled rfVIII was detected by sequential addition of avidin-peroxidase and OPD.fVIII inhibition in functional assays rfVIII (final concentration 0.2 µg/mL) was incubated with human IgG antibody at different concentrations for 2 hours at 37°C and the residual fVIII activity was assessed by a chromogenic assay (Coatest® Factor VIII, Chromogenix AB, Mölndal, Sweden). Inhibition of plasma fVIII activity was measured by the Bethesda method,25 in which a pool of normal plasma collected in buffered trisodium citrate was used as fVIII source. Residual fVIII activity was assessed by a chromogenic or by a 1-stage clotting assay.Cloning of normal and mutated fVIII cDNA fragments All DNA fragments encoding fVIII domains were generated by PCR using primers bound by the restriction sites HindIII (italic) and Not1 (underlined) sites. Sense primers, named according to the first fVIII amino acid residue encoded (codon in bold), were as follows:
Expression of fVIII recombinant fragments in reticulocyte transcription/translation system DNA 500 ng to 1 µg, linearized by Not1 digestion, was used as a template in a T7 RNA polymerase transcription system in micrococcal nuclease-treated reticulocyte lysates (Promega, Southampton, UK) according to the manufacturer's instructions in the presence of L-[35S]methionine (Amersham, Bucks, UK). The [35S]-methionine labeled fVIII fragments migrated on SDS-PAGE as bands matching the expected mass of corresponding fVIII polypeptides.Immunoprecipitation of L-[35S]methionine-labeled fVIII fragments Standard in vitro translation product 1 to 3 µL was added to 500 µL human antibody at 2 µg/mL in NET-gel buffer 50 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 0.1% Nonided NP-40;1 mmol/L EDTA (pH 8; 0.25% gelatin and 5% BSA). Tubes were gently rocked for 1 hour at 4°C. Twenty microliters of a 50% solution of Protein A Sepharose was then added to the antigen/antibody mixture, and incubated for 1 hour at 4°C on a rocking platform. The Sepharose beads were centrifuged and washed twice with Tris-NP40 10 mmol/L Tris-HCl (pH 7.5; 0.1% NP40). Bound antigen/antibody complexes were eluted from the beads by boiling for 4 minutes in 30 µL of SDS gel loading buffer. An aliquot of 15 µL was analyzed by 10% (w/v) polyacrylamide gel electrophoresis and visualized by autoradiography. The intensity of the bands was scored as plus (+) or minus ( ) by 2 independent
investigators. Control experiments were performed with the human
monoclonal antibody BO2C11, directed toward the fVIII C2
domain,19 a rabbit polyclonal IgG antibody (aA3) directed toward amino acid residues 1797-1815 of the fVIII A3 domain and purified by immunoadsorption on the corresponding insolubilized synthetic peptide (kind gift of M. Di Giambattista and R. Laub, Belgian
Red Cross), and normal donor's polyclonal IgG antibodies (Wi) purified
on Protein A Sepharose.
Production and characterization of the human monoclonal antibody LE2E9 The PBMC of a hemophilia A patient with inhibitor, LE, were immortalized by EBV infection concomitantly to activation by CD40 cross-linking. Four hundred and eighty cell lines were screened by ELISA for production of antibodies toward fVIII. One cell line, LE2E9, was successfully cloned by limiting dilution. Clonality was verified by RT-PCR amplification of mRNA coding for the V regions of the antibody heavy and light chains: a single sequence was obtained from 10 clones of PCR products. The V region of LE2E9 heavy chain gene was most homologous to DP-64, a member of the VH-4 gene family and the J region was most homologous to JH4b. Sequencing of the cloned light chain gene identified the V region gene as a V 3 and the J region gene as a
J4. Purified antibodies were obtained by passage of LE2E9 cell
culture supernatant on Protein-A Sepharose. An ELISA performed with IgG
subclass- and light chain-specific antibodies identified LE2E9 as an
IgG4.
LE2E9 inhibits wild-type allogeneic but not syngeneic fVIII LE2E9 inhibited fVIII with high specific activity: when 1 volume of LE2E9 was mixed with 1 volume of rfVIII at 0.2 µg/mL for an incubation of 2 hours at 37°C, the final LE2E9 concentration required to inhibit 50% of fVIII activity was 0.1 µg/mL (Figure 1). However, even in the presence of a large excess of LE2E9 (10 µg/mL), the inhibition remained incomplete (85%). In control experiments, fVIII activity was reduced by more than 95% after incubation with the human monoclonal antibody, BO2C11, at 1 µg/mL.19 Similar results were obtained when a pool of normal plasma was used as a source of fVIII (Figure 1) and when residual fVIII activity was evaluated either by chromogenic or 1-stage coagulation assays. By contrast, LE2E9 did not reduce the fVIII activity present in the plasma of patient LE's brother (LM), a mild hemophilia A patient with a fVIII level of 0.09 IU/mL (Figure 2). Likewise, no inhibition of fVIII activity was observed when LE2E9 was incubated with plasma of the patient from whom the LE2E9 cell line had been derived.
LE2E9 does not recognize a recombinant fVIII light chain carrying the mutation Arg2150His The absence of recognition of Arg2150His fVIII suggested that the epitope recognized by LE2E9 was located on the fVIII light chain. In addition, LE2E9 bound to purified isolated fVIII light chain but not to the fVIII heavy chain in ELISA (data not shown). To demonstrate that in the absence of plasma, LE2E9 did not recognize the mutated fVIII light chain, DNA fragments encoding wild-type and mutated fVIII light chains were synthesized. The corresponding proteins were expressed in reticulocyte lysates. The correct folding of native and mutated light chains was determined by immunoprecipitation with the human monoclonal antibody BO2C11, which recognizes a conformational epitope within the carboxy-terminal part of the fVIII light chain.19 Immunoprecipitation experiments indicated that BO2C11 bound wild-type and Arg2150His light chains, whereas LE2E9 captured exclusively the wild-type light chain (Figure 3). Prolonged exposure of SDS-PAGE gels to the autoradiography film failed to detect any significant binding of LE2E9 to the mutated light chain. Control experiments showed no binding to assay reagents other than fVIII or fVIII fragments, and preincubation with soluble rfVIII prevented the binding to methionine-labeled fVIII fragments, confirming the binding specificity.
LE2E9 binds to the C1 domain LE2E9 did not recognize fVIII in Western blotting (unpublished data, 1996), indicating that the epitope recognized was conformational. Further epitope mapping was therefore performed with fVIII fragments produced in reticulocyte lysates. Preliminary experiments had indicated that such an approach was efficient for the synthesis of fVIII domains. The immunoprecipitation procedure using labeled fVIII domains produced in reticulocyte lysate was validated by mapping the epitope recognized by the human monoclonal antibody BO2C11. A complete agreement was observed between the binding to fVIII C2 deletion fragments produced in reticulocyte lysates and the binding to recombinant fragments produced in Escherichia coli or COS cells (unpublished data, 1997, and Figure 4). LE2E9 bound to full-length light chain, to fragments corresponding to A3C1, C1C2, and the isolated C1 domain (Figure 4). By contrast, as shown in Figure 5, LE2E9 did not bind to the C1 or C1C2 domains with the substitution Arg2150His, although in a control experiment, the Arg2150His C1C2 domain was bound by BO2C11 like its normal counterpart.
A fVIII carrying a Pro2153Gln substitution is partially resistant to LE2E9 The observation that mutation Arg2150His in C1 completely prevented the binding of LE2E9 to fVIII prompted us to search for other mutations in the light chain that could alter the binding of LE2E9. As shown in the Table, LE2E9 inhibited the activity of all mutated fVIII molecules tested so far, except those carrying the mutation Arg2150His. Partial resistance to inactivation by LE2E9 was conferred by the Pro2153Gln substitution but not by the Arg2159Leu substitution in the C1 domain. As indicated in Figure 6, LE2E9 at a concentration of 3 µg/mL inhibited 50% of Pro2153Gln fVIII activity, whereas 15-fold less LE2E9 was required to inhibit 50% activity of normal rfVIII added in Pro2153Gln fVIII plasma. The prevention of the inhibition was not due to the interaction of vWf or anti-idiotypic antibodies, as rfVIII added to the patient's plasma containing Pro2153Gln fVIII was inactivated like fVIII in control plasma.
LE2E9 inhibits fVIII binding to vWf The capacity of LE2E9 to inhibit the binding of fVIII to vWf was assessed in ELISA. Figure 7 shows that LE2E9 inhibited the binding of fVIII to vWf in a dose-dependent manner. The concentration of LE2E9 required to achieve 50% inhibition (IC50) of fVIII binding was 0.25 µg/mL and more than 95% inhibition was obtained with 20 µg/mL of LE2E9.
LE2E9 binding to fVIII is inhibited by LE IgG To determine whether LE2E9 was representative of LE antibodies, a competitive assay was used. The binding of biotin-labeled LE2E9 to insolubilized fVIII was measured in the presence of increasing concentrations of either LE2E9, polyclonal LE IgG, or control polyclonal IgG. Polyclonal LE IgG dose-dependently inhibited LE2E9 binding to fVIII. The concentrations of LE2E9 or LE IgG inhibiting 50% of the binding of biotin-labeled LE2E9 to fVIII were 0.3 µg/mL and 170 µg/mL, respectively, whereas no inhibition was observed with control IgG (Figure 8).
The development of a fVIII inhibitor in mild hemophilia A patients
is a rare but severe complication of fVIII substitution therapy, which
is typically associated with the shift of the patient's bleeding
phenotype to that of severe hemophilia.13 Given the complexity inherent to the study of polyclonal anti-fVIII antibodies, we have analyzed at the clonal level the immune response of a mild
hemophilia A patient (Arg2150His) who presented with a high titer
inhibitor toward allogeneic but not self-fVIII.15 A human IgG4
We thank J. Arnout and M. Vanrusselt for performing the coagulation assays, J. M. Lavergne for identification of mutations in the fVIII gene, and J.-J. Pin, S. Lebecque, and J. Banchereau for their invaluable help in the production of the human monoclonal antibodies.
Submitted March 17, 1999; accepted August 24, 1999.
Supported in part by research grant G .0292.98 of the Flemish Research Foundation, by grant BR1/4-255/138 of the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, and by grant Schw 752/1-1 from the Deutsche Forschungsgemeinschaft. J. Vermylen is holder of the Dr Jean Choay Chair for Hemostasis Research.
Reprints: Marc Jacquemin, Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium.
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.
1. Vehar GA, Keyt B, Eaton D, et al. Structure of human factor VIII. Nature. 1984;312:337-342[Medline] [Order article via Infotrieve]. 2. Weiss HJ, Sussman II, Hoyer LW. Stabilization of: factor VIII in plasma by the von Willebrand factor: studies on posttransfusion and dissociated factor VIII and in patients with von Willebrand's disease. J Clin Invest. 1977;60:390-404. 3. Hamer RJ, Koedam JA, Beeser-Visser NH, Bertina RM, Van Mourik JA, Sixma JJ. Factor VIII binds to von Willebrand factor via its Mr 80,000 light chain. Eur J Biochem. 1987;166:37-43[Medline] [Order article via Infotrieve]. 4. 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]. 5. Peerlinck K, Arnout J, Gilles JGG, Saint-Remy JMR, Vermylen J. A higher than expected incidence of Factor VIII inhibitors in multitransfused haemophilia A patients treated with an intermediate purity pasteurized Factor VIII concentrate. Thromb Haemost. 1993;69:115-118[Medline] [Order article via Infotrieve]. 6. Peerlinck K, Arnout J, Di Giambatista M, et al. Factor VIII inhibitors in previously treated haemophilia A patients with a double virus-inactivated plasma derived factor VIII concentrate. Thromb Haemost. 1997;77:80-86[Medline] [Order article via Infotrieve]. 7. Hay CR, Ollier W, Pepper L, et al. HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A: UKHCDO Inhibitor Working Party. Thromb Haemost. 1997;77:234-237[Medline] [Order article via Infotrieve]. 8. Oldenburg J, Picard J, Schwaab R, Brackmann H-H, Tuddenham EGD, Simpson E. HLA-genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII. Thromb Haemost. 1997;77:238-242[Medline] [Order article via Infotrieve].
9.
Antonarakis SE, Rossiter JP, Young M, Horst J, de Moerloose P.
Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.
Blood.
1995;86:2206-2212 10. Schwaab R, Brackmann HH, Meyer C, et al. Haemophilia A: mutation type determines risk of inhibitor formation. Thromb Haemost. 1995;74:1402-1406[Medline] [Order article via Infotrieve].
11.
Tuddenham EGD, Schwaab R, Seehafer S.
Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.
Nucl Acids Res.
1994;22:4851-4868
12.
Lusher JM, Arkin S, Abildgaard CF, Schwartz RS.
Recombinant factor VIII for the treatment of previously untreated patients with hemophilia A: safety, efficacy, and development of inhibitors. Kogenate Previously Untreated Patient Study Group.
N Engl J Med.
1993;328:453-459 13. Hay CR, Ludlam CA, Colvin BT, et al. Factor VIII inhibitors in mild and moderate-severity haemophilia A: UK Haemophilia Centre Directors Organisation. Thromb Haemost. 1998;79:762-766[Medline] [Order article via Infotrieve].
14.
Fijnvandraat K, Turenhout EAM, van den Brink EN, et al.
The missense mutation Arg593
15.
Peerlinck K, Jacquemin M, Arnout J, et al.
Antifactor VIII antibody inhibiting allogeneic but not autologous factor VIII in patients with mild hemophilia A.
Blood.
1999;93:2267-2273
16.
Gilles JG, Arnout J, Vermylen J, Saint-Remy JMR.
Anti-factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction.
Blood.
1993;82:2452-2461 17. Sultan Y, Kazatchkine M, Maisonneuve P, Nydegger U. Anti-idiotypic suppression of auto-antibodies to factor VIII (anti-hemophilic factor) by high dose intravenous gammaglobulin. Lancet. 1984;2:765-768[Medline] [Order article via Infotrieve]. 18. Gilles JG, Saint-Remy JM. Healthy subjects produce both anti-factor VIII and specific anti-idiotypic antibodies. J Clin Invest. 1994;94:1496-1505.
19.
Jacquemin MG, Desqueper BG, Benhida A, et al.
Mechanisms and kinetics of fVIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor.
Blood.
1998;92:496-506 20. Suzuki H, Shima M, Arai M, et al. Factor VIII Ise (R2159C) in a patient with mild hemophilia A, an abnormal Factor VIII with retention of function but modification of C2 epitopes. Thromb Haemost. 1997;77:862-867[Medline] [Order article via Infotrieve]. 21. Vlot A, Koppelman SJ, Bouma BN, Sixma JJ. Factor VIII and von Willebrand factor. Thromb Haemost. 1998;79:456-465[Medline] [Order article via Infotrieve]. 22. Kaufman RJ. Post-translational modifications required for coagulation factor secretion and function. Thromb Haemost. 1998;79:1068-1079[Medline] [Order article via Infotrieve]. 23. Madec AM, Rousset F, Ho S, et al. Four IgG anti-islet human monoclonal antibodies isolated from a type 1 diabetes patient recognize distinct epitopes of glutamic acid decarboxylase 65 and are somatically mutated. J Immunol. 1996;156:3541-3549[Abstract].
24.
Bakkus MH, Heirman C, Van Riet I, Van Camp B, Thielemans K.
Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation.
Blood.
1992;80:2326-2335 25. Kasper CK, Aledort LM, Aronson D, et al. A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. 1975;34:612[Medline] [Order article via Infotrieve]. 26. Horton RM, Pease LR. Recombination and mutagenesis of DNA sequences using PCR. In: McPherson MJ, ed. Directed mutagenesis A practical approach. Oxford: IRL Press; 1991:217-246.
27.
Thompson AR, Murphy MEP, Liu ML, et al.
Loss of tolerance to exogenous and endogenous factor VIII in a mild hemophilia A patient with an Arg593 to Cys mutation.
Blood.
1997;90:1902-1910 28. Santagostino E, Gringeri A, Tagliavacca L, Mannucci P. Inhibitors to factor VIII in a family with mild hemophilia: molecular characterization and response to factor VIII and desmopressin. Thromb Haemost. 1995;74:619-621[Medline] [Order article via Infotrieve]. 29. Gilles JG, Desqueper B, Lenk H, Vermylen J, Saint-Remy JMR. Neutralising anti-idiotypic antibodies to factor VIII inhibitors following desensitization in patients with hemophilia A. J Clin Invest. 1996;97:1382-1388[Medline] [Order article via Infotrieve].
30.
Gawryl MS, Hoyer LW.
Inactivation of factor VIII coagulant activity by two different types of human antibodies.
Blood.
1982;60:1103-1109 31. Amano K, Arai M, Koshihara K, et al. Autoantibody to factor VIII that has less reactivity to factor VIII/von Willebrand factor complex. Am J Hematol. 1995;49:310-317[Medline] [Order article via Infotrieve]. 32. Gilles JG, Lavend'homme R, Peerlinck K, et al. Some factor VIII (FVIII) inhibitors recognise a FVIII epitope(s) that is present only on FVIII-vWF complexes. Thromb Haemost. 1999;82:40-45[Medline] [Order article via Infotrieve]. 33. Leyte A, Mertens K, Distel B, et al. Inhibition of human coagulation factor VIII by monoclonal antibodies: mapping of functional epitopes with the use of recombinant factor VIII fragments. Biochem J. 1989;263:187-194[Medline] [Order article via Infotrieve]. 34. Shima M, Scandella D, Yoshioka A, et al. Factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost. 1993;69:240-246[Medline] [Order article via Infotrieve].
35.
Saenko EL, Shima M, Rajalakshmi KJ, Scandella D.
A role for the C2 domain of factor VIII in binding to von Willebrand factor.
J Biol Chem.
1994;269:11,601-11,605
36.
Foster PA, Fulcher CA, Houghten RA, Zimmerman TS.
Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine.
Blood.
1990;75:1999-2004
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  S. Lacroix-Desmazes, A.-M. Navarrete, S. Andre, J. Bayry, S. V. Kaveri, and S. Dasgupta Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A Blood, July 15, 2008; 112(2): 240 - 249. [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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|
|
|
|
T.-C. Hsu, K. P. Pratt, and A. R. Thompson The factor VIII C1 domain contributes to platelet binding Blood, January 1, 2008; 111(1): 200 - 208. [Abstract] [Full Text] [PDF]
|
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|
|
|
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J. G. G. Gilles, S. C. Grailly, M. De Maeyer, M. G. Jacquemin, L. P. VanderElst, and J.-M. R. Saint-Remy In vivo neutralization of a C2 domain-specific human anti-Factor VIII inhibitor by an anti-idiotypic antibody Blood, April 1, 2004; 103(7): 2617 - 2623. [Abstract] [Full Text] [PDF]
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M. Jacquemin, V. Vantomme, C. Buhot, R. Lavend'homme, W. Burny, N. Demotte, P. Chaux, K. Peerlinck, J. Vermylen, B. Maillere, et al. CD4+ T-cell clones specific for wild-type factor VIII: a molecular mechanism responsible for a higher incidence of inhibitor formation in mild/moderate hemophilia A Blood, February 15, 2003; 101(4): 1351 - 1358. [Abstract] [Full Text] [PDF]
|
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|
|
K. Nogami, M. Shima, K. Nishiya, K. Hosokawa, E. L. Saenko, Y. Sakurai, M. Shibata, H. Suzuki, I. Tanaka, and A. Yoshioka A novel mechanism of factor VIII protection by von Willebrand factor from activated protein C-catalyzed inactivation Blood, May 13, 2002; 99(11): 3993 - 3998. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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I. Singh, A. Smith, B. Vanzieleghem, D. Collen, K. Burnand, J.-M. Saint-Remy, and M. Jacquemin Antithrombotic effects of controlled inhibition of factor VIII with a partially inhibitory human monoclonal antibody in a murine vena cava thrombosis model Blood, May 1, 2002; 99(9): 3235 - 3240. [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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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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K. Nogami, M. Shima, J. C. Giddings, K. Hosokawa, M. Nagata, S. Kamisue, H. Suzuki, M. Shibata, E. L. Saenko, I. Tanaka, et al. Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C-mediated proteolysis Blood, February 1, 2001; 97(3): 669 - 677. [Abstract] [Full Text] [PDF]
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M. Jacquemin, R. Lavend'homme, A. Benhida, B. Vanzieleghem, R. d'Oiron, J.-M. Lavergne, H. H. Brackmann, R. Schwaab, T. VandenDriessche, M. K. L. Chuah, et al. A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor Blood, August 1, 2000; 96(3): 958 - 965. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
> A and C
) or of normal plasma (
)
were incubated for 2 hours at 37°C. LE2E9 concentrations before
mixing with plasma were as indicated. The residual fVIII activity was
measured in a chromogenic assay.
, Pro2153Gln
plasma; 
, Pro2153Gln plasma supplemented with rfVIII; 
fragments in the Fab fragment preparation was further
excluded by ELISA. Thus, fVIII-coated plates were incubated with native
or Fab LE2E9. Binding of both Fab and native LE2E9 was detected by
addition of peroxidase-labeled anti-
Cys is related to antibody formation in a patient with mild hemophilia A.
Blood.
1997;89:4371-4377