A human factor VIII inhibitor tolerating the endogenous FVIII with a unique A2 domain substitution in CRM + hemophilia A

2003 ◽  
Vol 1 ◽  
pp. P0648-P0648
Author(s):  
D. Habart ◽  
M. Novotny ◽  
V. Komrska ◽  
M. Dobrovolna ◽  
Z. Vorlova
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viii Inhibitor ◽  
Human Factor Viii ◽  
Viii Inhibitor ◽  
A2 Domain

ANALYSIS OF HUMAN FACTOR VIII INHIBITOR EPITOPES TO FACTOR VIII POLYPEPTIDES BY IMMUNOBLOTTING

1987 ◽  
Author(s):  
A Yoshioka ◽  
M Shima ◽  
I Tanaka ◽  
T Fujiwara ◽  
H Nakai ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Factor Viii ◽  
Column Chromatography ◽  
Human Factor ◽  
Hemophilia A ◽  
Blue Staining ◽  
Factor Viii Inhibitor ◽  
Human Factor Viii ◽  
Viii Inhibitor ◽  
Group 3

In order to clarify human factor VIII inhibitor epitopes to factor VIII (F. VIII), we analyzed the inhibitor IgG developed in the patients with hemophilia A and autoimmune disease using immunoblotting of purified F. VIII and thrombin-degraded F. VIII. IgG fractions were obtained from 6 cases of severe hemophilia A and one autoimmune disease. The titer of the inhibitor plasma ranged from 50 to 3,000 Bethesda units/ml. Purification of F. VIII from commercial F. VIII concentrate was performed by immunoadsorbent column chromatography using anti-von Willebrand factor monoclonal antibody and anti-fibrinogen, anti-fibronectin and anti-IgM goat antibodies and subsequently by Aminohexyl Sepharose column chromatography essentially according to the method of Fulcher et al.. The specific activity of the purified F. VIII was 2,700 units/mg. On sodium-dodecylsulfate (SDS) 5 to 10% gradient polyacrylamide gel electrophoresis (PAGE), 80 kDa of a main fragment and a series of 90 to 210 kDa fragments were visualyzed by Coomassie blue staining. Immunoblotting of 5-10 ug of each of unreduced purified F. VIII and thrombin-degraded F. VIII. followed by reaction with inhibitor IgG samples, monoclonal antihuman IgG-3 and IgG-4 antibodies and radiolabeled rabbit antimouse IgG.All inhibitor IgG samples reacted with both purified F. VIII and thrombin-degraded F. VIII. The pattern of reactivity of inhibitor antibodies was divided into three groups; 1) inhibitor IgG which reacted with 80 and 70 kDa derived from carboxy-terminus of the F. VIII molecule, 2) inhibitor IgG which reacted with 90 to 210 kDa and 54 and/or 44 kDa derived from amino-terminus of the F. VIII, and 3) inhibitor IgG which reacted with both of N- and C-terminus of the F. VIII. One inhibitor IgG belonged to group 3 strongly reacted with a series of higher molecular polypeptides of F. VIII ranged from 180 .to 210 kDa. One autoantibody IgG from the patient with autoimmune disease was also belonged to group 3. There was no relationship between the titer and the pattern of reactivity of each inhibitor plasma. In summary, there is a heterogeneity of F. VIII inhibitor epitopes to F. VIII molecules.

Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1618-1626 ◽  
Author(s):  
D Scandella ◽  
M Mattingly ◽  
S de Graaf ◽  
CA Fulcher
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Factor Viii Inhibitor ◽  
Type I ◽  
Protein Fragments ◽  
Human Factor Viii ◽  
Circulating Antibodies ◽  
A1 Domain ◽  
A2 Domain

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

scholarly journals The diversity of the immune response to the A2 domain of human factor VIII

Blood ◽  
2013 ◽  
Vol 121 (14) ◽  
pp. 2785-2795 ◽  
Author(s):  
Rebecca C. Markovitz ◽  
John F. Healey ◽  
Ernest T. Parker ◽  
Shannon L. Meeks ◽  
Pete Lollar
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Entire Surface ◽  
Human Factor Viii ◽  
Key Points ◽  
A2 Domain

Key Points The Abs to the human fVIII A2 domain in a murine hemophilia A model inhibit fVIIIa and activation of fVIII Epitopes targeted by hemophilia A mouse Abs cover nearly the entire surface of the human fVIII A2 domain

Residues Glu2181-Val2243 Contain a Major Determinant of the Inhibitory Epitope in the C2 Domain of Human Factor VIII

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3701-3709 ◽  
Author(s):  
John F. Healey ◽  
Rachel T. Barrow ◽  
Hiba M. Tamim ◽  
Ira M. Lubin ◽  
Midori Shima ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Coagulation Factor ◽  
Major Determinant ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Differential Reactivity ◽  
Human Factor Viii ◽  
Viii Inhibitor ◽  
Von Willebrand

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.

scholarly journals Immunoblot cross-reactivity of factor VIII inhibitors with porcine factor VIII

Blood ◽  
1995 ◽  
Vol 86 (6) ◽  
pp. 2183-2190 ◽  
Author(s):  
K Koshihara ◽  
J Qian ◽  
P Lollar ◽  
LW Hoyer
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Cross Reactivity ◽  
The Other ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factor Viii Inhibitor ◽  
Human Factor Viii ◽  
Viii Inhibitor ◽  
A1 Domain ◽  
Inhibitor Titer

Porcine factor VIII has been used successfully to treat factor VIII inhibitor patients whose plasmas have minimal cross-reactivity to porcine factor VIII. However, some inhibitor plasmas do inhibit porcine factor VIII, and the extent of procoagulant inhibition often increases after treatment with porcine factor VIII. Because there is no information about the porcine factor VIII epitopes with which these antibodies react, we have compared the immunoblot and enzyme-linked immunosorbent assay (ELISA) reactivities with porcine and human factor VIII for 20 inhibitor plasmas (11 from hemophilia A patients and 9 autoantibodies). Immunoblots identified binding to porcine factor VIII for only 2 of the 12 plasmas from patients who had not received porcine factor VIII, but this reactivity could not be predicted from the inhibitor titer to porcine factor VIII. Immunoblot reactivity with porcine factor VIII was detected for 7 of 8 inhibitor plasmas from patients who had been previously treated with porcine factor VIII, and the strength of this reactivity was generally related to the inhibitor titer. Of the 5 plasmas that were immunoblot positive with the porcine factor VIII A2 domain, 4 had inhibitor titers greater than 45 Bethesda units when tested with porcine factor VIII, whereas only 1 of 15 of the other plasmas had this level of inhibitor activity with porcine factor VIII. In contrast, immunoblot reactivity to the porcine factor VIII A1 domain did not correlate with the antiporcine VIII inhibitor titer. We also determined the effect of preincubation with human or porcine factor VIII on immunoblot reactivity. In one case, immunoblot reactivity with porcine factor VIII was absorbed with porcine, but not human, factor VIII, which is consistent with antibody formation after treatment with porcine factor VIII. In no cases did human factor VIII reduce the reactivity of inhibitor plasmas with the porcine A1 domain, suggesting that these antibodies are directed at unique porcine factor VIII determinants. The reactivity to porcine A2 in 2 plasmas probably represented cross-reactivity of similar A2 determinants, because it was absorbed by both human and porcine factor VIII. Although the ELISA assays with porcine factor VIII detected antibodies in some plasmas that could not be identified by inhibitor assay or immunoblot, the level of ELISA reactivity was generally consistent with the titers of the other assays.

scholarly journals Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1618-1626 ◽  
Author(s):  
D Scandella ◽  
M Mattingly ◽  
S de Graaf ◽  
CA Fulcher
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Factor Viii Inhibitor ◽  
Type I ◽  
Protein Fragments ◽  
Human Factor Viii ◽  
Circulating Antibodies ◽  
A1 Domain ◽  
A2 Domain

Abstract Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

Residues Glu2181-Val2243 Contain a Major Determinant of the Inhibitory Epitope in the C2 Domain of Human Factor VIII

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3701-3709 ◽  
Author(s):  
John F. Healey ◽  
Rachel T. Barrow ◽  
Hiba M. Tamim ◽  
Ira M. Lubin ◽  
Midori Shima ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Coagulation Factor ◽  
Major Determinant ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Differential Reactivity ◽  
Human Factor Viii ◽  
Viii Inhibitor ◽  
Von Willebrand

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.

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