scholarly journals Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1811-1819 ◽  
Author(s):  
D Scandella ◽  
GE Gilbert ◽  
M Shima ◽  
H Nakai ◽  
C Eagleson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Intrinsic Factor ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Amino Acid Residues ◽  
Fviii Inhibitor ◽  
Human Factor Viii

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.

Human Factor VIII Inhibitor Alloantibodies with a C2 Epitope Inhibit Factor Xa-catalyzed Factor VIII Activation: A new Anti-factor VIII Inhibitory Mechanism

2002 ◽  
Vol 87 (03) ◽  
pp. 459-465 ◽  
Author(s):  
Keiji Nogami ◽  
Katsumi Nishiya ◽  
Yoshihiko Sakurai ◽  
Ichiro Tanaka ◽  
John Giddings ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Factor Viii Inhibitor ◽  
Inhibitory Mechanism ◽  
Fviii Inhibitor ◽  
Fviii Activity ◽  
Human Factor Viii ◽  
Sds Page ◽  
Viii Inhibitor ◽  
C1 Domains

SummaryFactor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-C1 domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in onestage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)’2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.

Non-Classical Anti-Factor VIII C2 Domain Inhibitors Are Present in the Plasmas of Most Factor VIII Inhibitor Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 786-786
Author(s):  
Shannon L. Meeks ◽  
John F. Healey ◽  
Rachel T. Barrow ◽  
Ernest T. Parker ◽  
Pete Lollar
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Proteolytic Activation ◽  
Autoimmune Antibodies ◽  
Fviii Inhibitor ◽  
Fviii Activity ◽  
Viii Inhibitor ◽  
Murine Mabs

Abstract Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. These inhibitors primarily are directed against the A2 or C2 domains of fVIII. The human response to the C2 domain of fVIII classically has been thought to inhibit fVIII activity by blocking its binding to phospholipid. We recently characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of antibodies. Groups A, AB, and B are classical anti-C2 antibodies. Groups BC and C consist of non-classical anti-C2 antibodies that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Most non-classical antibodies have inhibitor titers greater than 10,000 Bethesda units/mg IgG. To determine if non-classical antibodies are present in fVIII inhibitor patients, patient plasmas were tested in an ELISA for their ability to block the binding of representative antibodies from the different anti-human fVIII C2 antibody groups. Classical and non-classical monoclonal antibodies (MAbs) were biotinylated and serially diluted into either fVIII deficient plasma or patient inhibitor plasma and then added to microtiter wells coated with fVIII. The ability of patient plasma to block the binding of the murine MAbs to fVIII was determined. A total of 16 patient plasmas were assessed: 4 from patients with a C2 predominant response, 2 with a non-C2 predominant response, and 10 with unknown specificities. Three of the 4 patients with C2 predominant responses had non-classical anti-C2 antibodies, while the 2 with non-C2 predominant responses did not. In the unknown plasmas, 6 of 10 had evidence of non-classical antibodies. Figure 1 shows representative results of the effect of 3 patient plasmas on the binding of a biotinylated non-classical MAb to fVIII. Patient plasmas 1 and 2 blocked MAb binding while patient plasma 3 did not. This study indicates that the majority of patients with fVIII inhibitors have non-classical anti-C2 antibodies in their response to fVIII. Figure Figure

The preparation and phospholipid binding property of the C2 domain of human factor VIII

2003 ◽  
Vol 89 (05) ◽  
pp. 788-794 ◽  
Author(s):  
Kazuya Takeshima ◽  
Christina Smith ◽  
Jonathan Tait ◽  
Kazuo Fujikawa
Keyword(s):  
Factor Viii ◽  
Binding Affinity ◽  
Light Chain ◽  
Human Factor ◽  
Binding Activity ◽  
Exchange Chromatography ◽  
Binding Property ◽  
C2 Domain ◽  
Phospholipid Binding ◽  
Human Factor Viii

SummaryThe C2 domain of human factor VIII was expressed in a yeast secretion system and its binding properties were studied. A cDNA coding the C2 domain sequence of human factor VIII with a N-terminal six amino acids extension (C-C2) was constructed, transformed into Pichia pastoris cells and expressed. The product was purified by ammonium sulfate fractionation and anion exchange chromatography. It emerged as a single peak from both ion exchange and gel filtration columns, indicating C-C2 is a homogenous monomer. The binding activity of C-C2 to phosphatidylserine-containing phospholipid vesicles was measured by competitive binding with annexin V. The values of IC50were approximately 70nM for both factor VIII and its light chain, but were about 7000nM for C-C2. These results indicated C-C2 has 100-fold less binding affinity than factor VIII or the light chain. Direct binding to solidified phosphatidylserine-containing phospholipids also showed that C-C2 has ~50-fold less binding affinity than does the light chain. C-C2 poorly inhibited Xase activity. These results together clearly show that the C2 domain alone does not have full membrane binding activity, and suggest that the other light chain domains, A3 and/or C1, are also involved in the phospholipid binding activity of factor VIII.

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.

Reduced Affinity To VWF Induced By a Novel Mutation At Pro1809Leu (factor VIII-Tenri) Is The Cause Of Mild Hemophilia A Developing Anti-Factor VIII Inhibitor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2343-2343
Author(s):  
Koji Yada ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Hiroaki Minami ◽  
Philip J. Fay ◽  
...  
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Conformational Change ◽  
Binding Affinity ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Novel Mutation ◽  
Fold Increase ◽  
Factor Viii Inhibitor ◽  
C2 Domain

Abstract The development of inhibitory antibodies to factor (F)VIII in patients with mild hemophilia A is a rare, but significant event. We reported the mild hemophilia A associated with Pro(P)1809Leu(L) mutation in FVIII gene (FVIII-Tenri), developing the type II inhibitor (peak 5.6BU/ml), which inhibited allogeneic but not autologous FVIII recognizing the epitope(s) overlapping with that of anti-FVIII monoclonal antibody (mAb) ESH8 in the C2 domain remote from the mutated site in the A3 domain, at the 54th ASH meeting (#52283). However, the haemostatic characteristics of FVIII-Tenri remain unclear. In this study, to elucidate the characteristics of FVIII-Tenri, we prepared and stably expressed a recombinant, B-domainless FVIII mutant, P1809L. FVIII:C of the mutant reconstituted with FVIII-deficient plasma (0.3 nM) was 31.5 IU/dl, whilst that of the wild-type (WT) (0.3 nM) was 88.6 IU/dl, similar to the level of patient’s plasma. The polyclonal IgG immune-purified from this patient’s plasma decreased FVIII:C of WT by ∼60% at the maximum concentration of IgG, whilst any little affected in the mutant. The mutant was activated by thrombin and FXa, showing ∼16- and 7-fold increase in FVIII:C compared to baseline control, respectively, similar to WT. Furthermore, purified FXa generation assays showed that the mutant was any little different in Km for FIXa compared to WT (0.74 ± 0.07 and 0.72 ± 0.13 nM, respectively), supporting that this mutation little affected the association with FIXa. Interestingly, the mutant showed an ∼3-fold weak binding affinity (Kdapp 0.92 ± 0.23nM) to VWF compared to WT (Kdapp 0.33 ± 0.01nM), whilst any little difference was observed in binding affinity to phospholipid between the mutant and WT, in an ELISA. To further investigate the haemostatic mechanism(s) associated with less binding affinity to VWF, the residual FVIII:C of the mutant or WT (0.05-0.25nM) was measured in a one-stage clotting assay, after incubation with several anti-FVIII mAbs including anti-C2 mAbs ESH8 (residues 2248-2285) and ESH4 (2303-2332) with individual epitope overlapping the VWF-binding site(s) in the C2 domain. ESH8 conferred the ∼60% lower inhibition of FVIII:C in the mutant compared to that in WT, whilst ESH4 showed the similar inhibition of FVIII:C between in the mutant and WT. Anti-A2 mAb JR8 showed any little difference in the inhibition between in the mutant and WT. These results demonstrated that the mutation P1809L in the A3 domain did little affect upon activation by thrombin and FXa as well as association with FIXa and phospholipid, but attenuated the binding to VWF through the conformational change in the C2 domain. Taken together with the development of the allogeneic inhibitor associated with this mutation, these results strongly suggested that the conformational change occurred in the C2 domain remote from the mutated site in the A3 domain hampered the binding to VWF, resulting in the haemostatic impairment due to fragility of the mutant FVIII molecule as well as the change of the immunogenicity in the epitopes overlapping with the FVIII-VWF binding site (residue 2248-2285) presented on allogeneic FVIII. Disclosures: No relevant conflicts of interest to declare.

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.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

A 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

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

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