scholarly journals Inhibitors in German Hemophilia A Patients Treated with a Double Virus Inactivated Factor VIII Concentrate Bind to the C2 Domain of FVIII Light Chain

1999 ◽  
Vol 81 (01) ◽  
pp. 39-44 ◽  
Author(s):  
R. Laub ◽  
Di Giambattista ◽  
P. Fondu ◽  
H.-H. Brackmann ◽  
H. Lenk ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hepatitis A ◽  
Hemophilia A ◽  
Hepatitis A Virus ◽  
C2 Domain ◽  
Epitope Specificity ◽  
Previously Treated ◽  
Inhibitor Titer

SummaryTo reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

scholarly journals The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3663-3671 ◽  
Author(s):  
Richard Prescott ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Inge Scharrer ◽  
Inga Marie Nilsson ◽  
...  
Keyword(s):  
Immune Response ◽  
Antibody Response ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Epitope Specificity ◽  
Extensive Analysis ◽  
Number Of Patients ◽  
Recombinant Fviii ◽  
Inhibitor Titer

Abstract Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

scholarly journals The Inhibitor Antibody Response Is More Complex in Hemophilia A Patients Than in Most Nonhemophiliacs With Factor VIII Autoantibodies

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3663-3671 ◽  
Author(s):  
Richard Prescott ◽  
Hiroaki Nakai ◽  
Evgueni L. Saenko ◽  
Inge Scharrer ◽  
Inga Marie Nilsson ◽  
...  
Keyword(s):  
Immune Response ◽  
Antibody Response ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Epitope Specificity ◽  
Extensive Analysis ◽  
Number Of Patients ◽  
Recombinant Fviii ◽  
Inhibitor Titer

Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as ≥ 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

Dominant C2 Domain Epitope Specificity of Inhibitor Antibodies Elicited by a Heat Pasteurized Product, Factor VIII CPS-P, in Previously Treated Hemophilia A Patients without Inhibitors

1998 ◽  
Vol 79 (01) ◽  
pp. 62-68 ◽  
Author(s):  
Yoshikatsu Sawamoto ◽  
Richard Prescott ◽  
Degang Zhong ◽  
Evgueni Saenko ◽  
Eveline Mauser-Bunschoten ◽  
...  
Keyword(s):  
Factor Viii ◽  
Untreated Patient ◽  
Hemophilia A ◽  
C2 Domain ◽  
Epitope Specificity ◽  
Previously Treated ◽  
Heat Pasteurization ◽  
Recombinant Fviii ◽  
Product Factor ◽  
A2 Domain

SummaryFrom June, 1990, to November, 1991, in The Netherlands and Belgium, 16 previously treated severe hemophilia A patients (PTP) developed inhibitors after exposure to factor VIII CPS-P, a new heat pasteurized product. A previously untreated patient (PUP) also developed an inhibitor to CPS-P. In inhibitor neutralization assays with recombinant fVIII C2 and A2 domain polypeptides, plasmas from 14 PTPs were ≥79% neutralized by C2 and <10% by A2, but the PUP plasma was partially neutralized by C2 (48%) and A2 (28%). Immunoprecipitation assays of the PTP and PUP plasmas with the fVIII heavy chain and with recombinant C2 and A3-C1 polypeptides confirmed that the C2 dominant immune response to CPS-P was found only in the PTPs. Competition of the binding of 2 inhibitors to 125I-CPS-P by unlabeled CPS-P and another plasma fVIII was similar, demonstrating that the antibody response was not directed to epitopes only present in CPS-P. We propose that the immunogenicity of the CPS-P C2 domain was altered by heat pasteurization.

IMMUNOASSAY FOR FACTOR VIII-HEAVY CHAIN. AN INDICATOR FOR IMMUNE COMPLEXES DURING HIGH DOSE FVIII INHIBITOR TREATMENT

1987 ◽  
Author(s):  
O Nordfang ◽  
M Ezban ◽  
J B Knudsen
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Immune Complexes ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Test Sample ◽  
High Dose ◽  
Fviii Inhibitor ◽  
Normal Human

Specificity studies have shown that most hemophilia A inhibitor antibodies are directed towards the light chain of coagulation factor VIII (FVIII). Thus, conventional immunoassays for FVIII-antigen (FVIII:Ag) presumably have reactivity for FVIII-Light Chain (FVIII-LC) . Our sandwich FVIII :Ag assay has been shewn to be specific for only FVIII-LC. We have now developed a specific immunoassay for FVIII-Heavy Chain (FVIII-HC) . This has made it possible to investigate the FVIII-HC content in hemophilia A plasma, and to study the expression of FVIII-HC in culture medium frcm transfected cell lines.By adding purified FVIII-LC and FVIII-HC in coagulation inhibition assay, plasma frcm one of seven hemophilia A inhibitor patients was found to be reactive with both FVIII-LC and FVIII-HC. IgG frcm this plasma was used for a FVIII-HC specific inhibition radioimmunoassay. The polyspecific antibodies were coated to microplates with removable wells. The coated wells were incubated with test sample and with purified 125I-FVIII-HC. When normal human plasma pool contains 1 U/ml of FVIII-HC, the sensitivity of the assay was 0.004 U/ml.For normal plasma and plasma frcm non inhibitor hemophilia A patients, FVIII-HC measurements correlated with FVIII:C and FVIII-LC measurements. However, after FVIII injection hemophilia A inhibitor patients in high dose FVIII treatment showed a much higher FVIII-HC content (1-5 U/ml) than FVIII-LC and FVIII:C (< 0.05 U/ml). These patients have previously been shown to have antibodies towards FVIII-LC. Therefore the antigen measurements indicate that inhibitor patients in high dose FVIII treatment have FVIII/anti-FVIII-LC immune complexes. These circulating immune complexes may be the mediator of an antibody dependent immune tolerance, during the high dose FVIII treatment.

Prevalence and epitope specificity of non-neutralising antibodies in a large cohort of haemophilia A patients without inhibitors

2011 ◽  
Vol 105 (06) ◽  
pp. 954-961 ◽  
Author(s):  
Hervé Chambost ◽  
Christine Biron-Andreani ◽  
Pierre-Emmanuel Morange ◽  
Christophe Combescure ◽  
Alain Marques-Verdier ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Haemophilia A ◽  
Neutralising Antibodies ◽  
Epitope Specificity ◽  
Iatrogenic Complication ◽  
Severity Of The Disease ◽  
Recombinant Fviii

SummaryAntibodies (inhibitors and non-neutralising antibodies [NNA]) directed against factor VIII (FVIII) remain the main iatrogenic complication in haemophilia A (HA) patients. Inhibitors reduce FVIII procoagulant properties, whereas NNA are directed against non-functional epitopes. NNA are poorly studied and their prevalence, epitope specificity and physiopathology inadequately defined. The aim of this study was first to evaluate NNA prevalence in a French retrospective multicentric series of 210 patients without inhibitors, then to determine their epitope specificity (against the heavy chain [HC] or the light chain [LC] of FVIII) and particularly to assess the prevalence of anti-B domain NNA using specifically designed x-MAP assays. NNA occurred in 18.1% of patients (38/210) and their prevalence was not influenced by the severity of the disease. Among the 38 patients with NNA, 73.7% had anti-FVIII Abs against the HC, 13.2% against the LC and 13.2% had anti-FVIII Abs against both chains. There is thus a clear immuno-dominance of the HC of FVIII in the epitope profile of NNA, whatever the severity of HA. The proportion of NNA that recognised the B domain was 18.4% (n=7/38). A multivariate analysis did not highlight differences in NNA occurrence between patients treated with recombinant FVIII or with plasma-derived FVIII (19.6% vs. 14.9%, p=0.53).

Epitope Specificity of Factor VIII Inhibitor Antibodies

1998 ◽  
Vol 18 (03) ◽  
pp. 121-128 ◽  
Author(s):  
Dorothea Scandella
Keyword(s):  
Factor Viii ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Inhibitor Activity ◽  
Epitope Specificity ◽  
Factor Viii Concentrates ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
Viii Inhibitor ◽  
Acidic Region

SummaryAntibodies that inactivate factor VIII develop most frequently in patients with severe hemophilia A, and they present a serious complication in the therapy of such individuals. Most patients with inhibitors have two or more different antibodies capable of factor VIII inactivation in their plasma, as shown by assays in which several factor VIII domain fragments are tested for neutralization of the inhibitor activity. Such neutralization assays of 23 patient plasmas suggest that there are three common epitopes. These epitopes have been localized by several methods to the A2, A3, and C2 factor VIII domains. Less common inhibitor epitopes lie within the heavy chain acidic region residues 336-372 and in a second region of the C2 domain. The light chain acidic region, residues 1649-1689, may also contain an inhibitor epitope, but this remains to be confirmed. The inhibitor epitopes of autoantibody patients are more restricted as half of them have anti-C2 domain antibodies that make up 95% of the inhibitor activity. The remainder have several inhibitors similar to those of the hemophiliacs. The predominant C2 domain epitope specificity was also seen in rare cases of inhibitor development due to two heat pasteurized factor VIII concentrates in previously treated patients without inhibitors. Inhibitors in mild hemophiliacs are rare as these individuals are usually tolerant to factor VIII. When tolerance is overcome in such patients, the immune responses characterized were diverse. In some patients there was a complete loss of tolerance.

“In vitro” and in Animal Model Studies on a Double Virus- Inactivated Factor VIII Concentrate

1995 ◽  
Vol 74 (03) ◽  
pp. 868-873 ◽  
Author(s):  
Silvana Arrighi ◽  
Roberta Rossi ◽  
Maria Giuseppina Borri ◽  
Vladimir Lesnikov ◽  
Marina Lesnikov ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Animal Model ◽  
Factor Viii ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Inactivation ◽  
Enveloped Viruses ◽  
Model Studies ◽  
Heat Treated

SummaryTo improve the safety of plasma derived factor VIII (FVIII) concentrate, we introduced a final super heat treatment (100° C for 30 min) as additional virus inactivation step applied to a lyophilized, highly purified FVIII concentrate (100 IU/mg of proteins) already virus inactivated using the solvent/detergent (SID) method during the manufacturing process.The efficiency of the super heat treatment was demonstrated in inactivating two non-lipid enveloped viruses (Hepatitis A virus and Poliovirus 1). The loss of FVIII procoagulant activity during the super heat treatment was of about 15%, estimated both by clotting and chromogenic assays. No substantial changes were observed in physical, biochemical and immunological characteristics of the heat treated FVIII concentrate in comparison with those of the FVIII before heat treatment.

A Multicenter Pharmacosurveillance Study for the Evaluation of the Efficacy and Safety of Recombinant Factor VIII in the Treatment of Patients with Hemophilia A

1997 ◽  
Vol 78 (05) ◽  
pp. 1352-1356 ◽  
Author(s):  
Emel Aygören-Pürsün ◽  
Inge Scharrer ◽  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Parvovirus B19 ◽  
Subjective Evaluation ◽  
Recombinant Factor Viii ◽  
Fviii Inhibitor ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
Bleeding Episodes ◽  
Recombinant Fviii

SummaryIn this open multicenter study the safety and efficacy of recombinant factor VIII (rFVIII) was assessed in 39 previously treated patients with hemophilia A (factor VIII basal activity ≤15%).Recombinant FVIII was administered for prophylaxis and treatment of bleeding episodes and for surgical procedures. A total of 3679 infusions of rFVIII were given. Efficacy of rFVIII as assessed by subjective evaluation of response to infusion and mean annual consumption of rFVIII was comparable to that of plasma derived FVIII concentrates. The incremental recovery of FVIII (2.4 ± 0,83%/IU/kg, 2.12 ± 0.61%/IU/kg, resp.) was within the expected range. No clinical significant FVIII inhibitor was detected in this trial. Five of 16 susceptible patients showed a seroconversion for parvovirus B19. However, the results are ambiguous in two cases and might be explained otherwise in one further case. Thus, in two patients a reliable seroconversion for parvovirus B19 was observed.

Acidic Region Residues 1680–1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

2021 ◽  
Author(s):  
Yuto Nakajima ◽  
Hiroaki Minami ◽  
Keiji Nogami
Keyword(s):  
Surface Plasmon Resonance ◽  
Factor Viii ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
C2 Domain ◽  
Wild Type ◽  
Cross Link ◽  
Domain Specific ◽  
Cofactor Activity ◽  
Acidic Region

AbstractFactor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr1664 and residues 1675–1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.

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