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PubMed1983

Gel filtration patterns of factor VIII coagulant antigen and factor VIII related antigen in normal and von Willebrand's disease.

Gel filtration (sepharose 2B CL) patterns of factor VIII coagulant antigen (VIIIC:Ag) and factor VIII related antigen (VIIIR:Ag) were obtained using normal plasma and plasma from patients with von Willebrand's disease. The latter group consisted of five individuals with normal mobility of factor VIIIR:Ag on cross-immunoelectrophoresis (Type I) and five others with abnormal (increased) mobility (Type II). Results showed that the elution of VIIIC:Ag was delayed in those subjects whose ratio of VIIIR:Ag to VIIIC:Ag was reduced. It has previously been reported that VIIIR:Ag exerts a stabilizing influence on the coagulant activity of factor VIII (VIII:C); our data suggests that when VIIIR:Ag is deficient, abnormal (low molecular weight) forms of VIIIC:Ag circulate.

Antigens↗
PubMed1981

The effect of naturally occurring antibodies to factor VIII on an immunoradiometric assay for factor VIII coagulant antigen. Observation on a cross-reacting material-positive (CRM+) hemophiliac with a factor VIII inhibit.

An IRMA for VIII:CAg was performed on plasma samples from 13 hemophilic and four nonhemophilic subjects, containing naturally occurring antibodies against factor VIII. The VIII:C binding ligand was an iodinated Fab fraction of a high-titer factor VIII antibody arising in a hemophiliac. Since the IRMA is an equilibrium binding assay, the inclusion of an antibody against VIII:C in the test system would be expected to compete with the ligand for the available antigenic sites on the VII:C molecule: the higher the titer of the antibody expressed in BIU, the greater the degree of inhibition of the IRMA measuring VIII:CAg. In a mixture of equal parts of normal and inhibitor plasmas, the amount of residual VIII:C remaining after 2 hr incubation at 37 degrees C differed from the residual VIII:CAg in half (three of six) of the high-titer inhibitors (> 30 BIU) so studied. In the inhibitors of lower titer (< 20 BIU), correlation between BIU and the percentage of VIII:CAg neutralized was not observed. Three of these inhibitor plasmas did not significantly compete with the ligand. With this competitive protein-binding technique the four plasmas with spontaneous inhibitors could not be distinguished from those arising in hemophiliacs. In one hemophilic plasma the titer of VIII:C inhibitor over a 6-day span increased from 4 to 500 BIU as the VIII:CAg level dropped from 84% to 2%. The finding of circulating VIII:CAg in the presence of an VIII:C inhibitor further substantiates the heterogeneity of the naturally occurring VIII:C antibody and is evidence that factor VIII inhibitors can occur in CRM+ hemophilia.

Autoantibodies↗
PubMed1990

Characterization of recombinant factor VIII and a recombinant factor VIII deletion mutant using a rabbit immunogenicity model system.

The use of factor VIII prepared genetically engineered cell lines (rFVIII) may avoid some of the problems inherently associated with administering plasma-derived factor VIII (pdFVIII) concentrates to hemophilia A patients. Although rFVIII may represent an improvement over traditional therapeutics, the chance exists that protein production in cell culture may result in the presence of novel epitopes that may enhance the formation of inhibitor antibodies capable of neutralizing either rFVIII or pdFVIII. To assess the differences between rFVIII and its plasma-derived homologue, a rabbit immunogenicity model system was developed. Antibodies raised to rFVIII in rabbits were tested for the presence of antibodies capable of binding rFVIII but not pdFVIII, the presence of which would suggest that novel epitope(s) were present. This analysis was performed using a competitive enzyme-linked immunosorbent assay, as well as immunoadsorption. For either technique, rFVIII-specific antibodies were not detected, indicating that differences between rFVIII and pdFVIII were not found. When a rFVIII B-region deletion mutant was similarly tested, antibodies specific for this protein were found. These specific antibodies appeared to bind in the vicinity of the deletion site and their binding was not affected by carbohydrate removal. These results indicate that the rabbit immunogenicity model system is sensitive to alterations in the factor VIII molecule and suggest that full-length rFVIII will not be any more immunogenic in human patients than pdFVIII.

Animals↗
PubMed1989

Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid.

Most antibodies to factor VIII have recently been shown to react with discrete regions of the factor VIII light chain (within the C2 domain) and/or the factor VIII heavy chain (within the amino-terminal segment of the A2 domain). The mechanism by which these antibodies, usually designated "factor VIII inhibitors," interfere with factor VIII function has been examined by determining their effect on factor VIII binding to a phospholipid. Factor VIII-phosphatidylserine binding was prevented by all seven factor VIII inhibitors that had strong factor VIII light chain reactivity and reduced by two inhibitors with weak anti-light chain reactivity. None of four inhibitors with heavy chain reactivity prevented factor VIII-phosphatidylserine interaction, though a partial reduction (less than 50%) was noted for the intact IgG preparations. However, when Fab' fragments were substituted, no detectable reduction in factor VIII-phosphatidylserine binding was noted for the anti-heavy chain inhibitors and complete inhibition was retained by the anti-light chain inhibitors. These data suggest that a subset of factor VIII inhibitors, those that bind to light chain determinants, inactivate factor VIII by preventing its effective interaction with phospholipid.

Antigens↗
PubMed1981

The clinical relevance of factor VIII: C and factor VII R: Ag determination in newborns.

Higher levels of factor VIII: C and factor VIII R: Ag were found in healthy newborns (n = 60) as compared to adults. This could be explained as a stress reaction due to birth and the adaptation to extrauterine life. A further stress factor is disease. The highest values for factor VIII R: Ag were found in ill (n = 32) and in severely ill newborns (n = 21). The large ranges of factor VIII: C and of the ratio of factor VIII: C/VIII R: Ag in healthy newborns can be explained by an increased turnover of coagulation factors. Diseases in the newborn period lead to an increase of this process, resulting in even larger ranges of factor VIII: C and of the ratio of factor VIII: C/VIII R: Ag in ill and extremely ill newborns. Consumption of factor VIII: C with a low ratio of factor VIII: C/VIII R: Ag predominates in extremely ill newborns. The ratio of factor VIII: C/VIII R: Ag is more valuable than factor VIII: C for diagnosis of DIC in newborns. A diagnosis of hemophilia and von Willebrand's disease cannot be established with certainty in severely ill newborns. Stress and DIC may influence the characteristic changes of laboratory parameters.

Antigens↗
PubMed2000

Efficacy and safety of recombinant factor VIIa in the prophylaxis of bleeding in various surgical procedures in hemophilic patients with factor VIII and factor IX inhibitors.

Patients with hemophilia may develop alloantibodies (inhibitors) directed toward the substitution factor. In the long term, this complication significantly affects around 15% of hemophilia A patients and 2-5% of hemophilia B patients suffering the severe class of disease (critical functional factor level <0.01 IU/mL). The typical consequence of a newly developed inhibitor is that the patient can no longer take advantage of the principles of modern hemophilia management, including safe and early treatment of bleeding and prophylactic use of concentrate in prevention of bleeding. Moreover, problems that are normally solved by means of a surgical procedure covered by the usual concentrate will be regarded as difficult and are likely to be associated with risk of untoward bleeding if high-titer inhibitors are present. Over the past decade, considerable experience has been collected illustrating that surgery may be performed quite safely in the patient with inhibitors if hemostasis is assisted by a new hemostatic agent, an activated recombinant factor VII molecule (rFVIIa). Although only one controlled randomized surgical study has been presented, cumulated data from this and from other, less formal studies on the use of rFVIIa in hemophilic inhibitor surgery have illustrated that a vast variety of different surgical procedures can be accomplished with none of the life-threatening bleeding complications that would be anticipated if no hemostatic treatment was administered. The aim of the present review is to present a multiplicity of surgical procedures that have been carried out during the clinical development of rFVIIa. Further, an update of 21 surgical procedures in inhibitor patients from the center of the author will be reviewed and discussed.

Autoantibodies↗
PubMed1997

Analysis of factor VIII inhibitors using hybrid human/porcine factor VIII.

Recombinant hybrid human/porcine factor VIII molecules have been used to map a major determinant of the epitope recognized by human anti-factor VIII A2 domain inhibitory antibodies to a region bounded by human fVIII residues Arg484-Ile508. This approach is being used to characterize the C2 domain inhibitor epitope. The process of creating hybrid human/porcine factor VIII molecules to map inhibitor epitopes produces procoagulantly active fVIII with reduced reactivity with clinically significant factor VIII inhibitors. This suggests that it may be possible to develop of a hybrid human/porcine factor VIII that is useful in the management of hemophilia A and acquired hemophilia.

Amino Acid Sequence↗
PubMed1986

Isolation and characterization of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma.

Human factor VIII has been isolated from a high purity factor VIII concentrate by immunoaffinity chromatography and HPLC on Mono Q gel. Two fractions of factor VIII were obtained with a specific activity of approximately equal to 7000 units/mg. The major fraction contained eight peptide chains of 200, 180, 160, 150, 135, 130, 115, and 105 kDa plus one doublet chain of 80 kDa. The minor fraction contained one peptide chain of 90 kDa plus the chain of 80 kDa. Both fractions were activated by thrombin to the same extent. Amino-terminal amino acid sequence analysis was performed on the 180-kDa, 130-kDa, and 90-kDa chains and showed an identical amino-terminal sequence in these chains. Each chain from 200 kDa to 90 kDa was linked to one 80-kDa chain by a metal-ion bridge(s). Studies on factor VIII in plasma and cryoprecipitate, prepared and gel filtered in the presence of protease inhibitors, showed that one 200-kDa plus one 80-kDa chain were the only or dominating chains in the materials and may represent native factor VIII. The results indicated that all chains from 180 kDa to 90 kDa are fragments of the 200-kDa chain. All of these more or less fragmented chains form active factor VIII complexes with the 80-kDa chain.

Amino Acid Sequence↗
PubMedDate not supplied

A murine monoclonal anti-factor VIII inhibitory antibody and two human factor VIII inhibitors bind to different areas within a twenty amino acid segment of the acidic region of factor VIII heavy chain.

The factor VIII (FVIII) binding regions of the monoclonal anti-FVIII inhibitory antibody C5 and a human FVIII inhibitor antibody have previously been reported to be contained within amino acid residues 351-365 of FVIII. Localization of the binding regions of these two antibodies was based on their reactivity with four synthetic FVIII peptides. Nineteen synthetic FVIII peptides spanning the entire acidic region of the FVIII heavy chain have now been evaluated for the ability to inhibit the binding of C5 to FVIII in an ELISA assay. The smallest peptide tested that inhibited C5 binding to FVIII consisted of residues 351-361. Those peptides that were able to inhibit C5 binding in the ELISA assay were also able to neutralize the FVIII inhibitory activity of C5 in plasma. The FVIII inhibitory activity of two human FVIII inhibitor antibodies was also partially neutralized by peptides from this region. Evaluation of the pattern of peptides reactive with the three antibodies indicates that the binding regions of these antibodies are in very close proximity to each other, but are not identical. Their respective binding regions are contained within residues 351-361 (C5), 354-362 (inhibitor 1), and 342-354 (inhibitor 2). These results suggest that this 20 amino acid segment of the acidic region of the heavy chain of FVIII may be functionally important in the expression of FVIII procoagulant activity.

Amino Acid Sequence↗
PubMed2002

Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism.

Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 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 one-stage 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.

Antibody Specificity↗