Suppression of an antibody to factor VIII by a combination of factor VIII and cyclophosphamide.
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Antibodies↗
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Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules.
Factor VIII (fVIII) circulates as a heavy chain/light chain (A1-A2-B/ap-A3-C1-C2) heterodimer. The 41-residue light chain activation peptide, ap, is cleaved from fVIII during proteolytic activation by thrombin or factor Xa. We constructed 7 active recombinant hybrid B-domainless human/porcine fVIII molecules that contained combinations of porcine sequence replacements within the A2, ap-A3, and C2 domains. The cross-reactivity of 23 high-titer inhibitory antibodies between human fVIII and the hybrids was inversely related to the degree of porcine substitution. In all plasmas, the substitution of all 3 regions yielded cross-reactivities that were not significantly different from those of porcine fVIII. To differentiate between inhibitor binding to the ap region and the A3 domain, we constructed 2 additional hybrids that contained porcine A2 and C2 domain substitutions and either porcine A3 or porcine ap substitutions. The porcine ap segment was less antigenic than the human ap segment in several plasmas that had activity against the ap-A3 region. This indicates that some inhibitor plasmas contain antibodies directed against the fVIII ap segment in addition to A2, A3, and C2 domain epitopes identified in previous studies. Substitution of porcine sequences within the A2, A3, C2, and ap regions of human fVIII is necessary and sufficient to achieve a maximal reduction in antigenicity relative to porcine fVIII with respect to most inhibitory antibody plasmas. (Blood. 2000;95:564-568)
Animals↗
Factor VIII and human platelet aggregation. III. Further studies on aggregation of humna platelets by neuraminidase-treated human factor VIII.
When highly purified human factor VIII is submitted to agarose gel chromatography in the presence of 0.5 M CaCl2, the procoagulant activity (low molecular weight factor VIII, LMW-F VIII) is separated from the void volume protein (Vo-VIII). Upon incubation of human factor VIII with purfied neuraminidase, a very stable platelet aggregating activity develops in the "Vo-VIII' fraction, not in the "LMS-FVIII' part. Evidence is provided that the generated aggregating activity is a property of the 'carrier protein' for LMW-F VIII. Desialylated factor VIII retains its antigenic reactivity, its procoagulant or ristocetin cofactor properties and the capacity of its subunits to dissociate and recombine. Neuraminidase-treated human factor VIII, in contrast to intact bovine factor VIII or intact human factor VIII in the presence of restocetin, does not induce aggregation of EDTA-platelet rich plasma, of congenitally afibrinogenaemic platelet rich plasma, nor of washed platelets.
Chromatography, Agarose↗
Experience with highly purified porcine factor VIII in a patient with haemophilia A and a factor VIII inhibitor.
Highly purified porcine factor VIII has been advocated for the treatment of bleeding episodes in patients with haemophilia who have inhibitors to factor VIII. This approach has been successful in a patient with an intermediate potency inhibitor showing little cross-reactivity to porcine material. As in some other reported cases, a severe allergic reaction occurred on one occasion but did not preclude the subsequent use of the porcine concentrate in carefully controlled conditions.
Adult↗
Some human inhibitor antibodies interfere with factor VIII binding to factor IX.
Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.
Antibodies, Blocking↗
[High concentration of coagulation factor VIII as a risk factor for thrombosis].
Several case-control studies have shown that factor VIII levels > or = 150 IU/100 ml lead to a greatly increased risk of venous thrombosis. High levels of factor VIII are also associated with an increased risk of recurrences of thrombosis. The prevalence of high factor VIII levels is 25% in patients with a first episode of venous thrombosis and 11% among healthy controls. The contribution of high factor VIII levels to the onset of venous thrombosis in the population is 16%. The mechanism causing high factor VIII levels is largely unknown and probably consists of both genetic and acquired factors. At present, the routine screening of thrombosis patients for high factor VIII levels is not recommended.
Case-Control Studies↗
The type of factor VIII deficient plasma used influences the performance of the Nijmegen modification of the Bethesda assay for factor VIII inhibitors.
We have investigated the influence of the type of factor VIII deficient plasma used on the assay results of the Nijmegen modification of the Bethesda method for factor VIII inhibitors. Immuno depleted factor VIII deficient plasmas, lacking besides factor VIII also von Willebrand factor, gave decreased inhibitor titres compared to assay results with factor VIII deficient plasmas containing von Willebrand factor suggesting the need of the latter in the test system for the stability of factor VIII:C. Moreover the performance of the assay with immuno depleted plasma was contaminated in a certain type of this plasma by the presence of a factor VIII:C inhibitor. Chemically depleted factor VIII deficient plasma appeared to give falsely elevated titres when used in combination with other types of deficient plasmas as substrate plasma in the factor VIII:C assay due to the presence of activated factor Va in the preparation. Suggestions are described with respect to the observed limitations in order to obtain reliable results.
Blood Coagulation Tests↗
[Management of cesarean section under replacement therapy with factor VIII concentrates in a pregnant case with congenital combined deficiency of factor V and factor VIII].
The congenital combined deficiency of Factor V and Factor VIII, a rare bleeding disorder, was identified in a 25-year-old woman. She was admitted to our hospital with a complaint of genital bleeding. Her prothrombin time and activated partial thromboplastin time were prolonged. She had low levels of Factor V coagulant activity (F. V:C) 14%, and Factor VIII coagulant activity (F. VIII:C), 12%, and normal levels of von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (Rcof) and Protein C antigen. Her Protein C inhibitor level was slightly low. Her Rcof, vWF:Ag and F. VIII:C were elevated following administration of 1-deamino-8-D-arginine-vasopressin (DDAVP), but her F. V:C remained unchanged. Four years later, her F. VIII:C rose to 70% during the course of her pregnancy, but her F. V:C value remained low. It was expected that the vaginal delivery would be possible at the termination of pregnancy. Premature rupture of the membranes and an anomaly of rotation appeared in the course of delivery, however, and cesarean section was accomplished without excess bleeding under replacement therapy with Factor VIII concentrates. These findings suggested that DDAVP and Factor VIII concentrates were useful for management of her delivery. However the mechanisms of the rise of plasma F. VIII:C during pregnancy in a case with congenital combined deficiency of Factor V and Factor VIII are unclear.
Adult↗
Effects of chemical modifiers on recombinant factor VIII activity.
Factor VIII performs a critical role in the blood coagulation cascade but is extremely labile. We have carried out chemical studies on a fully functional recombinant Factor VIII (rFVIII) using a variety of protein modifying agents, some of which were bifunctional. Thiol-specific maleimides cause no activity loss despite a substantial decrease in rFVIII's free thiol content. Mild oxidation procedures had either neutral or adverse effects on activity. Amino-specific reagents led to significant losses of rFVIII procoagulant activity. It appears that free amino groups are essential for Factor VIII's function while some at least of its many free thiol groups are not. None of these derivatives was any less labile than unmodified rFVIII. Systematic chemical modification of important proteins in this way can give useful insights into appropriate protein engineering strategies.
Amines↗
No detectable alterations in immunogenicity following terminal severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX (HP9) concentrates.
We used monoclonal antibody ELISAs, antigen molecular size distribution, competition ELISA and neonatal mouse immune tolerance methods to detect potential neoantigen formation and increased immunogenicity following severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX concentrates. To provide positive controls, concentrates were heated in solution (70 degrees C for 2 h) to produce denaturation on purpose. The competition ELISA applied to factor IX proved particularly useful for quantifying differences between the positive control and the dry-heated/unheated concentrates. None of the test systems employed by us indicated any detectable neoantigen formation or any alteration in immunogenicity following terminal severe dry-heat treatment of the high-purity concentrates, and this finding is supported by clinical experience so far.
Antigens↗
[Determination of the factor VIII ristocetin cofactor (Willebrand factor) using a semi-quantitative slide test. Preliminary results].
The assay of factor VIII, co-factor of Ristocetin (VIIIR:Co) is a relatively delicate procedure which is presently reserved to specialized laboratories. It requires the use of an aggregometer and a long and difficult preparation of human platelets. In parallel with this classical method, we have used a new, rapid, semi-quantitative slide test whose advantages are: simple technique and rapid answer (2 minutes), small volume of plasma required for the test (50 microliters) and the possibility of using citrated or heparinized plasma taken form a venous or capillary blood sample. Using this test, we have assayed factor VIII co-factor of ristocetin: in 31 hospitalized adults patients with no previous history of bleeding and no disturbance of haemostasis and in 18 patients with Willebrand's factor deficiency in comparison with the standard technique using aggregometry (Allain's method); - in 28 normal neonates, in comparison with the VIIIR:Ag factor assay (Laurell's technique), only because of the small sample volume available; - in 17 patients with various disease associated with an abnormality of the VIII complex in comparison with the assay of VIIIR:Ag and VIIIC. The results obtained in the normal adults show a satisfactory correlation between the two methods. The mean level of factor VIII:Co is 100 +/- 10 per cent (M +/- SD) with the semi-quantitative slide test and 109 +/- 20 per cent (M +/- SD) with the method taken as the reference. The correlation is also satisfactory for patients with a deficit of Willebrand's factor. The test performed on the neonates gives a mean value of 101 +/- 37 per cent (M +/- SD) with good correlation between the factor VIIIR:Ag and the factor VIIIR:Co.
Adult↗
Standardization of Factor VIII. I. Calibration of British Standards for Factor VIII clotting activity.
Calibration of successive British Standards for Factor VIII clotting activity against the International Standard (concentrate) has brought to light substantial discrepancies among laboratories and between assay methods. These discrepancies were less in assays of concentrate standards than in calibration of plasma standards. Standardization of reagents in the two-stage assays substantially improved agreement among laboratories using this method. Standardization of the phospholipid reagent and haemophilic substrate separately had little effect on variation among laboratories performing one-stage assays. Standardization of reagents did not alter the basic discrepancy (approximately 20%) between the one-stage and two-stage assay methods. Omission of the aluminium hydroxide adsorption step from the two-stage method reduced the discrepancy between the two methods to less than 10%. Discrepancies did not occur when one plasma standard was assayed against another. Improvements in the stability of VIII:C in freeze-dried plasma now make it feasible to establish a long-term plasma reference standard.
Adsorption↗
Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain.
BACKGROUND: The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. OBJECTIVE: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. METHODS: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII-C2) to FVIII-deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII-C2, followed by a subcutaneous challenge of either FVIII-C2 or FVIII. The development of anti-FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. RESULTS AND CONCLUSIONS: Mucosal administration of FVIII-C2 decreases the titer of anti-FVIII-C2 inhibitors after FVIII-C2 challenge, and decreases the percentage of FVIII-C2 specific antibodies after challenge with full-length FVIII. Tolerance induction to FVIII-C2 is associated with increased IL-10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII-C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII-specific mucosal tolerance induction as an immunotherapy treatment for anti-FVIII inhibitor development warrants further investigation.
Administration, Intranasal↗
Variable inactivation of human factor VIII from different sources by human factor VIII inhibitors.
The source of human factor VIIII (FVIII) had a marked effect on the inhibitory activity of a panel of eight human FVIII inhibitors. Use of conventional FVIII concentrates gave lower inhibitor titres whereas a monoclonal antibody purified FVIII concentrate gave titres similar to or greater than those with plasma. Addition of phospholipid (PL) protected highly purified FVIII against inhibition. The content of PL-bound FVIII in concentrates may account for the observed differences.
Animals↗
Heterogeneity of autoantibodies to factor VIII: differences in specificity for apparently distinct antigenic determinants of factor VIII coagulant protein.
The interference of antibodies to factor VIII coagulant protein (VIII:C) of 9 nonhemophilic patients with the binding to factor VIII coagulant antigen (VIII:CAg) of a reference hemophilic 125I-Fab' reagent, used in a liquid phase VIII:CAg assay, was studied. The binding competition was estimated from immunoradiometric assay (IRMA) dose-response slope of VIII:CAg present in patient plasma, interference of antibodies with the 125I-Fab' binding to VIII:CAg in normal plasma, and the displacement of antibody from the complexes with VIII:CAg by the 125I Fab'. Antibody populations from three patients were studied in detail; in the VIII:CAg assay, two of them interfered with the 125I-Fab' binding, and one did not (patient 1). The formation of stable complexes between antibodies of each patient and VIII:CAg was demonstrated by protein-A-Sepharose adsorption. The 125I-Fab' binding to VIII:CAg-anti-VIII:CAg IgG complexes indicated that patient 1 antibodies and the 125I-Fab' recognized different antigenic determinants, whereas the other two patient antibodies and 125I-Fab' recognized closely related or identical VIII:CAg determinants. These results demonstrate an apparently selective recognition of at least two distinct VIII:CAg determinants by naturally occurring antibodies, suggesting a possibility of a wider use of these antibodies in studies of the structure and function of factor VIII.
Antigen-Antibody Complex↗
Preparation of factor-VIII-depleted plasma with antibodies and its use for the assay of factor VIII.
Standardized factor VIII (FVIII)-deficient plasma is necessary for the clotting assay of this protein. Ethical considerations and prevalence of HIV seropositivity in hemophilia A patients have required the replacement of severe hemophilia A plasma by another specific substrate for FVIII clotting assays. FVIII-deficient plasma was prepared by immunodepletion using polyclonal anti-von-Willebrand factor (vWF) and monoclonal anti-FVIII antibodies coated to agarose beads. This plasma, deficient in both FVIII and vWF, allows the evaluation of FVIII activity with a detection limit of 1% as compared to normal plasma. This immunodepleted plasma was compared to plasma from severe hemophilia A for the FVIII determination in plasma from normal subjects, severe and mild hemophiliacs, patients with von Willebrand disease and dicoumarol-treated patients. In all cases, similar values were obtained with both reagents at various levels of FVIII, with a correlation coefficient of 0.994. Such a good correlation was also obtained for the assay of FVIII in concentrates and in plasma from hemophiliacs following infusion. This immunodeficient plasma thus represents a suitable reagent for the FVIII clotting assay and provides an accurate alternative to the use of hemophilic plasma.
Antibodies↗
A method for detecting factor-VIII clotting activity associated with factor VIII-related antigen in agarose gels.
A method for detecting factor-VIII clotting activity in agarose is described. It is based on factor VIII promoting coagulation in a mixture of haemophilic plasma in agarose which is detected by a change in opacity. When this test was used to detect factor-VIII clotting activity in a one-dimensional Laurell electroimmunoassay for factor VIII-related antigen all the factor-VIII activity was found in the same position as the factor VIII-related antigen immunoprecipitate. Factor-VIII clotting activity did not appear to be simply trapped in this immunoprecipitate and therefore it has been concluded that the molecule containing factor-VIII clotting activity carries factor VIII-related antigen determinants.
Animals↗
Factor VIII:C and VIII:CAg response in patients with haemophilia A and von Willebrand's disease after administration of different factor VIII concentrates or plasma.
Factor VIII procoagulant activity (VIII:C) and factor VIII procoagulant antigen (VIII:CAg) were studied in seven patients with haemophilia A after administration of three different factor VIII concentrates or plasma. The in vivo recovery of VIII:CAg was less than that of VIII:C and the disappearance rate of VIII:CAg was much higher either when concentrates or plasma were given. The half-life of VIII:C was thus about 12 h but of VIII:CAg only about 3 h or less. Six patients with von Willebrand's disease were studied after administration of AHF-Kabi. In contrast to haemophilia A the discrepancy between VIII:C and VIII:CAg disappearance rates was not present in von Willebrand's disease, since both VIII:C and VIII:CAg showed a typical progressive increase. We conclude that factor VIII:C given to haemophilia patients does not behave like native VIII:C, not even when fresh plasma is used. Patients with von Willebrand's disease are capable of forming a normal VIII:C when appropriately stimulated.
Adult↗