A comparison of five different rabbit antisera to factor VIII and the demonstration of a factor VIII related antigen in normal and von Willebrand's disease platelets.
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Absorption↗
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Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII.
Coagulation factor VIII (FVIII) and factor V are homologous glycoproteins that have a domain structure of A1-A2-B-A3-C1-C2. FVIII is a heterodimer of the heavy chain (domains A1-A2-B) and the light chain (domains A3-C1-C2) in a metal ion-dependent association between the A1- and A3-domains. Previous studies identified a 110-amino acid region within the FVIII A1-domain that inhibits its secretion and contains multiple short peptide sequences that have potential to bind immunoglobulin-binding protein (BiP). FVIII secretion requires high levels of intracellular ATP, consistent with an ATP-dependent release from BiP. Site-directed mutagenesis was used to elucidate the importance of the potential BiP-binding sites in FVIII secretion. Mutation of Phe at position 309 to Ser or Ala enhanced the secretion of functional FVIII and reduced its ATP dependence. The F309S FVIII had a specific activity, thrombin activation profile, and heat inactivation properties similar to those of wild-type FVIII. However, F309S FVIII displayed increased sensitivity to EDTA-mediated inactivation that is known to occur through metal ion chelation-induced dissociation of the heavy and light chains of FVIII. The results support that Phe309 is important in high affinity heavy and light chain interaction, and this correlates with a high affinity BiP-binding site. Introduction of the F309S mutation into other secretion defective FVIII mutants rescued their secretion, demonstrating the ability of the this mutation to improve secretion of mutant FVIII proteins retained in the cell.
Adenosine Triphosphate↗
Changes in factor VIII binding capacity of von Willebrand factor and factor VIII coagulant activity in two patients with type 2N von Willebrand disease after hemostatic treatment and during pregnancy.
The changes of the FVIII binding capacity of vWF after the infusion of FVIII/vWF concentrate was studied in two patients with type 2N vWD, and also during pregnancy in one of them. After infusion of FVIII or DDAVP to the patients, FVIII:C in plasma increased as expected, but it then decreased, with a markedly short half-life, of about 2 h, due to the defect in the FVIII binding capacity of vWF in plasma. However, after infusion of FVIII/vWF concentrate (40 U of FVIII:C/kg), FVIII:C increased, from 4-6 to 100-160 U/dl, more than the expected values, and decreased with the half-life expected. The FVIII binding capacity of vWF in plasma changed in parallel with the concentration of exogenous normal vWF, with a half-life of more than 24 h. During pregnancy, no increase of FVIII:C was observed, although vWF:Ag increased from 40 (before pregnancy) to 90 U/dl in plasma at 35th week of gestation. The FVIII binding capacity of vWF in plasma showed no increase during pregnancy. Accordingly, the administration of FVIII/vWF concentrate to the patient at delivery resulted in adequate hemostasis.
Deamino Arginine Vasopressin↗
Purification of factor VIII and von Willebrand factor from human plasma by anion-exchange chromatography.
Factor VIII (anti-hemophilia A factor) is isolated from human plasma. Purification is carried out by a combination of precipitation and chromatographic procedures. After precipitation, the first step in virus inactivation is achieved through the effect of a non-ionic detergent such as Tween 80, and a solvent, e.g. tri-n-butylphosphate (TnBP). By subsequent anion-exchange chromatography, a highly enriched product is isolated, consisting of a complex formed by factor VIII and von Willebrand factor (FVIII-vWF). This treatment also removes the virus-inactivating reagents to quantities in the low ppm range. The second step in virus inactivation is aimed specifically at the non-enveloped viruses and consists of pasteurization at temperatures higher than 60 degrees C for 10 h. Through the addition of stabilizers, between 80% and 90% of the initial activity of FVIII is preserved during the modified pasteurisation. Along with the possibly denatured proteins the stabilizers, such as sugars, amino acids and bivalent cations, are subsequently removed by ion-exchange chromatography. The two-fold virus inactivation, by solvent/detergent treatment and subsequent pasteurisation, allows the destruction of both lipid-enveloped and non-enveloped viruses. During the procedure FVIII is stabilized through the high content of vWF. The complex consisting of FVIII and vWF can be dissociated by adding calcium ions. Subsequently both glycoproteins from this complex are separated from one another by further anion-exchange chromatography.
Biocompatible Materials↗
Mechanisms of action of immune tolerance induction against factor VIII in patients with congenital haemophilia A and factor VIII inhibitors.
In its most severe form, haemophilia A is a life-threatening haemorrhagic bleeding disorder that is caused by mutations in the factor VIII (FVIII) gene. About 25% of patients who receive replacement therapy with intravenous FVIII products develop neutralising antibodies (FVIII inhibitors) that inhibit the function of substituted FVIII. Long-term application of high or low doses of FVIII has evolved as an effective strategy for eradicating antibodies and inducing long-lasting immune tolerance. Despite clinical experience with the therapy, little is known about the immunological mechanisms that cause the down modulation of FVIII-specific immune responses or the induction of long-lasting immune tolerance against FVIII. This review summarises current knowledge of the immunological mechanisms that might be involved in the induction of immune tolerance against FVIII in patients with haemophilia A who have FVIII inhibitors. In addition to data from patients with haemophilia A, data from patients who have had organ transplants or have immune-related disorders, such as autoimmune diseases, are considered as well as data from animal models.
Animals↗
Anti-idiotypes to factor VIII antibodies and their possible role in the pathogenesis and treatment of factor VIII inhibitors.
Studies on the production and characterization of anti-idiotype antibodies (AId) to monoclonal factor VIII antibodies (McFVIIIAb) are reported. Two AIds were produced and one of these exhibited cross-reactivity with two other McFVIIIAb but showed no reactivity with haemophilic and non-haemophilic FVIIIAb. This AId was also active against McFVIIIAb which bound immunologically active forms of factor VIII but it did not neutralize McFVIIIAb directed against procoagulant factor VIII. The in vitro effect of therapeutic pooled human IgG concentrates upon haemophilic and non-haemophilic FVIIIAb was also assessed. Approximately 25% of non-haemophilic FVIIIAb were inhibited following incubation with human IgG whereas the latter had no effect upon haemophilic FVIIIAb activity. Studies on haemophilic sibling pairs of FVIIIAb and non-FVIIIAb producing individuals indicated FVIIIAb neutralizing activity in the non-FVIIIAb producing sibling's IgG fraction which may be of anti-idiotypic origin. These findings lend further support to suggestions that anti-idiotypes have a regulatory role in FVIIIAb production and are of potential therapeutic value.
Antibodies, Anti-Idiotypic↗
Natural antibodies to factor VIII.
Anti-factor VIII antibodies represent a unique model to study the relationship between natural autoreactivity (natural antibodies to factor VIII of healthy individuals), disease-associated autoimmunity ("spontaneous" factor VIII inhibitors of patients with anti-factor VIII autoimmune disease) and antigen-driven immune responses (immune inhibitors in multitransfused patients with hemophilia A) to a single human protein antigen. Although natural and disease-associated anti-factor VIII antibodies are not readily distinguished based on the comparison of their isotypic distribution and epitope mapping, available studies of cross-reacting idiotypes suggest that factor VIII inhibitors in patient's plasma encompass two populations of anti-factor VIII antibodies. Some antibodies result from the clonal expansion of B lymphocytes that exist before treatment with factor VIII and secrete anti-factor VIII antibodies with properties similar to those of natural anti-factor VIII antibodies present in healthy individuals; other inhibitors are produced by B cell clones that have undergone affinity maturation and hypermutation of the V regions of the antibodies they produce. The implications for the treatment of patients with anti-factor VIII inhibitors are discussed.
Antibodies, Anti-Idiotypic↗
Effect of desmopressin on plasma factor VIII and von Willebrand factor concentrations in Greyhounds.
OBJECTIVE: To determine the effect of 1-Deamino-8-D-arginine vasopressin on plasma concentrations of von Willebrand factor and factor VIII in Greyhound blood donors, and to compare the response of 1-Deamino-8-D-arginine vasopressin injection on plasma concentrations of von Willebrand factor between groups with different resting plasma concentrations of von Willebrand factor. ANIMALS: Fifteen Greyhound blood donors were used. Dogs were grouped into three categories depending on their von Willebrand factor concentrations. PROCEDURE: Desmopressin was administered subcutaneously at 1 microgram/kg [corrected] to all dogs. Plasma von Willebrand factor and factor VIII concentrations were measured before and 10, 20, 30, 45, 60, 90 and 120 min after desmopressin injection. RESULTS: The von Willebrand factor and factor VIII concentrations in all dogs increased significantly and remained higher than base-line throughout the 2 h period. CONCLUSION: Desmopressin is useful in increasing von Willebrand factor concentrations in Greyhound blood donors, including those with low resting concentrations.
Animals↗
Monoclonal antibodies against human factor VIII molecular neutralize antihemophilic factor and ristocetin cofactor activities.
A series of monoclonal antibodies have been raised against a preparation of the factor VIII/von Willebrand factor molecule. Of the seven hybridomas showing specific activity against the factor VIII molecule in a solid-phase radioimmunoassay, three (F4.55, F4.77, and F4.264) have been shown to partially inhibit ristocetin-induced platelet aggregation and two (F4.115 and F4.415) inhibit the antihemophilic activity of the factor VIII molecule. An additional monoclonal antibody was directed against a contaminant of the factor VIII preparation and is an antifibrinogen antibody.
Animals↗
High level expression of recombinant porcine coagulation factor VIII.
Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domain-deleted porcine factor VIII expression levels are 10- to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units x 10(6) cells(-1) x 24 h(-1) was observed from a baby hamster kidney-derived cell line stably expressing recombinant porcine factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state factor VIII mRNA levels. The identification of sequence(s) in porcine factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit factor VIII expression.
Animals↗
Cerebral venous thrombosis and plasma concentrations of factor VIII and von Willebrand factor: a case control study.
BACKGROUND: High plasma concentrations of factor VIII (FVIII) and von Willebrand factor (VWF) have been recently associated with a moderately increased risk of venous thrombosis, but their roles in cerebral sinus and venous thrombosis (CSVT) have not been addressed. To determine whether elevation of FVIII and VWF is more frequent in CSVT, we analysed plasma levels of FVIII and VWF in a case control study. METHODS: The study population consisted of 25 consecutive patients (of whom nine were excluded) admitted for CSVT to the Department of Neurology, Amiens University Hospital, France, from January 1997 to December 2002, for a general screening for thrombophilia. Sixty-four healthy subjects matched for age and sex formed the group control. RESULTS: Mean FVIII (CSVT: 167.3 (SD 48.8) IU/dl; control group: 117.9 (39.8) IU/dl; p = 0.001) and VWF levels (CSVT: 165.4 (76.5)%; control group: 108.5 (27.8)%; p = 0.01) were significantly higher in the CSVT group. Using the 95th percentile of the control group as the cut off value, elevated FVIII (>190 IU/dl) occurred in 25% (4/16) (p = 0.005) and elevated VWF (>168%) in 37.5% (6/16) of patients with CSVT (p<0.001). Using previously reported cut off values (>150 IU/dl or >150%) showed the same results (FVIII: p = 0.005; VWF: p = 0.009). CONCLUSION: Our study suggests that elevation of plasma factor VIII levels is the most common prothrombotic risk factor for CSVT. Elevation of VWF is also associated with an increased risk of CSVT but its effect seems to be partly mediated through FVIII.
Adult↗
Synthesis of intrinsic factor X activator. Inhibition of the function of formed activator by antibodies to factor VIII and to factor IX.
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Antibody Specificity↗
Successful management of massive intraperitoneal bleeding in a hemophilia A patient with inhibitor by surgical debridement of the incomplete hematoma and administration of recombinant factor VIII and activated factor VII.
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Autoantibodies↗
The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII.
von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VIII in the plasma. vWF is also required for the stable accumulation of recombinant factor VIII secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VIII subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VIII was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VIII or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VIII and that the presence of uncleaved vWF propeptide inhibits both factor VIII binding and stabilization.
Animals↗
[Activation of the antihemophilic globulin A (factor VIII). 2. Effect of ampholine on the course of blood coagulation and the molecular conformation of factor VIII].
Ampholine leads to an increase of activity of factor VIII and in vitro at the same time inhibits the activation of factor XII. The influence on factor XII takes place via influence on the contact surface. All the other coagulation factors remain uninfluenced. There are no differences between normal plasma and plasma of haemophilia with regard to the influence by ampholine. The molecular weight of factor VIII remains uninfluenced by ampholine. The activity of the various molecular forms of factor VIII is increased at the same level by ampholine.
Ampholyte Mixtures↗
Treatment of factor VIII inhibitors.
Factor VIII inhibitors are antibodies of the IgG class that neutralize the procoagulant activity of FVIII. They arise as alloantibodies in multitransfused haemophilia A patients and spontaneously in some individuals. Therapeutic strategy must be separated into two concepts: treatment of bleeding episodes and treatment of the inhibitor itself. Treatment of an haemorrhagic complication is dependent on the inhibitor titre and the status of the patient as a 'high' or 'low' responder. The choice has to be made between massive amounts of human FVIII concentrate, porcine FVIII and plasma preparations with 'bypassing' activity. Adjuvant therapy such as plasma exchange or extracorporeal passage of plasma over a Protein A sepharose column might be helpful. More recent products are still under evaluation such as FVIIa or recombinant tissue factor. Intravenous immunoglobulins have proven to be helpful mainly in patients with spontaneous inhibitors. Treatment of the inhibitor itself must be started as soon as possible. Several approaches are being used, immunosuppressive therapy or induction of immune tolerance using high intermediate or low doses of FVIII concentrate.
Antibodies↗
Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII.
Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3-C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA-VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.
Animals↗
Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia A mice compared with factor VIII protein infusion.
Hemophilia A patients are typically treated by factor VIII (FVIII) protein replacement, an expensive therapy that induces FVIII-specific inhibitors in approximately 30% of patients with severe hemophilia. FVIII gene therapy has the potential to improve the current treatment protocols. In this report, we used a hemophilia A mouse model to compare the humoral and cellular immune responses between an E1/E2a/E3-deficient adenovirus expressing human FVIII directed by a liver-specific albumin promoter and purified recombinant FVIII protein infusion. Adenovirus-mediated FVIII expression did not elicit detectable CD4+ or CD8+ T cell responses and induced a weak antibody immune response to FVIII. In contrast, FVIII protein administration resulted in a potent anti-FVIII antibody response and moderate CD4+ T cell response. Furthermore, hemophiliac mice preimmunized with FVIII protein infusion to induce anti-FVIII immunity, and subsequently treated by adenovirus-mediated FVIII gene therapy, expressed therapeutic levels of FVIII despite the presence of low levels of anti-FVIII antibodies. No FVIII was detected in the plasma of mice with intermediate or high antibody levels, although anti-FVIII antibody levels in some vector-treated animals declined. The data support the hypothesis that liver-specific gene therapy-mediated expression of FVIII may be less immunogenic than traditional protein replacement therapy.
Adenoviridae↗