Impact of different inhibitor reactivities with commercial factor VIII concentrates on thrombin generation

In order to describe the haemostatic role of a variation in inhibitor reactivity with different factor VIII (FVIII) concentrates, we have compared inhibitor titres against a panel of FVIII concentrates and correlated titre with the capacity to inhibit thrombin generation. Three plasma-derived concentrates were tested in vitro in mixing experiments with inhibitor plasmas from 11 patients with severe haemophilia A: Fanhdi, which contains von Willebrand factor (VWF) with a final ratio of approximately 1:1 (VWF IU per IU FVIII:C); Haemate-P with a ratio of 2.5:1 and Hemofil-M containing only trace amounts of VWF. In addition, the recombinant FVIII concentrate Kogenate Bayer containing no VWF was included. Inhibitor titres and the capacity to generate thrombin were measured. A statistically significant difference in measured titres was found with the highest titres recorded against Hemofil-M. The inhibitor titres needed to inhibit 50% maximum thrombin generation were the lowest for Kogenate Bayer and the highest and similar for Fanhdi and Haemate-P with intermediate titres needed for inhibition of Hemofil-M. In this study, the thrombin generation assay provides additional indications for the role of VWF in the treatment of patients with inhibitors. The VWF-containing concentrates Fanhdi and Haemate-P, added to FVIII-deficient plasma with the presence of inhibitor, generate more thrombin than do the purified concentrates Hemofil-M and Kogenate Bayer.     

In vitro reactivity of factor VIII inhibitors with von Willebrand factor in different commercial factor VIII concentrates

A relevant aspect in the treatment of patients with hemophilia A (HA) presenting inhibitor against factor VIII (FVIII) is the different antigenicity of FVIII used for replacement therapy. The aim of the study was to assess the effect of different products, with variable von Willebrand factor (vWF) concentration, in preventing the binding of inhibitor to FVIII. The reactivity of inhibitors from plasma of 18 patients with HA versus three commercial concentrates containing different amounts of vWF was compared. The results show that increasing amounts of vWF might have a protective effect on the transfused FVIII inactivation.     

Variation in factor VIII inhibitor reactivity with different commercial factor VIII preparations

Summary. During treatment of a haemophilia A patient with a high-responding inhibitor against factor VIII coagulant activity (VIII:C), we observed a difference in recovery of VIII:C depending upon which factor concentrate was infused. Inhibitor plasma samples or IgG fraction from seven patients were tested against a panel of seven different commercially available factor VIII concentrates of which five were plasma-derived and two recombinant. In two of the plasma samples, inhibitor titres manifested a wide range of values depending upon which concentrate was used in the test system. Thus, inhibitor neutralization was less and VIII:C recovery greater when factor VIII concentrates containing large amounts of von Willebrand factor were used than when highly purified concentrates containing no von Willebrand factor or only trace amounts were used. In both of these two patients the inhibitor was directed against the light chain of factor VIII, and it is possible that the epitope of the light chain with which the inhibitor reacts is partly blocked by the von Willebrand factor.
We conclude that inhibitors may differ in their reactivity with factor VIII molecules contained in clotting factor concentrates, and that there is factor VIII epitope variation between different concentrates. These findings have implications for the selection of concentrates for the treatment of inhibitor patients and the haemostatic effect may be improved if a concentrate giving the lowest inhibitor titre is chosen. Thus, in vitro testing of inhibitor reactivity with a panel of concentrates is recommended when treatment of inhibitor patients with factor VIII concentrates is considered.     

Inhibitor development in haemophilia A: the role of von Willebrand factor/factor VIII concentrates

Summary.  The presence of inhibitors that neutralize the function of factor VIII (FVIII) decreases the haemostatic efficacy of replacement clotting factor concentrate and increases morbidity among patients with haemophilia A. Certain genetic and environmental variables have been linked to a higher incidence of inhibitors. Conversely, the presence of von Willebrand factor (VWF) in some plasma-derived FVIII products may provide some measure of protection against inhibitor development, although the evidence is not conclusive. Clinical trials are needed to resolve this issue and determine the appropriate role of VWF-containing FVIII concentrates in the treatment of haemophilia A patients.     

Pharmacokinetics and hemostatic effect of different factor VIII/von Willebrand factor concentrates in von Willebrand's disease type III

Summary Four different plasma-derived concentrates composed of coagulation factor VIII (FVIII) and von Willebrand factor (vWF) of varying quality (Hemate-P, Behring; Profilate, Alpha; and FVIII-VHP-vWF, C.R.T.S Lille), or almost purified vWF (Facteur Willebrand, C.R.T.S Lille) and one recombinant FVIII concentrate (Recombinate, Baxter) were given, in doses of 30–60 IU VIII:C/kg or 70–110 IU RCof/kg, to five patients with von Willebrand's disease type III, in order to evaluate the role of the vWF in factor FVIII concentrates. All plasma concentrates except Profilate had a multimeric vWF pattern almost similar to that of normal plasma. Bleeding time (b.t.), VIII:C, vWF: Ag, ristocetin cofactor activity, and multimeric pattern of the plasma-vWF were followed for 72 h. Both Duke b.t. and the multimeric pattern in plasma normalized after infusion of Hemate-P, FVIII-VHP-vWF, and Facteur Willebrand and, to a lesser extent, after Profilate. As expected, in response to Recombinate there was no effect on primary hemostasis, and the half-life of FVIII procoagulant activity (VIII:C) was very short. Normalization of the vWF is important not only for improving the primary hemostasis, but also for maintaining the plasma FVIII concentration on a high level, both by reducing the elimination rate of infused FVIII and via a secondary release of endogenous FVIII. If a prompt hemostatic effect is required, we recommend a concentrate containing both FVIII and all vWF multimers, but for prophylactic treatment, pure vWF may be used.     

Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII

In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF-deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin-cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF-deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity.     

Factor VIII inhibitors: role of von Willebrand factor on the uptake of factor VIII by dendritic cells

Summary  In patients with haemophilia A, factor VIII (FVIII) therapy leads to the development of anti-FVIII alloantibodies that inhibit FVIII pro-coagulant activity, in up to 25% of the cases. At a time when efficient viral screening procedures are at place, development of inhibitors poses the greatest threat to haemophilia A patients. Various risk factors, both patient and product-related, are responsible for the development of inhibitory antibodies. The role of FVIII-specific CD4+ T lymphocytes in the initiation of the humoral immune response to exogenous FVIII has been well. In view of their capacity to stimulate naïve T cells, dendritic cells (DCs) play a central role in the initiation of the primary immune response. Thus, in the context of a primary alloimmunization against FVIII, i.e. when FVIII-specific B lymphocytes are not there to take up FVIII from the circulation and to serve as antigen presenting cells (APCs), DCs are the only cell type that internalize FVIII, leading to activation of FVIII-specific CD4+ T lymphocytes. von Willebrand factor (VWF) present in plasma-derived FVIII therapeutic concentrates, is known to act as a chaperone molecule for procoagulant FVIII. In addition to its role in reducing the 'antigenicity' of FVIII, the role of VWF in the reduction of the 'immunogenicity' of therapeutic FVIII in patients with haemophilia A has also been suggested. We have recently demonstrated that VWF protects FVIII from being endocytosed by human DCs and subsequently being presented to FVIII-specific T cells. We propose that VWF may reduce the immunogenicity of FVIII by preventing, upstream from the activation of immune effectors, the entry of FVIII in professional antigen presenting cells.     

Relationships between factor VIII:Ag and factor VIII in recombinant and plasma-derived factor VIII concentrates

A variety of plasma-derived (pd) and recombinant (r) factor VIII (FVIII) concentrates are used to prevent and treat bleeding in severe hemophilia A patients. A significant side effect of FVIII replacement is the development of FVIII neutralizing antibodies (inhibitors) in up to 30% of patients receiving FVIII concentrates. The FVIII protein content (FVIII:Ag) per unit of FVIII:C in FVIII concentrates, and how effectively the FVIII:Ag in FVIII concentrates binds to von Willebrand factor (VWF) may provide information relevant for the survival of FVIII:C in vivo and for estimating the risk for inhibitor development. The FVIII:Ag content of nine r-FVIII and nine pd-FVIII concentrates were quantified in this study using two enzyme-linked immunosorbent assay (ELISA) platforms. The two ELISA platforms were based on the use of a monoclonal anti-(FVIII light chain)-IgG and polyclonal anti-FVIII antibodies as capture antibodies and both ELISAs were equally able to detect > or =0.005 IU of FVIII:Ag. Measured in international units, the r-FVIII concentrates contained significantly higher FVIII:Ag per unit of FVIII:C than the pd-FVIII concentrates. The VWF-binding profiles of the r-FVIII and pd-FVIII concentrates were also determined by gel filtration chromatography. Unlike the plasma-derived products, the r-FVIII concentrates invariably contained a fraction of FVIII:Ag molecules (approximately 20%) which was unable to associate with VWF. Given that VWF regulates both factor VIII proteolysis and survival of FVIII:Ag in vivo, the fraction of FVIII:Ag unable to bind to VWF may have a reduced survival and be more susceptible to proteolytic degradation in vivo. The extent to which the fractions of FVIII:Ag in concentrates able and unable to bind to VWF contribute to inhibitor development in severe FVIII-deficient patients is unknown.     

von Willebrand factor containing factor VIII concentrates

There are several plasma derived von Wille-brand factors (vWF) containing factor (FVIII) concentrates that can be used in the treatment of von Willebrand disease (vWD). All concentrates are effective in attaining normal postinfusion levels or of FVIII:C but it is difficult to achieve normalization of the bleeding time even with concentrates containing almost all vWF multimers including those of high molecular weight. Haemate P (Centeon) may be considered as the golden standard concentrate available at present. However, the development of more purified vWF concentrates devoid of FVIII:C is the goal for future development.     

Optimizing therapy with factor VIII/von Willebrand factor concentrates in von Willebrand disease

In von Willebrand disease, the main goals of treatment are to correct the dual defect of haemostasis caused by a reduced or abnormal von Willebrand factor (vWF), i.e. the prolonged bleeding time (BT) and the deficiency of factor VIII coagulant activity (FVIII:C). The synthetic vasopressin analogue, desmopressin (DDAVP), has reduced the need for transfusions in most of the mild forms of von Willebrand disease but DDAVP is ineffective in type 3 and in other severe cases of types 1 and 2 von Willebrand disease. For many years cryoprecipitate has been the mainstay of replacement therapy but, after the introduction of virucidal methods, concentrates containing FVIII/vWF have been considered much safer than cryoprecipitate and proposed in von Willebrand disease management. FVIII/vWF concentrates have been produced and tested by many authors but there is only one report describing four virus-inactivated FVIII/vWF concentrates evaluated in a cross-over randomized trial. According to these in vitro and pharmacokinetic data, the following information can be derived: (a) no FVIII/vWF concentrate had an intact multimeric structure similar to that of normal plasma or of cryoprecipitate; (b) all FVIII/vWF concentrates were equally effective in attaining normal and sustained levels of FVIII:C postinfusion, although peak levels were more delayed in the concentrate devoid of FVIII:C; (c) no FVIII/vWF concentrate consistently normalized the BT in a sustained fashion. On the other hand, clinical haemostasis can be achieved in the management of bleeding episodes and of surgery for most of von Willebrand disease cases regardless of whether the BT is corrected; in the few rare cases with mucosal bleeding not controlled by FVIII/vWF concentrates, infusion of DDAVP or platelet concentrates can be administered in addition.     

Impact of inhibitor epitope profile on the neutralizing effect against plasma-derived and recombinant factor VIII concentrates in vitro

The inhibitory capacity of plasma samples from 24 patients with severe haemophilia A and high-responding inhibitors were evaluated in a concentrate-based assay using two plasma-derived (Haemate and Monoclate-P) and three recombinant (Helixate, Recombinate and ReFacto) factor VIII concentrates and correlated with the corresponding epitope profile. In most, but not all, inhibitor plasmas with a relatively low reactivity against the von Willebrand-containing product Haemate, the main epitopes were located in the FVIII light chain. The reactivities within the group of recombinant products varied in that the reactivity against the B-domain deleted ReFacto was in general higher than that against Recombinate and Helixate. This difference did not correlate with any particular epitope profile and indicates that the B-domain, type of formulation and/or purification procedures may have an impact on the inhibitor reactivity in vitro. The ratio between the inhibitor titres in the concentrate-based assay and the Bethesda assay was dependent on the inhibitor plasma and concentrate used. Taken together, our results show that the reactivity of inhibitor plasmas varies considerably between different FVIII concentrates and that it does not fully correlate with the epitope profile. Potential clinical implications of the observed differences in inhibitor reactivity are discussed.     

Acquired von Willebrand factor deficiency during high-dose infusion of recombinant factor VIII

Constant infusion of factor VIII (FVIII) into patients with haemophilia A after major surgery has been recommended as optimal treatment to avoid peaks and valleys in the circulating levels of FVIII and to allow the use of much lower doses of FVIII than are historically required.
One of our young patients with severe (<0.01 U/ml FVIII) haemophilia suffered a subdural haematoma for which he received treatment with 815 190 recombinant FVIII (rFVIII) units over a period of 52 d. 2 weeks after admission, because of low FVIII levels and the presence of FVIII inhibitors, the infusion rate was increased to >100 U/kg/h for 14 d. During this time the FVIII level fluctuated between 0.6 and 4.2 U/ml. For some period it was not possible to detect ristocetin co-factor activity in this patient's plasma and the von Willebrand factor (VWF) level and VWF multimer pattern resembled those of a patient with von Willebrand's disease. Subsequently, when the rFVIII dose was increased 2-fold, this was not reflected by the plasma level of FVIII although antibodies were not detected.
The data suggest that the prolonged infusion of very high levels of rFVIII which is deficient in von Willebrand factor can result in depletion of VWF from existing stores, producing a laboratory picture which is consistent with the diagnosis of von Willebrand's disease. Further, in the absence of complexing with VWF, FVIII appears to be cleared from the circulation at an increased rate. This is expensive and potentially compromising. Therefore, when administering very high doses of FVIII concentrates devoid of VWF for prolonged periods of time, ristocetin cofactor and VWF levels should be monitored.     

Product type and the risk of inhibitor development in nonsevere haemophilia A patients: a case‒control study

Inhibitor development is a major complication of treatment with factor VIII concentrates in nonsevere haemophilia A. It has been suggested that plasma-derived factor VIII (FVIII) concentrates elicit fewer inhibitors than recombinant FVIII concentrates, but studies in severe haemophilia A patients have shown conflicting results. We designed a case‒control study to investigate the clinical and genetic risk factors for inhibitor development in nonsevere haemophilia A patients. We investigated whether the type of FVIII concentrate was associated with inhibitor development in nonsevere haemophilia A patients. This nested case‒control study includes 75 inhibitor patients and 223 controls, from a source population of the INSIGHT study, including all nonsevere haemophilia A patients (FVIII:C 2–40%) that were treated with FVIII concentrates in 33 European and one Australian centre. Cases and controls were matched for date of birth and cumulative number of exposure days (CED) to FVIII concentrate. A conditional logistic regression model was used to calculate unadjusted and adjusted odds ratios. No increased risk for inhibitor development was found for any type of FVIII concentrate; either when comparing recombinant FVIII concentrates to plasma-derived FVIII concentrates (adjusted odds ratio 0·96, 95% confidence interval (CI) 0·36–2·52) or for specific types of FVIII concentrates.     

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.     

In vivo recovery and safety of human factor VIII product AAFACT® in patients with haemophilia A

AAFACT, a monoclonal purified, solvent/detergent treated human plasma-derived coagulation factor VIII concentrate obtained from plasma of voluntary, non-remunerated blood donors, is manufactured and marketed in the Netherlands by Sanquin Plasma Products since 1995. In a postmarketing surveillance study, 70 previously treated haemophilia A patients were included (73% severe, 14% moderate and 13% mild haemophilia A). Most of these patients were followed during 4 years for the appearance of adverse events, possible transmissions of blood-borne viruses and the occurrence of antibodies against FVIII. The efficacy of treatment was determined in each patient by the in vivo recovery of FVIII. During this study, only six adverse events, possibly related to the use of AAFACT, were reported. None of these were indicated as serious. Transmissions of HIV, HAV, HBV and HCV in the seronegative patients have not been observed. In none of the patients, inhibitors to FVIII were detected. The in vivo recovery of FVIII during this study was not different from the in vivo recovery observed in eight patients during the preregistration study. There was a correlation of in vivo recovery with age and body weight. From these results, we conclude that the clinical usage of this human plasma-derived FVIII product is efficient and safe.     

Product‐dependent anti‐factor VIII antibodies

The development of anti‐factor (F)VIII antibodies in haemophilia A (HA) subjects undergoing replacement therapy has been well documented. The correlation between antibody development and the FVIII product used for replacement therapy remains a subject of discussion. The aim of this study was to evaluate the presence of anti‐FVIII antibodies towards three commercial rFVIII products in 34 HA subjects’ plasmas. Antibodies were quantitated by a Multiplex Fluorescence Immunoassay. All plasmas contained anti‐FVIII antibodies at variable concentrations ranging from 50 nm to 570 μm . Eleven of the 20 HA subjects treated with one (r)FVIII product contained inhibitory anti‐FVIII antibodies (0.8‐3584 BU). The inhibitory antibody titre and the molar concentrations of total antibody were mildly correlated (r² = 0.6). Pronounced differences in antibody recognition with the three rFVIII products were observed. For the group treated with Product ‘A’, the titre towards this product was 2.4‐fold higher than that observed with another full‐length rFVIII‐containing product (Product ‘B’) and almost four‐fold higher than that measured with a B domain‐less rFVIII product (Product ‘C’). For the group of 14 HA subjects treated with FVIII other than Product ‘A’, only one showed higher antibody titre when measured with this product. Our data suggest that the development of anti‐FVIII antibodies is biased towards the product used for treatment and that a significant fraction of antibodies bind to the B domain of FVIII.     

Evaluation of a novel ELISA screening test for detection of factor VIII inhibitory antibodies in haemophiliacs

Treatment of patients with haemophilia A with coagulation factor concentrates may result in the development of inhibitory antibodies directed against factor VIII (FVIII). In this study, a previously unpublished ELISA test for FVIII inhibitor screening (Genetic Testing Institute [GTI] FVIII inhibitor, Brookfield, WI, USA) was evaluated in 131 blood samples (124 samples from patients with haemophilia A, and seven serial samples from one patient with an acquired FVIII inhibitor). Comparisons were made with the routine screening assay (based on recovery of FVIII) and confirmed where positive (< 90% recovery) with the New Oxford assay. The ELISA kit had a sensitivity of 97.7% and specificity of 78.4%. The high negative predictive value of this new test (98.6%) suggests it may be useful as a reliable, rapid (< 2 h) and flexible (microwell strip format) tool for inhibitor screening of samples from both patients with haemophilia A and those with suspected acquired FVIII inhibitors.     

Domain specificity of factor VIII inhibitors during immune tolerance induction in patients with haemophilia A

Summary. Introduction‐Frequent administration of high dosages factor VIII (FVIII), so‐called immune tolerance induction (ITI), provides an efficient strategy to eradicate inhibitory antibodies in patients with haemophilia A. At present, our knowledge on the characteristics of inhibitory antibodies in patients undergoing ITI is limited. Aim‐In this study we characterized the domain specificity of FVIII inhibitors in 11 haemophilia A patients during ITI. Results‐In three of six patients who were successfully tolerized, inhibitory antibodies were directed predominantly against the FVIII light chain. In two other patients within this group, a significant contribution of A2 antibodies was observed which did not change during treatment. In the sixth patient the relative contribution of A2 inhibitors declined which coincided with an increase in antilight chain antibodies. In four of five patients who failed ITI, A2 inhibitors were observed. In two patients the contribution of A2 inhibitors increased during treatment, while in two other patients the contribution of A2 inhibitor remained constant. The fifth patient had inhibitory antibodies predominantly directed against the FVIII light chain. Conclusion‐Overall, our findings revealed changes in domain specificity of FVIII antibodies in five of 11 patients analysed. Remarkably, antibodies exclusively directed towards the light chain of FVIII were predominantly observed in patients who were successfully tolerized.     

Prevalence of IgG antibodies to human parvovirus B19 in haemophilia children treated with recombinant factor (F)VIII only or with at least one plasma-derived FVIII or FIX concentrate: results from the French haemophilia cohort

Human parvovirus B19 (B19) has been transmitted by some brands of virally attenuated plasma-derived factor VIII (FVIII) or IX (FIX) concentrates. To quantify the differences of human parvovirus B19 risk transmission between albumin-stabilized recombinant factor and plasma-derived factor, we studied the prevalence of IgG antibodies to B19 (anti-B19) in 193 haemophiliac children between 1 and 6-years of age who had previously been treated with albumin-stabilized recombinant FVIII only (n = 104), and in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates (n = 89). Association between the prevalence of anti-B19 and the treatment group was analysed using multivariate logistic regression. Age, severity and type of haemophilia, number of cumulative days of exposure to factor VIII or IX, previous history of red blood cells or plasma transfusion were considered as potential confounding variables. A higher prevalence of anti-B19 was found in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates than in children treated with albumin- stabilized recombinant FVIII only (OR: 22.3; CI: 7.9-62.8), independently of the other factors studied.