Factor VIII and IX deficiencies related to acquired inhibitors in a patient with chronic hepatitis C virus infection receiving treatment with pegylated interferon plus ribavirin

The development of clotting factor inhibitor autoantibodies is rarely observed, but can result in a potentially life-threatening haemorrhagic disorder. These acquired inhibitors are most frequently against factor VIII (FVIII), whilst the detection of inhibitors against other clotting factors is rarer. Inhibitors against FVIII and FIX are mostly observed in patients with classical hereditary haemophilia after receiving factor replacement therapy. We report a rare case of acquired FVIII and factor IX (FIX) inhibitors in a single, non-haemophilic patient with chronic hepatitis C virus (HCV) infection who was receiving antiviral treatment with pegylated interferon plus ribavirin. The FVIII and FIX activities were <1% and high titres of inhibitors autoantibodies were found in his serum samples. After achieving a sustained virological response, combined immunosuppression with oral corticosteroids (prednisone) and azathioprine was introduced, eradicating the inhibitory autoantibodies. The development of these inhibitors in association with antiviral therapy for chronic hepatitis C is poorly understood, and particular attention must be given to HCV-infected patients with worsening coagulopathy, particularly if coexistent with treatment related thrombocytopenia.     

Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.     

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

Inhibitors are antibodies directed against haemophilia treatment products which interfere with their function. Factor VIII (FVIII) inhibitors in haemophilia A and factor IX (FIX) inhibitors in haemophilia B are significant clinically when they require a change in a patient's treatment regimen. Their persistence may increase morbidity and mortality. Multiple laboratory tests are now available for detecting and understanding inhibitors in haemophilia. Inhibitors are traditionally measured by their interference in clotting or chromogenic factor assays. They may also be detected using immunologic assays, such as enzyme‐linked immunosorbent assay or fluorescence immunoassay. Anti‐FVIII or anti‐FIX antibodies of IgG₄ subclass best correlate with the presence of functional inhibitors. Improvements in inhibitor measurement have been recently introduced. Preanalytical heat treatment of patient specimens allows testing of patients without delaying treatment. Use of chromogenic and immunologic assays may aid in identification of false‐positive results, which are frequent among low‐titre inhibitors. Validated reagent substitutions can be used to reduce assay cost. New methods for defining assay positivity and reporting low‐titre inhibitors have been suggested. Challenges remain in the areas of quality control, assay standardization, monitoring of patients undergoing immune tolerance induction therapy and testing in the presence of modified and novel treatment products.     

Characterization of Factor VIII Inhibitors

Factor VIII (FVIII) inhibitors develop as either alloantibodies against exogenous FVIII in patients with congenital hemophilia A after FVIII-replacement therapy or as autoantibodies against endogenous FVIII in previously healthy, nonhemophilic individuals. The predominant immunoglobulin G (IgG) subclass of FVIII inhibitors is IgG(4). The main epitopic regions are known to be located, however, in the A2, A3, and C2 domains. The A2 and A3 epitopes have been identified between amino acid residues 484 and 509 and residues 558 and 565, respectively. Both of these regions are close to the binding sites for activated FIX (FIXa). Two regions have been identified in the C2 domain, one in the amino-terminal portion of the domain (residues 2181-2243) and the other in the carboxy-terminal portion of the domain (residues 2248-2312 and residues 2315-2330). In addition, a crystallographic analysis of a complex of the C2 domain and a human monoclonal IgG(4)(K) Fab revealed that this type of antibody is in direct contact with hydrophobic and basic residues of the membrane-binding surface. Inactivated FVIII is rapidly cleared from the circulation in the presence of inhibitors. The inhibitors also bind to essential FVIII ligand proteins, including von Willebrand factor, FIXa, FXa, and thrombin, and to surface membrane phospholipid. Some type 2 inhibitors interfere with binding to activated protein C.     

Factor VIII and IX assays for post-infusion monitoring in hemophilia patients: Guidelines from the French BIMHO group (GFHT)

Replacement therapy with plasma-derived or recombinant FVIII and FIX (pdFVIII/pdFIX or rFVIII/rFIX) concentrates is the standard of treatment in patients with haemophilia A and B, respectively. Measurement of factor VIII (FVIII:C) or factor IX (FIX:C) levels can be done by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). The French study group on the Biology of Hemorrhagic Diseases (a collaborative group of the GFHT and MHEMO network) presents a literature review and proposals for the monitoring of FVIII:C and FIX:C levels in treated haemophilia A and B patients, respectively. The use of CSA is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. Except for rFVIII-Fc, great caution is required when measuring FVIII:C levels by OSA in patients substituted by EHL-rFVIII. The OSA is recommended for the monitoring of patients treated with pdFIX or rFIX. Large discordances in the FIX:C levels measured for extended half-life rFIX (EHL-rFIX), depending on the method and reagents used, must lead to great attention when OSA is used for measuring FIX:C levels in patients substituted by EHL-rFIX. Data of most of recent studies, obtained with spiked plasmas, deserve to be confirmed in plasma samples of treated patients.     

Acquired hemophilia in a patient with systemic lupus erythematosus: a case report and literature review

Abstract

We report the case of a 38-year-old female patient with systemic lupus erythematosus (SLE) who developed acquired hemophilia caused by factor VIII (FVIII) inhibitors. She manifested spontaneous bleeding symptoms such as ecchymoses and hematuria. Laboratory findings showed an isolated prolongation of the activated partial thromboplastin time, reduced FVIII activity, and a high titer of FVIII inhibitors. She was successfully treated with oral predonisolone and cyclosporine in combination with steroid and cyclophosphamide pulse therapy.     

Immune Tolerance Therapy in Patients with Acquired Hemophilia

Acquired hemophilia is a rare disorder with an estimated annual incidence of 0.2-1 cases per million individuals. The etiology of the disorder remains obscure, although approximately half of all cases are associated with other underlying conditions. In acquired hemophilia, the severe hemorrhagic diathesis is caused by the development of autoantibodies directed against a clotting factor, most commonly factor VIII. These autoantibodies inhibit normal coagulation and lead to bleeding complications, which can be life-threatening in a high percentage of cases. Prompt diagnosis and appropriate management of the disorder enable effective control; the short- and long-term aims of therapy are to terminate the acute bleed and eliminate or reduce the inhibitor, respectively. Immune tolerance therapy has been shown to successfully eradicate or suppress inhibitors in patients with congenital hemophilia A and may be applicable to patients with acquired hemophilia. Here we present preliminary data on the use of immune tolerance therapy in patients with acquired hemophilia and discuss possible treatment strategies.     

Hemarthrosis as acute presentation of acquired hemophilia in a patient with systemic lupus erythematosus: successful treatment and long-lasting remission

Hemorrhagic events due to production of antibodies directed against coagulation factors are rarely observed in systemic lupus erythematosus (SLE). We report the case of a patient with clinically quiescent SLE who developed factor VIII inhibitor in acquired hemophilia presenting as hemarthrosis. Initial treatment with FVII, FVIII and FIX plasma concentrate, metilprednisolone and immunoglobulins i.v. were started but new hemorrhagic manifestation occurred. Plasma exchange was also administered, but it was discontinued early due to partial efficacy. In addition, pulse cyclophosphamide 0.5 g/m² was started. Eight weeks later, FVIII and FIX activity returned within normal ranges, FVIII and FIX inhibitors decreased significantly and hemorrhagic manifestations disappeared. The rare occurrence of acquired hemophilia due to the presence of anti-factor VIII antibodies associated to SLE, which was reviewed, might explain the lack of therapeutic guide-lines; indeed therapeutic options are available but the outcome in each single patient is not predictable.     

Treatment of chronic hepatitis C: Anticipated impact of resistance in patients treated with protease inhibitors

A main target of specifically targeted antiviral therapy for hepatitis C (STAT-C) is the NS3-protease, which has key functions in the hepatitis C virus (HCV) replication cycle. HCV/NS3-protease inhibitors have shown high antiviral activity in vitro and in patients with chronic hepatitis C. Protease-resistant HCV variants occurred rapidly in patients receiving protease-inhibitor monotherapy. The development of resistance can be best explained by selection of preexisting resistant variants, which grow out under selective pressure. Numerous mutations associated with resistance were identified. Clinical trials showed that protease-resistant strains are sensitive to interferon and that a triple combination of protease inhibitors, peginterferon, and ribavirin may improve the sustained virologic response rate compared with standard peginterferon/ribavirin combination therapy. Overall, it can be anticipated that successful treatment with protease inhibitors will require either combination therapy with peginterferon/ribavirin or a combination of STAT-C compounds with distinct modes of action and resistance patterns.     

Laboratory measurement of factor replacement therapies in the treatment of congenital haemophilia: A United Kingdom Haemophilia Centre Doctors’ Organisation guideline

Assay discrepancies can occur with laboratory monitoring of FVIII and FIX replacement therapy, particularly for the extended half‐life products. This guideline collates current published data and provides advice on appropriate choice of assays for laboratory measurement of replacement therapy for patients with Haemophilia A and B without inhibitors. It is recommended that each haemophilia centre should ensure that appropriate laboratory assays are available for FVIII and FIX products in local clinical use. Patient samples should be assayed against calibrators traceable to WHO Plasma International Standards. Assay discrepancies are common especially for the extended half‐life FVIII and FIX products, and assays of these products may need to be verified with the specific CFC being used.     

Factor VIII inhibitors: a 50‐year perspective

Summary. Inhibitors of factor VIII (FVIII) have been studied for more than 50 years, but diagnostic and therapeutic challenges remain. To describe the features that distinguish alloantibodies from autoantibodies, list predisposing factors, and review methods for tolerance induction and autoantibody suppression. Review of key articles published during the past half‐century that have advanced knowledge in this field. Alloantibodies generally bind to the A2 or C2 domains of FVIII and disrupt the formation of the FVIII–FIX complex. They exhibit type 1 reaction kinetics, are saturable by FVIII, and display anamnesis. In contrast, autoantibodies usually bind to the C2 domain of FVIII, interfering with phospholipid and von Willebrand factor binding. They have type‐2 kinetics and are poorly neutralized by FVIII. Repeated exposures to FVIII induce tolerance in 70–80% of haemophiliacs with inhibitors, whereas drugs that deplete B‐lymphocytes restore self‐tolerance to FVIII in a similar percentage of non‐haemophiliacs. Future work should focus on improving assays that detect and quantify inhibitors, examining the pathophysiology of inhibitor formation using contemporary immunologic tools, and investigating new treatment modalities. These should include agents to control bleeding with less thrombotic risk, more specific immunomodulating drugs to curtail antibody formation, and, for haemophilia patients, genetic therapies to provide FVIII resistant to or protected from inactivation by inhibitors.     

Ribavirin mode of action in chronic hepatitis C: from clinical use back to molecular mechanisms

Ribavirin is an old broad‐spectrum antiviral that is highly effective when used in combination with interferon‐α and also as part of triple therapies containing new inhibitors of the hepatitis C virus (HCV) non‐structural (NS)3/4 protease or HCV NS5B polymerase for the treatment of patients with chronic hepatitis C. However, the molecular mechanisms by which ribavirin enhances early and sustained virological response rates during interferon‐based antiviral HCV therapy are still unknown. Several mechanisms including (i) immunomodulatory properties, (ii) inhibition of the inosine monophosphate dehydrogenase, (iii) direct inhibition of the HCV‐encoded NS5B RNA polymerase, (iv) induction of lethal mutagenesis and (v) modulation of interferon‐stimulated gene expression are currently proposed. Here, we discuss recent advances from in vitro data and their importance for the situation in patients with chronic hepatitis C. Furthermore, theoretical aspects from mathematical modelling of ribavirin action in chronic hepatitis C are reviewed.     

Inhibitors in haemophilia A and B: Management of bleeds,inhibitor eradication and strategies for difficult‐to‐treat patients

The standard therapy for patients with haemophilia is prophylactic treatment with replacement factor VIII (FVIII) or factor IX (FIX). Patients who develop inhibitors against FVIII/FIX face an increased risk of bleeding, and the likelihood of early development of progressive arthropathy, alongside higher treatment‐related costs. Bypassing agents can be used to prevent and control bleeding, as well as the recently licensed prophylaxis, emicizumab, but their efficacy is less predictable than that of factor replacement therapy. Antibody eradication, by way of immune tolerance induction (ITI), is still the preferred management strategy for treating patients with inhibitors. This approach is successful in most patients, but some are difficult to tolerise and/or are unresponsive to ITI, and they represent the most complicated patients to treat. However, there are limited clinical data and guidelines available to help guide physicians in formulating the next treatment steps in these patients. This review summarises currently available treatment options for patients with inhibitors, focussing on ITI regimens and those ITI strategies that may be used in difficult‐to‐treat patients. Some alternative, non‐ITI approaches for inhibitor management, are also proposed.     

Factor VIII inhibitor associated with peginterferon

We present a case of a 53-year-old black male who developed a factor VIII inhibitor during his treatment for hepatitis C virus infection with peginterferon. The development of inhibitors against factor VIII during peginterferon therapy has been rarely reported. We have reviewed the current literature and the following case presentation provides the first demonstration, to our knowledge, in which chronic exposure to peginterferon is associated with an acquired factor VIII inhibitor.     

Proteolytic antibodies activate factor IX in patients with acquired hemophilia

Acquired hemophilia is a rare bleeding disorder characterized by the spontaneous occurrence of inhibitory antibodies against endogenous factor VIII (FVIII). IgG from some patients with acquired hemophilia hydrolyze FVIII. Because of the complex etiology of the disease, no clinical parameter, including the presence of FVIII-hydrolyzing IgG, has been associated with patient's survival or death. Here, we demonstrate the presence of anti-FIX antibodies in acquired hemophilia patients. IgG from some patients were found to hydrolyze FIX. In most cases, IgG-mediated FIX-hydrolysis resulted in FIX activation. IgG-mediated hydrolysis of FIX thus led to the significant generation of activated FIX in 25 of 65 patients. Based on the estimated kinetic parameters, patients' IgG activated up to 0.3nM FIX in 24 hours, an amount that restored thrombin generation in vitro provided the presence of more than or equal to 3% residual FVIII activity in plasma. This work identifies proteolytic IgG as novel molecules able to activate FIX under pathologic conditions. IgG-mediated FIX activation is a prevalent phenomenon among acquired hemophilia patients. The presence of FIX-activating IgG may partly compensate for the antibody-mediated inhibition of endogenous FVIII in restoring thrombin generation. This clinical trial was registered at www.clinicaltrials.gov as #NCT00213473.     

Factor VIII assay mimicking in vivo coagulation conditions

Under certain circumstances, the determination of coagulation factor VIII (FVIII) is hampered by assay discrepancies between clotting and chromogenic approaches. These are observed in certain patients’ plasma as well as in certain concentrates. We intended to develop a novel assay for the quantification of coagulation FVIII which reflects the physiological situation better than the established assays. It is based on plasma without chelation of divalent cations and simultaneously minimizes the generation of activated factors which could function as uncontrolled triggers of coagulation. FVIII deficient plasma is prepared with the aid of biotinylated antibodies against FVIII from normal plasma in presence of inhibitors of contact activation. To start the assay only tiny amounts of activated FIX serve as trigger. The FVIII determination is performed in a kinetic experiment and is based on the cleavage of a fluorogenic substrate for activated FX. FVIII concentrations between 0.01 and 1 IU mL^?1 are easily determined. Plasma‐derived and recombinant FVIII concentrates were compared. All plasma‐derived concentrates were found to contain FVIII activities within the specification of the manufacturer. Recombinant concentrates yielded only 35–50% of the claimed potency. The novel in vivo‐like assay avoids the undue advantage or disadvantage of certain product characteristics by eliminating unphysiological assay conditions. Its usefulness could turn out in future experiments with plasma from haemophilia A patients.     

Successful response to rituximab in two cases of acquired haemophilia refractory to standard-therapy

Acquired autoantibodies against coagulation factors (acquired haemophilia) frequently constitute a life-threatening bleeding situation requiring a prompt therapeutic intervention, including control of bleeding and secondarily an attempt of eradication of the inhibitor by prolonged immunosuppressive therapy. The combination of oral corticosteroids and cyplophosphamide seems to be effective to eradicate the autoantibody, but some patients may be resistant. Another therapeutic approach, recently described, observes treatment with the chimeric anti-CD20 monoclonal antibody rituximab. We report two consecutively treated patients whose acquired FVIII inhibitors did not respond to standard immunosuppressive regimens, and only when rituximab was added to therapy, complete response and prolonged remission were obtained.     

Thyroid dysfunction in hepatitis C individuals treated with interferon-alpha and ribavirin: a review

Hepatitis C (HCV) is now the main cause of chronic hepatic disease, cirrhosis and hepatocellular carcinoma. Several extrahepatic diseases have been associated with chronic HCV infection, and in most cases appear to be directly related to the viral infection. Thyroid disorders are common in patients with chronic HCV. Some patients with chronic hepatitis C experience thyroid problems, and thyroid dysfunction may also be a side effect of interferon-based treatment. The principal risk factor for developing thyroid disease in the course of antiviral therapy is the previous positivity for anti-thyroid antibodies (anti-thyroid peroxidase) especially in older women. Screening for autoantibodies and serum thyroid-stimulating hormone is recommended before, during and after interferon-alpha treatment, and patients should be informed of the risk of thyroid dysfunction. This review includes a summary of thyroid disease associated with chronic HCV infection, interferon-alpha and ribavirin for treatment of HCV and potential to induce thyroid dysfunction.     

Improvement of renal dysfunction in a patient with hepatitis C virus‐related liver cirrhosis by daclatasvir and asunaprevir combination therapy: A case report

Recently, treatments for chronic hepatitis C virus (HCV) infection have been drastically improved by the development of direct‐acting antiviral agents. In September 2014, dual oral therapy using daclatasvir (DCV) and asunaprevir (ASV) was approved for the treatment of chronic HCV infection in Japan. We treated a patient with HCV‐related liver cirrhosis with severe leg edema due to chronic renal dysfunction using this dual oral therapy. Although serum alanine aminotransferase increased rapidly during the first week of treatment, the antiviral therapy was able to continue, and liver function recovered spontaneously. After 1 month of treatment, serum HCV RNA became continuously undetectable, and serum albumin level gradually increased. Throughout the therapy, serum creatinine level nearly normalized, and leg edema gradually improved. These improvements continued after the combination therapy was completed. HCV RNA remained undetectable following the end of therapy, and sustained virological response at 12 weeks was achieved. It has been reported that chronic HCV infection is associated with renal dysfunction and that HCV eradication can improve it. DCV and ASV combination therapy is safe for patients who have renal dysfunction and may be a suitable therapy for chronic hepatitis C patients with renal dysfunction.     

Hémophilie acquise auto-immune : quelle est la place du rituximab dans la stratégie thérapeutique ? Réflexion à partir d’une série monocentrique de 8 patients et revue de la littérature

IntroductionAutoimmune acquired haemophilia is a rare autoimmune disease. The purpose of immunosuppressive therapy is to stop the production of autoantibodies that inhibit clotting factors VIII or IX. A corticosteroids-cyclophosphamide combination is recommanded as first-line therapy. From our experience at the University Hospital of Nîmes, we discuss the place of rituximab in the therapeutic arsenal.MethodsWe report a monocentric observational retrospective study. Our data are discussed in light of literature data, in particular cohorts EACH2 and SACHA.ResultsEight patients (7 with FVIII anibodies) were consecutively included from 2005. The average age was 68.5 years with a male predominance (62.5%). Bleeding manifestations were usually spontaneous and superficial. A pathology, mostly autoimmune or neoplastic, was associated in 5/8 patients. A “by-pass” haemostatic treatment was prescribed for 3/8 patients. Rituximab was prescribed for 5/8 patients, three times as first-line therapy, and always associated with corticosteroids. Three patients received a cyclophosphamid/cortisone combination, two were treated exclusively with oral corticosteroids. Remission was obtained in all patients, without subsequent relapse. The average time to obtain remission under rituximab (after the first injection) was 32.5 days (10–143). The results observed in our series of patients are consistent with the data from the literature.ConclusionsRituximab appears to be an effective and well-tolerated treatment for autoimmune acquired haemophilia. However, its place remains to be specified.