RESUMO
The mechanisms of action of intravenous immunoglobulins (IVIg) in autoimmune diseases are not fully understood. The fixed duration of efficacy and noncumulative effects of IVIg in immune thrombocytopenia (ITP) and acquired von Willebrand disease (AVWD) suggest other mechanisms besides immunological ones. Additionally to the peripheral destruction of platelets in ITP, their medullary hypoproduction emerged as a new paradigm with rescue of thrombopoietin receptor agonists (TPO-RA). In an ITP mouse model, interleukin (IL)-11 blood levels increase following IVIg. IL-11 stimulates the production of platelets and other haemostasis factors; recombinant IL-11 (rIL-11) is thus used as a growth factor in post-chemotherapy thrombocytopenia. We therefore hypothesized that IVIg induces IL-11 over-production, which increases platelets, VWF and factor VIII (FVIII) levels in humans and mice. First, in an ITP mouse model, we show that IVIg or rIL-11 induces a rapid increase (72 h) in platelets, FVIII and VWF levels, whereas anti-IL-11 antibody greatly decreased this effect. Secondly, we quantify for the first time in patients with ITP, AVWD, inflammatory myopathies or Guillain-Barré syndrome the dramatic IL-11 increase following IVIg, regardless of the disease. As observed in mice, platelets, VWF and FVIII levels increased following IVIg. The late evolution (4 weeks) of post-IVIg IL-11 levels overlapped with those of VWF and platelets. These data may explain thrombotic events following IVIg and open perspectives to monitor post-IVIg IL-11/thrombopoietin ratios, and to assess rIL-11 use with or without TPO-RA as megakaryopoiesis co-stimulating factors to overcome the relative hypoproduction of platelets or VWF in corresponding autoimmune diseases, besides immunosuppressant.
Assuntos
Plaquetas/imunologia , Fator VIII/imunologia , Imunoglobulinas Intravenosas/imunologia , Interleucina-11/imunologia , Fator de von Willebrand/imunologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
INTRODUCTION: Haemophilia is a major bleeding disorder due to a deficiency of procoagulant factor VIII (type A) or IX (type B). The treatment is substitutive and based on infusion of factor concentrates. Main limitations of this therapy are cost, short factor half-life and the development of inhibitors (up to 30% of severe HA patients). An important aggravating factor of haemophilia is due to a premature fibrinolysis, directing attention to the therapeutic potential of suitable antifibrinolytics. Thrombomodulin (TM) is a key player of the coagulation cascade by activating protein C (an inhibitor of thrombin generation, thus antagonizing coagulation) and of the fibrinolytic cascade by activating thrombin activatable fibrinolysis inhibitor TAFI (thus reducing fibrinolysis). Solulin is a soluble form of TM that shows both capabilities. AIM: Here, we developed a new generation of solulin variants (F376A-, M388A- and F376A/M388A-solulin) with a decreased ability to activate protein C and a conserved capacity to activate TAFI. METHODS: We produced and characterized solulin variants in vitro. In addition, F376A/M388A-solulin was tested ex vivo, using blood samples of haemophilic A patients, with thromboelastography. RESULTS: The solulin variants (F376A, M388A and the double-mutant F376A/M388A) lost their abilities to activate protein C but are still capable to activate TAFI. Thrombelastography showed increased clot firmness and stability, that, as opposed to wild-type solulin, was maintained even at high concentrations of F376A/M388A-solulin (100 nm). CONCLUSION: In sum, these results open new opportunities for the development of specific medication for haemophilic patients.
Assuntos
Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Fibrinólise/fisiologia , HumanosAssuntos
Aneurisma da Aorta Abdominal/complicações , Aneurisma da Aorta Abdominal/cirurgia , Hemofilia A/complicações , Aneurisma da Aorta Abdominal/diagnóstico , Hemofilia A/diagnóstico , Hemofilia A/genética , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Tomografia Computadorizada Espiral , Resultado do Tratamento , Procedimentos Cirúrgicos VascularesRESUMO
The administration of therapeutic factor VIII (FVIII) to treat or prevent haemorrhages in haemophilia A patients results, in up to 30% of the cases, in the development of inhibitory anti-FVIII antibodies. Much debate has taken place on the relevance of the nature of the FVIII product as a risk factor for inhibitor development. Thus, the plasma-derived vs. recombinant origin, the second vs. third generation of the product, or the presence of the B domain have been controversially evoked. A few years ago, Refacto AF, a third-generation recombinant B domain-deleted FVIII was marketed. The aim of this study was to compare the immunogenicity of Refacto AF to that of two recombinant full-length FVIII products: Helixate and Advate. For the three recombinant FVIII products, we compared the binding to the mannose-sensitive endocytic receptor CD206, the dose-dependent endocytosis by immature monocyte-derived dendritic cells (DCs), the activation by FVIII-loaded DCs of a FVIII-specific HLA-DRB1*0101-restricted mouse T-cell hybridoma and the induction of inhibitory anti-FVIII IgG in FVIII-deficient mice. At elevated FVIII concentrations, Refacto AF was less endocytosed than full-length recombinant products. At lower concentrations, however, Refacto AF was endocytosed by DCs and activated T cells as well as Helixate and Advate. The levels of inhibitory anti-FVIII IgG induced by Refacto AF in FVIII-deficient mice were lower or equal to that induced by Helixate and Advate respectively. The predicted immunogenicity of Refacto AF is identical to or lower than that of the two recombinant full-length FVIII products available on the French market.
Assuntos
Fator VIII/efeitos adversos , Fator VIII/imunologia , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endocitose , Fator VIII/metabolismo , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemofilia A/imunologia , Humanos , Hibridomas/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Receptores de Superfície Celular/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Forty per cent of haemophilia A (HA) patients have missense mutations in the F8 gene. Yet, all patients with identical mutations are not at the same risk of developing factor VIII (FVIII) inhibitors. In severe HA patients, human leucocyte antigen (HLA) haplotype was identified as a risk factor for onset of FVIII inhibitors. We hypothesized that missense mutations in endogenous FVIII alter the affinity of the mutated peptides for HLA class II, thus skewing FVIII-specific T-cell tolerance and increasing the risk that the corresponding wild-type FVIII-derived peptides induce an anti-FVIII immune response during replacement therapy. Here, we investigated whether affinity for HLA class II of wild-type FVIII-derived peptides that correspond to missense mutations described in the Haemophilia A Mutation, Structure, Test and Resource database is associated with inhibitor development. We predicted the mean affinity for 10 major HLA class II alleles of wild-type FVIII-derived peptides that corresponded to 1456 reported cases of missense mutations. Linear regression analysis confirmed a significant association between the predicted mean peptide affinity and the mutation inhibitory status (P = 0.006). Significance was lost after adjustment on mutation position on FVIII domains. Although analysis of the A1-A2-A3-C1 domains yielded a positive correlation between predicted HLA-binding affinity and inhibitory status (OR = 0.29 [95% CI: 0.14-0.60] for the high affinity tertile, P = 0.002), the C2 domain-restricted analysis indicated an inverse correlation (OR = 3.56 [1.10-11.52], P = 0.03). Our data validate the importance of the affinity of FVIII peptides for HLA alleles to the immunogenicity of therapeutic FVIII in patients with missense mutations.
Assuntos
Fator VIII/genética , Fator VIII/imunologia , Hemofilia A/genética , Hemofilia A/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Isoanticorpos/imunologia , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Bases de Dados Genéticas , Epitopos/química , Epitopos/imunologia , Fator VIII/química , Hemofilia A/tratamento farmacológico , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Ligação Proteica/imunologia , Fatores de RiscoRESUMO
The purpose of the present work was to investigate the mechanisms by which glutathione depletion induced by treatment with buthionine sulfoximine (BSO) led within 24-30 h to PC 12 cells apoptosis. Our results showed that treatment by relatively low concentrations (10-30 µM) of deferoxamine (DFx), a natural iron-specific chelator, almost completely shielded the cells from BSO-induced toxicity and that DFx still remained protective when added up to 9-12h after BSO treatment. On the other hand, phosphopeptides derived from milk casein and known to carry iron across cell membranes, markedly potentiated the toxic action of BSO when loaded with iron but were ineffective in sodium form. Kept for 24 h in serum-free medium, the cells underwent a decrease in glutathione content after BSO treatment, but remained viable. However, these BSO-pre-treated cells showed a rapid (90-120 min) decrease in cell viability when incubated with low doses of iron, whereas a great proportion of them remained viable in the presence of higher concentrations of copper and zinc. We also observed in PC 12 cells an early (4-8 h) and transient increase in the expression of ferritin subunits following BSO addition. Taken together these results suggest that BSO-induced glutathione depletion leads to an alteration of cellular iron homeostasis, which may contribute to its toxicity.
Assuntos
Apoptose/efeitos dos fármacos , Butionina Sulfoximina/toxicidade , Desferroxamina/farmacologia , Glutationa/deficiência , Quelantes de Ferro/farmacologia , Neurônios/efeitos dos fármacos , Animais , Citoproteção , Relação Dose-Resposta a Droga , Homeostase , Ferro/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Células PC12 , Ratos , Fatores de TempoRESUMO
The major complication of the substitutive treatment of haemophilia A (HA) is the development of antifactor VIII (FVIII) antibodies. Most of these antibodies neutralize FVIII procoagulant activity, and are identified as FVIII inhibitor. A subgroup of these antibodies, 'catalytic antibodies', catalyses the FVIII hydrolysis. We investigated the frequency and the activity of catalytic antibodies, according to the phenotype of HA and the presence or absence of FVIII inhibitor. IgG from 16 patients with inhibitor and 17 patients without inhibitor were purified. Rates of FVIII hydrolysis and inhibitor titres were evaluated. Anti-FVIII catalytic antibodies were detected in 63.6% of patients with HA, irrespective of the HA phenotype and the presence of FVIII inhibitor. The frequency was significantly higher for severe HA patients (73.3%) and patients with inhibitor (87.5%), but their FVIII-proteolytic activity was not significantly different from patients with mild or moderate HA and patients without inhibitor. The evolution of both catalytic and inhibitory activities was studied for 11 patients with FVIII inhibitor. We observed two profiles. In the profile 1, 18.2% of patients, the catalytic activity and the inhibitor titre coevolved. In contrast, a dissociated evolution of these two parameters was observed in 72.8% patients in profile 2. These data confirm the importance of anti-FVIII catalytic activity in patients with severe, moderate and mild HA. Interestingly, most of the patients presented a dissociated profile, suggesting that anti-FVIII antibodies might not systematically act as FVIII inhibitors.
Assuntos
Anticorpos Neutralizantes/sangue , Fator VIII/imunologia , Hemofilia A/imunologia , Análise de Variância , Fator VIII/metabolismo , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemofilia A/metabolismo , Humanos , Hidrólise , Imunoglobulina G/sangue , Masculino , Estudos RetrospectivosRESUMO
BACKGROUND: Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme-binding proteins. Free heme has been proposed to affect coagulation and has been co-purified with the factor VIII (FVIII)-von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. OBJECTIVES: To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. METHODS AND RESULTS: Heme bound to several sites on FVIII with high apparent affinity. Heme-binding inhibited FVIII procoagulant activity in a dose-dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme-mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme-binding protein known for its protective activity in hemolytic conditions. CONCLUSIONS: Our data identify FVIII as a novel heme-binding protein. Occupation of high affinity heme-binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme-mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.
Assuntos
Coagulação Sanguínea , Fator IXa/metabolismo , Fator VIII/metabolismo , Heme/metabolismo , Sítios de Ligação , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Fator VIIIa/metabolismo , Hemina/metabolismo , Humanos , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície , Fator de von Willebrand/metabolismoRESUMO
The development of inhibitory anti-factor VIII (FVIII) antibodies in patients with haemophilia A following replacement therapy is associated with several types of risk factors. Among these, the purity of FVIII concentrates, and in particular the presence of von Willebrand factor (VWF), was controversially proposed to influence the immunogenicity of exogenous FVIII. We re-assessed in vivo and in vitro the immuno-protective effect of VWF towards FVIII. The immuno-protective effect of VWF towards FVIII was investigated in vivo, in a model of haemophilia A. We studied the endocytosis of FVIII by murine bone marrow-derived dendritic cells and evaluated the capacity of VWF to block the internalization of FVIII. We characterized the relevance of VWF for the accumulation of FVIII in the marginal zone of the spleen, a secondary lymphoid organ where the immune response to therapeutically administered FVIII initiates. Our results confirm that VWF reduces the immunogenicity of FVIII in FVIII-deficient mice. Paradoxically, VWF is important for the accumulation of FVIII in the marginal zone of the spleen. We propose that VWF exerts at least two non-mutually exclusive immunoprotective roles towards FVIII in haemophilic mice: VWF prevents the endocytosis of FVIII by professional antigen-presenting cells by blocking the interaction of FVIII with as yet unidentified endocytic receptor(s). Hypothetically, VWF, by virtue of increasing the half-life of FVIII in the circulation, may allow an increased contact time with tolerogenic marginal zone B cells in the spleen.
Assuntos
Fator VIII/imunologia , Hemofilia A/imunologia , Fator de von Willebrand/fisiologia , Animais , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Endocitose/imunologia , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: As the publication of the sequence of the factor VIII gene (FVIII) in 1984, a large number of mutations that cause hemophilia A (HA) have been identified. Thanks to the advances in the detection of mutations, it is now possible to identify a putative FVIII sequence alteration in the vast majority of patients with HA. OBJECTIVES: Our main objective was to report on the spectrum of FVIII mutations and their distribution throughout the gene in 120 patients with HA. METHODS: Screening of FVIII mutations was performed using direct sequencing. Newly described missense mutations were further studied by molecular modeling. RESULTS: A total of 47 different HA causative FVIII mutations have been identified, 26 of which are described for the first time. These novel mutations include 14 missense and six nonsense mutations, two small deletions, one large deletion and three splice-site mutations. We further investigated the development of FVIII-specific inhibitors in all patients with HA. We found that four novel mutations (Ser882X, Tyr1786Ser, Ala2218Thr and a splice-site defect in intron 22) were associated with inhibitor development. CONCLUSION: These data extend our insight into the mechanisms by which novel amino acid substitutions may lead to HA, and how HA patient genotypes influence the risk of FVIII inhibitor development.
Assuntos
Fator VIII/antagonistas & inibidores , Fator VIII/genética , Hemofilia A/sangue , Hemofilia A/genética , Mutação , Substituição de Aminoácidos , Códon sem Sentido , Análise Mutacional de DNA , Fator VIII/química , Genótipo , Humanos , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Fatores de Risco , Deleção de SequênciaRESUMO
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.