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Background: Free labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis., Objectives: To investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis., Methods: The effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively. Its effect on ferric-chloride (FeCl 3 )-induced arterial thrombosis and lung perfusion following hemin injection was assessed in wild-type mice., Results: Erythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited ferric-chloride-induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi., Conclusion: Hydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Emicizumab is a bispecific, chimeric, humanized immunoglobulin G (IgG)4 that mimics the procoagulant activity of factor (F) VIII (FVIII). Its long half-life and subcutaneous route of administration have been life-changing in treating patients with hemophilia A (HA) with or without FVIII inhibitors. However, emicizumab only partially mimics FVIII activity; it prevents but does not treat acute bleeds. Emergency management is particularly complicated in patients with FVIII inhibitors receiving emicizumab prophylaxis in whom exogenous FVIII is inefficient. We have shown recently that Imlifidase (IdeS), a bacterial IgG-degrading enzyme, efficiently eliminates human anti-FVIII IgG in a mouse model of severe HA with inhibitors and opens a therapeutic window for the administration of exogenous FVIII., Objectives: To investigate the impact of IdeS treatment in inhibitor-positive HA mice injected with emicizumab., Methods: IdeS was injected to HA mice reconstituted with human neutralizing anti-FVIII IgG and treated with emicizumab., Results: IdeS hydrolyzed emicizumab in vitro and in vivo, albeit, at slower rates than another recombinant human monoclonal IgG4. While F(ab') 2 fragments were rapidly cleared from the circulation, thus leading to a rapid loss of emicizumab procoagulant activity, low amounts of single-cleaved intermediate IgG persisted for several days. Moreover, the IdeS-mediated elimination of the neutralizing anti-FVIII IgG and restoration of the hemostatic efficacy of exogenous FVIII were not impaired by the presence of emicizumab and polyclonal human IgG in inhibitor-positive HA mice., Conclusion: Our results suggest that IdeS could be administered to inhibitor-positive patients with HA receiving emicizumab prophylaxis to improve and ease the management of breakthrough bleeds or programmed major surgeries., Competing Interests: Declaration of competing interests S.L.D. and J.D.D. are inventors on patent EP18305971.6 related to the use of IdeS in the context of AAV-mediated gene therapy. Other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Transplacental delivery of maternal immunoglobulin G (IgG) provides humoral protection during the first months of life until the newborn's immune system reaches maturity. The maternofetal interface has been exploited therapeutically to replace missing enzymes in the fetus, as shown in experimental mucopolysaccharidoses, or to shape adaptive immune repertoires during fetal development and induce tolerance to self-antigens or immunogenic therapeutic molecules., Objectives: To investigate whether proteins that are administered to pregnant mice or endogenously present in their circulation may be delivered through the placenta., Methods: We engineered monovalent immunoglobulin G (FabFc) specific for different domains of human factor VIII (FVIII), a therapeutically relevant model antigen. FabFc was injected with exogenous FVIII into pregnant severe hemophilia A mice or pregnant mice expressing human FVIII following AAV8-mediated gene therapy. FabFc and FVIII were detected in the pregnant mice and/or fetuses by enzyme-linked immunosorbent assay and immunohistochemistry., Results: Administration of FabFc to pregnant mice allowed the maternofetal delivery of FVIII in a FcRn-dependent manner. FVIII antigen levels achieved in the fetuses represented 10% of normal plasma levels in the human. We identified antigen/FabFc complex stability, antigen size, and shielding of promiscuous protein patches as key parameters to foster optimal antigen delivery., Conclusion: Our results pave the way toward the development of novel strategies for the in utero delivery of endogenous maternal proteins to replace genetically deficient fetal proteins or to educate the immune system and favor active immune tolerance upon protein encounter later in life., Competing Interests: Declaration of competing interests J.M. and A.C.N. are inventors on a patent licensed to BioMarin. The remaining authors have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. All rights reserved.)

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The standard assay for characterization of interaction of heme with proteins is absorbance spectroscopy. However, this approach demands relatively large quantities of proteins and it is difficult to perform in high-throughput manner. Here, we describe an immunosorbent assay based on the covalent in situ conjugation of heme to a pre-coated carrier. Advantage of this assay is that it allows both identification of heme-binding proteins and quantification of their binding avidity, using only minimal amounts of protein (1-10 μg). Importantly, the same approach can be used for covalent linkage of other natural or synthetic compounds and analyzing their interactions with proteins., (Copyright © 2020 Elsevier Inc. All rights reserved.)

B cells with regulatory properties (Bregs) were identified in human and in mice among different B-cell subsets. Their regulatory properties rely mainly on the production of anti-inflammatory cytokines, in particular IL10, IL-35 and TGFβ, and were extensively studied in mouse models of autoimmune and inflammatory diseases. However, the exact nature of the stimulatory signals conferring regulatory properties to B cells is still not clear. We serendipitously observed that fluorescein isothiocyanate (FITC) binds to a significant proportion of naïve mouse B cells. Binding of FITC to the B-cell surface implicated at least in part the B-cell receptor. It triggered IL-10 production and allowed the endocytosis of FITC-coupled antigens followed by their presentation to CD4 + T cells. In particular, B cells incubated with FITC-OVA polarized OTII T cells towards a Tr1/Th2 phenotype in vitro. Further, the adoptive transfer of B cells incubated with FITC-labeled myelin oligodendrocyte glycoprotein peptide protected mice from experimental autoimmune encephalomyelitis, a T-cell-dependent autoimmune model. Together, the data show that FITC-stimulated B cells polarize immune responses towards Tr1/Th2 and acquire immuno-modulatory properties., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N-acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N-acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Vascular endothelial cells (ECs) link hemostasis, thrombosis, and complement. ECs synthesize both the clotting initiator von Willebrand factor (VWF) and the complement regulator factor H (FH). VWF is stored in EC Weibel-Palade bodies (WPBs), but the intracellular location of FH is not well defined. We found that FH colocalizes with VWF in WPBs of human umbilical vein ECs. Moreover, FH bound to VWF with an apparent nanomolar affinity and the complex was present in normal plasma. The binding of VWF to FH enhanced FH cofactor activity toward factor I-mediated downregulation of complement activation. Besides, this interaction inhibited ADAMTS13-mediated proteolysis of VWF and promoted platelet aggregation. Here, we describe a novel interaction between complement and hemostasis. The simultaneous secretion of VWF and FH by activated ECs may promote adhesion of platelets to endothelial injury sites to assure wound healing, simultaneously dampening the proinflammatory effect of complement to limit bystander tissue damage.

Atypical hemolytic uremic syndrome (aHUS) is characterized by genetic and acquired abnormalities of the complement system leading to alternative pathway (AP) overactivation and by glomerular endothelial damage, thrombosis, and mechanical hemolysis. Mutations per se are not sufficient to induce aHUS, and nonspecific primary triggers are required for disease manifestation. We investigated whether hemolysis-derived heme contributes to aHUS pathogenesis. We confirmed that heme activates complement AP in normal human serum, releasing C3a, C5a, and sC5b9. We demonstrated that heme-exposed endothelial cells also activate the AP, resulting in cell-bound C3 and C5b9. This was exacerbated in aHUS by genetic abnormalities associated with AP overactivation. Heme interacted with C3 close to the thioester bond, induced homophilic C3 complexes, and promoted formation of an overactive C3/C5 convertase. Heme induced decreased membrane cofactor protein (MCP) and decay-accelerating factor (DAF) expression on endothelial cells, giving Factor H (FH) a major role in complement regulation. Finally, heme promoted a rapid exocytosis of Weibel-Palade bodies, with membrane expression of P-selectin known to bind C3b and trigger the AP, and the release of the prothrombotic von Willebrand factor. These results strongly suggest that hemolysis-derived heme represents a common secondary hit amplifying endothelial damage and thrombosis in aHUS.

Abzymes are immunoglobulins endowed with enzymatic activities. The catalytic activity of an abzyme resides in the variable domain of the antibody, which is constituted by the close spatial arrangement of amino acid residues involved in catalysis. The origin of abzymes is conferred by the innate diversity of the immunoglobulin gene repertoire. Under deregulated immune conditions, as in autoimmune diseases, the generation of abzymes to self-antigens could be deleterious. Technical advancement in the ability to generate monoclonal antibodies has been exploited in the generation of abzymes with defined specificities and activities. Therapeutic applications of abzymes are being investigated with the generation of monoclonal abzymes against several pathogenesis-associated antigens. Here, we review the different contexts in which abzymes are generated, and we discuss the relevance of monoclonal abzymes for the treatment of human diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The evaluation of antibody avidity by elution with chaotropic agents is a frequently used approach in research and diagnostics. It provides important information on the functional relevance of antibodies. However, in the literature, there is a large heterogeneity in the experimental settings for the determination of this important parameter. Here, we demonstrate that antibody concentration and the choice of chaotropic agent are critical for the reliable estimation of antibody avidity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Polyspecific antibodies represent a significant fraction of the antibody repertoires in healthy animals and humans. Interestingly, certain antibodies only acquire a polyspecific antigen-binding behavior after exposure to protein-modifying conditions, such as those found at inflammation sites, or used in small- and large-scale immunoglobulin purification. This phenomenon is referred to as "criptic polyspecificity". In the present study, we compare the potential of different chemical agents to induce IgG polyspecificity. Depending on the treatment used, quantitative and qualitative differences in the recognition of individual antigens from a standard panel were observed. Antibodies with cryptic polyspecificity utilized common mechanisms for the recognition of structurally unrelated antigens when exposed to a particular inductor of polyspecificity. Our study contributes to the understanding of the mechanisms underlying the cryptic polyspecificity., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Replacement therapy with exogenous factor VIII (FVIII) to treat hemorrhages induces anti-FVIII inhibitory immunoglobulin G in up to 30% of patients with hemophilia A. Chronic inflammation associated with recurrent bleedings is a proposed risk factor for FVIII inhibitor development. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory activity. Here, we demonstrate that induction of HO-1 before FVIII administration drastically reduces the onset of the anti-FVIII humoral immune response. The protective effect was specific for HO-1 because it was reproduced on administration of the end products of HO-1 activity, carbon monoxide, and bilirubin, and prevented by the pharmacologic inhibition of HO-1 using tin mesoporphyrin IX. HO-1 induction was associated with decreased major histocompatibility complex class II expression by splenic antigen-presenting cells and reduced T-cell proliferation. Triggering the endogenous anti-inflammatory machinery before FVIII administration may represent a novel therapeutic option for preventing the development of FVIII inhibitors in hemophilia A patients.

Complement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D(254)G and K(325)N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.

Reactive oxygen species (ROS) released from activated phagocytes are involved in the innate immune defense against pathogens. However, when released in excess and when the antioxidant systems are impaired, ROS may induce cellular and tissue damage and dissociation of iron ions or iron containing compounds (heme) from protein-bound state. Free iron ions and free heme are prooxidative. Immunoglobulins usually perform their biological functions at sites of inflammation, where they may encounter reactive oxygen species and/or redox active compounds. It has been demonstrated that the exposure of some antibodies to heme, to transition metal ions or to reactive oxygen species induces an appearance of new binding specificities for various autoantigens. This review article is devoted to the interplay between redox active agents and antibodies. The biological significance of the appearance of new antigen binding specificities on antibodies after exposure to redox-active agents is discussed.

A fraction of antibodies present in all healthy individuals begins to recognize large number of self-antigens only after a transient exposure to certain protein-destabilizing conditions, including low or high pH, high salt concentration, chaotropic factors and redox-active agents. Recent findings on the molecular mechanisms of the inducible antibody auto-reactivity, obtained by using kinetic and thermodynamic analysis on the interactions of single monoclonal antibody with its cognate antigen are discussed in the review.