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Bharath Wootla,
Olivier D Christophe,
Ankit Mahendra,
Jordan D Dimitrov, Yohann Repessé,
Véronique Ollivier,
Alain Friboulet,
Annie Borel-Derlon,
Hervé Levesque,
Jeanne-Yvonne Borg,
Sebastien Andre,
Jagadeesh Bayry,
Thierry Calvez,
Srinivas V Kaveri,
Sébastien Lacroix-Desmazes
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ABSTRACT: Factor VIII (FVIII) is a glycoprotein that plays an important role in the intrinsic pathway of coagulation. In circulation, FVIII is protected upon binding to von Willebrand factor (VWF), a chaperone molecule that regulates its half-life, distribution, and activity. Despite the biological significance of this interaction, its molecular mechanisms are not fully characterized. We determined the equilibrium and activation thermodynamics of the interaction between FVIII and VWF. The equilibrium affinity determined by surface plasmon resonance was temperature-dependent with a value of 0.8 nM at 35 °C. The FVIII-VWF interaction was characterized by very fast association (8.56 × 10(6) M(-1) s(-1)) and fast dissociation (6.89 × 10(-3) s(-1)) rates. Both the equilibrium association and association rate constants, but not the dissociation rate constant, were dependent on temperature. Binding of FVIII to VWF was characterized by favorable changes in the equilibrium and activation entropy (TΔS° = 89.4 kJ/mol, and -TΔS(++) = -8.9 kJ/mol) and unfavorable changes in the equilibrium and activation enthalpy (ΔH° = 39.1 kJ/mol, and ΔH(++) = 44.1 kJ/mol), yielding a negative change in the equilibrium Gibbs energy. Binding of FVIII to VWF in solid-phase assays demonstrated a high sensitivity to acidic pH and a sensitivity to ionic strength. Our data indicate that the interaction between FVIII and VWF is mediated mainly by electrostatic forces, and that it is not accompanied by entropic constraints, suggesting the absence of conformational adaptation but the presence of rigid "pre-optimized" binding surfaces.
Biochemistry 05/2012; 51(20):4108-16. · 3.42 Impact Factor
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The Journal of allergy and clinical immunology 02/2012; 129(4):1172-3; author reply 1174-5. · 9.17 Impact Factor
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Bharath Wootla,
Olivier D Christophe,
Ankit Mahendra,
Jordan D Dimitrov, Yohann Repessé,
Véronique Ollivier,
Alain Friboulet,
Annie Borel-Derlon,
Hervé Levesque,
Jeanne-Yvonne Borg,
Sebastien Andre,
Jagadeesh Bayry,
Thierry Calvez,
Srinivas V Kaveri,
Sébastien Lacroix-Desmazes
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ABSTRACT: 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.
Blood 02/2011; 117(7):2257-64. · 9.90 Impact Factor
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ABSTRACT: A large proportion of hemophilia A patients who receive replacement therapy, develop an immune response toward the infused factor VIII (FVIII). In this review, we discuss recent progress in several aspects of the anti-FVIII immune response, focusing on the sites of FVIII endocytosis (marginal zone of the spleen and bleeding site), the type of antigen-presenting cells (dendritic cells, macrophages and B cells) and endocytic receptors, implicated in FVIII presentation to T cells during primary and secondary immune response. Although it is becoming increasingly clear that regulatory T cells are involved in FVIII tolerance in healthy subjects and potentially in patients without inhibitors, we would like to demonstrate that little is known about the different T cells subsets and the cytokines network, which are also crucial for the development of allo- and autoimmune diseases. As more information on these issues becomes available, a better understanding of the role of each immune cells compartment in promoting FVIII tolerance or inhibitors development might lead to new strategies to promote FVIII tolerance in hemophilia A patients.
Clinical Reviews in Allergy & Immunology 02/2009; 37(2):105-13. · 3.68 Impact Factor
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Suryasarathi Dasgupta,
Ana Maria Navarrete,
Sebastien André,
Bharath Wootla,
Sandrine Delignat, Yohann Repessé,
Jagadeesh Bayry,
Antonino Nicoletti,
Evgueni L Saenko,
Roseline d'Oiron,
Marc Jacquemin,
Jean-Marie Saint-Remy,
Srini V Kaveri,
Sebastien Lacroix-Desmazes
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ABSTRACT: The development of factor VIII (FVIII) inhibitors remains the major hurdle in the clinical management of patients with hemophilia A. FVIII uptake by professional antigen-presenting cells (APC) is the first step involved in initiation of immune responses to FVIII. Studies on FVIII catabolism have highlighted the role played by CD91/LRP as a potential target for increasing FVIII half-life in patients and prolonging treatment efficiency. We investigated the involvement of CD91 in FVIII endocytosis by human dendritic cells (DC), a model of professional APC.
Immature DC were generated from circulating monocytes from healthy donors. Surface expression of CD91 was assessed by flow cytometry. Uptake of fluorescein isothiocyanate-conjugated ligands by immature DC was studied in the presence of various blocking agents.
CD91 was expressed on approximately 20% of DC and mediated the internalization of its model ligand, alpha2-macroglobulin. DC internalized FVIII and activated a human FVIII-specific T-cell clone in a dose-dependent manner. FVIII uptake by DC and subsequent T-cell activation were not inhibited by receptor-associated protein.
Our results indicate that CD91 and other members of the LDL receptor family are not strongly implicated in FVIII internalization by monocyte-derived DC, and suggest the involvement of alternative divalent ion-dependent endocytic receptors.
Haematologica 02/2008; 93(1):83-9. · 6.42 Impact Factor
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ABSTRACT: Up to 33% of patients with severe haemophilia A develop inhibitory antibodies to factor VIM (FVIII) that can significantly impair treatment with FVIII. The plasma protein von Willebrand factor (VWF) binds to FVIII and is known to be important for the functioning of FVIII. Accumulating data suggest that VWF may also be important for reducing the immunogenicity of therapeutically administered FVIII in patients with haemophilia A. Although contradictory results have been reported for studies in patients, studies in mice have shown that the levels of FVIII-binding antibodies induced following treatment with recombinant FVIII (rFVIII) are higher than those following treatment with plasma-derived FVIII preparations containing VWF, and that the addition of VWF to rFVIII reduces the levels of FVIII-binding antibodies induced. In in vitro studies, VWF has been shown to inhibit both the uptake of FVIII by immature dendritic cells and the activation of FVIII-specificT-cells in a dose-dependent manner. However, recombinant VWF (rVWF) lacking the FVIII-binding domain did not inhibit T-cell activation. These data suggest that VWF may reduce the immunogenicity of FVIII by inhibiting the uptake of FVIII by antigen presenting cells, the first step in the development of an immune response against a foreign antigen. Further studies are required to confirm the applicability of these results to patients with haemophilia. If confirmed, these data would encourage the use of VWF in conjunction with FVIII in the management of patients with haemophilia A.
Thrombosis Research 01/2008; 122 Suppl 2:S3-6. · 2.44 Impact Factor
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Suryasarathi Dasgupta, Yohann Repessé,
Jagadeesh Bayry,
Ana-Maria Navarrete,
Bharath Wootla,
Sandrine Delignat,
Theano Irinopoulou,
Caroline Kamaté,
Jean-Marie Saint-Remy,
Marc Jacquemin,
Peter J Lenting,
Annie Borel-Derlon,
Srinivas V Kaveri,
Sébastien Lacroix-Desmazes
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ABSTRACT: Von Willebrand factor (VWF) is a chaperone molecule for procoagulant factor VIII (FVIII). Its role in the reduction of the immunogenicity of therapeutic FVIII in patients with hemophilia A has been evoked but lacks clear cellular and molecular rationale. Here, we demonstrate that VWF protects FVIII from being endocytosed by human dendritic cells (DCs) and subsequently presented to FVIII-specific T cells. The immunoprotective effect of VWF requires a physical interaction with FVIII because the endocytosis of FVIII was significantly restored on hindering the formation of the VWF-FVIII complex. Interestingly, VWF had no direct inhibitory effect either on the ability of DCs to present antigenic peptides or on the activation potency of CD4+ T cells. We thus 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.
Blood 02/2007; 109(2):610-2. · 9.90 Impact Factor
