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Pete Lollar - One of the best experts on this subject based on the ideXlab platform.
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structure of the Factor VIII c2 domain in a ternary complex with 2 inhibitor antibodies reveals classical and nonclassical epitopes
Blood, 2013Co-Authors: Justin D Walter, Pete Lollar, John F Healey, Rachel A Werther, Shannon L Meeks, Caileen M Brison, Rebecca K Cragerud, Clint P. SpiegelAbstract:The Factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following Factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by 2 classes of anti-C2 inhibitory antibodies that bind to Factor VIII simultaneously was investigated by x-ray crystallography. The C2 domain/3E6 FAB/G99 FAB ternary complex illustrates that each antibody recognizes epitopes on opposing faces of the Factor VIII C2 domain. The 3E6 epitope forms direct contacts to the C2 domain at 2 loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, whereas the G99 epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282, and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charge present on both C2 epitopes and complementary negative charge on each antibody. A new model of membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. These results illustrate the potential complexities of the polyclonal anti-Factor VIII immune response and further define the “classical” and “nonclassical” types of antibody inhibitors against the Factor VIII C2 domain.
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high dose Factor VIII inhibits Factor VIII specific memory b cells in hemophilia a with Factor VIII inhibitors
Blood, 2005Co-Authors: Christina Hausl, Pete Lollar, Christopher B. Doering, Rafi U Ahmad, Maria Sasgary, Gunter Richter, Hans Peter Schwarz, Peter Turecek, Birgit M ReipertAbstract:Hemophilia A in its severe form is a life-threatening hemorrhagic disease that is caused by mutations in the Factor VIII (FVIII) gene (symbol F8). About 25% of patients who receive replacement therapy develop neutralizing antibodies that inhibit the function of substituted FVIII. Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. Little is known, however, about the immunologic mechanisms that cause the downmodulation of anti-FVIII antibodies by high doses of FVIII. We report that high doses of FVIII inhibit the restimulation of FVIIIspecific memory B cells and their differentiation into antibody-secreting plasma cells in vitro and in vivo in a murine model of hemophiliaA. The inhibition of memory B-cell responses is irreversible and not mediated by FVIII-specific T cells. Furthermore, it seems to involve the activation of caspases. We conclude that the inhibition of FVIII-specific memory B cells might be an early event in the down-modulation of anti-FVIII antibodies in patients with hemophilia A who receive high doses of FVIII. (Blood. 2005;106:3415-3422)
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identification of porcine coagulation Factor VIII domains responsible for high level expression via enhanced secretion
Journal of Biological Chemistry, 2004Co-Authors: Christopher B. Doering, Ernest T. Parker, John F Healey, Rachel T Barrow, Pete LollarAbstract:Blood coagulation Factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human Factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine Factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine Factor VIII. Novel hybrid human/porcine Factor VIII molecules were constructed to identify porcine Factor VIII domains that confer high level expression. Hybrid human/porcine Factor VIII constructs containing the porcine Factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine Factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine Factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human Factor VIII. Additionally, hybrid murine/porcine Factor VIII constructs containing the porcine Factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine Factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine Factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.
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high level expression of recombinant porcine coagulation Factor VIII
Journal of Biological Chemistry, 2002Co-Authors: Christopher B. Doering, Ernest T. Parker, John F Healey, Rachel T Barrow, Pete LollarAbstract:Recombinant human Factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine Factor VIII molecule. Recombinant B-domain-deleted porcine Factor VIII expression levels are 10- to 14-fold greater than recombinant B-domain-deleted human Factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units x 10(6) cells(-1) x 24 h(-1) was observed from a baby hamster kidney-derived cell line stably expressing recombinant porcine Factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand Factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state Factor VIII mRNA levels. The identification of sequence(s) in porcine Factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit Factor VIII expression.
J A Van Mourik - One of the best experts on this subject based on the ideXlab platform.
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a human alloantibody interferes with binding of Factor ixa to the Factor VIII light chain
Blood, 1998Co-Authors: K Fijnvandraat, Koen Mertens, J A Van Mourik, Patrick H N Celie, Ellen A M Turenhout, J Ten W Cate, M Peters, Jan VoorbergAbstract:Inhibitory antibodies directed against Factor VIII develop in a substantial number of patients with hemophilia A as a consequence of Factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of Factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled Factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized Factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a Factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of Factor IXa to immobilized Factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit Factor VIII activity by interfering with Factor IXa–Factor VIIIa complex assembly.
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the sequence glu1811 lys1818 of human blood coagulation Factor VIII comprises a binding site for activated Factor ix
Journal of Biological Chemistry, 1996Co-Authors: Peter J Lenting, M J S H Donath, J W H P Van De Loo, J A Van MourikAbstract:In previous studies have shown that the interaction between Factor IXa and VIII involves the light chain of Factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the Factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant Factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of Factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the Factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the Factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of Factor IXa to immobilized Factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit Factor X activation by Factor IXa in the presence of Factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the Factor IX zymogen and its activated form, Factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete Factor VIII light chain displays preferential binding to Factor IXa. In conclusion, our results demonstrate that peptides consisting of the Factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a Factor IXa binding site that is essential for the assembly of the Factor X-activating Factor IXa-Factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated Factor IX.
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identification of a binding site for blood coagulation Factor ixa on the light chain of human Factor VIII
Journal of Biological Chemistry, 1994Co-Authors: Peter J Lenting, M J S H Donath, J A Van MourikAbstract:The interaction between human Factor IXa and Factor VIII or its constituent units was investigated. Equilibrium binding studies were performed employing Factor VIII light chain that was immobilized on a monoclonal antibody. Factor VIII light chain was observed to bind Factor IXa with high affinity (Kd = 14.8 +/- 3.2 nM) and approximately 1:1 stoichiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution effectively inhibited binding of Factor IXa to the immobilized light chain (Ki = 10.9 +/- 1.9 nM). The isolated Factor VIII light chain and the Factor VIII heterodimer were equally effective in Factor IXa binding, demonstrating that this interaction did not require the Factor VIII heavy chain. Factor Xa and activated Protein C were found to be inefficient (Ki > or = 1.2 microM) in competing with Factor IXa, indicating that the high affinity for Factor VIII light chain was unique for Factor IXa. The Factor IXa-Factor VIII light chain interaction was inhibited by von Willebrand Factor, but this effect was abolished by cleavage of the Factor VIII light chain by thrombin. An antibody that inhibits von Willebrand Factor-Factor VIII complex formation did not compete for Factor IXa binding. In contrast, association of Factor IXa with the Factor VIII light chain was inhibited by an antibody directed against the Factor VIII region Gln1778-Asp1840. We propose that this sequence provides a Factor IXa binding site and that its exposure requires dissociation of the Factor VIII-von Willebrand Factor complex.
Ivan Peyron - One of the best experts on this subject based on the ideXlab platform.
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the c1 and c2 domains of blood coagulation Factor VIII mediate its endocytosis by dendritic cells
Haematologica, 2017Co-Authors: Bagirath Gangadharan, Mathieu Ing, Sandrine Delignat, Ivan PeyronAbstract:The development of inhibitory antibodies to therapeutic Factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-Factor VIII immune response is Factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naive CD4+ T cells. Recent studies indicate a role for the C1 domain in Factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic Factor VIII uptake. Co-incubation of Factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced Factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished Factor VIII immunogenicity in Factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent Factor VIII immunogenicity in mice. BO2C11 prevents Factor VIII binding to von Willebrand Factor, thus potentially biasing Factor VIII immunogenicity by perturbing its half-life. Interestingly, a Factor VIIIY1680C mutant, that does not bind von Willebrand Factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in Factor VIII-deficient mice. Co-incubation of Factor VIIIY1680C with BO2C11, however, resulted in decreased Factor VIII immunogenicity in vivo. In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand Factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of Factor VIII are implicated in immunogenic Factor VIII uptake, at least in vitro. Conversely, in vivo, the binding to endogenous von Willebrand Factor masks the reducing effect of mutations in the C domains on Factor VIII immunogenicity.
Christopher B. Doering - One of the best experts on this subject based on the ideXlab platform.
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high dose Factor VIII inhibits Factor VIII specific memory b cells in hemophilia a with Factor VIII inhibitors
Blood, 2005Co-Authors: Christina Hausl, Pete Lollar, Christopher B. Doering, Rafi U Ahmad, Maria Sasgary, Gunter Richter, Hans Peter Schwarz, Peter Turecek, Birgit M ReipertAbstract:Hemophilia A in its severe form is a life-threatening hemorrhagic disease that is caused by mutations in the Factor VIII (FVIII) gene (symbol F8). About 25% of patients who receive replacement therapy develop neutralizing antibodies that inhibit the function of substituted FVIII. Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. Little is known, however, about the immunologic mechanisms that cause the downmodulation of anti-FVIII antibodies by high doses of FVIII. We report that high doses of FVIII inhibit the restimulation of FVIIIspecific memory B cells and their differentiation into antibody-secreting plasma cells in vitro and in vivo in a murine model of hemophiliaA. The inhibition of memory B-cell responses is irreversible and not mediated by FVIII-specific T cells. Furthermore, it seems to involve the activation of caspases. We conclude that the inhibition of FVIII-specific memory B cells might be an early event in the down-modulation of anti-FVIII antibodies in patients with hemophilia A who receive high doses of FVIII. (Blood. 2005;106:3415-3422)
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identification of porcine coagulation Factor VIII domains responsible for high level expression via enhanced secretion
Journal of Biological Chemistry, 2004Co-Authors: Christopher B. Doering, Ernest T. Parker, John F Healey, Rachel T Barrow, Pete LollarAbstract:Blood coagulation Factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human Factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine Factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine Factor VIII. Novel hybrid human/porcine Factor VIII molecules were constructed to identify porcine Factor VIII domains that confer high level expression. Hybrid human/porcine Factor VIII constructs containing the porcine Factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine Factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine Factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human Factor VIII. Additionally, hybrid murine/porcine Factor VIII constructs containing the porcine Factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine Factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine Factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.
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high level expression of recombinant porcine coagulation Factor VIII
Journal of Biological Chemistry, 2002Co-Authors: Christopher B. Doering, Ernest T. Parker, John F Healey, Rachel T Barrow, Pete LollarAbstract:Recombinant human Factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine Factor VIII molecule. Recombinant B-domain-deleted porcine Factor VIII expression levels are 10- to 14-fold greater than recombinant B-domain-deleted human Factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units x 10(6) cells(-1) x 24 h(-1) was observed from a baby hamster kidney-derived cell line stably expressing recombinant porcine Factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand Factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state Factor VIII mRNA levels. The identification of sequence(s) in porcine Factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit Factor VIII expression.
Peter J Lenting - One of the best experts on this subject based on the ideXlab platform.
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the sequence glu1811 lys1818 of human blood coagulation Factor VIII comprises a binding site for activated Factor ix
Journal of Biological Chemistry, 1996Co-Authors: Peter J Lenting, M J S H Donath, J W H P Van De Loo, J A Van MourikAbstract:In previous studies have shown that the interaction between Factor IXa and VIII involves the light chain of Factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the Factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant Factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of Factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the Factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the Factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of Factor IXa to immobilized Factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit Factor X activation by Factor IXa in the presence of Factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the Factor IX zymogen and its activated form, Factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete Factor VIII light chain displays preferential binding to Factor IXa. In conclusion, our results demonstrate that peptides consisting of the Factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a Factor IXa binding site that is essential for the assembly of the Factor X-activating Factor IXa-Factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated Factor IX.
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identification of a binding site for blood coagulation Factor ixa on the light chain of human Factor VIII
Journal of Biological Chemistry, 1994Co-Authors: Peter J Lenting, M J S H Donath, J A Van MourikAbstract:The interaction between human Factor IXa and Factor VIII or its constituent units was investigated. Equilibrium binding studies were performed employing Factor VIII light chain that was immobilized on a monoclonal antibody. Factor VIII light chain was observed to bind Factor IXa with high affinity (Kd = 14.8 +/- 3.2 nM) and approximately 1:1 stoichiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution effectively inhibited binding of Factor IXa to the immobilized light chain (Ki = 10.9 +/- 1.9 nM). The isolated Factor VIII light chain and the Factor VIII heterodimer were equally effective in Factor IXa binding, demonstrating that this interaction did not require the Factor VIII heavy chain. Factor Xa and activated Protein C were found to be inefficient (Ki > or = 1.2 microM) in competing with Factor IXa, indicating that the high affinity for Factor VIII light chain was unique for Factor IXa. The Factor IXa-Factor VIII light chain interaction was inhibited by von Willebrand Factor, but this effect was abolished by cleavage of the Factor VIII light chain by thrombin. An antibody that inhibits von Willebrand Factor-Factor VIII complex formation did not compete for Factor IXa binding. In contrast, association of Factor IXa with the Factor VIII light chain was inhibited by an antibody directed against the Factor VIII region Gln1778-Asp1840. We propose that this sequence provides a Factor IXa binding site and that its exposure requires dissociation of the Factor VIII-von Willebrand Factor complex.
