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Alexander B. Meijer - One of the best experts on this subject based on the ideXlab platform.
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unique surface exposed hydrophobic residues in the C1 Domain of factor viii contribute to cofactor function and von willebrand factor binding
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Mariette Boonspijker, Alexander B. MeijerAbstract:Background The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. Objective The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. Methods Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. Results Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. Conclusion The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life cycle.
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Unique surface‐exposed hydrophobic residues in the C1 Domain of factor VIII contribute to cofactor function and von Willebrand factor binding
Journal of thrombosis and haemostasis : JTH, 2019Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Mariëtte Boon-spijker, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Alexander B. MeijerAbstract:BACKGROUND: The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and Von Willebrand Factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. OBJECTIVE: The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. METHODS: Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced into alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. RESULTS: Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, i.e.: F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130 and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. CONCLUSION: The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life-cycle.
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Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 Domain.
The international journal of biochemistry & cell biology, 2017Co-Authors: Lydia Castro-núñez, Carmen Van Der Zwaan, Koen Mertens, Alexander B. Meijer, Esther Bloem, Johanna M. Koornneef, Mariska G. Rondaij, Henriet MeemsAbstract:Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 Domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 Domain that have been implicated in lipid binding.
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Factor VIII C1-Domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake
The Journal of biological chemistry, 2013Co-Authors: Esther Bloem, Koen Mertens, Maartje Van Den Biggelaar, Aleksandra Wroblewska, Jan Voorberg, Johan H. Faber, Marianne Kjalke, Henning R. Stennicke, Alexander B. MeijerAbstract:The C1 Domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent “hot spot” on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091–2104 and 2157–2162 from hydrogen-deuterium exchange. These comprise the two C1 Domain spikes 2092–2093 and 2158–2159. Spike 2092–2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158–2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 Domain spikes 2092–2093 and 2158–2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 Domain in FVIII biology.
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Modification of an exposed loop in the C1 Domain reduces immune responses to factor VIII in hemophilia A mice
Blood, 2012Co-Authors: Aleksandra Wroblewska, Maartje Van Den Biggelaar, Simon D. Van Haren, Eszter Herczenik, Paul H. P. Kaijen, Aleksandra Ruminska, Sheng-yu Jin, X. Long Zheng, Anja Ten Brinke, Alexander B. MeijerAbstract:Development of neutralizing Abs to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by APCs can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional APCs is significantly blocked by mAb KM33, directed toward the C1 Domain of FVIII. We created a C1 Domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and retained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine BM-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/K2092A/F2093A have significantly lower anti-FVIII Ab titers and FVIII-specific CD4+ T-cell responses compared with mice treated with wild-type FVIII. These data show that alanine substitutions at positions 2090, 2092, and 2093 reduce the immunogenicity of FVIII. According to our findings we hypothesize that FVIII variants displaying a reduced uptake by APCs provide a novel therapeutic approach to reduce inhibitor development in hemophilia A.
Maartje Van Den Biggelaar - One of the best experts on this subject based on the ideXlab platform.
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unique surface exposed hydrophobic residues in the C1 Domain of factor viii contribute to cofactor function and von willebrand factor binding
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Mariette Boonspijker, Alexander B. MeijerAbstract:Background The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. Objective The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. Methods Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. Results Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. Conclusion The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life cycle.
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Unique surface‐exposed hydrophobic residues in the C1 Domain of factor VIII contribute to cofactor function and von Willebrand factor binding
Journal of thrombosis and haemostasis : JTH, 2019Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Mariëtte Boon-spijker, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Alexander B. MeijerAbstract:BACKGROUND: The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and Von Willebrand Factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. OBJECTIVE: The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. METHODS: Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced into alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. RESULTS: Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, i.e.: F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130 and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. CONCLUSION: The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life-cycle.
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Factor VIII C1-Domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake
The Journal of biological chemistry, 2013Co-Authors: Esther Bloem, Koen Mertens, Maartje Van Den Biggelaar, Aleksandra Wroblewska, Jan Voorberg, Johan H. Faber, Marianne Kjalke, Henning R. Stennicke, Alexander B. MeijerAbstract:The C1 Domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent “hot spot” on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091–2104 and 2157–2162 from hydrogen-deuterium exchange. These comprise the two C1 Domain spikes 2092–2093 and 2158–2159. Spike 2092–2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158–2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 Domain spikes 2092–2093 and 2158–2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 Domain in FVIII biology.
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Modification of an exposed loop in the C1 Domain reduces immune responses to factor VIII in hemophilia A mice
Blood, 2012Co-Authors: Aleksandra Wroblewska, Maartje Van Den Biggelaar, Simon D. Van Haren, Eszter Herczenik, Paul H. P. Kaijen, Aleksandra Ruminska, Sheng-yu Jin, X. Long Zheng, Anja Ten Brinke, Alexander B. MeijerAbstract:Development of neutralizing Abs to blood coagulation factor VIII (FVIII) provides a major complication in hemophilia care. In this study we explored whether modulation of the uptake of FVIII by APCs can reduce its intrinsic immunogenicity. Endocytosis of FVIII by professional APCs is significantly blocked by mAb KM33, directed toward the C1 Domain of FVIII. We created a C1 Domain variant (FVIII-R2090A/K2092A/F2093A), which showed only minimal binding to KM33 and retained its activity as measured by chromogenic assay. FVIII-R2090A/K2092A/F2093A displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine BM-derived dendritic cells. We subsequently investigated the ability of this variant to induce an immune response in FVIII-deficient mice. We show that mice treated with FVIII-R2090A/K2092A/F2093A have significantly lower anti-FVIII Ab titers and FVIII-specific CD4+ T-cell responses compared with mice treated with wild-type FVIII. These data show that alanine substitutions at positions 2090, 2092, and 2093 reduce the immunogenicity of FVIII. According to our findings we hypothesize that FVIII variants displaying a reduced uptake by APCs provide a novel therapeutic approach to reduce inhibitor development in hemophilia A.
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uptake of blood coagulation factor viii by dendritic cells is mediated via its C1 Domain
The Journal of Allergy and Clinical Immunology, 2012Co-Authors: Eszter Herczenik, Maartje Van Den Biggelaar, Alexander B. Meijer, Aleksandra Wroblewska, Simon D. Van Haren, Paul H. P. Kaijen, Anja Ten Brinke, Luisa Martinezpomares, Jan VoorbergAbstract:Background The uptake and processing of blood coagulation factor VIII (FVIII) by antigen-presenting cells and the subsequent presentation of FVIII-derived peptides to CD4 + T cells direct the immune response to FVIII in patients with hemophilia A. Multiple receptors including mannose receptor and low-density lipoprotein receptor–related protein-1 (LRP1) have been implicated in FVIII uptake. Objective This work studies the involvement of receptor candidates in FVIII uptake by dendritic cells (DCs). Furthermore, we explore FVIII residues that mediate endocytosis. Methods FVIII uptake was performed with human monocyte–derived and murine bone marrow–derived DCs. To investigate FVIII endocytosis, competition assays with soluble receptor ligands, binding studies with recombinant receptor fragments, and small-interfering RNA–induced gene silencing were performed. In addition, FVIII-targeting monoclonal antibodies KM33 and VK34 were used. To confirm in vitro results, hemophilic E17 knockout mice were pretreated with antibodies prior to FVIII injections and anti-FVIII titers were determined. Results Upon treatment of DCs with mannan or LRP ligand α2-macroglobulin, we observed only a minor decrease in FVIII internalization. In addition, small interfering RNA–mediated knockdown of LRP, mannose receptor, or DC-SIGN expression in monocyte-derived dendritic cells did not prevent FVIII uptake. Binding studies using Fc chimeras revealed that LRP, DC-SIGN, and mannose receptor can bind to FVIII; however, we did not observe a critical role for these receptors in FVIII uptake. Previous studies have shown that human antibodies targeting the C1 (KM33) and A2 (VK34) Domains of FVIII interfere with binding to endocytic receptors. Preincubation of FVIII with VK34 did not influence FVIII uptake; however, KM33 completely inhibited FVIII endocytosis by both monocyte-derived dendritic cells and bone marrow-derived dendritic cells. Accordingly, anti-FVIII antibody titers were greatly reduced following the preadministration of KM33 in vivo . Conclusion Together, our observations emphasize the physiological significance of KM33-targeted residues within the C1 Domain in the uptake of FVIII by DCs in vitro and in vivo .
Koen Mertens - One of the best experts on this subject based on the ideXlab platform.
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unique surface exposed hydrophobic residues in the C1 Domain of factor viii contribute to cofactor function and von willebrand factor binding
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Mariette Boonspijker, Alexander B. MeijerAbstract:Background The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. Objective The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. Methods Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. Results Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. Conclusion The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life cycle.
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Unique surface‐exposed hydrophobic residues in the C1 Domain of factor VIII contribute to cofactor function and von Willebrand factor binding
Journal of thrombosis and haemostasis : JTH, 2019Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Mariëtte Boon-spijker, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Alexander B. MeijerAbstract:BACKGROUND: The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and Von Willebrand Factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. OBJECTIVE: The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. METHODS: Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced into alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. RESULTS: Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, i.e.: F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130 and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. CONCLUSION: The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life-cycle.
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Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 Domain.
The international journal of biochemistry & cell biology, 2017Co-Authors: Lydia Castro-núñez, Carmen Van Der Zwaan, Koen Mertens, Alexander B. Meijer, Esther Bloem, Johanna M. Koornneef, Mariska G. Rondaij, Henriet MeemsAbstract:Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 Domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 Domain that have been implicated in lipid binding.
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Factor VIII C1-Domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake
The Journal of biological chemistry, 2013Co-Authors: Esther Bloem, Koen Mertens, Maartje Van Den Biggelaar, Aleksandra Wroblewska, Jan Voorberg, Johan H. Faber, Marianne Kjalke, Henning R. Stennicke, Alexander B. MeijerAbstract:The C1 Domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent “hot spot” on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091–2104 and 2157–2162 from hydrogen-deuterium exchange. These comprise the two C1 Domain spikes 2092–2093 and 2158–2159. Spike 2092–2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158–2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 Domain spikes 2092–2093 and 2158–2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 Domain in FVIII biology.
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Modification of An Interactive Surface in the C1 Domain Reduces Immune Responses to FVIII in a Mouse Model of Hemophilia A
Blood, 2011Co-Authors: Aleksandra Wroblewska, Koen Mertens, Simon D. Van Haren, Eszter Herczenik, Paul H. P. Kaijen, Anja Ten Brinke, Aleksandra Rusiecka, Jan VoorbergAbstract:Abstract 199 The X-chromosome-linked bleeding disorder hemophilia A is caused by the absence or dysfunction of blood coagulation factor VIII (FVIII) which can be corrected by regular intravenous administration of FVIII. Development of antibodies directed against FVIII (referred to as FVIII inhibitors) is common complication in hemophilia care. The formation of FVIII-neutralizing antibodies in hemophilia A patients is initiated by the endocytosis of FVIII by professional antigen-presenting cells. Endocytosis of FVIII by human monocyte-derived dendritic cells can be significantly blocked by monoclonal antibody KM33, directed towards the C1 Domain of FVIII. We have previously shown that C1 Domain residues 2092 and 2093 are required for KM33 binding to FVIII (Meems et al., 2009). However, replacement of both these residues by Ala did not completely abolish KM33 binding. We created an additional C1 Domain variant which included an arginine at position 2090. A FVIII variant in which three residues, 2090, 2092 and 2093 were substituted by alanine, showed only minimal binding to KM33. Functional analysis revealed that this variant designated FVIII-2090/2092/2093 retains pro-coagulant activity as measured by a chromogenic assay. FVIII-2090/2092/2093 displayed a strongly reduced internalization by human monocyte-derived dendritic cells and macrophages, as well as murine bone marrow-derived dendritic cells. These results emphasize a role for C1 Domain residues 2090, 2092 and 2093 in FVIII endocytosis by antigen-presenting cells. We subsequently investigated the in vivo ability of this variant to induce inhibitors in FVIII −/− mice (E17KO). We show that E17KO mice treated with the FVIII variant have significantly lower anti-FVIII antibody titers when compared to mice treated with wild type FVIII. In accordance with these findings reduced numbers of FVIII-specific antibody-secreting cells were detected in the spleen of mice treated with FVIII-2090/2092/2093. Also, FVIII-specific CD4 + T cell responses of splenocytes derived from FVIII-2090/2092/2093 infused mice were greatly reduced when compared to that of splenocytes derived from wild type FVIII infused mice. These findings show that alanine substitutions at positions 2090, 2092 and 2093 result in a FVIII molecule that is significantly less immunogenic when compared to wild type FVIII. Collectively, our data suggest that FVIII variants displaying a reduced uptake by antigen-presenting cells show a reduced immunogenicity in vivo. Based on our findings we hypothesize that FVIII variants displaying a reduced uptake by antigen-presenting cells provide a novel therapeutic approach to reduce inhibitor development in patients with hemophilia A. Disclosures: No relevant conflicts of interest to declare.
Marcelo G Kazanietz - One of the best experts on this subject based on the ideXlab platform.
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characterization of ajh 836 a diacylglycerol lactone with selectivity for novel pkc isozymes
Journal of Biological Chemistry, 2018Co-Authors: Mariana Cooke, Xiaoling Zhou, Peter M Blumberg, Victoria Casadomedrano, Cynthia Lopezhaber, Martin J Baker, Rachana Garg, Marcelo G KazanietzAbstract:: Diacylglycerol (DAG) is a key lipid second messenger downstream of cellular receptors that binds to the C1 Domain in many regulatory proteins. Protein kinase C (PKC) isoforms constitute the most prominent family of signaling proteins with DAG-responsive C1 Domains, but six other families of proteins, including the chimaerins, Ras-guanyl nucleotide-releasing proteins (RasGRPs), and MunC13 isoforms, also play important roles. Their significant involvement in cancer, immunology, and neurobiology has driven intense interest in the C1 Domain as a therapeutic target. As with other classes of targets, however, a key issue is the establishment of selectivity. Here, using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) competition binding assays, we found that a synthetic DAG-lactone, AJH-836, preferentially binds to the novel PKC isoforms PKCδ and PKCϵ relative to classical PKCα and PKCβII. Assessment of intracellular translocation, a hallmark for PKC activation, revealed that AJH-836 treatment stimulated a striking preferential redistribution of PKCϵ to the plasma membrane relative to PKCα. Moreover, unlike with the prototypical phorbol ester phorbol 12-myristate 13-acetate (PMA), prolonged exposure of cells to AJH-836 selectively down-regulated PKCδ and PKCϵ without affecting PKCα expression levels. Biologically, AJH-836 induced major changes in cytoskeletal reorganization in lung cancer cells, as determined by the formation of membrane ruffles, via activation of novel PKCs. We conclude that AJH-836 represents a C1 Domain ligand with PKC-activating properties distinct from those of natural DAGs and phorbol esters. Our study supports the feasibility of generating selective C1 Domain ligands that promote novel biological response patterns.
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a novel cross talk in diacylglycerol signaling the rac gap β2 chimaerin is negatively regulated by protein kinase cδ mediated phosphorylation
Journal of Biological Chemistry, 2010Co-Authors: Erin Griner, Harald Mischak, Cecilia M Caino, Maria Soledad Sosa, Francheska Colongonzalez, Michael J Chalmers, Marcelo G KazanietzAbstract:Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 Domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for β2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of β2-chimaerin on Ser169 located in the SH2-C1 Domain linker region via protein kinase Cδ, which retained β2-chimaerin in the cytosol and prevented its C1 Domain-mediated translocation to membranes. Furthermore, despite the fact that Ser169 phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable β2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts β2-chimaerin relocalization and activation.
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p23 tmp21 differentially targets the rac gap β2 chimaerin and protein kinase c via their C1 Domains
Molecular Biology of the Cell, 2010Co-Authors: Hongbin Wang, Marcelo G KazanietzAbstract:The C1 Domains in protein kinase C (PKC) isozymes and other signaling molecules are responsible for binding the lipid second messenger diacylglycerol and phorbol esters, and for mediating translocation to membranes. Previous studies revealed that the C1 Domain in alpha- and beta-chimaerins, diacylglycerol-regulated Rac-GAPs, interacts with the endoplasmic reticulum/Golgi protein p23/Tmp21. Here, we found that p23/Tmp21 acts as a C1 Domain-docking protein that mediates perinuclear translocation of beta2-chimaerin. Glu227 and Leu248 in the beta2-chimaerin C1 Domain are crucial for binding p23/Tmp21 and perinuclear targeting. Interestingly, isolated C1 Domains from individual PKC isozymes differentially interact with p23/Tmp21. For PKCepsilon, it interacts with p23/Tmp21 specifically via its C1b Domain; however, this association is lost in response to phorbol esters. These results demonstrate that p23/Tmp21 acts as an anchor that distinctively modulates compartmentalization of C1 Domain-containing proteins, and it plays an essential role in beta2-chimaerin relocalization. Our study also highlights the relevance of C1 Domains in protein-protein interactions in addition to their well-established lipid-binding properties.
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p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains
Molecular biology of the cell, 2010Co-Authors: Hongbin Wang, Marcelo G KazanietzAbstract:The C1 Domains in protein kinase C (PKC) isozymes and other signaling molecules are responsible for binding the lipid second messenger diacylglycerol and phorbol esters, and for mediating translocation to membranes. Previous studies revealed that the C1 Domain in alpha- and beta-chimaerins, diacylglycerol-regulated Rac-GAPs, interacts with the endoplasmic reticulum/Golgi protein p23/Tmp21. Here, we found that p23/Tmp21 acts as a C1 Domain-docking protein that mediates perinuclear translocation of beta2-chimaerin. Glu227 and Leu248 in the beta2-chimaerin C1 Domain are crucial for binding p23/Tmp21 and perinuclear targeting. Interestingly, isolated C1 Domains from individual PKC isozymes differentially interact with p23/Tmp21. For PKCepsilon, it interacts with p23/Tmp21 specifically via its C1b Domain; however, this association is lost in response to phorbol esters. These results demonstrate that p23/Tmp21 acts as an anchor that distinctively modulates compartmentalization of C1 Domain-containing proteins, and it plays an essential role in beta2-chimaerin relocalization. Our study also highlights the relevance of C1 Domains in protein-protein interactions in addition to their well-established lipid-binding properties.
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Identification of an autoinhibitory mechanism that restricts C1 Domain-mediated activation of the Rac-GAP α2-chimaerin
The Journal of biological chemistry, 2008Co-Authors: Francheska Colon-gonzalez, Federico Coluccio Leskow, Marcelo G KazanietzAbstract:Abstract Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 Domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in α2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type α2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize α2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the α2-chimaerin C1 Domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders α2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of α2-chimaerin in a phospholipase C-dependent manner, but not of a C1 Domain mutant with reduced affinity for DAG (P216A-α2-chimaerin). Therefore, DAG generation and binding to the C1 Domain are required but not sufficient for epidermal growth factor-induced α2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of α2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, α2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.
Carmen Van Der Zwaan - One of the best experts on this subject based on the ideXlab platform.
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unique surface exposed hydrophobic residues in the C1 Domain of factor viii contribute to cofactor function and von willebrand factor binding
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Mariette Boonspijker, Alexander B. MeijerAbstract:Background The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. Objective The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. Methods Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. Results Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. Conclusion The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life cycle.
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Unique surface‐exposed hydrophobic residues in the C1 Domain of factor VIII contribute to cofactor function and von Willebrand factor binding
Journal of thrombosis and haemostasis : JTH, 2019Co-Authors: Małgorzata A. Przeradzka, Nadia Freato, Mariëtte Boon-spijker, Josse Van Galen, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Alexander B. MeijerAbstract:BACKGROUND: The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and Von Willebrand Factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 Domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 Domain, this implies that the FVIII C1 Domain comprises unique surface-exposed elements involved in VWF and FIXa interaction. OBJECTIVE: The aim of this study is to identify residues in the FVIII C1 Domain that contribute to VWF and FIXa binding. METHODS: Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 Domain. The identified residues were replaced into alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. RESULTS: Five surface-exposed hydrophobic residues unique to the FVIII C1 Domain, i.e.: F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130 and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. CONCLUSION: The surface-exposed hydrophobic residues in C1 Domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 Domain in FVIII life-cycle.
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Cellular uptake of coagulation factor VIII: Elusive role of the membrane-binding spikes in the C1 Domain.
The international journal of biochemistry & cell biology, 2017Co-Authors: Lydia Castro-núñez, Carmen Van Der Zwaan, Koen Mertens, Alexander B. Meijer, Esther Bloem, Johanna M. Koornneef, Mariska G. Rondaij, Henriet MeemsAbstract:Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 Domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 Domain that have been implicated in lipid binding.
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C1 Domain residues Lys 2092 and Phe 2093 are of major importance for the endocytic uptake of coagulation factor VIII.
The international journal of biochemistry & cell biology, 2011Co-Authors: Henriet Meems, Carmen Van Der Zwaan, Koen Mertens, Maartje Van Den Biggelaar, Mariska G. Rondaij, Alexander B. MeijerAbstract:Factor VIII (FVIII) catabolism has been demonstrated to involve LDL receptor-related protein (LRP). We have established that antibody fragment KM33 inhibits cofactor function of FVIII by interacting with the membrane binding region 2092-2093 of the C1 Domain. As KM33 also inhibits LRP-dependent uptake of FVIII, we now assessed the role of region 2092-2093 for LRP-dependent endocytosis. For this purpose, we employed functional fluorescent FVIII-YFP or -GFP derivatives and U87MG cells which express high levels of LRP. Confocal microscopy studies and flow cytometry analysis combined with siRNA technology showed that the fluorescent FVIII derivatives are indeed effectively internalized by U87MG cells in a LRP-dependent manner. Competition experiments employing an antagonist of the LDL receptor family members revealed that there is a cell surface binding event for FVIII, which is independent of LRP. Cell surface binding proved to be less effective for the FVIII-YFP variants K2092A, F2093A and K2092A/F2093A. Surface plasmon resonance analysis showed that these substitutions affect LRP binding as well. Finally, flow cytometry analysis revealed a major reduction of endocytic uptake of these FVIII-YFP variants. Our results demonstrate that C1 Domain residues 2092-2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding.