Hydroxylation of aspartic acid to erythro-@-aspartic acid (Hya) occurs in epidermal growth factor... more Hydroxylation of aspartic acid to erythro-@-aspartic acid (Hya) occurs in epidermal growth factor (EGF)like modules in numerous extracellular proteins with diverse functions. Several EGF-like modules with the consensus sequence for hydroxylation bind Ca2+, and it has therefore been suggested that the hydroxyl group is essential for Ca2+ binding. To determine directly the influence of @-hydroxylation on calcium binding in the EGF-like modules from coagulation factors IX and X, we have now measured calcium binding to both the fully &hydroxylated and the non-@-hydroxylated modules of the two proteins. At low ionic strength, the Hyacontaining module of factor X has a slightly higher Caa+ affinity, but at physiological salt concentrations this difference is no longer significant for either factor IX or X. Analysis of the 'H NMR chemical shift differences between the hydroxylated and nonhydroxylated factor X modules show that hydroxylation has no effect on the domain fold. Furthermore, measurements on factor IX show that hydroxylation has no effect on the Ca2+/M8+ specificity of the ion binding site. We conclude that the hydroxyl group is not a direct ligand for the calcium ion in these EGF-like modules, nor is it essential for high-affinity Ca2+ binding. Hydroxylated aspartic acid has long been known as a microbial metabolite (Kornberg and Morris, 1965) and has been found in the urine of man and several animals (Ikegami, 1975). Recently, however, hydroxylated aspartic acid and *This work was supported in part by grants from the Swedish Natural Sciences Research Council, the Swedish Board of Technical Development, and the Swedish Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has demonstrat... more Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has demonstrated its clinical efficacy in treating bleeding in hemophilia patients with inhibitors. Using in vitro assays, we and others have previously shown that high dose rFVIIa shortens the onset of fibrin clot formation and normalizes the fibrin structure and porosity of clots formed under hemophilic conditions (absence of factors VIII and/or IX (Wolberg et al. (2001) 98(11): 826a). Recently, several superactive analogs of rFVIIa have been described, demonstrating increased thrombin-generating activity on platelets (Persson et al. (2001) 98(24): 13583–8), and increased capability to reduce the tail bleeding time and total blood loss in a mouse model of hemophilia A (Tranholm et al. (2003) 102(10): 3615–20). In the current study, we have used a cell-based reconstituted model system of coagulation to compare the abilities of rFVIIa and one of the superactive analogues, NN1731, to modulate the onset...
The formation of a complex between factor VIIa (FVIIa) and tissue factor (TF) triggers the blood ... more The formation of a complex between factor VIIa (FVIIa) and tissue factor (TF) triggers the blood coagulation cascade in response to a vascular injury. TF has the dual biological function of localizing FVIIa to the site of injury and converting the latent enzyme into an efficient catalyst of factor VII, IX, and X activation. The hemostatic effect of recombinant FVIIa in hemophilia patients, where FVIIa is present at supraphysiological concentrations, is however largely mediated by factor X activation on the surface of activated platelets to which FVIIa binds with relatively low affinity. In this setting, it would be pharmacodynamically advantageous to dispose of FVIIa variants with enhanced intrinsic activity. Moreover, such variants might shed light on how TF induces maturation of the active conformation of FVIIa. Intuitively, more active variants of FVIIa could be designed by exploiting structural differences between FVIIa before and after binding to TF. To get the most out of this...
Essentials Nonacog beta pegol (N9‐GP) activity is overestimated in clot method using silica‐based... more Essentials Nonacog beta pegol (N9‐GP) activity is overestimated in clot method using silica‐based reagents. Mimicking contact activation phase with silica reveals N9‐GP activation before recalcification. Localization of N9‐GP to silica facilitates activation by factor XIa and plasma kallikrein. Silica‐based reagents to be used with caution when monitoring N9‐GP therapy using clot method.
Factor VII (FVII) is a modular vitamin K-dependent plasma protein containing an N-terminal domain... more Factor VII (FVII) is a modular vitamin K-dependent plasma protein containing an N-terminal domain rich in posttranslationally γ-carboxylated Glu residues, γ-carboxyglutamic acid (Gla) [1]. These unique sidechains coordinate Ca 2 + and are crucial for the active conformation of activated FVII (FVIIa). The Gla domain interacts with injury-exposed cofactor, tissue factor (TF), and with the substrates factors IX (FIX) and X (FX), as well as with surrounding phospholipid surfaces, during initiation of blood clotting [2]. A natural FVII variant, FVII Tokushima, with very low procoagulant activity caused by a mutation (Cys-22 → Gly) in the Gla domain was recently described [3]. It was speculated that the polymorphism precludes proper folding of the Gla domain and, consequently, functionally essential Ca 2 + binding events. We expressed C22G-FVII and other variants of FVII and protein Z (PZ) with a disrupted disulphide bond in the Gla domain. N-terminal sequencing combined with functional analyses revealed a clear correlation between malfunction and lack of γ-carboxylation. Our data fuel the idea that the Cys residues in the Gla domain-to-be are part of a conserved γ-carboxylase recognition site [4], the disruption of which, like in FVII Tokushima, actually prevents γ-carboxylation. 1. Materials and methods 1.1. Proteins Plasma-derived PZ, FIXa, FXa and FX were from Haematologic Technologies (Essex Junction, VT, USA), and recombinant FVIIa produced in baby hamster kidney (BHK) cells was made in-house. Protein Z-dependent protease inhibitor (ZPI) was a gift from Dr. Alireza Rezaie (Oklahoma Medical Research Foundation, Oklahoma City, OK, USA) [5] and relipidated TF (Innovin) was from Dade Behring (Marburg, Germany). 1.2. Expression construct synthesis Constructs for protein expression in Chinese hamster ovary (CHO) cells, encoding PZ, FVII, C17S/C22S-FVII, and C22G-FVII (Genscript, Piscataway, NJ, USA), with a C-terminal HPC4 tag and stop codon, were inserted into a pQMCF5 vector flanked by BsiWI and NheI restriction sites. 1.3. Site-directed mutagenesis C18S/C22S-PZ forward primer 5′-GGAGAAGGAAAGCTACGAGGAAATCAGCGTGTACGAGG-3′ and reverse primer 5′-CCTCGTACACGCTGATTT CCTCGTAGCTTTCCTTCTCC-3′ were synthesised by Eurofins Genomics (Ebersberg, Germany), and used with the PZ plasmid as the template for sitedirected mutagenesis according to the QuikChange XL kit manual (Agilent Technologies, Santa Clara, CA, USA). XL10-Gold ultra-competent cells were transformed essentially as instructed, but eventually using SOC medium (superoptimal broth with catabolite repression (added glucose)), spread on kanamycin agar plates and left overnight at 37°C. Eight colonies were picked, smeared on a sectioned plate plus transferred to liquid kanamycin agar (agitation 250 rpm). They were both incubated overnight (37°C), the plates were refrigerated and plasmids prepared from liquid cultures (Qiaprep Spin Miniprep Kit, Qiagen, Valencia, CA, USA). Mutations were verified by sequencing using PZ-specific primers 5′-GGAATTCGGGACAAAAGTCG-3′ and 5′-CCTCAGACAGTGGTTCAAAG-3′.
Research and Practice in Thrombosis and Haemostasis, 2019
This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmerc... more This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Research and Practice in Thrombosis and Haemostasis, 2017
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-... more This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) a... more Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215–217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215–217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 60-fold ...
Pathophysiology of Haemostasis and Thrombosis, 2009
Des(1-38)- and des(1-44)-factor Vila (fVIIa) were inhibited with Phe-Phe-Arg-chloromethyl ketone ... more Des(1-38)- and des(1-44)-factor Vila (fVIIa) were inhibited with Phe-Phe-Arg-chloromethyl ketone (FFR-cmk). Des(1-38)-FFR-fVIIa inhibited tissue factor (TF)-enhanced fVIIa amidolytic activity with an IC50 value of 15 nM, whereas 3-and 6-fold higher values were obtained with des(1-44)-FFR-fVIIa using soluble and full-length TF, respectively. The value for FFR-fVIIa was 8-10 nM. Clotting time was prolongated with IC50 values for inactivated des(1-38)- and des(1-44)-
Hydroxylation of aspartic acid to erythro-@-aspartic acid (Hya) occurs in epidermal growth factor... more Hydroxylation of aspartic acid to erythro-@-aspartic acid (Hya) occurs in epidermal growth factor (EGF)like modules in numerous extracellular proteins with diverse functions. Several EGF-like modules with the consensus sequence for hydroxylation bind Ca2+, and it has therefore been suggested that the hydroxyl group is essential for Ca2+ binding. To determine directly the influence of @-hydroxylation on calcium binding in the EGF-like modules from coagulation factors IX and X, we have now measured calcium binding to both the fully &hydroxylated and the non-@-hydroxylated modules of the two proteins. At low ionic strength, the Hyacontaining module of factor X has a slightly higher Caa+ affinity, but at physiological salt concentrations this difference is no longer significant for either factor IX or X. Analysis of the 'H NMR chemical shift differences between the hydroxylated and nonhydroxylated factor X modules show that hydroxylation has no effect on the domain fold. Furthermore, measurements on factor IX show that hydroxylation has no effect on the Ca2+/M8+ specificity of the ion binding site. We conclude that the hydroxyl group is not a direct ligand for the calcium ion in these EGF-like modules, nor is it essential for high-affinity Ca2+ binding. Hydroxylated aspartic acid has long been known as a microbial metabolite (Kornberg and Morris, 1965) and has been found in the urine of man and several animals (Ikegami, 1975). Recently, however, hydroxylated aspartic acid and *This work was supported in part by grants from the Swedish Natural Sciences Research Council, the Swedish Board of Technical Development, and the Swedish Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has demonstrat... more Recombinant factor VIIa (rFVIIa, NovoSeven; Novo Nordisk A/S, Copenhagen, Denmark) has demonstrated its clinical efficacy in treating bleeding in hemophilia patients with inhibitors. Using in vitro assays, we and others have previously shown that high dose rFVIIa shortens the onset of fibrin clot formation and normalizes the fibrin structure and porosity of clots formed under hemophilic conditions (absence of factors VIII and/or IX (Wolberg et al. (2001) 98(11): 826a). Recently, several superactive analogs of rFVIIa have been described, demonstrating increased thrombin-generating activity on platelets (Persson et al. (2001) 98(24): 13583–8), and increased capability to reduce the tail bleeding time and total blood loss in a mouse model of hemophilia A (Tranholm et al. (2003) 102(10): 3615–20). In the current study, we have used a cell-based reconstituted model system of coagulation to compare the abilities of rFVIIa and one of the superactive analogues, NN1731, to modulate the onset...
The formation of a complex between factor VIIa (FVIIa) and tissue factor (TF) triggers the blood ... more The formation of a complex between factor VIIa (FVIIa) and tissue factor (TF) triggers the blood coagulation cascade in response to a vascular injury. TF has the dual biological function of localizing FVIIa to the site of injury and converting the latent enzyme into an efficient catalyst of factor VII, IX, and X activation. The hemostatic effect of recombinant FVIIa in hemophilia patients, where FVIIa is present at supraphysiological concentrations, is however largely mediated by factor X activation on the surface of activated platelets to which FVIIa binds with relatively low affinity. In this setting, it would be pharmacodynamically advantageous to dispose of FVIIa variants with enhanced intrinsic activity. Moreover, such variants might shed light on how TF induces maturation of the active conformation of FVIIa. Intuitively, more active variants of FVIIa could be designed by exploiting structural differences between FVIIa before and after binding to TF. To get the most out of this...
Essentials Nonacog beta pegol (N9‐GP) activity is overestimated in clot method using silica‐based... more Essentials Nonacog beta pegol (N9‐GP) activity is overestimated in clot method using silica‐based reagents. Mimicking contact activation phase with silica reveals N9‐GP activation before recalcification. Localization of N9‐GP to silica facilitates activation by factor XIa and plasma kallikrein. Silica‐based reagents to be used with caution when monitoring N9‐GP therapy using clot method.
Factor VII (FVII) is a modular vitamin K-dependent plasma protein containing an N-terminal domain... more Factor VII (FVII) is a modular vitamin K-dependent plasma protein containing an N-terminal domain rich in posttranslationally γ-carboxylated Glu residues, γ-carboxyglutamic acid (Gla) [1]. These unique sidechains coordinate Ca 2 + and are crucial for the active conformation of activated FVII (FVIIa). The Gla domain interacts with injury-exposed cofactor, tissue factor (TF), and with the substrates factors IX (FIX) and X (FX), as well as with surrounding phospholipid surfaces, during initiation of blood clotting [2]. A natural FVII variant, FVII Tokushima, with very low procoagulant activity caused by a mutation (Cys-22 → Gly) in the Gla domain was recently described [3]. It was speculated that the polymorphism precludes proper folding of the Gla domain and, consequently, functionally essential Ca 2 + binding events. We expressed C22G-FVII and other variants of FVII and protein Z (PZ) with a disrupted disulphide bond in the Gla domain. N-terminal sequencing combined with functional analyses revealed a clear correlation between malfunction and lack of γ-carboxylation. Our data fuel the idea that the Cys residues in the Gla domain-to-be are part of a conserved γ-carboxylase recognition site [4], the disruption of which, like in FVII Tokushima, actually prevents γ-carboxylation. 1. Materials and methods 1.1. Proteins Plasma-derived PZ, FIXa, FXa and FX were from Haematologic Technologies (Essex Junction, VT, USA), and recombinant FVIIa produced in baby hamster kidney (BHK) cells was made in-house. Protein Z-dependent protease inhibitor (ZPI) was a gift from Dr. Alireza Rezaie (Oklahoma Medical Research Foundation, Oklahoma City, OK, USA) [5] and relipidated TF (Innovin) was from Dade Behring (Marburg, Germany). 1.2. Expression construct synthesis Constructs for protein expression in Chinese hamster ovary (CHO) cells, encoding PZ, FVII, C17S/C22S-FVII, and C22G-FVII (Genscript, Piscataway, NJ, USA), with a C-terminal HPC4 tag and stop codon, were inserted into a pQMCF5 vector flanked by BsiWI and NheI restriction sites. 1.3. Site-directed mutagenesis C18S/C22S-PZ forward primer 5′-GGAGAAGGAAAGCTACGAGGAAATCAGCGTGTACGAGG-3′ and reverse primer 5′-CCTCGTACACGCTGATTT CCTCGTAGCTTTCCTTCTCC-3′ were synthesised by Eurofins Genomics (Ebersberg, Germany), and used with the PZ plasmid as the template for sitedirected mutagenesis according to the QuikChange XL kit manual (Agilent Technologies, Santa Clara, CA, USA). XL10-Gold ultra-competent cells were transformed essentially as instructed, but eventually using SOC medium (superoptimal broth with catabolite repression (added glucose)), spread on kanamycin agar plates and left overnight at 37°C. Eight colonies were picked, smeared on a sectioned plate plus transferred to liquid kanamycin agar (agitation 250 rpm). They were both incubated overnight (37°C), the plates were refrigerated and plasmids prepared from liquid cultures (Qiaprep Spin Miniprep Kit, Qiagen, Valencia, CA, USA). Mutations were verified by sequencing using PZ-specific primers 5′-GGAATTCGGGACAAAAGTCG-3′ and 5′-CCTCAGACAGTGGTTCAAAG-3′.
Research and Practice in Thrombosis and Haemostasis, 2019
This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmerc... more This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Research and Practice in Thrombosis and Haemostasis, 2017
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-... more This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) a... more Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215–217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215–217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 60-fold ...
Pathophysiology of Haemostasis and Thrombosis, 2009
Des(1-38)- and des(1-44)-factor Vila (fVIIa) were inhibited with Phe-Phe-Arg-chloromethyl ketone ... more Des(1-38)- and des(1-44)-factor Vila (fVIIa) were inhibited with Phe-Phe-Arg-chloromethyl ketone (FFR-cmk). Des(1-38)-FFR-fVIIa inhibited tissue factor (TF)-enhanced fVIIa amidolytic activity with an IC50 value of 15 nM, whereas 3-and 6-fold higher values were obtained with des(1-44)-FFR-fVIIa using soluble and full-length TF, respectively. The value for FFR-fVIIa was 8-10 nM. Clotting time was prolongated with IC50 values for inactivated des(1-38)- and des(1-44)-
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