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Darrel W. Stafford - One of the best experts on this subject based on the ideXlab platform.
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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Circulating and binding characteristics of wild-type factor IX and certain Gla Domain mutants in vivo
2016Co-Authors: Tong Gui, Hui Feng Lin, Maureane Hoffman, David L. Straight, Harold R. Roberts, Da-yun Jin, Darrel W. StaffordAbstract:tamic acid (Gla) Domain participates in binding endothelial cells/collagen IV. We injected recombinant factor IX containing mutations at residue 5 (K5A, K5R) into factor IX–deficient mice and compared their behavior with that of wild-type factor IX. The plasma concentration of factor IX that binds to endothelial cells/collagen IV (recombinant wild type and K5R) was consistently lower than that of the one that does not bind (K5A). Mice treated with wild type or K5R had 79 % of the injected factor IX in the liver after 2 min-utes, whereas 17 % remained in circula-tion. In mice injected with K5A, 59 % of the injected factor IX was found in liver and 31 % was found in plasma. When we blocked the liver circulation before factor IX injection, 74 % of K5A and 64 % of K5R remained in the blood. When we treated the mouse with EDTA after injecting exog-enous factor IX, the blood levels of factor IX that bind to endothelial cells/collagen IV increased, presumably because of re-lease from endothelial cell/collagen IV binding sites. In contrast, the levels of the mutants that do not bind were unaffected by EDTA. In immunohistochemical stud-ies, factor IX appears on the endothelial surfaces of mouse arteries after factor IX injection and of human arteries from sur-gical specimens. Thus, we have demon-strated that factor IX binds in vivo to endothelial cell–collagen IV surfaces. Our results suggest that factor IX Gla-Domain mediated binding to endothelial cells/ collagen IV plays a role in controlling factor IX concentration in the blood
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Binding of the factor IX γ-carboxyglutamic acid Domain to the vitamin K-dependent γ-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product
The Journal of biological chemistry, 2003Co-Authors: Pen Jen Lin, David L. Straight, Darrel W. StaffordAbstract:Abstract Propeptides of the vitamin K-dependent proteins bind to an exosite on γ-glutamyl carboxylase; while they are bound, multiple glutamic acids in the γ-carboxyglutamic acid (Gla) Domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla Domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla Domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla Domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nm and the koff values were 273 × 10-5,9 × 10-5, and 37 × 10-5 s-1, respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla Domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s-1 in enzyme excess and 0.016 s-1 in substrate excess. The steady state rate in substrate excess is 4.5 × 10-4s-1. These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla Domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.
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Identification of a gene encoding a typical γ‐carboxyglutamic acid Domain in the tunicate Halocynthia roretzi
Journal of thrombosis and haemostasis : JTH, 2003Co-Authors: Yagi K, Da-yun Jin, Ping Lin, Darrel W. StaffordAbstract:Summary. We report the identification of a gene capable of encoding a novel Gla (γ-carboxyglutamic acid) protein from the tunicate Halocynthia roretzi, a primitive member of the phylum Chordata. We call this new hypothetical protein Gla-RTK; it has a Gla Domain typical of human vitamin K-dependent coagulation factors, a transmembrane Domain, and a receptor tyrosine kinase Domain. The receptor tyrosine kinase Domain is very similar to the ARK (adhesion-related kinase) family of receptor tyrosine kinases. The ARK family includes Axl, Tyro3, and c-Mer. This gene also encodes a propeptide that binds to the human gamma-glutamyl carboxylase within a range of affinities observed for mammalian propeptides. The cDNA for this putative protein is found distributed throughout the oocyte and embryo but the cDNA is apparently not transcribed except during oogenesis. One of the most interesting aspects of this hypothetical protein is that its Gla Domain is highly homologous to the Gla Domain of Gas6, a ligand for Axl, while its receptor tyrosine kinase Domain is highly homologous to Axl.
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identification of a gene encoding a typical γ carboxyglutamic acid Domain in the tunicate halocynthia roretzi
Journal of Thrombosis and Haemostasis, 2003Co-Authors: C P Wang, K Yagi, K W Makabe, Darrel W. StaffordAbstract:Summary. We report the identification of a gene capable of encoding a novel Gla (γ-carboxyglutamic acid) protein from the tunicate Halocynthia roretzi, a primitive member of the phylum Chordata. We call this new hypothetical protein Gla-RTK; it has a Gla Domain typical of human vitamin K-dependent coagulation factors, a transmembrane Domain, and a receptor tyrosine kinase Domain. The receptor tyrosine kinase Domain is very similar to the ARK (adhesion-related kinase) family of receptor tyrosine kinases. The ARK family includes Axl, Tyro3, and c-Mer. This gene also encodes a propeptide that binds to the human gamma-glutamyl carboxylase within a range of affinities observed for mammalian propeptides. The cDNA for this putative protein is found distributed throughout the oocyte and embryo but the cDNA is apparently not transcribed except during oogenesis. One of the most interesting aspects of this hypothetical protein is that its Gla Domain is highly homologous to the Gla Domain of Gas6, a ligand for Axl, while its receptor tyrosine kinase Domain is highly homologous to Axl.
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Circulating and binding characteristics of wild-type factor IX and certain Gla Domain mutants in vivo.
Blood, 2002Co-Authors: Tong Gui, Hui Feng Lin, Da Yun Jin, Maureane Hoffman, David L. Straight, Harold R. Roberts, Darrel W. StaffordAbstract:Residue K5 in factor IX γ-carboxyglutamic acid (Gla) Domain participates in binding endothelial cells/collagen IV. We injected recombinant factor IX containing mutations at residue 5 (K5A, K5R) into factor IX–deficient mice and compared their behavior with that of wild-type factor IX. The plasma concentration of factor IX that binds to endothelial cells/collagen IV (recombinant wild type and K5R) was consistently lower than that of the one that does not bind (K5A). Mice treated with wild type or K5R had 79% of the injected factor IX in the liver after 2 minutes, whereas 17% remained in circulation. In mice injected with K5A, 59% of the injected factor IX was found in liver and 31% was found in plasma. When we blocked the liver circulation before factor IX injection, 74% of K5A and 64% of K5R remained in the blood. When we treated the mouse with EDTA after injecting exogenous factor IX, the blood levels of factor IX that bind to endothelial cells/collagen IV increased, presumably because of release from endothelial cell/collagen IV binding sites. In contrast, the levels of the mutants that do not bind were unaffected by EDTA. In immunohistochemical studies, factor IX appears on the endothelial surfaces of mouse arteries after factor IX injection and of human arteries from surgical specimens. Thus, we have demonstrated that factor IX binds in vivo to endothelial cell–collagen IV surfaces. Our results suggest that factor IX Gla-Domain mediated binding to endothelial cells/collagen IV plays a role in controlling factor IX concentration in the blood.
Takashi Morita - One of the best experts on this subject based on the ideXlab platform.
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Use of Snake Venom Inhibitors in Studies of the Function and Tertiary Structure of Coagulation Factors
International Journal of Hematology, 2004Co-Authors: Takashi MoritaAbstract:C-type lectin-like proteins (CLPs) of snake venom have a variety of biological properties, acting for example as anticoagulants, procoagulants, and agonists/antagonists of platelet activation. The structural and functional studies of the first identified venom CLP, factors IX/X-binding protein (IX/X-bp), have contributed to our understanding of the roles of magnesium ions in the blood coagulation cascade reaction. The crystal structures of γ-carboxyglutamic acid (Gla) Domains of coagulation factors X and IX have recently been clarified in structural studies of complexes between the Gla Domain of factor X and X-bp (a venom CLP) and between the Gla Domain of factor IX and IX-bp (a venom CLP).
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Crystal structure of Mg2+- and Ca2+-bound Gla Domain of factor IX complexed with binding protein.
The Journal of biological chemistry, 2003Co-Authors: Yasuo Shikamoto, Takashi Morita, Zui Fujimoto, Hiroshi MizunoAbstract:Abstract Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a γ-carboxyglutamic acid (Gla) Domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla Domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla Domain (IXGD1–46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1–46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1–46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1–46 and IX-bp, forced IXGD1–46 to rotate 4° relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla Domain during blood coagulation.
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Crystal structure of an anticoagulant protein in complex with the Gla Domain of factor X
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Hiroshi Mizuno, Zui Fujimoto, Hideko Atoda, Takashi MoritaAbstract:The γ-carboxyglutamic acid (Gla) Domain of blood coagulation factors is responsible for Ca2+-dependent phospholipid membrane binding. Factor X-binding protein (X-bp), an anticoagulant protein from snake venom, specifically binds to the Gla Domain of factor X. The crystal structure of X-bp in complex with the Gla Domain peptide of factor X at 2.3-Å resolution showed that the anticoagulation is based on the fact that two patches of the Gla Domain essential for membrane binding are buried in the complex formation. The Gla Domain thus is expected to be a new target of anticoagulant drugs, and X-bp provides a basis for designing them. This structure also provides a membrane-bound model of factor X.
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coagulation factor x binding protein from deinagkistrodon acutus venom is a Gla Domain binding protein
Biochemistry, 1998Co-Authors: Hideko Atoda, Hiroshi Mizuno, Midori Ishikawa, Takashi MoritaAbstract:Factor IX/factor X-binding protein (IX/X-bp) is an anticoagulant isolated from the venom of Trimeresurus flavoviridis (habu snake) and binds predominantly to factor IX. In this study, we isolated IX/X-bp-like proteins from the venom of Deinagkistrodon acutus (hundred pace snake) with binding characteristics different from those of IX/X-bp. The complete amino acid sequence and binding characteristics of the main anticoagulant protein, named X-bp, were investigated. The concentrations of X-bp at half-maximal binding to solid-phase factors X and IX were 0.4 and 3 nM, respectively. The binding of X-bp to solid-phase factor X was inhibited by 50% by 6- and 9-fold excess concentrations of factor X and Gla Domain (GD) peptide 1-44, respectively, but was not influenced by GD peptide 1-41 and Gla Domainless factor X. X-bp bound two Ca2+ ions per molecule with Kd values of 16 ± 0.7 (mean ± SE, n = 6) and 103 ± 10 μM. X-bp was a heterodimer of C-type lectin-like subunits. The 16 kDa chain (A chain) consisted of 129 ...
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Localization of the specific binding site for magnesium(II) ions in factor IX
FEBS letters, 1996Co-Authors: Fujio Sekiya, Misa Yoshida, Toshiko Yamashita, Takashi MoritaAbstract:We demonstrated recently that coagulation factor IX has a specific binding site(s) for Mg2+ ions, independent of the Ca2+-binding sites, and that binding of Mg2+ ions is very important for expression of the functional conformation of this protein. We report here the localization of this Mg2+-specific binding site. We prepared three Gla-containing fragments of bovine factor IX, namely GlaEGFNC (residues 1–144+286–296), GlaEGFN (1–83) and the Gla Domain peptide (1–46). Fragments GlaEGFNC and GlaEGFN retained the ability to undergo a conformational change upon binding of Mg2+ ions in the presence of excess Ca2+ ions. This change could be detected by a conformation-specific antibody. Furthermore, the Gla Domain peptide was capable of binding Mg2+ ions, as determined by the metal ion-induced quenching of the intrinsic fluorescence. It appears that the Mg2+-specific binding site of factor IX is located in the N-terminal Gla Domain.
Midori Shima - One of the best experts on this subject based on the ideXlab platform.
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interactions between residues 2228 2240 within factor viiia c2 Domain and factor ixa Gla Domain contribute to propagation of clot formation
Thrombosis and Haemostasis, 2011Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Kenichi Ogiwara, Midori ShimaAbstract:Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 Domain (residues 2228–2240) interacts with the FIXa Gla Domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228–2240. The peptide 2228–2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804–1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228–2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228–2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 Domain (residues 2228–2240) with the FIXa Gla Domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
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Interactions between residues 2228–2240 within factor VIIIa C2 Domain and factor IXa Gla Domain contribute to propagation of clot formation
Thrombosis and Haemostasis, 2011Co-Authors: Tetsuhiro Soeda, Kenichi Ogiwara, Midori Shima, Keiji NogamiAbstract:SummaryFactor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 Domain (residues 2228–2240) interacts with the FIXa Gla Domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228–2240. The peptide 2228–2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804–1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228–2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228–2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 Domain (residues 2228–2240) with the FIXa Gla Domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
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the factor viiia c2 Domain residues 2228 2240 interacts with the factor ixa Gla Domain in the factor xase complex
Journal of Biological Chemistry, 2009Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Katsumi Nishiya, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Midori ShimaAbstract:Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 Domain of factor VIIIa and the Gla Domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these Domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 Domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla Domain of factor IXa inhibited binding by approximately 95%, and Gla Domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 Domain interacted with the Gla Domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 Domain constitutes an interactive site for the Gla Domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
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The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex
The Journal of biological chemistry, 2008Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Katsumi Nishiya, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Midori ShimaAbstract:Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 Domain of factor VIIIa and the Gla Domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these Domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 Domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla Domain of factor IXa inhibited binding by approximately 95%, and Gla Domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 Domain interacted with the Gla Domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 Domain constitutes an interactive site for the Gla Domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
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Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.
Blood, 2007Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Kazuhiko Tomokiyo, Midori ShimaAbstract:Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 Domains of factor VIII interact with the catalytic Domain and EGF2 Domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla Domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) Domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 Domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl 2 . This binding was competitively inhibited by the addition of excess of factor VIII or rC2 Domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity ( K d ) of rC2 Domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 Domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 Domain binding to EGR-factor IXa in a dose-dependent manner (IC 50 ; 758 nM), suggesting the authentic of the C2 Domain and factor IXa-GD interaction. The addition of rC2 Domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC 50 ; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 Domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 Domain in the association between factor VIII and factor IXa in the factor Xase complex.
Roger J S Preston - One of the best experts on this subject based on the ideXlab platform.
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platelet factor 4 impairs the anticoagulant activity of activated protein c
Journal of Biological Chemistry, 2009Co-Authors: Roger J S Preston, Bjorn Dahlback, Sinh Tran, Jennifer A Johnson, Fionnuala Ni Ainle, Shona Harmon, B White, Owen P Smith, Vince P Jenkins, James S OdonnellAbstract:Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) Domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla Domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla Domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.
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a single amino acid substitution i18v in the Gla Domain of activated protein c markedly impairs ability of pe and glycer to enhance anticoagulant activity
Blood, 2008Co-Authors: Jennifer A Johnson, Fionnuala Ni Ainle, Shona Harmon, James S Odonnell, Roger J S PrestonAbstract:Phosphatidylethanolamine (PE) and glucosylceramide (GlyCer) are cell surface lipids that preferentially enhance anticoagulant, rather than procoagulant pathways. In particular, both PE and GlyCer enhance the anticoagulant activity of activated protein C (APC). Previous studies have indicated that specific APC Gla Domain residues may mediate APC interaction with PE and GlyCer. To investigate whether specific APC residues mediate PE and GlyCer enhanced APC anticoagulant activity, we expressed a series of APC variants in which APC Gla Domain residues not shared with the human prothrombin Gla Domain were substituted with their prothrombin amino acid equivalent. The anticoagulant activity of each APC Gla Domain variant was assessed in a tissue factor-initiated thrombin generation assay containing phospholipid vesicles of differing composition (80% PC/20% PS); or PC/PS/PE (60%/20%/20%); or PC/PS/GlyCer (60%/20%/20%). For each of these lipid mixtures, thrombin generation (endogenous thrombin potential, ETP) was not significantly different in the absence of APC. In the presence of PC/PS vesicles, APC reduced thrombin generation by 63±3% at the highest APC concentration tested (6nM). However, APC impairment of thrombin generation was enhanced 3-fold in the presence of PC/PS/PE compared with vesicles containing PC/PS alone, and in the presence of PC/PS/GlyCer was enhanced 4.3-fold. Enhancement of anticoagulant function by PE and GlyCer was similar for the majority of the APC Gla Domain variants tested. Interestingly, one APC variant (APC-I18V) exhibited similar anticoagulant activity to that of wild type APC with PC/PS vesicles, but was not enhanced by the presence of PE- or GlyCer-containing vesicles. Phospholipid vesicles containing PE or GlyCer have been previously described to enhance protein S cofactor enhancement of APC. Therefore, to further characterize APC-I18V, we assessed the ability of wild type APC and APC-I18V to be enhanced by protein S in the presence of PC/PS, PC/PS/PE or PC/PS/GlyCer using a protein S-sensitive thrombin generation assay. In the presence of PC/PS, increasing protein S concentration in protein S-deficient plasma resulted in an APC-mediated slow decrease in thrombin generation, irrespective of whether wild type or APC-I18V was used (IC50 for protein S-mediated APC inhibition of thrombin generation with PC/PS, 130nM). However, in the presence of PC/PS/PE or PC/PS/GlyCer, thrombin generation was impaired by wild type APC at 3–4-fold lower protein S concentration than that observed when PC/PS vesicles alone were used (IC50, PC/PS/PE=31.5nM and PC/PS/GlyCer=37.5nM). APC-I18V, however, did not exhibit a similarly increased sensitivity to protein S in the presence of PE or GlyCer, as the anticoagulant activity of this variant was the same as when only PC/PS was included. To investigate whether the loss of specific neutral lipid enhancement in APC-I18V affected its ability to initiate cytoprotective signaling via EPCR-PAR-1 on endothelial cells, the capacity of APC-I18V to inhibit thrombin-induced endothelial cell barrier permeability was assessed. When cells were pre-treated with either wild type APC or APC-I18V, there was a significant enhancement in barrier integrity and attenuation of thrombin-induced permeability (P<0.05), demonstrating that loss of PE/GlyCer enhancement of APC anticoagulant activity does not adversely affect EPCR binding and EPCR/PAR-1 cytoprotective signaling. Collectively, these results suggest PE and GlyCer enhancement of APC anticoagulant activity is mediated by increased sensitivity to protein S, and that Ile-18 in the APC Gla Domain is critical for mediating APC-specific functional enhancement by PE/GlyCer.
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A Single Amino Acid Substitution (I18V) in the Gla Domain of Activated Protein C Markedly Impairs Ability of PE and GlyCer to Enhance Anticoagulant Activity
Blood, 2008Co-Authors: Jennifer A Johnson, Fionnuala Ni Ainle, Shona Harmon, James S. O’donnell, Roger J S PrestonAbstract:Phosphatidylethanolamine (PE) and glucosylceramide (GlyCer) are cell surface lipids that preferentially enhance anticoagulant, rather than procoagulant pathways. In particular, both PE and GlyCer enhance the anticoagulant activity of activated protein C (APC). Previous studies have indicated that specific APC Gla Domain residues may mediate APC interaction with PE and GlyCer. To investigate whether specific APC residues mediate PE and GlyCer enhanced APC anticoagulant activity, we expressed a series of APC variants in which APC Gla Domain residues not shared with the human prothrombin Gla Domain were substituted with their prothrombin amino acid equivalent. The anticoagulant activity of each APC Gla Domain variant was assessed in a tissue factor-initiated thrombin generation assay containing phospholipid vesicles of differing composition (80% PC/20% PS); or PC/PS/PE (60%/20%/20%); or PC/PS/GlyCer (60%/20%/20%). For each of these lipid mixtures, thrombin generation (endogenous thrombin potential, ETP) was not significantly different in the absence of APC. In the presence of PC/PS vesicles, APC reduced thrombin generation by 63±3% at the highest APC concentration tested (6nM). However, APC impairment of thrombin generation was enhanced 3-fold in the presence of PC/PS/PE compared with vesicles containing PC/PS alone, and in the presence of PC/PS/GlyCer was enhanced 4.3-fold. Enhancement of anticoagulant function by PE and GlyCer was similar for the majority of the APC Gla Domain variants tested. Interestingly, one APC variant (APC-I18V) exhibited similar anticoagulant activity to that of wild type APC with PC/PS vesicles, but was not enhanced by the presence of PE- or GlyCer-containing vesicles. Phospholipid vesicles containing PE or GlyCer have been previously described to enhance protein S cofactor enhancement of APC. Therefore, to further characterize APC-I18V, we assessed the ability of wild type APC and APC-I18V to be enhanced by protein S in the presence of PC/PS, PC/PS/PE or PC/PS/GlyCer using a protein S-sensitive thrombin generation assay. In the presence of PC/PS, increasing protein S concentration in protein S-deficient plasma resulted in an APC-mediated slow decrease in thrombin generation, irrespective of whether wild type or APC-I18V was used (IC50 for protein S-mediated APC inhibition of thrombin generation with PC/PS, 130nM). However, in the presence of PC/PS/PE or PC/PS/GlyCer, thrombin generation was impaired by wild type APC at 3–4-fold lower protein S concentration than that observed when PC/PS vesicles alone were used (IC50, PC/PS/PE=31.5nM and PC/PS/GlyCer=37.5nM). APC-I18V, however, did not exhibit a similarly increased sensitivity to protein S in the presence of PE or GlyCer, as the anticoagulant activity of this variant was the same as when only PC/PS was included. To investigate whether the loss of specific neutral lipid enhancement in APC-I18V affected its ability to initiate cytoprotective signaling via EPCR-PAR-1 on endothelial cells, the capacity of APC-I18V to inhibit thrombin-induced endothelial cell barrier permeability was assessed. When cells were pre-treated with either wild type APC or APC-I18V, there was a significant enhancement in barrier integrity and attenuation of thrombin-induced permeability (P
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multifunctional specificity of the protein c activated protein c Gla Domain
Journal of Biological Chemistry, 2006Co-Authors: Roger J S Preston, Eva Ajzner, Cristina Razzari, Stalo Karageorgi, Sonia Dua, Bjorn Dahlback, D A LaneAbstract:Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla Domain is implicated in both interactions. We sought to identify how the protein C Gla Domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla Domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla Domain. We hypothesized that the unique protein C/APC Gla Domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla Domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla Domains.
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selective modulation of protein c affinity for epcr and phospholipids by Gla Domain mutation
FEBS Journal, 2004Co-Authors: Roger J S Preston, Bjorn Dahlback, Ana Villegasmendez, Yonghui Sun, Jose Hermida, Paolo Simioni, Helen Philippou, D A LaneAbstract:Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla Domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla Domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23x10(5) M-1.s(-1), a dissociation rate constant of 7.61x10(-2) s(-1) and equilibrium binding constant (K-D) of 147 nM. It was activated by thrombin over endothelial cells with a K-m of 213 nM and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla Domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (
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interactions between residues 2228 2240 within factor viiia c2 Domain and factor ixa Gla Domain contribute to propagation of clot formation
Thrombosis and Haemostasis, 2011Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Kenichi Ogiwara, Midori ShimaAbstract:Factor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 Domain (residues 2228–2240) interacts with the FIXa Gla Domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228–2240. The peptide 2228–2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804–1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228–2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228–2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 Domain (residues 2228–2240) with the FIXa Gla Domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
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Interactions between residues 2228–2240 within factor VIIIa C2 Domain and factor IXa Gla Domain contribute to propagation of clot formation
Thrombosis and Haemostasis, 2011Co-Authors: Tetsuhiro Soeda, Kenichi Ogiwara, Midori Shima, Keiji NogamiAbstract:SummaryFactor (F)VIII functions as a cofactor in the tenase complex responsible for phospholipid (PL)-dependent FXa generation by FIXa. We have recently reported that the FVIIIa C2 Domain (residues 2228–2240) interacts with the FIXa Gla Domain in this complex. We examined the role of this interaction in the generation of tenase activity during the process of clot formation, using a synthetic peptide corresponding to residues 2228–2240. The peptide 2228–2240 inhibited FVIIIa/FIXa-mediated FX activation dose-dependently in the presence of PL by >95% (IC50; ~10 μM). This effect was significantly greater than that obtained by peptide 1804–1818 (IC50; ~180 μM) which corresponds to another FIXa-interactive site in the light chain that provides the majority of binding energy for FIXa interaction. Peptide 2228–2240 had little effect on the prothrombin time and did not inhibit FIX activation in the coagulation process mediated by FVIIa/tissue factor or FXIa, suggesting specific inhibition of the intrinsic tenase complex. Clot waveform analysis, a plasma based-assay used to evaluate the process of intrinsic coagulation, demonstrated that peptide 2228–2240 significantly depressed both maximum coagulation velocity (|min1|) and acceleration (|min2|), reflecting the propagation of clot formation, although the clotting time was only marginally prolonged. Thromboelastography, an alternative whole blood based-assay, demonstrated that the peptide inhibited clot formation time, α-angle and maximal clot firmness, but had little effect on the clotting time. Interactions of the FVIIIa C2 Domain (residues 2228–2240) with the FIXa Gla Domain in the tenase complex appeared to contribute essentially to the propagation of clot formation.
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the factor viiia c2 Domain residues 2228 2240 interacts with the factor ixa Gla Domain in the factor xase complex
Journal of Biological Chemistry, 2009Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Katsumi Nishiya, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Midori ShimaAbstract:Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 Domain of factor VIIIa and the Gla Domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these Domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 Domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla Domain of factor IXa inhibited binding by approximately 95%, and Gla Domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 Domain interacted with the Gla Domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 Domain constitutes an interactive site for the Gla Domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
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The Factor VIIIa C2 Domain (Residues 2228–2240) Interacts with the Factor IXa Gla Domain in the Factor Xase Complex
The Journal of biological chemistry, 2008Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Katsumi Nishiya, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Midori ShimaAbstract:Factor VIIIa functions as a cofactor for factor IXa in the phospholipid surface-dependent activation of factor X. Both the C2 Domain of factor VIIIa and the Gla Domain of factor IXa are involved in phospholipid binding and are required for the activation of factor X. In this study, we have examined the close relationship between these Domains in the factor Xase complex. Enzyme-linked immunosorbent assay-based and surface plasmon resonance-based assays in the absence of phospholipid showed that Glu-Gly-Arg active site-modified factor IXa bound to immobilized recombinant C2 Domain (rC2) dose-dependently (Kd = 108 nm). This binding ability was optimal under physiological conditions. A monoclonal antibody against the Gla Domain of factor IXa inhibited binding by approximately 95%, and Gla Domainless factor IXa failed to bind to rC2. The addition of monoclonal antibody or rC2 with factor VIIIa inhibited factor IXa-catalyzed factor X activation in the absence of phospholipid. Inhibition was not evident, however, in similar experiments in the absence of factor VIIIa, indicating that the C2 Domain interacted with the Gla Domain of factor IXa. A fragment designated C2-(2182-2259), derived from V8 protease-cleaved rC2, bound to Glu-Gly-Arg active site-modified factor IXa. Competitive assays, using overlapping synthetic peptides encompassing residues 2182-2259, demonstrated that peptide 2228-2240 significantly inhibited both this binding and factor Xa generation, independently of phospholipid. Our results indicated that residues 2228-2240 in the factor VIIIa C2 Domain constitutes an interactive site for the Gla Domain of factor IXa. The findings provide the first evidence for an essential role for this interaction in factor Xase assembly.
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Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.
Blood, 2007Co-Authors: Tetsuhiro Soeda, Keiji Nogami, Masahiro Takeyama, Kenichi Ogiwara, Yoichi Sakata, Akira Yoshioka, Kazuhiko Tomokiyo, Midori ShimaAbstract:Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 Domains of factor VIII interact with the catalytic Domain and EGF2 Domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla Domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) Domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 Domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl 2 . This binding was competitively inhibited by the addition of excess of factor VIII or rC2 Domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity ( K d ) of rC2 Domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 Domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 Domain binding to EGR-factor IXa in a dose-dependent manner (IC 50 ; 758 nM), suggesting the authentic of the C2 Domain and factor IXa-GD interaction. The addition of rC2 Domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC 50 ; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 Domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 Domain in the association between factor VIII and factor IXa in the factor Xase complex.