The Propeptides of Human Protein C, Factor VII, and Factor IX Are Exchangeable with Regard to Directing Gamma-Carboxylation of these Proteins

1996 ◽  
Vol 76 (02) ◽  
pp. 205-207
Author(s):  
Jie-Ping Geng ◽  
Francis J Castellino
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Amino Acid Residues ◽  
Intact Proteins ◽  
Coagulation Factor Vii ◽  
Carboxyglutamic Acid ◽  
Gla Domain

SummaryThe specificity of the propeptide sequence in directing vitamin Independent post-translational γ-carboxylation has been assessed by examination of the extent of processing of chimeric constructs of blood coagulation factor VII (fVII), factor IX (fIX) and protein C (PC). One chimera consisted of a protein in which the γ-carboxyglutamic acid (Gla)/helical stack domain of PC (amino acid residues 1 to 46) was replaced by that of fIX (residues 1 to 47) in an otherwise intact PC. Another consisted of the same construction of a fVII/PC Gla domain-based mutant protein. The final chimera contained the leader/propeptide sequence of PC (residues -42 to -1) replaced by that of fIX (residues -46 to -1). In each case, all Glu-precursor Gla residues in the Gla domains of the proteins were fully processed to Gla. These results demonstrate that the propeptides of fIX and PC are capable of directing γ-carboxylation of the Gla regions of either protein, that the propeptide of PC can fully function in γ-carboxylation of the Gla region of fVII, and further suggest that, with regard to γ-carboxylation, communications between the propeptides and Gla domains in intact proteins are general in nature.

Properties of a Recombinant Chimeric Protein in which the gamma-Carboxyglutamic Acid and Helical Stack Domains of Human Anticoagulant Protein C Are Replaced by those of Human Coagulation Factor VII

1997 ◽  
Vol 77 (05) ◽  
pp. 0926-0933 ◽  
Author(s):  
Jie-Ping Geng ◽  
Francis J Castellino
Keyword(s):  
Metal Ion ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Chimeric Protein ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Wild Type ◽  
Coagulation Factor Vii ◽  
Carboxyglutamic Acid ◽  
Wild Type Counterpart

SummaryA chimeric cDNA, encoding residues 1-46 (the γ-carboxyglutamic acid module and its trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419 of human anticoagulant protein C (PC), was constructed and expressed. The resulting protein, r-[∆GD-HSPC/∇GD-HSfVII]PC, was properly processed with regard to signal/ propeptide release, cleavage of the K156R dipeptide, Gla and Hya contents, and the presence of glycosylation.The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to acidic phospholipid vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for the Ca2+-induced conformation of the Gla domain (GD) of PC, but did react with another MAb directed in part to the Ca2+-dependent conformation of the GD of fVII. Further, this chimeric protein possessed similar steady state constants as wild-type r-PC toward activation by thrombin and thrombin/thrombomodulin. The activated form of the chimera was very similar to that of its wild- type counterpart in its whole plasma anticoagulant activity, as well as its activity toward inactivation of coagulation factor VIII. The chimeric protein did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region of fVII is insufficient for that particular interaction.The results demonstrate that the GD/HS of fVII, when present in the PC and APC background, serves to maintain the Ca2+/PL-related functions of these latter proteins, and suggest that the Ca2+ and PL- dependent interactions of the GD-HS of PC are sufficiently general in nature such that the GD-HS regions of other proteins of this type can satisfy most of the requirements of PC and APC. The data presented also offer support for the independent nature of the domain unit consisting of the GD/HS module.

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952 ◽  
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

Novel Sites of Gamma Carboxylation in Human Factor VII

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3759-3759
Author(s):  
Emily M Wilkerson ◽  
Barbara Bates ◽  
Kraig T Kumfer ◽  
Nicholas M Riley ◽  
Brad S Schwartz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer ◽  
Protein Function ◽  
Synthetic Peptides ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Post Translational Modification ◽  
Coagulation Factor Vii ◽  
Gla Domain

Abstract Introduction Gamma- (γ-) carboxylation of glutamate residues is a vitamin K-dependent post-translational modification critical to the function of several plasma proteins. Found in the N-terminal domains of specific proteins, most of which are involved in hemostasis, these γ-carboxyglutamate residues (Gla) help mediate binding of divalent cations and are essential to protein function. Coagulation factor VII(a) bears 10 known Gla residues as characterized by N-terminal sequencing, yet Thim et al.( Biochemistry 27:7785 1988) used amino acid analysis to quantify 11.0 mol of Gla/mol of protein for plasma-derived factor VII(a). We used mass spectrometry to map and validate Gla residues of coagulation factor VII(a) to potentially identify Gla residues outside of the Gla domain. Methods Four sources of factor VII(a) (2 plasma-derived and 2 recombinant) were extracted, digested, and analyzed by tandem mass spectrometry (LC-MS/MS). Each sample was digested with trypsin and chymotrypsin to provide orthogonal coverage. Peptides derived from factor VII(a) proteolysis were analyzed on a nanoLC coupled to a quadrupole-Orbitrap-quadrupole linear ion trap mass spectrometer (Orbitrap Fusion Lumos Thermo Scientific). Multiple fragmentation methods were used to map and validate the sites including collisional based dissociation (CAD), higher energy collisional activated dissociation (HCD), electron transfer dissociation (ETD), and electron-transfer/higher-energy electron transfer activation (EThcD). Tandem MS spectra were collected at resolution 30K at 200 m/z, and data were processed using MaxQuant, COMPASS, and Proteome Discoverer. All identified sites were validated through manual annotation of spectra. We estimate that any site with >1% occupancy will be identified as Gla with this method. To validate select sites of novel gamma carboxylation, synthetic peptides were made for 4 different sites that were consistently identified in factor VII(a) from all 4 sources. Synthetic peptides were analyzed using MS methods described above, generating "true positives" to match with peptides identified from the factor VII(a) sources. Spectra from the synthetic peptides and factor VII from each source were compared using manual spectral annotation. Results In addition to identifying known Gla residues at positions 6, 7, 19, 20, 29, and 35 of factor VII(a), we detected and validated 9 novel Gla residues outside of the N-terminal Gla domain. Novel sites include residues 94, 116, 132, 219, 215, 229, 265, 196 and 385. Four of these residues (210, 220, 296 and 385) were identified as Gla in all 4 sources of factor VII(a) and were validated with synthetic peptides using a combination of fragmentation methods, providing high confidence in their characterization. Published crystallographic data suggest that residues 210 and 220 of factor VIIa-tissue factor are closely approximated to a Ca2+ ion complexed to the C-terminal protease domain; this is not the case for residues 296 and 385. We continue to refine the technique to map the Gla residues (novel and known), and to quantify the fraction of factor VII(a) molecules from each source that contain the modification at each site, in order to better incorporate our data with established studies showing >90% occupancy at each of the 10 Gla domain sites. These data suggest there is room to expand our understanding of how carboxylation contributes to specific protein function, in order to provide more comprehensive understanding of this post-translational modification, and refine our understanding of hemostatic mechanisms. Disclosures No relevant conflicts of interest to declare.

scholarly journals A candidate activation pathway for coagulation factor VII

2019 ◽  
Vol 476 (19) ◽  
pp. 2909-2926
Author(s):  
Tina M. Misenheimer ◽  
Kraig T. Kumfer ◽  
Barbara E. Bates ◽  
Emily R. Nettesheim ◽  
Bradford S. Schwartz
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Contact Activation ◽  
Coagulation Factor Vii ◽  
Factor Viiia

Abstract The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

scholarly journals Sequence Determinants of Mouse Protein C Interaction with Mouse Endothelial Protein C Receptor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4227-4227
Author(s):  
Giulia Pavani ◽  
Katherine A. Stafford ◽  
Paris Margaritis
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Critical Role ◽  
Single Amino Acid ◽  
Factor Vii ◽  
Endothelial Protein C Receptor ◽  
Coagulant Activity ◽  
Single Amino Acid Residue ◽  
Gla Domain ◽  
Single Substitution

Abstract The Endothelial Protein C Receptor (EPCR) is an important component of the Protein C anticoagulant pathway. The interaction of Protein C (PC) through its Gla domain with EPCR enhances PC activation, thus down-regulating thrombin production. EPCR can also bind human activated Factor VII (FVIIa) and modulate its activity and localization. The residues involved in receptor recognition in both PC and FVIIa are Phe4 and Leu8, located in the first portion of the Gla domain. The importance of Phe8 is indicated by the lack of EPCR binding of a PC variant that contains a Val8 from human prothrombin. Remarkably, the similarity between PC and FVIIa for EPCR binding is lacking in the mouse. Others and we have shown that mouse FVIIa (mFVIIa, which contains a Leu4 and a Leu8) interacts with mouse EPCR (mEPCR) poorly, thus failing to model the spectrum of known human FVIIa properties. In previous work, we generated mFVIIa chimeras that contain parts of the mouse PC (mPC) Gla domain and determined that three residues in the mPC Gla domain can confer mEPCR binding to mFVIIa. Specifically, molecule mFVIIa-FMR that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from mPC was functionally similar to mFVIIa and could bind mEPCR as a true gain-of-function. However, little is known on the contribution of any/all of these positions in mPC binding to mEPCR. Here, we wanted to understand the sequence determinants that dictate this interaction. For this, we generated single amino acid mutants of mPC at position 4, 8 or 9 from the corresponding residues of mFVIIa. Using conditioned medium from transiently transfected cells, we tested the ability of each mPC mutant to bind to mEPCR expressed on the surface of CHO-K1 cells. A single substitution of Phe4 with Leu abolished mEPCR binding of mPC, in contrast to modifications at position 8 (Met to Leu) or 9 (Arg to Trp). The importance of Phe4 for the mPC-mEPCR interaction was confirmed in a reverse experiment modifying mFVIIa (that has poor mEPCR affinity) individually at position 4 (Leu to Phe), 8 (Leu to Met) or 9 (Trp to Arg) according to the mPC sequence. We found that Leu4->Phe was the sole modification that could confer mEPCR binding to mFVIIa. We have previously shown that the interaction of mFVIIa-FMR with mEPCR enhances its hemostatic function (vs. mFVIIa) after administration in hemophilic mice that have undergone injury (Pavani G et al, Blood 2014). To further explore the contribution of position 4 (Leu->Phe [L4F]) in these effects, recombinant mFVIIa-L4F was purified. Titration of mFVIIa-L4F on CHO-K1 cells expressing mEPCR showed a specific and dose-dependent receptor binding, in contrast to mFVIIa, confirming our previous data (see above). Moreover, mFVIIa-L4F showed no difference in clotting activity compared to mFVIIa in a prothrombin time-based assay. In order to compare mFVIIa-L4F to mFVIIa in its ability to generate mouse thrombin, we used a thrombin generation assay using hemophilia B plasma spiked with either procoagulant. We found that addition of either mFVIIa or mFVIIa-L4F generated similar amounts of thrombin at all concentrations tested (3.1 - 25 nM). Therefore, mFVIIa-L4F exhibited similar coagulant activity to mFVIIa but gained mEPCR binding capacity, a feature shared with mFVIIa-FMR. Further experiments are underway to determine whether the single substitution in mFVIIa-L4F is sufficient to recapitulate the enhanced hemostatic properties observed in vivo with mFVIIa-FMR. In conclusion, our findings identify a single amino acid residue (Phe4) in the Gla domain of mouse PC that plays a critical role in the binding to its natural receptor. This property can also be transplanted into mFVIIa, without affecting its coagulant activity. These observations reveal another difference between human and mouse systems and may have implications for EPCR-dependent functions or properties of other vitamin K-dependent proteins. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

scholarly journals The Impacts of G4S Mutation on N-glycosylation and conformation of the Human Coagulation Factor IX GLA domain: In silico and In vitro Analysis

2021 ◽  
Author(s):  
Fahimeh Ghasemi ◽  
Mina Maddah ◽  
Hourieh Kalhor ◽  
Mohsen Khorashadizadeh ◽  
Alireza Zomorodipour
Keyword(s):  
Amino Acid ◽  
Coagulation Factor ◽  
Glycosylation Site ◽  
Factor Ix ◽  
Hemophilia B ◽  
Phospholipid Bilayer ◽  
Single Amino Acid ◽  
Missense Mutations ◽  
Coagulation Factor Ix ◽  
Gla Domain

Abstract Missense mutations are the most prevalent form of mutation in hemophilia B patients. These alterations may result in the creation of novel and non-native N-glycosylation sites (Asn-X-Ser/Thr) through single amino acid substitutions. The pathogenic mechanisms of N-glycosylation mutations in hemophilia B patients have not been extensively studied yet. By survey among known missense mutations, we found only one N-glycosylation mutation in the γ-carboxyglutamic-rich (GLA) domain of the human coagulation factor IX (hFIX). This mutation that was reported in patients with mild and moderate hemophilia B, is caused by G4S amino acid substitution. To investigate the possibility of glycan attachment to the novel N-glycosylation site in G4S-mutant hFIX and the occurrence of hyperglycosylation, site-directed mutagenesis was applied to introduce the selected mutation into the coding sequence of the hFIX. The nucleotide sequences of the both native and G4S-mutant hFIX were separately cloned into the pcDNA3.1 expression plasmid and transiently expressed in HEK293T cells. Our results from gradient SDS-PAGE and western blotting analysis of the both recombinant native and mutant hFIX demonstrated no glycan attachment to the new N-glycosylation site in the G4S-mutant hFIX. Molecular dynamics (MD) simulation was also conducted to provide atomistic insights into structure and behavior of the native and G4S-mutant GLA domains in the both free and membrane-bound states. The results revealed that the mutation slightly affected the dynamic behavior of the mutant GLA domain. The conformational analysis proved that the native GLA domain had less fluctuation and more stability than the mutant GLA domain. The slight conformational changes may influence the binding capacity and interaction of the mutant GLA domain to phospholipid bilayer which is necessary for coagulation activity of the hFIX. These findings were in accordance with the nature of the G4S mutation which causes mild hemophilia B.

scholarly journals Modification of the N-terminus of human factor IX by defective propeptide cleavage or acetylation results in a destabilized calcium-induced conformation: effects on phospholipid binding and activation by factor XIa

1997 ◽  
Vol 323 (3) ◽  
pp. 629-636 ◽  
Author(s):  
Emiel G. C. WOJCIK ◽  
Marieke VAN DEN BERG ◽  
Swibertus R. POORT ◽  
Rogier M. BERTINA
Keyword(s):  
Metal Ion ◽  
Three Dimensional ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Dimensional Structure ◽  
Amino Terminus ◽  
Human Factor Ix ◽  
Carboxyglutamic Acid ◽  
Factor Xia ◽  
Gla Domain

The propeptide of human coagulation factor IX (FIX) directs the γ-carboxylation of the first 12 glutamic acid residues of the mature protein into γ-carboxyglutamic acid (Gla) residues. The propeptide is normally removed before secretion of FIX into the blood. However, mutation of Arg-4 in the propeptide abolishes propeptide cleavage and results in circulating profactor IX in the blood. We studied three such genetic variants, factor IX Boxtel (Arg-4 → Trp), factor IX Bendorf (Arg-4 → Leu) and factor IX Seattle C (Arg-4 → Gln). These variant profactor IX molecules bind normally to anti-FIX:Mg(II) antibodies, which indicates that the mutations do not seriously affect γ-carboxylation. Metal ion titration of the binding of variant profactor IX to conformation-specific antibodies demonstrates that the calcium-induced conformation is destabilized in the variant molecules. Also the binding of FIX Boxtel to phospholipids and its activation by factor XIa requires a high (> 5 mM) calcium concentration. The three-dimensional structure of the Gla domain of FIX in the presence of calcium indicates that the acylation of the amino-terminus, rather than the presence of the propeptide, was responsible for the destabilization of the calcium-induced conformation. In order to confirm this, the α-amino group of Tyr1 of FIX was acetylated. This chemically modified FIX showed a similar destabilization of the calcium-induced conformation to variant profactor IX. Our data imply that the amino-terminus of FIX plays an important role in stabilizing the calcium-induced conformation of the Gla domain of FIX. This conformation is important for the binding to phospholipids as well as for the activation by factor XIa. Our results indicate that mutations in FIX that interfere with propeptide cleavage affect the function of the protein mainly by destabilizing the calcium-induced conformation.

A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates

2002 ◽  
Vol 363 (2) ◽  
pp. 411-416 ◽  
Author(s):  
Raffaella TOSO ◽  
Mirko PINOTTI ◽  
Katherine A. HIGH ◽  
Eleanor S. POLLAK ◽  
Francesco BERNARDI
Keyword(s):  
Tissue Factor ◽  
Coagulation Factor ◽  
Catalytic Efficiency ◽  
Fibrin Clot ◽  
Enzymic Activity ◽  
Factor Ix ◽  
Site Directed Mutagenesis ◽  
Factor Vii ◽  
Factor X ◽  
Coagulation Factor Vii

Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa—TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa—TF complex, but also in the interaction of FVII with both its activators and cofactor TF.

Functional Characterization of a Recombinant FVIIa Drug Candidate

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4653-4653
Author(s):  
Peter L Turecek ◽  
Katalin Varadi ◽  
Susanne Vejda ◽  
Ernst Boehm ◽  
Hanspeter Rottensteiner ◽  
...  
Keyword(s):  
Factor Viii ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Animal Origin ◽  
Drug Candidate ◽  
Coagulation Factor Vii ◽  
Fibrin Formation

Abstract Abstract 4653 Human coagulation factor VII (FVII) is a vitamin-K-dependent protein with a molecular weight of 50 kDa. Activation of FVII occurs by cleavage of the arginine 152 – isoleucine 153 peptide bond resulting in two disulfide-linked peptide chains. The therapeutic utility of rFVIIa is based on its capacity to trigger hemostasis independently from factor VIII and factor IX and thus even in the presence of factor VIII or factor IX inhibitors. Baxter has developed a recombinant FVIIa (rFVIIa) that is synthesized by a genetically engineered Chinese hamster ovary (CHO) cell line. No materials of human or animal origin are employed in the manufacture, purification, or formulation of the final product, thus reducing the risk of transmission of adventitious agents. The growth medium is a chemically defined medium, and the downstream process does not use monoclonal antibodies for the purification of rFVIIa. The rFVIIa drug candidate was functionally characterized in vitro and its features were compared with those of a commercially available rFVIIa. The overall hemostatic potency of rFVIIa was assessed by its FVIII bypassing activity in a human FVIII-deficient plasma with high-titer inhibitor by measuring the kinetics of thrombin generation and fibrin formation. The thrombin-generating capacity of the rFVIIa drug candidate was similar to that of commercial rFVIIa. The time and rate of fibrin formation was measured by thromboelastography, where a dose-dependent normalization of the impaired clotting times and fibrin formation was observed. A similar FXa-generating potency was found for rFVIIa and commercial rFVIIa when measured on the surface of TF-expressing fibroblasts, suggesting a full capability to bind to TF-bearing cells and trigger hemostasis on their surfaces. rFVIIa could be inactivated by anti-thrombin III-heparin in solution with relipidated TF with no relevant difference to the comparator product. TF pathway inhibitor effectively inhibited FXa generation in a cell-based activity assay, with a similar IC50 for rFVIIa and the comparator. In summary, the functional characteristics of the rFVIIa drug candidate were very similar to those of commercially available rFVIIa. Disclosures: Turecek: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Boehm:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Reiter:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Mundt:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

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