Cloning and Characterization of the Murine Coagulation Factor X Gene

2000 ◽  
Vol 83 (05) ◽  
pp. 732-735 ◽  
Author(s):  
Adrian Cooper ◽  
Zhong Liang ◽  
Francis Castellino ◽  
Elliot Rosen
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Factor Viii ◽  
Start Codon ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Coagulation Factor Vii ◽  
Coagulation Factor V

SummaryThe gene encoding murine coagulation factor X (fX) was isolated and characterized from a λFIX II library generated from murine genomic DNA. The 20130 bp sequence contains 18049 nucleotides that extend from the initiating methionine to the polyadenylation site. 1056 nucleotides 5’ of the start codon were determined and contain putative start sites for the FX mRNA as well as sites for binding of putative transcription factors. The sequence extends 1024 3’ of the polyadenylattion site.The gene contains 8 exons and 7 introns which were determined by comparing the mouse FX cDNA and gene sequences. The exonic structure of the gene is similar to that of the other mammalian vitamin K-dependent serine proteases of the coagulation system. These include an exon encoding the prepropepetide, the gladomain, a short helical stack, two exons for the two EGF domains, the activation pepetide, and two exons encoding the serine protease domain. The 5’ sequence of the mouse FX gene overlaps with the 3’ region of the FVII gene indicating that the murine FVII and FX gene are arranged in a head to tail arrangement as they are in humans. Abbreviations: fVII, coagulation factor VII; fIX, coagulation factor IX; fX, coagulation factor X; PC, Protein C; fV, coagulation factor V; fVa, activated coagulation factor V; fVIII, coagulation factor VIII; fVIIIa, activated coagulation factor VIII.

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.

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.

Generation Of Coagulation Factors By The Isolated Rat Liver Perfused With A Synthetic Blood Substitute

1981 ◽  
Author(s):  
C A Owen ◽  
E J W Bowie
Keyword(s):  
Factor Viii ◽  
Rat Liver ◽  
Blood Substitute ◽  
Factor Ix ◽  
Factor Vii ◽  
Clotting Factor ◽  
Isolated Perfused Rat Liver ◽  
Factor Xii ◽  
Factor V ◽  
Factor X

Measuring the release of small amounts of a clotting factor from an isolated perfused rat liver is difficult if the perfusate already contains some of the factor. Further, platelet-containing perfusates generate a coagulant activity that may invalidate clotting assays.We have successfully employed a completely synthetic blood substitute for rat liver perfusions. The perfusate is “Fluosol-43” generously furnished by Alpha Therapeutic Corporation. The oxygen-carrying perfluorochemical is FC-43 (perfluorotributylamine) and the substitute for albumin is hydroxyethyl starch. Using the Brauer perfusion technique, we found that rat livers in 5 hours released an average of 2.3% of the normal plasma concentration of prothrombin, 8.4% factor V, 16.2% factor VII, 7.0% factor IX, 3.7% factor X, 28.3% factor XI and 12.3% factor XII. Antithrombin III and plasminogen were also generated.Only minute amounts of factor VIII were released unless serum, cryoprecipitate or cryoprecipitate-free plasma was added; then the yield was 8.8% on average. The more “venom factor” (platelet aggregability with Bothrops alternata venom) added to the synthetic perfusate, the more factor VIII was released.

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.

scholarly journals Inhibitors in Patients with Congenital Bleeding Disorders Other Than Hemophilia

2017 ◽  
Vol 44 (06) ◽  
pp. 595-603
Author(s):  
Giuseppe Marano ◽  
Carlo Mengoli ◽  
Vanessa Piccinini ◽  
Simonetta Pupella ◽  
Stefania Vaglio ◽  
...  
Keyword(s):  
Considerable Increase ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor V ◽  
Bleeding Disorders ◽  
The Past ◽  
Congenital Deficiency ◽  
Coagulation Factor V

AbstractThe most worrying complication of replacement therapy for severe hemophilia A and B is currently the occurrence of inhibitory alloantibodies against infused factor VIII and factor IX, respectively. Inhibitors compromise the management of hemorrhage in affected patients, with a considerable increase in complications, disability, and costs. While these alloantibodies have been extensively studied in the past years in hemophilia A and B, those occurring in patients with other inherited bleeding disorders are less well characterized and still poorly understood, mostly due to the rarity of these hemorrhagic conditions. This narrative review will deal with inhibitors arising in patients with inherited bleeding disorders other than “classical” hemophilia, focusing in particular on those developing in patients with congenital deficiency of coagulation factor V, factor VII, factor XI, and factor XIII.

Prothrombin Activation Is Increased among Asymptomatic Carriers of the Prothrombin G20210A and Factor V Arg506Gln Mutations

2000 ◽  
Vol 84 (09) ◽  
pp. 396-400 ◽  
Author(s):  
Steve Humphries ◽  
Belinda Smillie ◽  
Lily Li ◽  
Jacqueline Cooper ◽  
Samad Barzegar ◽  
...  
Keyword(s):  
Factor Viia ◽  
Conclusive Evidence ◽  
Factor Ix ◽  
Factor Vii ◽  
Prothrombin G20210a ◽  
Factor V ◽  
Factor X ◽  
Coagulation Proteins ◽  
Prothrombin Activation

SummaryThe risk of venous thrombosis is increased in individuals who carry specific genetic abnormalities in blood coagulation proteins. Among Caucasians, the prothrombin G20210A and factor V Arg506Gln (FV R506Q) mutations are the most prevalent defects identified to date. We evaluated their influence on markers of coagulation activation among participants in the Second Northwick Park Heart Study, which recruited healthy men (aged 50–61 years) from nine general medical practices in England and Wales. They were free of clinical vascular disease and malignancy at the time of recruitment. Genotypes for the two mutations were analyzed using microplate array diagonal gel electrophoresis, and coagulation markers (factor XIIa; activation peptides of factor IX, factor X, and prothrombin; fibrinopeptide A) were measured by immunoassay. Factor VII coagulant activity and factor VIIa levels were determined by a functional clotting assay. Among 1548 men genotyped for both mutations, 28 (1.8%) and 52 (3.4%) were heterozygous for prothrombin G20210A and FV R506Q, respectively. The only coagulation marker that was significantly associated with the two mutations was prothrombin activation fragment F1+2 [mean ± SD, 0.88 ± 0.32 nmol/L in men with prothrombin G20210A (p = 0.002) and 0.89 ± 0.30 in men with FV R506Q (p = 0.0001) versus 0.72 ± 0.24 among non-carriers for either mutation]. This data provides conclusive evidence that heterozygosity for the prothrombin G20210A as well as the FV R506Q mutations in the general population leads to an increased rate of prothrombin activation in vivo.

Multidimensional Similarity of Letters

1969 ◽  
Vol 28 (1) ◽  
pp. 3-12 ◽  
Author(s):  
Teodor Kuennapas ◽  
Anne-Jeanette Janson
Keyword(s):  
Factor Viii ◽  
Factor Ix ◽  
Factor Vii ◽  
Similarity Analysis ◽  
Factor V ◽  
Factor I ◽  
Considerable Portion ◽  
Lower Case ◽  
Factor Ii

28 lower-case letters of the Swedish alphabet were studied by the method of multidimensional similarity analysis. 57 Ss participated in the experiment. 9 factors were found. Factor I is called ‘t’ or ‘Vertical linearity,’ Factor II: ‘o’ or ‘Roundness,’ Factor III: ‘n’ or ‘Parallel vertical linearity,’ Factor IV: ‘i’ or “Vertical linearity with dot,’ Factor V: ‘p’ or ‘Roundness attached to vertical linearity,’ Factor VI: ‘k’ or ‘Vertical linearity with crossness,’ Factor VII: ‘a’ or ‘Roundness attached to a hook,’ Factor VIII: V or ‘Angularity open upward’ and Factor IX: ‘z’ or ‘Zigzaggedness.’ ‘Vertical linearity’ and ‘Roundness’ are the most important of these factors and account for a considerable portion of the similarity among many letters.

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