Generation of Transgenic Mice Expressing High Levels of Activated Murine Coagulation Factor VII.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5253-5253
Author(s):  
Majed N. Aljamali ◽  
Paris Margaritis ◽  
Alexander Schlachterman ◽  
Katherine A. High
Keyword(s):  
Transgenic Mice ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Wild Type ◽  
Extrinsic Pathway ◽  
Coagulation Activity ◽  
Coagulation Factor Vii ◽  
Gene Therapy Approach

Abstract Treatment of acute bleeding episodes in hemophilic patients with inhibitors can be successfully managed by the infusion of recombinant human factor VIIa (rhFVII, NovoSeven“). We have recently shown the efficacy of a gene transfer approach to treat hemophilia B (HB) mice by adeno-associated virus (AAV) expressing activated murine FVII (mFVIIa) (J Clin Invest. 2004 Apr; 113(7): 1025–31). To assess the consequences of long-term expression of different levels of mFVIIa, we generated transgenic mice expressing mFVIIa driven by a liver-restricted (transthyretin) promoter. Results from four founders have been analyzed. The levels of mFVII antigen in both founders and their offspring were 3.5–7.5 microgram/ml, about 2.5–5 fold the baseline compared to their non-transgenic littermates. Moreover, the expressed protein retains its coagulation activity in the extrinsic pathway as demonstrated by shortening of the prothrombin time (PT) from 22.3±0.6 sec in the non-transgenic mice to 12.3±1.6 sec in the transgenic littermates. We found two male HB mice that were also transgenic for mFVIIa, resulting from the breeding of one male founder and an HB heterozygote female. The high levels of mFVII antigen (7.5 and 5.5 microgram/ml) were accompanied by significantly shorter PTs (9.8 and 12.3, respectively) compared to wild-type baseline of 22 sec, and most importantly, by a shorter activated partial thromboblastin time (aPTT) compared to their two HB littermates (36.3 and 28.8 versus 61.3 and 61.8 sec, respectively), i.e., mice transgenic for mFVIIa show aPTT similar to their wild type littermates. Additionally, kinetics and general characteristics of in vivo clot formation after laser-induced injuries to the arteries of the cremaster muscle were similar in an HB-mFVIIa transgenic mouse (the only one tested) and in normal mice, while clots were absent in HB control mice. On the other hand, thrombin anti-thrombin (TAT) levels in the transgenic mice were comparable to their HB and wild type littermates. These findings support the efficacy and safety of a gene therapy approach for the expression of mFVIIa and should further allow us to assess the risk of continuous expression of elevated levels of mFVIIa in mouse plasma.

scholarly journals The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII

1990 ◽  
Vol 265 (2) ◽  
pp. 327-336 ◽  
Author(s):  
V J J Bom ◽  
R M Bertina
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Reaction Rate ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Fluid Phase ◽  
Fold Increase ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.

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 Funksionele beskrywing van ’n faktor VIIa inhiberende peptied, IP-7, geselekteer deur faagblootleggingstegnologie

2006 ◽  
Vol 25 (4) ◽  
pp. 209-220
Author(s):  
S.M. Meiring ◽  
C.E. Roets ◽  
P.N. Badenhorst
Keyword(s):  
Phage Display ◽  
Tissue Factor ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Competitive Inhibitor ◽  
Factor Vii ◽  
Antithrombotic Agent ◽  
Phage Display Technology ◽  
Current Form ◽  
Coagulation Factor Vii

Die tegniek van faagblootlegging is gebruik om ’n sikliese heptapeptied te selekteer wat met weefselfaktor(WF) kompeteer vir binding aan stollingsfaktor VIIa. Die aminosuurvolgorde van die peptied is Cys-Ala- Trp-Pro-His-Thr-Pro-Asp-Cys (C-AWPHTPD-C) en dit verleng die protrombientyd (PT) op ’n konsentrasie-afhanklike wyse. Die peptied beperk plaatjieklewing aan beide menslike endoteelsel- en weefselfaktormatrikse in ’n vloeikamermodel onder arteriële vloeitoestande. Die peptied funksioneer as ’n volledig mededingende inhibeerder van faktor VIIa met ’n inhibisiekonstante (Ki) van 123,2 μM. In sy huidige vorm is die peptied waarskynlik nie sterk genoeg om verder as antitrombotiese middel ontwikkel te word nie, maar verskillende strategieë kan gevolg word om die werking daarvan te versterk. AbstractFunctional characterisation of a factor VIIa inhibiting peptide, IP-7 selected by phage display technology By using the technique of phage display, we selected a cyclic heptapeptide sequence Cys-Ala-Trp-Pro-His-Thr-Pro-Asp-Cys (C-AWPHTPD-C) that competes with tissue factor for binding to coagulation factor VII. This peptide prolongs the prothrombin time (PT) in a concentration dependent way. It also reduces platelet adhesion to both human endothelial cell and tissue factor matrixes in a flow chamber under arterial flow conditions. Furthermore, it acts as a full competitive inhibitor of factor VIIa with an inhibition constant (Ki) of 123,2 μM. In its current form the peptide is probably not sufficiently potent for development as an antithrombotic agent, but different strategies could be followed to reinforce its performance.

scholarly journals Modulating the Antithrombin-Mediated in Vivo Clearance of Coagulation Factor VIIa

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4233-4233 ◽  
Author(s):  
Henrik Østergaard ◽  
Lene Hansen ◽  
Hermann Pelzer ◽  
Henrik Agersø ◽  
Anette A. Pedersen ◽  
...  
Keyword(s):  
Complex Formation ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Sprague Dawley ◽  
Resistant Variant ◽  
Opposite Behavior

Abstract The short half-life of coagulation factor VIIa (FVIIa) in circulation is the result of elimination through multiple pathways of which inactivation by the plasma inhibitor antithrombin (AT) accounts for as much as 65% of the total clearance in humans. Remarkably, the rate of inhibition in vivo is about 30 times greater than the uncatalyzed rate of inhibition in vitro suggesting the presence of rate enhancing components in vivo (Agersø et al. (2011) J Thromb Haemost, 9:333-338). Prime candidates include endogenous heparin-like glycosaminoglycans (GAGs) potentiating the reactivity of antithrombin, or tissue factor (TF) which upon binding to FVIIa increases its susceptibility to inhibition. In the present study site-directed mutagenesis of FVIIa was undertaken to identify variants with altered AT reactivity in order to explore the relationship between the reactivity of FVIIa with AT in vitro and in vivo as well as the nature of endogenous rate enhancing components. The pharmacokinetic properties of FVIIa variants were determined in Sprague Dawley rats as this model recapitulates the aspects of AT-mediated FVIIa clearance observed in humans and allows for interaction of human FVIIa with endogenous rat TF. Similar to the human situation, inactivation of wild-type FVIIa in rat is evident as an accumulation of circulating FVIIa-AT complexes and a progressive divergence of the pharmacokinetic profiles representing FVIIa clot activity and total FVIIa antigen. Initially, the ability to modulate the in vivo complex formation with AT was investigated using two FVIIa variants exhibiting enhanced (>200%) or reduced (<10%) in vitro reactivity with AT, respectively, regardless of the type of cofactor present. Reflecting the in vitro reactivity, clot activity and antigen PK profiles in rats were found to coincide for the AT resistant variant along with essentially no detectable AT complex formation, whereas exacerbated AT complex formation and clot activity:antigen discrepancy was observed for the variant exhibiting enhanced in vitro reactivity. Interestingly, among the generated FVIIa variants with altered AT reactivity, two subsets were identified that displayed differential in vitro reactivity with AT depending on the type cofactor present. Accordingly, one group exhibited a greater susceptibility to inhibition relative to wild-type FVIIa in the presence of heparin but not in the presence of TF, while the other group demonstrated the opposite behavior. Endowed with the ability to report on the cofactor identity from the rate of inhibition relative to wild-type FVIIa, variants from each group were tested for their tendency to accumulate as complexes with AT following intravenous administration to rats. Supporting a contribution from endogenous GAGs to the in vivo inactivation of FVIIa, the measured in vivo peak levels of accumulated FVIIa-AT complexes were found to directly correlate with the in vitro rate constants determined for the variants in the presence of heparin, but not when the cofactor was TF or the combination of TF and heparin. Altogether, these results 1) demonstrate a direct relationship between the in vitro reactivity of FVIIa with AT in the presence of heparin and the clearance of FVIIa through this pathway in vivo, and 2) identify heparin-like GAGs as the likely rate enhancing component of FVIIa inhibition in vivo. Disclosures Østergaard: Novo Nordisk A/S: Employment. Hansen:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Agersø:Novo Nordisk A/S: Employment. Pedersen:Novo Nordisk A/S: Employment. Glue:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Andersen:Novo Nordisk A/S: Employment. Bjelke:Novo Nordisk A/S: Employment. Breinholt:Novo Nordisk A/S: Employment. Stennicke:Novo Nordisk A/S: Employment. Gandhi:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Hermit:Novo Nordisk A/S: Employment.

scholarly journals Monitoring of Recombinant Activated Coagulation Factor VII (rFVIIa) Therapy in Patients (pts) with Inherited Factor VII (FVII) Deficiency

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2412-2412
Author(s):  
Gennadii M. Galstian ◽  
Olesya A. Polevodova ◽  
Elena Yakovleva ◽  
Antonina Shchekina ◽  
Igor Davydkin ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Screening Tests ◽  
Factor Vii ◽  
Extrinsic Pathway ◽  
Coagulation Factor Vii ◽  
Hemostatic Effect ◽  
Normal Ranges ◽  
Rfviia Administration ◽  
Fvii Deficiency

Introduction and Objectives: Inherited FVII deficiency is a rare hemorrhagic disorder.Clinical bleeding does not correlate with the level of FVII plasma activity. FVII involves in the extrinsic coagulation pathway. ROTEM is point of care method. EXTEM is one of ROTEM screening tests. EXTEM analyses the extrinsic pathway of coagulation. Aim of the study was to evaluate hemostatic effect and safety of rFVIIa treatment in pts with inherited FVII deficiency. Materials and Methods: The results of rFVIIa treatment were investigated in 4 pts (1 male, 3 females) with inherited FVII deficiency. Pts were treated with rFVIIa before gynecological (2), abdominal (1) and orthopedic surgery (1). Pts received 30 mcg/kg of rFVIIa (Koagil VII, Generium, Russia) before surgery. Plasma FVII activity, fibrinogen, INR, APTT, ROTEM and thromboelastography (TEG) parameters were investigated before rFVIIa administration, then in 15 minutes, in 2 hours, in 6 hours and in 12 hours. Results: Before rFVIIa administration median plasma FVII activity was low, CT EXTEM was long and INR was greater than 3. TEG, APTT and fibrinogen were in normal ranges (table 1, fig. 1). Fifteen minutes after rFVIIa administration FVII:C activity increased, INR and CT EXTEM decreased. Hemostatic effect of rFVIIa remained during 12 hours. During this period CT EXTEM correlated with INR (r = 0.97, p<0.001). Other parameters did not change significantly. None patients had hemorrhagic complications. Conclusion: CT EXTEM allowed to evaluate hemostatic effect of rFVIIa in operating room and good correlated with INR in pts with inherited FVII deficiency. Disclosures No relevant conflicts of interest to declare.

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.

Novel aberrant splicings caused by a splice site mutation (IVS1a+5g>a) in F7 gene

2005 ◽  
Vol 93 (06) ◽  
pp. 1077-1081 ◽  
Author(s):  
Qihua Fu ◽  
Xuefeng Wang ◽  
Yiqun Hu ◽  
Zhenyi Wang ◽  
Qiulan Ding ◽  
...  
Keyword(s):  
Splice Site ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Heterozygous Mutation ◽  
Alternative Transcript ◽  
Compound Heterozygous ◽  
Nucleotide Position ◽  
Coagulation Activity ◽  
Exon 2 ◽  
Coagulation Factor Vii

SummaryLow FVII coagulant activity (FVII:C 8.2%) and antigen level (FVII:Ag 34.1%) in a 46-year-old Chinese male led to a diagnosis of coagulation factor VII (FVII) deficiency. Compound heterozygous mutations were identified in his F7 gene:a G to A transition in the 5’ donor splice site of intron 1a (IVS1a+5g>a) and a T to G transition at the nucleotide position 10961 in exon 8, resulting in a His to Gln substitution at amino acid residue 348. An analysis of ectopic transcripts of F7 in the leukocytes of the patient reveals that the mutation (IVS1a+5g>a) is associated with two novel aberrant patterns of splicing. The predominant alternative transcript removes exon 2, but retains intron 3, which shifts the reading frame and predicts a premature translation termination at the nucleotide positions 2–4 in intron 3. The minor alternative transcript skips both exon 2 and exon 3 (FVII Δ2, 3), leading to an in-frame deletion of the propeptide and γ-carboxylated glutamic acid (Gla) domains of mature FVII protein. In vitro expression studies of the alternative transcript FVII Δ2, 3 by transient transfection of HEK 293 cells with PcDNA 3.1(-) expression vector showed that although the mutant protein could be secreted, no pro-coagulation activity was detected. The coexistence of the two abnormal transcripts and a heterozygous mutation His348Gln, explained the patient’s phenotype.

scholarly journals Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 549
Author(s):  
Jesper J. Madsen ◽  
Ole H. Olsen
Keyword(s):  
Factor Viia ◽  
Conformational Dynamics ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Atomistic Simulations ◽  
Protease Domain ◽  
Proteolytic Activation ◽  
Coagulation Factor Vii ◽  
Conformational Plasticity ◽  
And Function

The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics.

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