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.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

Formation of FVIIa-Antithrombin Complexes After Administration of rFVIIa to Hemophilia Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1295-1295
Author(s):  
Mirella Ezban ◽  
Erika Martin ◽  
John Christian Barrett ◽  
Janice Kuhn ◽  
Mindy Nolte ◽  
...  
Keyword(s):  
Complex Formation ◽  
Animal Studies ◽  
Factor Viia ◽  
Plasma Concentrations ◽  
Volume Of Distribution ◽  
Rfviia Administration ◽  
The Impact ◽  
D Dimer

Abstract Abstract 1295 Poster Board I-317 Introduction Recent animal studies suggest that measurable amounts of factor VIIa and antithrombin (AT) complexes are formed and accumulate following rFVIIa administration. The in vivo rate of inhibition has been reported to be faster than the un-stimulated in vitro reaction between AT and free rFVIIa and of the same order of magnitude as the rate determined in the presence of tissue factor. To study the impact of AT inhibition on the elimination of rFVIIa in humans, we measured the pharmacokinetics (PK) of rFVIIa and rFVIIa-AT complex formation in 10 hemophilia A or B patients. Patients and Methods The PK of single-dose rFVIIa 90 μg/kg (Novo Nordisk A/S) was evaluated in 10 severe FVIII- or FIX-deficient patients in a non-bleeding state. The plasma concentrations of FVIIa activity (FVII:C), FVII antigen (FVII:Ag), FVIIa-AT, D-dimer and F1+2 fragment were determined immediately before, and at 0.5, 1, 2, 4 and 6 hours following rFVIIa dosing. Results Significant amounts of FVIIa–AT complex were formed in vivo after rFVIIa administration, and reached a maximum of 5.4 ± 0.8 nmol/L [mean ±SD] at 2 hours following rFVIIa administration and declined to 4.4 ± 0.9 nmol/L at 6 hours, as compared to 0.1 ± 0.05 nmol/L at baseline. While the FVII:C PK data in this study were consistent with previous data, there was greater total body clearance (Cltot), a larger volume of distribution (Vdss) and a shorter plasma half-life (T1/2) of FVII:C relative to FVII:Ag (Table). No change in D-dimer was observed after the administration of rFVIIa, while a slight increase in F1+2 fragment levels to 258 ± 73 pmol/L was measured 4 hours after rFVIIa dosing, as compared to 141 ± 45 pmol/L at baseline. Conclusion A significant divergence between the clearance of rFVIIa, as determined by either FVII:C or FVII:Ag measurements, can be accounted for by AT complex formation. Inhibition by AT appears thus to have a significant impact on the elimination of FVII:C activity from the circulation when rFVIIa is administered at a therapeutic dose. Similar to animal data, the formation of the FVIIa-AT complexes in vivo was faster than anticipated from in vitro studies, indicating that the exposure to the vessel wall stimulates the FVIIa inhibition by AT. Analyses of coagulation parameters did not indicate induction of systemic coagulation. Disclosures Ezban: NovoNordisk A/S: Employment. Pelzer:NovoNordisk: Employment. Agerso:NovoNordisk: Employment. Petersen:NovoNordisk: Employment. Hedner:NovoNordisk: Employment. Carr:NovoNordisk: Employment.

scholarly journals cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 in Growing C2 Myoblasts: Role in Modulating MyoD Half-Life and Myogenic Activity

1999 ◽  
Vol 19 (4) ◽  
pp. 3167-3176 ◽  
Author(s):  
Magali Kitzmann ◽  
Marie Vandromme ◽  
Valerie Schaeffer ◽  
Gilles Carnac ◽  
Jean-Claude Labbé ◽  
...  
Keyword(s):  
Half Life ◽  
Site Directed Mutagenesis ◽  
Cyclin B ◽  
Wild Type ◽  
E Box ◽  
Integral Role ◽  
Phosphopeptide Mapping

ABSTRACT We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

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 A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2335-2340
Author(s):  
Toufik Abache ◽  
Alexandre Fontayne ◽  
Dominique Grenier ◽  
Emilie Jacque ◽  
Alain Longue ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Viiia

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 μg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.

Reduced Activation of the Gla19Ala FX Variant via the Extrinsic Coagulation Pathway Results in Symptomatic CRMred FX Deficiency

2002 ◽  
Vol 88 (08) ◽  
pp. 236-241 ◽  
Author(s):  
M. Pinotti ◽  
G. Marchetti ◽  
M. Baroni ◽  
F. Cinotti ◽  
M. Morfini ◽  
...  
Keyword(s):  
Factor Viia ◽  
Activity Levels ◽  
Factor X ◽  
Wild Type ◽  
Rich Domain ◽  
Factor Viiia ◽  
Domain Interactions ◽  
Vitro Model

SummaryWe characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the γ-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19AlaFX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation.The study of this FX deficiency provides an “in vivo” and “in vitro” model for the investigation of Gla domain interactions.

scholarly journals Prevalent Polymorphisms in Wild-Type HIV-1 Integrase Are Unlikely To Engender Drug Resistance to Dolutegravir (S/GSK1349572)

2013 ◽  
Vol 57 (3) ◽  
pp. 1379-1384 ◽  
Author(s):  
Cindy Vavro ◽  
Samiul Hasan ◽  
Heather Madsen ◽  
Joseph Horton ◽  
Felix DeAnda ◽  
...  
Keyword(s):  
Additional Data ◽  
Integrase Inhibitor ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Functional Roles ◽  
Hiv 1

ABSTRACTThe majority of HIV-1 integrase amino acid sites are highly conserved, suggesting that most are necessary to carry out the critical structural and functional roles of integrase. We analyzed the 34 most variable sites in integrase (>10% variability) and showed that prevalent polymorphic amino acids at these positions did not affect susceptibility to the integrase inhibitor dolutegravir (S/GSK1349572), as demonstrated bothin vitro(in site-directed mutagenesis studies) andin vivo(in a phase IIa study of dolutegravir monotherapy in HIV-infected individuals). Ongoing clinical trials will provide additional data on the virologic activity of dolutegravir across subject viruses with and without prevalent polymorphic substitutions.

Prolonged in-vivo half-life of factor VIIa by fusion to albumin

2008 ◽  
Vol 99 (04) ◽  
pp. 659-667 ◽  
Author(s):  
Thomas Weimer ◽  
Wilfried Wormsbächer ◽  
Ulrich Kronthaler ◽  
Wiegand Lang ◽  
Uwe Liebing ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Half Life ◽  
Mammalian Cells ◽  
Factor Viia ◽  
Therapeutic Option ◽  
Human Albumin ◽  
Pharmacokinetic Studies ◽  
Wild Type

SummaryFor the treatment of haemophilia patients with inhibitors, recombinant factor VIIa (rFVIIa) is available as a therapeutic option to control bleeding episodes with a good balance of safety and efficacy. However, the short in-vivo half-life of approximately 2.5 hours makes multiple injections necessary, which is inconvenient for both physicians and patients. Here we describe the generation of a recombinant FVIIa molecule with an extended half-life based on genetic fusion to human albumin. The recombinant FVII albumin fusion protein (rVII-FP) was expressed in mammalian cells and upon activation displayed a FVII activity close to that of wild type FVIIa. Pharmacokinetic studies in rats demonstrated that the half-life of the activated recombinant FVII albumin fusion protein (rVIIa-FP) was extended six- to sevenfold compared with wild type rFVIIa. The in-vitro and in-vivo efficacy was evaluated and was found to be comparable to a commercially available rFVIIa (NovoSeven®). The results of this study demonstrate that it is feasible to develop a half-life extended FVIIa molecule with haemostatic properties very similar to the wild-type factor.

scholarly journals Cochaperone Interactions in Export of the Type III Needle Component PscF of Pseudomonas aeruginosa

2010 ◽  
Vol 192 (14) ◽  
pp. 3801-3808 ◽  
Author(s):  
Sophie Plé ◽  
Viviana Job ◽  
Andréa Dessen ◽  
Ina Attree
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Complex Formation ◽  
Hot Spot ◽  
Protein Stabilization ◽  
Site Directed Mutagenesis ◽  
Surface Stability ◽  
Type Iii

ABSTRACT Type III secretion (T3S) systems allow the export and translocation of bacterial effectors into the host cell cytoplasm. Secretion is accomplished by an 80-nm-long needle-like structure composed, in Pseudomonas aeruginosa, of the polymerized form of a 7-kDa protein, PscF. Two proteins, PscG and PscE, stabilize PscF within the bacterial cell before its export and polymerization. In this work we screened the 1,320-Å2 interface between the two chaperones, PscE and PscG, by site-directed mutagenesis and determined hot spot regions that are important for T3S function in vivo and complex formation in vitro. Three amino acids in PscE and five amino acids in PscG, found to be relevant for complex formation, map to the central part of the interacting surface. Stability assays on selected mutants performed both in vitro on purified PscE-PscG complexes and in vivo on P. aeruginosa revealed that PscE is a cochaperone that is essential for the stability of the main chaperone, PscG. Notably, when overexpressed from a bicistronic construct, PscG and PscF compensate for the absence of PscE in cytotoxic P. aeruginosa. These results show that all of the information needed for needle protein stabilization and folding, its presentation to the T3 secreton, and its export is present within the sequence of the PscG chaperone.

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