An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice

2005 ◽  
Vol 94 (12) ◽  
pp. 1138-1147 ◽  
Author(s):  
Megan E. Begbie ◽  
Asif Mamdani ◽  
Sharon Gataiance ◽  
Louise J. Eltringham-Smith ◽  
Varsha Bhakta ◽  
...  
Keyword(s):  
Specific Activity ◽  
Factor Ix ◽  
Cleavage Sites ◽  
Post Translational Modification ◽  
Hek 293 ◽  
Haemophilia B ◽  
293 Cells ◽  
Serine Protease Activity ◽  
B Mice ◽  
Activation Peptide

SummaryThe factors responsible for the removal of injected factor IX (fIX) from the blood of individuals with haemophilia B are only partly understood, and may include binding to endothelial or subendothelial sites, passive extravasation related to size or charge, or interactions requiring fIX activation. To investigate these issues, we have produced and characterised recombinant fIX proteins with amino acid changes: Δ155–177, an internal deletion which removes most of the activation peptide while retaining the activation cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and Δ155–177 exhibited 72 and 202% of the specific activity of fIX (WT), respectively; S365A was without activity. Following intravenous injection in haemophilia B (fIX knockout) mice, recoveries did not differ for fIX (WT) and Δ155–177, but were higher for K5A and S365A. The terminal catabolic halflife of Δ155–177, alone among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection, the observed areas under the clearance curve (AUCs) of Δ155–177 and K5, but not S365A, were elevated 2-fold. Δ155–177 was equally effective as fIX (WT) in reducing blood loss following tail vein transection in haemophilia B mice. Our results suggest that deletion of the multiple sites of fIX post-translational modification found within the activation peptide eliminated important fIX clearance motifs.

scholarly journals Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2333-2341 ◽  
Author(s):  
Henrik Østergaard ◽  
Jais R. Bjelke ◽  
Lene Hansen ◽  
Lars Christian Petersen ◽  
Anette A. Pedersen ◽  
...  
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Duration Of Effect ◽  
B Mice ◽  
Bleeding Episodes ◽  
Activation Peptide ◽  
Extended Circulation

Abstract Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the Km for activation by FVIIa–tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood–based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Factor IX variants improve gene therapy efficacy for hemophilia B

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2316-2323 ◽  
Author(s):  
Joerg Schuettrumpf ◽  
Roland W. Herzog ◽  
Alexander Schlachterman ◽  
Antje Kaufhold ◽  
Darrel W. Stafford ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Intramuscular Injection ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Promising Strategy ◽  
B Mice ◽  
Higher Doses ◽  
Vector Delivery

Abstract Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis

2010 ◽  
Vol 104 (08) ◽  
pp. 355-365 ◽  
Author(s):  
Chung-Yang Kao ◽  
Chia-Ni Lin ◽  
I-Shing Yu ◽  
Mi-Hua Tao ◽  
Hua-Lin Wu ◽  
...  
Keyword(s):  
Mouse Models ◽  
Specific Activity ◽  
Factor Ix ◽  
Wild Type ◽  
Haemophilia B ◽  
Virus Particles ◽  
Thrombosis Risk ◽  
Increased Risk ◽  
Recombinant Factor Ix ◽  
Fold Excess

SummaryEngineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976–1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

scholarly journals A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

Blood ◽  
2014 ◽  
Vol 123 (4) ◽  
pp. 582-589 ◽  
Author(s):  
Jamil A. Haque ◽  
Matthew G. McDonald ◽  
John D. Kulman ◽  
Allan E. Rettie
Keyword(s):  
Vitamin K ◽  
Activity Analysis ◽  
Factor Ix ◽  
Hek 293 ◽  
Vitamin K Cycle ◽  
Structure Activity ◽  
Activity Structure ◽  
293 Cells ◽  
Key Points

Key Points Factor IX glutamyl carboxylation in engineered HEK 293 cells recapitulates in vivo anticoagulant inhibition of vitamin K cycle activity. Warfarin metabolite structure-activity analysis on vitamin K cycle antagonism determines their contributions to in vivo anticoagulation.

Galectin-3 Binding Protein Contaminates the Purification of Recombinant Factor IX.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1038-1038
Author(s):  
Mark D. Blostein ◽  
Jessica Cuerquis ◽  
Jacques Galipeau
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Biologically Active ◽  
Hek293 Cells ◽  
Post Translational Modification ◽  
Hek 293 ◽  
Galectin 3 ◽  
Recombinant Factor Ix

Abstract The hemophilas are the most common severe hereditary bleeding disorders. The standard treatment of these disorders is to infuse plasma concentrates of the missing coagulation factor. However, the high cost of replacement therapy and the serious morbidity from the transmission of infectious agents have mandated a need for alternative therapeutic strategies. One of these strategies is gene therapy and we embarked on a novel gene therapeutic strategy involving the use of factor IX-transduced bone marrow stroma to treat hemopihlia B, a deficiency of the gamma-carboxylated protein, factor IX. The rate limiting step for the production of factor IX is gamma-carboxylation, a post translational modification carried out only in mammalian cells. In order to test the carboxylation efficiency of our gene therapeutic platform, we first transfected Human Embryonic Kidney (HEK) 293 cells with a retroviral construct containing factor IX. Factor IX was purified by immunoaffinity chromatography and then subject to hydroxyapatite chromatography to isolate biologically active properly carboxylated factor IX from inactive uncarboxylated factor IX. Unexpectedly, during hydroxyapatite chromatography, we discovered that purified factor IX was contaminated by a heretofore unknown protein. Further analysis by mass spectrometry sequencing revealed this protein to be galectin-3 binding protein (G3BP).G3BP contaminates media of HEK293 cells regardless of whether factor IX is transfected into these cells. Furthermore, the contamination of purified media is not specific for factor IX as G3BP is isolated regardless of the antibody used for purification. In summary, the above results provide important information regarding the production of recombinant factor IX and uncover a potentially new contaminant in recombinant products produced in mammalian cells.

Glycopegylated Factor IX Enables Prolonged Efficacy After Subcutaneous Injection But Requires a Higher Than Expected Plasma Activity To Prevent Bleeding In Hemophilia B Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1104-1104
Author(s):  
Derek S Sim ◽  
Alan Brooks ◽  
Cornell Mallari ◽  
Yifan Xu ◽  
Rick I Feldman ◽  
...  
Keyword(s):  
Half Life ◽  
Subcutaneous Injection ◽  
Specific Activity ◽  
Periodate Oxidation ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
B Mice ◽  
Trough Levels ◽  
Plasma Activity

Abstract Background Reduced frequency of administration as well as subcutaneous (s.c.) injection would improve the treatment of Hemophilia B. Conjugation to polyethylene glycol (PEG) has been shown to increase the half-life of i.v. dosed Factor IX (FIX), but s.c. dosing of PEGylated FIX was not previously evaluated. Because s.c. dosing is limited by volume and bioavailability, we evaluated the combination of PEGylation with the increased specific activity variant R338A to reduce the amount of protein needed to provide therapeutic levels of FIX. Methods FIX-R338A was PEGylated on N-linked glycans in the activation peptide by periodate oxidation of sialic acid residues followed by conjugation to amino-oxy functionalized PEG. Pharmacokinetic (PK) profiles were determined in hemophilia B mice and cynomologous monkeys. Allometric scaling was used to predict dose regimens in humans. Prophylactic efficacy was determined in a Hemophilia B mouse tail bleeding model. Results 60kDaPEG-R338A had prolonged terminal half-life in mice (3-fold) and monkeys (5-fold) and the s.c. bioavailability was 44% and 35%, respectively. The volume of distribution was reduced 5-fold. To achieve a trough level of 3% FIX activity, s.c. dosing at weekly, bi-monthly and monthly intervals was predicted to require doses of 7, 25 and 220 IU/kg in patients. However, in a tail vein transection injury model, approximately 10-fold higher plasma FIX activity levels of PEGylated proteins were found to be needed to protect hemophilia B mice against bleeding than was required for i.v. dosed un-PEGylated recombinant FIX. This difference was observed for PEGylated wild-type and R338A proteins, dosed i.v or s.c. We hypothesize that this is related to the reduced distribution of PEGylated FIX to the extravascular compartment. Trough levels of 30% FIX activity were predicted to be achievable in humans after weekly and bi-monthly s.c. dosing at 70 and 260 IU/kg. Conclusions The PEGylation of FIX led to a significant improvement in both i.v. and s.c. PK. Unexpectedly, a 10-fold higher plasma activity was needed for PEGylated FIX to provide protection against bleeding in Hemophilia B mice, suggesting that trough levels of 10 to 30% of PEGylated FIX activity may be needed in patients to provide efficacy equivalent to current therapy of recombinant or plasma derived FIX. Nevertheless, 60kDaPEG-R338A has the potential to treat hemophilia B patients with once weekly or twice monthly subcutaneous injection. Disclosures: Sim: Bayer HealthCare: Employment. Brooks:Bayer HealthCare: Employment. Mallari:Bayer HealthCare: Employment. Xu:Bayer HealthCare: Employment. Feldman:Bayer HealthCare: Employment. Schneider:Bayer HealthCare: Employment. Patel:Bayer HealthCare: Employment. Blasko:Bayer HealthCare: Employment. Ho:Bayer HealthCare: Employment. Su:Bayer HealthCare: Employment. Liu:Bayer HealthCare: Employment. Laux:Bayer HealthCare: Employment. Murphy:Bayer HealthCare: Employment.

scholarly journals Immune responses to human factor IX in haemophilia B mice of different genetic backgrounds are distinct and modified by TLR4

Haemophilia ◽  
2014 ◽  
Vol 21 (1) ◽  
pp. 133-139 ◽  
Author(s):  
B. K. Sack ◽  
X. Wang ◽  
A. Sherman ◽  
G. L. Rogers ◽  
D. M. Markusic
Keyword(s):  
Immune Responses ◽  
Human Factor ◽  
Factor Ix ◽  
Haemophilia B ◽  
Human Factor Ix ◽  
B Mice

High Purity Factor IX and Prothrombin Complex Concentrate (PCC): Pharmacokinetics and Evidence that Factor IXa Is the Thrombogenic Trigger in PCC

1996 ◽  
Vol 76 (01) ◽  
pp. 023-028 ◽  
Author(s):  
H Philippou ◽  
A Adami ◽  
D A Lane ◽  
I R MacGregor ◽  
E G D Tuddenam ◽  
...  
Keyword(s):  
High Purity ◽  
Prothrombin Complex Concentrate ◽  
Factor Ix ◽  
Coagulation System ◽  
Factor X ◽  
Haemophilia B ◽  
System Activation ◽  
Activation Peptide ◽  
Factor X Activation ◽  
Thrombin Antithrombin Iii Complex

SummaryRecent studies using assays for surrogate markers of thrombogenic-ity in man have demonstrated that activation of the coagulation system occurs following infusion of clinical doses of prothrombin complex concentrates (PCC) but not after the same doses of high-purity factor IX concentrates (HP-FIX) in patients with haemophilia B. Here we have investigated the mechanism of such thrombogenesis by applying assays that detect early-through to late-events in coagulation system activation in a pharmacokinetic cross-over study of 50 IU/kg PCC and a new HP-FIX product in haemophilia B patients. Satisfactory recoveries and half-lives were observed for both concentrates.HP-FIX caused no increases in thrombin-antithrombin III complex (TAT), prothrombin activation peptide fragment F1+2 (F1+2), factor X activation peptide (FXAP) or factor Vila (FVIIa). In contrast the same dose of factor IX in the form of PCC was followed by significant increases over pre-infusion levels of TAT, F1+2 and FXAP, but not FVIIa. Elevations of FIXAP occurred after both HP-FIX and PCC but did not reach normal levels and were attributed to normalisation of the FIX concentration in those patients whose levels of FIXAP were initially low. We conclude that the thrombogenic trigger associated with PCC infusion occurs at the level of factor X activation. In the absence of any increase in FVIIa, we would attribute this to the likely presence of FIXa in the PCC.

Performances of an Artificial Reagent for the One-stage Factor IX Assay

1975 ◽  
Vol 33 (03) ◽  
pp. 547-552 ◽  
Author(s):  
L Meunier ◽  
J. P Allain ◽  
D Frommel
Keyword(s):  
Human Plasma ◽  
Factor Ix ◽  
Severe Haemophilia ◽  
Normal Human Plasma ◽  
Haemophilia B ◽  
One Stage ◽  
Normal Human ◽  
The One

SummaryA mixture of adsorbed normal human plasma and chicken plasma was prepared as reagent for factor IX measurement using a one-stage method. The substrate was found to be specific for factor IX. Its performances tested on samples displaying factor IX activity ranging from <l%–2,500% compared favorably with those obtained when using the plasma of severe haemophilia B patients as substrate.

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