Functional characteristics of N8, a new recombinant FVIII

Christiansen, M. L. S.; Balling, Kristoffer W.; Persson, Egon; Hilden, Ida; Bagger-Sørensen, A.; Sørensen, Brit B.; Viuff, Dorthe; Segel, Stine; Klausen, Niels Kristian; Ezban, Mirella; Lethagen, Stefan; Steenstrup, Thomas D.; Kjalke, Marianne

doi:10.1111/j.1365-2516.2010.02333.x

Cited by 42 publications

(58 citation statements)

References 22 publications

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“…A residual binding of both recombinant FVIII variants to the CR doublets non-active for plasma-derived FVIII was attributed to a nonspecific component. Also, our data suggest that the B-domain of FVIII does not contribute to its binding to LDLR, similarly to that indicated for LRP (41). In conclusion, our data demonstrate that the LDLR-binding site is essentially the same for each FVIII variant, which supports the results of the mapping study.…”

Section: Expression Of Ldlr Fragments-supporting

confidence: 80%

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Kurasawa

Shestopal

Karnaukhova

et al. 2013

Journal of Biological Chemistry

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Background: Low density lipoprotein receptor (LDLR) mediates clearance of blood coagulation factor VIII (FVIII). Results:The region of complement-type repeats 2-5 in LDLR was identified as the binding site for FVIII and for ␣-2-macroglobulin receptor-associated protein (RAP). Conclusion: Binding sites of LDLR for FVIII, and also for RAP, were characterized. Significance: This provides new data on LDLR structure and function and on FVIII catabolism.

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Section: Expression Of Ldlr Fragments-supporting

confidence: 80%

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Kurasawa

Shestopal

Karnaukhova

et al. 2013

Journal of Biological Chemistry

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“…44,45 Anti-TFPI antibody 5 and polysaccharide 8,9 TFPI inhibitor studies provide a conceptual foundation for anti-TFPI therapy as a novel hemophilia therapy. These studies suggest that TFPI inhibition has the potential to be useful in improving coagulation in hemophilia patients, 5,6,9 both as a single agent and in combination with current replacement factors or bypassing agents.…”

Section: Discussionmentioning

confidence: 99%

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

Waters

Genga

Schwartz

et al. 2011

Blood

129

109

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Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

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“…FX activation experiments were performed as described by Christiansen et al 18 (see supplemental Methods for a full description).…”

Section: In Vitro Functional Characterizationmentioning

confidence: 99%

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

Østergaard¹,

Bjelke²,

Hansen

et al. 2011

Blood

129

133

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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 K m 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 hemo- IntroductionFactor IX (FIX) is a vitamin K-dependent glycoprotein and an essential protease of the hemostatic system. The domain organization of FIX is shared with factors VII, X, and protein C and comprises an N-terminal domain rich in ␥-carboxyglutamic acid (Gla), 2 epidermal growth factor-like repeats and a C-terminal trypsin-like protease domain. 1 Together they form a 55-kDa single-chain protease precursor circulating in plasma at a concentration of approximately 90nM (5 g/mL), defined as 1 IU/mL. FIX is converted to the 2-chain activated form by the tissue factor (TF)-factor VIIa (FVIIa) complex or factor XIa (FXIa). Activation occurs by limited proteolysis at Arg145 and Arg180 in the protease domain and liberates a 35-amino acid activation peptide that carries the only 2 N-linked glycans in the protein. 2,3 Subsequent assembly of FIXa with the cofactor VIIIa on the activated platelet surface greatly enhances the proteolytic activity of FIXa toward its substrate factor X (FX) and is essential for propagation of the coagulation response. 4 The importance of this activity is reflected by the occurrence of the bleeding disorder hemophilia B (HB) in individuals carrying mutations in the FIX gene. The prevalence of HB is approximately 1 in 25 000 males, and it has been estimated that approximately 84 000 people are affected worldwide. 5 The mainstay in HB treatment is substitution therapy by infusion of plasma-derived or recombinant FIX (rFIX). The therapeutic goal is to prevent bleeding episodes and to provide safe and efficacious treatment of bleedings when they occur. Because of the relatively short half-life of FIX (18-24 hours [6][7][8] ), the recommended prophylaxis regimen consists of 2 to 3 weekly infusions of 40-100 IU/kg 9 FIX to maintain trough levels above 1% and thus shifting patients from a severe to a milder phenotype. When adhered to, prophylaxis in patients without severe joint disorder is efficacious with a frequency of only 0-2 breakthrough bleeds per year in the majority of patients. 8,10 However, the need for multiple weekly infusions present challen...

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Functional characteristics of N8, a new recombinant FVIII

Cited by 42 publications

References 22 publications

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

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

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