Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A

Tranholm, Mikael; Kristensen, Kim; Kristensen, Annemarie T.; Pyke, Charles; Røjkjær, Rasmus; Persson, Egon

doi:10.1182/blood-2003-05-1369

Cited by 101 publications

(120 citation statements)

References 22 publications

(24 reference statements)

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“…11,12 Several of these analogs also possess increased thrombin-generating activity on activated platelets 11 and have been shown to reduce the tail bleeding time and total blood loss in a mouse model of hemophilia A. 13 In the current study we have used a cell-based reconstituted model system of coagulation to fully characterize the effect of one of these rFVIIa analogs, rFVIIa with mutations V158D/ E296V/M298Q (FVIIa IIa , NN1731), on factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability. This model system has previously been used to examine the effect of elevated prothrombin on fibrin clot formation and structure, as well as the effects of hemophilia and rFVIIa on fibrin clot formation, structure, and stability.…”

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confidence: 99%

A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia

Allen

Persson²,

Campbell³

et al. 2007

ATVB

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Objective-Recombinant factor VIIa (rFVIIa, NovoSeven) has proven efficacy in treating bleeding in hemophilia patients with inhibitors. A rFVIIa analog with mutations V158D/E296V/M298Q (NN1731) exhibits increased procoagulant activity in in vitro and in vivo models. The aim of this work was to define the effects of NN1731 toward factor X activation, platelet activation, thrombin generation, and fibrin clot formation and stability. Methods and Results-In a cell-based in vitro model of hemophilia, rFVIIa and NN1731 similarly increased factor X activation on tissue factor-bearing cells; however, NN1731 exhibited 30-fold higher factor Xa generation on platelets than similar rFVIIa concentrations. NN1731-mediated thrombin generation depended on platelet activation, but NN1731 did not directly activate platelets. NN1731 produced 4-to 10-fold higher maximal thrombin generation rates than equal rFVIIa concentrations. Both rFVIIa and NN1731 shortened clotting times in the absence of factors IX and VIII; however, NN1731 did so at 50-fold lower concentrations than were required of rFVIIa. In fibrinolytic conditions, both rFVIIa and NN1731 increased fibrin formation and stability; however, NN1731 was effective at 50-fold lower concentrations than were required of rFVIIa. Conclusions-By increasing factor Xa generation, NN1731 promotes the formation of thrombin and a stable clot to a greater degree than rFVIIa.

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confidence: 99%

A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia

Allen

Persson²,

Campbell³

et al. 2007

ATVB

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“…Histopathological analysis of kidneys didn't reveal any changes like fibrin deposition. The variants showed a 3-4 times higher potency above rFVIIa with reduced doses (3 mg/ kg vs. 10 mg/kg rFVIIa) at the same time [19]. However this was much less than observed in vitro [16].…”

Section: Engineering Fviia Variants With Increased Intrinsic Activitymentioning

confidence: 79%

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

2012

J Genet Syndr Gene Ther

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FVII/FVIIaPatients with hemophilia A and B have deficient protein concentrations of FVIII and FIX, respectively and therefore are treated by protein substitution with plasma-derived or recombinant factor FVIII or FIX concentrates. In about 20-30% of the patients with hemophilia A and in 3-5% of those with hemophilia B this can lead to the development of neutralizing antibodies against the infused proteins which makes therapy inefficient [1,2]. Recombinant activated factor VII (rFVIIa) protein is currently an alternative therapy available for inhibitor patients to treat excessive bleeding. However, supraphysiological concentrations and repeated administration are required to induce hemostasis [3].Consequently, FVIIa analogs were created for future protein replacement therapy or gene delivery approaches based on crystalline structure analysis of free and TF-bound FVIIa or on sequence homology of other more potent proteases [4,5].Like the other vitamin K dependent coagulation factors, FVII is physiologically synthesized in the liver. FVII is secreted into the circulation as a 406-residue single-chain polypeptide which contains an N-terminal γ-carboxyglutamic acid (Gla) domain in which all ten Glu residues are post-translationally carboxylated followed by two regions homologous to epidermal growth factor domains and a serine protease domain [3,6,7]. Upon vascular injury, the cofactor tissue factor (TF) is exposed to FVII and triggers the extrinsic pathway of the coagulation cascade by activation of FIX, factor X and FVII auto-activation on the TF-bearing cells resulting in large-scale thrombin generation and a fibrin clot. FVII is activated to FVIIa by internal proteolysis due to a cleavage of a single Arg152-Ile153 peptide bond. The physiological plasma concentration of FVII is around 10 nM, however, only about 1% of FVII is circulating in its free and active form. FVIIa has a plasma halflife of approximately 2.5 hours [8]. FVII activation results in connected FVII light and heavy chains which form a one-to-one complex with TF in the presence of Ca 2+ ions. The complex formation is an essential process in which TF allosterically leads to a fully active FVIIa [9]. The cleavage alone leaves FVIIa in a zymogen-like conformation of quite low specific activity [3,10]. Several residues in the first EGF-like domain of FVII and in the protease domain, especially methionine at position 306, seem to be pivotal for the cofactor-mediated allosteric stimulation [11]. TF stabilizes the active conformation of FVIIa for accelerated activation of its substrates FIX and FX [12,13] by inducing a conformational change and establishing a stable salt bridge between the residues Ile153 and Asp343 [14] and stabilization of the interaction between the residues Leu305 and Phe374 which in turn stabilizes S 1 and S 3 substrate pocket and the activation pocket [15]. These findings contributed to the development of FVIIa variants with enhanced TFindependent (intrinsic) specific activity by mimicking the effect of TF binding [16]. Additional fin...

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“…The second definition led to the development of rFVIIa [6], which stimulates thrombin generation at the site of bleeding in hemophiliac patients with FVIII inhibitors and does not itself elicit FVIII inhibitors. However, the efficacy and halflife of rFVIIa will have to be improved [7] if it is to be used by inhibitor-free hemophiliacs. The third definition would provide the key to most current problems, if we could understand how FIXa activates FX on activated platelets, and if we could find a way to replace FVIII/VIIIa in this enzymatic reaction.…”

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confidence: 99%

“…) [7]. The VWF: Ag was performed by ELISA (Diagnostica Stago; 50%-150%); VWF: RCo was performed by aggregometry using Ristocetin (1.5 mg mL ) (normal; 60%-150%) [8].…”

mentioning

confidence: 99%

“…In all patients, screening tests for coagulation were performed by using standard method [6,7], which included platelet count (PC), bleeding time (BT), prothrombin time (PT), activated partial thromboplastin time (APTT), clot solubility (CS) and factor VIII assay if required. The diagnosis of VWD was confirmed by specific tests such as Ristocetin-induced platelet aggregation (RIPA), von Willebrand factor antigen (VWF : Ag), Ristocetin co-factor activity (VWF : RCo).…”

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Future perspective for the treatment of hemophilia A

Laurian

2005

Journal of Thrombosis and Haemostasis

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Improved hemostasis with superactive analogs of factor VIIa in a mouse model of hemophilia A

Cited by 101 publications

References 22 publications

A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia

A Variant of Recombinant Factor VIIa With Enhanced Procoagulant and Antifibrinolytic Activities in an In Vitro Model of Hemophilia

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

Future perspective for the treatment of hemophilia A

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