Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro

Bunce, Matthew W.; Toso, Raffaella; Camire, Rodney M.

doi:10.1182/blood-2010-08-300756

Cited by 73 publications

(68 citation statements)

References 49 publications

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“…As shown in Figure 4, the activity in plasma decreased rapidly, with a half-life of approximately 90 sec, and reached a plateau at 20 min for GDXa or FXa, confirming results previously found for FXa. 26 Nevertheless, the effect on thrombin generation was maintained over time, because, at 1 h, when the residual activity of GDXa was roughly 10% of its initial activity ( Figure 4B), the restoration of thrombin generation was maintained. After 1 min of incubation, endogenous thrombin potential increased from 0 to 610 nM and remained at 478 nM after 60 min (Table 4).…”

Section: Half-life Of Gla-domainless Activated Factor X and Normal Acmentioning

confidence: 99%

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Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex

Marlu¹,

Polack²

2012

Haematologica

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Section: Half-life Of Gla-domainless Activated Factor X and Normal Acmentioning

confidence: 99%

“…TFPI is a slow, tight binding inhibitor of FXa 26,27 and, at a low concentration, a weak inhibitor of GDXa. [28][29][30] We, therefore, compared TFPI inhibition of FXa and GDXa (Table 1).…”

Section: © F E R R a T A S T O R T I F O U N D A T I O Nmentioning

confidence: 99%

Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex

Marlu¹,

Polack²

2012

Haematologica

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“…28 Thrombin generation assay, coagulation assays, mFVIIa antigen determination, rotational thromboelastometry, and factor Xa generation assay Thrombin generation was determined as previously described. 29,30 Prothrombin time (PT) assays were determined as previously described 22 using human factor VII-deficient plasma (George King Bio-Medical, Overland Park, KS) and Innovin (Siemens Healthcare, Malvern, PA). When necessary, quantification was performed using standard curves of increasing concentration of each recombinant protein.…”

Section: Binding Assaysmentioning

confidence: 99%

The endothelial protein C receptor enhances hemostasis of FVIIa administration in hemophilic mice in vivo

Pavani

Ivanciu

Faella

et al. 2014

Blood

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“…This critical event is accompanied by various rearrangements of several surface loops at the catalytic domain which contribute to the maturation of the active state of FXa [73]. Bunce et al could show that the amino acid substitutions I195L and V196A are crucial for the zymogen-like properties in FXa [65]. Both FXa variants exhibited only a poor activity in absence of FVa and were not affected by inactivation of antithrombin III and tissue factor pathway inhibitor (TFPI).…”

Section: Engineering Fxa Variants With Zymogen-like Propertiesmentioning

confidence: 99%

“…During the last years, two major strategies based on either FXa or FX variants were envisaged for bypassing of FVIII-and FIX-deficiencies in hemophiliacs. In the first approach FXa variants with zymogen-like properties are generated which display a higher stability and extended half-life in comparison to wild-type FXa [65] whereas the second approach relies on the generation of zymogenic FX chimera proteins which can be activated by thrombin [66].…”

Section: Fx/fxamentioning

confidence: 99%

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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Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro

Cited by 73 publications

References 49 publications

Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex

Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex

The endothelial protein C receptor enhances hemostasis of FVIIa administration in hemophilic mice in vivo

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

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