Substitution of Valine for Leucine 305 in Factor VIIa Increases the Intrinsic Enzymatic Activity

Persson, Egon; Bak, Helle; Olsen, Ole H.

doi:10.1074/jbc.m102187200

Cited by 46 publications

(61 citation statements)

References 25 publications

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“…Under these conditions, the apparent velocity of FX hydrolysis by FVIIaWT was significantly higher than that of FVIIa303T. This finding suggests that the conformational equilibrium FVIIa « FVIIa*, where FVIIa is the zymogen-like form and FVIIa* is the functionally active form of the FVIIa molecule (Dennis et al, 2000Persson et al, 2001a;Roberge et al, 2001;Sichler et al, 2002), is severely shifted to the right. The perturbation of this conformational equilibrium is even more evident in the presence of TF, whose binding is unable to shift the conformational transition toward a fully active FVIIa form.…”

Section: Discussionmentioning

confidence: 53%

“…Globally, these exosites are referred to as the 'activation domain' and encompass residues 284-294, 295-304, 332-345 and 363-374 (Eigenbrot et al, 2001). Studies of the mutations of FVII showed that the substitutions M298Q (Petrovan & Ruf, 2001), R304Q (O'Brien et al, 1991), R304W (Matsushita et al, 1994), L305V (Persson et al, 2001a), and M306D (Persson et al, 2001b) in the serine protease domain had adverse effects on the enzyme's activity and TF interaction. Indeed, the R304Q and R304W mutations were associated with reduced affinity for human TF and reduced FVIIa activity, while the L305V, M306D and M298Q mutations increase FVIIa activity even in the absence of TF, without altering the TF affinity.…”

Section: Discussionmentioning

confidence: 99%

“…With this background, we report our experiments on the expression, purification and characterization of the mutant FVIIP303T protein (Peyvandi et al, 2000). The P303 residue in the FVIIa-TF complex is located at the interface between the heavy and the light chains of FVIIa and is close to L305 (Persson et al, 2001a) and M306 (Persson et al, 2001b), residues previously demonstrated to be involved in the TF-linked activation of the enzyme. We hypothesized that the Pro303Thr (P303T) substitution may alter the stabilization of the active form of FVIIa by TF binding, causing a severe dysfunction of the enzyme.…”

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confidence: 99%

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The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

et al. 2004

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SummaryWe report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild‐type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (Kd from 4·4 nmol/l for FVIIaWT to 17·3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (kcat/Km from 2·3 × 107/mol/l s for FVIIaWT to 8·7 × 105/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen‐like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF‐catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the Km value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.

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Section: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

et al. 2004

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“…Following substitution of amino acids in vicinity to this region, namely Leu305Val and Phe374Pro, slightly increased amidolytic and proteolytic activity by 4-fold and 1.5-fold, respectively, mediating a local reorganization of the helix [10]. More potent variants were generated by introducing amino acids which can be found in thrombin possessing a considerable constitutive activity.…”

Section: Engineering Fviia Variants With Increased Intrinsic Activitymentioning

confidence: 99%

“…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].…”

Section: Fvii/fviiamentioning

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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Active antithrombin glycoforms are selectively physiosorbed on plasma extracellular vesicles

Radeghieri

Alacqua

Zendrini

et al. 2022

J of Extracellular Bio

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Antithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the "EV-protein corona." The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency-affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms.

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Substitution of Valine for Leucine 305 in Factor VIIa Increases the Intrinsic Enzymatic Activity

Cited by 46 publications

References 25 publications

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

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

Active antithrombin glycoforms are selectively physiosorbed on plasma extracellular vesicles

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