Ca2+ Binding to the First Epidermal Growth Factor-like Domain of Factor VIIa Increases Amidolytic Activity and Tissue Factor Affinity

Persson, Egon; Olsen, Ole H.; Østergaard, Anette; Nielsen, Lene

doi:10.1074/jbc.272.32.19919

Cited by 33 publications

(35 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, the decreased HX rates in the EGF1 domain correlate with studies of an antibody specific for the EGF1 domain, which binds to FVIIa with increased affinity in the presence of a covalent active site inhibitor known to induce the active conformation (26). Furthermore, Ca 2ϩ -binding to the EGF1 domain has been shown to increase the amidolytic activity (27). The results presented in this study provide a structural rationale for these previously inexplicable observations.…”

Section: Discussionsupporting

confidence: 48%

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

Rand

Jørgensen

Olsen

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TFbound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50 Å and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases. Coagulation factor VII (FVII)2 circulates in the blood with ϳ1% in a two-chain form (FVIIa) and the remainder as singlechain zymogen FVII. FVIIa consists of a trypsin-like protease domain and an N-terminal light chain composed of a membrane-binding ␥-carboxyglutamate-rich domain (Gla domain) and two epidermal growth factor-like domains (EGF1 and EGF2) (1). Upon tissue injury, tissue factor (TF) becomes exposed, and the TF⅐FVIIa complex forms and serves as the initiator of the blood coagulation cascade (2). Numerous coagulation proteases function optimally only when complexed to cofactors, and substantial biochemical evidence supports the concept that several of these cofactors induce allosteric changes in the conformation of their cognate enzymes (1, 2). TF functions to localize FVIIa and as an allosteric regulator, which dramatically enhances the activity of FVIIa. Similar to the trypsinogen-trypsin pair, zymogen FVII is converted to FVIIa by proteolysis of an internal peptide bond. A canonical "activation domain" was defined in trypsin, which includes the N terminus created upon endoproteolytic activation and three loops referred to as the activation loops (3). In trypsin, the newly generated N-terminal tail spontaneously inserts itself into a cavity close to the three activation loops, termed the activation pocket, resulting in the formation of a critical salt bridge between the N-terminal Ile-16 and Asp-194 of the active site or and in FVIIa (the chymotrypsin numbering is denoted in superscript with parentheses). This salt bridge leads to the formation of a correctly assembled S1 pocket of the active site and full activity. FVIIa, however, has very low activity following endoproteolytic activation and does not spontaneously rearrange into the active form. The three-dimensional structure of FVIIa bound to TF has been solved providing important structural details of the complex and the active form of FVIIa (4). In the complex, parts of the light chain a...

show abstract

Section: Discussionsupporting

confidence: 48%

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

Rand

Jørgensen

Olsen

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Surface Plasmon Resonance Measurements-The conditions for sTF immobilization (ϳ1000 resonance units) in the Biacore 1000 instrument (Biacore AB, Uppsala, Sweden), regeneration of the sTF-coated surface, and evaluation of binding data were as described previously (18,19). Wild-type, L305V-, and L305V/M306D/D309S-FVIIa were injected for 7 min at a concentration of 30 -150 nM in 50 mM Hepes, pH 7.5, containing 0.15 M NaCl, 5 mM CaCl 2 , and 0.02% Tween 80, followed by a 10-min dissociation phase.…”

Section: Methodsmentioning

confidence: 99%

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

Persson¹,

Bak²,

Olsen³

2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Factor VII requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active factor VIIa (FVIIa) conformation. The former event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. We have designed a number of FVIIa mutants with the aim of mimicking the effect of TF, that is, creating molecules with increased intrinsic (TF-independent) enzymatic activity.

show abstract

“…The conditions for sTF immobilization (Ϸ500 resonance units) in the Biacore 1000 instrument (Biacore, Uppsala, Sweden), regeneration of the sTF-coated surface, and global evaluation of binding data were as described (21,22). Wild-type or mutant FVIIa, in 50 mM Hepes, pH 7.5, containing 0.15 M NaCl, 5 mM CaCl 2 , and 0.02% Tween 80, was injected for 7 min at concentrations of 30 and 50 nM followed by a 10-min dissociation phase.…”

Section: Mutagenesis and Preparation Of Fviia Mutantsmentioning

confidence: 99%

Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity

Persson

Kjalke

Olsen

2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

119

218

View full text Add to dashboard Cite

A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa (EC 3.4.21.21), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation with zymogen-like properties. Association with tissue factor (TF), locally exposed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by replacing residues believed to function as determinants for the inherent zymogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appear to retain the substrate specificity of the parent enzyme and can be further stimulated by TF. Insights into the mechanism behind the increased activity of the mutants, presumably also pertinent to the TFinduced, allosteric stimulation of FVIIa activity, were obtained by studying their calcium dependence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficacious treatment of bleeding episodes especially in hemophiliacs with inhibitory antibodies precluding conventional replacement therapy.

show abstract

Ca2+ Binding to the First Epidermal Growth Factor-like Domain of Factor VIIa Increases Amidolytic Activity and Tissue Factor Affinity

Cited by 33 publications

References 56 publications

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

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

Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity

Contact Info

Product

Resources

About