Generation of Gla‐domainless FVIIa by cathepsin G‐mediated cleavage

Nicolaisen, Else Marie; Petersen, Lars C.; Thim, Lars; Jacobsen, Jes K.; Christensen, Morten H.; Hedner, Ulla

doi:10.1016/0014-5793(92)80989-t

Cited by 28 publications

(23 citation statements)

References 20 publications

(11 reference statements)

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“…Since binding of F6A4 to factor VIIa requires an intact peptide bond between Lys38 and Leu39, which is close to Trp41, our results suggest that Ca2+ binding site(s) with an apparent K<, of 0.6-0.8 mM changes the structure of factor VIla in-a way that affects the hydrophobic stack. It should also be noted that factor VIIa is susceptible to proteolytic cleavage at the Lys38-Leu39 and Tyr44-Ser45 peptide bonds in the absence of Ca" but not in its presence (Sakai et al, 1990;Nicolaisen et al, 1992). The second transition in the Gla domain of factor VIIa occurred with a midpoint around 1.3-1.4 mM Ca2+.…”

Section: Discussionmentioning

confidence: 99%

“…Human recombinant factor VIIa was expressed and purified as previously described (Thim et al, 1988). Des(1-38)-factor VIIa was produced by factor VlIa autoproteolysis (Sakai et al, 1990), whereas des(1-44)-factor VIIa was produced by cathepsin-G-mediated cleavage (Nicolaisen et al, 1992). Cathepsin G was purified from human neutrophiles by the method of Baugh and Travis (1976).…”

Section: Methodsmentioning

confidence: 99%

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Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Persson

Petersen

1995

European Journal of Biochemistry

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The structural and functional effects of Ca'+ binding to vitamin-K-dependent coagulation factor VIIa were investigated. Conformational changes with a midpoint around 0.7 mM Ca2+ quenched the intrinsic protein fluorescence of a fragment of factor VIIa comprising only the light chain and this coincided with an increase in factor VIIa amidolytic activity in the absence of tissue factor. Ca'+ binding to sites in factor VIIa and in the fragment with an apparent dissociation constant of 1.3 -1.4 mM induced binding to phospholipids. A similar Caz+ dependency was not observed with factor VIIa lacking the N-terminal 38 or 44 residues of the light chain and the observed effects could thus be attributed to y-carboxyglutamicacid-dependent Ca2+ binding. Mg" appeared to bind to the site(s) of relatively higher affinity since, although it was less efficient than Ca", it stimulated the amidolytic activity and induced quenching of the intrinsic fluorescence. In contrast, Mg'+ did not induce expression of the phospholipid-interactive structure. The binding properties of two monoclonal antibodies that recognized epitopes in the y-carboxyglutamic-acid-rich domain of factor VIIa corroborated the occurrence of two Ca' ' -induced, sequential structural changes and only one of the antibodies recognized the Mg2' -induced structure. Thus Ca2+ binding to the y-carboxyglutamic-acid-containing domain appeared to result in at least two distinct structural transitions with different functional consequences. The two (sets of) sites responsible for the observed effects could be distinguished based upon differences in Ca'+ affinity and metal ion selectivity. The interaction between factor VIIa and tissue factor was monitored by means of a direct binding assay and an amidolytic assay. In both systems, half-maximal Ca'+ enhancement was observed at 0.25 mM. This coincided with a Ca'+-induced conformational change in factor VIIa associated with fluorescence quenching. The same effect on amidolytic activity was observed with the two N-terminally truncated forms of factor VIIa and it is presumably mediated by Ca" binding to a site located in the serine protease part.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Persson

Petersen

1995

European Journal of Biochemistry

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“…The residual FVIIa activity in FFR-FVIIa was Ͻ0.1% when measured in an FVIIa-specific amidolytic assay. Des-(1-44)-FVIIa and des-(1-44)-FFR-FVIIa (19), factors X (FX) (20) and Xa (FXa) (21), human brain TF apoprotein (22), sTF (23), and polyclonal rabbit anti-human TF IgG (3) were prepared as described previously. TF apoprotein was reconstituted into 60% phosphatidylcholine, 40% phosphatidylserine vesicles as described (25).…”

Section: Methodsmentioning

confidence: 99%

Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor

Sørensen

Persson

Freskgård

et al. 1997

Journal of Biological Chemistry

135

140

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Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active siteinhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.The in vivo initiation of the coagulation cascade is triggered by the binding of plasma factor VIIa (FVIIa) 1 to the cell surface receptor tissue factor (TF) (1). TF is normally expressed in adventitial cells and pericytes surrounding blood vessels but not in cells that come in contact with blood, such as monocytes and endothelial cells (2, 3). Tissue injury disrupting the endothelial cell barrier is normally required for FVIIa to come in contact with TF. However, under pathological conditions monocytes and endothelial cells could be perturbed to induce TF (4 -7).Factor VII circulates as a single chain zymogen, and the binding to TF markedly increases the susceptibility of factor VII to cleavage at Arg 152 resulting in formation of a two-chain serine protease, FVIIa. TF is a glycoprotein consisting of 263 amino acids with a 219-amino acid extracellular part. The extracellular part is structured in two fibronectin type III-like domains (8, 9). The crystal structure of D-Phe-L-Phe-L-Arg (FFR)-FVIIa-soluble TF (sTF) complex has recently been determined (10). In this complex, FVIIa has been shown to adopt an extended confirmation and wrap around TF with the Gla domain near the cell membrane and the catalytic domain distal to it.Administration of inactivated FVIIa was shown to reduce angiographic restenosis and decrease neointimal hyperplasia in a rabbit atherosclerotic injury model (11) and also to reduce thrombus formation at sites of vascular injury in a baboon femoral balloon artery angioplasty model (12). In a preliminary study, it was shown that infusion of a low concentration of inac...

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“…Des(I-38) factor VIIm, kindly provided by Dr. T. Jergensen (Novo Nordisk A/S, Denmark), was prepared as described by Sakai et al [15]. Des(I-44) factor VII,, kindly provided by Dr. E.M. Nicolaisen (Novo Nordisk A/S, Denmark), was prepared as described [21]. Ca 2+ ion depleted factor ViIa preparations were obtained after two desalting steps: (i) gel filtration on a PD-10 column equilibrated with 10 mM glycylglycine, 0.1 M NaCI pH 6.5, essentially as described by the manufacturer (Pharmacia AB, Upsala, Sweden), and (ii) by ion-exchange using Chelex 100 (Bio-Rad, Richmond, CA).…”

Section: Proteinsmentioning

confidence: 99%

“…Some studies showed, however, that truncation of the Gladomain resulted in a complete loss of the TF-dependent stimulation of activity [17], whereas others observed that some augmentation was still preserved in des-Gla factor VII~ [16]. Chymotryptic cleavage [17], and also cleavage by cathepsin G [21], result primarily in generation of the des(I-44) factor VIIa form, whereas auto-cleavage primarily results in the formation of the des(I-38) form [ 16]. Previous studies did not consider the possiblity of distinct functional properties of the two des-Gla forms, which may, according to our present findings, at least partly explain the controversy.…”

Section: The 'Hydrophobic Stack'mentioning

confidence: 99%

Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

1994

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Des(l-38) factor VII, and des(l--44) factor VII, were obtained by limited proteolysis. The binding of tissue factor to these factor VII,-derivatives was assessed from its stimulation of the proteolytic activity on chromogenic oligopeptide substrates. Compared to native factor VII, (Kay = 0.6 + 0.1 riM), Tissue factor binds to des(l-38) factor VII, with a lower, but still signiticant affinity (Kw = 4.8 + 0.3 nM). The activity of des(I-44) factor VII, was only slightly stimulated by TF (KTF --200 nlVO. Binding of TF depends critically on the presence of Ca 2+ ions. Ca 2+ ions stimulated the activity of factor VIIJTF with an apparent K~ = 0.16 + 0.02 raM. Factor VII, in the absence of tissue factor was stimulated by Ca 2+ with an apparent K~ = 0.05 + 0.01 raM, and similar K~ values were obtained for the truncated derivatives of factor VIIa. Measurements of Ca2+-induced changes in intrinsic protein fluorescence suggest a conformational change. The Ca 2+ ion concentration at which this change occurred was higher for des(I-44) factor VII, (apparent Kc~ = 0.14 mM) than for des(I-38)-and native factor VII, (apparent K~ = 0.04 mM). The Tb 3+ ion luminescence technique was used to further investigate the Ca 2+ binding sites. Tb 3+ ions bound with a lower affinity to des(l-44) factor VII, than to des(1-38)-and native factor VIIa. The observed drastic decrease in affinity for tissue factor as a result of truncation of the 'hydrophobic stack' residues 39--44, suggest that this region of factor VII~ provides a structural determinant that together with other regions participates in tissue factor binding.

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Generation of Gla‐domainless FVIIa by cathepsin G‐mediated cleavage

Cited by 28 publications

References 20 publications

Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor

Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions

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Generation of Gla‐domainless FVIIa by cathepsin G‐mediated cleavage

Cited by 28 publications

References 20 publications

Structurally and Functionally Distinct Ca2+ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Structurally and Functionally Distinct Ca2+ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor

Involvement of the hydrophobic stack residues 39–44 of factor VIIa in tissue factor interactions

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Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Structurally and Functionally Distinct Ca²⁺ Binding Sites in the γ‐Carboxyglutamic Acid‐Containing Domain of Factor VIIa

Involvement of the hydrophobic stack residues 39–44 of factor VII_a in tissue factor interactions