Antibody-induced Enhancement of Factor VIIa Activity through Distinct Allosteric Pathways

Andersen, Lone B.; Andreasen, Peter A.; Svendsen, I.; Keemink, Janneke; Østergaard, Henrik; Persson, Egon

doi:10.1074/jbc.m111.312330

Cited by 11 publications

(14 citation statements)

References 35 publications

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“…[11][12][13] Soon after the development of monoclonal antibody technology, antibodies against FVII and FVIIa were generated. [14][15][16][17][18] Of note, many of them recognize both the active and zymogen forms of FVII.…”

Section: Introductionmentioning

confidence: 99%

“…Philippou et al generated a polyclonal antibody directed against the C-terminal part of the FVIIa light chain, which displayed >3000-fold greater reactivity to FVIIa than FVII. 19 Andersen et al 18 reported on antibodies that show preferential binding to FVIIa and stimulated FVIIa activity. Most recently, a llama-derived single-domain antibody (sdAb) that binds to FVIIa but not to FVII has been described.…”

Section: Introductionmentioning

confidence: 99%

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A single‐domain antibody that blocks factor VIIa activity in the absence but not presence of tissue factor

Ferrière

Kawecki

Ottavi³

et al. 2019

Journal of Thrombosis and Haemostasis

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Background Activated factor VII (FVIIa) is pertinent to the initiation of blood coagulation. Proteolytic and amidolytic activity of FVIIa are greatly enhanced by its cofactor, tissue factor (TF). Objective We aimed to generate a single‐domain antibody (sdAb) that recognizes free FVIIa rather than TF‐bound FVIIa. Methods A llama‐derived phage library was used to screen for anti‐FVIIa sdAbs. Results One sdAb, KB‐FVIIa‐004, bound to FVIIa, but not to its precursor FVII or to homologous proteins (prothrombin, factor X, or their activated derivatives). FVIIa amidolytic activity was inhibited by KB‐FVIIa‐004 (Ki = 28‐45 nM) in a competitive manner. KB‐FVIIa‐004 also inhibited FVIIa‐mediated FX activation (Ki = 26 nM). In contrast, KB‐FVIIa‐004 was inefficient in prolonging the clotting time of the prothrombin time‐test, which was prolonged by a maximum of 10 s at high sdAb concentrations (10 μM). Furthermore, FVIIa/TF amidolytic activity or FVIIa/TF‐mediated FX activation remained unaffected up to a 50‐fold to 1000‐fold molar excess of KB‐FVIIa‐004. These data suggest that KB‐FVIIa‐004 loses its inhibitory activity in the presence of TF. A KB‐FVIIa‐004/albumin fusion‐protein (004‐HSA) was generated for in vivo testing. By using 004‐HSA, we observed that this sdAb blocked the therapeutic capacity of FVIIa to correct bleeding in FVIII‐deficient mice. Discussion This observation is compatible with the view that FVIIa functions independently of TF under these conditions. In conclusion, we have generated a sdAb that specifically blocks TF‐independent activity of FVIIa. This antibody can be used to gain insight into the roles of TF‐bound and TF‐free FVIIa.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A single‐domain antibody that blocks factor VIIa activity in the absence but not presence of tissue factor

Ferrière

Kawecki

Ottavi³

et al. 2019

Journal of Thrombosis and Haemostasis

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“…On TF, a membrane proximal patch has been defined and distinct recognition sites have been localized on FVIIa ( 17 – 19 ). Since the enhancement of proteolytic activity by sTF in solution is only a few hundred-fold, of which allosteric activation accounts for the major part ( 15 , 20 ), exosite-mediated FX recognition appears to be realized primarily on the membrane surface.…”

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confidence: 99%

Engineering of a membrane-triggered activity switch in coagulation factor VIIa

Nielsen

Sørensen

Holmberg

et al. 2017

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

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SignificanceCoagulation factor VIIa (FVIIa) is an intrinsically poor serine protease that requires assistance from its cofactor tissue factor (TF) to trigger the extrinsic pathway of blood coagulation. TF stimulates FVIIa through allosteric maturation of its active site and by facilitating substrate recognition. The surface dependence of the latter property allowed us to design a potent membrane-triggered activity switch in FVIIa by engineering a disulfide cross-link between an allosterically silent FVIIa variant and soluble TF. These results show that optimization of substrate recognition remote from the active site represents a promising new route to simultaneously enhance and localize the procoagulant activity of FVIIa for therapeutic purposes.

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“…The amidolytic activity of FVIIa was measured using the chromogenic substrates Chromozym t-PA or Spectrozyme FXa, as previously described (Andersen et al, 2012). In brief, a 96-well plate was blocked overnight in 200 µL Assay Buffer (AB) containing; 20 mM HEPES (pH 7.8), 150 mM NaCl, 5 mM CaCl 2 , 0.1 % PEG 8000, 0.5 % BSA, at 4 °C.…”

Section: Fvii and Fx Enzyme Activity Assaysmentioning

confidence: 99%

“…FX activation by TF-FVIIa complex was also measured using chromogenic assay as described (Andersen et al, 2012). The activity of FX was determined by incubating 100 µL AB containing 200 nM FX with 100 nM rFVIIa, or with 10 nM rFVIIa and 200 nM TF (Thromborel ® S), incorporated into phospholipid as recommended (Kelley et al, 1997) of 6 µL TriniCLOT ™ aPTT HS, for 1 hr at 37 °C.…”

Section: Fvii and Fx Enzyme Activity Assaysmentioning

confidence: 99%

Beyond antibodies: development of a novel molecular scaffold based on human chaperonin 10

Alsultan¹

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This study reports the development of a new molecular scaffold based on human Chaperonin 10 (hCpn10), for the development of protein-based new molecular entities (NMEs) of diagnostic and therapeutic potential. The aims were to establish the fundamental basis of a molecular design for the scaffold, to enable the display of non-native peptides with binding activity to selected targets, while maintaining structural integrity and native heptameric conformation. Recently there has been much global interest in developing NMEs of protein and peptide origin, as therapeutic agents and diagnostic reagents. A class of NMEs are based on molecular scaffolds, whereby peptides with binding activity to a given target are displayed on

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Antibody-induced Enhancement of Factor VIIa Activity through Distinct Allosteric Pathways

Cited by 11 publications

References 35 publications

A single‐domain antibody that blocks factor VIIa activity in the absence but not presence of tissue factor

A single‐domain antibody that blocks factor VIIa activity in the absence but not presence of tissue factor

Engineering of a membrane-triggered activity switch in coagulation factor VIIa

Beyond antibodies: development of a novel molecular scaffold based on human chaperonin 10

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