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

Nielsen, Anders L.; Sørensen, Annette; Holmberg, Heidi L.; Gandhi, Prafull S.; Karlsson, Johan; Buchardt, Jens; Lamberth, Kasper; Kjelgaard‐Hansen, Mads; Ley, Carsten Dan; Sørensen, Brit B.; Ruf, Wolfram; Olsen, Ole H.; Østergaard, Henrik

doi:10.1073/pnas.1618713114

Cited by 6 publications

(9 citation statements)

References 54 publications

(74 reference statements)

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“…Half‐maximal inhibition was obtained at a sdAb/FVIIa molar ratio of 1.9 ± 0.7 (Figure C). Also for this inhibition, a K i could be calculated from the Cheng‐Prusoff equation, by using a K M of 0.6 μM for FVIIa‐mediated FX activation . This approach generated a K i value of 26 ± 10 nM.…”

Section: Resultsmentioning

confidence: 99%

“…Also for this inhibition, a K i could be calculated from the Cheng-Prusoff equation, by using a K M of 0.6 μM for FVIIa-mediated FX activation. 26 This approach generated a K i value of 26 ± 10 nM. Apparently, KB-FVIIa-004 displays a similar efficacy in blocking FVIIa activity toward small and macromolecular substrates.…”

Section: Effect Of Kb-fviia-004 On Fx Activationmentioning

confidence: 92%

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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: Resultsmentioning

confidence: 99%

Section: Effect Of Kb-fviia-004 On Fx Activationmentioning

confidence: 92%

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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“…This facilitates the insertion of the N-terminus and the proper salt bridge formation between I16 and D194. Knowledge about the activation mechanism and allostery has already yielded successful engineering of FVIIa variants with enhanced activity independent of stimulation by endogenous TF [ 37 , 38 , 39 ]. Of particular interest is a recently engineered FVIIa-trypsin variant, FVIIa VYT , which is completely TF-independent due to its grafted trypsin 170-loop (as previously described [ 40 , 41 , 42 ]) in addition to another mutation, L163V [ 39 ].…”

Section: Introductionmentioning

confidence: 99%

Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain

Madsen

Olsen

2021

Biomolecules

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The vast majority of coagulation factor VII (FVII), a trypsin-like protease, circulates as the inactive zymogen. Activated FVII (FVIIa) is formed upon proteolytic activation of FVII, where it remains in a zymogen-like state and it is fully activated only when bound to tissue factor (TF). The catalytic domains of trypsin-like proteases adopt strikingly similar structures in their fully active forms. However, the dynamics and structures of the available corresponding zymogens reveal remarkable conformational plasticity of the protease domain prior to activation in many cases. Exactly how ligands and cofactors modulate the conformational dynamics and function of these proteases is not entirely understood. Here, we employ atomistic simulations of FVIIa (and variants hereof, including a TF-independent variant and N-terminally truncated variants) to provide fundamental insights with atomistic resolution into the plasticity-rigidity interplay of the protease domain conformations that appears to govern the functional response to proteolytic and allosteric activation. We argue that these findings are relevant to the FVII zymogen, whose structure has remained elusive despite substantial efforts. Our results shed light on the nature of FVII and demonstrate how conformational dynamics has played a crucial role in the evolutionary adaptation of regulatory mechanisms that were not present in the ancestral trypsin. Exploiting this knowledge could lead to engineering of protease variants for use as next-generation hemostatic therapeutics.

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“…The goal of this Neighborhood Watch article is to provide some context on the use of factor VIIa (FVIIa) in management of hemophilic bleeding and discuss a recent article on engineering a novel FVIIa variant .…”

Section: Introductionmentioning

confidence: 99%

The next best thing in factor VIIa

Hoffman

2018

Journal of Thrombosis and Haemostasis

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

Cited by 6 publications

References 54 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

Conformational Plasticity-Rigidity Axis of the Coagulation Factor VII Zymogen Elucidated by Atomistic Simulations of the N-Terminally Truncated Factor VIIa Protease Domain

The next best thing in factor VIIa

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