WEDGE: an anticoagulant thrombin mutant produced by autoactivation

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Therapeutic protease production often relies on external enzymes, posing regulatory challenges for availability and purity.
• Recent research explored engineering protease precursors for spontaneous autoactivation, bypassing external enzyme requirements.
• Autoactivation offers a novel approach to simplify protease production, but requires practical validation.

Purpose of the Study:

• To directly test the feasibility and efficacy of the autoactivation strategy in a practical application.
• To engineer an autoactivating version of a therapeutically relevant protease for testing.

Main Methods:

• Engineered an autoactivating variant of the W215A/E217A (WE) thrombin mutant.
• Assessed large-scale production of the autoactivating WE variant.
• Evaluated the functional properties of the autoactivating WE variant in vitro and in vivo.

Main Results:

• The autoactivating WE variant was produced in large quantities, comparable to conventional methods.
• The engineered autoactivating protease maintained all critical functional properties.
• Both in vitro and in vivo assessments confirmed the retained functionality.

Conclusions:

• Autoactivation is a viable and effective strategy for producing therapeutically relevant trypsin-like proteases.
• This proof-of-principle study validates autoactivation as an innovative production method.
• The engineered autoactivating WE variant demonstrates the potential for simplified manufacturing of anticoagulants.