Conversion of thrombin into an anticoagulant by protein engineering

Area of Science:

• Biochemistry
• Vascular Biology
• Hematology

Background:

• Thrombin plays a dual role in hemostasis, mediating both procoagulant and anticoagulant functions.
• Endothelial thrombomodulin binding shifts thrombin's activity towards anticoagulation via protein C activation.
• Previous studies suggested functional dissociation of thrombin's procoagulant and anticoagulant activities.

Purpose of the Study:

• To engineer a thrombin variant with selective anticoagulant activity.
• To develop a novel antithrombotic agent with a potentially improved safety profile.

Main Methods:

• Protein engineering using site-directed mutagenesis to create a thrombin variant (E229A).
• In vitro and in vivo studies in non-human primates to assess anticoagulant and procoagulant activities.
• Evaluation of bleeding risk using template bleeding time.

Main Results:

• The E229A mutation significantly enhanced thrombin's specificity for protein C activation.
• In vivo, the modified thrombin demonstrated dose-dependent, reversible anticoagulation.
• No procoagulant activity was observed, and template bleeding times were not prolonged.

Conclusions:

• The E229A thrombin variant effectively functions as an endogenous protein C activator.
• This engineered thrombin represents a promising antithrombotic strategy with a potentially reduced risk of bleeding complications.