Potent anticoagulant aptamer directed against factor IXa blocks macromolecular substrate interaction

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Factor IXa is a key enzyme in the coagulation cascade.
• Aptamers are being investigated as novel antithrombotic agents.
• Understanding the mechanism of aptamer action is crucial for therapeutic development.

Purpose of the Study:

• To elucidate the molecular mechanism of an aptamer targeting factor IXa.
• To determine how this aptamer functions as an anticoagulant.

Main Methods:

• In vitro biochemical assays measuring clotting times.
• Assays to assess factor X activation by factor IXa.
• Small synthetic substrate cleavage assays.

Main Results:

• The aptamer binds factor IXa and prolongs plasma clotting time.
• It completely inhibits factor X activation by factor IXa, independent of factor VIIIa.
• The aptamer slows, but does not fully block, small synthetic substrate cleavage.

Conclusions:

• The aptamer likely binds the catalytic domain of factor IXa, sterically hindering an extended substrate-binding site.
• This mechanism differs from small molecule inhibitors, suggesting aptamers can target sites outside the active site.
• Aptamers may serve as valuable tools for probing and inhibiting enzyme activity at non-active sites.