In silico discovery of a compound with nanomolar affinity to antithrombin causing partial activation and increased...

Area of Science:

• Biochemistry
• Pharmacology
• Hematology

Background:

• Thromboembolic disorders represent a significant medical and socioeconomic burden, necessitating novel anticoagulant therapies.
• Heparin is a common anticoagulant but is associated with adverse effects, driving the search for alternatives.
• Antithrombin is a key protein in the coagulation cascade, and its activation is crucial for anticoagulation.

Purpose of the Study:

• To identify novel non-polysaccharide molecules that can activate antithrombin.
• To characterize the interaction of a predicted molecule, d-myo-inositol 3,4,5,6-tetrakisphosphate (TMI), with antithrombin.
• To evaluate the potential of TMI as an anticoagulant therapeutic.

Main Methods:

• In silico screening of a large molecular database to identify potential antithrombin activators.
• Isothermal titration calorimetry to determine binding affinity of TMI to antithrombin.
• Functional assays, fluorescence analysis, and citrullination experiments to assess antithrombin activation.
• Evaluation of TMI efficacy in patient plasma with antithrombin deficiency and in an endothelial cell model.

Main Results:

• D-myo-inositol 3,4,5,6-tetrakisphosphate (TMI) was identified as a potent binder to antithrombin with a high affinity (45 nM), exceeding that of heparin (273 nM).
• TMI induces partial activation of antithrombin, enhancing its interaction with heparin and low-affinity heparins.
• TMI demonstrated improved antithrombin inhibitory function in plasma from patients with antithrombin deficiency and in an endothelial cell model.

Conclusions:

• A novel non-polysaccharide scaffold, TMI, effectively interacts with the heparin-binding domain of antithrombin.
• TMI's ability to partially activate antithrombin suggests its potential as a therapeutic anticoagulant.
• Experimental characterization supports the potential clinical application of TMI in managing thromboembolic disorders.