Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

Area of Science:

• Biochemistry
• Pharmacology
• Biotechnology

Background:

• Targeted antithrombotic drug delivery aims to concentrate effects at thrombosis sites, minimizing hemorrhage in healthy vessels.
• Previous research engineered a single Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5) for platelet targeting, reducing bleeding risk compared to non-targeted AcAP5.
• Increasing platelet-binding sites on Factor Xa (FXa) inhibitors could enhance adhesion to activated platelets and further reduce bleeding.

Purpose of the Study:

• To engineer a recombinant protein with multiple platelet-binding sites to improve antithrombotic drug targeting efficiency.
• To evaluate the anti-FXa activity, platelet-targeting capability, and bleeding risk of the engineered variant NR4.

Main Methods:

• Engineered a variant NR4 by introducing three Arg-Gly-Asp (RGD) motifs into AcAP5, retaining anti-FXa activity.
• Utilized protein-protein docking and molecular dynamics simulations to assess binding interactions with platelet receptor αIIbβ3.
• Employed flow cytometry and rat arterial thrombosis models to confirm enhanced platelet targeting and evaluated tail bleeding time in mice.

Main Results:

• NR4 demonstrated retained anti-FXa activity and all three RGD motifs were predicted to bind platelet receptor αIIbβ3.
• Molecular dynamics simulations indicated NR4 has greater interaction potential with αIIbβ3 compared to single-RGD variants.
• Flow cytometry and thrombosis models confirmed NR4's enhanced platelet targeting. NR4-treated mice showed a trend towards reduced tail bleeding time.

Conclusions:

• Engineering multiple binding sites into a single recombinant protein is an effective strategy to enhance platelet-targeting efficiency.
• The NR4 variant exhibits improved platelet targeting and a potential for reduced bleeding complications compared to single-RGD variants.
• This approach offers a promising avenue for developing safer and more effective antithrombotic therapies.