Structure-based rational design has been considered as a promising approach to design novel proteins. For this
purpose, we designed artificial anticoagulant proteins that are able to target Factor Xa (FXa) using a functional motifgrafting
approach. The motif corresponded to the residues Cys15 to Cys42 of Ancylostoma caninum anticoagulant peptide
5 (AcAP5), a potent FXa inhibitor. By screening of the Protein Data Bank (PDB) using Vector Alignment Search Tool
(VAST, search for three-dimensional scaffolds in protein structures), we screened scaffolds as hosts to reproduce the functional
topology of this motif. Three designed artificial chimeric proteins were expressed and purified to test their FXainhibiting
ability. One of the recombinant proteins, pep3, was found to inhibit FXa with strong activity (IC50 of 152 nM)
in vitro. Moreover, pep3 inhibited arterial thrombosis formation in rats with uniform potency compared with natural
AcAP5. Therefore, our data demonstrate that motif-grafting is a useful tool to engineer novel artificial anticoagulant proteins.
Keywords: AcAP5, anticoagulant, Factor Xa, inhibitor, motif-grafting, VAST.
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