title: Engineering novel anticoagulant proteins by motif grafting.
abstract: 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.
authors.forename: Yuanjun
authors.initials: Y
authors.lastname: Zhu
authors.forename: Aihua
authors.initials: A
authors.lastname: Liu
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing, 100191, China. wangyinye@bjmu.edu.cn.
authors.forename: Yinye
authors.initials: Y
authors.lastname: Wang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D002339:Carotid Arteries / Q000187:drug effects; D030562:Databases, Protein; D065427:Factor Xa Inhibitors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology / Q000627:therapeutic use; D013927:Thrombosis / Q000188:drug therapy
chemical_list: D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D010455:Peptides; D011993:Recombinant Fusion Proteins