title: Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting.
abstract: Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Yuanjun
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Ruyi
authors.initials: R
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Mengyang
authors.initials: M
authors.lastname: Shui
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
authors.forename: Yinye
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000722:Ancylostoma / Q000737:chemistry / Q000235:genetics; D000818:Animals; D000925:Anticoagulants / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001777:Blood Coagulation / Q000187:drug effects* / Q000235:genetics; D001792:Blood Platelets / Q000187:drug effects / Q000473:pathology; D065427:Factor Xa Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D005818:Genetic Engineering; D006801:Humans; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D013927:Thrombosis / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology
chemical_list: D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; C000627691:anticoagulant peptide 5, Ancylostoma caninum; C047981:arginyl-glycyl-aspartic acid