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-Giy-Asp(RGD)motif into Ancylostoma caninum anticoagulant peptide 5(AcAPS),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 αⅡbβ3than 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 Iess 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.