Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Sciences, Beijing, China.
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.
Sci Rep. 2016 Jul 19;6:29895. doi: 10.1038/srep29895.
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.
靶向递送抗血栓药物可将作用集中在血栓部位，并减少未受伤血管的出血副作用。我们最近报道称，血小板靶向因子 Xa（FXa）抑制剂，通过将一个 Arg-Gly-Asp（RGD）基序工程改造到Ancylostoma caninum抗凝肽 5（AcAP5）中构建，可以降低系统性出血风险，而非靶向 AcAP5 在小鼠动脉损伤模型中。增加 FXa 抑制剂与血小板结合的位点数量可能有助于它们与活化的血小板结合，从而进一步降低出血风险。为此，我们在 AcAP5 中引入了三个 RGD 基序，生成了一个含有三个血小板结合位点的变体 NR4。NR4 保留了其固有的抗 FXa 活性。蛋白-蛋白对接显示，所有三个 RGD 基序都能够与血小板受体 αIIbβ3 结合。分子动力学模拟表明，NR4 与 αIIbβ3 相互作用的机会多于含有单个 RGD 的 NR3。流式细胞术分析和大鼠动脉血栓形成模型进一步证实，NR4 具有增强的血小板靶向活性。此外，在颈动脉内皮损伤模型中，NR4 处理的小鼠的尾巴出血时间比 NR3 处理的小鼠有减少的趋势。因此，我们的数据表明，在一个重组蛋白中设计多个结合位点是提高其血小板靶向效率的有用工具。
