治疗性蛇毒抗血小板溶栓素抗栓与溶栓分子机制研究

Snake venom Agkisacucetin (AGK) is a novel and promising antiplatelet drug candidate targeted GPIb. AGK, which is a C-type like lectins protein (CLP), binds to GPIb to prevent its binding to von Willebrand factor (vWF) and thus inhibits downstream platelet adhension and aggregation. Although the phase II clinical studies have demonstrated encouraging results of AGK on treatment of thrombosis with little bleeding risk recently, the molecular mechanisms of AGK are still unclear. Recent studies show that antithrombotic effect of AGK might be through multiple mechanisms beyond GPIb-vWF pathway, which could be significantly different from other antiplatelet drugs with bleeding risk. Further understanding of the antithrombotic and thrombolytic mechanisms of AGK could greatly promote the research and development of novel antithrombotic agents. So the study on the molecular mechanisms of AGK by parsing the key binding sites between AGK and GPIb is proposed here base on the existing clues and results from preliminary research.

蛇毒抗血小板溶栓素Agkisacutacin (AGK)是由中国科学技术大学于20世纪90年代初从皖南尖吻蝮蛇毒液中发现的C型凝集素类似蛋白，是目前全球唯一进入IIb期临床研究阶段的GPIb靶向蛇毒抗血小板药物，药物疗效令人非常鼓舞，且无显著出血副作用。然而AGK的抗栓与溶栓机制却并不十分清楚。虽然以往研究显示AGK通过与GPIb结合，干扰GPIb与vWF的结合而阻断血小板粘附和聚集，但近期研究表明，在vWF-/-基因敲除小鼠上，AGK仍然能够发挥抗血栓的效果。提示，AGK还可能通过其它相关机制发挥抗栓与溶栓作用。为此，本研究拟利用丙氨酸扫描突变法，解析AGK与GPIb复合物的关键结合位点及结合界面，揭示AGK抑制GPIb相关的凝血通路的分子机制，阐明AGK抗栓与溶栓作用机制，为开发新型抗血栓药物奠定理论基础。

蛇毒抗血小板溶栓素Agkisacutacin (AGK)是蛇毒C型凝集素类似蛋白。作为 GPIb靶向的抗血小板血栓药物，AGK药物疗效令人非常鼓舞，且无显著出血副作用。然而AGK的抗栓与溶栓机制却并不十分清楚。虽然以往研究显示AGK通过与GPIb结合，干扰GPIb与vWF的结合而阻断血小板粘附和聚集，然而两者的结合界面上的关键氨基酸并不清楚。此外，AGK还可能通过vWF以外的相关机制发挥抗栓与溶栓作用。为此，本研究利用丙氨酸扫描突变法，初步解析AGK与GPIb的部分关键结合位点，从而进一步探讨AGK有可能会抑制GPIb与α-thrombin或Thrombospondin-1 (TSP-1) 的结合，进而阻断其介导的血小板活化和聚集过程。本研究初步揭示AGK抑制GPIb相关的凝血通路的分子机制，为开发新型抗血栓药物奠定理论基础。同时，本研究还开展了重组蛇毒AGK异源二聚体蛋白表达平台应用扩展研究，以免疫激动剂超级IL-15/IL-15R复合物为研究模型，成功制备具有生物活性的超级IL-15/IL-15R复合物。这些结果提示本项目开发的蛇毒蛋白AGK异源二聚体蛋白表达系统可能同样适合于表达其他异源二聚体蛋白，具有一定应用推广前景。