Chang Chien-Hsin, Chung Ching-Hu, Tu Yi-Shu, Tsai Cheng-Chieh, Hsu Chun-Chieh, Peng Hui-Chin, Tseng Yufeng J, Huang Tur-Fu
From the Graduate Institute of Pharmacology, College of Medicine (C.-H.C., C.-C.H., H.-C.P., T.-F.H.), Graduate Institute of Biomedical Electronics and Bioinformatics (Y.-S.T., C.-C.T., Y.J.T.), and Department of Computational Science and Information Engineering (Y.J.T.), National Taiwan University, Taipei; and Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (C.-H.C.).
Arterioscler Thromb Vasc Biol. 2017 Jul;37(7):1307-1314. doi: 10.1161/ATVBAHA.116.308604. Epub 2017 Jun 8.
Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic.
Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time.
We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.
目前常用的抗血小板药物有一个共同的副作用——出血风险增加和血小板减少。相反，与糖蛋白VI（GPVI）表达缺陷相关的出血缺陷通常表现为出血时间略有延长。然而，临床上缺乏GPVI拮抗剂。
利用反相高效液相色谱和测序技术，我们揭示了强效特异性靶向GPVI的蛇毒C型凝集素蛋白（snaclec）——trowaglerix α亚基的部分序列。从trowaglerix衍生的六肽（Troα6 [trowaglerix a链六肽，CKWMNV]）和十肽（Troα10）通过阻断血小板GPVI受体特异性抑制胶原诱导的血小板聚集。计算肽设计有助于设计一系列Troα6/Troα10肽。对这些十肽与GPVI进行的蛋白质对接研究表明，Troα10结合于D1结构域的下表面和D2结构域的外表面，这与胶原结合位点和单链可变片段（scFv）D2结合位点的位置不同。多克隆抗体对胶原诱导的血小板聚集的抑制作用证实了这个新发现的位点。这表明GPVI的D2结构域是GPVI介导的血小板聚集中一个新的重要结合表位。在荧光素钠诱导的肠系膜小静脉血小板血栓形成和氯化铁诱导的颈动脉损伤血栓形成模型中，Troα6/Troα10对血栓形成显示出显著的抑制作用，且不延长体内出血时间。
我们开发了一种源自trowaglerix的新型抗血栓肽，其通过拮抗GPVI发挥作用，安全性更高——无严重出血。多肽在GPVI上的结合表位新颖且重要。这些六肽/十肽在开发用于治疗动脉血栓形成性疾病的理想小质量GPVI拮抗剂方面具有治疗潜力。
