Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination
Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination
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DOI：
10.1111/jth.13617
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发表时间：
2017-03-01
影响因子：
10.4
通讯作者：
Fujimura, Y.
中科院分区：
文献类型：
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作者：
Matsui, T.;Hori, A.;Fujimura, Y.
背景 Botrocetin-2 (Bot2) 是一种类似 botrocetin 的蛋白质，由从蛇 Bothrops jararaca 中克隆的亚基组成。 Bot2 与血管性血友病因子 (VWF) 特异性结合，该复合物诱导糖蛋白 (GP) Ib 依赖性血小板凝集。 目的 利用 Bot2 的 VWF 结合能力，尝试创建与 VWF 结合但抑制血小板凝集的突变 Bot2。 方法与结果 Bot2 cDNA 中引入几个点突变，并在 Bot2 上纯化重组蛋白（重组 Bot2 [rBot2]）。抗 Botrocetin 柱。亚基中 Asp70 处具有 Ala (Asp70Ala) 或 Arg115Ala 和 Lys117Ala 的突变体 rBot2 显示出血小板凝集诱导活性降低。具有 Asp70Ala 的 rBot2 在 ELISA 板上显示出对固定化 VWF 的结合活性很小，而具有 Arg115Ala/Lys117Ala 的 rBot2 在与 VWF 形成复合物后显示出对 GPIb（糖运载蛋白）的结合活性降低。 rBot2 在 Arg115 和 Lys117 处点突变为带相反电荷的 Glu，显示出对 VWF 的正常结合活性，但没有血小板凝集活性。此外，该双重突变蛋白可抑制瑞斯托菌素诱导或高剪切应力诱导的血小板聚集，并抑制流动条件下的血栓形成。结论 Botrocetin 亚基中的 Asp70 对于 VWF 结合很重要，亚基中的 Arg115 和 Lys117 对于与 GPIb 的相互作用至关重要。带有 Arg115Glu 和 Lys117Glu 的双突变 rBot2 可排斥 GPIb，并可能具有作为特异性阻断 VWF 功能的抗血栓试剂的潜力。这是关于可抑制 VWF-GPIb 相互作用的人工 Botrocetin 的第一份报告。
Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of and subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination.Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination.Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the subunit (Asp70Ala), or Arg115Ala and Lys117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Asp70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Arg115Ala/Lys117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Arg115 and Lys117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions.Conclusions Asp70 in the subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Arg115Glu and Lys117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction.