New Infestin-4 Mutants with Increased Selectivity against Factor XIIa
New Infestin-4 Mutants with Increased Selectivity against Factor XIIa
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DOI：
10.1371/journal.pone.0144940
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发表时间：
2015-12-15
期刊：
影响因子：
3.7
通讯作者：
Panteleev, Mikhail A.
中科院分区：
文献类型：
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作者：
Kolyadko, Vladimir N.;Lushchekina, Sofya V.;Panteleev, Mikhail A.
因子XIIa（fXIIa）是一种丝氨酸蛋白酶，其触发凝血接触途径并在血栓形成中起作用。因为它干扰凝血试验，在许多情况下需要抑制fXIIa。Infestin-4（Inf 4）是fXIIa的Kazal型抑制剂。其对fXIIa的特异性可以通过蛋白酶结合环中的点突变来增强。我们试图使Inf 4适应于在各种体外条件下选择性抑制接触途径，例如，在血液收集期间和组织因子（TF）依赖性凝血的“整体”测定中。首先，我们设计了一组新的Inf 4突变体，与野生型Inf 4相比，在分子动力学模拟过程中稳定了典型构象。如显色试验所示，在这些重组突变体中，针对Xa因子（fXa）、纤溶酶和其他凝血蛋白酶的脱靶活性降低或消除。还使用蛋白质-蛋白质对接分析了与fXIIa和fXA的相互作用。接下来，在血浆中测试突变体B，最有效的突变体之一（其对fXIIa的Ki为0.7 nM）。在5-20 μ M的浓度下，该突变体延迟了凝血酶的接触激活产生，以及在血栓弹力图和血栓动力学测定中的凝血。在这些试验中，突变体B不影响TF引发的凝血，因此证明了足够的选择性及其作为凝血诊断试剂的潜在实际意义。
Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 mu M, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.