Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Brussels, Belgium.
J Thromb Haemost. 2014 Feb;12(2):229-36. doi: 10.1111/jth.12473.
Down-regulation of fibrinolysis due to cleavage of C-terminal lysine residues from partially degraded fibrin is mainly exerted by the carboxypeptidase activity of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recently, some intrinsic carboxypeptidase activity (i.e. zymogen activity) was reported for the proenzyme (TAFI); however, there is some discussion about its ability to cleave high molecular weight substrates.
We aimed to identify and characterize nanobodies toward mouse TAFI (mTAFI) that stimulate the zymogen activity and to test their effect in an in vitro clot lysis assay and an in vivo mouse thromboembolism model.
Screening of a library of nanobodies toward mTAFI revealed one nanobody (VHH-mTAFI-i49) that significantly stimulates the zymogen activity of mTAFI from undetectable (< 0.35 U mg⁻¹) to 4.4 U mg⁻¹ (at a 16-fold molar ratio over mTAFI). The generated carboxypeptidase activity is unstable at 37 °C. Incubation of mTAFI with VHH-mTAFI-i49 revealed a time-dependent reduced activatability of mTAFI. Epitope mapping revealed that Arg227 and Lys212 are important for the nanobody/mTAFI interaction and suggest destabilization of mTAFI by disrupting the stabilizing interaction between the activation peptide and the dynamic flap region. In vitro clot lysis experiments revealed an enhanced clot lysis due to a reduced activation of mTAFI during clot formation. In vivo application of VHH-mTAFI-i49 in a mouse thromboembolism model decreased dose-dependently the fibrin deposition in the lungs of thromboembolism-induced mice.
The novel, nanobody-induced, reduced activatability of mTAFI demonstrates to be a very potent approach to enhance clot lysis.
由于部分降解纤维蛋白的 C 末端赖氨酸残基的裂解，纤溶的下调主要由激活的血栓调节蛋白可激活纤溶抑制物（TAFIa）的羧肽酶活性发挥作用。最近，有人报道了该前体酶（TAFI）具有一些内在的羧肽酶活性（即酶原活性）；然而，关于其切割高分子量底物的能力仍存在一些争议。
我们旨在鉴定和表征针对小鼠 TAFI（mTAFI）的纳米抗体，以刺激酶原活性，并在体外血栓溶解测定和体内小鼠血栓栓塞模型中测试其效果。
筛选针对 mTAFI 的纳米抗体文库发现了一种纳米抗体（VHH-mTAFI-i49），它能显著刺激 mTAFI 的酶原活性，使 mTAFI 的酶原活性从检测不到（<0.35 U mg⁻¹）增加到 4.4 U mg⁻¹（在 16 倍 mTAFI 摩尔比下）。在 37°C 时，产生的羧肽酶活性不稳定。mTAFI 与 VHH-mTAFI-i49 孵育后，mTAFI 的可激活能力随时间呈下降趋势。表位作图显示 Arg227 和 Lys212 对纳米抗体/mTAFI 相互作用很重要，并表明通过破坏激活肽和动态瓣区之间的稳定相互作用来破坏 mTAFI 的稳定性。体外血栓溶解实验显示，由于在血栓形成过程中 mTAFI 的激活减少，血栓溶解增强。在血栓栓塞模型中体内应用 VHH-mTAFI-i49 可剂量依赖性地减少血栓栓塞诱导小鼠肺部的纤维蛋白沉积。
新型纳米抗体诱导的 mTAFI 降低可激活能力是增强血栓溶解的一种非常有效的方法。
