Laboratory for Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
J Thromb Haemost. 2011 Nov;9(11):2268-77. doi: 10.1111/j.1538-7836.2011.04495.x.
Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co-administration of t-PA and a TAFIa inhibitor enhances t-PA treatment.
We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t-PA induced clot lysis.
Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A(147) -C(305) -I(325) -I(329) -Y(333) -Q(335) ), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16-fold molar excess of nanobody over TAFIa, IC(50) 's range between 0.38- and > 16-fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t-PA concentration. The nanobodies were found to bind in the active-site region of TAFIa and their time-dependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition.
These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.
由于激活的凝血酶可激活的纤溶抑制物（TAFIa）具有非常强大的抗纤溶作用，因此 t-PA 与 TAFIa 抑制剂联合使用可增强 t-PA 的治疗效果。
我们旨在生成专门抑制 TAFIa 活性的纳米抗体，并测试其对 t-PA 诱导的血栓溶解的影响。
描述了针对一种激活的更稳定的 TAFIa 突变体（TAFIa A(147)-C(305)-I(325)-I(329)-Y(333)-Q(335)）产生的 5 种纳米抗体。这些纳米抗体特异性抑制 TAFIa 活性，在纳米抗体与 TAFIa 的摩尔比达到 16 倍时，抑制率高达 99%，IC50 范围在 0.38-16 倍摩尔比之间。在缺乏血栓调节蛋白（TM）的体外血栓溶解实验中，纳米抗体表现出促纤溶作用。然而，在存在 TM 的情况下，一种纳米抗体表现出抗纤溶作用，而其他纳米抗体在低浓度下表现出轻微的抗纤溶作用，在高浓度下表现出明显的促纤溶作用。这种双相模式高度依赖于 TM 和 t-PA 浓度。这些纳米抗体被发现结合在 TAFIa 的活性部位，它们在 TAFIa 失活过程中的时变差异结合行为揭示了一个未知的中间构象转变的发生。
这些纳米抗体是非常有效的 TAFIa 抑制剂，是加速纤溶作用的有用工具。我们的数据还表明，TM 的存在可能会逆转 TAFIa 抑制的促纤溶作用。新构象转变的识别为不稳定 TAFIa 的构象失活提供了新的见解。
