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首页> 外文期刊>Thrombosis and Haemostasis: Journal of the International Society on Thrombosis and Haemostasis >Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide.
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Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide.

机译：靶向血纤蛋白的直接因子Xa抑制：由抗血纤蛋白单链抗体和壁虱抗凝血肽组成的重组因子Xa抑制剂的构建和表征。

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摘要

We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv(59D8)). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clot-bound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications.TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for anti-fXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule.Two step affinity chromatography with Ni(2+) and beta(15-22)-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv(59D8)-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv(59D8)-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.
机译：我们研究了直接的fXa抑制剂tick抗凝肽（TAP）是否可以在N末端偶联对血纤蛋白（scFv（59D8））特异性的凝块靶向单链抗体。由于其在凝血级联早期处于内在和外在途径的汇合点的独特位置，因此因子Xa（fXa）代表了有吸引力的治疗靶标。与间接抑制剂相反，直接fXa抑制剂可有效抑制血凝块结合和凝血酶原相关的fXa。将直接的fXa抑制剂靶向血凝块有望增强局部抗凝能力并减少全身性抗凝作用，从而可能减少出血并发症.TAP是一种高效的fXa抑制剂。由于其N末端对于抗fXa活性至关重要，因此TAP是否作为N末端偶联的融合分子具有活性是一个具有挑战性的问题.Ni（2+）和beta（15-22）两步亲和色谱人血纤蛋白的β-肽产生纯的36 kDa蛋白，并对其靶向功能和抗fXa活性进行了测试。重组融合体没有破坏融合伴侣的功能。抗体结合功能与亲本分子相当。 TAP活性被部分降低，认为抗fXa活性不需要游离的N端，但对最大效价很重要。在人全血凝块中，scFv（59D8）-TAP在200到500 nM的浓度下显示出抗凝特性，而非靶向TAP根本没有显示出抗凝活性。总之，scFv（59D8）-TAP构成了有希望的新型抗凝剂，具有纤维蛋白靶向因子Xa抑制作用。在大肠杆菌中生产和建立的纯化方法是低成本，可重复生产的现代大规模生产的坚实基础。

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