Egler C, Albert T, Brokemper O, Zabe-Kühn M, Mayer G, Oldenburg J, Schwaab R
Center of Advanced European Studies And Research, Bonn, Germany.
J Mol Recognit. 2009 Jul-Aug;22(4):301-6. doi: 10.1002/jmr.947.
The murine monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 specifically bind and inhibit the procoagulant activity of human coagulation factor VIII (FVIII). They are frequently used as a model of inhibitors which are raised against injected FVIII in about 25% of hemophiliacs as a serious side effect of substitution therapy. However, binding kinetics of the interaction of these antibodies with FVIII and their influence on FVIII activity (inhibition) have not yet been examined systematically. For this, we examined association and dissociation of protein:antibody interaction using surface plasmon resonance (SPR) and determined their ability to inhibit the FVIII activity in a one-stage and a two-stage assay. SPR-analysis revealed that the equilibrium dissociation constants (K(D)) of ESH8 and ESH4 are low and in a similar range (ESH8: K(D(ESH8)) = 0.542 nM; ESH4: K(D(ESH4)) = 0.761 nM). A 5.7 times higher K(D) than for ESH4 was observed for ESH2 (4.33 nM), whereas ESH5 showed the highest K(D) of 28.8 nM. In accordance with the lowest K(D), ESH8, and ESH4 reduced FVIII activity of normal human plasma almost completely in a one-stage clot inhibition assay (ESH8: 91.9%; ESH4: 90.1%). However, ESH8 inhibited FVIII activity more efficiently as only 1.0 microg/ml ESH8 was sufficient to obtain maximum inhibition compared to up to 600 microg/ml of ESH4. Despite its attenuated K(D), ESH2 inhibits FVIII:C still efficiently, reducing 61.3% of FVIII activity at a concentration of 9 microg/ml in the one-stage clotting assay. However, a discrepancy of inhibitory efficiency was found depending on the method used to measure FVIII activity. These effects seem to be mainly caused by differences of activation time of FVIII during both FVIII activity assays. The systematic assessment of these results should support FVIII interaction studies, and can provide data to rationally test peptides/mimotopes to remove or neutralize inhibitors of FVIII activity.
鼠单克隆抗体ESH2、ESH4、ESH5和ESH8可特异性结合并抑制人凝血因子VIII（FVIII）的促凝血活性。它们常被用作抑制剂模型，在约25%的血友病患者中，作为替代疗法的严重副作用，会产生针对注射的FVIII的抑制剂。然而，这些抗体与FVIII相互作用的结合动力学及其对FVIII活性（抑制作用）的影响尚未得到系统研究。为此，我们使用表面等离子体共振（SPR）研究了蛋白质与抗体相互作用的结合和解离，并在单阶段和双阶段试验中测定了它们抑制FVIII活性的能力。SPR分析显示，ESH8和ESH4的平衡解离常数（K(D)）较低且处于相似范围（ESH8：K(D(ESH8)) = 0.542 nM；ESH4：K(D(ESH4)) = 0.761 nM）。ESH2的K(D)比ESH4高5.7倍（4.33 nM），而ESH5的K(D)最高，为28.8 nM。与最低的K(D)一致，在单阶段凝血抑制试验中，ESH8和ESH4几乎完全降低了正常人血浆的FVIII活性（ESH8：91.9%；ESH4：90.1%）。然而，ESH8抑制FVIII活性的效率更高，因为与高达600 μg/ml的ESH4相比，仅1.0 μg/ml的ESH8就足以实现最大抑制。尽管ESH2的K(D)有所减弱，但在单阶段凝血试验中，它在9 μg/ml的浓度下仍能有效抑制FVIII:C，降低61.3%的FVIII活性。然而，根据用于测量FVIII活性的方法，发现抑制效率存在差异。这些影响似乎主要是由两种FVIII活性试验中FVIII的激活时间差异引起的。对这些结果的系统评估应有助于FVIII相互作用研究，并可为合理测试肽/模拟表位以去除或中和FVIII活性抑制剂提供数据。
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