Division of Cardiovascular and Diabetes Research, Leeds Institute for Genetics Health and Therapeutics, University of Leeds, Leeds, UK.
Bioscreening Technology Group in the School of Molecular and Cellular Biology, University of Leeds, Leeds, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S57. doi: 10.1016/S0140-6736(15)60372-5.
Enhanced complement C3 incorporation into the fibrin network in diabetes is one mechanism for impaired fibrinolysis and increased thrombosis risk in this condition. Our aim was to develop new strategies to modulate fibrinolysis in diabetes by interfering with fibrin-C3 interaction.
To modulate interaction between fibrinogen and C3 we used a novel technique by screening fibrinogen with a phage display library of 3 billion random, conformational 9AA peptides (termed adhirons). The effect of high affinity fibrinogen binding adhirons, released by the addition of excess C3, on fibrin clot lysis and structure was assessed in turbidimetric assays. Fibrinogen-C3 interactions were further studied by peptide microarray techniques and modelled with the website PepSite2.
Ten high affinity fibrinogen binding adhirons, released by C3, were available for turbidimetric analysis. One adhiron (A6) was found to have a sequence homology with C3 and studied further. In the absence of C3, adhiron A6 failed to modulate fibrin clot lysis time (mean 644 s [SE 13] and 620 [14] without and with adhiron A6, respectively). However, adhiron A6 abolished C3-induced prolongation of clot lysis, reducing mean lysis time from 728 s (SE 25) to 632 (24) (p=0·01). The peptide microarray screening of C3 identified two peptide motifs within the β chain of fibrinogen (residues 424-433, 435-445) that bound to C3. PepSite2 predicted that adhiron A6 binds to similar areas on the β chain of fibrinogen.
Using a novel phage display system, we discovered an adhiron that shared sequence homology with C3 and abolished C3-induced prolongation of fibrin clot lysis by interfering with C3-fibrinogen interaction within the β chain. This technique offers a unique method to identify new therapeutic targets for the reduction of diabetes-specific thrombosis risk.
Sir Jules Thorn Charitable Trust.
糖尿病患者的纤维蛋白网络中补体 C3 的含量增加是纤溶受损和血栓形成风险增加的机制之一。我们的目的是通过干扰纤维蛋白-C3 相互作用来开发新的策略来调节糖尿病中的纤溶。
为了调节纤维蛋白原和 C3 之间的相互作用，我们使用了一种新的技术，即用包含 30 亿个随机构象 9AA 肽（称为 adhirons）的噬菌体展示文库筛选纤维蛋白原。通过添加过量的 C3 释放的高亲和力纤维蛋白原结合 adhirons 在光散射测定中评估对纤维蛋白凝块溶解和结构的影响。通过肽微阵列技术进一步研究纤维蛋白原-C3 相互作用，并使用 PepSite2 网站进行建模。
有 10 个高亲和力纤维蛋白原结合 adhirons 通过 C3 释放，可用于光散射分析。发现一个 adhiron（A6）与 C3 具有序列同源性，并进一步研究。在没有 C3 的情况下，adhiron A6 未能调节纤维蛋白凝块的溶解时间（分别为 644s[SE 13]和 620[14]）。然而，adhiron A6 消除了 C3 诱导的凝块溶解延长，使平均溶解时间从 728s（SE 25）减少到 632（24）（p=0·01）。C3 的肽微阵列筛选鉴定出纤维蛋白原 β 链上的两个肽基序（残基 424-433、435-445）与 C3 结合。PepSite2 预测 adhiron A6 与纤维蛋白原 β 链上的类似区域结合。
我们使用一种新的噬菌体展示系统发现了一个与 C3 具有序列同源性的 adhiron，并通过干扰β 链上的 C3-纤维蛋白原相互作用，消除了 C3 诱导的纤维蛋白凝块溶解延长，从而为降低糖尿病特异性血栓形成风险提供了新的治疗靶点。
朱勒斯·索恩慈善信托基金。
