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ChemMedChem:开发出一种荧光肽更准确地测定凝血酶活性

来源:生物谷 2012-02-29 23:26

凝血酶(thrombin)在凝血系统的许多疾病中发挥着关键性作用。过量表达凝血酶能够导致血栓形成,但是它若表达不足则可能导致血友病。因此,准确地监控凝血酶活性对于确定给定病人的合适治疗方法是至关重要的,因它的活性与血液凝结能力相关联。监控凝血酶活性随时间变化的工具就是所谓的凝血酶生成测试实验(thrombin generation test, TGT)。

在荷兰内梅亨大学Floris Rutjes教授领导的一项合作性项目中,研究人员开发出一种新的凝血酶特异性荧光肽(fluorogenic peptid),它能够用于TGT中准确地测定凝血酶浓度。

当把这种肽加入到凝血血浆样品(clotting plasma sample)中,它被凝血酶分解后释放出荧光团。

对释放的荧光团进行分光光度测量能够增强TGT的灵敏性,因而也就增加该方法的准确度,从而测出许多种重要的凝血参数。

将荧光肽与TGT结合在一起有望广泛地用于止血和血栓领域。(生物谷:towersimper编译)

Fluorogenic Peptide-Based Substrates for Monitoring Thrombin Activity

Dr. Sander S. van Berkel, Bas van der Lee1, Dr. Floris L. van Delft, Dr. Rob Wagenvoord, Prof.Dr. H. Coenraad Hemker, Prof. Dr. Floris P. J. T. Rutjes

The synthesis of a series of peptides containing C-terminal 7-amino-4-methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H-Gly-Gly-Arg-AMC, of which the water solubility and kinetic parameters (KM and kcat) are greatly improved over those of the substrate in current use in the TGT: Cbz-Gly-Gly-Arg-AMC. A series of N-terminally substituted Gly-Gly-Arg-AMC derivatives were synthesized, as well as implementation of structural changes at either the P2 or P3 position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3-triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the KM and kcat values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et-malonate-Gly-Arg-AMC and N-functionalized Gly-Gly-Arg-AMC derivatives, are suitable candidates for replacement of the substrate currently in use.

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