RGD-hirudin-based low molecular weight peptide prevents blood coagulation via subcutaneous injection
RGD-hirudin-based low molecular weight peptide prevents blood coagulation via subcutaneous injection
复制标题
RGD-水蛭素低分子量肽通过皮下注射预防血液凝固
DOI：
10.1038/s41401-019-0347-0
复制
发表时间：
2020-01-16
影响因子：
8.2
通讯作者：
Mo, Wei
中科院分区：
文献类型：
--
作者：
Li, Ya-ran;Huang, Yi-nong;Mo, Wei
血栓栓塞性疾病是一种常见的心脑血管疾病，严重威胁着人类的生命和健康。凝血酶不仅影响外源性凝血途径，还影响内源性凝血途径。因此，它成为抗凝药物的最重要的靶点之一。RGD-hirudin是一种靶向凝血酶的抗凝药物，但只能静脉注射。我们设计了一种基于RGD-水蛭素的低分子量肽，可以防止血液凝块。我们首先使用NMR来鉴定与凝血酶相互作用的RGD-水蛭素的关键氨基酸残基。然后，我们基于RGD-hirudin的结构和功能，利用同源模建设计了一个新的直接凝血酶抑制肽（DTIP）。分子对接结果表明，DTIP与凝血酶的靶向性和结合作用与RGD-hirudin相似，表明DTIP与凝血酶直接相互作用。通过丙氨酸扫描鉴定了DTIP的活性氨基酸，并成功构建了突变体。在体外凝血时间试验中，我们发现添加DTIP的大鼠血浆中的aPTT、PT和TT比添加突变体的大鼠血浆中的aPTT、PT和TT显著延长。皮下注射DTIP可明显延长大鼠凝血时间。血栓弹力图分析显示DTIP显著延迟凝血。生物层干涉法研究表明，DTIP与突变体之间的凝血酶亲和常数无显著差异，表明它可能与凝血酶的其他位点结合，而不是与其活性中心结合。结果表明，低分子量的DTIP经皮下注射可预防血栓形成。
Thromboembolic disease is a common cardio-cerebral vascular disease that threatens human life and health. Thrombin not only affects the exogenous coagulation pathway, but also the endogenous pathway. Thus, it becomes one of the most important targets of anticoagulant drugs. RGD-hirudin is an anticoagulant drug targeting thrombin, but it can only be administered intravenously. We designed a low molecular weight peptide based on RGD-hirudin that could prevent blood clots. We first used NMR to identify the key amino acid residues of RGD-hirudin that interacted with thrombin. Then, we designed a novel direct thrombin inhibitor peptide (DTIP) based on the structure and function of RGD-hirudin using homology modeling. Molecular docking showed that the targeting and binding of DTIP with thrombin were similar to those of RGD-hirudin, suggesting DTIP interacted directly with thrombin. The active amino acids of DTIP were identified by alanine scanning, and mutants were successfully constructed. In blood clotting time tests in vitro, we found that aPTT, PT, and TT in the rat plasma added with DTIP were greatly prolonged than in that added with the mutants. Subcutaneous injection of DTIP in rats also could significantly prolong the clotting time. Thrombelastography analysis revealed that DTIP significantly delayed blood coagulation. Bio-layer interferometry study showed that there were no significant differences between DTIP and the mutants in thrombin affinity constants, suggesting that it might bind to other sites of thrombin rather than to its active center. Our results demonstrate that DTIP with low molecular weight can prevent thrombosis via subcutaneous injection.