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.

血栓栓塞性疾病是威胁人类生命健康的常见心脑血管疾病。凝血酶不仅影响外源性凝血途径，也影响内源性途径。因此，它成为抗凝药物最重要的靶点之一。RGD-水蛭素是一种针对凝血酶的抗凝药物，但只能静脉内给药。我们设计了一种基于 RGD-水蛭素的低分子量肽，可以预防血栓。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-水蛭素的结构和功能，使用同源模型设计了一种新型的直接凝血酶抑制肽（DTIP）。分子对接显示DTIP与凝血酶的靶向结合与RGD-水蛭素相似，表明 DTIP 直接与凝血酶相互作用。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。在体外凝血时间测试中，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显着延长。大鼠皮下注射DTIP也能显着延长凝血时间。血栓弹力图分析显示 DTIP 显着延迟血液凝固。生物层干涉研究表明，DTIP与突变体的凝血酶亲和力常数没有显着差异，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。我们的研究结果表明，低分子量的 DTIP 可以通过皮下注射预防血栓形成。