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.