RGD-hirudin-based low molecular weight peptide prevents blood coagulation via subcutaneous injection

Abstract

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.

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

“…Therefore, Li et al designed a novel direct thrombin inhibitor peptide (DTIP) based on the structure and function of RGD-hirudin. It was shown that DTIP could significantly delay blood coagulation and prevent thrombosis via subcutaneous injection (Li et al, 2020). Besides, HV12p-rPA is a fusion protein composed of C-terminal 12-residue of hirudin-PA (HV12p) and reteplase (rPA), which combines the biological specialties of its components and thus possesses both fibrinolytic and anticoagulant properties.…”

Inhibitory role of recombinant neorudin on canine coronary artery thrombosis

“…A direct inhibitor of thrombin, hirudin, can effectively inhibit both free and bound thrombin. It is a more beneficial antithrombotic drug than heparin in some animal models of deep vein injury 7,8 . However, the primary adverse effect of hirudin, the increased risk of systemic bleeding, also limited its use in the clinic 9,10 …”

“…It is a more beneficial antithrombotic drug than heparin in some animal models of deep vein injury. 7,8 However, the primary adverse effect of hirudin, the increased risk of systemic bleeding, also limited its use in the clinic. 9,10 Therefore, to overcome the bleeding disadvantage of hirudin, a new-generation anticoagulant, recombinant neorudin (EPRhirudin, EH) that is a targeted hirudin variant 2-Lys47(HV2) fusion protein, was developed as a prodrug of HV2 by introducing EPR (Glu-Pro-Arg).…”

Anti-thrombotic Effects Mediated by a Novel Dual-Target Peptide Inhibiting Both Platelet Aggregation and Thrombin Activity without Causing Bleeding

“…In previous studies, we analyzed the structure of DTIP and its interaction with thrombin, 30 and determined the key amino acid residues of DTIP that inhibit thrombin activity. 31 DTIP binds to exosite I and to the apolar region of thrombin via its N-terminal moiety, thus blocking access to the thrombin active site. Therefore, it is not appropriate to add RGD sequences to both ends of DTIP.…”