The Antithrombotic Effect of Recombinant Neorudin on Thrombi

Abstract

Introduction: Recombinant neorudin (EPR-hirudin, EH) was developed through the addition of an EPR (Glu-Pro-Arg) peptide to the amino terminus of hirudin, which can be recognized and cut by coagulation factors XIa (FXIa) and/or Xa (FXa). In this study, the low-bleeding antithrombotic effects of EH were evaluated utilizing experimental models of thrombosis in rabbits and rats to provide a test basis for clinical trials.

Methods: The bleeding risks of EH and hirudin were first compared in mice by the tail-clipping method, and then the antithrombotic activity of EH was investigated in a rabbit model of arteriovenous bypass thrombosis and a rat model of thrombotic cerebral infarction.

Results: In mice, intravenous administration of EH at 1.5 mg/kg and 3 mg/kg did not affect the bleeding time compared with normal saline, while the administration of hirudin at 1.5 mg/kg prolonged the bleeding time by over 3 times the administration of normal saline. Furthermore, intravenous administration of EH had a significant dose-dependent inhibitory effect on the formation and development of arteriovenous bypass thrombosis and thrombotic cerebral infarction. Compared with an equimolar dose of hirudin, the antithrombotic effect of EH was similar, while the bleeding side effects were significantly attenuated. Moreover, when the antithrombotic effects were similar, EH had a shorter bleeding time and was associated with less bleeding than low molecular weight heparin (LMWH). EH had a therapeutic effect on thrombotic cerebral infarction without increasing the occurrence of cerebral hemorrhage.

Conclusion: The findings from the preclinical animal models used in this study showed that EH could not only effectively inhibit thrombus formation but also reduce the risk of bleeding.

Keywords: antithrombotic effect; bleeding; hirudin; recombinant neorudin.

Figure 1

Antithrombotic experiment design. (A) Experimental process in rabbit model of arteriovenous bypass thrombosis. (B) Experimental process in rat model of thrombotic cerebral infarction.

Figure 2

The tail bleeding time in healthy Kunming mice. Twenty minutes after injection with EH or hirudin through caudal vein, 6 mm of tail tip was cut off and the tail was immediately put into normal saline to record the bleeding time. **P < 0.01, in comparison with normal saline.

Figure 3

Effect of EH on thrombosis and bleeding in rabbit model of arteriovenous bypass thrombosis. At the beginning of arteriovenous bypass thrombus induction in rabbit, EH was intravenously infused for 30 minutes. Sixty minutes after induction, the thrombus was removed for analysis and the side-effect of bleeding was evaluated. (A) Effect of EH on the wet and dry weight of thrombus. The wet weight of thrombus was record first and cut two parts and one part was baked in oven at 50°C for three days to calculate the dry weight of thrombus. (B) Effect of EH on the quantity of thrombus matrix fibrin. The other part of thrombus was used to detect the amount of fibrin by digestion with 1% NaOH. (C) Effect of EH on auricular bleeding time and coagulation time of whole blood. After a fixed length wound (avoiding large blood vessels) was cut at the ear edge of the rabbit the bleeding time was recorded, and a drop of whole blood was taken in a glass dish to record the coagulation time by needle picking. *P < 0.05, **P < 0.01, ***P < 0.001, in comparison with normal saline; ⁺P < 0.05, in comparison with equimolar hirudin.

Figure 4

The effect of EH on neurological impairment scores at 4 h and 24 h after operation in rat with cerebral infarction. Thirty minutes after drug administration, the cerebral infarction in rats was induced with FeCl₃, and the degree of neurological damage was scored with a blinded method at 4 h and 24 h after drug treatment according to the evaluation criteria in Table 1. *P < 0.05, **P < 0.01, ***P < 0.001, in comparison with normal saline.

Figure 5

Effects of EH on cerebral infarction in rats. After treatment for 24 h, the rat brain was removed and brain section with 2 mm thick was cut. (A) Rat brain sections staining with TTC. Normal brain tissue was stained red by TTC, while the ischemic brain tissue was not stained. (B) Cerebral infarction volume calculated by the image collection and analysis system software v4.0. (C) Cerebral edema volume calculated by the image collection and analysis system software v4.0. (D) Free hemoglobin content in brain tissue determined by micro free hemoglobin (FHb) determination kit. ^ΔΔΔP < 0.001, in comparison with sham operation group, *P < 0.05, **P < 0.01, ***P < 0.001, in comparison with normal saline.