Study On The Pharmacodynamics Of Recombinant Hirudin-staphylokinase Fusion Protein And PEG-hirudin And Study Of The Antagonistic Action Of The Enzymatic Haemostatic Agent Against Hirudin

Objective: By taking advantage of the affinity of hirudin towards thrombin and its precursors and constructed a fusion protein between the 12 amino acid peptide from the hirudin C terminus and the thrombolytic agent staphylokinase. Through examining the binding selectivity of the resultant fusion protein towards the fibrin fribrinogen, we intended to study the feasibility of developing drug agents with dual thrombolytic and anti-thrombosis functions. In addition, we investigate whether the snake venoms have the effects of pro- and anti-coagulation, and how batroxobin antagonizes hirudin.Methods: Based upon the specific affinity of hirudin towards thrombin and its precursors, we constructed a fusion gene encoding the protein with a fusion point between the 12 amino acid peptide from the hirudin C terminus and the thrombolytic agent staphylokinase. Affinity measurement was used to identify the targeting effct of recombinant staphylokinase-hirudin fusion protein, and a mice tail thrombosis model was applied to assay its thrombolytic effect. In addition, we also investigated the bleeding liability induced by the fusion protein in a rabbit ear model and the length of acting time of PEG-Hir in vivo. The fibrinogen method was applied to study the pro-and anti-coagulation effects of snake venoms in vitro. And the fibrinogen method in vitro and a canine blood coagulation model were used to identify how batroxobin antagonize the bleeding effect of hirudin.Results: The results showed that the recombinant fusion protein possessed an obvious target affinity toward thrombin and a significantly thrombolytic effect on micetail thrombosis. The targeting thrombolytic effect of the recombinant fusion protein was superior to that of either staphylokinase or hirudin or the mixture of staphylokinase and hirudin at the same concentration. The recombinant fusion protein didn't affect the blood coagulation indices, but hirudin and heparin did obviously in the normal rabbits when administered alone at the same dose. In addition, the bleeding liability induced by the recombinant fusion protein was less than that of hirudin. We also found that PEG-Hir had a longer acting time than hirudin in rabbits.Snake venoms contain many kinds of proteins. Some have the effect of pro-coagulation of blood, and others can antagonize blood coagulation. These enzymatic haemostatic agents can be used to antagonize the side effect of hirudin. We take the batraxobin as an example to illustrate that the enzymatic haemostatic can antagonizes hirudin. Agglutination reaction occurred after the tubes containing hirudin were added with batroxobin , i.e., batroxobin can antagonize hirudin. In a canine model,, the reduction extent of TT and APTT in the test group was larger than that in the control group; the bleeding time was decreased obviously and it was shorter in the experimental group than in the control group at lh, 2h and 3h after hirudin injection, indicating batroxobin can antagonize the bleeding side effects of hirudin.