Design And Characterization Of Recombinant Targeting Bifunctional Fusion Protein For Therapy Of Thrombosis

Despite successful lytic therapy of thromboembolic disorder, reocclusion of the damaged vessels or bleeding complication frequently reduces the therapeutic effect. Making efforts to combine the benefits of thrombolytics and anticoagulants for prevention of vessel reocclusion and to alleviate their side effect of bleeding complication, we designed a targeting bifunctional fusion protein, termed as SFH (Staphylokinase with hirudin linked by FXa recognition peptide).A fusion protein of staphylokinase with hirudin was expressed in methylotrophic yeast Pichia pastoris. The fusion protein yield was up to 1 g/L broth, which expression was induced by fed-batch methanol for 120 hours. Considering the effect of glycosylation of Asn28 of staphylokinase on its activity of activating plasminogen, and to remove the sequence of glycosylation, mutants were constructed and expressed. It was observed that the mutation of amino acid 28-30 dramatically decreased the fibrinolytic of staphylokinase. It was concluded that the fusion protein could be expressed at high level in Pichia pastoris and that the residues of 26-30 amino acid played key role for staphylokinase activating plasminogen.To overcome the dilemma between glycosylation and mutation, the fusion protein without mutation was constructed and expressed in E.coli. The fusion protein retained plasminogen-activating and fibrinolytic activity from the domain of staphylokinase but no anticoagulant activity due to the extension of the N-terminus of hirudin. However, the fusion protein showed effectively anticoagulant activity when fresh thrombus and activated FXa were present due to the local liberation of intact hirudin by FXa. At equimolar concentrations, an enhanced higher anticoagulant activity of the fusion protein than staphylokinase was observed in experiments in a mouse tail thrombosis model induced by kappa-carrageenin. Therefore, the fusion protein SFH is a targeting-released bifunctional molecule being able both to activate fibrinolysis via plasminogen activation and to inhibit clot growth via direct thrombin inhibition at thrombus where the inhibition activity is required. In conclusion, the newly designed chimeric protein has three advantages: targeting to thrombin, fibrinolytic and anticoagulant bifunction, and minimal anticoagulant activity at thrombus-free sites, maximal local concentration and anticoagulant activity in the vicinity of thrombus. It was showed that the fusion protein was a promising drug candidate for targeted therapy of thrombosis.