Construction and functional evaluation of hirudin derivatives with low bleeding risk.

The purpose of this study was to design and evaluate hirudin (HIR) derivatives with low bleeding risk. In these derivatives, the factor (F) XIa, FXa, and thrombin recognition peptides (EPR, GVYAR, and LGPR, respectively) were linked to the N-terminus of HIR. The intact derivatives have no anticoagulant activity because of the extension of the N-terminus of HIR. After cleavage by the corresponding coagulation factor that occurs on the activation of the coagulation system and in the presence of the thrombus, its activity is released. This limited the anticoagulant activity of these derivatives to the vicinity of the thrombus, and as a result, systemic bleeding complications were avoided. The definite antithrombotic effect and low bleeding parameters of these derivatives were investigated in rat carotid artery and inferior vena cava thrombosis models. In both models, the three derivatives showed significant antithrombotic effects, indicating that anticoagulant activity could be successfully released in vivo. Moreover, the bleeding parameters of these derivatives were lower than that of HIR as indicated by the values of activated partial thromboplastin time (APTT) and thrombin time (TT). To further assess the safety of these derivatives, bleeding time was measured in a mouse tail-cut model. Although the derivatives had obvious effects on bleeding at a dose of 6 mg/kg, the effect of these derivatives on bleeding was significantly weaker than that of HIR at a dose of 1.5 mg/kg. Thus, the benefit-to-risk profiles of the derivatives were superior to that of HIR.

Two characteristics of a recombinant fusion protein composed of staphylokinase and hirudin: high thrombus affinity and thrombus-targeting release ofanticoagulant activity.

To improve thrombolytic effect, a fusion protein SFH composed of staphylokinase (SAK) and hirudin (HV) with blood coagulation factor Xa (FXa) recognition peptide as a linker, was designed. SFH showed improved thrombolytic effect and low bleeding in vivo. Two thrombus-targeting mechanisms might account for the above features of SFH. This study was designed to study the two thrombus-targeting mechanisms of SFH. ELISA and immunohistochemistry assay were used to study the improved thrombus selectivity of SFH and the results showed that SFH, compared with SAK, displayed higher affinity for thrombin and thrombin-rich thrombus. To verify the thrombus-targeting release of anticoagulant activity of SFH, FH-a derivative of HV with only FXa recognition sequence at N terminus of HV was designed and used in animal tests. In inferior vena cava thrombosis model, FH showed equal antithrombotic effect as HV, indicating that HV could be successfully released from FH by FXa cleavage in vivo. More importantly, no prolongation of plasma TT, APTT and PT were found in FH group, but significant prolongations were discovered in HV group. This revealed that the anticoagulant activity of FH was released in thrombus-targeting way and limited in the vicinity of the thrombus, and this could be extrapolated to SFH. In conclusion, the high thrombus affinity and thrombus-targeting release of anticoagulant activity of SFH assigned low bleeding risk to SFH.

Locally activity-released bifunctional fusion protein enhances antithrombosis and alleviates bleeding risk.

Despite the fact that lytic therapy of thromboembolic disorder has been achieved, reocclusion of the damaged vessels and bleeding complication frequently reduce the therapeutic effect. In order to prevent the vessel reocclusion and enhance the therapeutic effect, combining the anticoagulant with the thrombolytic was assumed. Herein, we propose that restraining but locally releasing anticoagulant activity in the vicinity of thrombus is a way to alleviate the bleeding risk. A bifunctional fusion protein, termed as SFH (Staphylokinase (SAK) linked by FXa recognition peptide at N-terminus of Hirudin (HV)), was designed. SFH retained thrombolytic activity but no anticoagulant activity in thrombus-free blood due to the extension of the N-terminus of HV. However, it could locally liberate intact HV and exhibit anticoagulant activity when FXa or fresh thrombus was present. At equimolar dose, both improved antithrombotic and thrombolytic effects of SFH were observed in kappa-carrageenin inducing mouse-tail thrombosis model and rat inferior vena cava thrombosis model, respectively. Moreover, we observed significantly lower bleeding risk in mice and rats treated with SFH than with the mixture of SAK and HV with monitoring TT (P < 0.01), aPTT (P < 0.05) and PT (P < 0.05), and bleeding time (P < 0.05). In conclusion, SFH is a promising bifunctional therapeutic candidate with lower bleeding risk.

[Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris].

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.