A Novel Fusion Protein TAP-SSL5 Function As Anti-inflammatory And Anticoagulant

Background and ObjectiveAtherosclerosis is a chronic inflammatory disease. Inflammation plays an important role in the development of atherosclerosis. The activation of platelets and coagulation contributes to thrombosis and atherosclerosis as well. Staphylococcal superantigen-like protein-5 (SSL5) can bind to P-selectin glycoprotein ligand-1(PSGL-1) and inhibit monocyte's adhesion on endothelial cells. Tick anticoagulant peptide (TAP) is an inhibitor of factor Xa (FXa). In this study, a fusion protein TAP-SSL5 was constructed to inhibit inflammation and FXa simultaneously, which may be a promising new class of anti-inflammatory and anti-thrombosis drug.Methods1. The genetic fragments of'ssl5'and'tap'were obtained by PCR, and both were cloned into pET32a vector. The tap-ssl5 gene were synthesized according to its sequence and were cloned into pET22b vector. Recombinant proteins were expressed in E. coli BL21 (DE3).2. The binding of TAP-SSL5 to HL60 cells and the competitive inhibition of KPL1 ( a mouse anti-human PSGL-1 monoclonal antibody) binding to leukocytes were examined using flow cytometry. To investigate the adhesion of leukocytes on P-selectin-coated surface, neutrophils or HL60 cells were labeled with calcein-AM, and adherent cells were quantified using fluorescent microplate reader. The inhibitory effect of TAP-SSL5 on FXa activation were assayed by chromogenic substrate, using purified FXa or FX in human and mouse plasma activated with russell viper venom-X (RVV-X)..3. Ferric chloride-induced rat vena cava thrombus formation model was used to study antithrombotic effect of TAP-SSL5. Rat carotid artery balloon injury model was used to observe the effect of TAP-SSL5 on vascular intimal hyperplasia. ApoE gene knockout (apoE-/-) mice fed with high-cholesterol diet was used as atherosclerotic model to observe the effect of TAP-SSL5 on atherosclerotic plaque formation, the level of inflammatory cytokines in the mouse aortic wall was determined by protein chips. In both animal experiments above, the rats or apoE-/- mice was randomizely grouped into control group and TAP-SSL5 treatment group.Results1. TAP-SSL5 could bind to leukocytes or HL60 cells and competitively inhibit KPL-1 binding to leukocytes or HL60 cells, and reduce the adhesion of neutrophils or HL60 cells on immobilized P-selectin, the inhibition rates were 67.9% and 81.6% for neutrophils and HL60 cells respectively (p <0.001). The inhibition effect of TAP-SSL5 on FXa activity was obvious and in a concentration-dependent manner, 3μmol/L TAP-SSL5 reduced purified FXa activity by 93.7%, reduced by 75.5% in mouse plasma, and 94.7% in human plasma (p <0.001).2. TAP-SSL5 reduced ferric chloride-induced venous thrombus formation significantly. In the rat carotid artery balloon injury model, after 21 days treatment of TAP-SSL5, the neointimal hyperplasia reduced by 31.5% (p <0.001) compared with controls. In apoE-/- mice, atherosclerotic plaque was significantly reduced by TAP-SSL5 compared with controls, and the expression of many inflammatory cytokines in the aortic wall decreased in the TAP-SSL5 treatment group compared with controls.ConclusionsThe recombinant fusion protein TAP-SSL5 could inhibit the adhesion of leukocytes on P-selectin-coated surface by binding to PSGL-1 on the leukocytes. It could inhibit the thrombus formation as well by its anti-FXa activity. The neointimal hyperplasia after rat carotid artery balloon injury and the atherosclerotic plaque formation in apoE-/- mice were both inhibited by TAP-SSL5. These effects were associated with the function of TAP-SSL5 including its anti-inflammatory and anticoagulant activity. TAP-SSL5 may be a promising protein drug for the treatment of injured vascular disease and prevention of thrombosis.