融合蛋白TAP-SSL5抑制动静脉血栓形成

TAP-SSL5 fusion protein inhibits arterial and venous thrombosis

目的:探讨重组抗炎、抗凝双效融合蛋白蜱抗凝血肽(TAP)-金黄色葡萄球菌超抗原样蛋白-5(SSL5)对动静脉血栓形成的影响和机制。方法:采用基因重组技术构建TAP-SSL5基因,进而制备融合蛋白TAPSSL5,通过流式细胞检测技术研究重组蛋白TAP-SSL5与粒细胞表面P-选择素糖蛋白配体1(P-selectin glycoprotein ligand-1,PSGL-1)结合的特性;采用反应底物法在体外检测融合蛋白TAP-SSL5对活化的凝血因子Xa(factor Xa,FXa)活性的抑制作用。采用Fe Cl3诱导的SD大鼠下腔静脉血栓模型,研究TAP-SSL5对静脉血栓形成的影响;采用Fe Cl3诱导的C57BL/6J小鼠肠系膜动脉血栓模型,在双光子显微镜下连续检测TAP-SSL5对动脉血栓形成的影响。结果:TAP-SSL5可与粒细胞表面PSGL-1结合,并抑制粒细胞与P-selectin和血小板的结合。TAP-SSL5呈剂量依赖性地抑制FXa活性(P <0. 01)。体内实验中TAP-SSL5可明显抑制Fe Cl3诱导的SD大鼠下腔静脉及C57BL/6J小鼠肠系膜动脉血栓的形成。结论:融合蛋白TAP-SSL5具有抗炎和抗凝双重效果,可以明显抑制动静脉血栓的形成。

AIM:To study the effect of tick anticoagulant peptide(TAP)-staphylococcal-like protein 5(SSL5),an anti-inflammatory and anticoagulant fusion protein,on the arterial and venous thrombosis.METHODS:In this study we engineered TAP,an inhibitor of activated coagulation factor Xa(FXa),with SSL5 to construct the fusion protein TAP-SSL5,and its effects on the interaction between platelets and neutrophils were investigated by flow cytometry.The anti-FXa activity of TAP-SSL5 was also measured using both pure FXa protein and FXa in plasma derived from the factor X activated by Russell’s viper venom-factor X activator(RVV-X).Finally,the effect of TAP-SSL5 on venous thrombosis in SD rats induced by FeCl 3 was studied,and the FeCl 3-induced mouse mesenteric arterial thrombosis model was used to evaluate the effects of TAP-SSL5 on thrombi formation by real-time intravital microscopy.RESULTS:TAP-SSL5 inhibited the binding of activated platelets to neutrophils.TAP-SSL5 also suppressed the activity of FXa in a concentration dependent manner in vitro.TAP-SSL5 at 10 mg/kg significantly inhibited the initial platelet interaction and adhesion response to vascular injury and completely prevented vessel occlusion in vivo.CONCLUSION:TAP-SSL5 inhibits thrombosis by its anticoagulative and anti-platelet adhesion properties.TAP-SSL5 may serve as a potential therapeutics against the vascular thrombotic diseases.