Wang Chengtao, Ji Baoping, Cao Yanping, Sun Baoguo, Liu Xudong
College of Chemical and Environmental Engineering, Beijing Technology & Business University, Beijing 100048, China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2010 Apr;27(2):332-6, 378.
A novel fibrinolytic enzyme subtilisin FS33, which exhibits much higher activity for decomposing fibrin than urokinase, was purified from Douchi, a traditional soybean-fermented food in China. In order to increase bio-utilization and thrombus targetability of subtilisin FS33 labeled by fluorescein isothiocyanate (FITC), the surface modified liposomes encapsulating subtilisin FS33 and FITC with a synthetic peptide Arg-Gly-Asp-Ser (RGDS), being putatively a specific antagonist of fibrinogen receptor on platelet membrane, were prepared and used to evaluate the therapeutic efficacy in a rat model thrombotic carotid artery. The arterial thrombosis was induced by applying two pieces of filter paper (1 x 2 cm) saturated with 10% of ferric chloride (FeCl3). The rats were infused via the jugular vein with either liposomes carrying BSA (control group) or RGDS-liposomes carrying subtilisin FS33 at doses of 2000 and 4000 U/kg. The plasma of the group infused with RGDS-liposomes showed higher antithrombotic and fibrinolytic activity than did the control group within 15-120 min after infusing. The higher the dose was gived, the higher the activity was shown. APTT(activiated partial thromboplastin time), PT (prothrombin time) and TT (thrombin time) were extended remarkably (P < 0.05, P < 0.01), and FDP (fibrinogen degradation products) also increased greatly (P < 0.01), while ELT (euglobulinlysis time) decreased obviously (P < 0.05). FITC content in heart and brain evidently increased (P < 0.05), and results of D-dimer test were all positive. In addition, the venous thrombi in brain and kidney were dissolved totally or partly as observed by patholgical section. All these indicated that subtilisin FS33 enhanced the antithrombotic and fibrinolytic activities in rat, and RGDS-liposomes improved, in a certain degree, the thrombolytic specificity for targeting to thrombus.
一种新型纤溶酶枯草杆菌蛋白酶FS33,其分解纤维蛋白的活性比尿激酶高得多,是从中国传统大豆发酵食品豆豉中纯化得到的。为了提高异硫氰酸荧光素(FITC)标记的枯草杆菌蛋白酶FS33的生物利用率和血栓靶向性,制备了用合成肽Arg-Gly-Asp-Ser(RGDS)包裹枯草杆菌蛋白酶FS33和FITC的表面修饰脂质体,RGDS被认为是血小板膜上纤维蛋白原受体的特异性拮抗剂,并用于评估大鼠颈总动脉血栓形成模型中的治疗效果。通过应用两片浸有10%氯化铁(FeCl3)的滤纸(1×2厘米)诱导动脉血栓形成。通过颈静脉给大鼠输注携带牛血清白蛋白的脂质体(对照组)或剂量为2000和4000 U/kg的携带枯草杆菌蛋白酶FS33的RGDS-脂质体。输注RGDS-脂质体组的血浆在输注后15至120分钟内显示出比对照组更高的抗血栓和纤溶活性。给药剂量越高,活性越高。活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和凝血酶时间(TT)显著延长(P<0.05,P<0.01),纤维蛋白原降解产物(FDP)也大幅增加(P<0.01),而优球蛋白溶解时间(ELT)明显缩短(P<0.05)。心脏和大脑中的FITC含量明显增加(P<0.05),D-二聚体检测结果均为阳性。此外,通过病理切片观察到脑和肾中的静脉血栓全部或部分溶解。所有这些表明枯草杆菌蛋白酶FS33增强了大鼠的抗血栓和纤溶活性,并且RGDS-脂质体在一定程度上提高了血栓靶向溶栓的特异性。