Choi Jun-Hui, Sapkota Kumar, Kim Seung, Kim Sung-Jun
Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University, Gwangju 501-759, Republic of Korea.
Central Department of Zoology, Tribhuvan University, Kirtipur, Kathmandu, Nepal.
Biochimie. 2014 Oct;105:45-57. doi: 10.1016/j.biochi.2014.06.012. Epub 2014 Jun 27.
A bi-functional fibrinolytic serine protease, Starase exhibiting thrombolytic potency was purified from hepatic caeca of Asterina pectinifera. Starase showed a single band of approximately 48 kDa by SDS-PAGE and fibrin zymography. The N-terminal sequence of Starase was AIPTEFDARTKKHNN, which does not match with any known fibrinolytic enzyme. Starase had optimum amidolytic activity at 50 °C and pH 8.0 and the activity was inhibited by PMSF and APMSF. Starase showed the highest specificity toward the substrate H-D-Val-Leu-Lys-pNA for plasmin followed by pyroGlu-Gly-Arg-pNA for urokinase. The apparent Km and Vmax values of Starase toward a chromogenic substrate for plasmin H-D-Val-Leu-Lys-pNA were determined as 1.37 mM and 6.8 mM/min/mg respectively. The fibrinolytic activity of Starase by fibrin plate assay displayed that it could not only directly degrade fibrin clot but also activate plasminogen. Starase showed a strong fibrinogenolytic activity, cleaving all three major chains of fibrinogen rapidly. In addition, Starase with more than 1 μg could cleave extracellular matrix component type VII collagen, and plasma proteins such as bovine albumin and bovine gamma globulin. It could also inhibit factor Xa and thrombin activity. Starase at a dose of 0.8 mg/kg was devoid of hemorrhagic activity and it demonstrated antithrombotic effect in three animal models; FeCl2-induced carotid arterial thrombus model, carrageenan-induced tail thrombosis model and collagen and epinephrine induced pulmonary thromboembolism mice model. These results suggest that Starase has the potential to be a potent thrombolytic agent due to its bi-functional properties (containing both direct-acting and plasminogen-activating activities) and lack of hemorrhagic activity. Although Starase might interfere with the normal composition of the plasma proteins, it may be used not only for the treatment and prevention of thrombosis, but also in a number of biomedical applications.
一种具有溶栓效力的双功能纤维蛋白溶解丝氨酸蛋白酶——海星酶，是从栉孔扇贝的肝盲囊中纯化得到的。通过SDS - PAGE和纤维蛋白酶谱分析，海星酶显示出一条约48 kDa的单一蛋白条带。海星酶的N端序列为AIPTEFDARTKKHNN，与任何已知的纤维蛋白溶解酶均不匹配。海星酶在50°C和pH 8.0时具有最佳酰胺水解活性，其活性受到苯甲基磺酰氟（PMSF）和氨苯甲基磺酰氟（APMSF）的抑制。海星酶对纤溶酶的底物H - D - 缬氨酸 - 亮氨酸 - 赖氨酸 - 对硝基苯胺（H - D - Val - Leu - Lys - pNA）表现出最高特异性，其次是对尿激酶的焦谷氨酸 - 甘氨酸 - 精氨酸 - 对硝基苯胺（pyroGlu - Gly - Arg - pNA）。海星酶对纤溶酶的显色底物H - D - Val - Leu - Lys - pNA的表观米氏常数（Km）和最大反应速度（Vmax）值分别测定为1.37 mM和6.8 mM/分钟/毫克。通过纤维蛋白平板试验检测，海星酶的纤维蛋白溶解活性表明它不仅能直接降解纤维蛋白凝块，还能激活纤溶酶原。海星酶表现出很强的纤维蛋白原溶解活性，能迅速切割纤维蛋白原的所有三条主要链。此外，浓度超过1μg的海星酶能切割细胞外基质成分VII型胶原蛋白以及血浆蛋白，如牛血清白蛋白和牛γ球蛋白。它还能抑制因子Xa和凝血酶的活性。剂量为0.8 mg/kg的海星酶没有出血活性，并且在三种动物模型中均显示出抗血栓作用；分别是氯化亚铁诱导的颈动脉血栓模型、角叉菜胶诱导的尾部血栓模型以及胶原蛋白和肾上腺素诱导的肺血栓栓塞小鼠模型。这些结果表明，由于其双功能特性（兼具直接作用和纤溶酶原激活活性）且缺乏出血活性，海星酶有潜力成为一种有效的溶栓剂。尽管海星酶可能会干扰血浆蛋白的正常组成，但它不仅可用于治疗和预防血栓形成，还可用于许多生物医学应用。
