Yeon Seung Ju, Chung Goo Yong, Hong Jae Sang, Hwang Jin Ha, Shin Hwa Sung
Department of Biological Engineering, Inha University, Incheon, South Korea.
ST Pharm Co., Ltd., Gyeonggi-do, South Korea.
In Vitro Cell Dev Biol Anim. 2017 Jun;53(6):494-501. doi: 10.1007/s11626-017-0137-2. Epub 2017 Mar 10.
Ischemic stroke and cardiovascular disease can occur from blockage of blood vessels by fibrin clots formed naturally in the body. Therapeutic drugs of anticoagulant or thrombolytic agents have been studied; however, various problems have been reported such as side effects and low efficacy. Thus, development of new candidates that are more effective and safe is necessary. The objective of this study is to evaluate fibrinolytic activity, anti-coagulation, and characterization of serine protease purified from Lumbrineris nipponica, polychaeta, for new thrombolytic agents. In the present study, we isolated and identified a new fibrinolytic serine protease from L. nipponica. The N-terminal sequence of the identified serine protease was EAMMDLADQLEQSLN, which is not homologous with any known serine protease. The size of the purified serine protease was 28 kDa, and the protein purification yield was 12.7%. The optimal enzyme activity was observed at 50°C and pH 2.0. A fibrin plate assay confirmed that indirect fibrinolytic activity of the purified serine protease was higher than that of urokinase-PA, whereas direct fibrinolytic activity, which causes bleeding side effects, was relatively low. The serine protease did not induce any cytotoxicity toward the endothelial cell line. In addition, anticoagulant activity was verified by an in vivo DVT animal model system. These results suggest that serine protease purified from L. nipponica has the potential to be an alternative fibrinolytic agent for the treatment of thrombosis and use in various biomedical applications.
缺血性中风和心血管疾病可能由体内自然形成的纤维蛋白凝块阻塞血管引起。人们已经对抗凝剂或溶栓剂等治疗药物进行了研究；然而，也有各种问题被报道，如副作用和疗效低等。因此，开发更有效、更安全的新候选药物是必要的。本研究的目的是评估从日本刺沙蚕（多毛纲）中纯化的丝氨酸蛋白酶的纤溶活性、抗凝作用及特性，以用于新型溶栓剂的研发。在本研究中，我们从日本刺沙蚕中分离并鉴定了一种新的纤溶丝氨酸蛋白酶。所鉴定的丝氨酸蛋白酶的N端序列为EAMMDLADQLEQSLN，与任何已知的丝氨酸蛋白酶均无同源性。纯化的丝氨酸蛋白酶大小为28 kDa，蛋白质纯化产率为12.7%。在50°C和pH 2.0时观察到最佳酶活性。纤维蛋白平板试验证实，纯化的丝氨酸蛋白酶的间接纤溶活性高于尿激酶型纤溶酶原激活剂（urokinase-PA），而导致出血副作用的直接纤溶活性相对较低。该丝氨酸蛋白酶对内皮细胞系未诱导任何细胞毒性。此外，通过体内深静脉血栓形成（DVT）动物模型系统验证了抗凝活性。这些结果表明，从日本刺沙蚕中纯化的丝氨酸蛋白酶有潜力成为治疗血栓形成的替代溶栓剂，并用于各种生物医学应用。
