Department of Biotechnology, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea.
Biochimie. 2013 Jun;95(6):1266-77. doi: 10.1016/j.biochi.2013.01.023. Epub 2013 Feb 9.
Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. In this study, codiase, a new bi-functional fibrinolytic serine protease having thrombolytic, anticoagulant, and antiplatelet activities was purified from marine green alga, Codium fragile. The molecular weight of the enzyme was estimated to be 48.9 kDa by SDS-PAGE, and mass spectrometry. Fibrin zymography analysis showed an active band with similar molecular weight. The N-terminal sequence was found to be APKASTDQTLPL, which is different from that of other known fibrinolytic enzymes. Codiase displayed maximum activity at 30 °C and pH 6.0, and the activity was inhibited by Zn(2+) and Fe(2+). Moreover, the enzyme activity was strongly inhibited by serine protease inhibitor such as PMSF. Codiase exhibited high specificity for the substrate S-2288, and the Km and Vmax values for this substrate were found to be 0.24 mM and 79 U/ml respectively. Fibrin plate assays revealed that it was able to hydrolyze fibrin clot either directly or by activation of plasminogen. Codiase effectively hydrolyzed fibrin and fibrinogen, preferentially degrading α- and Aα chains, followed by γ-γ, and γ-chains. However, it provoked slower degradation of Bβ and β-chains. The structural change of fibrin clot and fibrinogen by codiase was also detected by FTIR-ATR spectroscopy analysis. In vitro and in vivo studies revealed that codiase reduces thrombosis in concentration-dependent manner. Codiase was found to prolong activated partial thromboplastin time (APTT), and prothrombin time (PT). PFA-100 studies showed that codiase prolonged the closure time (CT) of citrated whole human blood. These favorable antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to mice and NIH-3T3 cells, make it a potential agent for thrombolytic therapy.
血栓形成是全世界发病率和死亡率的主要原因。溶栓剂对于血栓形成的预防和治疗都很重要。在这项研究中，从海洋绿藻脆江蓠中纯化得到了一种新型双功能纤维蛋白溶酶丝氨酸蛋白酶 codiase，具有溶栓、抗凝和抗血小板作用。SDS-PAGE 和质谱法估计该酶的分子量为 48.9 kDa。纤维蛋白酶谱分析显示具有相似分子量的活性带。N 端序列被发现为 APKASTDQTLPL，与其他已知的纤维蛋白溶解酶不同。Codiase 在 30°C 和 pH 6.0 时显示最大活性，并且活性被 Zn(2+)和 Fe(2+)抑制。此外，该酶的活性被丝氨酸蛋白酶抑制剂如 PMSF 强烈抑制。Codiase 对 S-2288 底物表现出高特异性，Km 和 Vmax 值分别为 0.24 mM 和 79 U/ml。纤维蛋白平板测定表明，它能够直接或通过激活纤溶酶原水解纤维蛋白凝块。Codiase 有效地水解纤维蛋白和纤维蛋白原，优先降解α-和 Aα链，然后是γ-γ和γ-链。然而，它引起 Bβ和β-链的降解速度较慢。FTIR-ATR 光谱分析也检测到 codiase 引起的纤维蛋白凝块和纤维蛋白原的结构变化。体外和体内研究表明，codiase 以浓度依赖的方式减少血栓形成。发现 codiase 延长活化部分凝血活酶时间（APTT）和凝血酶原时间（PT）。PFA-100 研究表明，codiase 延长柠檬酸化全人血的闭合时间（CT）。这些有利的抗血栓形成谱，以及其抗凝和血小板解聚特性，以及对小鼠和 NIH-3T3 细胞没有毒性，使其成为溶栓治疗的潜在药物。
