Tang Song-Shan, Wang Xiao-Hua, Zhang Juan-Hui, Tang Bo-Shan, Qian Li, Li Pei-Ying, Luo Lie-Wei
Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Department of Biochemistry, Guangzhou Medical College, Guangzhou 510009, China.
Eur J Pharm Sci. 2013 Apr 11;49(1):90-8. doi: 10.1016/j.ejps.2013.02.002. Epub 2013 Feb 18.
A number of snake venom thrombin-like enzymes (TLEs) have already been characterized. Some TLEs play significant roles in vessel injury hemostasis. A novel TLE (Agacutase) was purified from Deinagkistrodon acutus snake venom by the means of Sephadex G-75, DEAE-Sepharose FF, and Sephadex G-25 column chromatography. Structural analysis indicated that Agacutase is a single-chain glycoprotein with a molecular mass of 31,084 Da, isoelectric point of 4.38, optimal activity at 37 °C and pH 6.6, sugar content of 7.6%. Its N-terminal 44 amino acid sequence was determined to be VIGGNECDTNEHRFLAAFFTSRPWIFQCAGTLIHEEWVLAAAHC, showing maximum identity of 80% with that of Dav-X protease. The Agacutase-induced clotting activity was not influenced by heparin, hirudin, or Dextran 40, but activated by Ca(2+) and inhibited by PMSF or lactose, which suggests that Agacutase is a serine protease and the coagulation activity is independent of Thrombin. Agacutase with arginine esterase activity specifically cleaves the α-chain of fibrinogen. Agacutase iv (0.03-0.12 U/kg) shortened 16-68% of the rabbit blood clotting time. No significant influence was indicated on platelet, Factor II and XIII, or fibrinolytic system. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound. Pharmacological comparison showed the hemostatic effect of Agacutase lasted 24h while Reptilase did 8h. Its maximum tolerated, abnormal toxicity, allergic, and hemorrhagin doses were 80 U/kg, 1 U, 2 U, and 50 U, respectively, whereas those of Reptilase or Agacutin were 35 U/kg, 0.25 U, 0.25 U, and 0.2 U, respectively. The results indicated that Agacutase may be a predominant coagulant.
多种蛇毒类凝血酶（TLEs）已被鉴定。一些TLEs在血管损伤止血中发挥重要作用。通过Sephadex G - 75、DEAE - Sepharose FF和Sephadex G - 25柱色谱法从尖吻蝮蛇毒中纯化出一种新型TLE（尖吻蝮蛇凝血酶）。结构分析表明，尖吻蝮蛇凝血酶是一种单链糖蛋白，分子量为31,084 Da，等电点为4.38，在37℃和pH 6.6时活性最佳，糖含量为7.6%。其N端44个氨基酸序列被确定为VIGGNECDTNEHRFLAAFFTSRPWIFQCAGTLIHEEWVLAAAHC，与Dav - X蛋白酶的序列显示出80%的最大同源性。尖吻蝮蛇凝血酶诱导的凝血活性不受肝素、水蛭素或右旋糖酐40的影响，但受Ca(2+)激活，受PMSF或乳糖抑制，这表明尖吻蝮蛇凝血酶是一种丝氨酸蛋白酶，其凝血活性独立于凝血酶。具有精氨酸酯酶活性的尖吻蝮蛇凝血酶特异性切割纤维蛋白原的α链。静脉注射尖吻蝮蛇凝血酶（0.03 - 0.12 U/kg）可使兔凝血时间缩短16 - 68%。对血小板、因子II和XIII或纤溶系统无显著影响。它将纤维蛋白原转化为可溶性纤维蛋白，加速伤口止血。药理学比较显示，尖吻蝮蛇凝血酶的止血作用持续24小时，而巴曲酶为8小时。其最大耐受量、异常毒性、过敏和出血剂量分别为80 U/kg、1 U、2 U和50 U，而巴曲酶或尖吻蝮蛇血凝酶的相应剂量分别为35 U/kg、0.25 U、0.25 U和0.2 U。结果表明，尖吻蝮蛇凝血酶可能是一种主要的促凝剂。
