Nishida S, Fujimura Y, Miura S, Ozaki Y, Usami Y, Suzuki M, Titani K, Yoshida E, Sugimoto M, Yoshioka A
Department of Blood Transfusion, Nara Medical College, Japan.
Biochemistry. 1994 Feb 22;33(7):1843-9. doi: 10.1021/bi00173a030.
A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)
从巴西矛头蝮蛇毒中纯化出一种纤维蛋白原凝血酶（双蝰蛇凝血酶）。在SDS聚丙烯酰胺凝胶电泳中，双蝰蛇凝血酶在非还原条件下的M(r)值为33,000，在还原条件下为35,000，其纤维蛋白原凝血活性相当于814 - 904 NIHα-凝血酶单位/毫克。二异丙基氟磷酸完全消除其活性，但水蛭素（一种特异性α-凝血酶抑制剂）对双蝰蛇凝血酶活性影响可忽略不计。与α-凝血酶不同，双蝰蛇凝血酶可裂解掉纤维蛋白肽A而不释放纤维蛋白肽B。双蝰蛇凝血酶可激活血液凝固因子VIII，但其活性约比α-凝血酶低950倍。双蝰蛇凝血酶自身不会诱导洗涤后的正常血小板聚集或释放5-羟色胺，但在外源纤维蛋白原存在时可使血小板聚集。后一种活性可被抗糖蛋白（GP）IIb/IIIa单克隆抗体（阻断纤维蛋白原与GP IIb/IIIa结合）或抗GP Ib单克隆抗体（特异性抑制α-凝血酶与GP Ib结合）完全抑制。前列腺素E1（1 microM）和EDTA（10 mM）也可消除血小板聚集而不影响凝血活性。来自一名伯纳德-索利尔综合征患者的洗涤血小板即使在外源纤维蛋白原存在时也对双蝰蛇凝血酶无反应，这表明双蝰蛇凝血酶在血小板上的初始结合位点是GP Ib，而非最近克隆的凝血酶受体。通过分别分析（S）-吡啶基乙基化蛋白以及用溴化氰和无色杆菌蛋白酶I消化产生的肽段，确定了双蝰蛇凝血酶的完整氨基酸序列。双蝰蛇凝血酶由232个氨基酸残基组成，含有三条天冬酰胺连接的寡糖链。（摘要截短于250字）
