Dunwiddie C, Thornberry N A, Bull H G, Sardana M, Friedman P A, Jacobs J W, Simpson E
Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
J Biol Chem. 1989 Oct 5;264(28):16694-9.
Antistasin is a 119-amino acid protein isolated from the salivary glands of the Mexican leech Haementeria officinalis. The determination of the primary structure of antistasin revealed that the protein is highly disulfide-bonded with a 2-fold internal homology. Antistasin exhibits a potent anticoagulant activity purportedly due to the selective inhibition of Factor Xa (Tuszinsky, G. P., Gasic, T. B., and Gasic, G. J. (1987) J. Biol. chem. 262, 9718-9723). In the present study a detailed kinetic analysis of the inhibitory interaction between antistasin and Factor Xa was performed. In addition, the specificity of antistasin was examined by testing its ability to inhibit a variety of serine proteinases. Utilizing purified antistasin and a tripetidyl p-nitroanilide substrate, antistasin was shown to act as a reversible inhibitor of Factor Xa which exhibits slow-tight binding kinetics. Antistasin reacts stoichiometrically with Factor Xa with inhibition displaying a mixed, primarily competitive type. The inhibition is partial in the presence of Ca2+ and becomes complete in the absence of Ca2+. The estimated dissociation constant for the enzyme-inhibitor complex is between 0.31 and 0.62 nM. After binding to Factor Xa, antistasin is cleaved at a single site to yield a modified inhibitor. Automated gas-phase sequence analysis of the modified inhibitor indicates the arginine residue at position 34 in antistasin occupies the P1 position of the reactive site. These data indicate that the leech has evolved a highly selective and potent inhibitor of coagulation Factor Xa that shares several mechanistic similarities with other serine proteinase inhibitors.
抗凝血酶抑制剂是一种从墨西哥药用水蛭（Haementeria officinalis）唾液腺中分离出的由119个氨基酸组成的蛋白质。抗凝血酶抑制剂一级结构的测定表明，该蛋白质高度二硫键结合，具有2倍的内部同源性。抗凝血酶抑制剂表现出强大的抗凝血活性，据推测这是由于它对Xa因子的选择性抑制作用（图辛斯基，G.P.，加西克，T.B.，和加西克，G.J.（1987年）《生物化学杂志》262卷，9718 - 9723页）。在本研究中，对抗凝血酶抑制剂与Xa因子之间的抑制相互作用进行了详细的动力学分析。此外，通过测试其抑制多种丝氨酸蛋白酶的能力来检验抗凝血酶抑制剂的特异性。利用纯化的抗凝血酶抑制剂和三肽对硝基苯胺底物，结果表明抗凝血酶抑制剂作为Xa因子的可逆抑制剂，呈现缓慢紧密结合动力学。抗凝血酶抑制剂与Xa因子按化学计量反应，抑制作用表现为混合型，主要是竞争性类型。在有Ca2 +存在时抑制作用不完全，而在无Ca2 +时则完全抑制。酶 - 抑制剂复合物的估计解离常数在0.31至0.62 nM之间。与Xa因子结合后，抗凝血酶抑制剂在单个位点被切割，产生一种修饰的抑制剂。对修饰抑制剂的自动气相序列分析表明，抗凝血酶抑制剂中第34位的精氨酸残基占据反应位点的P1位置。这些数据表明，水蛭进化出了一种对凝血因子Xa具有高度选择性和强效的抑制剂，它与其他丝氨酸蛋白酶抑制剂在几个机制上具有相似性。
