Theunissen H J, Dijkema R, Swinkels J C, de Poorter T L, Vink P M, van Dinther T G
Department of Biotechnology and Biochemistry, N. V. Organon, Oss, The Netherlands.
Thromb Res. 1994 Jul 1;75(1):41-50. doi: 10.1016/0049-3848(94)90138-4.
Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.
抗凝血酶是一种Xa因子抑制剂，存在于墨西哥水蛭药用南美水蛭的唾液腺中。抗凝血酶蛋白由119个氨基酸组成，其中第1至55位残基（结构域I）与第56至110位残基（结构域II）有56%的相似性。在九个C端氨基酸（第111至119位残基；结构域III）中，有四个带正电荷。Xa因子的反应位点位于结构域I中。在本研究中，我们评估了各个结构域和反应位点中单个氨基酸对抑制Xa因子的作用。构建了一系列突变体并在中国仓鼠卵巢（CHO）细胞中表达。Xa因子的体外显色分析表明，结构域I足以抑制Xa因子。结构域II和III既不具有任何内在的Xa因子抑制活性，也不影响结构域I的活性。此外，结构域II不会通过部分使其序列适应结构域I中反应位点的序列而成为Xa因子抑制剂。第33位半胱氨酸的突变对Xa因子抑制并不关键，这表明反应位点环结构相对刚性。
