MUTATIONAL ANALYSIS OF ANTISTASIN, AN INHIBITOR OF BLOOD-COAGULATION FACTOR-XA DERIVED FROM THE MEXICAN LEECH HAEMENTERIA-OFFICINALIS

Antistasin is a Factor Xa inhibitor that is present in the salivary gl ands of the Mexican leech Haementeria officinalis. The antistasin prot ein 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 am ino 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 acid s in the reactive site for the inhibition of Factor Xa. A series of mu tants was constructed and expressed in Chinese hamster ovary (CHO) cel ls. In vitro chromogenic assays for Factor Xa show that domain I is su fficient for inhibition of Factor Xa. Domains II and III neither conta in 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 re active site loop structure.