Antistasin, a leech-derived inhibitor of factor Xa. Kinetic analysis of enzyme inhibition and identification of the reactive site.

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.