Isolation and characterization of americanin, a specific inhibitor of thrombin, from the salivary glands of the lone star tick Amblyomma americanum (L.)

A thrombin (EC 3.4.21.5) inhibitor (americanin) was isolated from the salivary glands of the lone star tick Amblyomma americanum (L.) using reversed-phase chromatography and anion-exchange chromatography. Americanin did not inhibit any other protease tested, including factor Xa, plasmin, trypsin, chymotrypsin, elastase, papain, pepsin, and carboxypeptidase. The inhibition of thrombin by americanin decreased dramatically with dilution of the reaction mixture including thrombin, its substrate, and americanin. When thrombin assays were performed in the presence of americanin, the reaction curve showed a time-dependent inhibition. Significant inhibition was observed when americanin concentration was approximately equal to that of thrombin, with a Ki of 0.073 nM. The results suggest that americanin is a specific, reversible, competitive, slow, tight-binding inhibitor of thrombin.