Isolation of thrombin inhibitor from the leech Hirudinaria manillensis

The leech Hirudinaria manillensis belongs to the same family as the medicinal leech Hirudo medicinalis, which has been widely used for the study of hirudin, a specific thrombin inhibitor. A similar inhibitor has now been isolated from the heads of the Hirudinaria leech by acetone/acid extraction and further purified to near homogeneity by ion exchange chromatography followed by affinity chromatography on thrombin-agarose and reverse phase HPLC. The purified material was recovered at about 10-15% yield and had a specific activity of about 12,000-14,000 ATU/mg, similar to other hirudin variants. The inhibitor was shown to be homogenous by sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of 8 M urea with an apparent molecular mass of about 7000 daltons under reducing conditions. Comparison of the anticoagulant effect on human plasma by partial thromboplastin time assay have shown that the inhibitor from Hirudinaria has similar potency as hirudin variant 1 at equivalent dosage. However, it does not cross-react with monoclonal antibodies towards recombinant hirudin variant 1. Comparison of the N-terminal amino acid sequence up to residue 25 also indicates differences at positions 2, 13, 17 and 24 between the two thrombin inhibitors. These findings indicate that the primary anticoagulant present in the leech Hirudinaria is a potent thrombin inhibitor (Bufrudin) with biological activity similar to hirudin, but differs in its structural and immunological properties.