It has long been known that medicinal leeches (Hirudo medicinalis) contain a substance with anticoagulant properties. In early studies, aqueous extracts and dry preparations from leeches containing minute amounts of the active substance were used to prevent blood from clotting. Only after the development of protein chemistry and the elucidation of the biochemistry of blood coagulation was the anticoagulant agent, hirudin, isolated and its chemical nature and mode of action clarified.The historical background of hirudin isolation as well as the individual steps of its biochemical and pharmacological characterization have been described in several reviews (36,68,70,72,74,79).
BIOCHEMISTRYHirudin is extracted from the homogenized heads of medicinal leeches and enriched by precipitation procedures followed by ion-exchange chromatography and gel filtration (2,65,98,137). Affinity chromatography on matrix-bound thrombin can be used to obtain highly purified hirudin preparations (139,140). The pure anticoagulant agent obtained is a carboxyhydrate-free single chain miniprotein containing 65 amino acids with a molecular weight of about 7 kDa. The amino acid composition is characterized by a remarkably high content of acidic amino acids at the C-terminal, the absence of arginine, methionine, and tryptophan, and the presence of a sulfated tyrosine residue and three intramolecular disulfide bridges (62,65,70).Considerable progress in the isolation, purification, and analysis of hirudin has shown the medicinal leech generates several variants of the miniprotein in trace amounts. The isoinhibitor forms share 85-90% amino acid sequence homology. Their main structural elements, which include N-terminal hydrophobic amino acids, followed by a core region linked by three disulfide bridges and the unique clustering of acidic amino acid residues in the C-terminal portion remain constant (4,17,18,113). The N-terminal amino acids Val-Val can be replaced by Ile-Thr (Fig. 1).