Molecular basis for the inhibition of thrombin by hirudin.

Abstract

The amino acid sequence of the 65 residue major form of hirudin is shown in Figure 1. Hirudin was originally isolated from the salivary glands of the European medicinal leech Hirudo medicinalis by Markwardt1. The anticoagulant properties of leech saliva were, however, first described over 100 years ago by Haycraft2. The N-terminal region of the hirudin contains 6 cysteines that form three disulphide bridges: Cys6′-Cysl4′, Cys16′-Cys28′ and Cys22′-Cys39′. (Residues in hirudin are distinguished from those in thrombin by the use of primed numbers and the numbering of the sequence of thrombin is that of Bode et al.3 which is based on chymotrypsin numbering). The C-terminal region of hirudin is particularly rich in acidic residues (Figure 1) and the acidic nature of this region of hirudin is important for the formation of the thrombin-hirudin complex as will be discussed later. One of the charged residues found in this region of the natural hirudin is a sulphated tyrosine residue at position 63. This post-translational modification is not, however, found in recombinant molecules expressed in Escherichia coli or yeast. More than 20 different isoforms of hirudin have been isolated from H. medicinalis and sequenced4–6 and in each of these isoforms the position of the disulphide bridges and the acidic nature of the C-terminal region is conserved. The residues that are conserved between the different isoforms are indicated in Figure 1 and many of these residues are involved in the binding of hirudin to thrombin7–9.