Abstract
Hirudin, a thrombin-specific inhibitor, comprises a compact amino-terminal core domain (residues 1-52) and a disordered acidic carboxyl-terminal tail (residues 53-65). An array of core fragments were prepared from intact recombinant hirudin by deletion of various lengths of its carboxyl-terminal tail on selective enzymatic cleavage. Hir1-56 and Hir1-53 were produced by pepsin digestion at Phe56-Glu57 and Asp53-Gly54. Hir1-52 was generated by Asp-N cleavage at Asn52-Asp53. Hir1-49 was prepared by cleavage of Gln49-Ser50 by chymotrypsin, elastase, and thermolysin. In addition, Hir1-62 (containing part of the carboxyl-terminal tail) was derived from Hir1-65 by selective removal of the three carboxyl-terminal amino acids using carboxypeptidase A. Hirudin amino-terminal core fragments were stable at extreme pH (1.47 and 12.6), high temperature (95 degrees C), and resistant to attack by various proteinases. For instance, following 24-h incubation with an equal weight of pepsin, the covalent structure of Hir1-52 remained intact and its anticoagulant activity unaffected. Unlike intact hirudin (Hir1-65) the inhibitory potency of which is a consequence of concerted binding of its amino-terminal and carboxyl-terminal domains to the active site and the fibrinogen recognition site of thrombin, the core fragments block only the active site of thrombin with binding constants of 19 nM (Hir1-56), 35 nM (Hir1-52), and 72 nM (Hir1-49). As an anticoagulant Hir1-56 is about 2-, 4-, and 30-fold more potent (on a molar basis) than Hir1-52, Hir1-49, and Hir1-43, respectively. Hir1-56 was also about 15-fold more effective than the most potent carboxyl-terminal fragment of hirudin, sulfated-Hir54-65, although they bind to independent sites on thrombin. The potential advantages of hirudin core fragments as antithrombotic agents are discussed in this report.
Highlights
Production of Hirudin Amino-terminal Core Fragments-Hirudin core fragments were produced from intact recombinant hirudin by selective cleavage at the hinge region of its two functional domains (Fig. 1)
It has been proposed that hirudin contains two functional domains (Chang, 1983a, 1986; Markwardt, 1985) that bind to different sites in thrombin (Fenton, 1981)
The amino-terminal core fragments of hirudin were isolated and demonstrated to bind to the active site of thrombin (Chang et al, 1990a; Dennis et al, 1990; Dodt et al, 1990). These findings were subsequently verified by x-ray analysis of the hirudin-thrombin complex (Rydel et al, 1990; Gruetter et al, 1990)
Summary
A thrombin-specific inhibitor, comprises a compact amino-terminal core domain (residues l-52). (b) Unlike most serine protease inhibitors which are compact molecules, hirudin has a tadpole-like shape, consisting of a compact amino-terminal domain and a disordered carboxyl-terminal tail (Chang, 1983a; Folkers et al, 1989). In fragment form, both domains act as anticoagulants but through different mechanisms (Krstenansky and Mao, 1987; Mao et al, 1988; Chang et al, 1990a, 199Ob; Dennis et al, 1990; Dodt et al, 1990). One major goal of this study is to tailor a derivative of hirudin which is resistant to digestive enzymes
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