Production, properties, and thrombin inhibitory mechanism of hirudin amino-terminal core fragments.
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.
Chang
J Y
JY
Pharmaceuticals Research Laboratories, Ciga-Geigy Ltd., Bssel, Switzerland.
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D005340:Fibrinogen / Q000378:metabolism; D006629:Hirudins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*
D006629:Hirudins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005340:Fibrinogen; D013917:Thrombin