title: Deciphering the structural elements of hirudin C-terminal peptide that bind to the fibrinogen recognition site of alpha-thrombin.
abstract: The C-terminal peptide of a hirudin acts as an anticoagulant by binding specifically to a noncatalytic (fibrinogen recognition) site of thrombin. This binding has been shown to shield five spatially distant lysines of the thrombin B-chain (Lys21, Lys65, Lys77, Lys106, and Lys107). It was also demonstrated that modification of the sequence of the hirudin C-terminal peptide invariably diminished its anticoagulant activity. The major object of this study is to investigate how the decreased activity of the modified hirudin C-terminal peptide is reflected by the change of its binding properties to these five lysines of thrombin. A synthetic peptide representing the last 12 C-terminal amino acids of hirudin (Hir54-65) was (1) truncated from both its N-terminal and its C-terminal ends, or (2) substituted with Gly along residues 57-62, or (3) chemically modified to add (sulfation at Tyr63) or abolish (Asp and Glu modification with carbodiimide/glycinamide) its negatively charged side chains. The binding characteristics of these peptides to thrombin were investigated by chemical methods, and their corresponding anticoagulant activities were studied. Our results demonstrated the following: (1) the anticoagulant activities of hirudin C-terminal peptides were quantitatively related to their abilities to shield the five identified lysines of thrombin. The most potent peptide was sulfated Hir54-65 (S-Hir54-65) with an average binding affinity to the five lysines of 120 nM. A heptapeptide (Hir54-60) also displayed anticoagulant activity and thrombin binding ability at micromolar concentrations. (2) All active hirudin C-terminal peptides regardless of their sizes and potencies were shown to be capable of shielding the five lysines of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Pharmaceuticals Research Laboratory, Ciba-Geigy Ltd., Basel, Switzerland.
authors.forename: J Y
authors.initials: JY
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D000925:Anticoagulants; D002851:Chromatography, High Pressure Liquid; D005340:Fibrinogen / Q000378:metabolism*; D006629:Hirudins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D010449:Peptide Mapping; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin
chemical_list: D000925:Anticoagulants; D006629:Hirudins; D010446:Peptide Fragments; D005340:Fibrinogen; D014357:Trypsin; D013917:Thrombin