title: The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen.
abstract: Hirudin, a potent 65-residue polypeptide inhibitor of alpha-thrombin found in the saliva of the leech Hirudo medicinalis, and fragments thereof are potentially useful as antithrombotic agents. Hirugen, the synthetic N-acetylated COOH-terminal dodecapeptide (Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3)-Leu) of hirudin was shown in the present study to behave as a pure competitive inhibitor (Ki = 0.54 microM) of human alpha-thrombin-catalyzed release of fibrinopeptide A from human fibrinogen. In contrast to this inhibitory activity, hirugen slightly enhanced (increased kcat/Km 1.6-fold) alpha-thrombin-catalyzed hydrolysis of the fluorogenic tripeptide substrate N-p-Tosyl-Gly-Pro-Arg-7-amino-4-methylcoumarin. These observations indicate that hirugen binds to alpha-thrombin at an exosite distinct from the active site, and that interaction with this exosite is a major determinant of the competence of alpha-thrombin to bind fibrinogen. Consistent with this view, hirugen blocked binding of fibrin II to alpha-thrombin. Studies of the effect of hirugen on the rate of inactivation of alpha-thrombin by antithrombin III (AT), the major plasma inhibitor of alpha-thrombin, indicated that binding of hirugen to alpha-thrombin results in less than a 2.5-fold decrease in the rate of inactivation of alpha-thrombin by AT, both in the absence and presence of heparin. This behavior is distinct from that of active site-directed competitive inhibitors of alpha-thrombin which bind to alpha-thrombin and block both conversion of fibrinogen to fibrin and inactivation of alpha-thrombin by AT. Hirugen, an exosite-directed competitive inhibitor, blocks the interaction of alpha-thrombin with fibrinogen while leaving alpha-thrombin competent to react with AT. Thus, unlike active site-directed competitive inhibitors, hirugen should act in concert with AT and heparin to reduce the amount of fibrinogen that is processed during the lifetime of alpha-thrombin in plasma.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
authors.forename: M C
authors.initials: MC
authors.lastname: Naski
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
authors.forename: J M
authors.initials: JM
authors.lastname: Maraganore
authors.forename: S T
authors.initials: ST
authors.lastname: Olson
authors.forename: J A
authors.initials: JA
authors.lastname: Shafer
mesh_terms: D000595:Amino Acid Sequence; D005340:Fibrinogen / Q000378:metabolism*; D006629:Hirudins / Q000138:chemical synthesis / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D006629:Hirudins; D009842:Oligopeptides; D005340:Fibrinogen; D013917:Thrombin