title: Thrombin exosite for fibrinogen recognition is partially accessible in prothrombin.
abstract: The procoagulant alpha-thrombin is produced by the proteolytic cleavages of a minimum of two peptide bonds Arg274-Thr275 and Arg323-Ile324 in prothrombin. The Arg323-Ile324 cleavage is required for the expression of the active site of thrombin (Morita, T., Iwanaga, S. Suzuki, T. (1976) J. Biochem. (Tokyo) 79, 1089-1108; Hibbard, L. S., Nesheim, M. E., and Mann, K. G. (1982) Biochemistry 21, 2285-2292). It is not yet clear to what extent the proteolytic events are responsible for exposing protein recognition exosites on thrombin. We employed high resolution NMR spectroscopy to examine interactions of prothrombin and thrombin with synthetic hirudin peptides targeted toward the fibrinogen recognition exosite of thrombin. The hirudin tail synthetic analogues (acetyl-Asp55-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln65/G ly65-OH) exhibited similar NMR relaxation enhancements (line broadening patterns and transferred nuclear Overhauser effects) with human prothrombin as with human alpha-thrombin, indicating that both proteins bind the peptide in a similar manner. The protein-induced relaxation enhancements are specific to the interaction of the hirudin peptides with the fibrinogen recognition exosite of thrombin since no significant effects were observed with either human serum albumin or with human gamma-thrombin, which has an impaired recognition exosite. The binding affinities were determined from NMR relaxation time measurements, which gave approximate Kd values of 500 microM and < 100 microM for prothrombin and alpha-thrombin, respectively. Since the hirudin tail fragment binds specifically to the fibrinogen recognition exosite in alpha-thrombin, this exosite appears to be partially accessible in prothrombin in a proenzyme form.
authors.affiliation: Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
authors.forename: F
authors.initials: F
authors.lastname: Ni
authors.forename: Q
authors.initials: Q
authors.lastname: Ning
authors.forename: C M
authors.initials: CM
authors.lastname: Jackson
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005340:Fibrinogen / Q000378:metabolism*; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011516:Prothrombin / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
chemical_list: D006629:Hirudins; D011516:Prothrombin; D005340:Fibrinogen; D013917:Thrombin