title: Binding of hirudin to meizothrombin.
abstract: Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C-terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F-thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.
authors.affiliation: Biomedical Research Center, Immuno AG, Orth an der Donau, Austria.
authors.forename: B E
authors.initials: BE
authors.lastname: Fischer
authors.forename: U
authors.initials: U
authors.lastname: Schlokat
authors.forename: M
authors.initials: M
authors.lastname: Himmelspach
authors.forename: F
authors.initials: F
authors.lastname: Dorner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism; D006629:Hirudins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine; D011516:Prothrombin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D006629:Hirudins; D011994:Recombinant Proteins; D005344:Fibrinopeptide A; D010649:Phenylalanine; D011516:Prothrombin; D013917:Thrombin; C026812:meizothrombin