title: Evidence that both exosites on thrombin participate in its high affinity interaction with fibrin.
abstract: Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gammaA/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gammaA/gammaA-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gammaA/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gammaA/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gammaA/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gammaA/gamma'-fibrin, but not to gammaA/gammaA-fibrin; (b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gammaA/gamma'-fibrin via low affinity sites; and (c) exosite 2-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gammaA/gamma'-fibrin. First, the affinity of gamma-thrombin for gammaA/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gammaA/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1-directed ligands reduced alpha-thrombin binding to gammaA/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gammaA/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gammaA/gamma'-fibrin.
authors.affiliation: Department of Medicine, McMaster University and the Henderson Research Centre, Hamilton, Ontario L8V 1C3, Canada.
authors.forename: Caroline H
authors.initials: CH
authors.lastname: Pospisil
authors.forename: Alan R
authors.initials: AR
authors.lastname: Stafford
authors.forename: James C
authors.initials: JC
authors.lastname: Fredenburgh
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Weitz
mesh_terms: D001665:Binding Sites; D001669:Biochemical Phenomena; D001671:Biochemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
chemical_list: D008024:Ligands; D010455:Peptides; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin