title: Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate.
abstract: Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.
authors.affiliation: Department of Haematology, Imperial College London, United Kingdom. medhp@medphysics.leeds.ac.uk
authors.forename: Helen
authors.initials: H
authors.lastname: Philippou
authors.forename: James
authors.initials: J
authors.lastname: Rance
authors.forename: Timothy
authors.initials: T
authors.lastname: Myles
authors.forename: Scott W
authors.initials: SW
authors.lastname: Hall
authors.forename: Robert A
authors.initials: RA
authors.lastname: Ariens
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Grant
authors.forename: Lawrence
authors.initials: L
authors.lastname: Leung
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D001667:Binding, Competitive; D004789:Enzyme Activation; D005176:Factor XIII / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
chemical_list: D005176:Factor XIII; D013917:Thrombin