title: Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII.
abstract: Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
authors.forename: M C
authors.initials: MC
authors.lastname: Naski
authors.forename: L
authors.initials: L
authors.lastname: Lorand
authors.forename: J A
authors.initials: JA
authors.lastname: Shafer
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects; D005337:Fibrin / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000737:chemistry; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*
chemical_list: D006629:Hirudins; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010455:Peptides; C061895:factor XIII activation peptide; C062510:hirugen; D005345:Fibrinopeptide B; D005337:Fibrin; D013917:Thrombin