title: Kinetics of the inhibition of human factor Xa by full-length and truncated recombinant tissue factor pathway inhibitor.
abstract: The inhibition equilibrium and kinetics of association and dissociation of the binding of three types of recombinant tissue factor pathway inhibitor (TFPI), namely full-length TFPI, C-terminal-truncated TFPI, and TFPI without the third Kunitz domain (TFPI1-161), to factor Xa have been measured. Formation and dissociation of the complexes were monitored by continuous measurement of the changes in the rate of hydrolysis of a peptidyl-p-nitroanilide substrate. Progress curves of product formation were fitted to a set of equations describing a one-step bimolecular inhibitory reaction in the presence of a competing substrate. For full-length TFPI the rate constants of association (kon) and dissociation (koff) were (5.1 +/- 0.7) x 10(6) M-1.s-1 and (2.6 +/- 0.9) x 10(-4)s-1 respectively. Thus, although the inhibition constant (50 pM) is far below the plasma concentration (2.5 nM) of TFPI, the half-time for transition to equilibrium in plasma is rather long (66s). The truncated forms of TFPI differ in that they have a 4-fold lower kon value but a similar dissociation rate constant. Therefore the inhibition constant, Ki, is 4-fold higher (0.2 nM) and the half-time to achieve equilibrium is prolonged to 250 s. The kon values of full-length and C-terminal-truncated TFPI, but not that of TFPI1-161, were found to decrease with increasing ionic strength.
authors.affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
authors.forename: T
authors.initials: T
authors.lastname: Lindhout
authors.forename: G
authors.initials: G
authors.lastname: Willems
authors.forename: R
authors.initials: R
authors.lastname: Blezer
authors.forename: H C
authors.initials: HC
authors.lastname: Hemker
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D006801:Humans; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D015951:Factor Xa