Kinetics of the inhibition of human factor Xa by full-length and truncated recombinant tissue factor pathway inhibitor

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), 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 (k(on)) and association (k(off)) were (5.1+/-0.7)x10(6) M(-l.) s(-l) and (2.6+/-0.9)x10(-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 (66 s). The truncated forms of TFPI differ in that they have a 4-fold lower k(on) value but a similar dissociation rate constant. Therefore the inhibition constant, K-i, is 4-fold higher (0.2 nM) and the half-time to achieve equilibrium is prolonged to 250 s. The k(on) values of full-length and C-terminal-truncated TFPI, but not that of TFPI1-161, were found to decrease with increasing ionic strength.