Kinetics of the Inhibition of Tissue Factor-Factor Vila by Tissue Factor Pathway Inhibitor

Abstract

SummaryTissue factor-factor VIIa catalysed activation of factor X and factor IX is inhibited by the complex of tissue factor pathway inhibitor (TFPI) and factor Xa. At present, no information is available as to what extent the kinetics of complex formation between TFPI and factor Xa during factor X activation contribute to the overall rate of inactivation of the factor X converting complex. We have determined the kinetic parameters of the individual reactions, i. e. factor X activation, formation of the TFPI-factor Xa complex, and inactivation of tissue factor-factor VIIa by the TFPI-factor Xa complex. We modelled the overall reaction by assuming a two-step reaction: factor Xa generated by tissue factor-factor VIIa forms a reversible complex with TFPI and in the second step this complex forms a reversible quaternary complex with tissue factor- factor VIIa. The validity of the model was demonstrated by analysis of factor Xa generation curves in the presence of TFPI. Independently determined constants for factor X activation (kcat= 12 s-1, Km = 70 nM) and inhibition of tissue factor-factor VIIa by TFPI-factor Xa complex (rate constant of inhibition of 1.1 × 108 M-1s-1) were used. The association rate constant of the formation of the TFPI-factor Xa complex was estimated by fitting the model to the data. The rate constants of association of the complex between factor Xa and the variants full length TFPI, TFPI 1-247 and TFPI1-61 were very close to the values determined independently in a kinetic study on the inhibition of factor Xa in the presence of phospholipids, namely 3.4 × 106 M-1s-1, 0.4 × 106 M-1s-1 and 0.3 × 106 M-1s-1, respectively. These results indicate that the factor Xa-dependent inhibition of tissue factor-factor VIIa-catalysed factor X activation by TFPI can be adequately described by the two-step reaction sequence. We found that phospholipids (25 mol % phosphat-idylserine/75 mol % phospatidylcholine) increased the rate constant of association with factor Xa for full length TFPI, but not for the C-ter- minus truncated TFPI. Our results further indicate that optimal inhibition of tissue factor-factor VIIa activity is obtained with full length TFPI because of the higher rate of TFPI-factor Xa complex formation.

Regulation of Extrinsic Pathway Factor Xa Formation by Tissue Factor Pathway Inhibitor

“…All three sets of analyses consistently required values of k INH, VIIa⅐TF in excess of 10 8 M Ϫ1 ⅐s Ϫ1 for adequate description (Table I). Interestingly, the values for k INH,VIIa⅐TF determined by numerical analyses are in remarkable agreement with the same rate constant inferred from kinetic studies of VIIa⅐TF inhibition during X activation using picomolar concentrations of Xa and TFPI (22).…”

“…However, since inhibition of both factor Xa and VIIa⅐TF by physiological (nanomolar) concentrations of TFPI is not instantaneous, the regulation of this pathway is likely to be heavily influenced by the kinetics determining the individual steps. Although the individual reactions of TFPI have been studied by a number of groups, there is uncertainty as to which inhibition reaction corresponds to the rate-limiting step (21,22). In addition, the effects of TFPI on active factor Xa formation by the extrinsic pathway have not been experimentally documented.…”

“…Complex-Previous studies have relied on measurements of factor X activation to infer the kinetics of inhibition of VIIa⅐TF by the preformed Xa⅐TFPI binary complex (21,22). Because these measurements involve the generation of factor Xa in situ and usually require the use of low concentrations of Xa⅐TFPI relative to the overall equilibrium dissociation constant, it is difficult to distinguish the contribution of the kinetics of inhibition of VIIa⅐TF by preformed Xa⅐TFPI from the other reactions of TFPI.…”

Inhibition of Tissue Factor-Factor VIIa-catalyzed Factor X Activation by Factor Xa-Tissue Factor Pathway Inhibitor

“…The effect of TFPI on TF⅐FVIIa activity in the absence of FXa is negligible (21,22), implying that the true inhibitor of TF⅐FVIIa activity is the FXa⅐TFPI complex. The rate of complex formation of FXa and TFPI is enhanced by negatively charged phospholipids for full-length TFPI (TFPI FL ) but not for TFPI 1-161 , a truncated variant lacking the third Kunitz domain and the potential phospholipid binding C-terminal tail (16,23).Recently (24), we demonstrated that TFPI FL in complex with FXa has a much higher affinity for anionic phospholipid membranes compared with that of either protein alone. It is well recognized that the binding of blood coagulation enzymes as well as their cofactors and substrates to membranes containing anionic phospholipids may result in an immense increase of the catalytic efficiency of these enzymes.…”

“…The effect of TFPI on TF⅐FVIIa activity in the absence of FXa is negligible (21,22), implying that the true inhibitor of TF⅐FVIIa activity is the FXa⅐TFPI complex. The rate of complex formation of FXa and TFPI is enhanced by negatively charged phospholipids for full-length TFPI (TFPI FL ) but not for TFPI , a truncated variant lacking the third Kunitz domain and the potential phospholipid binding C-terminal tail (16,23).…”