Kinetics of the Inhibition of Factor Xa and the Tissue Factor-Factor VIIa Complex by the Tissue Factor Pathway Inhibitor in the Presence and Absence of Heparin

Biochemistry ◽  
1994 ◽  
Vol 33 (42) ◽  
pp. 12686-12694 ◽  
Author(s):  
Jolyon Jesty ◽  
Tze-Chein Wun ◽  
Ann Lorenz
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Tissue Factor Pathway ◽  
Presence And Absence ◽  
Kinetics Of

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

1995 ◽  
Vol 74 (03) ◽  
pp. 910-915 ◽  
Author(s):  
Theo Lindhout ◽  
Jo Franssen ◽  
George Willems
Keyword(s):  
Tissue Factor ◽  
Rate Constant ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Full Length ◽  
Factor X ◽  
Tissue Factor Pathway ◽  
Factor X Activation ◽  
Kinetics Of

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.

Protamine Neutralization of the Release of Tissue Factor Pathway Inhibitor Activity by Heparins

1993 ◽  
Vol 70 (06) ◽  
pp. 0942-0945 ◽  
Author(s):  
Job Harenberg ◽  
Marietta Siegele ◽  
Carl-Erik Dempfle ◽  
Gerd Stehle ◽  
Dieter L Heene
Keyword(s):  
Tissue Factor ◽  
Binding Sites ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Factor X ◽  
Rebound Phenomenon ◽  
Tissue Factor Pathway ◽  
Kinetics Of

SummaryThe present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa- activity were taken, in order to detect a possible relation between the remaining anti-factor X a activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs.There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamineIt is assumed that protamine displaces heparins from the binding sites of TFPI. There were no differences between UF- and LMW-heparin. The data indicate that the sustained antifactor Xa activity after antagonization of LMW-heparins as well as heparin rebound phenomena are not mediated by TFPI activity.

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

1994 ◽  
Vol 297 (1) ◽  
pp. 131-136 ◽  
Author(s):  
T Lindhout ◽  
G Willems ◽  
R Blezer ◽  
H C Hemker
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Product Formation ◽  
Dissociation Rate ◽  
Full Length ◽  
Inhibition Constant ◽  
Half Time ◽  
Tissue Factor Pathway ◽  
Kinetics Of

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.

scholarly journals Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

2013 ◽  
Vol 289 (3) ◽  
pp. 1732-1741 ◽  
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Markus Fries ◽  
M. Christella L. G. D. Thomassen ◽  
Alexandra Heinzmann ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Tight Binding ◽  
Factor Xa ◽  
Factor X ◽  
Intermediate Segment ◽  
Tissue Factor Pathway ◽  
Α Helix

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

Localization of tissue factor pathway inhibitor to lipid rafts is not required for inhibition of factor VIIa/tissue factor activity

2003 ◽  
Vol 89 (01) ◽  
pp. 65-73 ◽  
Author(s):  
Garnet Jack ◽  
Keith Page ◽  
Tina Tetzloff ◽  
Connie Hall ◽  
Alan Mast ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tissue Factor ◽  
Lipid Rafts ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Hek293 Cells ◽  
Membrane Domains ◽  
Gpi Anchor ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) abrogates coagulation initiated by the factor VIIa/tissue factor catalytic complex. While the gene structure of TFPI suggests that it is a secreted protein, a large pool of TFPI is associated with the vascular endothelium through its affinity for a glycosylphosphatidylinositol (GPI)-linked membrane protein. Inhibition of tissue factor by TFPI coincides with the translocation of quaternary complexes containing tissue factor, factor VIIa, factor Xa, and TFPI to detergent-insoluble plasma membrane domains rich in cholesterol, sphingomyelin, and GPI-linked proteins known as lipid rafts and caveolae. It is not known if localization of TFPI to these membrane domains is required for its inhibition of tissue factor procoagulant activity. We generated chimeric TFPI molecules linked directly to the plasma membrane via a GPI anchor or hydrophobic transmembrane domain and expressed these in HEK293 cells that produce tissue factor but not endogenous TFPI. The GPI-anchored chimera was exclusively enriched in detergent-insoluble membrane fractions while the transmembrane molecule was not. Transfectants expressing equal levels of the GPI-linked or transmembrane TFPI displayed equal anticoagulant potency as assessed by tissue factor-mediated conversion of factor X to factor Xa. Disruption of lipid rafts with cyclodextrin likewise had no effect on the inhibitory activity of the transmembrane or GPI-linked TFPI chimeras in HEK293 cells, nor on endogenous TFPI expressed by ECV304 cells. Thus, we conclude that the GPI anchor and membrane localization to lipid rafts does not enhance inhibition of factor VIIa/ tissue factor by cell-surface associated TFPI.

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

Blood ◽  
2011 ◽  
Vol 117 (20) ◽  
pp. 5514-5522 ◽  
Author(s):  
Emily K. Waters ◽  
Ryan M. Genga ◽  
Michael C. Schwartz ◽  
Jennifer A. Nelson ◽  
Robert G. Schaub ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Intrinsic Pathway ◽  
Extrinsic Pathway ◽  
Tissue Factor Pathway

Abstract Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

scholarly journals Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2004-2010 ◽  
Author(s):  
R Wesselschmidt ◽  
K Likert ◽  
T Girard ◽  
TC Wun ◽  
GJ Jr Broze
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Reversible Inhibitor ◽  
Normal Plasma ◽  
Encounter Complex ◽  
Factor Xa Inhibition ◽  
Tissue Factor Pathway ◽  
Inhibit Factor

Abstract Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N- terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254–265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1–252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.

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

1999 ◽  
Vol 274 (40) ◽  
pp. 28225-28232 ◽  
Author(s):  
Irene Salemink ◽  
Jo Franssen ◽  
George M. Willems ◽  
H. Coenraad Hemker ◽  
Theo Lindhout
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor X ◽  
Tissue Factor Pathway ◽  
Factor X Activation

scholarly journals Catabolism of Factor VIIa Bound to Tissue Factor in Fibroblasts in the Presence and Absence of Tissue Factor Pathway Inhibitor

1999 ◽  
Vol 274 (52) ◽  
pp. 36995-37003 ◽  
Author(s):  
Alexei Iakhiaev ◽  
Usha R. Pendurthi ◽  
Jason Voigt ◽  
Mirella Ezban ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Tissue Factor Pathway ◽  
Presence And Absence
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