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.

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.

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.

Recombinant Tissue Factor Pathway Inhibitor Enhances the Binding of Factor Xa to Human Monocytes

2001 ◽  
Vol 85 (05) ◽  
pp. 830-836
Author(s):  
Anguo Li ◽  
Alvin Chang ◽  
Glenn Peer ◽  
Tze-Chen Wun ◽  
Fletcher Taylor
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Factor Xa ◽  
Human Monocytes ◽  
Factor X ◽  
Peripheral Blood Mononuclear ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a kunitz-type inhibitor of activated factor X (Xa). TFPI was reported to mediate Xa binding to a few of carcinoma cell lines. In this study it was observed that the Xa activity associated with human peripheral blood mononuclear cells (PBMC) incubated with Xa in the presence of recombinant TFPI (rTFPI) was much higher than with Xa alone. Xa activity on PBMC was also observed after whole blood was incubated with pre-formed Xa/TFPI complex. Further studies with flow cytometric analysis demonstrate that rTFPI enhances the binding of Xa to human monocytes. Western blot analysis showed that rTFPI was cleaved into a few of fragments after its incubation with monocytes either in the presence or absence of Xa. Based on these results and the observations reported by others, we speculate that Xa/TFPI complex may bind to human monocytes by a yet unidentified mechanism. The recovery of Xa activity from Xa/TFPI complex on PBMC may be related to the cleavage of rTFPI by Xa and/or monocyte proteases. This observation suggests a new mechanism by which monocytes become procoagulant in some pathological conditions in addition of the well known tissue factor expression on proinflammatic monocytes.

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.

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 Factor Xa Cleavage of Tissue Factor Pathway Inhibitor Is Associated with Loss of Anticoagulant Activity

1998 ◽  
Vol 80 (08) ◽  
pp. 273-280 ◽  
Author(s):  
Irene Salemink ◽  
Jo Franssen ◽  
George Willems ◽  
Coenraad Hemker ◽  
Anguo Li ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Inhibitory Activity ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Peptide Bond ◽  
Factor X ◽  
Tissue Factor Pathway ◽  
Factor Assay

SummaryTissue factor : factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa : TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa : TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa : TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Arg199-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa : TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa : TFPI towards TF : factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF : factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

Inhibition of Factor X Activation at Extracellular Matrix of Fibroblasts During Flow Conditions: A Comparison Between Tissue Factor Pathway Inhibitor and Inactive Factor VIIa

1995 ◽  
Vol 74 (06) ◽  
pp. 1478-1485 ◽  
Author(s):  
Sanne Valentin ◽  
Chris P M Reutlingsperger ◽  
Ole Nordfang ◽  
Theo Lindhout
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Flow Chamber ◽  
Factor X ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Tissue Factor Pathway ◽  
Factor X Activation

SummaryTissue factor pathway inhibitor (TFPI) is a naturally occurring factor Xa-dependent inhibitor of factor VIIa/tissue factor activity. In the present study, we examined the importance of the TFPI C-terminus and 3rd Kunitz-like domain for the inhibitory capacity of TFPI towards factor VIIa/tissue factor-catalyzed factor X activation and compared the inhibition with that of inactivated factor VIIa (factor VIIai). The extracellular matrix of fibroblasts, mounted in a parallel-plate flow chamber, were perfused with reaction mixtures that contained factors X, VIIa, and varying amounts of TFPI or factor VIIai. Inhibition was evaluated from the time course of factor Xa production at the outlet of the flow chamber. The factor VIIa/tissue factor-catalyzed factor Xa production was inhibited by factor VIIai and compatible with a direct competition between factor VIIai and factor VIIa for tissue factor. In contrast, TFPI showed a progressive inhibition of factor Xa production; the initial rate of factor X activation, however, was not inhibited by TFPI. Inhibition of factor Xa generation already in progress was seen for TFPI but not factor VIIai. In both cases we found that the truncated TFPI variants were as potent as full length TFPI. As to the stability of the enzyme- inhibitor complexes, TFPI/Xa/VIIa/tissue factor and factor VIIai/tissue factor, marked differences were observed. About 60% of the factor VIIa/tissue factor activity was recovered from the truncated TFPI/Xa/VIIa/tissue factor complex after 150 min of perfusion with reaction mixtures that contained factors X and VIIa. In contrast, full length TFPI did not dissociate from the complex, nor could factor VIIai be displaced by a large excess of factor VIIa.

Inhibition of endothelial cell tube formation by the low molecular weight heparin, tinzaparin, is mediated by tissue factor pathway inhibitor

2004 ◽  
Vol 92 (09) ◽  
pp. 627-633 ◽  
Author(s):  
S. Mohamed ◽  
Shaker Mousa
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Tube Formation ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Endothelial Tube Formation ◽  
Tissue Factor Pathway

SummaryHeparin and low molecular weight heparins (LMWHs) have both antithrombotic and anti-angiogenic activities. The antiangiogenic activity of LMWH may be associated with the release of endothelial tissue factor pathway inhibitor (TFPI), an important endogenous inhibitor of tissue factor/Factor VIIa (TF/fVIIa).To evaluate the effects of LMWH, tinzaparin, and TFPI in a model of angiogenesis-mediated processes, we compared the effects of tinzaparin, and recombinant TFPI in inhibiting either basic fibroblast growth factor-2 (FGF2) or TF/fVIIainduced endothelial cell tube formation in human umbilical vein endothelial cells (HUVEC).Our results show that tinzaparin and recombinant TFPI both blocked endothelial tube formation induced by either FGF2 or TF/fVIIa, in a concentration-dependent manner. Endothelial tube formation was only marginally inhibited by a potent and specific anti-Factor Xa, recombinant tick anticoagulant protein (rTAP). A monoclonal anti-TFPI antibody reversed the inhibitory effects of either tinzaparin or recombinant-TFPI on HUVEC tube formation. Tinzaparin fractions in the range of 8,000 to 12,600 Da were most effective in stimulating the release of TFPI from HUVEC. These results suggest that the inhibitory effect of the LMWH tinzaparin on endothelial tube formation is associated with stimulation of the release of TFPI, but not to anti-Factor Xa activity.

scholarly journals Activation of factor X and its regulation by tissue factor pathway inhibitor in small-diameter capillaries lined with human endothelial cells

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2909-2916 ◽  
Author(s):  
T Lindhout ◽  
R Blezer ◽  
P Schoen ◽  
O Nordfang ◽  
C Reutelingsperger ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Vii ◽  
Factor X ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Tissue Factor Pathway

Abstract The activation of factor X at the surface of endothelial cells was investigated under controlled flow conditions. A method is described for preparing polyethylene capillaries whose inner walls are covered with a confluent layer of human umbilical vein endothelial cells. To obtain a stable and unperturbed layer of endothelial cells it was essential to pre-perfuse the endothelialized capillaries with medium for about 18 hours. At this stage no tissue factor activity could be detected, but when the seeded cells were perfused with medium containing tumor necrosis factor (TNF) a maximum steady-state rate of factor Xa production (16 fmol factor Xa/min/cm2) was observed within 8 hours. Further experiments were performed with endothelial cells incubated for 4 hours with TNF. Factor Xa was produced at a rate of 7 fmol factor Xa/min/cm2 on perfusion of the capillaries with factor X (100 nmol/L) and factor VII (0.1 U/mL) at a shear rate of 34 s-1. The extracellular matrix preparations of these cells produced factor Xa at a 20-fold higher rate (150 fmol factor Xa/min/cm2). In both cases factor Xa formation was dependent on the presence of factor VII and was completely inhibited when the perfusate also contained 5 nmol/L recombinant tissue factor pathway inhibitor (rTFPI). Pre-perfusion with factor Xa-TFPI complex in the absence of factor VIIa caused a much lesser inhibitory effect, suggesting that TFPI-mediated neutralization of endothelial cell and matrix tissue factor activity requires the presence of factor VIIa in addition to the presence of factor Xa.

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