Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity

Blood ◽  
1995 ◽  
Vol 85 (1) ◽  
pp. 121-129 ◽  
Author(s):  
LV Rao ◽  
O Nordfang ◽  
AD Hoang ◽  
UR Pendurthi
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Antithrombin Iii ◽  
Factor Viia ◽  
Factor Xa ◽  
Cell Surfaces ◽  
Tissue Factor Activity ◽  
At Iii ◽  
Tissue Factor Pathway

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.

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.

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.

Co-Interaction and Increased Release of Tissue Factor Pathway Inhibitor by Heparanase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4038-4038
Author(s):  
Yona Nadir ◽  
Benjamin Brenner ◽  
Anna Zetser ◽  
Flonia Levy-Adam ◽  
Victoria Kaplan ◽  
...  
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Tissue Factor ◽  
Cell Lines ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Hek 293 ◽  
A Cell ◽  
Tissue Factor Pathway

Abstract Background and objectives. Tissue factor pathway inhibitor (TFPI) is a potent direct inhibitor of factor Xa and factor VIIa-tissue factor complex. In addition, TFPI was shown to be an inhibitor of angiogenesis and metastasis. Heparanase is an endo-beta-D-glucuronidase of 65 kDa that cleaves heparan sulfate chains on cell surfaces and in the extra-cellular matrix an activity that closely correlates with cell invasion, angiogenesis and tumor growth. The study hypothesis was that heparanase may reduce the level of TFPI or release it from the cell surface in an attempt to increase heparanase prometastatic potential. Material and methods. The effect of exogenous heparanase on TFPI expression and release to the medium was studied in HUVEC by immunoblotting, real time RT-PCR, and flow-cytometry. Human cell lines (MDA-MB-435 breast carcinoma; U87 glioma; HEK-293 embryonic kidney) were transfected to over express heparanase and the effect on TFPI was studied. TFPI expression was explored in heparanase transgenic mice by immunoblotting and immunostaining. Transfections with various modified forms of heparanase were used to further explore the effect of heparanase. Interaction between TFPI and heparanase was studied by co-immunoprecipitation analysis. Results. Heparanase was found to increase the release of TFPI to the medium, reduce the level of TFPI at the cell surface, and to up-regulate its expression in the cells. These results were verified in HUVEC, tumor cell lines, and in the animal model. The effect was independent of heparanase activity or interaction with heparan sulfate, and dependent on heparanase secretion. A protein co-interaction between TFPI and heparanase was found. Conclusions. Overall, a cell surface interaction is suggested in which heparanase impose increased release of TFPI from the cell surface to the medium, providing a local procoagulant and a systemic anticoagulant environment.

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

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.

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.

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