scholarly journals Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 676-682 ◽  
Author(s):  
Y Kazama ◽  
Y Komiyama ◽  
W Kisiel
Keyword(s):  
Cell Surface ◽  
Hepg2 Cell ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Factor V ◽  
Surface Binding ◽  
Cell Surface Binding ◽  
Tissue Factor Pathway ◽  
Protease Nexin ◽  
Prothrombinase Activity

Abstract Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti- factor V IgG and to a large extent (approximately 70%) was calcium- independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2- bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L- arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti- tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti- factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti- TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.

scholarly journals Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 676-682 ◽  
Author(s):  
Y Kazama ◽  
Y Komiyama ◽  
W Kisiel
Keyword(s):  
Cell Surface ◽  
Hepg2 Cell ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Factor V ◽  
Surface Binding ◽  
Cell Surface Binding ◽  
Tissue Factor Pathway ◽  
Protease Nexin ◽  
Prothrombinase Activity

Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti- factor V IgG and to a large extent (approximately 70%) was calcium- independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2- bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L- arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti- tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti- factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti- TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Susan A Maroney ◽  
Rodney M Camire ◽  
Alan E Mast
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Platelet Rich Plasma ◽  
Anticoagulant Activity ◽  
Factor V Leiden ◽  
Factor Xa ◽  
Factor V ◽  
Activation Threshold ◽  
Tissue Factor Pathway

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα

2019 ◽  
Vol 120 (01) ◽  
pp. 055-064
Author(s):  
Peter van Doorn ◽  
Jan Rosing ◽  
Elena Campello ◽  
Saskia Middeldorp ◽  
Paolo Simioni ◽  
...  
Keyword(s):  
Tissue Factor Pathway Inhibitor ◽  
Bleeding Risk ◽  
Factor V ◽  
Prothrombinase Complex ◽  
C Terminus ◽  
Normal Individuals ◽  
Splicing Variants ◽  
Tissue Factor Pathway ◽  
Fv Leiden ◽  
Prothrombinase Activity

Abstract Background Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. Objective This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. Materials and Methods FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). Results The TFPIr (0.25–0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28–0.31) to FVL heterozygotes (0.35, 95% CI 0.34–0.37) and homozygotes (0.39, 95% CI 0.37–0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. Conclusion We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.

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.

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.

scholarly journals Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1845-1850 ◽  
Author(s):  
AE Mast ◽  
GJ Jr Broze
Keyword(s):  
Tissue Factor ◽  
Calcium Ions ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor V ◽  
Dependent Manner ◽  
Tissue Factor Pathway

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.

scholarly journals Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPIα: Difference between FV1 and FV2

2018 ◽  
Vol 118 (07) ◽  
pp. 1194-1202 ◽  
Author(s):  
Peter van Doorn ◽  
Jan Rosing ◽  
Connie Duckers ◽  
Tilman Hackeng ◽  
Paolo Simioni ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Model System ◽  
Factor V ◽  
Prothrombinase Complex ◽  
Tissue Factor Pathway ◽  
Cofactor Activity

Background Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype. Objective This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2. Materials and Methods Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition. Results In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors. Conclusion FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF.

Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1845-1850 ◽  
Author(s):  
AE Mast ◽  
GJ Jr Broze
Keyword(s):  
Tissue Factor ◽  
Calcium Ions ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor V ◽  
Dependent Manner ◽  
Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.

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