The functional expression of tissue factor by fibroblasts and endothelial cells under flow conditions

Abstract The expression of tissue factor (TF) by a variety of vascular cell types under physiologic flow conditions is critical to factor X activation and in vivo clotting. Therefore, in a parallel-plate flow chamber (volume 40 microL) we mounted monolayers of human embryonic fibroblasts (FBs) or interleukin-1 alpha (IL-1 alpha) (5 U/mL x 4 hours)-stimulated human umbilical vein endothelial cells (ECs). Inflow buffer contained 10 nmol/L factor VIIa, 100 nmol/L factor X, and 2.0 mmol/L CaCl. With FBs, production of factor Xa (product of outflow concentration of factor Xa-and flow rate) increased 200-fold over the range of shear stress from 0 to 2.7 dynes/cm2. Production values (mean +/- SE (N)) were 7.93 +/- 0.024 (6), 312 +/- 7.3 (6), 688 +/- 33.1 (8), 1,033 +/- 119 (6), and 1,601 +/- 183 (7) fmol/cm2.minute at shear stresses of 0, 0.27, 0.68, 1.35, and 2.7 dynes/cm2, respectively. Further experiments at 0.68 dynes/cm2 indicated that factor Xa production increased with factor X concentration over the range from 3 to 100 nmol/L, but changed little from 300 to 1,000 nmol/L. With ECs, production was 0.13 +/- 0.86 (6), 8.17 +/- 1.65 (13), and 1.66 +/- 1.66 (5) fmol/cm2.minute at 0, 0.68, and 2.7 dynes/cm2, respectively. However, in the presence of an antibody directed against tissue factor pathway inhibitor (TFPI) production with ECs was augmented to 16.46 +/- 0.80 (8), 149.8 +/- 18.6 (8), and 48.9 +/- 10.3 (10), respectively, at these same shear stresses. Control experiments with factor VIIa, factor X, or both absent confirm for both cell types the specificity of the reaction for the TF pathway. Similarly, specificity for TF itself is shown by the virtual absence of factor Xa generation in the presence of the monoclonal antibody HTF1–7B8 directed against human TF. We conclude that ECs, even when activated, are normally unable to generate significant quantities of factor Xa in the presence of factors X and VIIa. However, significant quantities of factor Xa are possible in the presence of an inhibitor of TFPI. On the other hand, production of factor Xa by fibroblasts is markedly augmented by shear stress, yet independent of the availability of substrate factor X above an inflow concentration of 100 nmol/L. The latter suggests a direct effect of flow on the fibroblast monolayers, not substrate limitation by convective diffusion.

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The functional expression of tissue factor by fibroblasts and endothelial cells under flow conditions

Blood ◽

10.1182/blood.v81.12.3265.bloodjournal81123265 ◽

1993 ◽

Vol 81 (12) ◽

pp. 3265-3270 ◽

Cited By ~ 3

Author(s):

EF Grabowski ◽

DB Zuckerman ◽

Y Nemerson

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Tissue Factor ◽

Factor Viia ◽

Factor Xa ◽

Cell Types ◽

Shear Stresses ◽

Factor X ◽

Flow Conditions ◽

Production Values

The expression of tissue factor (TF) by a variety of vascular cell types under physiologic flow conditions is critical to factor X activation and in vivo clotting. Therefore, in a parallel-plate flow chamber (volume 40 microL) we mounted monolayers of human embryonic fibroblasts (FBs) or interleukin-1 alpha (IL-1 alpha) (5 U/mL x 4 hours)-stimulated human umbilical vein endothelial cells (ECs). Inflow buffer contained 10 nmol/L factor VIIa, 100 nmol/L factor X, and 2.0 mmol/L CaCl. With FBs, production of factor Xa (product of outflow concentration of factor Xa-and flow rate) increased 200-fold over the range of shear stress from 0 to 2.7 dynes/cm2. Production values (mean +/- SE (N)) were 7.93 +/- 0.024 (6), 312 +/- 7.3 (6), 688 +/- 33.1 (8), 1,033 +/- 119 (6), and 1,601 +/- 183 (7) fmol/cm2.minute at shear stresses of 0, 0.27, 0.68, 1.35, and 2.7 dynes/cm2, respectively. Further experiments at 0.68 dynes/cm2 indicated that factor Xa production increased with factor X concentration over the range from 3 to 100 nmol/L, but changed little from 300 to 1,000 nmol/L. With ECs, production was 0.13 +/- 0.86 (6), 8.17 +/- 1.65 (13), and 1.66 +/- 1.66 (5) fmol/cm2.minute at 0, 0.68, and 2.7 dynes/cm2, respectively. However, in the presence of an antibody directed against tissue factor pathway inhibitor (TFPI) production with ECs was augmented to 16.46 +/- 0.80 (8), 149.8 +/- 18.6 (8), and 48.9 +/- 10.3 (10), respectively, at these same shear stresses. Control experiments with factor VIIa, factor X, or both absent confirm for both cell types the specificity of the reaction for the TF pathway. Similarly, specificity for TF itself is shown by the virtual absence of factor Xa generation in the presence of the monoclonal antibody HTF1–7B8 directed against human TF. We conclude that ECs, even when activated, are normally unable to generate significant quantities of factor Xa in the presence of factors X and VIIa. However, significant quantities of factor Xa are possible in the presence of an inhibitor of TFPI. On the other hand, production of factor Xa by fibroblasts is markedly augmented by shear stress, yet independent of the availability of substrate factor X above an inflow concentration of 100 nmol/L. The latter suggests a direct effect of flow on the fibroblast monolayers, not substrate limitation by convective diffusion.

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

Blood ◽

10.1182/blood.v79.11.2909.2909 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2909-2916 ◽

Cited By ~ 17

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

Blood ◽

10.1182/blood.v79.11.2909.bloodjournal79112909 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2909-2916 ◽

Cited By ~ 1

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.

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Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647472 ◽

1991 ◽

Vol 65 (02) ◽

pp. 139-143 ◽

Cited By ~ 7

Author(s):

Cynthia H Gemmell ◽

Vincet T Turitto ◽

Yale Nemerson

Keyword(s):

Steady State ◽

Tissue Factor ◽

Factor Viia ◽

Transmembrane Protein ◽

Strong Association ◽

Tubular Reactor ◽

Factor Xa ◽

Phospholipid Bilayer ◽

Factor Vii ◽

Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

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TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

Blood ◽

10.1182/blood-2011-10-388512 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1256-1262 ◽

Cited By ~ 37

Author(s):

Thomas J. Girard ◽

Elodee Tuley ◽

George J. Broze

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Feedback Inhibition ◽

Culture Media ◽

Factor Viia ◽

Factor Xa ◽

Gpi Anchor ◽

C Terminus ◽

Before And After ◽

Tissue Factor Pathway

Abstract Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ.

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Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation

Blood ◽

10.1182/blood.v69.2.645.645 ◽

1987 ◽

Vol 69 (2) ◽

pp. 645-651 ◽

Cited By ~ 137

Author(s):

LV Rao ◽

SI Rapaport

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Factor Xa ◽

Factor Ix ◽

Factor Vii ◽

Factor X ◽

Extrinsic Pathway ◽

Mechanism Of Inhibition ◽

Peptide Release ◽

Release Assay

Abstract We have extended earlier studies (Blood 66:204, 1985) of a mechanism of inhibition of factor VIIa/tissue factor activity that requires a plasma component (called herein extrinsic pathway inhibitor or EPI) and factor Xa. An activated peptide release assay using 3H-factor IX as a substrate was used to evaluate inhibition. Increasing the tissue factor concentration from 20% to 40% (vol/vol) overcame the inhibitory mechanism in normal plasma but not in factor VII-deficient plasma supplemented with a low concentration of factor VII. A second wave of factor IX activation obtained by a second addition of tissue factor to plasma with a normal factor VII concentration was almost abolished by supplementing the reaction mixture with additional EPI and factor X. Factor Xa's active site was necessary for factor Xa's contribution to inhibition, but preliminary incubation of factor Xa with EPI in the absence of factor VIIa/tissue factor complex or of factor VIIa/tissue factor complex in the absence of EPI did not replace the need for the simultaneous presence of factor Xa, factor VIIa/tissue factor, calcium, and EPI in an inhibitory reaction mixture. Inhibition of factor VIIa/tissue factor was reversible; both tissue factor and factor VIIa activity could be recovered from a dissociated, inhibited factor VIIa/tissue factor complex. EPI appeared to bind to a factor VIIa/tissue factor complex formed in the presence of factor Xa but not to a factor VIIa/tissue factor complex formed in the absence of factor Xa.

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Activity and Regulation of Factor VIIa Analogs with Increased Potency at the Endothelial Cell Surface.

Blood ◽

10.1182/blood.v108.11.1751.1751 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1751-1751

Author(s):

Samit Ghosh ◽

Mirella Ezban ◽

Egon Persson ◽

Ulla Hedner ◽

Usha Pendurthi ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Surface ◽

Proteolytic Activity ◽

Thrombin Generation ◽

Factor Viia ◽

Factor Xa ◽

Factor Ix ◽

Factor X ◽

Wild Type

Abstract High doses of recombinant factor VIIa (FVIIa) have been found to bypass factor IX or factor VIII deficiency and ameliorate the bleeding problems associated with hemophilia patients with inhibitors. Recent studies show that FVIIa also acts as an effective hemostatic agent in other categories of patients, and thus has become a promising candidate for prevention and treatment of excessive bleeding associated with many other diseases/injuries. Although recombinant FVIIa has proven to be a very effective and safe drug in the treatment of bleeding episodes in hemophilia patients with inhibitors and other indications, a small fraction of patients may be refractory to FVIIa treatment. The reason for this is unclear at present, but it is possible that administration of very high pharmacological doses of FVIIa or use of genetically modified FVIIa molecules with increased potencies may circumvent the problem. The most dramatic effect on the activity (a 40-fold increase in proteolytic activity) of FVIIa was obtained by occupying the corresponding positions in thrombin/factor IXa for those positions 158, 296 and 298 of FVIIa (FVIIaDVQ). A FVIIa mutant in which the hydrophobic residue Met 298 was replaced with Gln (FVIIaQ) has 7-fold higher proteolytic activity. In the present study, we investigated the interactions of FVIIaQ and FVIIaDVQ with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT) in solution and at the vascular endothelium. Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type FVIIa in the absence of TF. In the presence of TF, TFPI, TFPI-Xa and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the wild-type FVIIa failed to generate significant amounts of factor Xa on unperturbed endothelial cells, FVIIa variants, particularly FVIIaDVQ, generated a substantial amount of factor Xa on unperturbed endothelium (1 nM of factor VIIa generated 0.3 ± 0.15 nM factor Xa/h whereas FVIIaQ and FVIIaDVQ generated 1.26 ± 0.1 nM/h and 9.48 ± 1.32 nM/h, respectively). Annexin V fully attenuated the FVIIa-mediated activation of factor X on unperturbed endothelial cells whereas anti-TF IgG had no effect. On stimulated HUVEC, FVIIa and FVIIa variants activated factor X at similar rates (30–40 nM/h). AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. AT inhibition of FVIIa bound to stimulated endothelial cells requires exogenous heparin. Interestingly, TFPI-Xa was found to inhibit the activities of both FVIIa and FVIIa analogs bound to unperturbed endothelial cells. Despite significant differences observed in factor Xa generation on native endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions, probably due to limited availability of anionic phospholipids and/or putative factor Xa and Va binding sites on their cell surface. Over all, our present data suggest that although FVIIa variants may generate factor Xa on native endothelium, the resultant factor Xa does not lead to enhanced thrombin generation on native endothelium compared to FVIIa. These data should reduce potential concerns about whether the use of FVIIa variants triggers unwanted coagulation on native endothelium, and may facilitate the development of FVIIa analogs as effective therapeutic agents in near future for treatment of patients with bleeding disorders.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m113.533836 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1732-1741 ◽

Cited By ~ 28

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.

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Fluid Shear Stress Attenuates Tumor Necrosis Factor-α–Induced Tissue Factor Expression in Cultured Human Endothelial Cells

Blood ◽

10.1182/blood.v91.11.4164 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4164-4172 ◽

Cited By ~ 26

Author(s):

Yutaka Matsumoto ◽

Yohko Kawai ◽

Kiyoaki Watanabe ◽

Kazuo Sakai ◽

Mitsuru Murata ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Tumor Necrosis Factor ◽

Tissue Factor ◽

Shear Force ◽

Tumor Necrosis ◽

Shear Stresses ◽

Dependent Manner ◽

Tnf Α ◽

Necrosis Factor

Abstract Hemodynamic forces modulate various endothelial cell functions under gene regulation. Previously, we have shown that fibrinolytic activity of endothelial cells is enhanced by the synergistic effects of shear stress and cytokines. In this study, we investigated the effect of shear stress on tumor necrosis factor (TNF)-α–induced tissue factor (TF) expression in cultured human umbilical vein endothelial cells (HUVECs), using a modified cone-plate viscometer. Shear stresses at physiological levels reduced TNF-α (100 U/mL)–induced TF expression at both mRNA and antigen levels, in a shear-intensity and exposure-time dependent manner, whereas shear stress itself did not induce TF expression in HUVECs. TF expressed on the cell surfaces measured by flow cytometry using an anti-TF monoclonal antibody (HTF-K180) was also decreased to one third by shear force applied at 18 dynes/cm2 for 15 hours before and 6 hours after TNF-α stimulation. Furthermore, functional activity of TF, as assessed by the activation of factor X in the presence of FVIIa and Ca2+, was also decreased by shear application. However, the stability of TF mRNA was not decreased in the presence of shear stress. These results suggest that shear force acts as an important regulator of TF expression in endothelium at the transcriptional level.

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Studies of the activation of factor VII bound to tissue factor [see comments]

Blood ◽

10.1182/blood.v87.9.3738.bloodjournal8793738 ◽

1996 ◽

Vol 87 (9) ◽

pp. 3738-3748 ◽

Cited By ~ 29

Author(s):

LV Rao ◽

T Williams ◽

SI Rapaport

Keyword(s):

Tissue Factor ◽

Factor Viia ◽

Tissue Injury ◽

Factor Xa ◽

Inhibitory Effect ◽

Factor Ix ◽

Factor Vii ◽

Activate Factor ◽

Factor X ◽

Ovarian Carcinoma Cell Line

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I- factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.

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