Inhibition Of Tissue Factor Activity Research Articles

Tissue factor pathway inhibitor production by human proximal tubular epithelial cells in culture

Fibrin deposition in the peritubular capillaries and along the tubular basement membrane is commonly observed in several renal diseases and suggests the involvement of blood coagulation in tubulointerstitial damage. It has been demonstrated that tissue factor (TF) is present in tubular epithelial cells of animal models of nephritis. Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation through its ability to inhibit TF activity and it is now thought to be produced mainly by the vascular endothelial cells. We examined whether human proximal tubular epithelial cells (PTEC) could produce TFPI and attempted to clarify the regulatory factors affecting TFPI production. Cultured human PTEC were used. The procoagulant activity (PCA) in PTEC lysate was quantified by measurement of the one-stage recalcification time. TFPI in the cell supernatants was measured by ELISA. The mRNA of TF and TFPI in PTEC was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). PCA which is compatible with TF activity was present in the PTEC lysate. TF mRNA and TFPI mRNA were detected in PTEC. The amount of TFPI increased over time in the cell supernatants. Immnoblot analysis revealed 40 kD protein of TFPI, and TFPI antigen was demonstrated in PTEC by immunofluorescence. The concentration of TFPI was significantly increased following incubation with thrombin and heparin in a dose- and time-dependent manner, although the amount of TFPI mRNA was not changed. Our study showed that TFPI is produced in cultured PTEC and added one more cell type that produced TFPI other than endothelial cells. Thrombin and heparin stimulated TFPI secretion from PTEC. TFPI of PTEC may act against generation of thrombin and tubular fibrin formation induced by tissue factor activation. The augmentation of TFPI secretion by heparin may play an important role in the modulation of anticoagulant properties of PTEC.

Tissue factor in acute coronary syndromes.

Thrombosis at the site of atherosclerotic plaque disruption is the principal cause of acute coronary syndromes. The severity of the clinical consequences is determined by the extent and the progression of the thrombus that are caused by local and systemic factors. In atherosclerotic lesions mediators induce tissue factor (TF) in macrophages, smooth muscle cells, and endothelial cells. Procoagulant microparticles in the lipid core further enhance the thrombogenicity of the plaque. In addition, in acute coronary syndromes circulating monocytes and microparticles express TF and, thereby, contribute to systemic procoagulant activity. As a regulatory mechanism surface-bound, endogenous tissue factor pathway inhibitor-1 (TFPI) inhibits TF activity by translocation of the quaternary complex TF-FVIIa-FXa-TFPI into glycosphingolipid-rich microdomains more efficiently than exogenously added TFPI. This inhibition occurs not only in endothelial cells but also on circulating monocytes and presumably microparticles. Because therapeutic thrombolysis in acute myocardial infarction degrades TFPI, a prothrombotic state due to unopposed TF activity may occur. Several studies have demonstrated a contribution of local and bloodborne TF to thrombus formation; a direct relationship with the clinical outcome, however, awaits further studies. This article discusses the current understanding of the role of TF and its regulation by TFPI in acute coronary syndromes.

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis

Tissue factor (TF) is a transmembrane glycoprotein that was originally recognized for its ability to initiate the extrinsic pathway of coagulation. More recently, additional functions of TF in cellular signalling have emerged, notably the role of TF in vasculogenesis and angiogenesis. We have described previously the ability of a peptide derived from the apolipoprotein B100 (apoB100) moiety of low-density lipoproteins (KRAD14) to inhibit the procoagulant function of TF. In this study, we demonstrate the ability of the KRAD14 peptide to attenuate the density of cellular network structures of T24 cells grown on specialized matrix (MatrigelTM). In addition, an alternative inhibitor of TF activity, the TF8 5G9 antibody, also reduces the density of cellular network formation. Targeted use of a stable structural equivalent of the KRAD14 peptide may thus prove useful in the prophylactic treatment of diseases whose pathologies feature the formation of neovascular tissue, e.g. tumour growth and metastasis, rupture of atherosclerotic plaques and retinopathy secondary to diabetes.

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis

The Role of Tissue Factor in Renal Ischemic Reperfusion Injury of the Rat

Background. Tissue factor (TF) is a membrane-bound glycoprotein that is the primary cellular initiator of the blood clotting cascade and its expression is induced on macrophages and endothelial cells during the inflammatory or immune response. Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation through its ability to inhibit tissue factor activity. We studied the role of TF in the kidney following warm ischemic reperfusion and studied the effect of TFPI in vivo.Materials and Methods. After laparotomy of Lewis rats, the right kidney was harvested and left renal artery and vein were clamped. The kidney was reperfused after 60, 120, and 180 min of ischemia. Rats were sacrificed at 0, 1.5, 5, 12, and 24 h after reperfusion with or without TFPI treatment, and the kidney was harvested. Blood samples were collected at 0, 5, 12, and 24 h after reperfusion from the abdominal aorta. Blood urea nitrogen and kalium were monitored. TF expression was also studied by immunohistochemical staining with a monoclonal antibody (HTF-K108).Results. Histologically, the necroses of the tubular epithelial cells were observed 1.5 h after reperfusion. Immunohistochemically, TF staining was positive on the glomerular endothelial cells and stimulated monocytes but negative on the tubular epithelial cells. The necrotic area extended and encompassed almost all of the ischemic kidney by 12 h after reperfusion. TF was stained on the glomerular base membrane, the glomerular endothelial cells, and the stimulated monocytes but was not evident on the necrotic tubular epithelial cells. Fibrinogen was also observed in the glomerular endothelial cells at 5–12 h after reperfusion, while it was slight in normal tissue. With TFPI treatment, the necrotic area was narrow and TF was slightly stained on the endothelial cells.Conclusions. These results suggest that TF plays an important role in the development of renal injury after ischemia and reperfusion. The microcirculatory incompetence due to microthrombus might cause the formation and development of the necrosis. These results also suggest that TFPI plays a key role in modulating tissue factor-dependent blood coagulation, therefore TFPI is a strong medication for prevention of ischemic reperfusion injury.

Deficiency of tissue factor pathway inhibitor promotes atherosclerosis and thrombosis in mice.

Tissue factor initiates blood coagulation after atherosclerotic plaque disruption. Tissue factor pathway inhibitor (TFPI) inhibits tissue factor activity and may reduce thrombus formation in this setting. We evaluated the effect of heterozygous TFPI deficiency on the development of atherosclerosis and thrombosis in atherosclerosis-prone mice. Mice with a combined heterozygous TFPI deficiency and homozygous apolipoprotein E deficiency (TFPI(+/-)/apoE(-/-)) were generated by crossbreeding, and they were analyzed for atherosclerosis throughout the vascular tree. Compared with mice with a normal TFPI genotype (TFPI(+/+)/apoE(-/-)), mice with a TFPI deficiency exhibited a greater atherosclerotic burden involving the carotid and common iliac arteries. Staining for active tissue factor within the plaque revealed more activity in TFPI(+/-)/apoE(-/-) mice compared with TFPI(+/+)/apoE(-/-) mice. Consistent with increased plaque tissue factor activity, the time to occlusive thrombosis after photochemical carotid plaque injury was significantly decreased in TFPI(+/-)/apoE(-/-) mice. These observations indicate that TFPI protects from atherosclerosis and is an important regulator of the thrombosis that occurs in the setting of atherosclerosis.

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis

Inhibition of tissue factor activity reduces the density of cellular network formation in an in vitro model of angiogenesis

Homocysteine and Oxidized Low Density Lipoprotein Enhance Platelet Adhesion to Endothelial Cells under Flow Conditions: Distinct Mechanisms of Thrombogenic Modulation

We investigated the effects of two well established risk factors for cardiovascular disease, homocysteine and oxidized low density lipoprotein (ox-LDL), on endothelial cell thrombogenicity. For this purpose we studied platelet adhesion to human endothelial cells (EC) under flow conditions at a shear rate of 350 s(-1) following EC treatment with either homocysteine or ox-LDL. Treatment of EC with either homocysteine (1 or 10 mmol/L for 16 h) or ox-LDL (100 microg/ml for 16 h) resulted in a 2-3 fold enhancement in platelet adhesion. The enhancement in platelet adhesion induced by 1 mmol/L homocysteine, but not that induced by 10 mmol/L homocysteine, was absolutely dependent on fibrin formation. Homocysteine treatment has significantly increased the cell surface tissue factor (TF) activity and slightly reduced the expression of the intercellular adhesion molecule I (ICAM-1). In contrast, ox-LDL treatment upregulated ICAM-1 expression and had no significant effect on endothelial TF activity. Neither homocysteine nor Ox-LDL affected surface expression of the alpha(v)beta3 integrin. The homocysteine-induced enhancement in platelet adhesion was almost completely abolished by blockade of the EC TF activity by a polyclonal antibody. The enhancing effect of homocysteine was also greatly reduced by inhibition of the EC alpha(v)beta3 integrin, but was not affected by blockade of EC ICAM-1. On the other hand, ox-LDL-induced enhancement in platelet - EC adhesion was greatly inhibited by blocking ICAM-1 or alpha(v)beta3, but remained unaffected by inhibition of TF activity. Preincubation of platelets with the glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist Reo-Pro has virtually abolished the enhancing effect of both homocysteine and ox-LDL. Our results suggest that homocysteine and ox-LDL might increase endothelial thrombogenicity by distinct mechanisms: homocysteine - by inducing TF activity, and ox-LDL - by upregulating ICAM-1, both of which enhance GPIIb-IIIa/fibrinogen dependent platelet adhesion to EC. The alpha(v)beta3 integrin, although not affected by EC stimulation, seems to play a crucial role in platelet-EC interaction regardless of the mechanism of EC perturbation.

I. Suppression by compound 48/80 of bacterial endotoxin-inducible monocytic tissue factor activity: direct blockade of factor VII binding to THP-1 monocytes

Hypercoagulation with upregulated monocytic tissue factor (TF) activity often occurs under a variety of inflammatory conditions including endotoxemia. The antagonism to bacterial endotoxin (LPS) signaling often results in the depression in TF upregulation. We herein report that compound 48/80 (48/80) significantly depressed LPS-induced TF activity in human and cebus monkey peripheral blood monocytes. Employing a model monocyte-like cell line (THP-1), we explored the regulatory mechanism to identify the inhibitory site(s) of 48/80. We determine whether the inhibition results from the blockade of LPS signaling. 48/80 dose-dependently inhibited LPS-induced TF activity. Chase of LPS-challenged cells with 48/80 also significantly offset TF upregulation. In immunofluorescent approaches, FACScan analysis revealed that 48/80 had no effect on either LPS recognition or the expression of its receptors (CD14 and CD11b). Moreover, LPS-induced TF expression as well as synthesis remained unaffected in the presence of 48/80. Consistent with the independence of LPS action, 48/80 was also able to inhibit TF activity induced by A23187, ionomycin, or Quin-2 AM. Interestingly, 48/80 significantly decreased the FVII binding to either resting or LPS-challenged cells. In conclusion, our results elucidate that the inhibitory action of 48/80 was independent of LPS signaling including recognition, receptor expression, and the induced TF expression/synthesis. However, 48/80 was able to directly block FVII binding to monocytic TF, thereby resulting in such antagonism to LPS-induced TF-initiated extrinsic coagulation.

Tissue factor pathway inhibitor expression in human crescentic glomerulonephritis

Tissue factor (TF) pathway inhibitor (TFPI), the major endogenous inhibitor of extrinsic coagulation pathway activation, protects renal function in experimental crescentic glomerulonephritis (GN). Its glomerular expression and relationship to TF expression and fibrin deposition in human crescentic GN have not been reported. Glomerular TFPI, TF, and fibrin-related antigen (FRA) expression were correlated in renal biopsies from 11 patients with crescentic GN. Biopsies from 11 patients with thin basement membrane disease and two normal kidneys were used as controls. TFPI was undetectable in control glomeruli but was detectable in interstitial microvessels. In crescentic biopsies, TFPI was detected in cellular crescents and was more prominent in fibrous/fibrocellular crescents, indicating a correlation with the chronicity of crescentic lesions. TFPI appeared to be associated with macrophages but not endothelial or epithelial cells. TFPI was generally undetectable in regions of the glomerular tuft with minimal damage. In contrast, TF and FRA were strongly expressed in regions of minimal injury, as well as in more advanced proliferative and necrotizing lesions. Despite prominent TF expression, FRA was less prominent in fibrous/fibrocellular crescents in which TFPI expression was maximal. These data suggest that TFPI is strongly expressed in the later stages of crescent formation and is inversely correlated with the presence of FRA in human crescentic GN. This late induction of TFPI may inhibit TF activity and favor reduced fibrin deposition in the chronic stages of crescent formation.

Adenosine Regulates Tissue Factor Expression on Endothelial Cells

The aim of this study was to evaluate the inhibitory activity of adenosine on tumor necrosis factor-α (TNF), thrombin-, or phorbol 12-myristate 13-acetate (PMA)-induced tissue factor (TF) expression on human umbilical vein endothelial cells (HUVECs). This inhibitory effect of adenosine was found to be counteracted by the non-selective adenosine receptor (AR) antagonist, 8-(p-sulfophenyl) theophylline. To clarify the role of ARs (A1, A2a, A2b, and A3) in this regulation, we evaluated the effect of several agonists and antagonists specific for AR-subclass on TF expression. The selective A2aAR agonist, 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamido adenosine hydrochloride (CGS 21680), the A3AR agonist, N6-2-(4-aminophenyl) ethyladenosine (APNEA), and the A1AR antagonist, 1,3-dipropyl-8-(2-amino-4-chlorophenyl) xanthine (PACPX) each inhibited TF activity expression induced by TNF, thrombin, or PMA on HUVECs. In contrast, the selective A1AR agonist, chloro-N6-cyclopentyladenosine (CCPA) and the A2AR antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX) enhanced each stimulant-induced TF activity expression. All agonist or antagonist alone did not alter the basal TF expression on HUVECs. Our results suggest that stimulation of A2aAR and A3AR down-regulates and that of A1AR up-regulates the endothelial cell TF expression induced by TNF, PMA, or thrombin. Thus, it appears that adenosine itself may exert anticoagulant activity on vascular endothelial cells via its A2a and A3 receptors, particularly during ischemic or atherosclerotic processes which are known to be associated with local increased levels of adenosine.

The Influence of β2-Glycoprotein I on Tissue Factor Activity

The beta2-glycoprotein I (beta2-GPI) is known for its procoagulant as well as anticoagulant properties. The influence of beta2-GPI on tissue factor (TF) activity was investigated by chromogenic assays for both factor Xa generation and factor VIIa activity. When 1.36 mg/mL beta2-GPI was added to phospholipids prior to the addition of TF and factor VIIa, the TF activity was moderately inhibited by beta2-GPI in a concentration-dependent manner. Inhibition of TF activity by beta2-GPI required incubation for at least 20 min. When beta2-GPI was added to the mixture of phospholipids and TF followed by the addition of factor VIIa, the TF activity was little affected by the addition of beta2-GPI. The TF activity was inhibited when beta2-GPI was added after forming a complex with TF and factor VIIa. The results suggest that beta2-GPI inhibits both the reconstitution of TF with phospholipids and factor Xa generation by the TF factor-VIIa complex but does not affect the formation of a TF factor-VIIa complex.

Dilazep, an Antiplatelet Agent, Inhibits Tissue Factor Expression in Endothelial Cells and Monocytes

AbstractDilazep, an antiplatelet agent, is generally used as an antithrombotic drug in clinical practice. Dilazep is also known to exert cytoprotective and antioxidant effects on endothelial cells. However, its effect on the endothelial or monocyte procoagulant activity is unknown. In the current study, the effect of dilazep on the expression of tissue factor (TF ) in human umbilical vein endothelial cells (HUVECs) after the stimulation with tumor necrosis factor-α (TNF ), thrombin, or phorbol 12-myristate 13-acetate (PMA) was evaluated. We also evaluated the effect of dilazep on TNF (1,000 U/mL)-induced TF expression on monocytes. Dilazep inhibited TF activity induced on HUVECs by each stimulant, TNF (1000 U/mL), thrombin (25 nmol/L), or PMA (5 nmol/L) in a dose-dependent fashion (1 to 100 μg/mL). TF activity decreased to approximately 10% after treating with 100 μg/mL of dilazep. Dilazep also blocked the expression of TF antigen induced by each stimulant on the surface of HUVECs as determined by flow cytometric analysis. In addition, in HUVECs, it significantly decreased the expression of TF mRNA and the total TF antigen induced by thrombin or PMA, but not those induced by TNF, suggesting that dilazep blocks the TF expression induced by PMA or thrombin at a transcriptional level and that induced by TNF at a posttranscriptional level. Western blot analysis showed that dilazep reduces the accumulation of native TF but increases that in lower molecular weight TF derivatives. The adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline, partially counteracted the anticoagulant activity of dilazep on HUVECs, thereby suggesting that the inhibitory effect of dilazep on TF expression in HUVECs depends, at least in part, on its adenosine potentiating activity. Dilazep also inhibited TNF-induced TF expression on monocytes in a dose-dependent fashion (0.1 to 100 μg/mL). In brief, the current study showed for the first time that dilazep, a commonly used antiplatelet drug, strongly inhibits the TF expression in HUVECs and monocytes. Dilazep may have a potent therapeutic value in patients with hypercoagulable state for its inhibitory property on the procoagulant activity of endothelial cells and monocytes.

Dilazep, an Antiplatelet Agent, Inhibits Tissue Factor Expression in Endothelial Cells and Monocytes

Dilazep, an antiplatelet agent, is generally used as an antithrombotic drug in clinical practice. Dilazep is also known to exert cytoprotective and antioxidant effects on endothelial cells. However, its effect on the endothelial or monocyte procoagulant activity is unknown. In the current study, the effect of dilazep on the expression of tissue factor (TF ) in human umbilical vein endothelial cells (HUVECs) after the stimulation with tumor necrosis factor-α (TNF ), thrombin, or phorbol 12-myristate 13-acetate (PMA) was evaluated. We also evaluated the effect of dilazep on TNF (1,000 U/mL)-induced TF expression on monocytes. Dilazep inhibited TF activity induced on HUVECs by each stimulant, TNF (1000 U/mL), thrombin (25 nmol/L), or PMA (5 nmol/L) in a dose-dependent fashion (1 to 100 μg/mL). TF activity decreased to approximately 10% after treating with 100 μg/mL of dilazep. Dilazep also blocked the expression of TF antigen induced by each stimulant on the surface of HUVECs as determined by flow cytometric analysis. In addition, in HUVECs, it significantly decreased the expression of TF mRNA and the total TF antigen induced by thrombin or PMA, but not those induced by TNF, suggesting that dilazep blocks the TF expression induced by PMA or thrombin at a transcriptional level and that induced by TNF at a posttranscriptional level. Western blot analysis showed that dilazep reduces the accumulation of native TF but increases that in lower molecular weight TF derivatives. The adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline, partially counteracted the anticoagulant activity of dilazep on HUVECs, thereby suggesting that the inhibitory effect of dilazep on TF expression in HUVECs depends, at least in part, on its adenosine potentiating activity. Dilazep also inhibited TNF-induced TF expression on monocytes in a dose-dependent fashion (0.1 to 100 μg/mL). In brief, the current study showed for the first time that dilazep, a commonly used antiplatelet drug, strongly inhibits the TF expression in HUVECs and monocytes. Dilazep may have a potent therapeutic value in patients with hypercoagulable state for its inhibitory property on the procoagulant activity of endothelial cells and monocytes.

Antithrombotic and antilesion benefits without hemorrhagic risks by inhibiting tissue factor pathway.

The effects of inhibiting tissue factor-dependent thrombus formation on vascular neointimal lesion formation have been evaluated by inhibiting tissue factor activity using intravenous injections of active-site inactivated recombinant factor VIIa (FVIIai) administered to baboons immediately prior to initiating bilateral femoral balloon artery angioplasty and surgical carotid endarterectomy. FVIIai abolished thrombus formation at sites of vascular injury and decreased vascular lesion formation by approximately 50 percent at 30 days. We conclude that thrombus formation at sites of vascular injury is predominately tissue factor-dependent and that transient inhibition of tissue factor activity prevents both vascular thrombosis and vascular lesion formation, which implies that transiently inhibiting tissue factor at the time of elective mechanical vascular procedures may be useful in reducing clinical restenosis.

Tissue Factor Pathway Inhibitor Production by Human Mesangial Cells in Culture

Fibrin formation within the glomeruli has been observed in various forms of human and experimental glomerulonephritis and it may play an important role in progressive glomerular injury. Furthermore it has been hypothesized that glomerular fibrin deposition may occur through activation of either the intrinsic or extrinsic coagulation pathway. It has been demonstrated that a procoagulant activity (PCA) which is compatible with tissue factor is present in the glomeruli and becomes increased in human proliferative glomerulonephritis and in animal models of nephritis. Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation through its ability to inhibit tissue factor activity. TFPI is present in plasma and in platelets, and it is now thought to be produced mainly by endothelial cells. We examined whether human mesangial cells (HMC) could produce TFPI and attempted to clarify regulatory factors which affect TFPI production. Cultured HMC were used and TFPI in the cell supernatants was measured by ELISA using a specific antibody. Cultured HMC showed the production of TFPI. Immunoblot analysis revealed 40 kD protein of TFPI. The concentration of TFPI was significantly increased following the incubation with thrombin and heparin, including low molecular weight heparin, in a dose- and time-dependent manner. However, fetal calf serum, phorbol myristate acetate, lipopolysaccharide, IL-1 beta and tissue factor did not stimulate TFPI synthesis. Our data show that cultured HMC have the ability to produce TFPI which inhibits fibrin formation. It is possible that thrombin-induced enhancement of TFPI synthesis may be caused by the autoregulatory system of blood coagulation and that with heparin it may represent another anticoagulatory effect of heparin.

An unusual antibody that blocks tissue factor/factor VIIa function by inhibiting cleavage only of macromolecular substrates

Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8–11D12 and TF9–9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9–9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8–11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8–11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8–11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.

An Unusual Antibody That Blocks Tissue Factor/Factor Vila Function by Inhibiting Cleavage Only of Macromolecular Substrates

Tissue factor (TF), the cell surface receptor and cofactor for factor VIIa (FVIIa), is considered the major physiologic trigger of the coagulation cascade. Most monoclonal antibodies to TF have been reported to inhibit TF activity by blocking association of FVII(a) with TF. Using solution-phase kinetic analyses, we have reexamined two strongly inhibitory anti-TF monoclonal antibodies (TF8-11D12 and TF9-9C3) previously reported to block FVII binding in cell-binding assays. Kinetic analysis of TF9-9C3 was consistent with direct competition with FVIIa for binding to TF. However, antibody TF8-11D12 did not block FVIIa binding to TF as measured by ability of the TF:FVIIa complex to cleave a small peptide substrate or by enhanced reactivity of FVIIa with a tripeptidyl-chloromethylketone. Interestingly, TF8-11D12 strongly inhibited cleavage of all three known macromolecular substrates (factors VII, IX, and X) of the TF:FVIIa complex. We hypothesize that TF8-11D12 blocks access of macromolecular substrates to the active site of FVIIa by steric hindrance. This study identifies a useful probe for TF function and provides insights into the inhibitory mechanism of an unusual class of antibody proposed for therapeutic intervention in thrombotic disease.

Human fibroblast tissue factor is inhibited by lipoprotein-associated coagulation inhibitor and placental anticoagulant protein but not by apolipoprotein A-II

Studies of proteins that inhibit tissue factor activity have generally been conducted using either an extracted tissue homogenate (“thromboplastin”) or tissue factor protein reconstituted into phospholipid vesicles rather than with tissue factor expressed in cell membranes (its physiological environment). In the present study, a human fibroblast cell strain was used to evaluate the effects of lipoprotein associated coagulation inhibitor (LACI), placental anticoagulant protein (PAP), and apolipoprotein A-II (apo A-II) on human tissue factor in cell membranes. LACI was tested from 7.8 to 500 pmol/L on fibroblasts cultured at cell densities ranging from 3,500 to 9,925 cells/well, and caused a progressive inhibition of tissue factor activity. PAP was tested from 3.9 nmol/L to 1 mumol/L at cell densities ranging from 4,500 to 15,400 cells/well and caused up to 83% inhibition of tissue factor activity. Inhibition by these proteins appeared to be influenced by cell density as well as whether the cells were intact or disrupted. Apo A-II, up to 1 mumol/L, did not inhibit the tissue factor activity of intact or disrupted fibroblasts at any cell density examined even though it did inhibit the activity of tissue factor in phospholipid vesicles. Of these inhibitors of tissue factor-dependent activation of factor X, LACI was the most effective in suppressing the generation of factor Xa activity. The effects obtained with apo A-II are clearly dependent on the nature of the tissue factor preparation with which it is tested. The disparity between the inhibitory effect of apo A-II on the activity of tissue factor reconstituted into lipid vesicles and the absence of effect on the activity of tissue factor remaining in cell membranes serves to reemphasize the necessity of reexamining results obtained with model systems using as nearly physiological reagents as possible.