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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1121-1130.)
© 1995 American Heart Association, Inc.

Articles

Tissue Factor Pathway Inhibitor Activity Associated With LDL Is Inactivated by Cell- and Copper-Mediated Oxidation

Philippe Lesnik; Christine Dentan; Alin Vonica; Martine Moreau; M. John Chapman

From the Institut National de la Santé et de la Recherche Médicale, Unité de Recherches sur les Lipoprotéines et l'Athérogénèse, Hôpital de la Pitié, Paris, France.

Correspondence to Philippe Lesnik and M. John Chapman, Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Recherches sur les Lipoprotéines et l'Athérogénèse, U-321, Pavillon Benjamin Delessert, Hôpital de la Pitié, 83, Bd de l'Hôpital, 75651 Paris Cedex 13, France.

	Abstract

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Abstract Human plasma contains a multivalent, Kunitz-type proteinase inhibitor termed tissue factor pathway inhibitor (TFPI), which is a specific inhibitor of the action of the factor VII(a)–tissue factor complex in coagulation. A major fraction of plasma TFPI is transported in association with LDL. Because LDL may undergo oxidation in the arterial wall during atherogenesis, we examined the effect of copper- and cell-mediated oxidative modification on TFPI activity associated with LDL. Oxidation mediated by copper ions resulted in a significant inactivation of LDL-associated TFPI (60% to 72% at 24 hours with 2.5 µmol/L CuCl₂). The inactivation of TFPI was strongly negatively correlated with both an increase in the net electrical charge of LDL (r=-.80, P.0001) and with the production of thiobarbituric acid–reactive substances (r=-.78, P.0001) and lipid peroxides (r=-.80, P.0001). Cell-mediated oxidation, involving incubation of LDL for 48 hours with either monocyte-like THP1 cells or human monocytes in Ham's F-10 medium, effected a significant decrease (64% and 75%, respectively) in LDL-associated TFPI activity. By contrast, prolonged exposure of LDL to purified soybean lipoxygenase (5000 U/mL) was less effective in inactivating TFPI (47% reduction after incubation for 72 hours at 37°C). We subsequently investigated the mechanism(s) that may underlie such inactivation. Oxidation of LDL is accompanied by the generation of various aldehydes, including malondialdehyde and 4-hydroxynonenal. Chemical modification with these aldehydes revealed a significant inverse correlation between the progressive loss of TFPI activity and both the increase in net electrical charge (r=-.93, P.0001) and the derivatization of free amino acid residues of LDL (r=-.90, P.0001). Specific chemical modification of lysine amino groups by acetylation similarly led to inactivation of LDL-associated TFPI activity. TFPI activity was almost totally abolished (<1.4%) when the TNBS reactivities of acetylated LDL, malondialdehyde-modified LDL, and 4-hydroxynonenal–modified LDL were 31%, 21%, and 43% that of native LDL, respectively. Our data demonstrate that expression of LDL-associated anticoagulant activity is markedly decreased as a consequence of the oxidative process, and suggest that the progressive aldehydic derivatization of apo B of LDL, and of the associated TFPI protein, may contribute to this phenomenon. Because tissue factor is overexpressed in the atheromatous plaque, it may exert a marked local procoagulant effect. The oxidative inactivation of LDL-associated TFPI will therefore effectively neutralize its inhibitory action on tissue factor activity, resulting in a disequilibrium in favor of coagulation.

Key Words: anticoagulant activity • thrombosis • monocytes

	Introduction

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TFPI* is a natural blood coagulation inhibitor that is confined to at least four different pools; one is associated with the endothelial cell surface, a second is stored in platelets, a third is carrier free, and a fourth is bound to circulating lipoprotein particles.^{1 2} More than 90% of the TFPI in the plasma of normal individuals circulates in association with lipoproteins.⁵ Among the plasma lipoproteins, some 40% to 80% of TFPI in plasma is transported by apo B–containing particles, principal among which are LDLs.^{5 6 7 8 9} Circulating TFPI, which exists primarily in two major forms of M_r 35 kD and 41 kD, appears to afford protection against thrombosis.^{10 11} Indeed, TFPI is a Kunitz-type protease inhibitor that regulates extrinsic pathway–induced coagulation by blocking the proteolytic activity of factor Xa and factor VIIa/TF.¹²

It is becoming increasingly clear that the oxidative modification of Nat-LDL underlies the atherogenicity of these cholesterol-rich particles.^{13 14} Among the factors capable of inducing the oxidative modification of LDL are metal ions such as Cu²⁺,¹⁵ cellular lipoxygenase,^{16 17 18} soybean lipoxygenase,^{19 20} endothelial cells,²¹ smooth muscle cells,²² and monocyte-macrophages.^{23 24} The cell-mediated oxidative modification of LDL is dependent on the presence of small amounts of copper or iron in the medium and can be prevented by exposure to chelators of these metals.^{21 22} In this context, it is relevant that significant amounts of copper have been detected in atherosclerotic lesions.²⁵

The oxidative modification of LDL alters its biologic properties, leading to endocytic uptake by macrophages by means of scavenger receptors²⁶ ; such uptake results in cellular cholesterol accumulation and foam cell formation. Equally, Ox-LDLs are chemotactic to monocytes²⁷ and, in addition, are cytotoxic to endothelial cells and smooth muscle cells.²⁸ Furthermore, Ox-LDLs can activate multiple functions of macrophages and endothelial cells that may contribute to the formation of atherosclerotic lesions.²⁹

TFPI activity in plasma is closely correlated with LDL cholesterol and apo B levels.^{9 30} Moreover, therapeutic lowering of LDL cholesterol in hypercholesterolemia decreases TFPI level without affecting free TFPI concentrations. These observations indicate a strong biologic association between apo B and TFPI. In this context, the association of TFPI with LDL may be of considerable significance with respect to the thrombotic state potentially associated with atherosclerotic plaques. Indeed, TF is overexpressed in human atherosclerotic plaques and in foam cells.^{31 32} Because TFPI-bearing LDL may penetrate the subendothelial space,^{33 34} the accumulation of such LDL in the intima might therefore exert an antithrombotic effect. Several lines of evidence suggest that the biologic oxidation of LDL occurs within the arterial wall.¹⁴ Because the oxidation of LDL results in profound alteration of both its lipid and protein components, including the hydrolysis of phospholipids, loss of esterified cholesterol, the covalent modification of apo B by the decomposition products of lipid peroxidation such as MDA and 4-HNE, and the fragmentation of apo B, our aim was to evaluate whether LDL-associated TFPI activity might be affected by cell-, copper-, or lipoxygenase-mediated LDL oxidation. Our present findings suggest that the oxidative inactivation of LDL-associated TFPI may occur in vivo in the arterial wall as a consequence of the formation of reactive aldehydes; such aldehydes may modify lysine and/or other amino acid residues of LDL-associated TFPI, resulting in alteration of the structural and functional properties of TFPI.

	Methods

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Materials
Purified coagulation factors were of human origin except for TF, which was prepared from rabbit brain (Sigma Diagnostics). Factors VIIa, X, and Xa and rabbit polyclonal antibody against factor X were obtained from Diagnostica Stago. These reagents were dissolved according to the instructions of the manufacturer and further diluted in a buffer (TSC buffer) containing 0.05 mol/L Tris-HCl, 0.1 mol/L NaCl, 0.01 mol/L sodium citrate, and 0.02% NaN₃. BSA (Sigma) was added to this solution (final concentration, 2 mg/mL) to constitute TSC/BSA buffer. Rabbit TF was adjusted to optimal concentration with distilled water, and under such conditions the clotting time of normal plasma was 16 seconds (equal volumes of plasma, 35 mmol/L CaCl₂, and TF). The chromogenic substrate S2222 (N-benzoyl-Ile-Glu-Arg-p-nitroanilide; Kabi) was dissolved in distilled water at a final concentration of 2.7 mmol/L.

A rabbit polyclonal antibody to human TFPI was supplied by Novo Nordisk A/S. 4-HNE was the kind gift of Dr Gree (University of Rennes, France) and Dr F. Bellamy (Fournier Laboratories). Purified lipoxygenase type V from soybean and BHT were obtained from Sigma. CHOD iodide was obtained from Merck.

Purification of LDL
The most abundant subspecies of LDL (d=1.024 to 1.050 g/mL) in normolipidemic human plasma was isolated by sequential preparative ultracentrifugation.³⁵ All density solutions contained EDTA (0.01%) and gentamicin (0.005%) at pH 7.4. To avoid any contribution of Lp(a) to LDL preparations, and because significant amounts of Lp(a) may be present over the density interval corresponding to dense LDL (d=1.050 to 1.063 g/mL),⁸ we chose an upper limiting density interval of 1.050 g/mL for LDL isolation. It is relevant that LDLs of d=1.024 to 1.050 g/mL contain approximately 56% of the total TFPI activity associated with LDLs of d=1.019 to 1.063 g/mL.⁸ The purity and integrity of LDL and apo B were established on the basis of criteria described earlier.³⁶ In this way, the potential contamination of LDL with other lipoproteins and plasma proteins was excluded. The concentrations of lipids (total cholesterol, 187±39 mg/dL; total triglycerides, 56±17 mg/dL) in plasmas used for LDL isolation corresponded to values typical of a normolipidemic population.³⁵ The mean concentration of Lp(a) was 13±10 mg/dL plasma. The isolated LDL were dialyzed in Spectrapor membrane tubing (M_r cutoff, 3.500) against 0.01 mol/L PBS containing 0.3 mmol/L EDTA at pH 7.4, filtered through a 0.22-µm filter, and stored at 4°C. The protein content of lipoprotein fractions was determined by the procedure of Lowry et al³⁷ with BSA (Sigma) used as the working standard.

Chemical Modification of Lipoproteins
Ac-LDLs were prepared according to the procedure of Basu et al³⁸ ; ratios between 5 and 40 moles of acetic anhydride per mole of lysine were used. LDLs were modified with 4-HNE–LDL under reducing conditions as described by Palinski et al.³⁹ 4-HNE, at concentrations ranging from 0.31 to 10 mmol/L (determined by absorbance at 222 nm, with =13 600 L · mol^-1 · cm^-1), was incubated with LDL for 5 hours at 37°C. MDA-LDL was prepared by incubation of LDL for 3 hours at 37°C with MDA concentrations ranging from 3 to 50 mmol/L (final concentration) according to the protocol of Palinski et al.³⁹

Before oxidation, LDLs were dialyzed against 0.01 mol/L PBS at pH 7.4 and at 4°C to remove EDTA. Copper-oxidized LDLs were prepared by incubation of 0.5 mg LDL protein/mL with 2.5 µmol/L of aqueous CuCl₂ in PBS at 37°C for 0 to 48 hours. In control experiments, BHT was added at a final concentration of 60 µmol/L. Ox-LDL and modified LDL were extensively dialyzed against PBS containing 3 mmol/L EDTA to remove unreacted chemical products.

As described in "Results," Ox-LDLs used in dot blot analysis for immunological detection of associated TFPI were either reisolated by ultracentrifugation and then dialyzed against PBS-EDTA or were dialyzed only without prior ultracentrifugation.

Monocyte Isolation and Culture
Human monocytic THP1 cells obtained from the American Type Culture Collection (TIB 202) were cultured in RPMI 1640 (Eurobio) containing 20 µmol/L 2-mercaptoethanol, 10% fetal calf serum (Eurobio), and 40 µg/mL of gentamycin. Cell viability was assessed by the trypan blue exclusion method.

Buffy coat cells were separated from the fresh anticoagulated blood of healthy normolipidemic volunteers and the subsequent isolation of monocytes was performed as described by Rouis et al.⁴⁰ Cell viability was typically more than 95% by the trypan blue exclusion method.

Cell-Induced Modification
THP1 cells and freshly isolated human monocytes were washed three times with Ham's F-10 medium (Eurobio) to remove serum. Cells were plated at 3x10⁶ cells/35x10-mm dishes (Primaria) in 1.0 mL Ham's F-10 supplemented with gentamycin (40 µg/mL). LDLs were added to the culture medium to a final concentration of 0.5 mg LDL protein/mL, and the medium was supplemented in defined experiments with CuCl₂ (2.5 µmol/L). Incubation was then carried out for 0 to 48 hours (unless otherwise indicated) at 37°C in a humidified atmosphere containing 5% CO₂. Control dishes, from which either cells or LDLs were omitted, were incubated under identical conditions. At the end of the incubation period, EDTA (3 mmol/L) was added to stop oxidation. The media and the cells were immediately separated by centrifugation at 500g for 10 minutes at 4°C. Cell-free culture supernatant was divided into two parts. In the first portion, the medium content of TBARS was measured. The second portion was adjusted to a density of approximately 1.060 g/mL with NaCl-KBr solution containing EDTA (0.03 mmol/L) and ultracentrifuged in a TLA-100.3 rotor at 100 000 rpm in a Beckman TL100 ultracentrifuge for 210 minutes at 10°C. LDL was recovered as a 1-mL fraction at the meniscus of the tube and extensively dialyzed against PBS-EDTA (0.3 mmol/L). The electrophoretic mobility and TFPI activity of such isolated LDL fractions were measured. To verify whether THP1 cells and monocytes secreted TFPI into the medium under our experimental conditions, we performed control experiments without LDL. Cell-derived TFPI might bind to LDL and therefore lead to underestimation of its oxidative inactivation. The cells and supernatant in control dishes were subsequently collected and the cells disrupted by sonication; the possible content of TFPI was then evaluated in both cells and supernatant. We did not detect TFPI antigen in either THP1 or human monocyte supernatants (data not shown).

Assessment of LDL Modification
The time course of the copper-induced oxidation of LDL (62 µg protein/mL) in PBS containing 2.5 µmol/L CuCl₂ was monitored continuously, for periods of up to 6 hours, by the formation of conjugated dienes measured as the increase in absorbance at 236 nm.¹⁵ Kinetic studies of the formation of lipid peroxides during copper-induced oxidation were determined by use of the lipid peroxide assay of El-Saadani et al.⁴¹ The principle of this assay is based on the oxidative capacity of lipid peroxides to convert iodide to iodine, which can be measured spectrophotometrically at 365 nm. Lipid peroxidation products of copper- and cell-oxidized LDL were estimated before dialysis as the fluorescent products obtained upon reaction with thiobarbituric acid as described by Buege and Aust⁴² ; results are expressed as nanomolar equivalents of MDA per milligram of LDL protein. The content of nonmodified amino groups in Ac-LDL, 4-HNE–LDL, MDA-LDL, and Nat-LDL were estimated by use of TNBS.⁴³ TNBS reactivity was not determined in experiments conducted in Ham's F-10 culture medium to avoid interference by free amino groups present in components of the medium. Because we observed that the TNBS reactivity and the electrophoretic mobility of LDL evolved in parallel in cell-mediated oxidation studies, we determined the electrophoretic mobility of LDL as a measure of the degree of protein modification. The total (net) electrical charge on both Nat-LDL and modified LDL was assessed by electrophoresis in agarose gel (Corning).⁴⁴ The electrophoretic mobility of modified LDL was compared with that of the Nat-LDL from which it was derived and expressed as the REM.

Assay of TFPI Activity
TFPI activity was measured by a slight modification of the method described by Sandset and coworkers.^{2 7 45} The assay was performed in microtitration plates in a 37°C water bath. Triplicate aliquots (25 µL) of the sample of LDL or standard were first distributed into wells. The amidolytic assay was performed in two stages by incubation of 100 µL of combined reagent I for 20 minutes followed by incubation of 50 µL of combined reagent II for 25 minutes. Reagent I contained TF, factor Xa, factor VIIa, and CaCl₂ at final concentrations of 1% (vol/vol), 5 mU/mL (0.8 nmol/L), 2.5 mU/mL (25 pmol/L), and 15 mmol/L, respectively. Reagent II was a mixture (vol/vol) of factor X (0.4 U/mL; 60 nmol/L) and S2222 (2.7 mmol/L). The reaction was stopped by the addition of 50 µL of 50% acetic acid. Absorbance was read at 405 nm by use of a microtiter plate reader (Dynatech). We prepared standard curves by assaying dilutions (0% to 1%) of pooled, heated, citrated plasma diluted in TSC/BSA buffer containing 2 µg/mL hexadimethrine bromide (Sigma) to prevent any influence of heparin, as described earlier.⁴⁵ TFPI activity in 1 mL of plasma was arbitrarily defined as 100% activity (or 1 U/mL). A plot of TFPI activity (as OD=405 nm) as a function of progressive dilutions of a pooled plasma sample was used for calibration purposes. TFPI activity in LDL was then expressed relative to the protein content of each Nat-LDL or modified LDL. Our dilutions ranged from 0 to 25 pmol/L in our standard when a pooled plasma was used at a final concentration of 2.5 nmol/L.⁴ When samples were incubated with a specific polyclonal antibody to human TFPI, proteolytic cleavage of S2222 was not inhibited, demonstrating the TFPI dependence of the reaction.

Dot Blot Analyses of Lipoprotein-Associated TFPI
Lipoprotein samples (40 µg LDL protein), taken either before or after ultracentrifugation, were applied to wells of a Bio-Dot apparatus (Bio-Rad). The nitrocellulose membranes were first equilibrated in 0.01 mol/L PBS buffer containing EDTA (3 mmol/L) at pH 7.4. Samples were blotted onto the nitrocellulose membrane and subsequently washed three times with PBS/EDTA. Immunoblotting was used as a method for detection of TFPI in the various preparations of Nat-LDL and Ox-LDL. The nitrocellulose paper was blocked with 2.5% nonfat milk in TS buffer (0.05 mol/L Tris HCl, 0.15 mol/L NaCl) at pH 7.5 for 1 hour at room temperature and incubated overnight at 4°C either with a rabbit polyclonal anti-TFPI IgG (diluted 1:2500) or with factor Xa (diluted 1:500) and a rabbit polyclonal anti–factor X IgG (diluted 1:1500) for 2 hours. All antibodies were diluted in a buffer containing 0.05 mol/L Tris HCl, 0.15 mol/L NaCl, and 0.17% BSA. The nitrocellulose papers were washed three times between each incubation in TS buffer containing 2.5% nonfat dry milk; the blots were subsequently incubated for 1 hour with a goat anti-rabbit horseradish peroxidase–conjugated IgG (diluted 1:10 000). A chemiluminescent substrate of this enzyme was used to reveal the presence of TFPI.⁴⁶ Control wells were not incubated with the anti-TFPI and anti–factor X antibodies. Quantitation of relative peroxidase activities was assessed by densitometry (Preference, Sebia, Paris). No background absorbance was detected in control wells.

Statistical Analysis
Statistical analysis was by Student's t test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Copper-Induced Oxidation on LDL-Associated TFPI Activity
Fig 1 shows the time course of LDL oxidation at 37°C in PBS in the presence of 2.5 µmol/L CuCl₂. Our data demonstrate a significant decrease (60% to 72% at 24 hours) in TFPI activity in each of the three different LDL preparations isolated from three donors (Fig 1A); reduction (16% to 19%) in TFPI activity was detected as early as the first hour of incubation. In parallel, the REM of Ox-LDL on agarose gel was increased up to approximately fourfold compared with that of Nat-LDL (Fig 1B). Finally, the generation of lipid peroxides, and products of peroxidation (MDA and other aldehydes) were estimated by the lipid peroxide assay of El-Saadani et al⁴¹ (Fig 1C), and the TBARS assay of Buege and Aust,⁴² respectively (Fig 1D). Rapid lipoperoxide and TBARS production occurred within the first 4 hours of oxidation, reaching a maximum between 10 and 15 hours (275 to 350 nmol lipid peroxides/mg LDL protein and 80 to 100 nanomolar equivalents of MDA per milligram of LDL protein) and decreasing thereafter. Within the first 15 hours of oxidation, a strong negative correlation was detected between the degree of LDL oxidation and the decrease in TFPI activity. Indeed, correlation coefficients of r=-.80 (P.0001), r=-.78 (P.0001), and -r=.80 (P.0001) were found for the relationships between decrease in TFPI activity and the increase in lipid peroxides, TBARS content, and relative electrophoretic mobility, respectively. Moreover, because we observed that the generation of lipid peroxides and products of peroxidation evolved in parallel, we thereafter determined only TBARS content as a parameter of lipid oxidation in LDL.

View larger version (20K): [in this window] [in a new window]   Figure 1. Graphs show results of kinetic studies of the copper-stimulated oxidation of human LDL and the effect on LDL-associated TFPI activity (A), REM of LDL (B), lipid peroxide content of LDL (C), and TBARS content of LDL (D). LDLs were isolated from three different normolipidemic human plasmas and are represented by symbols (, , and ). LDLs (0.5 mg protein/mL) were incubated at 37°C in the presence of 2.5 µmol/L CuCl2 in 0.01 mol/L PBS, pH 7.4. Control LDLs that were not treated with CuCl2 had TFPI activity similar to that of LDLs treated with CuCl2 at time 0. Each data point represents the mean of duplicate determinations for each LDL preparation.

Several additional experiments were performed to establish whether the oxidative mechanism was indeed responsible for the observed decrease in TFPI activity. Incubation of LDL for 24 hours under the same conditions as described above (PBS containing 2.5 µmol/L CuCl₂ at pH 7.4 and 37°C) but in the presence of a lipophilic antioxidant, BHT (60 µmol/L), which is known to protect LDL from oxidation (REM=1.20±0.1; TBARS=12±4 nanomolar equivalents of MDA per milligram of LDL protein), resulted in approximately 91% (n=3) recovery of TFPI activity compared with the initial activity. Moreover, TFPI activities associated with LDL incubated for 24 hours in the absence of CuCl₂ were similar to those observed at time 0. These results argue indirectly for a non–temperature- and a non–time-dependent loss of LDL-associated TFPI activity during our experiments performed at 37°C for up to 24 hours. These correlations were strengthened by the observation that the LDL preparation that showed the most rapid onset of oxidation (Fig 1B, 1C, and 1D) also showed a similar rapid loss in TFPI activity (Fig 1A). It is equally important to note that the susceptibility to oxidation differed, as assessed by continuous monitoring of conjugated diene formation for periods of up to 6 hours, among individual LDL preparations (data not shown). Thus, preparations that were most resistant to copper-induced oxidation were equally most resistant to TFPI inactivation. Furthermore, the TNBS reactivity of the protein moiety of LDL decreased progressively with copper-mediated oxidation from 91.2±2.2% at 1 hour to 72.9±8.2% at 5 hours and to 50.5±8.7% at 24 hours of incubation.

To assess whether TFPI was released from LDL particles during the process of oxidation and lost after the subsequent reisolation and dialysis of the LDL samples, we performed dot blotting followed by immunodetection. The presence of TFPI protein was detected in Ox-LDL by polyclonal anti-TFPI IgG or by the functional capacity of TFPI to bind factor Xa, which was then revealed by a polyclonal anti–factor X IgG (Fig 2). No significant loss of the immunological reactivity of TFPI occurred either during reisolation (Fig 2; before and after ultracentrifugation) or during the short time period of oxidation (12 hours), whereas there was already a progressive loss in the recognition of TFPI by factor Xa that became significant after 8 hours of oxidation: approximately 40% (P.03) and approximately 80% (P.006) of immunoreactivity was lost after 8 hours and 12 hours of oxidation, respectively. In immunoblots in which an anti–TFPI IgG was used, the densitometric absorbance decreased progressively and significantly with prolonged exposure of LDL to copper oxidation (60% of immunoreactivity was lost after 24 hours; P.01).

View larger version (15K): [in this window] [in a new window]   Figure 2. Graph shows quantitative association of TFPI bound to Nat-LDL and Ox-LDL, assessed by densitometric scanning of immunoblots. Oxidation of LDL was performed as described in Fig 1 legend. Aliquots of LDL (40 µg protein) were applied to each well and allowed to blot after undergoing extensive washing with a vacuum system (see "Methods"). The TFPI content of individual fractions was immunodetected as described in "Methods." Squares represent immunodetection of TFPI antigen in LDL by a polyclonal anti-TFPI IgG. Circles represent the functional capacity of TFPI to bind to factor Xa, which was then revealed by a polyclonal anti–factor X IgG. 100% represents the densitometric absorbance of Nat-LDL blots. and indicate LDLs that were not ultracentrifuged after oxidation; , LDLs that were reisolated by ultracentrifugation and dialyzed. Values are the means (n=3) of duplicate determinations. Student's t test was calculated for LDL recognized by anti-TFPI IgG versus LDL recognized by anti–factor X IgG (*P.03, **P.006) and for LDL recognized by anti-TFPI IgG before and after 24 hours of oxidation (***P.01).

Relationships Between Chemical Modification of LDL and LDL-Associated TFPI Activity
Subsequent experiments were designed to obtain more insight into the mechanism of the oxidative reduction in TFPI activity. During oxidation, the lipid and protein moieties of LDL are chemically modified. To discriminate between lipid-dependent or protein-dependent inactivation of LDL-associated TFPI activity, we conducted specific chemical modifications that principally affect the protein moiety, and more precisely the free amino groups of lysine, histidine, and arginine residues. These chemical modifications were acetylation, MDA modification, and 4-HNE modification^{38 47 48 49 50} (Table 1). Modification of LDL was assessed by the increase in electrophoretic mobility and by the decrease in TNBS reactivity. The TBARS content was also verified after extensive dialysis of modified LDL. The results in Table 1 indicate that each of the three types of modification affects TFPI activity. Furthermore, the degree of LDL acetylation as assessed by electrophoretic mobility (1.45 to 4.71, respectively) and TNBS reactivity (free amino groups, 84.8% to 31%, respectively) is proportional to the loss of TFPI activity (60.8% to 1.4%, respectively). Progressive modification with increasing concentrations of MDA also resulted in a loss in LDL-associated TFPI activity (42.9% to 0.6%, respectively). Finally, we performed a third chemical modification with 4-HNE, which led to a dramatic diminution of LDL-associated TFPI activity of almost 100%. Because TFPI activity progressively decreased as a function of the degree of LDL oxidation in vitro (Fig 1), we investigated the potential relationship between TFPI inactivation and the progressive chemical modification of amino acid residues with 4-HNE. The results (Fig 3A and 3B) indicate a strong and negative correlation between LDL-associated TFPI inactivation and both the electrophoretic mobility (r=-.93; P.0001) and TNBS reactivity (r=.80; P.0001) of LDL. In Table 1, for the same TNBS reactivity (44%) of Ac-LDL, MDA-LDL, and 4-HNE–LDL, both the relative electrophoretic mobilities (3.14, 1.92, and 2.05, respectively) and the percentage of TFPI activity (28.5%, 3.2%, and 0.4%, respectively) were distinct. The discrepancies between the degree of charge modification of LDL on the one hand and of the loss of TFPI activity on the other may possibly be accounted for by differences in the amino acid residues that constitute the targets for each of these chemical modifications.

View this table: [in this window] [in a new window]   Table 1. Chemical Modification of LDL and Effect on LDL-Associated TFPI Activity

View larger version (16K): [in this window] [in a new window]   Figure 3. Graphs show TFPI activity associated with LDL modified with 4-HNE. LDLs (1 mg protein/mL) isolated from three separate donors were incubated for 5 hours at 37°C with different concentrations of 4-HNE ranging from 0.31 to 10 mmol/L (final concentration) under reducing conditions. Modified LDLs were then extensively dialyzed against PBS containing 3 mmol/L EDTA. TFPI activity was determined on dialyzed samples of 4-HNE–LDL. REM (A) and TNBS reactivity (B) were determined as described in "Methods." LDL incubated with 10 mmol/L 4-HNE for 5 hours was partially aggregated; however, the REM could be measured for a small quantity that migrated from the origin during electrophoresis. Values are the means of triplicate determinations (except for REM, for which duplicate determinations were made).

Effect of Cell-Induced Oxidation on LDL-Associated TFPI Activity
Because TFPI activity decreased as a function of copper-mediated LDL oxidation in vitro, we performed similar experiments in the presence of both human monocytes (Table 2) and a monocyte-like cell line, THP1, in culture (Fig 4). Cultured human monocytes and THP1 cells were chosen because TFPI is not secreted constitutively by these cells⁵¹ (control, Fig 4A and 4D). LDLs were incubated in Ham's F-10 medium without any addition, or supplemented with 2.5 µmol/L CuCl₂, and in the presence or absence of THP1 cells (Fig 4). After the incubation period, LDLs were reisolated from the cell supernatants by ultracentrifugation and then extensively dialyzed. This step allowed quantification of TFPI activity associated with LDL. The TFPI activity contained in LDL incubated with THP1 cells decreased slowly to approximately 21% of the initial value over a 72-hour period of incubation (Fig 4A), and was inversely related to the parameters of oxidation (Fig 4B and 4C). Diminution of TFPI activity was also observed, although to a significantly lesser degree (43% at 72 hours), when LDL were incubated with Ham's F-10 medium alone; this medium is known to contain micromolar amounts of iron and nanomolar amounts of copper ions. Such levels of transition metal ions cause only minimal oxidation of LDL in the absence of cells, as already shown by Steinbrecher et al.²¹ Quantification of TBARS in the incubation medium (Fig 4C) revealed a rapid increase between 24 hours and 48 hours in the presence of THP1 cells with a maximum at 48 hours.

View this table: [in this window] [in a new window]   Table 2. Human Monocyte–Induced Modification of LDL and Effect on LDL-Associated TFPI Activity

View larger version (23K): [in this window] [in a new window]   Figure 4. Graphs show time course of THP1 cell-mediated oxidation of LDL and effect on LDL-associated TFPI activity (A and D), REM (B and E), and TBARS content of LDL (C and F). The cellular modification of LDL was performed by incubation of 0.5 mg LDL protein from 0 hours up to 72 hours at 37°C with monocyte-like cells (THP1) (in six-well plates) in Ham's F-10 medium (1 mL/well) supplemented with gentamicin (40 µg/mL) but in the absence (A, B, and C) or in the presence (D, E, and F) of CuCl2.Values are mean±SD of duplicate determinations from three distinct experiments. indicates LDL incubated with cells; , LDL incubated without cells; and , a control experiment containing cells but in the absence of LDL. *P.036, **P.022, ***P.003 for LDL incubated with or without cells.

The reduction in TFPI activity and the evolution in parameters of oxidation upon incubation of LDL with cells in the presence of added copper ions were more rapid; thus, in the presence of CuCl₂ and cells, TBARS content reached a plateau at 24 hours (Fig 4F), with the minimum of LDL-associated TFPI activity being attained within 24 hours (Fig. 4D). The electrophoretic mobility of LDL oxidized by cells and added copper was elevated (REM=3) after incubation for 48 hours and was comparable to that in the presence of cells alone after incubation for 72 hours (Fig 4B and 4E). Oxidation of LDL with CuCl₂ was slower in Ham's F-10 medium (Fig 4 control without cells) than in previous experiments in PBS and in the presence of copper ions (Fig 1), possibly because of the presence of some components that may act as antioxidants in the culture medium. The oxidation parameters measured under these conditions (Fig 4B, 4C, 4E, and 4F) always paralleled the decay in TFPI activity (Fig 4A and 4D).

Finally, no contribution of cellular synthesis to TFPI activity was observed during the prolonged incubation period (Fig 4A and 4D). Furthermore, to investigate whether human monocytes might oxidize and inactivate TFPI, we performed experiments similar to those with THP1 cells but in which human monocytes derived from circulating blood were used. In Table 2, we show that LDL-associated TFPI activity in the presence of monocytes was reduced by approximately 75% at 48 hours, and that both the electrophoretic mobility and the TBARS content of LDL were elevated, involving a twofold increase in REM and a fivefold increase in TBARS content.

LDL-Associated TFPI Activity During Lipoxygenase-Mediated Modification
Previous studies have indicated that lipoxygenase-dependent pathways may be of physiological relevance to LDL oxidation. Indeed, LDL oxidation mediated by cells of the atherosclerotic lesion may implicate lipoxygenase activity.^{16 17 18} Because soybean lipoxygenase may catalyze transformation of LDL to oxidized forms, we performed a set of experiments with this lipoxygenase (Fig 5).²⁰ Modification was carried out in PBS medium free of copper or iron ions, and was done in the presence of soybean lipoxygenase. The time course of enzymatic modification demonstrated a slow but significant loss (47%) of LDL-associated TFPI activity (Fig 5A). Such inactivation was inversely correlated with elevation in electrophoretic mobility (Fig 5B) (r=-.90, P.01) and in TBARS production (Fig 5C) (r=-.93, P.006).

View larger version (12K): [in this window] [in a new window]   Figure 5. Graphs show time course of oxidative modification of LDL mediated by soybean lipoxygenase and effects on LDL-associated TFPI activity (A) and on parameters of LDL oxidation (REM [B] and TBARS [C]). Three different LDL preparations (0.5 mg protein/mL) were incubated in PBS with 5000 U/mL of lipoxygenase type V from soybean at 37°C for various periods of time in PBS. Values are mean±SD of duplicate determinations. indicates LDL incubated in the absence of lipoxygenase; , LDL incubated in the presence of lipoxygenase. *P.09, **P.04.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present studies demonstrate for the first time that biologic oxidation of LDL leads to a significant decrease in associated TFPI activity, thereby suggesting that Ox-LDL lose their capacity to inhibit the extrinsic pathway of coagulation initiated by TF. These investigations involved use of a cell-free system containing copper ions, cellular systems of cultured THP1 cells and of human monocytes, and an enzymatic system involving a purified soybean lipoxygenase in copper-free medium. All oxidation systems effected a progressive and marked loss of TFPI activity that was correlated with increase in the net negative electric charge, with decrease in total free amino groups and increase in the TBARS content of LDL. Insight into the mechanism that underlies TFPI inactivation during oxidation was obtained by chemical modification of LDL by acetylation and with MDA and 4-HNE. Such specific modifications principally affect the protein moiety of LDL, ie, apo B100. The progressive chemical derivatization of free amino groups of apo B100 by acetylation and by free aldehydes led to a parallel loss of TFPI activity. All these modifications affected TFPI activity, suggesting that inactivation of TFPI may occur in vivo. Indeed, it is known that the oxidation of LDL that may occur during atherogenesis involves several complex mechanisms,⁵² and that such oxidative modification may be mediated by various cell types present in the arterial wall.¹⁴

Earlier studies demonstrated that modification of LDL by oxidative processes or by specific chemical modification (by acetylation or with MDA or 4-HNE) lead to increase in the electronegativity of apo B100 in the LDL particle. One of the characteristics of the oxidation of LDL is the production of aldehydes such as MDA and HNE as a result of the decomposition of lipid hydroperoxides.^{53 54 55} Both HNE and MDA interact with exposed lysine residues of apo B.^{50 55 56} When 15% to 20% or more of the lysine residues in apo B are blocked by MDA or HNE, recognition by the scavenger receptor occurs.^{50 54 57 58 59} Equally, acetylation of LDL neutralizes the positive charge of -amino groups of lysine, and thereby converts a weakly anionic lipoprotein into a strongly anionic one.³⁸ Our results also argue for an alteration of a physiological function associated with LDL particles but distinct from the physiological role of their major apolipoprotein component, apo B100. Indeed, we demonstrated that during the oxidative process and during chemical modification, the anticoagulant activity associated with LDL is subject to dramatic inactivation. Furthermore, our data indicate that the decrease in TFPI activity that we observed is independent of the nature of the modification, but is always correlated with changes in the electrophoretic mobility of LDL (which correlated with decrease in reactive amino groups^{54 58} ) and TBARS production. Such loss of TFPI activity may be related directly to derivatization of its amino groups by lipid adducts that neutralize the positive charge on this protein. Because oxidation leads to an alteration in electric charge, thereby resembling modification by acetylation or with MDA or 4-HNE, it seems reasonable to suspect that lysine residues may be directly involved in TFPI inactivation.

In the three experimental modifications we used, total inactivation of TFPI associated with Ac-LDL, MDA-LDL, and 4-HNE–LDL was linked to distinct electrophoretic mobilities (4.71, 3.76, and 2.05, respectively; Table 1), and to the degree of free amino group modification (31%, 21.5%, and 43.6%, respectively). Such differences may be explained by the distinct nature of the chemical modifications, because they affect several amino acid residues (lysine, arginine, histidine, and tyrosine) in different relative proportions.^{49 50 56} Indeed, not only lysine but also tyrosine, arginine, and to a minor extent histidine are involved in the formation of HNE-derived epitopes on apo B100.^{48 49} In addition, Fong et al⁶⁰ found that copper ion–induced oxidation resulted in a consistent decrease in the histidine, lysine, and proline contents of apo B100.

The molecular features of the determinant(s) of the inactivation of TFPI have yet to be defined. TFPI is rich in lysine and arginine (9% and 5.5%, respectively).^{1 61} It is therefore tempting to speculate that specific cationic residues may be affected by oxidative and chemical modifications. The HNE modification of LDL led to a marked decrease in TFPI activity; nevertheless, HNE-mediated inactivation of TFPI activity was associated with an electrophoretic mobility lower (REM=2; Table 1) than that associated with Ac-LDL and MDA-LDL. This finding may be explained by the fact that HNE modification of LDL is drastic compared with that involving MDA.^{39 48 49 62} Thus, modification with HNE can lead to aggregation and formation of cross-links between LDL particles. Similarly, 4-HNE modification reacts with a large spectrum of amino acids as well as the phospholipid moiety of this cholesterol-rich lipoprotein.⁴⁹

Several experiments have demonstrated that inhibition of the factor VIIa/TF complex by TFPI involves the formation of a TF–factor VIIa–factor Xa–TFPI complex,¹ and that this inhibitory complex results from the initial and prerequisite binding of factor Xa to TFPI. The production of mutant forms of TFPI allowed the attribution of a specific role to each of the Kunitz-type domains 1 and 2. The active site clefts for each of the Kunitz-type inhibitory domains 1 and 2 are composed of lysine 35 and arginine 107.^{1 61} Under these conditions, the active site of TFPI contains the amino acids that are most sensitive to modification and may also be more exposed to the aqueous environment. This hypothesis is supported by the observation of Warn-Cramer et al,⁶³ who used cyclohexanedione to block positively charged arginine residues of TFPI. This modification blocked the binding of factor Xa to TFPI, thereby preventing the formation of the inhibitory complex with factor VIIa/TF.

Most studies of the biologic properties of LDL in atherogenesis have involved the use of lipoprotein preparations that have been subjected to strong oxidizing conditions. Indeed, in our experiments a loss of TFPI activity was clearly demonstrated in even "minimally modified LDL" (ie, LDL that had undergone short-term incubation with copper or lipoxygenase-modified LDL, which we assume to represent forms that are not recognized by scavenger receptors¹⁹ ). Our data are insufficient to determine the molecular details of TFPI-mediated inactivation during oxidation. Nevertheless, our results support the hypothesis that the enzymatic activity of TFPI is progressively lost as a result of the derivatization of accessible and essential cationic amino acids. Indeed, the TFPI activity (data not shown; see "Methods") and TFPI antigen (Fig 2) associated with minimally oxidized LDL were recovered either before or after ultracentrifugal separation. This experiment therefore excluded uncoupling of TFPI from the LDL particle as an explanation for the loss of TFPI activity. Moreover, the major decrease in TFPI activity occurred in the first 8 hours of oxidation (Fig 1), whereas the loss of TFPI–factor Xa binding was not significant until after 8 hours (Fig 2). Therefore, it seems unlikely that impaired TFPI–factor Xa interaction might account for the reduced TFPI activity in the early phase of oxidation.

Upon more extensive oxidation, we hypothesize, fragmentation of TFPI protein may lead to uncoupling of TFPI bound to LDL and/or to lack of recognition of TFPI by polyclonal antibodies as a result of the destruction of the native epitope structure. This hypothesis may indirectly argue that the native conformation of TFPI is required for its efficient association with LDL. An alternative hypothesis may result from modification of apo B or lipid components of LDL that give rise to altered surface structure in the particle and thus to impairment of TFPI binding.

Dense LDL particles, the major carriers of TFPI in plasma,⁸ are particularly sensitive to oxidative stress that may occur in the arterial intima and are in consequence potentially the most atherogenic subpopulation of LDLs.⁶⁴ Indeed, it is noteworthy that the penetration of the arterial wall by such lipoprotein particles is inversely proportional to their size.³³ Clearly, then, the infiltration of small LDLs into the intimal space and the inactivation of LDL-bound TFPI would result in the neutralization of the antithrombotic role associated with TFPI. These data emphasize the pathophysiological consequences of the loss of the protective, procoagulant role of LDL in the microenvironment of the atheromatous plaque.

In blood, Nat-LDL also transports PAF-degrading acetylhydrolase,⁶⁵ which controls the levels of PAF-acether and may regulate the biologic effects of this potent inflammatory and thrombotic mediator.⁶⁶ The loss of TFPI activity shown in this study and the loss of PAF-degrading acetylhydrolase activity in Ox-LDL recently demonstrated by our group⁶⁷ confers two new proatherogenic and prothrombogenic properties to these oxidized lipoproteins.

	Selected Abbreviations and Acronyms

 

4-HNE–LDL = 4-hydroxynonenal–modified LDL Ac-LDL = acetylated LDL MDA-LDL = malondialdehyde-modified LDL Nat-LDL = native LDL Ox-LDL = oxidatively modified LDL PAF = platelet-activating factor REM = relative electrophoretic mobility TBARS = thiobarbituric acid–reactive substances TF = tissue factor TFPI = tissue factor pathway inhibitor

	Acknowledgments

 
Dr Lesnik and Mlle Dentan were the recipients of a research fellowship from the French Ministry of Research and Technology. These studies were supported by INSERM and by the "Concerted Action" research grant of the European Community No. PL 931790. We are indebted to Dr J. Thillet for stimulating discussion and to C. Debets-Albertini (Centre Départemental de Transfusion Sanguine, Créteil, France) for the generous gift of thrombopheresis residues. We are most grateful to Novo Nordisk A/S for the gift of the anti-TFPI antibody.

	Footnotes

 
¹ The Scientific and Standardisation Committee of the International Society on Thrombosis and Hemostasis recommended use of the term "tissue factor pathway inhibitor." TFPI has been previously termed "lipoprotein-associated coagulation inhibitor,"¹ "extrinsic pathway inhibitor,"^{2 3} or "tissue factor inhibitor."⁴

Received February 3, 1995; accepted May 5, 1995.

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