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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:309-315.)
© 1999 American Heart Association, Inc.

Original Contributions

Tissue Factor Pathway Inhibitor Is Expressed by Human Monocyte–Derived Macrophages

Relationship to Tissue Factor Induction by Cholesterol and Oxidized LDL

Laure Petit; Philippe Lesnik; Christiane Dachet; Martine Moreau; M. John Chapman

From 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, Paris, France.

Correspondence to Dr 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—Lipid-laden macrophages express tissue factor (TF), which may activate the extrinsic coagulation pathway on rupture of the atherosclerotic plaque. Tissue factor pathway inhibitor (TFPI) is a major regulator of TF-induced coagulation. We evaluated the possibility that monocyte-derived macrophages express this protein, thereby contributing to regulation of TF activity (TFact). Equally, we investigated the effect of cholesterol and of oxidized LDL (Ox-LDL) on the expression of TFPI and TF by human monocyte–derived macrophages (HMDMs). Northern blot analysis of TFPI mRNA from cultured HMDMs revealed a single band at 4.2 kb with weak intensity; this finding was confirmed by reverse transcription–polymerase chain reaction. Gel filtration of HMDM supernatants showed the presence of an active 100-kDa form of TFPI, which was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under nonreducing conditions; under reducing conditions, however, the immunoblot revealed a 40-kDa form of TFPI. The TFPI in HMDM supernatants possessed heparin-binding affinity, suggesting potential interaction of TFPI with heparan sulfate proteoglycans. Stimulation of foam cell formation by incubation of macrophages for 48 hours with exogenous free cholesterol indicated that neither the biological activity nor the de novo synthesis of TFPI protein was affected. In contrast, cholesterol loading with exogenous free cholesterol induced significant upregulation of total TFact (2.6-fold: 25.0 versus 9.4 mU/mg cell protein, cholesterol-treated versus control cells; P<0.05); such induction was not correlated with an elevation in TF antigen (8.5 versus 7.8 ng/mg cell protein, cholesterol-treated versus control cells). Similarly, cholesterol-rich Ox-LDL induced an increase in TFact (1.9-fold: 18.9 versus 10.0 mU/mg cell protein, Ox-LDL–treated versus control cells; P<0.05); by contrast, the amount of TF antigen remained unchanged (7.1 versus 7.9 ng/mg cell protein, Ox-LDL–treated versus control cells). Our data indicate that enhancement of the procoagulant activity of TF in macrophage-derived foam cells is not counterbalanced by upregulation of TFPI activity, suggesting that lesion foam cells are in a procoagulant state; they may therefore contribute to thrombus generation on plaque rupture.

Key Words: anticoagulant activity • atherothrombosis • foam cells • heparin • procoagulant activity

	Introduction

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One of the earliest events in the atherogenic process involves the adherence of circulating blood monocytes to the endothelium and their subsequent infiltration into the intimal space of the arterial wall, where they mature into macrophages.¹ Exposure of macrophages to oxidatively modified LDL (Ox-LDL), a major component of human atherosclerotic plaques,² leads to intracellular cholesterol deposition and formation of lipid-laden foam cells; the accumulation of such cells is characteristic of vulnerable atherosclerotic plaques. Indeed, unstable lipid-rich plaques may rupture, leading to thrombus-mediated acute coronary syndromes.³ The component(s) responsible for the elevated thrombogenicity of the ruptured plaque is indeterminate, but tissue factor (TF) has been suggested as a potential candidate.^{4 5} TF has been detected in relative abundance within the atherosclerotic core, a location that probably reflects its origin in macrophage foam cells that have undergone necrosis.⁶ In vitro, macrophage expression of TF is positively regulated by Ox-LDL⁷ and cholesterol.⁸ These observations raise the question as to whether cholesterol-loaded foam cell macrophages may be critical in the thrombotic response to plaque rupture.

Exposure of TF to plasma circulating factor VII/VIIa results in the activation of factors IX and X, thereby leading to thrombin formation.⁹ This pathway is regulated by a specific inhibitor of TF, the tissue factor pathway inhibitor (TFPI).^{10 11} The functional properties of TFPI have led to the formulation of a revised theory of coagulation¹⁰ in which factor VIIa/TF is responsible for the initiation of coagulation; owing to TFPI-mediated inhibition of TF-activated coagulation, sustained hemostasis requires the persistent and amplified procoagulant action of the intrinsic pathway.

TFPI consists of 3 tandem Kunitz-type inhibitory domains.^{11 12} Initially, TFPI binds to factor Xa and subsequently to the TF/factor VIIa complex or a preformed factor Xa/factor VIIa/TF complex.¹⁰ TFPI is synthesized by several cell lines and cell types, including HepG2 hepatoma cells, the U937 monocytic cell line, and vascular endothelium and is equally present in platelets.^{10 11 13 14} Stimulation of monocytes increases production of TFPI antigen (TFPIag), whereas nonactivated circulating human monocytes are deficient in TFPI activity (TFPIact).¹⁵ Moreover, in situ hybridization studies have demonstrated the presence of TFPI in macrophages within the villi of term placentas.¹⁶

In the present study, we initially evaluated the possibility that TFPI is produced by human monocyte–derived macrophages (HMDMs) in primary culture. Second, we investigated the relationship between the expression of TF relative to that of TFPI in human macrophages exposed to Ox-LDL or exogenous cholesterol. Our data reveal overexpression of TFact relative to that of TFPI in cholesterol-loaded macrophages, thereby suggesting that these cells may promote thrombosis on plaque rupture.

	Methods

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Purification and Chemical Modification of LDL
LDLs were isolated in the density interval 1.024 to 1.050 g/mL by sequential preparative ultracentrifugation from normolipidemic human plasma^{17 18} and dialyzed as previously described.¹⁷ Copper-oxidized LDLs were prepared by incubating 500 µg LDL protein per mL in PBS containing 2.5 µmol/L CuCl₂ for 48 hours at 37°C.¹⁷ The degree of oxidative modification of LDL was characterized by determination of the content of lipid peroxides, thiobarbituric acid–reactive products of peroxidation, and oxysterols.¹⁹ Typically, Ox-LDL preparations contained 54 to 116 nmol lipid peroxides per mg LDL protein and 12 to 22 nmol equivalents of malondialdehyde per mg LDL protein. The major oxysterols found were 7-ketocholesterol (62 µg/mg LDL protein), 7ß-hydroxycholesterol (41 µg/mg LDL protein), 7-hydroxycholesterol (33 µg/mg LDL protein), and 5ß,6ß-epoxycholesterol (28 µg/mg LDL protein). The protein content of LDL and Ox-LDL was determined by the procedure of Lowry et al.²⁰ To prevent endotoxin contamination during LDL isolation and oxidative modification, we used pyrogen-free commercial plastic at all critical steps; under these conditions, endotoxin content was <1 pg/µg LDL protein as monitored by the Limulus amebocyte lysate chromogenic assay (Biogenic).

Isolation, Culture, and Treatment of HMDMs
Monocytes were isolated from the blood of healthy, normolipidemic volunteers (thrombopheresis residues) as previously described.^{18 21} The cells were cultured in RPMI-1640 medium (Bio-Whittaker) containing 10% heat-inactivated, pooled human serum from fasting subjects (ATGC Biotechnology) and 40 µg/mL gentamicin (Schering-Plow).²¹ At day 14 of culture, all cells were positive for the macrophage-specific marker CD68 but were negative for the lymphocyte-specific marker CD3.¹⁸ To obtain in vitro foam cells, we incubated mature macrophages with Ox-LDL (100 µg protein per mL) or exogenous free cholesterol (EFC; 100 µg/mL) for 48 hours according to the protocol that we described earlier^{8 18} ; the resulting intracellular accumulation of cholesterol (in the free and esterified forms) represented 32 and 85 µg/mg cell protein, respectively, and 5.14 and 30 µg/mg cell protein, respectively, for cholesteryl ester. Typically, cholesteryl ester is present in trace amounts (<1 µg/mg cell protein) in control macrophages.⁸ Gas chromatographic analysis failed to detect contamination of commercial preparations of free cholesterol with oxysterols. Polymyxin B (60 U/mL) was added to the medium to avoid cellular activation mediated by potential contamination with lipopolysaccharides.^{8 22} Macrophage viability was assessed by the release of lactate dehydrogenase into the extracellular medium (kit LDH, Boehringer Mannheim). The results indicated no statistical difference in the level of cytotoxicity between control and treated cells (viability >95%).

Macrophage mRNA Analysis
Total RNA was extracted from HMDMs maintained in culture for 15 days by the guanidine isothiocyanate method.²³ Northern blotting was performed as described previously²⁴ using a specific 751-bp cDNA probe (the kind gift of Dr C.P.E. Van der Logt, University Hospital Leiden, Leiden, Netherlands). For quantification of TFPI mRNA, we used the reverse transcription–polymerase chain reaction (RT-PCR) procedure. TFPI cDNAs were synthesized by incubating 10 µg of total HMDM RNA with 200 U of Superscript BRL reverse transcriptase (Gibco/BRL) and poly(dT) and primer that spanned bases 940 to 959 of the TFPI cDNA as previously described.¹² After incubation at 37°C for 1 hour, the newly generated cDNA was amplified for 30 cycles by using Taq DNA polymerase (Dynazyme) and 2 different primers that spanned bases 209 to 228 and 864 to 884 of TFPI cDNA.¹² The PCRs were performed under the following conditions: 1-minute denaturation at 94°C, 1-minute primer annealing at 55°C, and 2-minute extension at 72°C. We calibrated the cDNA content of macrophages on the basis of their content of ß-actin cDNA, which was quantified by competitive PCR.^{25 26} Increasing concentrations of TFPI cDNA were then amplified on the basis of a linear relation between cDNA concentrations and the PCR products.²⁷

Immunoprecipitation of ³⁵S-Labeled TFPI
TFPI synthesized de novo in 14-day-adherent HMDMs was immunoprecipitated and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).⁸ For immunoprecipitation, supernatants from macrophage culture media were incubated with a rabbit polyclonal antibody against human TFPI (American Diagnostica Inc). For calibration, colored protein markers (Amersham) were electrophoresed in parallel.

Western Blot Analysis of TFPI From HMDMs
Concentrated supernatants from macrophage cultures were electrophoresed on 10% SDS-PAGE gels,²⁸ and proteins were electrophoretically blotted onto nitrocellulose sheets (Bio-Rad). A monoclonal antibody directed against human TFPI (dilution 1:500; 4903, American Diagnostica) was used as the primary antibody. The ECL chemiluminescent method (Amersham) was used for signal detection.

Chromatographic Analysis of TFPI
Gel filtration was performed on a column (1.5 cmx1 m) of Sephadex G200 (Pharmacia) calibrated with molecular weight standards (Sigma). The column was equilibrated at 4°C and eluted with a buffer (20 mmol/L Tris-HCl and 10 mmol/L MgCl₂, pH 7.5) containing 0.3 mol/L NaCl at a flow rate of 8 mL/h. Eluted fractions were analyzed for TFPIag and TFPIact. Heparin affinity chromatography was performed as described previously.²⁹ HMDM supernatant (70 mL, containing 0.015 mol/L NaCl, 2 mmol/L Tris-HCl, 0.1 mmol/L MgCl₂, 0.2 mmol/L PMSF, 0.2 µg/mL leupeptin, 0.2 µmol/L aprotinin, 0.2 mmol/L benzamidine, and 0.2 µg/mL trypsin inhibitor at pH 7.4) was applied at 25 mL/h to a column of heparin–Sepharose CL-6B (1.5x14 cm) at room temperature. The unabsorbed fraction was eluted with 10 mmol/L NaH₂PO₄ buffer, and subsequent fractions were eluted successively with 0.3, 0.55, and 1.0 mol/L NaCl in the buffer. On rechromatography, TFPI present in the unbound fraction was not retained. The eluted fractions were analyzed for TFPIag.

Quantification of TFPI and TF by ELISA
TFPI (intact and truncated forms) and TF antigens were quantified by use of the Imubind total TFPI ELISA kit and the Imubind TF ELISA kit (American Diagnostica), respectively. TFPI and TF levels were determined by measuring absorbance at 405 nm and comparing values to those of a standard curve established by use of native TFPI or standard TF.

TFPI and TF Activity Assays
TFPIact was measured by the amidolytic assay described by Sandset et al³⁰ with minor modifications.¹⁷ TFact associated with the cell surface or present in the culture supernatant was measured by the amidolytic assay described by Archipoff et al.³¹ The cells were first rinsed 3 times with PBS and then incubated for 5 minutes in culture medium M199 (Eurobio) containing 0.5% BSA at room temperature. M199 containing BSA was subsequently removed and replaced by 60 µL of M199. Fifty microliters of M199 containing purified human factors VIIa (5 nmol/L final concentration) and X (100 nmol/L final concentration) were then added to each well. After 20 minutes, the supernatants were removed and the amidolytic activity of generated factor Xa was measured in the supernatant by adding 50 µL of the specific chromogenic substrate S2765 to give a final concentration of 0.2 mmol/L. The initial increase in optical density at 405 nm was determined over a time course of 5 minutes.

Statistical Analysis
Results are expressed as mean±SD. Mean values were compared by the Student's t test with significance defined as P<0.05.

	Results

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TFPI Production by Cultured HMDMs
Human monocytes in culture undergo morphological and functional changes throughout their maturation into macrophages.³² Therefore, we evaluated the amount of TFPI that accumulated within 48 hours in serum-free media after 2 or 12 days of adherence culture of monocytes or macrophages, respectively. The results revealed a marked variability from one donor to another; the most frequent concentration observed was 0.66 ng TFPI per mg cell protein at 2 days of culture. However, at 12 days of culture, TFPI concentration attained an average of 8 ng/mg cell protein (range, 4 to 11). Freshly isolated monocytes in nonadherent culture express minor amounts of TFPI (0.25 ng TFPI per mg cell protein).¹⁵ The discrepancy between data in freshly isolated compared with adherent monocytes may be related mainly to the process of cellular adherence. In fact, earlier studies have shown that the attachment of circulating monocytes to plastic dishes stimulates the expression of TFPI.³³

Because atheromatous plaques have been reported to contain mainly mature macrophages rather than monocytes, we investigated the time course of the release of TFPI protein into the supernatant of adherent macrophages at 14 days. The cells were incubated with serum-free medium. At 6 and 18 hours of incubation, cells expressed low levels of TFPI (4 ng/mg cell protein). Levels of TFPI increased progressively to attain levels of 6 and 8 ng/mg cell protein, respectively, at 36 and 48 hours of culture (data not shown).

Western blot analysis of TFPI produced by adherent HMDMs revealed a single band with an apparent M_r of 80 kDa under nondenaturing conditions. However, on disulfide reduction, the 80-kDa complex was dissociated into a major component with an apparent M_r of 40 kDa and a minor protein of 25 kDa (Figure 1).

View larger version (57K): [in this window] [in a new window]   Figure 1. Western blot analysis of TFPI released by HMDMs. Fourteen-day-adherent macrophages were incubated with serum-free medium for 48 hours. Supernatants were collected and centrifuged to remove cellular debris. Samples of culture medium (10 µg protein) were applied to SDS-PAGE gels with (+ßme) or without (-ßme) ß-mercaptoethanol and transblotted onto nitrocellulose membranes, and TFPI protein was detected using a specific monoclonal antibody. SDS-PAGE standards (Broad range, Bio-Rad) were electrophoresed in parallel.

To elucidate whether TFPI secreted by macrophages was biologically active, serum-free media from HMDMs cultured for 36 hours were collected, concentrated, and fractionated on a G200 column. TFPIag and TFPIact were eluted in a major peak corresponding to an apparent M_r of 100 kDa and a minor peak of 36 kDa (Figure 2). In addition, the size and abundance of TFPI mRNA isolated from 12-day-cultured macrophages were analyzed by Northern blotting. We detected a single band at 4.2 kb with weak intensity (data not shown). In view of the low abundance of macrophage TFPI mRNA seen on Northern blot analysis, we performed detection of TFPI mRNA in HMDMs by RT-PCR²⁵ (Figure 3). On electrophoresis in agarose gel, the PCR product migrated as a unique specific band of 675 bp.

View larger version (17K): [in this window] [in a new window]   Figure 2. Elution profiles of TFPIag and TFPIact on gel filtration chromatography. Fourteen-day-adherent macrophages were incubated with serum-free medium for 36 hours. Supernatants were collected and centrifuged. Samples were dialyzed against (NH4)2CO3-EDTA buffer at pH 7.4, concentrated, and then dialyzed against elution buffer (see Methods). Two milliliters (110 ng TFPIag) was applied to the gel in the absence of a reducing agent. The eluted fractions were tested for TFPIag and TFPIact (see Methods).

View larger version (76K): [in this window] [in a new window]   Figure 3. Detection of macrophage TFPI mRNA by RT-PCR by electrophoresis in agarose gel. The PCR product from TFPI cDNA of macrophages was loaded in lane 2. As positive controls, PCR products from a TFPI cDNA control and from the TFPI cDNA of a human endothelial cell line (ECV 304) were loaded on lanes 1 and 3, respectively. DNA was revealed by ethidium bromide staining.

Effect of Heparin on the Release of TFPI by Cultured HMDMs
Heparin has been shown to release TFPI from endothelial cell surfaces into plasma.³⁰ We therefore investigated the effect of heparin on the secretion and cell-surface association of TFPI by HMDMs. Cells were incubated in the absence or presence of heparin (10 U/mL heparin, Choay) in serum-free medium for 36 hours. Under these conditions, heparin mediated a marked increase in TFPI mass (70%) in the culture medium. The heparin binding affinity of secreted TFPI was next estimated by heparin affinity chromatography. Figure 4 shows that 11% of the total eluted TFPI protein was in the flow-through volume and 56%, 21%, and 12% of TFPI protein in these forms displayed a low, intermediate, and a high heparin binding affinity, respectively.

View larger version (9K): [in this window] [in a new window]   Figure 4. Elution profiles of culture media on fractionation by heparin affinity chromatography. Fourteen-day-adherent macrophages were incubated with serum-free medium for 48 hours. Supernatants were collected and centrifuged; 70 mL (35 ng TFPIag) was applied to a heparin-Sepharose column. Elution was first performed with NaCl-free buffer; further elutions were performed successively with 0.3 mol/L, 0.3 to 0.55 mol/L, and 0.55 to 1 mol/L solutions of NaCl.

Effect of Cholesterol and Ox-LDL on the Expression of TFPI and TF by HMDMs
Macrophages treated with EFC (100 µg/mL) or ethanol (control cells) produced similar amounts of TFPIact and TFPIag (the Table). In addition, Ox-LDL (100 µg protein per mL) was without effect on the production of TFPI by HMDMs. These experiments necessitated the subtraction of background values for TFPIact associated with Ox-LDL; to avoid inaccuracy in estimation of TFPIact due to LDL-associated TFPI,¹⁷ newly synthesized TFPI was immunoprecipitated. A protein with an M_r of 36 kDa was immunoprecipitated with a specific polyclonal antibody to TFPI from untreated cells and from cells treated for 36 hours with native LDL (Figure 5). Amounts of immunoprecipitable TFPI in the supernatant from Ox-LDL–treated cells were slightly but not significantly lower than those from control cells.

View this table: [in this window] [in a new window]   Table 1. Effect of Cholesterol and Ox-LDL on the Expression of TFPI and TF by Adherent HMDMs

View larger version (79K): [in this window] [in a new window]   Figure 5. Autoradiogram of 35S-labeled TFPI immunoprecipitated from macrophage supernatants. TFPI was immunoprecipitated from the conditioned medium of cultured macrophages with an antibody to TFPI from control cells (lane 1) and from cells incubated with native LDL (lane 2) or Ox-LDL (lane 3). In lane 4, immunoprecipitation was performed using a nonimmune IgG control serum.

In view of previous studies that have identified separate pools of TF in different cell types (ie, monocytes and fibroblasts),^{34 35} we measured both TFact and TFag in intact HMDMs and HMDM lysates and in the supernatants of HMDMs, respectively. We observed that the sum of cell surface TFact and disseminated TFact (total TFact) was significantly increased in the presence of cholesterol (2.6-fold: 9.41 versus 24.98 mU/mg cell protein; calculated from the Table). Similarly, Ox-LDL increased total TFact under the same experimental conditions (1.9-fold: 10.03 versus 18.92 mU/mg cell protein). During cholesterol treatment, the upregulation of total TFact was mainly due to an increase in cell surface–associated TFact. By contrast, disseminated TFact was responsible for the increase in total TFact of HMDMs on Ox-LDL treatment (the Table). Macrophages treated with EFC (100 µg/mL) or ethanol (control cells) produced similar amounts of TFag (7.84 versus 8.53 ng/mg cell protein). In addition, Ox-LDL (100 µg protein per mL) was without effect on the production of total TFag by HMDMs; nevertheless, we observed a 2-fold increase in TFag levels in cell supernatants (the Table).

The CD11b/18 receptor (Mac-1), which is expressed by monocytes, murine macrophages, and murine foam cells, can directly coordinate the activation of factor X.³⁶ To assess whether TFact was specifically dependent on TF and not due to the presence of a direct activator of factor X, we measured TFact in the absence of factor VIIa or in the presence of a monoclonal antibody against human TF. Under these conditions, no activation of factor X was observed in HMDMs treated under all of our experimental conditions.

Quantification of Macrophage TFPI mRNA by RT-PCR
We quantified TFPI mRNA in control and in Ox-LDL–treated cells by quantitative RT-PCR.²⁵ A nonsignificant decrease of some 25% in the content of TFPI mRNA in macrophages treated for 48 hours with Ox-LDL (100 µg protein/mL) was observed (Figure 6).

View larger version (14K): [in this window] [in a new window]   Figure 6. Effect of Ox-LDL on TFPI mRNA produced by macrophages. Macrophages were treated with Ox-LDL for 48 hours, and TFPI mRNA content was determined by RT-PCR. Samples were calibrated on the basis of their content of ß-actin (ACTINE) cDNA (see Methods).

	Discussion

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The present study demonstrates, for the first time, the expression and production of TFPI, a key inhibitor of TFact, in HMDMs and in cholesterol-loaded foam cells. However, TFPI expression was not modified in macrophage foam cells on cellular cholesterol loading with either EFC or Ox-LDL; by contrast, total TFact was significantly upregulated (2-fold) in cholesterol-laden cells, thereby indicating that procoagulant TFact predominates. These findings are of special relevance to the potential proatherogenic activity of the atherosclerotic plaque, because macrophages in our culture system displayed a secretory phenotype that closely resembled that of in situ plaque macrophages.^{37 38 39 40} Indeed, these cells express abundant amounts of apolipoprotein E,⁴¹ lipoprotein lipase,²⁶ an elastase-like enzyme,²¹ and TF.⁸

TFPI synthesized within 48 hours of HMDM culture was detected in the medium in 2 forms, 1 with an M_r close to 40 kDa and a second of M_r 80 to 100 kDa. Under reducing conditions, TFPI presented as 2 forms, 1 with an apparent M_r of 40 kDa and a second as a minor protein of 25 kDa. The amino acid composition of TFPI predicts an M_r of 32 kDa. However, owing to multiple glycosylation sites,¹² the apparent M_r determined by SDS-PAGE was 43 kDa.⁴² Such M_r values are similar to those of TFPI secreted by HMDMs. Furthermore, immunoreactive forms of TFPI with a higher M_r under nonreducing conditions have been observed.^{11 13} These forms were recovered after concentration of the culture medium and may thus represent an in vitro artifact. The high-M_r species were also found in concentrated supernatants of HMDMs and may represent a heterodimer or homodimer complex of TFPI. It is unknown whether the 25-kDa form is a degradation product of the 40-kDa form. Nevertheless, proteolysis of TFPI typically occurs in the supernatant of HepG2 cells, as reported earlier.¹¹ Such a phenomenon may readily occur in macrophage supernatants in the absence of a protease inhibitor. Macrophages and foam cells synthesize several proteinases^{21 43} that may cleave TFPI, such as leukocyte elastase.⁴⁴

Macrophage TFPI mRNA presented as a single band with weak intensity at 4.2 kb; such low expression may explain the absence of the 1.4-kb mRNA species described previously.⁴⁵ Our data confirm those of McGee et al,¹⁵ however, in which TFPI mRNA was expressed at low levels in freshly isolated monocytes and in 48-hour-adherent monocytes. Induction of TFPI during monocyte maturation revealed a 10-fold increase in expression of TFPI at day 12 compared with day 2 of culture (8 and 0.7 ng/mg cell protein, respectively) in adherent and unstimulated cells. However, the TF and TFPI proteins were produced in almost equimolar concentrations at 12 days of macrophage culture, suggesting a tight and coordinated control of the coagulant/anticoagulant phenomenon under normal conditions.

The release of TFPI into the culture medium was 2-fold elevated when HMDMs were incubated with heparin for a period of 36 hours, suggesting that TFPI contains heparin binding sites. Indeed, the retention of TFPI by a heparin-Sepharose column confirms the presence of such sites.⁴⁶ Therefore, heparin may afford protection to TFPI against proteolytic degradation. However, such protective action disappeared within 48 hours of incubation, possibly because heparin loses its functional properties. Furthermore, an inhomogeneity in TFPI binding to heparin has been previously demonstrated and is associated with truncation at the carboxy terminus.^{29 46} Because the C-terminus domain is responsible for both high-affinity binding to heparin and optimal anticoagulant activity, the TFPI that eluted at NaCl concentrations of <0.3 mol/L almost certainly represents mainly C-terminus–truncated forms.⁴⁶ Under these conditions, 2/3 of TFPI in the supernatant at 48 hours of cell culture would be predicted to display weak anticoagulant activity. The remaining 1/3 of the TFPI pool may be present as either full-length or truncated forms of intermediate size. Such forms have been demonstrated to possess optimal anticoagulant activity. We hypothesize that the presence of such size heterogeneity in TFPI reflects the presence of elevated proteolytic activity in macrophage culture media. Cell-associated TFPIag may in fact mainly represent cellular degradation products, thereby accounting for the low levels of TFPIact associated with HMDMs.

A minor fraction of TFPIag (10% to 20% of secreted TFPI) was detectable at the cell surface, whereas cell-associated TFPIact represented from 4% to 9% of the total secreted TFPI pool. Cell-associated TFPI may originate from the binding of TFPI to an as-yet-unidentified macrophage surface molecule that may subsequently mediate TFPI binding to the LDL receptor–related protein.^{47 48} The LDL receptor–related protein system may thus represent a loop for regulation of TFPI in the cellular microenvironment of macrophages in the arterial intima.

Macrophage TFPI expression was not modulated by Ox-LDL or EFC. Furthermore, Van der Logt et al¹⁴ found no variation in TFPI synthesis after endotoxin or phorbol ester stimulation. Despite production of equivalent amounts of TF and TFPI proteins, however, we detected an imbalance between TF and TFPI activities in HMDMs stimulated by EFC or Ox-LDL (the Table). Similar discrepancies between TFact and TFag have been described recently in monocytoid cells.⁴⁹ Additional procoagulant mechanisms may underlie this discrepancy and that are independent of the expression of the CD11b/18 receptor (Mac-1),³⁶ as demonstrated by the use of a factor VII–deficient TF assay. We speculate that Ox-LDL and EFC can influence the membrane distribution of anionic phospholipids, which are key cofactors for the surface assembly of procoagulant complexes and are essential for the acceleration of TF-dependent initiation of blood coagulation.^{50 51} EFC may insert into the membrane phospholipid bilayer and thus promote the ability of phospholipids to accelerate TF/factor VII/VIIa activity. In the same manner, the lipid and oxysterol content of Ox-LDL can enhance TF procoagulant activity.⁵² It is equally relevant that TF protein is subject to posttranslational modification, including glycosylation, phosphorylation (via protein kinase C⁵³ ), and disulfide bond formation.⁵⁴ Therefore, the possibility cannot be excluded that Ox-LDL and/or cholesterol may modulate such posttranslational modification and thereby modify TFact, especially because Ox-LDL is a potent stimulator of protein kinase C.^{55 56}

TF is a transmembrane glycoprotein that is almost exclusively cell associated. Nevertheless, disseminated forms of TF procoagulant activity have been detected in the supernatants of adherent human monocytes and fibroblasts,^{34 35} and these arise from the release of membrane vesicles containing TFag and phospholipids. The latter particles are capable of disseminated procoagulant activities, as suggested by the presence of TFag in plasma and urine.⁵⁷ Some 20% of total TFact was released by HMDMs. Therefore, microparticles shed from resting HMDMs and cholesterol-treated HMDMs may disseminate TF-dependent procoagulant activity.

Because TFPIact associated with native LDL is progressively inhibited during oxidative modification,¹⁷ the contribution of Ox-LDL–associated TFPI to the local hemostatic equilibrium in the plaque is probably minor. We conclude that human macrophages and foam cells constitute a source of TFPI that may locally contribute to regulation of coagulation in their extracellular microenvironment. Furthermore, our data confirm earlier studies^{48 58} that reported localization of TFPI mRNA and protein to lipid-laden, macrophage-rich areas of human atherosclerotic lesions and that hypothesized that such cells are responsible for local TFPI synthesis. Nevertheless, the present studies suggest that overexpression of TF in macrophage foam cells^{6 8} is not counterbalanced by upregulation of TFPI synthesis during foam cell formation. Considered together, our data strengthen the thesis that upregulation of macrophage TFPI production may represent an important future therapeutic target and that such upregulation could counteract the thromboembolic complications associated with plaque rupture.

	Acknowledgments

 
L. Petit gratefully acknowledges the award of a Research Fellowship by the Fondation Sanofi. These studies were supported by INSERM, by the Faculté de Médecine Pitié-Salpétrière (Paris Université VI), and by the award of a "Bourse d'aide à la Recherche" by Laboratoires Fournier (to Dr P. Lesnik). We thank Dr D. Stengel for her contribution to quantitative RT-PCR and Dr M. Rouis for stimulating discussion.

Received October 28, 1997; accepted June 29, 1998.

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