thrombin modulates synthesis of plasminogen activator ......overnight at room temperature. the u-pa...

Thrombin Modulates Synthesis of Plasminogen Activator Inhibitor Type 2 by Human Peripheral Blood Monocytes

By Helen Ritchie, Alec Jamieson, and Nuala A. Booth

Fibrin deposition is characteristic of inflammatory diseases. The monocyte is central to the inflammatory response and can affect fibrinolysis by expression of urokinase (U-PA) and plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2, respectively). This study examines whether thrombin, which promotes fibrin deposition, can contribute to fibrin persistence by modulating expression of proteins of the fibrinolytic system. Monocytes were isolated from human peripheral blood and analyzed for PAI-2, PAI-l, and U-PA anti- gens by enzyme-linked immunosorbent assay (ELISA). Monocytes responded to thrombin by increased expression of PAI-2 in a dose- and time-dependent manner, with maxi-

HE MONOCYTE is a central cell type in inflammatory diseases and, in particular, in atherosclerosis. Blood-

borne monocytes migrate into the artery wall and are then believed to become localized due to an increased expression of chemotactic agents. The monocyte can be activated by cytokines present in the plaque. The activated monocyte it- self is a rich source of cytokines that influence the progres- sion and development of the atherosclerotic plaque. The monocyte differentiates into a macrophage and ultimately forms a foam cell, after the uptake of modified low-density lipoprotein. '

Fibrin deposition is a characteristic of the atherosclerotic plaque,' where fibrin has been suggested to act as a chemoat- tractant and as a scaffold for migrating cells. Fibrin deposition results from activation of the extrinsic coagulation pathway, and tissue factor (TF) expression is regarded as the major initiator of the extrinsic coagulation pathway, leading to the generation of thrombin.' Thrombin then cleaves fibrinogen to form fibrin. Fibrin degradation is mediated by the fibrinolytic system; specifically, the serine protease, plasmin. Plasmin is generated by the cleavage of plasminogen, a reaction catalyzed by plasminogen activators (PAS), of which there are two types, tissue-type PA (t-PA) and urokinase (U-PA). Both t-PA and U-PA are inhibited by plasminogen activator inhibitors types 1 and 2 (PAL1 and PAI-2), both members of the serpin (serine protease inhibitor) family. Fibrin deposition and persistence is, therefore, dependent on

T

From the Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen; and the Vascular Injlammatory and Mus- culoskeletal Research Department, ZENECA Pharmaceuticals, Alderley Park, Macclesfield, UK.

Submitted February 17, 1995; accepted June 27, 1995. Supported by a Medical Research Council Collaborative student-

ship with ZENECA Pharmaceuticals. Address reprint requests to Nuda A. Booth, PhD, Department

of Molecular and Cell Biology, University of Aberdeen, Marischal College, Aberdeen, AB9 IAS Scotland, UK.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/8609-0011$3.00/0

3428

mal synthesis at a concentration of 1 UlmL to 10 U/mL. This trend was also evident for PAI-1, which was present at much lower levels. Thrombin and lipopolysaccharide (LPS) stimulated comparable levels of PAI-2, studied at the antigen and mRNA level. The dose effect of LPS on PAI-2 and PAI-1 was found to differ from that of thrombin. The level of U-PA was undetectable by ELSA and zymography in all samples. Thrombin stimulates PAI-2 synthesis by human monocytes, therefore creating an imbalance in the fibrinolytic system. This may contribute to persistence of fibrin, deposited during inflammation. 0 1995 by The American Society of Hematology.

the local balance between and within the coagulation and fibrinolytic systems.

The monocyte has the ability to affect both coagulation and fibrinolytic systems. Activated monocytes have been shown to synthesize U-PA, t-PA, PAI-I, PAI-2, and TF, depending on the s t i m u l u ~ . ~ ~ ~ PAI-2 is the major plasminogen activator inhibitor produced by the monocyte," and its synthesis is highly regulated. PAI-2 is induced by cytokines such as tumor necrosis factor (TNF)-a, interleukin-lP, interleukin-2, and interferon-y but is down-regulated by interleukin-4.'"-'* Lipopolysaccharide (LPS) is a potent stimulator of both PAI-2 and TF production by the monocyte, rendering it capable of modulating fibrin deposition and per- ~ is tence .~ .~ PAI-2 exists in two forms, an intracellular non- glycosylated species and a secreted glycosylated protein; both forms are translated from one mRNA transcript.13 PAI- 2 is, therefore, a member of the emerging family of intracellular serpins. The role of intracellular serpins is unclear, but PAL2 has been implicated in protection from TNF-a- mediated cytolysis in the fibrosarcoma cell line, HT1080.I4

Thrombin, a serine protease, acts to modulate many bio- logic functions, in addition to its main role in cleavage of fibrinogen to form fibrin, a reaction that links the coagulation and fibrinolytic systems. Thrombin is capable of modulating chemotaxis of the monocyte and also induces expression of monocyte chemotactic protein, MCP-I.'5.'h The effect of thrombin on other cell types includes stimulation of smooth muscle cell proliferation and induction of PAI-l and t-PA in endothelial cells and PAL1 in smooth muscle ~ e l 1 s . l ~ ~ ~ "

Here we examine the effect of thrombin on PAI-2, PAI- 1, and U-PA expression by human peripheral blood monocytes. Thrombin upregulated PAI-2 in a dose- and a time-dependent manner, with no counterbalancing effect on U-PA. This indicates that thrombin mediates a change in the balance of the fibrinolytic system favoring fibrin deposition.

MATERIALS AND METHODS

Monocyte isolation and culture. Peripheral blood was collected from healthy volunteers with their informed consent. Blood was drawn into a 0.1 volume of 3.8% wt/vol trisodium citrate, and mononuclear cells were isolated by centrifugation on Ficoll-Paque (Phar- macia, Uppsala, Sweden). Monocytes were purified further by adher- ence, and 5 X 105/mL were plated into 24-well tissue culture plates (Nunc, Roskilde, Denmark) by incubation at 37°C for 1 hour in a 5% CO, environment. Nonadherent cells were aspirated off, and the

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THROMBIN STIMULATES MONOCYTE PAL2 SYNTHESIS 3429

monocyte-rich population was washed three times in RPMI (GIBCO BRL, Paisley, UK) supplemented with 4 mmol/L glutamine (GIBCO BRL) and 50 pg/mL gentamycin (Sigma, Poole, UK). Monocytes were characterized using the nonspecific esterase stain2’ (Sigma) and were found to constitute greater than 90% of the population, with platelets being the major contaminating cell type in freshly isolated cells; these platelets were lost by 24 to 48 hours in culture.

Monocytes were cultured in RPMI containing 10% fetal calf serum (GIBCO BRL) with bovine thrombin (Leo Pharmaceuticals, Ballerup, Denmark) or LPS (Sigma), for specified times. Supernatant was removed, and cells were lysed in 1 mL water before storage at -70°C. LPS-free conditions were routinely maintained using plastic, disposable pyrogen-free equipment. Reagents were filtered using a 0.2-pm filter (Sartorius, Gottingen, Germany); LPS was not detectable in any reagents by the limulus amoebocyte assay (Sigma). U937 cells (American Type Culture Collection, Rockville, MD) were cultured in RPMI containing 10% fetal calf serum, 4 mmol/L glutamine, 100 IU/mL penicillin, and 1 0 0 ,ug/mL streptomycin (GIBCO BRL) in a 5% CO2 environment. Samples were treated in the same manner as monocyte samples.

Lactate dehydrogenase (LDH) was assayed in culture supernatants by the CytoTox 96 assay (Promega, Southampton, UK).22

Quantijcation of total cellular protein. Total protein was measured in cell lysates as de~cribed.’~ All data are expressed as nano- grams of antigen per milligram of total cellular protein.

Measurement of PAI-2. PAI-2 was measured quantitatively using a new specific enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Costar, Cambridge, MA) were coated overnight at 4°C with a monoclonal antibody to PAL2 at 2 pg/mL (MAI-21; Biopool, Umel, Sweden) in 50 mmoVL carbonate-bicarbonate buffer, pH 9.6. All steps involved addition of 100 pL per well. Plates were blocked using coating buffer containing 1% wt/vol bovine serum albumin. Samples or standards were added to the wells for 2 hours at 37°C. Recombinant PAL2 (rPAI-2), provided by Delta Biotechnology (Nottingham, UK), was diluted to give a standard curve of 0 to 10 ng/~nL.’~ The IgG fraction of a rabbit polyclonal antibody to rPAI-2 (10 pg/mL) was incubated for 2 hours at 37T, followed by a goat anti-rabbit IgG linked to alkaline phosphatase (Boehringer Mannheim, Mannheim, Germany; diluted 1 in 750). Color was developed using p-nitrophenol phosphate (2 mg/mL; Sigma) for 30 minutes at 37°C. Absorbance of 405 nm was read using a Titertek spectrophotometer (Lugano, Switzerland) with a reference filter of 692 nm. The limit of sensitivity of the ELISA was 0.2 ng/mL, and the coefficient of variance was 6.4%.

Measurement of PAI-1 and U-PA. PAI-I and U-PA were measured by ELISA.25 Microtiter plates (Costar) were coated for 3 hours at 37°C with a monoclonal antibody to U-PA (2.5 pg/mL Muk-l; Biopool). In subsequent steps, 100 p1 of solution was added to each well. The plate was blocked, and sampleslstandards were incubated overnight at room temperature. The U-PA was diluted to give a standard curve of 0 ng/mL to 21 .O ng/mL. A rabbit polyclonal antibody to U-PA conjugated to horseradish peroxidase (1.3 pg/mL) was incubated at room temperature for 3 hours. Color was developed using 100 mmoVL sodium acetate citrate buffer, pH 6.0, urea hydro- gen peroxide (0.005%), and tetramethyl benzidine (0.1 mg/mL) in dimethylsulfoxide, incubated at room temperature for 15 minutes. The reaction was stopped with 2.5 m o m sulfuric acid and read at 450 nm with a reference filter of 692 nm using a Titertek spectrophotometer. The limit of sensitivity was 0.2 ng/mL.

Plasminogen activator activity was also assessed by zymography as described after sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (SDS-PAGE).26

Isolation and detection of total RNA. RNA was prepared from 5 X lo6 monocytes using the guanidinium-phenol-chloroform extraction method.z7 Briefly, monocytes were washed with phosphate-buf-

fered saline and 2 mL of 4 moVL guanidinium thiocyanate solution containing 0.83% wt/vol N-lauryl sarcosine, 25 mmom sodium citrate pH 7.1, and 13 mmol/L p-mercaptoethanol (GIT C solution; Sigma) was added to the monolayer. Sodium acetate, pH 4.0 (300 pL of a 2 moVL solution), was then added, followed by 3 mL of phenol-chloroform-isoamylalcohol (25:24: 1). Samples were left on ice for I5 minutes and centrifuged at 10,000g for 20 minutes at 4°C. The aqueous layer was removed, and RNA was precipitated at -20°C. RNA was pelleted by centrifugation, the pellet was dissolved in GIT C solution, and RNA was reprecipitated as above. The RNA pellet was dried and dissolved in water treated with diethylpyrocar- bonate (DEFT). The RNA pellet was not washed with ethanol, which was found to decrease the yield of RNA. RNA (approximately 3 to 4 pg total RNA from 5 X lo6 cells) was glyoxalated with an equal volume of 6 moVL glyoxal as described, electrophoresed on a 1% agarose gel containing 10 mmoVL iodoacetamide, and transferred to Hybond N+ nylon membrane (Amersham, Little Chalfont, UK).2R The membrane was prehybridized at 60°C for 16 hours in a 0.5 moll L phosphate buffer, pH 7.2, containing 10 mmol/L EDTA and 7% SDS. A 1.4-kb EcoRI-digested fragment of human PAI-2 cDNAZ3 was used as a probe and was labeled with 50 pCi deoxycytidine triphosphate (dCTP) using a random primer kit (Boehringer Mann- heim) and added to the prehybridization buffer for 24 hours. The membrane was then washed in sequence with 2X saline sodium phosphate EDTA (SSPE), 0.1% SDS, l x SSPE, 0.1% SDS, and 0.1X SSPE, 0.1% SDS. Membranes were exposed to X-ray film at -70°C. Membranes were also probed for 18 S ribosomal RNA2’ using the same conditions.

RESULTS

Effect of thrombin on PAL2 production by monocytes. PAI-2 accumulation in cells and culture medium was mark- edly stimulated by thrombin. Concentrations of thrombin from 0.1 mU/mL to 25 U/mL thrombin significantly increased secreted PAI-2 levels above the unstimulated control ( P < .001 from 0.1 to 25 U/mL; Fig 1A). The response curve was bell-shaped, with maximal secretion of PAI-2 at a concentration of 1 U/mL thrombin; this concentration was chosen for all subsequent experiments. A similar response to thrombin was observed in the case of intracellular PAI-2 (Fig lB), with significant increases occurring over the range of 0.1 mU/mL to 50 U/mL (P < .001). PAI-1 was detected at only very low levels, 3 to 8 ng/mg total cell protein, and was only detected in the supernatants; some stimulation was observed at 1 U/mL (P < .OOOl). No U-PA was detected in these monocyte cultures. All data shown are from one representative donor, typical of four replicate experiments.

The time course of PAI-2 in response to thrombin was observed in both supernatants and cell lysates of monocytes (Fig 2). Cell lysate samples were removed in the same volume as supernatants, so that quantities of PAI-2 could be directly compared. Levels of intracellular PAI-2 were much higher than those of secreted PAI-2, and secreted PAI-2 was not detectable at the earliest time point (4 hours). Thrombin stimulated PAI-2 production, as detected in both supernatant and cell lysate, with a marked increase in the 24-hour samples that was maintained up to 72 hours after stimulation ( P < .0001 at 24, 48, and 72 hours; cell lysates and supernatants). The small increase over time in unstimulated cells may reflect monocyte activation or trace thrombin activity in the medium.




3430

The failure of high concentrations of thrombin to stimulate PAI-2 synthesis may reflect cytotoxic effects, and this was investigated by measurement of LDH as a marker of leakage from the cytoplasm. No difference was seen in supernatant LDH activity between control cultures in the absence of thrombin and those in which thrombin was present at 0.001

A

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Thrombin (U/mL)

B 500 I T

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8 300- e - c)

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Thrombin (U/mL)

Fig 1. Monocytes were stimulated with a range of thrombin concentrations from 0 t o 50 UlmL. Supernatant and cell lysate samples were removed after 48 hours'stimulation and were analyzed for PAI- 2 and PAI-I by ELISA. Values represent the mean ? SEM, determined using Student's t-test, of four replicate tissue culture wells of monocytes from a representative donor. The data shown are typical of four experiments. (A) PAI-2 in culture supernatant. PAI-2 was significantly increased above control (no thrombin) after stimulation with 0.1, 1, IO, and 25 UlmL thrombin ( P < ,001). Thrombin at 0.001 UlmL, 0.01 UlmL, and 50 UlmL did not significantly increase PAI-2. (B) PAL2 in cell lysate. The increase in PAI-2 was significant above control at thrombin concentrations of 0.1, 1, IO, 25, and 50 UlmL ( P < .001).

RITCHIE, JAMIESON, AND BOOTH

A 500 1 I

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B 2 ; 100

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Fig 2. Monocytes were stimulated with 1 U/mL thrombin. Super- natant and cell lysate samples were removed at 4. 24. 48, and 72 hours and analyzed for PAL2 by ELISA. Values represent mean ? SEM, determined using Student's t-test, of triplicate tissue culture wells from a representative donor. The data shown are typical of four experiments. (A) PAI-2 in culture supernatant in the presence (0) and absence (0) of thrombin. (B) Intracellular PAI-2 in the presence (0) and absence (0) of thrombin.

to SO U/mL, nor was there any observed increase in LDH in culture supernatants over time (data not shown).

Donor variability in levels of PAI-2 produced by monocytes is a well-reported phenomen~n .~ , '~ The response to thrombin was studied in a total of 10 individual donors. Thrombin stimulated PAL2 antigen in all monocyte samples throughout the time course as in Fig 2. PAI-2 was present in monocyte supernatants at concentrations of 0 to 88.4 ng PAI-2 per milligram of intracellular protein (unstimulated), increasing to 25.7 to 388 ng/mg after 48 hours' stimulation, which represented a level of induction of 2-fold to 16-fold above unstimulated controls. Corresponding values of intra-




THROMBIN STIMULATES MONOCYTE PAL2 SYNTHESIS 3431

cellular PAI-2 were higher in all donors, in the range of 20 to 534 ng/mg (control) and 170 to 1,389 nglmg after stimulation, a 2-fold to 15-fold increase (Fig 3).

Thrombin stimulation of PAI-2 is comparable with LPS stimulation. LPS is a potent stimulator of PAI-2 synthesis by monocytes, and its effect on upregulation of PAL2 mRNA was compared with that of thrombin (1 UlmL). The response of monocyte PAI-2 and PAI-l production to LPS is shown in Fig 4. LPS, at concentrations from 1 ng/mL to 1 pg/mL, significantly increased secreted PAI-2 above control levels (Fig 4A; P < .O001). Similarly, all concentrations studied ( 1 0 0 pg/mL to 1 pg/mL) significantly increased intracellular PAI-2 levels above control levels (Fig 4B; P < .OOOS). It was notable that at the highest concentration of LPS used (1 p@&), PAI-2 secretion was dramatically increased. This increase was at the expense of intracellular PAI-2, which decreased, presumably reflecting cell death.

A 400,

Q 3 lm]

Y

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A 400 1 T

0 0.1 1 10 100 lo00

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Fig 4. Monocytes were stimulated with a range of LPS concentrations from 0 to 1 pglmL and were analyzed for PAI-2 and PAI-1 by EUSA. Supernatant and cell lysate samples were removed after stimulation for 48 hours. Values represent mean k SEM. determined using Student's t-test, of four replicate tissue culture wells from a representative donor. The data shown are typical of three experiments. (A) PAI-2 in culture supernatant. LPS Significantly increased PAI-2 above control (no LPS) at concentrations of 1, 10, 100, and 1.000 nglmL (P C .0001), but not at 0.1 nglmL. (B) Intracellular PAI-2. All concentrations of LPS stimulated accumulation of PAL2 above control (P < .01).

For subsequent experiments, an LPS concentration of 10 ng/ mL was chosen. In contrast with PAI-2, PAI-1 production was decreased in response to increasing concentrations of LPS, and the decrease in PAI-1 below control levels was significant at LPS concentrations from l ngl& to 1 pg/mL (P < .O l ) .

Direct comparison of monocyte stimulation by thrombin (1 U/mL) and LPS (10 ng/mL) was achieved by studying




3432

the sanlc donor at 48 hours (Fig S). Both agents had similar effects, with sixfold increases in secreted PAI-2. Intracellular PAI-2 levels increased by fivefold after stimulation with either thrombin or LPS. The increases in response to both stimulants were again at the significance level of P < .001, but there was no significant difference between the two agents.

Thrombin and LPS stimulation of monocyte PAL2 were also studied at the mRNA level in the same donor. RNA was extracted from monocytes after stimulation with thrombin ( 1 U/mL) and LPS ( I O ng/mL) for S hours. and the resulting Northern blot was probed for PAI-2 mRNA and for 18 S ribosomal RNA (Fig SB). A threefold increase in PAL2 mRNA. in both thrombin- and LPS-stimulated monocytes over unstimulated monocytes, was observed relative to I8 S ribosomal RNA.

The variation among donors in terms of PAI-2 antigen in response to thrombin (Fig 3) was extended by examining variability in levels of PAL2 mRNA as determined by scan- ning densitometry. Stimulation by thrombin ( 1 U/mL for S hours) of monocytes from four donors increased PAI-2 mRNA by a factor of 1 A-, 3-. 3-, and 4.5-fold above control levels. Control levels of PAL2 mRNA also varied (data not shown). in agreement with determinations of antigen.

U937 cells respond to throtnhitt by increasing PAL2 pro- duc~iot~. The response of the monocyte-like cell line U937 was studied after stimulation with thrombin ( 1 U/mL) over time. PAI-2 production in U937 cells in response to thrombin was similar to that of peripheral blood monocytes. A significant increase in secreted PAL2 and intracellular PAL2 (both P < .03) was evident at 24 hours and was more marked at 48 and 72 hours ( P < .OOI; Fig 6). As seen in peripheral blood monocytes, intracellular PAI-2 was consistently present at higher concentrations than was secreted PAI-2. Sig- nificant variation in PAI-2 production by U937 cells was evident from experiment to experiment: the data shown are typical of four experiments. Levels of PAL2 produced by U937 cells were higher than those by monocytes, both in the basal. unstimulated state and after thrombin stimulation. As with monocytes, PAI-I production by U937 cells was at very low levels, detectable in supernatants only at 96 hours at approximately 8 ng/mg total protein. Thrombin stimulated a significant increase in PAI-I, which was then detectable at 72 hours (P < .02).

DISCUSSION

This study shows that monocytes, a predominant cell type in inflammation and atherosclerosis, respond to thrombin by

b

Fig 5. Stimulation of monocytes from the same donor by thrombin (1 UlmL) and LPS ( l0 nglmL) was compared. (A) PAL2 was determined by ELISA after 48 hours' stimulation. Values represent mean 2 SEM, determined using Student's t-test, of four replicate tissue culture wells from a representative donor. The data are typical of those from four experiments. Secreted PAL2 (HI and intracellular PAI- 2 (B) was measured in unstimulated monocytes and after stimulation with thrombin or LPS. (B) Thrombin and LPS stimulation of monocytes from the same donor were compared at the mRNA level, and the subsequent Northern blot was probed for PAW and 18 S ribosomal RNA. Total RNA was extracted from (a) unstimulated monocytes and after following stimulation with (b) thrombin or (c) LPS.

RITCHIE, JAMIESON, AND BOOTH

T

0

Control Thrombin LPS

B

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C




THROMBIN STIMULATES MONOCYTE PAI-2 SYNTHESIS 3433

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h

"l -1 r

I i

0 20 40 60 80

Time (h)

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0 20 40 60 80

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Fig 6. U937 cells were stimulated with 1 UlmL thrombin. PAI-2 and PAL1 were analyzed by ELISA. Values represent mean ? SEM, datermined using Student's t-test, of four replicate tissue culture wells from a representative experiment, typical of four such experiments. (A) PAL2 in supernatant of control (0) and thrombin-stimulated (0) U937 cells. The increase in PAI-2 after thrombin stimulation was significant over control levels at 24 hours (P c .05), 48 hours, and 72 hours (P c .OOOl), but not at 8 hours. (61 Intracellular PAl-2 in control (0) and thrombin-stimulated (0) U937 cells. The increase in PAL2 wes significant at 24 hours (P < .OM), at 48 hours (P < .0001), and at 72 hours ( P < .M).

increasing PAI-2 synthesis, thus potentially contributing to inhibition of fibrinolysis. Thrombin stimulation of peripheral blood monocytes resulted in increased levels of both secreted and intracellular PAI-2, reaching maximal concentrations at 24 hours. The regulation of PAL2 was shown to occur at the level of mRNA. The effect on PAI-2 did not merely reflect increased general cellular synthesis, as measured in terms of total protein and 18 S ribosomal RNA. Furthermore, only a minor effect on PAI-1 synthesis was observed.

Stimulation of PAL2 synthesis was optimal at a thrombin concentration of 1 U/mL, a concentration that has been shown to be effective in stimulating other monocyte proteins, such as MCP-1, and also has profound effects in other cell types, such as smooth muscle cells.'6,20 This concentration of thrombin is of physiologic rele~ance.~' Thrombin consistently stimulated PAI-2 production by all monocytes studied; the baseline value and extent of stimulation by thrombin varied. Similar variation was noted at the level of mRNA for PAI-2. Such donor variability in production of PAI-2 by monocytes is a well-recognized phenomen~n.~, '~

It is noteworthy that very high concentrations of thrombin (25 and 50 U/mL) were less effective in stimulating PAL2 synthesis. This lack of response may be due to receptor desensitization3' or downregulation, as has been reported for human mesangial cells exposed to lower concentrations of thombin for 24 hours.32 It appears that it was not due to cytotoxicity, a possibility examined by measurement of leakage of intracellular LDH, which did not vary with time or with thrombin concentration.

Stimulation of monocyte PAI-2 production by thrombin was comparable in extent to that observed with LPS and was similarly detectable at the level of mRNA at 5 hours.33 Both stimulated accumulation of intracellular PAL2 to levels that were consistently higher than those of secreted PAI-2. High concentrations of LPS (1 ,ugh&) resulted in very high levels of supernatant PAI-2, while intracellular PAL2 was seen to decrease, suggesting that cell death and release of intracellular PAI-2 may have occurred. LPS decreased PAI-l production by monocytes, in agreement with other reports.34

The increase in secreted and intracellular PAL2 after stimulation by thrombin occurred in the absence of any dramatic change in plasminogen activators. No U-PA was detectable by ELISA in cell lysates or supernatants. This possibly re- flects the relative insensitivity of the ELISA used here, be- cause U-PA has previously been detected in monocyte^.'^ Our failure to detect any U-PA by zymography may be due to its occurrence in the single-chain form. Only trace amounts of U-PA are required to generate plasmin activity on the surface of the monocyte, where U-PA is bound to the U-PA receptor.36 PAL2 is capable of modulating U-PA activity, and PAI-2, at a 20-fold molar excess, is capable of inhibiting of 75% of u-PA-directed plasminogen activation on the surface of the monocyte. This is in contrast with fluid- phase U-PA; which is inhibited by only a twofold excess of inhibitor.37 It is not yet clear if single-chain U-PA is present on the monocyte surface after thrombin stimulation. Single- chain U-PA is not inhibited by PAI-2, unlike two-chain u- PA, thus allowing invasion of the monocyte even in areas where PAL2 is predominant.38

PAI-2 upregulation in response to thrombin was observed in U937, a cell line of monocyte lineage.39 Levels of PAL2 were higher in resting U937 cells than in peripheral blood monocytes, indicating differences between the cell line and primary culture. Variability in PAI-2 production was also seen in both control and thrombin-stimulated U937 cells. This may be attributable to a number of experimental vari- ables, including the differentiation state of the cells.40

Levels of secreted and intracellular PAI-2 in peripheral




3434 RITCHIE, JAMIESON, AND BOOTH

blood monocytes were increased 24 hours after stimulation. At this time point, the levels of PAI-2 reached a plateau, suggesting that the monocyte was synthesizing PA[-2 at its maximal rate. Intracellular PAI-2 remained at a high level throughout stimulation. Maintenance of PAI-2 within the cell may simply be due to the poor secretion signal found within PAI-2, or an active system for control of accumulation or secretion of PAL2 may be present. The system responsible for glycosylation and secretion of PAI-2 may also be func- tioning at a maximal rate. The function of intracellular PAI- 2 is not clear, and therefore, the relevance of increased intracellular accumulation on stimulation is unknown. Secreted PAL2 can actively inhibit U-PA and modulate u-PA-dependent migration of monocyte^.^' It is possible that intracellular PAL2 serves as a pool available for controlled or uncon- trolled release from the monocyte. This may be relevant in the artery wall, where oxidized lipoproteins have been reported to be cytotoxic to monocyte^.^' Cell death and subsequent release of PAI-2 may contribute to stability and fibrin deposition in local areas of the plaque.

PAI-2 has been classically associated with pregnancy, where plasma levels are found to be in~reased.~' PAL2 was also characterized as a major protein produced by the histiocytic cell line U937, particularly after stimulation, and PAI- 2 is the major plasminogen activator inhibitor produced by activated monocyte^.^,^^ It is now apparent that PAI-2 can be synthesized by many cell types, such as endothelial cells, smooth muscle cells, the epidermis, the fibrosarcoma cell line HT1080, and various leukemic cell lines.'4.20~4n.44-46 In most of these situations, PAI-2 is an intracellular protein.

Other functions have been proposed for intracellular PAI- 2, a member of the growing family of intracellular serpin^.^'.^' The fibrosarcoma cell line HT1080 produces PAI- 2, and most of the PAL2 is retained intracellularly; in this situation, PAI-2 has been implicated in protection from TNF- a-mediated c y t ~ l y s i s . ' ~ More recently, PAI-2 has been suggested to act as a marker of cell apoptosis in a leukemic cell line.4y The contribution of PAI-2 to prevention of cell metastasis and also its characterization as a tumor suppressor protein due to its chromosomal position add to the growing evidence that intracellular PAL2 has another function in addition to the inhibition of plasminogen activation.'".'' Bio- chemical studies of transfected PAI-2 in HT1080 cells sug- gest that intracellular PAI-2 is capable of self-polymerizing when levels are high enough, thus preventing secretion of PAI-2."

The contribution of synthesis of PAI-2 by the monocyte to atherosclerosis has not been well established. Circulating monocytes are recruited from the bloodstream and are the major infiltrating cell type found in the atherosclerotic plaque. Accumulation of activated monocytes in a local area may result in significant levels of PAI-2. PAL2 has a role in modulation of u-PA-dependent migration of monocytes, a system that requires PAI-2 to exert its effect in a local environment." Production of antifibrinolytic activity by the monocyte in response to thrombin may contribute to fibrin persistence in the local environment within the plaque. Thrombin is known to be active in environments such as the subendothelial space, where it is protected from inhibition.'3

Atherosclerotic plaques have been demonstrated to have increased staining for PAI-I comparable data on PAI-2 have not been reported, but we have observed that atherosclerotic plaques also show staining for PAI-2 that colocal- ized with macrophage/foam cells (Robbie et al. unpublished data, May 1994). This suggests that monocyte production of PAI-2 has a potential localized role in the atherosclerotic plaque.

ACKNOWLEDGMENT

We thank Dr DJ. Ballance for purified recombinant PAI-2 and for the cDNA probe.

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Engl J Med 314:488, 1986 2. Collen D, Juhan-Vague I: Fibrinolysis and atherosclerosis.

Semin Thromb Hemost 14: 180, 1988 3. Rapaport SI, Rao LVM: Initiation and regulation of tissue

factor-dependent blood coagulation. Arterioscler Thromb 14: 1202. I994

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1995 86: 3428-3435

H Ritchie, A Jamieson and NA Booth type 2 by human peripheral blood monocytesThrombin modulates synthesis of plasminogen activator inhibitor

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