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REPORT

Activation of cytosolic phospholipase A₂ and fatty acid transacylase is essential but not sufficient for thrombin-induced smooth muscle cell proliferation

Department of Biochemistry, Hebrew University Faculty of Medicine, Jerusalem, Israel

Submitted 21 May 2007 ; accepted in final form 25 March 2008

ABSTRACT

Thrombin is a potent stimulant of smooth muscle cell (SMC) proliferation in inflammatory conditions, leading to pathological thickening of vascular walls in atherosclerosis and airway remodeling in asthma. Cell proliferation requires the formation and remodeling of cell membrane phospholipids (PLs), involving the activation of PL-metabolizing enzymes. Yet, the role of specific PL-metabolizing enzymes in SMC proliferation has hardly been studied. To bridge this gap, in the present study, we investigated the role of key enzymes involved in PL metabolism, the PL-hydrolyzing enzyme phospholipase A₂ (PLA₂) and the PL-synthesizing enzyme lysophosphatidic acid-fatty acid transacylase (LPAAT), in thrombin-induced proliferation of bovine aortic SMCs (BASMCs). Concomitantly with the induction of BASMC proliferation, thrombin activated cytosolic PLA₂ (cPLA₂-), expressed by selective release of arachidonic acid and mRNA expression, as well as LPAAT, expressed by nonselective incorporation of fatty acid and mRNA expression. Specific inhibitors of these enzymes, arachidonyl-trifluoromethyl-ketone for cPLA₂ and thimerosal for LPAAT, suppressed their activities, concomitantly with suppression of BASMC proliferation, suggesting a mandatory requirement for cPLA₂ and LPAAT activation in thrombin-induced SMC proliferation. Thrombin acts through the protease-activated receptor (PAR-1), and, accordingly, we found that thrombin-induced BASMC proliferation was suppressed by the PAR-1 inhibitor SCH-79797. However, the PAR-1 inhibitor did not prevent thrombin-induced mRNA expression of cPLA₂ and LPAAT, implying that the activation of cPLA₂ and LPAAT is essential but not sufficient for thrombin-induced proliferation of BASMCs.

lysophosphatidic acid acyltransferase; protease-activated receptor-1

Cell proliferation requires the formation and remodeling of cell membranes, especially the metabolism of phospholipids (PLs) that form the membrane bilayer. Membrane remodeling involves two key enzymatic activities: PL hydrolysis by phospholipase A₂ (PLA₂), which provide lysophospholipids for the incorporation of fatty acids (FAs) by lysophosphatidic acid-FA transacylase (LPAAT), to form new PLs required for membrane remodeling and cell growth. This process thus assigns a pivotal role to PL-metabolizing enzymes in cell proliferation. Nonetheless, scant research has been devoted to the role of PL metabolism in thrombin-induced SMC proliferation (27).

The PLA₂ family consists of secretory (sPLA₂) and intracellular enzymes, which include cytosolic PLA₂ (cPLA₂) and Ca²⁺-independent PLA₂ (iPLA₂) (11). iPLA₂ is generally considered a housekeeping enzyme, maintaining the membrane PL composition, whereas the other PLA₂s have been reported to be activated in inflammatory conditions. While iPLA₂ and sPLA₂ have no preference for specific fatty acyl chains, cPLA₂ is specific to arachidonic acid (AA)-carrying PLs and is thus considered a major contributor of AA for the production of inflammatory eicosanoids.

Enhanced activities of both thrombin and PLA₂ are characteristic of inflammatory conditions. Yet, the role of PLA₂ isoenzymes in thrombin-induced SMC proliferation has been little studied, despite the key role of PLA₂ in membrane PL metabolism. A previous study reported that cPLA₂-mediated AA release is critical for the growth of human vascular SMCs in medium supplemented with growth factors (other then thrombin). However, only a single study (27) has directly addressed PLA₂ involvement in thrombin-induced SMC growth, suggesting the requirement of iPLA₂ in thrombin-induced proliferation of rat vascular SMCs, whereas the other PLA₂s have not been studied this context.

At the reverse pathway of membrane PL metabolism, FA transacylation into lysophospholipid has been reported to be involved in cancer cell survival and growth, and its inhibition induced their apoptosis (17), but its role in SMC proliferation has not been studied.

The present study was undertaken to elucidate the role of PL metabolic enzymatic activities, specifically those involved in FA release and reacylation, during thrombin-induced SMC proliferation using bovine aortic SMCs (BASMCs). We report that the activation of cPLA₂, releasing AA, and of LPAAT, which incorporates different FA types into membrane PLs, is required but not sufficient for thrombin-induced BASMC proliferation.

MATERIALS AND METHODS

Materials. Culture medium, FCS, antibiotics, and trypsin (solution A) were purchased from Bet Haemek Biological Industries. [³H]thymidine, AA, and oleic acid (OA) were purchased from Amersham (Aylesbury, UK). Human thrombin was purchased from OMRIX Biopharmaceuticals (Brussels, Belgium). The protease-activated receptor (PAR)-1 inhibitor N³-cyclopropyl-7-{[4-(1-methylethyl)phenyl]methyl}-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine dihydrochloride (SCH-79797) was purchased from Tocris Bioscience. BSA (essentially FA free), trypan blue solution, bromoenolactone (BEL; iPLA₂ inhibitor), arachidonyl-trifluoromethyl-ketone (AACOCF₃; cPLA₂ inhibitor), and all other buffers and chemicals were purchased from Sigma (St. Louis, MO).

Cell cultures. SMCs harvested from the bovine aorta (BASMCs) were generously provided by the laboratory of I. Vlodawsky (Hadassah University Hospital, Jerusalem, Israel). Cells were grown at 5% CO₂ at 37°C in DMEM (4.5% glucose) with 10% FCS, glutamine, penicillin, and streptomycin. Medium was changed every 3–4 days. Experiments were conducted using cells from generations 4–12. To synchronize the cell cycle, cells were subjected to starvation by an incubation with 0.2% FCS for 48 h.

Cell viability was determined by vital staining using 0.1% trypan blue.

Determination of BASMC proliferation. The cell growth capacity was determined by the conventional method of incorporation of radioactively labeled thymidine into DNA (14). Confluent cells were synchronized by "starvation" as described above and incubated under the different treatments for the desired times, and [³H]thymidine (2 µCi/well in 96-well plates) was added to the culture medium for 2 h. Cells were then washed with PBS, incubated in 10% trichloroacetic acid (TCA) at 4°C for 1 h, washed twice with 5% TCA, and incubated for 1 h in 0.2 N NaOH at 37°C. The fluid was collected, and its radioactivity was measured in a scintillation counter.

Determination of PLA₂ activity by the release of AA and OA. cPLA₂ is specific to AA-carrying PLs, whereas sPLA₂ and iPLA₂ have no preference for FAs and can release both AA and OA. Therefore, monitoring of the production of AA and OA by cells is conventionally used for the differentiation of cPLA₂ from sPLA₂ and iPLA₂ activities. To this end, confluent synchronized cells were metabolically labeled by an overnight incubation in culture medium supplemented with [³H]AA or [³H]OA (2.5 µCi/well in 24-well plates). Cells were then washed twice with medium containing 1% BSA and further incubated for the desired times under the different treatments. Medium was then separately collected from each well, and its labeled AA or OA content was determined by scintillation counting. For total labeling, cells were washed to remove free [³H]FAs and then lysed with 1 ml of 1% triton, and the total AA or OA content was determined by scintillation counting.

Measurement of FA acylation by incorporation of [³H]OA or [³H]AA into membrane PLs. Confluent synchronized cells were incubated under the different treatments, after which [³H]OA or [³H]AA was added for up to 2 h. Medium was subsequently removed, and cells were washed twice with medium containing 1% BSA. Cells were lysed with 1 ml of 1% triton, and the labeled FA content was determined by scintillation counting.

mRNA expression of cPLA₂ and LPAAT by RT-PCR. Cells were grown to confluence in six-well plates and synchronized as described above. On the day of the experiment, cells were incubated with thrombin-supplemented or -deficient (control) fresh medium (with 0.2% serum) for 1, 4, or 12 h at 37°C. Cells were then washed in PBS and lysed with 0.5 ml TriReagent (T-9424, Sigma). RNA was isolated according to the manufacturer's instructions, and the RNA concentration and purity were determined by spectrophotometry. RNA was reverse transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase (M1701, Promega). RNA (5 µg) was mixed with RT buffer containing 50 mM Tris (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgCl₂, and 10 mmol/l DTT, 200 units reverse transcriptase, 40 units RNAsin, 0.2 µg oligo dT, and 1 mmol/l dNTP, for a total volume of 20 µl. Samples were incubated for 2 h at 37°C followed by 5 min at 95°C, supplemented with 80 µl of PCR water, and stored at –20°C until subjected to PCR amplification.

For cPLA₂, PCR was performed using bovine-compatible human cPLA₂- primers (sense: 5'-TGTTCAACAGAGTTTTGG-3' and antisense: 5'-AACAGAGCAACGAGATGG-3') to amplify a 900-bp fragment (15). cDNA (2.5 µl) was mixed with ReddyMix PCR MasterMix (1.5 mmol/l MgCl₂) (ABgene) for a total volume of 25 µl. PCRs were carried out in a PT-100 (MJ Research, Watertown, MA) programmable thermal controller with an initial 5-min denaturation at 94°C followed by the cycled program of 30 s at 94°C (denaturation), 30 s at 50°C (annealing), and 2 min at 72°C (extension). PCRs were carried out for 32 cycles, and a final extension of 10 min at 72°C ended the reaction. PCR products (20 µl) were analyzed by 1.5% agarose gel electrophoresis and visualized with ethidium bromide.

For LPAAT, PCR was performed using primers (sense: 5'-CTGTGTGTGCGTGCGAGGAC-3' and antisense: 5'-CAGCATGGCGCCGTTGTGGTT-3') to amplify a 402-bp fragment (16). PCRs were carried out with an initial 5-min denaturation at 94°C followed by the cycled program of 30 s at 94°C (denaturation), 30 s at 62°C (annealing), and 30 s at 72°C (extension). PCRs were carried out for 26 cycles, and a final extension of 10 min at 72°C ended the reaction. Results were normalized to 28S rRNA.

cPLA₂ phosphorylation by Western blot analysis. Cells were grown to confluence in six-well plates and synchronized in medium with 0.2% serum for 24 h. On the day of the experiment, cells were stimulated with thrombin (10 U/well) for 1 h. Cells, on ice, were washed twice with 1.5 ml cold PBS, and 150 µl of lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 µg/ml leupeptin] were added for 5 min. Cells were scraped, transferred to microcentrifuge tubes, and sonicated four times for 5 s on ice. Insoluble cellular components were removed by centrifugation at 10,000 rpm for 10 min at 4°C. The protein content of the supernatant was determined according to the Bradford method using BSA as the standard. Extracts (40 µg protein) were mixed with 4x loading buffer [125 mM Tris (pH 6.8), 20% glycerol, 10% mercaptoethanol, 4.6% SDS, and 0.02% bromophenol blue] and boiled for 5 min. Proteins were separated by 10% Tris-glycine SDS-PAGE and transferred onto nitrocellulose membranes (Protran BA85, Schleicher & Schuell Bioscience) at 150 mA for 2 h. For cPLA₂ (no. 2832, Cell Signaling) and phospho-cPLA₂ (no. 2831, Cell Signaling), membranes were blocked for 1 h in Tris-buffered saline-0.1% Tween 20 (TBST) containing 5% nonfat milk powder, washed three times for 5 min in TBST, and incubated with 1:1,000 diluted antibodies overnight at 4°C in TBST containing 5% BSA. Membranes were washed three times for 5 min in TBST, incubated with a 1:7,500 dilution of goat anti-rabbit secondary antibody (no. 111-035-144, Jackson ImmunoResearch) conjugated to horseradish peroxidase for 1 h, and washed three times for 5 min in TBST before detection using SuperSignal Chemiluminescent substrate (Pierce, Rockford, IL).

Statistical analysis. Data were examined for statistical significance using SPSS 8.02 software for Student's t-test, and differences were considered significant at P < 0.05.

RESULTS

Thrombin-induced proliferation of BASMCs. To assess the induction of SMC proliferation by thrombin and calibrate its effect, we determined thymidine incorporation into confluent SMCs under increasing concentrations of thrombin and compared it with SMC proliferation in serum-containing (10%) medium. As shown in Fig. 1, thrombin enhanced SMC proliferation in a concentration-dependent manner, reaching FCS-induced growth at 2 U/ml, which is conventionally used in other studies (8). However, the proliferation-inducing activity differed significantly between thrombin batches, and, therefore, every new batch was calibrated independently against FCS, and the thrombin concentration used in the following experiments was at least 2 U/ml.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Induction of bovine aortic smooth muscle cell (BASMC) proliferation by thrombin. Confluent/synchronized cells were incubated with increasing thrombin concentrations for 48 h, and thymidine incorporation was determined as described in MATERIALS AND METHODS. The proliferation was compared with that in FCS (10%)-containing medium. Each data point is the mean ± SE for 4 replicates.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effect of thrombin on fatty acid (FA) release. Confluent/synchronized cells were metabolically labeled overnight with either [3H]arachidonic acid ([3H]AA) or [3H]oleic acid ([3H]OA). On the day of the experiment, cells were washed with label-free medium, preincubated with or without thimerosal for 20 min, and then stimulated with thrombin for 1 h. The culture medium was then collected, and its [3H]FA content was determined. Each data point is the mean ± SE for 5 replicates. *P = 0.002; **P = 0.0002.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Inhibition of thrombin-induced-AA release (A) and BASMC proliferation (B) by cytosolic phospholipase A2 (cPLA2) inhibitor. A: confluent/synchronized cells were labeled overnight with [3H]AA. Cells were washed and then incubated for 30 min with or without thimerosal in the presence of increasing concentrations of the cPLA2 inhibitor arachidonyl-trifluoromethyl-ketone (AACOCF3). Cells were then stimulated with thrombin for 1 h, and the [3H]AA content in the culture medium was determined. Each data point is the mean ± SE for 5 replicates. *P = 0.02; **P = 0.2 x 10–4; P = 0.4 x 10–4; P = 0.4 x 10–3. B: confluent/synchronized cells were incubated for 30 min with or without the cPLA2 inhibitor (AACOCF3) or the iPLA2 inhibitor [bromoenolactone (BEL)] and then incubated overnight in either control or thrombin-supplemented medium, and [3H]thymidine incorporation was measured during the following 2 h. Cells were washed and then lysed for the determination of the cellular label content. Each data point is the mean ± SE for 5 replicates. *P = 0.04; **P = 0.001.

Thrombin-induced cPLA₂ mRNA expression. After the above experiments, which suggested the requirement of cPLA₂ activation for thrombin-induced BASMC proliferation, cPLA₂ mRNA expression was determined after the induction of proliferation by thrombin. As shown in Fig. 4A, consistent with an increase in enzymatic activity (Figs. 2 and 3), thrombin elevated cPLA₂ mRNA expression up to 2.5-fold.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Induction of mRNA expression (A) and phosphorylation (B) of cPLA2 by thrombin. A: confluent/synchronized cells were incubated for the indicated times in either control or thrombin-supplemented medium, and enzyme mRNA expressions were determined by RT-PCR. Each data point is the mean ± SE for 3 replicates. *P = 0.003. B: cells were incubated for 1 h with or without thrombin, lysed, and then subjected to Western blot analysis using antibodies to cPLA2 or its phosphorylated form (cPLA2-P) as described in MATERIALS AND METHODS. The Shown are representative blots of 3 reproducible experiments in which treatment with thrombin increased cPLA2-P by 757%, 427%, and 149%, as determined by densitometry.

Activation of FA incorporation into membrane PLs of thrombin-induced proliferating BASMCs. As discussed in the Introduction, cell growth obviously requires increased membrane production, and if this involves AA release from PL by PLA₂, as described above, then FA reacylation into lysophospholipids would follow. To examine the requirement for transacylase activity in thrombin-induced BASMC growth, we determined the thrombin effect on AA and OA incorporation into cell membranes. As shown in Fig. 5A, thrombin enhanced OA incorporation into cell membrane PLs. However, whereas thrombin elevated the release of AA (Fig. 2), it did not increase the net AA incorporation into cell PLs (not shown). These findings suggest that during cell proliferation thrombin exerts membrane PL remodeling by increasing the level of PL-containing OA (and possibly other non-AA FAs) while releasing AA.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Effect of thrombin on OA incorporation into BASMC phospholipids (A) and mRNA expression of lysophosphatidic acid-fatty acid transacylase (LPAAT; B). A: confluent/synchronized cells were incubated for the indicated times in control or thrombin-supplemented medium, and [3H]OA was then added to the medium for 45 min. Cells were washed with medium and then harvested, and their [3H]OA content was determined. Each data point is the mean ± SE for 4 replicates. *P < 0.05; **P < 0.02. B: confluent/synchronized cells were incubated for the indicated times in either control or thrombin-supplemented medium, and enzyme mRNA expressions were determined by RT-PCR. Each data point is the mean ± SE for 3 replicates. *P = 0.002.

View larger version (12K): [in this window] [in a new window]   Fig. 6. Inhibition of BASMC proliferation by the FA acylation inhibitor thimerosal. Confluent/synchronized cells were incubated overnight in control or thrombin-supplemented medium, and [3H]thymidine incorporation was determined in the absence or presence of thimerosal. Each data point is the mean ± SE for 5 replicates. *P = 0.002.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Inhibition of thrombin-induced BASMC by the protease-activated receptor (PAR)-1 inhibitor. Confluent/synchronized cells were incubated with the PAR-1 inhibitor SCH-79797 for 30 min prior to overnight treatment with thrombin. Proliferation was then determined by thymidine incorporation. Each data point is the mean ± SE for 5 replicates. *P = 2.4 x 10–6.

View larger version (13K): [in this window] [in a new window]   Fig. 8. PAR-1 inhibitor does not affect mRNA expression of cPLA2 and LPAAT. Confluent/synchronized cells were incubated with the PAR-1 inhibitor SCH-79797 for 30 min prior to treatment with thrombin for 12 h. mRNA levels of the enzymes were determined as described in MATERIALS AND METHODS. Each data point is the mean ± SE for 2 replicates. No significant differences were observed between the bars denoted with * or **.

SMC proliferation is a key process in pathological thickening of the vascular wall in the development of atherosclerosis and stenosis and in airway remodeling in asthma. Both processes are facilitated by inflammatory conditions. Cell growth involves the formation of new cell membrane components, PLs in particular. The formation of membrane PLs can be done by de novo synthesis and/or PL remodeling involving lipolysis and reconstitution. Both thrombin and products of PL hydrolysis are markedly enhanced in inflammatory conditions. Products of PL hydrolysis, commonly denoted inflammatory lipid mediators, such as lysophospholipid, lysophosphatidic acid in particular, as well as AA and its eicosanoid derivatives, induce SMC proliferation, leading to related pathology (2, 9, 10, 13, 18). PL lipolysis and the subsequent production of inflammatory lipid mediators can occur by multiple enzymatic pathways (20), but PLA₂ enzymes play a major role in these processes. In addition, extracellular sPLA₂s, which are secreted by activated inflammatory cells, can act as receptor ligands, independent of their lipolytic activity, to activate cell signaling and subsequent cell proliferation (25). Yet, the possible involvement of PL-metabolizing enzymes in thrombin-induced SMC proliferation has not been well investigated, as discussed in the Introduction.

The present study shows that the thrombin-induced proliferation of BASMCs requires 1) the activation of lipolytic enzyme cPLA₂ (but not iPLA₂ or sPLA₂), as shown by its elevated mRNA expression and enzyme phosphorylation, selective AA release, and the concomitant inhibition of cell proliferation by the selective cPLA₂ inhibitor AACOCF₃; and 2) the activation of the FA-acylating enzyme LPAAT, as shown by its elevated mRNA expression, incorporation of FAs, and the concomitant inhibition of cell proliferation by the acylation inhibitor thimerosal.

Previous studies have demonstrated the involvement of iPLA₂ (also called Ca²⁺-independent cPLA₂-) in membrane remodeling (4) and FA transacylation (26). It was also reported that thrombin-stimulated activation of MAPKs in rabbit ventricular myocytes is dependent on iPLA₂ activity (5). Of particular relevance to the present study is the report of Yellaturu and Rao (27), which showed that thrombin-induced proliferation of rat thoracic aorta SMCs required increased activity of iPLA₂, and this was inhibited by BEL (at 10 µM). In variance to that, in the present study, we could not verify the involvement of iPLA₂ in thrombin-induced proliferation of BASMCs, since, except for AA, no release of other FA could be detected, even in the presence of the transacylation inhibitor (which further increased AA release). In addition, application of the same procedure of Yellaturu and Rao, namely, treatment with the iPLA₂ inhibitor BEL (up to 50 µM in some experiments), did not affect the thrombin-induced AA release. Although we do not have a satisfactory explanation for the discrepancy between our findings and those of Yellaturu and Rao, this might be due to differences in species (rats vs. cows), or methodology, e.g., inhibition of FA reacylation, as applied in the present study. Also, a later study (21), using HEK-293 cells and Caki-1 cells, showed that iPLA₂-β and iPLA₂- may differ in their effect on cell growth and can be selectively inhibited by R- and S-enantiomers of BEL. This might suggest that the distinction between iPLA₂ isoforms and their selective inhibition (by BEL enantiomers) should be considered when studying the role of iPLA₂ in cell function.

On the other hand, we clearly found that thrombin-induced BASMC proliferation is associated with cPLA₂ expression and selectively enhanced AA release. Both AA release and cell proliferation were suppressed by the cPLA₂ inhibitor AACOCF₃. These findings strongly suggest that cPLA₂ activation is required for thrombin-induced BASMC proliferation. This conclusion is in accord with the report of Anderson and Marshall (2), which showed that the cPLA₂-mediated (AACOCF₃- inhibited) release of AA is critical for the proliferation of human artery vascular SMCs (although in growth medium, not thrombin-supplemented medium).

At the pathway of PL formation, the present study also showed that thrombin-induced BASMC proliferation requires the activation of FA transacylase, as shown by the enhanced FA incorporation and expression of LPAAT mRNA, and inhibition of all of the above by the acylation inhibitor. As noted above, both PL hydrolysis and reconstitution may occur by more than one enzymatic pathway. The present study shows that in thrombin-induced BASMC proliferation, membrane PL formation involves their hydrolysis by cPLA₂ and FA incorporation by LPAAT.

Taken together, these findings suggest that by activation of AA-specific cPLA₂ and non-FA-specific LPAAT, thrombin-induced BASMC proliferation is associated with the increased production of AA, a precursor for diverse proinflammatory eicosanoids. Since elevated thrombin levels and subsequent SMC proliferation are characteristic of inflammatory conditions, it is probable that the thrombin-induced AA and subsequent eicosanoid production are important mechanisms by which thrombin exerts its proinflammatory effect.

Of particular interest in this study is the finding that the PAR-1 inhibitor suppressed thrombin-induced cell proliferation without affecting the thrombin-induced activity of the above PL-metabolizing enzymes (Figs. 7 and 8). Yet, both cPLA₂ and LPAAT inhibitors independently suppressed thrombin-induced BASMC proliferation (Figs. 3 and 6). Taken together, these findings appear to suggest that thrombin induces SMC proliferation by two independent mechanisms: 1) via its proteolytic action on PAR-1 and 2) via PAR-1-independent activation of PL-metabolizing enzymes cPLA₂ and LPAAT. It is possible, of course, that the thrombin-induced activation of PL-metabolizing enzymes is mediated by other PARs or another mechanism(s) altogether, which is yet to be explored. Nevertheless, the results shown in Figs. 3B, 6, and 7 demonstrate that each of the inhibitors tested, namely, the cPLA₂ inhibitor (Fig. 3B), the LPAAT inhibitor (Fig. 6), and PAR-1 inhibitor (Fig. 7), abolished thrombin-induced BASMC proliferation, bringing it to that of control, thrombin-untreated cells. This implies that the effects of the two mechanisms are not additive. Instead, both are essential since blockade of either of them eliminates the induction of BASMC proliferation by thrombin.

The PAR-mediated mechanism has been given considerable attention, whereas the other mechanism has hardly been studied, and its extensive investigation is clearly of interest. The findings of the present study thus open a new avenue in the study of thrombin function and might introduce new insights into the control of thrombin-associated pathological conditions by the regulation of cPLA₂ and/or LPAAT.

FOOTNOTES

Address for reprint requests and other correspondence: S. Yedgar, Dept. of Biochemistry, Hebrew Univ.-Hadassah Medical School, Jerusalem, Israel 91120 (e-mail: yedgar{at}md.huji.ac.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* N. Gluck and O. Schwob contributed equally to this work.

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