Advertisement
J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M600480-JLR200 on April 24, 2007

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600480-JLR200v1
48/7/1533    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ibrahim, S.
Right arrow Articles by Ponsin, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ibrahim, S.
Right arrow Articles by Ponsin, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 48, 1533-1538, July 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity

Salam Ibrahim*,{dagger},§,**,{dagger}{dagger},§§, Catherine Calzada*,{dagger},§,**,{dagger}{dagger},§§, Valérie Pruneta-Deloche*,{dagger},§,**,{dagger}{dagger},§§, Michel Lagarde*,{dagger},§,**,{dagger}{dagger},§§ and Gabriel Ponsin1,*,{dagger},§,**,{dagger}{dagger},§§

* Université de Lyon, Lyon, F-69003 France
{dagger} Institut National de la Santé et de la Recherche Médicale, U870, Institut Fédératif de Recherche 62, Lyon, F-69008 France
§ Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1235, Lyon, F-69008 France
** Institut National des Sciences Appliquées-Lyon, Régulations Métaboliques, Nutrition et Diabètes, Villeurbanne, F-69621 France
{dagger}{dagger} Université Lyon 1, Lyon, F-69003 France
§§ Hospices Civils de Lyon, Lyon, F-69008 France

Published, JLR Papers in Press, April 24, 2007.

1 To whom correspondence should be addressed. e-mail: gabriel.ponsin{at}insa-lyon.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 µM PL) to platelets (2 x 108/ml) was measured after incubations of 1 h at 37°C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 µM); esculetin, a 12-lipoxygenase inhibitor (20 µM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor (100 µM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca2+ chelator (20 µM); bromoenol lactone (BEL), a Ca2+- independent phospholipase A2 (iPLA2) inhibitor (100 nM); and 1-[6-[[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 µM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A2 (cPLA2) and iPLA2, only cPLA2 is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA2-dependent process. The inhibition of iPLA2 by BEL had no effect on PL transfers.

Supplementary key words thrombin • lipoprotein lipase • metabolic inhibitors • Ca2+- independent phospholipase A2 • very low density lipoprotein

Abbreviations: BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester; BEL, bromoenol lactone; cPLA2, cytosolic phospholipase A2; iPLA2, Ca2+-independent phospholipase A2; MAFP, methyl arachidonyl fluorophosphonate; [14C]PAPC, 1-palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PL, phospholipid; PLA2, phospholipase A2; PLC, phospholipase C; PLTP, phospholipid transfer protein; TXB2, thromboxane B2; U73122, 1-[6-[[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]-1H-pyrrole-2,5-dione


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In platelets, phospholipids (PLs) are involved in several signal transduction pathways, including those that depend upon the activities of phospholipase A2 (PLA2) and phospholipase C (PLC) enzymes (1–4). Platelet activation stimulates the activity of PLA2, which cleaves fatty acids from the sn-2 position of PLs. In particular, the cytosolic phospholipase A2 (cPLA2) reaction favors the release of arachidonic acid, which is the precursor of prostaglandins and leukotrienes generated through the actions of cyclooxygenase and lipoxygenase, respectively (1, 2). In addition, in activated platelets, the formation of diacylglycerols resulting from the action of PLC stimulates several metabolic cascades leading to various effects, including protein phosphorylation, granule secretion, and release of fatty acids by diacylglycerol and monoacylglycerol lipases (3, 4). Thus, platelets actively degrade PLs, which necessitates their permanent regeneration. Although PLs may be resynthesized in platelets (5), a substantial part has been shown to be imported from circulating lipoproteins. In vitro, LDL and HDL, the two major human plasma lipoprotein fractions, transfer various PL species to platelets, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (6–8).

More recently, we considered the possibility that VLDL-associated PLs also could be transferred to platelets (9). Albeit less abundant than LDL and HDL in fasting conditions, VLDL may have increased postprandial concentrations. VLDLs are secreted by the liver in the circulation, where they undergo hydrolysis of their core triglyceride content through the successive actions of LPL and hepatic lipase, which ultimately results in the formation of LDL (10, 11). During this process, the excess of VLDL surface components, including apolipoproteins, cholesterol, and PLs, is released from the particles. Although a large part of cholesterol and PL is transferred to HDL (12, 13), our work demonstrated that VLDL-associated PLs can also be transferred to platelets and that these transfers are favored by LPL and platelet activation (9). This effect of LPL results from two different actions. First, the LPL-mediated lipolysis of VLDL destabilizes the particle surface, thereby favoring the release of PLs. Second, the fatty acids released during lipolysis favor platelet activation, as judged from their increased thromboxane production. That the transfer of PL from VLDL to platelets depended upon platelet activation was confirmed by our observation that thrombin stimulates both platelet thromboxane production and PL transfer.

Although platelets are able to import PL from various lipoprotein fractions, the underlying mechanisms of these transfers appeared to be complex. They are independent of lipoprotein binding and internalization (7). In agreement with this concept, the scavenger receptor class B type 1, which can mediate the specific import of PL into various cells, was shown to be absent in platelets (8). However, major differences emerge when comparing the PL transfers obtained from the different lipoprotein fractions. The transfer of LDL- or HDL-derived PE into platelets, but not that of PC or sphingomyelin, was stimulated by platelet activators, including thrombin, collagen, and ADP, and was dependent upon the secretion of an unidentified cellular protein factor (14). In contrast, there was no apparent specificity of the PL species transferred from VLDL to platelets (9). Both LPL and thrombin stimulated the import by platelets of VLDL-derived palmitoyl-arachidonyl-PC, palmitoyl-arachidonyl-PE, and dipalmitoyl-PC with similar efficiencies. Thus, the regulation of PL uptake by platelets appears to dramatically depend upon the lipoprotein used as the donor. Although LDL might preferentially transfer certain PL species, namely PE, by a specific mechanism, VLDL could supply all types of PLs to platelets without consideration of their nature. This concept prompted us to further characterize the metabolic pathway governing the transfer of PL from VLDL to platelets. In this work, using a variety of metabolic inhibitors, we present in vitro evidence showing that this transfer results from a cPLA2-dependent process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
[1-14C]arachidonic acid (40–60 mCi/mmol) and 1-palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine ([14C]PAPC; 40–60 mCi/mmol) were purchased from Perkin-Elmer (Boston, MA). Thrombin, bovine milk LPL (EC 3.1.1.34), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), and polyoxyethylen-9-laurylether were obtained from Sigma Chemical (St. Louis, MO). PLs were assayed using enzymatic kits from Wako Chemicals GmbH (Neuss, Germany). Thromboxane B2 (TXB2) concentrations were determined using the enzyme immunoassay Biotrak system from Amersham Biosciences (Orsay, France). Esculetin, methyl arachidonyl fluorophosphonate (MAFP), 1-[6-[[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), and bromoenol lactone (BEL) were from Biomol (Plymouth Meeting, PA).

Isolation and labeling of lipoproteins
VLDL (d < 1.006 g/ml) and lipoprotein-deprived plasma (d > 1.21 g/ml) were isolated from human plasma by preparative ultracentrifugation (15). Depending upon the volume of plasma, the ultracentrifugation was performed either in a Beckman LE 80K using a 50.2 fixed-angle rotor or in a Beckman TL-100 tabletop ultracentrifuge using a TLA 100.3 fixed-angle rotor. The resulting preparations were then dialyzed extensively against a buffer containing 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, and 10 mM NaN3, pH 7.4. VLDLs were labeled with [14C]PAPC (1 µCi/10 µmol lipoprotein PL) as reported previously (16). The desired amount of radioactive label was dried under nitrogen, solubilized in ethanol, and added to VLDL under vortexing. To avoid destruction of the lipoprotein structure, the final proportion of ethanol in the samples was maintained at <1% (v/v). The samples were then incubated for 3 h at 37°C, and the labeled VLDLs were reisolated by ultracentrifugation.

Platelet isolation
Fresh blood was collected at the local blood bank (Etablissement Français du Sang) from healthy volunteers. Blood was drawn into a one-seventh volume of a solution containing 19.6 mM citric acid, 89.4 mM sodium citrate, 16.1 mM NaH2PO4, and 128.7 mM dextrose, pH 5.6. The platelet isolation procedure was essentially based on that described previously (17). Briefly, platelet-rich plasma was obtained after blood centrifugation at 200 g for 17 min at 20°C and acidified to pH 6.4 with 0.15 M citric acid. Platelets were immediately pelleted by centrifugation at 900 g for 12 min and washed in acidified lipoprotein-deprived plasma. After repelleting, the platelets were finally washed and resuspended in a Tyrode-HEPES buffer solution containing 137 mM NaCl, 2.7 mM KCl, 0.41 mM NaH2PO4, 11.9 mM NaHCO3, 1 mM MgCl2, 5.5 mM glucose, and 5 mM HEPES, pH 7.35. Platelet suspensions were left for 1 h at room temperature before experiments were started.

Platelet aggregation
To avoid abnormal experimental data that could result from unknown medical treatment taken by blood donors, each platelet preparation was controlled for its functional ability to aggregate before being used in our studies. Aggregations were induced by arachidonic acid and performed in a Chronolog dual-channel aggregometer (Coulter, Margency, France) according to the turbidimetric method of Born (18).

Transfers of labeled PLs from VLDL to platelets
Labeled VLDLs (200 nmol/ml PL) were incubated with platelets (3 x 108 cells) in a final volume of 1.5 ml at 37°C for 1 h (9). For each experiment, the VLDLs and platelets were each isolated from a single donor. At the end of the incubations, platelets were separated from the medium by centrifugation. The pelleted platelets were first washed in plasma to remove nonspecifically adsorbed labeled VLDL and then in Tyrode-HEPES buffer. The final pellets were dissolved by overnight incubation in 0.25 ml of 0.4% polyoxyethylen-9-laurylether and counted for radioactivity. The results were finally corrected for nonspecifically adsorbed radioactivity at time 0 (nonincubated platelets).

When platelet activation was desired, LPL (500 ng/ml) or thrombin (0.1 U/ml) was added at the beginning of the incubations. In contrast, to inhibit specific metabolic pathways, various inhibitors were added to platelets during preincubation periods as follows: 2 min for U73122 (20 µM), a PLC inhibitor (19); 5 min for aspirin (300 µM), a cyclooxygenase inhibitor (20), and esculetin (20 µM), a lipoxygenase inhibitor (21); 10 min for MAFP (100 µM), a cPLA2 and Ca2+-independent phospholipase A2 (iPLA2) inhibitor (22, 23), and BEL (100 nM), an iPLA2 inhibitor (22, 24); and 20 min for BAPTA-AM (20 µM), an intracellular Ca2+ chelator (25, 26).

Determination of TXB2 production
The production of TXB2 by platelets was determined at the beginning and end of incubations carried out under various conditions as described above. However, platelets were not separated from their media before the TXB2 assay, thereby permitting the measurement of total TXB2.

Determination of the [1-14C]arachidonic acid content of platelet PLs
PL hydrolysis was assayed in platelets prelabeled with [1-14C]arachidonic acid (20 nCi/ml) for 1 h at 37°C (27). After separation from the medium by centrifugation, platelets were washed in Tyrode-HEPES buffer and incubated for 5 min in the absence or presence of thrombin or cPLA2 inhibitors. For these particular experiments, no VLDL was added to the incubation medium to avoid PL transfers. At the end of the incubations, platelets were washed and resuspended in 1 ml of buffer. Lipids were then extracted and separated by thin-layer chromatography in a solvent composed of hexane-diethyl ether-acetic acid (80:20:1). The PL spots were finally scraped off and counted for radioactivity.

Determination of plasma phospholipid transfer protein activity
Phospholipid transfer protein (PLTP) activity was determined using an in vitro assay in which we measured the transfer of radiolabeled PL from VLDL to HDL using delipidated plasma as the source of PLTP, as described previously (16). Basal as well as PLTP-facilitated PL transfers were measured in the absence or presence of cPLA2 inhibitors at the same concentrations as those used with platelets. The results are expressed as percentages of PL transferred during 2 h incubations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To explore the dependence of VLDL-associated PL transfer to platelets upon platelet activation, we performed a first series of experiments in which various inhibitors were used to block different metabolic pathways involved in platelet activation. The transfers of [14C]PAPC from VLDLs to platelets were measured after incubations of 1 h at 37°C. They were stimulated by ~2.5- and 3.5-fold when platelets were activated by either thrombin (0.1 U/ml) or LPL (500 ng/ml), respectively (Fig. 1 ). When the experiments were performed in the presence of aspirin, a cyclooxygenase inhibitor, at a concentration (300 µM) known to block the formation of thromboxane, no change was observed in the transfers of PL whether stimulated or not by either thrombin or LPL. Similarly, the transfers of PL remained unchanged when the 12-lipoxygenase-dependent metabolic pathway was inhibited by esculetin (20 µM). In contrast, when the incubations were carried out in the presence of MAFP, a PLA2 inhibitor, the stimulating effects of both thrombin and LPL on PL transfers were abolished, resulting in values comparable to those of controls.


Figure 1
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 1. Effect of metabolic inhibitors on the transfers of 1-palmitoyl-2-[1-14C]arachidonyl-phosphatidylcholine ([14C]PAPC) from VLDL to platelets. Platelets were incubated with [14C]PAPC-labeled VLDL [200 nmol of phospholipids (PLs)/ml] for 1 h at 37°C, in a total volume of 1.5 ml, in the presence or absence of thrombin (0.1 U/ml) or LPL (500 ng/ml). To study the putative dependence of PL transfers upon various metabolic pathways, the results obtained in basal conditions (white bars) were compared with those resulting from the effects of aspirin (300 µM), a cyclooxygenase inhibitor (black bars); esculetin (20 µM), a 12-lipoxygenase inhibitor (hatched bars); or methyl arachidonyl fluorophosphonate (100 µM), a phospholipase A2 (PLA2) inhibitor (gray bars). Transfers are expressed as percentages of radioactivity incorporated by 3 x 108 platelets. Values shown are means ± SEM from four to six independent experiments.

 
Three different PLA2 enzymes are present in human platelets: cPLA2, iPLA2, and secretory PLA2. Only the two former may be affected by MAFP. However, their activities can be discriminated on the basis of their Ca2+ dependence, because cPLA2 but not iPLA2 is a Ca2+-dependent enzyme. This prompted us to study the thrombin- or LPL-stimulated transfers of [14C]PAPC from VLDL to platelets in the presence of either BAPTA-AM, a Ca2+ chelator, or U73122, a PLC inhibitor (Fig. 2 ). The results showed that both BAPTA-AM and U73122 inhibited the stimulating effects of thrombin and LPL, clearly suggesting that PL import into platelets was controlled by a cPLA2-dependent process rather than an iPLA2-dependent process. This was confirmed by the results obtained when the incubations were performed in the presence of 100 nM BEL. The latter, which at this concentration inhibits iPLA2 but not cPLA2 (28), had no effect on PL transfers, whether stimulated or not by either thrombin or LPL. To control that metabolic inhibitors did not directly modify the ability of VLDL to transfer PLs, in particular by inhibiting the activity of PLTP that could be present at the lipoprotein surface, we performed in vitro assays in which we measured the transfers of radiolabeled PLs from VLDL to HDL (Table 1 ). None of MAFP, BAPTA-AM, or U73122 had any effect on basal or PLTP-facilitated PL transfers.


Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 2. Comparison of the effects of cytosolic Ca2+-dependent phospholipase A2 (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2) on the transfer of [14C]PAPC from VLDL to platelets. Platelets were incubated with [14C]PAPC-labeled VLDL (200 nmol PL/ml) for 1 h at 37°C, in a total volume of 1.5 ml, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml). To discriminate between the putative effects of cPLA2 and iPLA2, the results obtained in basal conditions (white bars) were compared with those observed in the presence of 1-[6-[[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; 20 µM), a phospholipase C (PLC) inhibitor (black bars); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM; 20 µM), a Ca2+ chelator (hatched bars); or bromoenol lactone (BEL; 100 nM), an iPLA2 inhibitor (gray bars). Transfers are expressed as percentages of radioactivity incorporated by 3 x 108 platelets. Values shown are means ± SEM from four independent experiments.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. Effects of metabolic inhibitors on PLTP-facilitated PL transfer

 
Finally, we determined the metabolic effects of BAPTA-AM and U73122 on platelet PL hydrolysis and TXB2 production. In the absence of inhibitors, we observed a decrease of the arachidonic content of PLs that was stimulated by thrombin, clearly showing a major hydrolysis (Table 2 ). In the presence of U73122, which inhibits both cPLA2 and PLC, PL hydrolysis was strongly inhibited, whereas it was only partially decreased in the presence of BAPTA-AM, which inhibits only cPLA2. In addition, clear stimulating effects of thrombin and LPL emerged when the platelet production of TXB2 was studied (Fig. 3 ). The thrombin-stimulated production of TXB2 was totally inhibited by BAPTA-AM or U73122, whereas they only partially decreased the stimulating effect of LPL. As expected, BEL, which specifically inhibits iPLA2, had no effect on TXB2 production.


View this table:
[in this window]
[in a new window]

  		TABLE 2. PL arachidonic acid content

 

Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 3. Effects of cPLA2 and iPLA2 inhibitors on the production of thromboxane B2 (TXB2) by platelets. Platelets were incubated with VLDL (200 nmol PL/ml) for 1 h at 37°C, in a total volume of 1.5 ml, in the absence (white bars) or presence of U73122 (20 µM), a PLC inhibitor (black bars); BAPTA-AM (20 µM), a Ca2+ chelator (hatched bars); or BEL (100 nM), an iPLA2 inhibitor (gray bars). The thrombin-stimulated (0.1 U/ml) or LPL-stimulated (500 ng/ml) productions of TXB2 are expressed as percentages of those obtained in basal conditions. TXB2 concentrations were measured using a commercial kit, as described in Materials and Methods. Values shown are means ± SEM from three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We recently showed that platelets are able to import PL from VLDL and that this process is stimulated when platelets are activated through the action of thrombin or LPL (9). The present work was intended to characterize the relationship between the transfer of PL from VLDL to platelets and platelet activation. The latter results from the combination of complex metabolic pathways that can be summarized as follows. Plasma membrane PL may be hydrolyzed by cPLA2, resulting in the release of fatty acids. Among these, arachidonic acid can be metabolized through two different pathways (29–31). The first, which depends upon cyclooxygenase activity, leads to the formation of prostaglandins and thromboxane A2, a very potent platelet activator. The second pathway is governed by the action of 12-lipoxygenase, which transforms arachidonic acid in 12-hydroperoxy-eicosatetraenoic acid, a metabolite also able to favor platelet activation. In addition, phosphatidylinositol diphosphate may also be hydrolyzed by PLC, leading to the formation of diacylglycerols and inositol triphosphate. Although diacylglycerols stimulate metabolic cascades resulting in protein phosphorylation, granule secretion, and the release of fatty acids by diacylglycerol and monoacylglycerol lipases, inositol triphosphate favors the increase of Ca2+ intracellular concentration (3, 4).

To study the putative involvement of these metabolic pathways, we measured the transfer of PL from VLDL to platelets in the presence of aspirin or esculetin, which inhibit cyclooxygenase and 12-lipoxygenase, respectively (20, 21). We observed no changes in the transfers of PL to platelets, whether stimulated or not by thrombin or LPL, indicating that neither cyclooxygenase- nor 12-lipoxygenase-dependent processes were directly responsible for the magnitude of PL transfers. In contrast, these PL transfers were decreased when the incubations were performed in the presence of MAFP, an inhibitor of PLA2. PLA2 is a superfamily of enzymes consisting of secretory and intracellular species. The latter comprise cPLA2 and iPLA2, which are both inhibited by MAFP (22, 23). To discriminate between these two enzymes, we took advantage of the differences in their mechanism of action. cPLA2 but not iPLA2 is a Ca2+-dependent enzyme (28). The activation of cPLA2 depends upon two synergistic processes: the catalytic activity of the enzyme requires a previous phosphorylation, and its translocation to the membrane necessitates the mobilization of cytosolic Ca2+ (32, 33). Because the latter may be regulated by the PLC pathway, we measured the thrombin- and LPL-stimulated transfers of PL from VLDL to platelets in the presence of U73122, a PLC inhibitor (19). The PL transfers were clearly inhibited. However, as mentioned above, in addition to inositol triphosphate-stimulated Ca2+ mobilization, the action of PLC generates diacylglycerols that stimulate different metabolic cascades (3, 4). Thus, to distinguish between these various effects, we performed experiments in which U73122 was substituted for BAPTA-AM, a Ca2+ chelator (25, 26). The PL transfers were decreased similarly, showing that the effect of PLC was attributable to its ability to mobilize cytosolic Ca2+. Overall consideration of these results clearly suggests that the transfer of PL from VLDL to platelets depends upon a cPLA2-dependent process rather than an iPLA2-dependent process. This concept was finally confirmed by our observation that PL transfers remained unaffected when iPLA2 was specifically inhibited in the presence of BEL (24).

In addition to PL transfers, the importance of cPLA2 in platelet metabolism was assessed by measuring the arachidonic acid content of PLs in platelets and their TXB2 production. A strong PL hydrolysis was observed during platelet incubations, which was in part dependent upon cPLA2 activity. As shown previously (9), both thrombin and LPL stimulated platelet TXB2 production. The stimulating effect of thrombin was totally inhibited by BAPTA-AM or U73122, whereas that of LPL was only partially decreased. This was expected, because LPL-stimulated platelet activation is initially attributable to the uptake by platelets of the fatty acids released during LPL-mediated lipolysis of VLDL (9).

Although the question of how the transfers of PL are precisely affected by cPLA2 activity has not been addressed directly in this work, a likely mechanism may be considered. Because the cPLA2-stimulated hydrolysis of PL occurs at the inner leaflet of the plasma membrane, the enzyme activity necessarily results in disequilibrium of the PL concentrations between the membrane inner and outer leaflets. Several physiological processes are known to lead to comparable disequilibrium, which is compensated by flip-flop mechanisms (34, 35). Thus, on the basis of a similar mechanism, we speculate that, in our case, the net translocation of PL from the outer to the inner leaflet of the membrane would cause a PL deficit in the outer leaflet that might be compensated by the import of PL from VLDL.


   ACKNOWLEDGMENTS
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale. S.I. was the recipient of a grant from the French Ministry of Education and Research.

Manuscript received November 8, 2006 and in revised form March 8, 2007 and in re-revised form April 18, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Samuelsson, B., M. Goldyne, E. Granström, M. Hamberg, S. Hammarström, and C. Malmsten. 1978. Prostaglandins and thromboxanes. Annu. Rev. Biochem. 47: 997–1029.[CrossRef][Medline]

  2. Clark, J. D., A. R. Schievella, E. A. Nalefski, and L. L. Lin. 1995. Cytosolic phospholipase A2. J. Lipid Mediat. Cell Signal. 12: 83–117.[CrossRef][Medline]

  3. Ha, K. S., and J. H. Exton. 1993. Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor. A possible function for phosphatidylcholine-derived diacylglycerol. J. Biol. Chem. 268: 10534–10539.[Abstract/Free Full Text]

  4. Hug, H., and T. F. Sarre. 1993. Protein kinase C isoenzymes: divergence in signal transduction? Biochem. J. 291: 329–343.[Medline]

  5. Kent, C. 1995. Eukaryotic phospholipid biosynthesis. Annu. Rev. Biochem. 64: 315–343.[CrossRef][Medline]

  6. Engelmann, B., C. Kögl, R. Kulschar, and B. Schaipp. 1996. Transfer of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin from low- and high-density lipoprotein to human platelets. Biochem. J. 315: 781–789.[Medline]

  7. Dobner, P., E. Koller, and B. Engelmann. 1999. Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids. FEBS Lett. 444: 270–274.[CrossRef][Medline]

  8. Urban, S., S. Zieseniss, M. Werder, H. Hauser, R. Budzinski, and B. Engelmann. 2000. Scavenger receptor B1 transfers major lipoprotein-associated phospholipids into the cells. J. Biol. Chem. 275: 33409–33415.[Abstract/Free Full Text]

  9. Ibrahim, S., A. Djimet-Baboun, V. Pruneta-Deloche, C. Calzada, M. Lagarde, and G. Ponsin. 2006. Transfer of very low density lipoprotein-associated phospholipids to activated human platelets. J. Lipid Res. 47: 341–348.[Abstract/Free Full Text]

  10. Jackson, R. L., L. R. McLean, and R. A. Demel. 1987. Mechanism of action of lipoprotein lipase and hepatic triglyceride lipase. Am. Heart J. 113: 551–554.[CrossRef][Medline]

  11. Deckelbaum, R. J., S. Eisenberg, M. Fainaru, Y. Barenholz, and T. Olivecrona. 1979. In vitro production of human low density lipoprotein-like particles: a model for very low density lipoprotein catabolism. J. Biol. Chem. 254: 6079–6087.[Free Full Text]

  12. Glomset, J. A. 1968. The plasma lecithin:cholesterol acyl transferase reaction. J. Lipid Res. 9: 155–167.[Abstract]

  13. Patsch, J. M., A. M. Gotto, Jr., T. Olivecrona, and S. Eisenberg. 1978. Formation of high density lipoprotein 2-like particles during lipolysis of very low density lipoproteins in vivo. Proc. Natl. Acad. Sci. USA. 75: 4519–4523.[Abstract/Free Full Text]

  14. Engelmann, B., B. Schaipp, P. Dobner, M. Stoeckelhuber, C. Kögl, W. Siess, and A. Hermetter. 1998. Platelet agonists enhance the import of phosphatidylethanolamine into human platelets. J. Biol. Chem. 273: 27800–27808.[Abstract/Free Full Text]

  15. Havel, R. J., H. A. Eder, and J. H. Bragdon. 1955. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34: 1345–1354.[Medline]

  16. Elchebly, M., T. Pulcini, B. Porokhov, F. Berthezène, and G. Ponsin. 1996. Multiple abnormalities in the transfer of phospholipids from VLDL and LDL to HDL in non-insulin-dependent diabetes. Eur. J. Clin. Invest. 26: 216–223.[CrossRef][Medline]

  17. Lagarde, M., P. A. Bryon, M. Guichardant, and M. Dechavanne. 1980. A simple and efficient method for platelet isolation from their plasma. Thromb. Res. 17: 581–588.[CrossRef][Medline]

  18. Born, G. V. R. 1962. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 194: 927–929.[Medline]

  19. Bleasdale, J. E., N. R. Thakur, R. S. Gremban, G. L. Bundy, F. A. Fitzpatrick, R. J. Smith, and S. Bunting. 1990. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J. Pharmacol. Exp. Ther. 255: 756–768.[Abstract/Free Full Text]

  20. Vane, J. R., and R. M. Botting. 2003. The mechanism of action of aspirin. Thromb. Res. 110: 255–258.[CrossRef][Medline]

  21. Sekiya, K., H. Okuda, and S. Arichi. 1982. Selective inhibition of platelet lipoxygenase by esculetin. Biochim. Biophys. Acta. 713: 68–72.[Medline]

  22. Balsinde, J., M. A. Balboa, P. A. Insel, and E. A. Dennis. 1999. Regulation and inhibition of phospholipase A2. Annu. Rev. Pharmacol. Toxicol. 39: 175–189.[CrossRef][Medline]

  23. Lio, Y. C., L. J. Reynolds, J. Balsinde, and E. A. Dennis. 1996. Irreversible inhibition of Ca2+ independent phospholipase A2 by methyl arachidonyl fluorophosphonate. Biochim. Biophys. Acta. 1302: 55–60.[Medline]

  24. Ackermann, A. J., K. Conde-Frieboes, and E. A. Dennis. 1995. Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J. Biol. Chem. 270: 445–450.[Abstract/Free Full Text]

  25. Börsch-Haubold, A. G., R. M. Kramer, and S. P. Watson. 1995. Cytosolic phospholipase A2 is phosphorylated in collagen- and thrombin-stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase. J. Biol. Chem. 270: 25885–25892.[Abstract/Free Full Text]

  26. Roberts, D. E., A. McNicol, and R. Bose. 2004. Mechanism of collagen activation in human platelets. J. Biol. Chem. 279: 19421–19430.[Abstract/Free Full Text]

  27. Bills, T. K., J. B. Smith, and M. J. Silver. 1977. Selective release of arachidonic acid from the phospholipids of human platelets in response to thrombin. J. Clin. Invest. 60: 1–6.[Medline]

  28. HaZen, S. L., L. A. Zupan, R. H. Weissg, D. P. Getmang, and R. W. Gross. 1991. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2: mechanism-based discrimination between calcium-dependent and -independent phospholipases A2. J. Biol. Chem. 266: 7227–7232.[Abstract/Free Full Text]

  29. Blockmans, D., H. Deckmyn, and J. Vermylen. 1995. Platelet activation. Blood Rev. 9: 143–156.[CrossRef][Medline]

  30. Balsinde, J., M. V. Winstead, and E. A. Dennis. 2002. Phospholipase A2 regulation of arachidonic acid mobilization. FEBS Lett. 531: 2–6.[CrossRef][Medline]

  31. Coulon, L., C. Calzada, P. Moulin, E. Vericel, and M. Lagarde. 2003. Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets. Free Radic. Biol. Med. 35: 616–625.[CrossRef][Medline]

  32. Yedgar, S., D. Lichtenberg, and E. Schnitzer. 2000. Inhibition of phospholipase A2 as a therapeutic target. Biochim. Biophys. Acta. 1488: 182–187.[Medline]

  33. McNicol, A., and T. S. Shibou. 1998. Translocation and phosphorylation of cytosolic phospholipase A2 in activated platelets. Thromb. Res. 92: 19–26.[CrossRef][Medline]

  34. Bevers, E. M., P. Comfurius, D. W. Dekkers, and R. F. Zwaal. 1999. Lipid translocation across the plasma membrane of mammalian cells. Biochim. Biophys. Acta. 1439: 317–330.[Medline]

  35. Sims, P. J., and T. Wiedmer. 2001. Unraveling the mysteries of phospholipids scrambling. Thromb. Haemost. 86: 266–275.[Medline]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600480-JLR200v1
48/7/1533    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ibrahim, S.
Right arrow Articles by Ponsin, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ibrahim, S.
Right arrow Articles by Ponsin, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Advertisement
spacer
Advertisement
Advertisement