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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1907-1913
Published online before print May 25, 2006, doi: 10.1161/01.ATV.0000228821.79588.ef
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1907.)
© 2006 American Heart Association, Inc.

Thrombosis

Human Platelets Secrete the Plasma Type of Platelet-Activating Factor Acetylhydrolase Primarily Associated With Microparticles

John V. Mitsios; Maria P. Vini; Dominique Stengel; Ewa Ninio; Alexandros D. Tselepis

From the Laboratory of Biochemistry, Department of Chemistry (J.V.M., M.P.V., A.D.T.), University of Ioannina, Greece; and INSERM U525 (D.S., E.N.), Université Pierre et Marie Curie-Paris 6 and Faculté de Médecine Pierre et Marie Curie, Paris, France.

Correspondence to Dr Alexandros D. Tselepis, MD, PhD, Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece, 45110. E-mail atselep{at}uoi.gr


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Objective— Platelet-activating factor acetylhydrolase (PAF-AH) expresses a Ca2+-independent phospholipase A2 activity and hydrolyzes platelet-activating factor as well as oxidized phospholipids. Two major types of PAF-AH have been described: the plasma type, which is associated with lipoproteins, and the intracellular type II PAF-AH.

Methods and Results— We investigated the type(s) of PAF-AH expressed in human platelets as well as the mechanism and the enzyme type secreted from platelets during activation. The majority of the enzyme activity (75.1±14.3% of total) is found in the cytosol, whereas 24.9±7.3% is associated with the membranes. Immunofluorescence microscopy studies and Western blotting analysis showed that platelets contain the plasma type as well as the intracellular type II PAF-AH. Furthermore, platelets contain high levels of the mRNA of plasma PAF-AH, whereas only a small quantity of the type II PAF-AH mRNA was detected. On activation, platelets secrete the plasma type of PAF-AH mainly associated with platelet-derived microparticles (PMPs). The enzyme activity was also detected on circulating PMPs in plasma from normolipidemic healthy subjects.

Conclusion— This is the first indication that in addition to lipoproteins, PAF-AH in human plasma is carried by PMPs, suggesting that the PMP-associated PAF-AH may play a role in the dissemination of biological activities mediated by these particles.

Activated platelets secrete the plasma type of platelet-activating factor acetylhydrolase (PAF-AH) primarily associated with platelet-derived microparticles (PMPs) in vitro. Importantly, the enzyme activity is also detected on circulating PMPs in normolipidemic plasma, suggesting that the PMP-associated PAF-AH may play a role in the dissemination of biological activities mediated by these particles.


Key Words: atherosclerosis • inflammation • PAF • PAF-acetyhydrolase • PMPs


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Platelet-activating factor (PAF) is a biologically active phospholipid involved in diverse pathologies such as inflammation and atherosclerosis.1 PAF can activate various cell types including platelets.2 In the presence of PAF, platelets aggregate and degranulate, releasing biologically potent agents.2 Furthermore, platelets activated by other stimuli, such as thrombin, synthesize and secrete PAF.2,3 A prominent role in the regulation of PAF activity in vivo plays PAF acetylhydrolase (PAF-AH), an enzyme that expresses a Ca2+-independent phospholipase A2 activity and degrades PAF into the inactive metabolite lyso-PAF.4

Two major types of PAF-AH have been described, namely the extracellular (plasma) and the intracellular (cytosolic) type. The plasma enzyme is a single polypeptide that originates mostly from cells of the hematopoietic lineage,5 primarily from monocytes/macrophages.6 In human plasma, PAF-AH is predominantly associated with low-density lipoprotein (LDL), whereas a small proportion also binds to high-density lipoprotein (HDL).4 Thus, PAF-AH is also denoted as lipoprotein-associated phospholipase A2.7 Apart from PAF, plasma PAF-AH can effectively hydrolyze biologically potent oxidized phospholipids containing short-chain peroxidized sn-2 residues.8 Several types of the intracellular form of PAF-AH have been described.9 The intracellular type II PAF-AH consists of a single 40-kDa polypeptide chain that exhibits a similar substrate specificity and a 42% amino acid identity with that of the plasma enzyme. A multimeric PAF-AH (IB), which is composed of {alpha}, ß, and {gamma} subunits, was also described in the brain.10

It has been shown previously that type II PAF-AH is the major enzyme type present in human platelets.11 On activation, platelets secrete PAF-AH activity;12 however, the mechanism and the type of PAF-AH secreted remain to be established. Platelet activation either by shear stress or by the combination of thrombin plus collagen leads to the secretion of PAF, which is mainly associated with microparticles.13,14 Indeed, platelet activation by shear stress or various agonists results in the shedding of submicroscopic membrane vesicles (platelet-derived microparticles [PMPs]), which are enriched in bioactive lipids.15,16 PMPs are also enriched in bioactive platelet-derived proteins and express several platelet receptors such as the integrin receptor {alpha}IIbß3.17 Because of the above characteristics, PMPs express a wide range of biological actions.18–20

In the present study, we show for the first time that platelets contain both the plasma type (mRNA and protein) as well as the intracellular type II PAF-AH protein only. On activation, platelets secrete the plasma type of PAF-AH, which is primarily associated with PMPs.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
For detailed Materials and Methods, please see the online data supplement, available at http://atvb.ahajournals.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
PAF-AH Secretion From Activated Platelets
Thrombin induced a dose- and time-dependent secretion of the enzyme, with a maximum of 716±144 pmol/109 cells per hour observed at 30 minutes of platelet activation with a thrombin concentration of 0.2 IU/mL (supplemental Figure I, available online at http://atvb.ahajournals.org). Importantly, no enzyme activity was detected in the supernatants from resting platelets, whereas no cell lysis was observed during platelet activation because lactate dehydrogenase was not released during aggregation. In contrast to thrombin, ADP failed to induce PAF-AH secretion at any concentration used, although it induced a dose-dependent platelet aggregation (maximum aggregation 41±8% at a concentration of 5 µmol/L; n=5) and {alpha}-granule secretion because it was determined by the surface expression of P-selectin (34.8±17.0% CD62p-positive cells in the presence of 5 µmol/L ADP versus 1.7±0.3% for nonactivated platelets; n=5).

Characterization of the Platelet PAF-AH
Thrombin-activated platelets secreted 22±5% of the total PAF-AH activity measured in the lysate of resting platelets (3254±654 pmol/109 cells per hour). The total enzyme activity in the platelet lysate after activation (3048±598 pmol/109 cells per hour) was similar to that measured before activation. On lysate ultracentrifugation, the majority of the enzyme activity (2444±306 pmol/109 cells per hour), representing 75.1±14.3% of total, remained in the supernatant (cytosolic fraction), whereas 24.9±7.3% was associated with the membrane-rich pellet. PAF-hydrolyzing activities in the cytosolic fraction, on the membrane-rich pellet, and secreted into the medium were characterized as PAF-AH because they were Ca2+ independent and were inhibited either by 1 mmol/L of the serine esterase inhibitor Pefabloc21 or by 30 µmol/L CV-3988, a specific PAF receptor antagonist22 that also inhibits PAF-AH.23 Furthermore, PAF-AH activity was specific for short acyl chains at the sn-2 position because it was not inhibited in the competition studies by the sn-2 long-chain phosphatidylcholine. Importantly, the secreted and the membrane-associated enzyme activities were resistant to treatment with 1 mmol/L of the sulfydryl agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). In contrast, the cytosolic enzyme activity was significantly reduced by DTNB treatment, suggesting the presence of a free cysteine residue essential for catalysis,24 thus resembling type II PAF-AH,24 (supplemental Table II).

The platelet-associated PAF-AH was also characterized by immunofluorescence microscopy. Platelets are positively stained with the monoclonal antibodies against the plasma type and the intracellular type II of PAF-AH, although the staining for the plasma type of PAF-AH is weaker compared with that of type II PAF-AH (supplemental Figure II). To further investigate whether platelets contain both types of PAF-AH, we performed Western blotting analysis of the PAF-AH semipurified by 117-fold (9% of recovery) from the total platelet lysate. The semipurified PAF-AH was stained with both monoclonal antibodies against type II and the plasma type of PAF-AH (Figure 1A and 1B). Additionally, these results revealed that the plasma type of PAF-AH might not be glycosylated because it does not exhibit a broadband characteristic for the N-glycosylated PAF-AH associated with LDL (Figure 1A).25


Figure 1
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  		Figure 1. Western blot analysis of semipurified platelet PAF-AH. Platelet PAF-AH was semipurified 117-fold and subjected to 5% to 19% gradient SDS-PAGE followed by immunoblotting. Recombinant plasma PAF-AH (Rec) and LDL were concomitantly electrophoresed. A, Immunoblotting with the monoclonal antibody against plasma PAF-AH. B, Immunoblotting with the monoclonal antibody against the type II PAF-AH. C, RT-PCR classical detection of plasma type of PAF-AH in human platelets, expression of ß-actin, and PAF-Receptor served as positive controls. D, Quantification in human platelets by QPCR (20 ng of cDNA) of various PAF-AH mRNA, ß-actin (positive control), and leukocyte-specific but nonplatelet transcripts: CD11c/ß2 integrin and CD14. CT values are inversely correlated to the relative abundance of the specific transcripts. Experiments were performed in triplicate.

We next searched for the mRNA of both types of PAF-AH in platelets; platelets contain high levels of mRNA corresponding to the plasma type of PAF-AH, with the cycle threshold (CT) value in quantitative polymerase chain reaction (QPCR) of 24.3 when using 20 ng of cDNA (Figure 1C and 1D). The mRNA of the type II PAF-AH was undetectable in classical polymerase chain reaction (PCR; data not shown), whereas an extremely low quantity with a CT value of 30.4 was detected by using a more sensitive method of QPCR (Figure 1D). Finally, among the brain type of PAF-AH (IB) subunits, only the {gamma} subunit was slightly detectable in 2 separate platelet preparations (Figure 1D) because the CT values for the {alpha} and ß subunits were >31. Of importance, the specific leukocyte transcripts CD11c/ ß2 integrin and CD14 were not found in the RNA preparations of these platelets (Figure 1D), thus excluding the possibility of contamination by other circulating cells. The mRNA extracted from human cultured monocyte/macrophages, which express all types of PAF-AH and both the CD11c/ß2 integrin and CD14, served as a positive control for all PCR procedures (CT ranging from 24 to 26).

The PAF-AH Secreted From Platelets Is Associated With PMPs
Based on previous studies indicating that PAF secreted by platelets is associated with PMPs,13,14 we asked whether such PMPs could also be the carriers of PAF-AH secreted from thrombin-activated platelets. Indeed, thrombin (0.2 IU/mL) induced a time-dependent shedding of PMPs, denoted by an increase in the population in the lower left quadrant of the forward side scatter (FSC) versus side scatter (SSC) dot plot (Figure 2), which paralleled the secretion of PAF-AH as it is shown in supplemental Figure IB.


Figure 2
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  		Figure 2. Representative flow cytometric FSC vs SSC dot plots showing the time-dependent production of PMPs from platelets activated with 0.2 IU/mL of thrombin. Data are representative of 10 different platelet preparations.

To investigate whether platelet aggregation is a prerequisite step for PAF-AH secretion, we activated platelets in the presence of Arg-Gly-Asp-Ser (RGDS), which inhibits platelet aggregation by preventing the binding of fibrinogen to the activated form of the integrin receptor {alpha}IIbß3.26 RGDS (0.2 mmol/L) completely inhibited thrombin-induced platelet aggregation (Figure 3A); however, it failed to significantly inhibit either PAF-AH secretion (Figure 3B) or PMP production: 79.2% total expression of both annexin V and CD61 in the absence of RGDS versus 65.5% total expression of both annexin V and CD61 in the presence of RGDS (Figure 3C). To further investigate whether PAF-AH secretion is independent of platelet aggregation, we activated platelets in the presence of Ca2+-ionophore A23187 under nonstirring conditions that induces platelet vesiculation as opposed to platelet aggregation,27 and therefore, this treatment is considered one of the best inducers of PMP production.18 Ca2+-ionophore A23187 (10 µmol/L) induced a time-dependent shedding of PMPs in parallel to the secretion of PAF-AH, similarly to that obtained with thrombin stimulation. Importantly, RGDS neither influenced PAF-AH secretion nor PMP production (Figure 4A and 4B).


Figure 3
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  		Figure 3. A, Representative aggregation tracing showing the inhibitory effect of 0.2 mmol/L RGDS on platelet aggregation induced by 0.2 IU/mL thrombin. B, Secretion of PAF-AH from platelets activated with 0.2 IU/mL thrombin in the presence or absence of 0.2 mmol/L RGDS. FITC indicates fluorescein isothiocyanate. C, Flow cytometric profile indicating the production of PMPs in the presence of RGDS. PMPs were positively stained with both annexin V and anti-CD61, and results are expressed as the percentage of total events expressing both antigens in the top right quadrant. Experiments were performed in triplicate.


Figure 4
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  		Figure 4. The effect of RGDS (A) or calpeptin (C) on PAF-AH secretion from washed platelets activated with Ca2+-ionophore A23187 and the flow cytometric profile indicating the effect of RGDS (B) or calpeptin (D) on Ca2+-ionophore A23187-induced PMPs production from washed platelets. Experiments were performed in triplicate. Data in A and C represent the mean ±SD from 3 different platelet preparations. *P<0.01 compared to that of the absence of calpeptin.

To show whether the secretion of PAF-AH induced by Ca2+-ionophore A23187 is associated with the production of PMPs; we used calpeptin, a specific inhibitor of calpain and PMP formation.27,28 Calpeptin (300 µmol/L) significantly inhibited Ca2+-ionophore A23187-induced PAF-AH secretion (Figure 4C), along with PMP production; in the presence of calpeptin, 15.0% of total events expressing CD61 were found in the lower left quadrant of the FSC versus SSC dot plot, which corresponds to the PMP population, versus 61.1% of total events found in the absence of calpeptin (Figure 4D). This suggests that vesiculation of the platelet membrane is a prerequisite to PAF-AH secretion.

Characterization of the PAF-AH Associated With PMPs
Subsequently, we characterized the PAF-AH associated with the PMPs produced on platelet activation with Ca2+-ionophore A23187. PMPs were isolated by ultracentrifugation of the supernatant of activated platelets and characterized by flow cytometry. The PMPs population was found to be ≥98% positive for CD61. PMPs were characterized further by their staining with annexin V and anti-CD41a (supplemental Figure IIIA). Importantly, all PAF-AH activity secreted in the supernatant was recovered in isolated PMPs. PMPs were enriched in PAF-AH, exhibiting a higher specific activity compared with the platelet lysate (10.1±2.4 nmol/mg protein per hour versus 4.9±0.3 nmol/mg protein per hour, respectively; P≤0.01; n=7). The PMP-associated PAF-AH activity was inhibited by 30 µmol/L CV-3988, but it was not affected by 1 mmol/L DTNB, suggesting that this enzyme is of the plasma type (supplemental Figure IIIB). Additionally, the PMP-associated PAF-AH was immunoprecipitated with a rabbit polyclonal antiserum raised against the recombinant plasma PAF-AH (supplemental Figure IIIC).

Association of PAF-AH With Circulating PMPs
The above results provide strong evidence that PMPs produced by activated platelets in vitro contain the plasma type of PAF-AH. However, it remains to be established whether circulating PMPs are also carriers of PAF-AH in plasma. To explore this possibility, we prepared plasma from peripheral blood of 10 normolipidemic apparently healthy volunteers and investigated whether a proportion of circulating plasma PAF-AH is associated with PMPs. Circulating microparticles were detected in plasma by flow cytometry as a population of particles that exhibited a typical FSC versus SSC profile; 10±2% of the total population was positive for annexin V. Among the gated annexin V–positive particles, 89±3% express both CD41a and CD31 whereas 11±2% express only CD41a. There were no detectable amounts of CD45 or CD14 in the annexin V–positive population. Because CD41a is a specific marker of PMPs, whereas CD31 (platelet-endothelial cell adhesion molecule-1) characterizes both the PMPs as well as the endothelial cell-derived microparticles, we suggest that all of the annexin V–positive particles found in plasma are PMPs, a finding that is in accordance with previously published results.29 In contrast, in the annexin V–negative population, 5±1% were positive for CD31 (endothelial cell-derived microparticles). No detectable amounts of CD41a, CD45, or CD14 were observed in this population. This finding is in accordance with previously published observations30 showing that most of the plasma endothelial cell-derived microparticles are negative for annexin V. To assess the possibility of nonspecific binding, fluorescein isothiocyanate– and phycoerythrin-labeled isotype-matched mouse monoclonal IgG antibodies were tested. Unspecific binding was negligible. Representative dot plots showing the flow cytometric profile of the gated annexin V–positive particles are shown in supplemental Figure IV.

We next studied whether PMPs are carriers of PAF-AH in plasma by using an ELISA method. PAF-AH activity was detected only in the wells that were coated with anti-CD61 (Figure 5), showing that exclusively platelet-derived {alpha}IIbß3-containing microparticles that were captured by ELISA contained PAF-AH activity. Similar results were obtained when isolated PMPs were used as a positive control for PAF-AH. These data provide for the first time evidence that circulating PMPs are carriers of PAF-AH in human plasma.


Figure 5
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  		Figure 5. Detection of PAF-AH activity on PMPs in vivo using a captured ELISA method. ELISA plates were coated with monoclonal antibodies anti-CD61, anti-CD45, or the isotype-matched mouse monoclonal IgG. Coating was performed overnight at 4°C. A total of 100 µL of the plasma-containing microparticles or PMPs (150 µg of protein/mL) prepared from activated washed platelets were placed into the wells and incubated for 2 hour at 37°C. The plate was washed 3 times with a HEPES buffer supplemented with 0.01% EDTA (PAF-AH assay buffer), and then PAF-AH assay was performed in each well using the trichloroacetic acid precipitation procedure. Data represent the mean±SD from 10 different plasma preparations.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study shows that human platelets contain 2 types of PAF-AH: the plasma type as well as the intracellular type II. Importantly, 75% of total enzyme activity is found in the cytosolic fraction and resembles type II PAF-AH, a finding that is in accordance with previously published data,11 whereas 25% of enzyme activity is associated with the cell membranes and shares similar characteristics with that of the plasma type PAF-AH. Platelets are capable of absorbing several proteins from plasma, such as fibrinogen or albumin on endocytosis and are able to store them in their granules.31 In addition, it has been shown that tissue factor–bearing microvesicles (of monocyte origin) are able to fuse with activated platelets and transfer their protein as well as their lipid content to the platelet surface.32 Thus, it might be envisioned that platelets absorb the plasma type of PAF-AH either from circulating lipoproteins or from microvesicles derived from monocytes/macrophages, the main cellular source of the plasma type of PAF-AH. However, the above possibilities are unlikely because both the plasma and the monocytes/macrophage-secreted PAF-AH are N-glycosylated,25 whereas we show here that platelet PAF-AH lacks glycosylation. Furthermore, we showed that platelets contain high levels of mRNA corresponding to the plasma type of PAF-AH, suggesting that these cells may synthesize de novo plasma PAF-AH. Previous reports have provided evidence that platelets can synthesize proteins such as interleukin-1ß in an activation-dependent manner.33 In our studies, the total enzyme activity determined in the platelet lysate before activation was similar to that measured after activation with thrombin. This suggests that translation of the mRNA for the plasma type of PAF-AH by platelets is independent of their activation state.

A substantial proportion of the platelet PAF-AH is secreted during thrombin-induced aggregation, a finding that is in accordance to our previous findings.12 Importantly, PAF-AH secretion occurred in parallel to the shedding of PMPs observed during platelet aggregation, although the latter is not required either for PAF-AH secretion or for PMP production, as documented in the experiments with the peptide RGDS, which inhibited platelet aggregation but did not effect PAF-AH secretion or PMP production. This suggestion is further supported by the observation that ADP induced platelet aggregation and {alpha}-granule secretion, but it failed to promote PAF-AH secretion and PMP production; the latter is in accordance with previously published results showing that ADP is unable to promote PMP production.27,34

We clearly show that the secretion of PAF-AH from platelets depends on the shedding of PMPs from their surface, and this could be the major mechanism for PAF-AH secretion on platelet activation. This is supported by the observation that the Ca2+-ionophore A23187, which induces platelet vesiculation but not platelet aggregation,27 promoted PAF-AH secretion in parallel to the production of PMPs. Furthermore, calpeptin, a specific inhibitor of calpain, significantly inhibited the generation of PMPs and the secretion of PAF-AH. Indeed, it is accepted that the proteases calpain I and II play an important role in the formation of PMPs, because their activation affects the structural integrity of the platelet, leading to vesiculation of its membrane and to the formation of PMPs.27 Overall, vesiculation of platelet membrane and formation of PMPs are necessary for PAF-AH secretion. Importantly, the PAF-AH associated with PMPs shares similar characteristics with that of the plasma type, and its concentration is higher than in the platelet lysates, as already observed for other key platelet bioactive compounds,18 including PAF.13,14 Most important, by using an immunocaptured ELISA method, we demonstrated that not only PMPs generated on platelet activation in vitro but also PMPs existing in plasma of normolipidemic volunteers contain the plasma type of PAF-AH. This is the first indication that in addition to lipoproteins, circulating PMPs are carriers of PAF-AH in human plasma.

The presence of PAF-AH in PMPs may be pathophysiologically important because PMPs express several protein receptors and ligands and contain biologically active lipids including PAF, which allows their interaction with various cells,35,36 especially PAF, because it plays an important role in cell-to-cell interactions, as observed in models of acute and chronic inflammation.37,38 Thus, it has been suggested that most signaling by PAF may occur between closely juxtaposed cells (endothelial cells, neutrophils, monocytes), and that PAF can be recognized by its receptor on target cells while associated with the plasma membrane of the signaling cell.37,38 Consequently, the PMP-associated PAF-AH may be important in regulating the activity of PMP-associated PAF in the juxtacrine signaling. It has been shown recently that PMPs are elevated in the circulation of patients with acute coronary syndromes.39,40 In this context, we demonstrated that the balance between PAF production and secretion as well as PAF-AH secretion from platelets of patients undergoing coronary angioplasty is significantly altered before angioplasty as well as 48 hours afterward.41 This suggests that alterations in the balance between PAF and PAF-AH secreted by activated platelets may be of importance in coronary atherothrombosis and in the inflammatory response elicited during intracoronary injury induced by angioplasty.41 Recent data from large population studies consistently report a positive association between the risk for atherosclerotic events and the mass or activity of total plasma PAF-AH (denoted as lipoprotein-associated phospholipase A2), which mainly reflects the LDL-associated enzyme.42,43 In contrast, the HDL-associated PAF-AH may exhibit antiatherogenic properties.4 Therefore, the role of PMPs in disseminating PAF-AH on inflammation and atherosclerosis remains to be elucidated. In addition, the usefulness for quantitation of PMP-associated PAF-AH in plasma as a prognostic or diagnostic tool in atherosclerotic diseases should be investigated in large clinical studies.

In conclusion, the present study shows for the first time that human platelets contain 2 types of PAF-AH: type II PAF-AH, primarily found in the cytosol, and plasma PAF-AH, which was associated with membranes. Only the mRNA coding for plasma PAF-AH was detected in platelets. On activation with thrombin or Ca2+-ionophore A23187, platelets secreted exclusively plasma PAF-AH that was mainly associated with PMPs. Collectively, we provide strong evidence that in addition to plasma lipoproteins, the platelet-borne PMPs are also efficient carriers of PAF-AH in circulation.


*    Acknowledgments
 
Sources of Funding

A portion of this research was cofunded by the European Union in the framework of the program "Heraklitos" of the "Operational Program for Education and Initial Vocational Training" of the Third Community Support Framework of the Hellenic Ministry of Education, funded by 25% from national sources and by 75% from the European Social Fund (ESF) awarded to J.V.M. This work was partially supported by the Institut National de la Santé et de la Recherche Médicale, in which E.N. is director of research of Centre National de la Recherche Scientifique.

Disclosures

None.


*    Footnotes
 
Original received March 13, 2006; final version accepted May 10, 2006.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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