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NDT Advance Access originally published online on May 3, 2007
Nephrology Dialysis Transplantation 2007 22(9):2623-2629; doi:10.1093/ndt/gfm212
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

L-carnitine inhibits a subset of platelet activation responses in chronic uraemia

Mario Bonomini1, Vittorio Sirolli1, Secondo Dottori2, Luigi Amoroso1, Lorenzo Di Liberato1 and Arduino Arduini3

1Institute of Nephrology, Department of Medicine, G. d’Annunzio University, Chieti-Pescara, 2Department of Metabolism and Endocrinology, Sigma-Tau Pharmaceuticals, Pomezia, Rome and 3Department of Research and Development, Iperboreal Pharma S.r.l., Chieti, Italy

Correspondence and offprint requests to: Prof. Mario Bonomini, MD, Clinica Nefrologica – Emodialisi Ospedale Clinicizzato ‘SS. Annunziata’, Via dei Vestini 66013 Chieti, Italy. Email: m.bonomini{at}nephro.unich.it



   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Activated uraemic platelets expose the aminophospholipid phosphatidylserine (PS) at their outer surface, which generates a cell procoagulant phenotype and seems at least partly due to an increase in cell caspase-3 activity. L-Carnitine (LC) may decrease surface-exposed PS in stored apheresis platelets and inhibit the activity of recombinant caspases, but its effects on platelet activation response with PS externalization have not been ascertained in chronic renal failure. In the present study, we investigated in vitro and in vivo the effects of LC on PS exposure in platelets from chronic uraemic patients.

Methods. Platelet PS-exposure was assayed by flow cytometry using annexin V. Caspase activity in platelets was determined by the cleaving activity of the specific substrate DEVD-pNA and by a flow cytometric assay using rhodamine-fluorescence. The effects of LC in vivo were examined in a prospective cross-over trial including 10 patients on maintenance haemodialysis (HD) who were randomly allocated to two different treatment groups: LC (2 g i.v.) for 4 months followed by placebo (2 g i.v.) for another 4 months (group A), or placebo followed by LC (group B).

Results. PS-exposing platelets in blood samples obtained from HD patients were significantly higher than in healthy subjects (P < 0.001) under both unstimulated and agonist-stimulated conditions. When uraemic platelets were pre-incubated with LC before agonist stimulation, platelet PS exposure proved to be significantly reduced (–13.7% for 0.5 mM LC and –25% for 5 mM LC). Pre-incubation of uraemic platelets with LC again significantly decreased the cells’ caspase activity (P < 0.05). In HD patients (Group A), LC supplementation was associated with a significant decrease (P < 0.05) in platelet PS exposure followed by a progressive increase during treatment with placebo. In the other group of patients, while no change in platelet PS exposure was observed during the first 4 months of treatment with placebo, a significant reduction (P < 0.05) in PS-positive platelets occurred after 2 and 4 months of LC therapy.

Conclusion. Our data show that LC may reduce, possibly via inhibition of caspase activity, the exposure of PS in activated uraemic platelets. These findings may have implications for the thrombophilic tendency of uraemia.

Keywords: caspase; end-stage renal disease; L-carnitine; phosphatidylserine; platelet; uraemia



   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 
Several studies have shown that platelets from end-stage renal disease (ESRD) patients on maintenance haemodialysis (HD) may be in a state of chronic activation related to both uraemia and the dialysis procedure [1–4]. Though the consequences of platelet activation in ESRD remain to be definitely established, it might contribute to the thrombotic tendency which is a cause of substantial morbidity and mortality [5], as well as being involved in biological reactions of potential pathophysiological significance [3,6,7].

Among their other responses to a variety of different stimuli, activated platelets expose the aminophospholipid phosphatidylserine (PS) at their outer surface [8]. PS is normally confined to the inner leaflet of cell plasma membrane; when redistributed to the cell surface, it may cause several physiological and pathological phenomena [9,10], with particular regard to the processes of cell–cell interaction, haemostasis and cell activation. PS exposure is significantly enhanced in platelets from ESRD patients and is associated with increased procoagulant activity by those cells [4], a finding that may point to a pathogenic mechanism linking platelets to the thrombophilic susceptibility of such patients [11].

Increased PS exposure in uraemic platelets seems at least partly due to an increase in cell caspase-3 activity [4]. Caspase-3 is a key effector enzyme of nucleate cell apoptosis, a process marked by phenomena (PS redistribution to the cell surface, cell shrinkage and plasma membrane blebbing and microvesiculation), which resemble those occurring during late platelet activation response. Caspase involvement in platelet PS exposure is suggested by most [12–14] though not all [15] studies. In our recent investigations, we found from two independent assays that the enzymatic activity of caspase-3 is significantly greater in platelets from ESRD patients than in those of healthy controls, and that inhibition of caspase-3 activity by a specific cell-permeant inhibitor causes a marked reduction of PS exposure in agonist-stimulated uraemic platelets [4].

It has recently been shown that surface-exposure of PS in extended stored apheresis platelets is decreased by the presence of L-carnitine (LC) in the storage bag, whereas in the absence of LC a higher percentage of surface-exposed PS is found [16]. LC is a naturally occurring compound which is best known for its role in facilitating the transport of long-chain fatty acids across the mitochondrial membrane. It has also been demonstrated that LC protects Jurkat cells against Fas-mediated apoptosis and inhibits the activity of recombinant caspases including caspase-3 [17]. Such anti-caspase activity has been suggested as a mechanism for LC's anti-apoptotic effect, and this may partly explain the lower percentage of surface PS expression in stored platelets.

LC has largely been studied, and is also frequently used, in patients on maintenance HD, but its effects on platelet activation response with PS externalization have not been ascertained as yet. In the present study, we first examined the in vitro effects of LC on the exposure of PS in platelets obtained from ESRD patients. In view of the favourable results observed, we next carried out a randomized crossover trial in vivo, to assess the effects of LC supplementation on platelet PS exposure in patients on maintenance HD.



   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 Acknowledgements
 References
 
Study population
Platelets were obtained from ESRD patients on chronic maintenance HD presenting at the dialysis centre of the University of Chieti, who had been on regular dialysis treatment for more than 6 months. Exclusion criteria included use of LC in the past 4 months, diabetes mellitus, active infection, malignant or inflammatory disease, unstable clinical conditions and unstabilized erythropoietin dosage. None of the patients was known to have a pre-existing haemostatic disorder unrelated to uraemia and all had been free of medications known to affect platelet function for at least 1 month prior to the study.

Healthy control subjects enrolled in the study (five men and five women, aged 48 ± 11 years) had normal renal function and platelet count and were not receiving any medication.

The study protocol was approved by the Ethics Committee of the University of Chieti, and all subjects gave informed written consent.

Materials
Fluorescein isothiocyanate-labelled annexin V (FITC-AnV) was purchased from Bender MedSystems (San Bruno, CA, USA). Prostacyclin and calpeptin were from Calbiochem (San Diego, CA, USA). N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (DEVD-pNA) was obtained from Enzyme System Products (Livermore, CA, USA). Anti-CD62P phycoerythrin (PE)-labelled monoclonal antibody (MoAb) and isotypic IgG-FITC control MoAb were acquired from Cymbus Biotechnology (Chandlers Ford, Hampshire, UK). Flow cytometric caspase activity assay was from Oncogene Research Products (San Diego, CA, USA). The chromogenic substrate for thrombin CBS 34.47 (H-D-cyclohekylglycyl-{alpha}-aminobutyryl-arginyl-paranitroanilide) and purified prothrombin were purchased from Diagnostica Stago (Asnieres, France). Russels’ Viper Venom (RVV) was obtained from Enzyme Research Laboratories (South Bend, IN, USA). All other reagents were from Sigma.

Preparation of cells
Blood samples obtained from HD patients before the start of the mid-week session were collected into evacuated tubes containing ethylenediamine-tetraacetic acid (EDTA). The blood was centrifuged at 50 x g for 30 min at 22°C temperature to obtain platelet-rich plasma (PRP). The platelets were re-suspended in platelet buffer [10 mmol/l Hepes-Na, pH 7.4, 136 mmol/l NaCl, 2.7 mmol/l KCl, 2 mmol/l MgCl2, 1 mmol/l NaH2PO4, 5 mmol/l glucose and 5 mg/ml bovine serum albumin (BSA)] and, after the addition of prostacyclin (1 µg/ml), centrifuged at 800 x g for 10 min. The PRP was measured with an automated cell counter and was found to be 99% platelets. For activation experiments, platelets suspended in platelet buffer were allowed to recover for 90 min at 37°C to ensure they were in a resting state.

For preparation of cell samples, both substance concentrations and incubation times were set up according to our previous studies [4] and to preliminary experiments (data not shown). LC was used at concentrations of 0.5, 5 and 10 mM. Because preliminary analysis of data showed similar effects between LC concentrations of 5 and 10 mM, results are reported for LC 0.5 and 5 mM only.

To assess platelet PS exposure, 107 platelets in 200 µl of platelet buffer with 2.5 mmol l–1 CaCl2 were combined in polystyrene tubes with 10 µl of FITC-AnV. For stimulated platelet experiments, platelets were treated with thrombin (1 U ml–1) plus collagen (20 µg ml–1) at 37°C for 20 min before the above step. In some experiments, platelets were pre-incubated with LC or with an equal amount of saline (control) for 3 h before agonist stimulation. Samples were then incubated for 20 min at room temperature in the dark, washed in platelet buffer, and analysed by flow cytometry. Non-specific FITC-labelled monoclonal antibody was used for the negative controls.

To measure platelet surface expression of CD62P (P-selectin), an early platelet activation marker, 10 µl of PRP were incubated with thrombin (1 U ml–1) and the reaction was stopped after 0, 30 or 60 s by adding an equal volume of 2% paraformaldehyde. Aliquots of the fixed platelets were then incubated with 10 µl of anti-CD62P PE MoAb for 20 min at room temperature in the dark, washed in 1 ml of platelet buffer and examined by flow cytometry.

For the flow cytometric caspase activity assay based on cleavage of the cell-permeable caspase substrate (aspartyl)2-rhodamine 110 (D2R), platelets (1 x 105) in 0.3 ml incubation buffer (provided by the kit) were incubated with 3 µl of 1 M dithiothreitol (10 mM final concentration) and 1 µl of D2R for 20 min in the dark at 37°C, and then analysed by flow cytometry.

For studies with the calpain inhibitor calpeptin, 50 µg ml–1 of such inhibitor was added to platelets during the final 10 min of pre-incubation and then co-incubated without or with LC (5 mM) for 3 h. Samples were next washed with platelet buffer, treated with thrombin (1 U ml–1) plus collagen (20 µg ml–1) at 37°C for 20 min and labelled with FITC-AnV for analysis by flow cytometry.

Measurements
Samples were analysed immediately after preparation. Flow cytometry (Epics Elite equipped with a 4.5 version software; Coulter, Hialeah, FL, USA) was used to measure platelet PS exposure, CD62P expression, and caspase activity (rhodamine-positive cells), as previously described [3,4].

Platelet caspase activity was also determined [4] by a caspase-3-like-peptidase assay (cleavage of DEVD-pNA) in uraemic platelets either untreated or pre-incubated with LC or saline for 3 h. After lysis of platelets (5 x 108 in 1 ml of platelet buffer in polystyrene tubes) by addition of 1:3 volumes of 4% Triton X-100, 8 mmol/l EGTA, 20 mmol/l dithiothreitol, 200 µg/ml aprotinin, 200 µg/ml benzamidine and 200 µg/ml leupeptin, 140 µl of the lysate were aliquoted into a flat-bottomed 96-well microplate, and 50 µl of platelet buffer was added to the sample wells. Ten µl of 2 mmol l–1 DEVD-pNA was added to the proper wells and the plate was then incubated for 2 h at 37°C, optical density (OD) being monitored at 405 nm by a Victor 2 spectrophotometer (Wallac, Turku, Finland). The mean {Delta}OD per minute was calculated from the linear range of the reaction.

For the prothrombinase assay, an established assay for the measurement of phospholipid-dependent thrombin generation, fresh plasma from normal subjects was used as a source of factors V and X, prothrombin was diluted in a 1:50 ratio in Tris-buffered saline, and factor X was activated by the addition of 1 µg ml–1 of RVV. The reaction mixture consisted of 200 µl of diluted plasma, 100 µl of RVV, 100 µl of PRP-derived platelets and 250 µl of CaCl2. After incubation for 10 min at 37°C, the reaction was stopped by adding 10 µl EDTA, and the chromogenic substrate (CBS 34.47) was added. The reaction supernatant was then transferred to a Beckman DU-64 spectrophotometer, and light absorption was read at a wavelength of 405 nm. A standard curve of authentic thrombin was used as reference to measure the amount of thrombin released during incubation.

Plasma levels of LC fractions (total LC, free LC, acyl-carnitine) were determined by high performance liquid chromatography tandem mass spectrometry, as reported [18].

In vivo study
The in vivo study was prospective, randomized, placebo-controlled, double-blind and cross-over. Ten patients on maintenance HD were enlisted (Table 1). Patients were randomly allocated to receive either 4 months of LC treatment followed by 4 months of placebo treatment (Group A), or to receive 4 months of placebo treatment followed by 4 months of LC treatment (Group B). Both LC and placebo were used at a dosage of 2 g and were administered intravenously at the end of each dialysis session.


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  		Table 1. Baseline characteristics of the ESRD study population

 
Data analyses
Statistical analyses were performed using the statistical software Sigmastat version 2.0 for Windows (Jandel Scientific Software, San Rafael, CA, USA). Data were analysed by Student's t-test or by analysis of variance (ANOVA) followed by the Tukey multiple comparison test, as appropriate. Results are expressed as a mean ± SEM and P < 0.05 was considered significant.



   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 Acknowledgements
 References
 
In vitro studies
The mean percentage of PS-exposing platelets in blood samples obtained from HD patients (n = 10) was significantly higher than in healthy control subjects: 10.5 ± 0.5% annexin-V positive platelets vs 3.5 ± 0.3% annexin-V positive platelets, respectively; P < 0.001. Altered membrane phospholipid asymmetry with enhanced PS externalization in uraemic platelets is confirmed by the prothrombinase assay, which measures the conversion of prothrombin to thrombin induced by PS exposure on the platelet surface. Indeed, platelets from HD patients generated more thrombin (4.3 ± 0.2 µml–1) than platelets from healthy subjects (1.7 ± 0.1 µml–1) (n = 7 for each group; P < 0.001), supporting increased exposure of PS.

Annexin-V expressing uraemic platelets (%) increased still further following stimulation with thrombin plus collagen (11.9 ± 0.7 for untreated samples and 34.5 ± 1.3 for agonist-stimulated platelets; n = 4; P < 0.001). When uraemic platelets were pre-incubated with LC before agonist stimulation, however, PS exposure proved to be significantly reduced (Figure 1). Inhibition brought about by LC in PS-exposing platelets was 13.7% for 0.5 mM LC and 25% for 5 mM LC. Pre-incubation of platelets with saline as a control had no inhibitory effect on PS exposure (Figure 1).


Figure 1
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  		Fig. 1. Effects of LC on uraemic platelet PS exposure following agonist stimulation. Platelets from HD patients (n = 4) were either stimulated with thrombin (1 U ml–1) plus collagen (20 µg ml–1) for 20 min (None) or pre-incubated for 3 h with saline or LC at the concentrations indicated before being agonist-stimulated as above, and then examined by flow cytometry for annexin V-binding. * Significantly different from None and Saline; o P < 0.02 vs Carnitine 0.5 mM.

 
Because the activity of caspase-3 in uraemic platelets is significantly higher than in normal controls and could cause platelet PS exposure [4], we next examined whether LC, which inhibits the activity of recombinant caspases 3, 7 and 8 in the Fas ligation pathway [17], may affect the increased activity of caspases in platelets from ESRD patients. Two independent assays were used to determine the caspase activity of uraemic platelets: a caspase-3-like peptidase assay based on the cleavage of DEVD-pNA, a specific substrate for effector caspases including caspase-3 [19], and a flow cytometric assay based on the use of (aspartyl)2-rhodamine 110 (D2R), a reported substrate for members of the caspase family proteases which gives rise to fluorescence upon cleavage by activated cellular caspases [20]. Both the cleaving activity of DEVD-pNA and the percentage of D2R-positive platelets proved to be significantly (P < 0.05) reduced when uraemic platelets were pre-incubated with LC as compared with untreated or placebo-treated cells (Figure 2), suggesting an inhibitory effect of LC on caspase activity in ESRD platelets.


Figure 2
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  		Fig. 2. Effects of LC on uraemic platelet caspase activity. Platelets from HD patients (n = 4) were either used untreated or incubated with saline or LC (5 mM) before the assay. (A) Caspase-3-like peptidase assay (cleavage of DEVD-pNA). (B) Aspartyl2-rhodamine 110 flow cytometric assay. * P < 0.05 vs other groups.

 
Evidence that LC might decrease platelet PS exposure via caspase inhibition is also provided by experiments with the calpain inhibitor calpeptin. Calpeptin increases platelet PS externalization, and such an increase is significantly and substantially abolished by inhibition of caspase activity in either normal [12] or ESRD [4] platelets. We observed that PS exposure in agonist-stimulated uraemic platelets significantly increased following calpeptin pre-treatment but was significantly inhibited when 5 mM LC was added to calpeptin during platelet pre-treatment (PS-positive platelets, %, were 54.4 ± 1.3 and 40.4 ± 1.6, respectively; P < 0.02).

We next examined whether LC may inhibit other platelet activation events such as {alpha} granule secretion. Secretion of platelet {alpha} granule, an early platelet activation response, was assessed by surface expression of CD62P, a specific platelet activation marker [21]. Treatment of uraemic platelets with thrombin up-regulated the expression of CD62P (Figure 3). Pre-incubation of uraemic platelets with LC (5 mM) did not prevent thrombin-induced CD62P up-regulation (Figure 3).


Figure 3
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  		Fig. 3. Effects of LC on uraemic platelet {alpha} granule secretion (CD62P surface expression). Platelets from HD patients (n = 5) were pre-incubated with no LC or with LC (5 mM) for 3 h and stimulated for the times indicated with thrombin (1 U ml–1). *P < 0.001 vs 0 s.

 
Taken together, these in vitro results suggest that LC may decrease late activation response with PS externalization in uraemic platelets, likely via a caspase-targeted inhibitory mechanism.

In vivo studies
The effects of LC on platelet PS exposure were examined in 10 chronic HD patients in a prospective crossover fashion. Patients were randomly allocated to two different treatment groups: LC followed by placebo (group A) or placebo followed by LC (group B). Treatment was well tolerated, and no attributable side effect was noted.

Blood LC fraction levels (total, free and its acyl esters) rose during the 4 months of treatment with LC in both study groups (Table 2). In group A patients, plasma levels of LC fractions declined during the placebo period following LC administration, returning to pre-study levels after 4 months from LC suspension.


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  		Table 2. Initial and bi-monthly values (µmol/l) for plasma carnitine fractions (total, free, and acyl-carnitines) during the study period

 
The results of platelet PS exposure during the study period are shown in Figure 4. Compared with the beginning of treatment, LC supplementation was associated in group A patients with a significant decrease (P < 0.05) in platelet PS exposure. This was followed by a progressive increase in platelet PS positivity during treatment with placebo, arriving after 4 months at values comparable with those observed before the beginning of treatment (time 0, Figure 4). In group B patients, no change was observed in platelet PS exposure during the 4 months of placebo treatment. However, when patients subsequently received LC, a significant reduction (P < 0.05) in platelet PS externalization was observed after both 2 and 4 months of LC therapy (Figure 4).


Figure 4
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  		Fig. 4. Effects of LC on in vivo platelet PS exposure in uraemic patients on maintenance HD (n = 5 for each group). * P < 0.01 vs time 0 and time 8 months; o P < 0.01 vs time 0, 2 and 4 months.

 


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
Acknowledgements
 References
 
Chronic uraemia is often associated with several signs of platelet activation [1–3] including increased surface exposure of the aminophospholipid PS [4]. Platelet surface-exposed PS may promote both the coagulation process and the cell propensity to be recognized and removed by macrophages [9,10]. We have previously observed that the thrombophilic susceptibility of ESRD patients may be partly ascribed to increased PS exposure on the outer membrane leaflet of platelets [4]. It has also been shown that platelet survival is shortened in dialysis patients [2], resulting in an increased cell turnover that may contribute to the acquired platelet defect associated with renal failure. Thus, abnormal platelet exposure of PS might play a role in the complex pathophysiology of the uraemic syndrome.

The results of the present study indicate that, in chronic uraemia, LC may reduce a subset of platelet activation response with PS externalization. Pre-incubation of platelets with LC significantly reduced PS exposure following agonist stimulation, an apparently dose-dependent inhibitory effect saturable at an LC concentration of 5 mM. Perhaps more important is the observation that LC therapy was able to reduce in vivo platelet PS exposure in patients on regular HD, as observed in a randomized cross-over study during the 4 months of LC treatment. Interestingly, a statistically significant increase in plasma LC concentration was always associated with a statistically significant reduction in PS exposure (see also treatment groups A and B of Figure 4).

The effect of LC on uraemic platelet PS exposure might be mediated by inhibition of the activity of the caspase enzyme. A role for caspase-3 in apoptosis-like events such as PS externalization during platelet activation has been demonstrated in models of agonist-stimulated platelets [12], stored platelet concentrates [13], and rat models in vivo [14], as well as in platelets from ESRD patients [4]. In keeping with the observation that LC may inhibit caspase activity in Jurkat cells [17], we observed here by two independent assays that LC may significantly reduce the activity of caspase in platelets from uraemic patients.

Our in vitro data on LC effect on platelet caspase activity does not necessarily mean that the reduced PS exposure found in LC-treated patients is linked to a reduced caspase activity in circulating platelets. Direct measurement of caspase activity in platelets from patients treated with LC would not have been helpful since LC would most likely act as a reversible inhibitor [17]. However, it is worth noting that a significant reduction in PS exposure was observed in uraemic platelets pre-incubated with 0.5 mM LC, a concentration not far from the one achieved in the plasma of HD patients treated with LC (between 0.173 and 0.259 mM). Plasma LC concentrations higher than 0.5 mM have been shown to be safe in HD patients [22]. In addition, considering that in vitro platelets were exposed to LC for only a short period of time (3 h), whereas in patients treated with LC platelets were exposed to LC for much longer (days), it may be further argued the LC intra-platelet concentration should not be very dissimilar in the two sets of experiments. Note that LC does not move freely across biological membranes and, with the exception of carrier-mediated transport [23], LC passive diffusion across membranes is a relatively slow process [24]. Irrespective of the precise LC mechanism in limiting PS exposure, the addition of LC in the millimolar range to platelets stored in the liquid state has been shown to significantly counteract several detrimental metabolic and morphological changes occurring in platelets stored for more than 5 days at 20–24°C [25], suggesting that LC may improve stored platelet quality through mechanisms other than direct intervention on caspase activity.

Studies examining the effects of LC administration on platelet activity in ESRD are few and contradictory. Supplementation of LC (3 g) was associated with a rise in platelet aggregation in six HD patients [26]. However, such results were not subsequently confirmed in a larger study which showed no difference between LC and placebo on platelet aggregation or plasma concentration of various factors involved in platelet activation [27]. The results of the present study suggest that in patients on chronic HD, LC may have a favourable effect on late platelet activation response, which is characterized by externalization of PS. In contrast, exposure of P-selectin, which occurs early in the platelet activation response, was not affected by LC, supporting a restricted rather than global role for carnitine in platelet activation events. Our study has however some limitations. The study population is small, and the active treatment period might have been too short to fully examine the LC effects, considering (as shown in Figure 4) the progressive decrease in platelet PS expression over the 4 consecutive months of LC supplementation. In addition, thrombotic markers (e.g. F1 + 2, TAT complexes but also platelet release products as PF4) were not monitored.

In conclusion, our data confirm [4] a highly significant increase in the exposure of negatively charged aminophospholipid PS by uraemic platelets, a finding shown to be procoagulant in vitro [4]. Our data also show that LC may reduce PS exposure in activated uraemic platelets, possibly via inhibition of caspase activity. Such an effect of LC on platelet function may benefit ESRD patients by reducing the risk of thromboembolic episodes. Whether this conjecture holds true requires further investigation, of course.



   Acknowledgements
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was presented in part at the XLIII Annual Meeting of the European Renal Association–European Dialysis Transplantation Association, Glasgow, 2006. This work was supported by a research grant from Sigma-Tau Pharmaceuticals, Pomezia, Rome.

Conflict of interest statement. S.D. is an employee of Sigma-Tau Pharmaceuticals.



   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 23. 1.07
Accepted in revised form: 19. 3.07


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