|Year : 2012 | Volume
| Issue : 2 | Page : 57-63
Platelet aggregation in children with end-stage kidney disease in relation to level of L-arginine as a risk factor for vascular thrombosis
Doaa Youssef1, Mervate Atfy1, Maha Atfy2, Amal Ghareeb3
1 Faculty of Medicine, Zagazig University, Sharkeya, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Sharkeya, Egypt
3 Department of Biochemistry, Faculty of Medicine, Zagazig University, Sharkeya, Egypt
|Date of Web Publication||21-Sep-2013|
Department of Pediatrics, Zagazig University, Sharkeya
Source of Support: None, Conflict of Interest: None
Background: L-arginine is the precursor for nitric oxide (NO) synthesis in vascular cells. The sources of L-arginine are endogenous and exogenous. Although macrophages and endothelial cells can generate L-arginine, most synthesis takes place in the liver and kidney. Level of L-arginine as of other amino acids is affected in malnourished patients; NO regulates platelet activation by inhibiting adhesion and aggregation.
Objectives: The aim of the study is to evaluate the level of L-arginine in patients of end-stage kidney disease (ESKD) under regular hemodialysis and to determine its relation to platelet aggregation as a risk factor for vascular thrombosis which is a very important complication that affects the life span of those patients. In addition, we also tried to establish the relationship between L-arginine, platelet aggregation, and nutritional state of patients with ESKD.
Subjects and Methods: Our study was "a case-control study", comparing data between 30 patients with ESKD under treatment with regular hemodialysis-group (A); group A1 (16)-patients with ESKD on regular hemodialysis with malnutrition, group A2 (14)-patients with ESKD on regular hemodialysis with normal nutrition state, and group B 10 subjects as healthy control group, each patient was evaluated for serum arginine level by high-performance liquid chromatography, and platelet aggregation, as well as other routine investigations. A thorough clinical history and examination were performed for all the patients.
Results: Our study shows significant higher level of platelet aggregation to ADP in group A than group B (119±11.9% and 81.1±6.5% respectively, P < 0.05), a lower level of serum arginine in group A (71±45.3 μg/L) than in group B (120.5±14.6 μg/L) with P< 0.05. Also our study shows a lower level of serum arginine in group A1 (60.5±12.3 μg/L) than group A2 (83±27 μg/L) and a lower level in group A2 than group B (120.5±14.6 μg//l), and we found a negative correlation between serum arginine level and platelet aggregation expressing a more risk for thrombosis in patients with ESKD specially those with malnutrition.
Conclusions: We concluded that patients with ESKD has a low level of L-arginine specially in those with malnutrition and this leads to increase in platelet aggregation and may increase the possibility of thrombus formation in malnourished patients with ESKD. We suggest the use of nutritional supplementation of L-arginine to suppress this process of atherothrombosis.
Keywords: Arginine, end-stage kidney disease, nutrition
|How to cite this article:|
Youssef D, Atfy M, Atfy M, Ghareeb A. Platelet aggregation in children with end-stage kidney disease in relation to level of L-arginine as a risk factor for vascular thrombosis. J Acad Med Sci 2012;2:57-63
|How to cite this URL:|
Youssef D, Atfy M, Atfy M, Ghareeb A. Platelet aggregation in children with end-stage kidney disease in relation to level of L-arginine as a risk factor for vascular thrombosis. J Acad Med Sci [serial online] 2012 [cited 2019 Dec 14];2:57-63. Available from: http://www.e-jams.org/text.asp?2012/2/2/57/118660
| Introduction|| |
Uremia is defined as an irreversible and long-standing loss of renal function causing ill-health, and patients with end-stage chronic renal failure are treated principally by renal dialysis and transplant; however, in underdeveloped countries, renal transplantation is not a viable option for the majority of patients on hemodialysis. The most common form of dialysis is hemodialysis, which allows a 10-year mean survival rate of uremic patients in developed countries. 
Although L-arginine is a semiessential amino acid for most mammals and is required during periods of growth, severe stress, and injury and since the total amino acid pool seems depleted in uremia, L-arginine may become an essential amino acid in this pathological state. Indeed, L-arginine metabolic pathways are directly involved in the pathophysiology of the uremic syndrome, and by-products of L-arginine metabolism, such as polyamines and urea, are potential uremic toxins. 
The sources of L-arginine are endogenous and exogenous. Although macrophages and endothelial cells can generate L-arginine, most synthesis takes place in the liver and kidney by the transfer of an amino group from L-aspartic acid or L-glutamic acid to L-citrulline in a reaction mediated by the enzyme argininosuccinic acid synthetase. Since the liver utilizes most of the L-arginine produced in the urea cycle, the kidney, together with dietary intake (1-2 g/day), is responsible for maintaining normal plasma levels of L-arginine (80-100 μg//L) [Figure 1]. 
L-arginine is the physiological precursor for nitric oxide (NO) synthesis, and availability and transport of L-arginine modulate the rates of NO biosynthesis in circulating blood cells and the vasculature. NO is involved in many vascular functions such as vasodilatation and inhibition of platelet aggregation and adhesion. 
NO synthesis is activated during adhesion of platelets to collagen and aggregation induced by ADP and arachidonic acid. L-arginine, the substrate for NO production, inhibits platelet aggregation both in vitro and in vivo. 
In healthy young men, oral supplementation with L-arginine increases platelet cGMP production, impairs platelet aggregation without affecting endothelium-dependent vasodilatation, and results in platelet-specific increases in NO synthesis. [ 5 ] Moreover, oral administration of L-arginine inhibits platelet aggregation through intraplatelet NO synthesis in healthy young adults and in hypercholesterolemic patients. 
Atherogenesis in advanced chronic renal failure may be the consequence of a synergism of malnutrition, inflammation, oxidative stress, and impaired platelet and endothelial signaling, as endothelial dysfunction is a common pathological feature of patients with mild-to-severe renal failure; this abnormality together with platelet activation can lead to atherothrombosis associated with a dysfunction of the L-arginine-NO pathway. 
Many factors are implicated in the upregulation of system y+L and NO synthesis in platelets in uremia. Among these, the low concentration of plasma L-arginine seems to be a key factor involved in the activation of L-arginine uptake in platelets in well-nourished CRF patients. Increased NO production in platelets from well-nourished uremic patients is consistent with the prolonged bleeding time observed in CRF, notably reversed in uremic rats following L-NMMA infusions, Platelets from well-nourished uremic patients on hemodialysis synthesize more NO than controls, interestingly, platelets from uremic patients seem to have a diminished response to NO, supporting the hypothesis that NO synthesis in these cells may already be upregulated. It is possible that activation of the high-affinity y+L transport system in uremia provides a unique mechanism for maintaining L-arginine supply for platelet NO synthesis in a milieu of reduced arginine availability and prothrombotic factors such as increased fibrinogen concentration. 
| The Aim of the Work|| |
The purpose of the study is to evaluate the level of L-arginine in patients of end-stage kidney disease (ESKD) under regular hemodialysis and to detect its relation to platelet aggregation as a risk factor for vascular thrombosis which is a very important complication that affects the life span of those patients, also we tried to find the relationship between L-arginine, platelet aggregation, and nutritional state of patients with ESKD.
| Subjects and Methods|| |
Our study is a "case-control study", we studied 30 patients under regular hemodialysis for ESKD group (A), and 10 healthy children as a control group (B).
Group ( A ) was 12 males and 18 females with age ranging from 11.1±4.5 years, with duration of disease (mean: 25.8±15 m) ranging from 2 to 60 months and duration of dialysis (mean: 22.7±15.4 m) months ranging from 2 to 60 months.
We divided this group into two subgroups (A1) (16) patients which are those with malnutrition, that is, body mass index (BMI) below 18.5 kg/m 2 and subgroup (A2) (14) patients with normal nutrition state with BMI equal or above 18.5 kg/m 2 .
While group (B) comprised of four males and six females with age ranging 10.4±2.8 years, they were all well-nourished with BMI equal or above 18.5 kg/m 2 .
All subjects were subjected to complete history taking, thorough clinical examination (including nutritional state assessment), and routine investigations (complete blood count, blood urea nitrogen, serum creatinine), platelet aggregation, serum level of L arginine.
For group (A), samples were taken before dialysis session.
Nutritional State Assessment
The assessment of nutritional status in ESKD patients can be performed using an anthropometric parameter, BMI, that is, the ratio of postdialysis body weight (kg) divided by height square (meters), considering malnourished values as less than 18.5 kg/m 2 . ,
Platelet aggregation studies were carried out in a four-channel aggregometer (platelet aggregation profiler model PAP-4, Bio/Data Corporation, Hatboro, PA, USA), while stirring PRP aliquots at 37°C. ADP (10 μM), collagen (1.9 μg/mL), sodium arachidonate (500 μg/L) and ristocetin (1.5 mg/mL) were used as inducers (Bio/Data Corporation, Hatboro, PA, USA). Platelet aggregation with the four platelet agonists was assessed by determining the maximal platelet aggregation percentage. Collagen aggregation was also assessed by the latent period between addition of the aggregating agent and the onset of the aggregation phase, expressed in minutes. 
Determinations of Serum Arginine Level Using High-Performance Liquid Chromatography
Derivatized arginine was analyzed by high-performance liquid chromatography (HPLC).  Instrument used was Perkin Elmer model LC 1020 plus, carried out on a gradient pump (binary LC pump 250) LC 18 column (particle size 3 μm, 3 cm Χ 4.6 mm inner diameter, Supelco Inc., Supelco Park, Bellefonte, PA, USA). The separation of arginine carried out using 0.1 mol/L potassium dihydrogen phosphate buffer (pH 2.1) adjusted with orthophosphoric acid containing 4% acetonitrile as a mobile phase at a flow rate of 1 mL/min.
The present work was carried out according to method described by Ubbink et al.,  but with the use of ultraviolet (UV)/visible spectrophotometer detector (model LC 290, Perkin Elmer) instead of fluorescence detector.
UV detector was set at different wavelengths and a wavelength 270 nm was selected as it gives the maximum absorption of standards.
Separate standard for each arginine was injected alone to determine its retention time, and then a mixed pool of these standards was injected for the calibration of the system. Then, 20 μL of each sample was injected. The arginine was separated and peak was identified on the basis of their retention time on comparison, with the standard compounds. Identity of the resolved arginine was confirmed by enrichment technique that means addition of small amount of arginine to both control and patient sample where peak enlargement was observed. The retention time of arginine was observed be 4.5 min.
Statistical analysis was performed using a computer-based program (SPSS version 11). The quantitative data are presented as mean±standard deviation unpaired independent t-test is statistical test of significance for two groups, paired t-test for paired quantitative data for more than four groups analysis of variance test is the test of statistical significance. P value less than 0.05 indicated statistical significance. 
| Results|| |
Our results expressed in Tables below as follow; [Table 1] shows matched age, sex-matched group of our two study groups, significantly higher level of urea and creatinine in group A than group B as level of urea in group A (131.1±21.1 mg/dL), while it is (16.7±4 mg/dL) in group B, serum creatinine level of (8.1±2.2mg/dL) in group A and (0.4±0.17 mg/dL) in group B, and it shows, no significant difference as regard platelet count, but there was significant higher level of platelet aggregation to ADP in group A than group B (119%±11.9%) and (81.1%±6.5%) respectively with P<0.05, and lower level of both serum albumin in group A (3.3±0.5 g/dL) than group B (4.2±0.2 g/dL) and a significantly lower level of serum arginine in group A (71±45.3 μg/L) than group B (120.5±14.6 μg/L) with P <0.05.
[Table 2] shows comparison between group A1 (patients with ESKD malnourished) and group A2 (patients with ESKD normal nourished), and it shows no significant difference between both groups as regard patient age group, duration of disease, duration of dialysis, blood urea level, serum creatinine level, and platelet number, while it shows increase in platelets aggregation in group A1 (128.7%±5.7%) than group A2 (107.9%±5.4%) although it is not the statistically significant level as P>0.05 but it should be taken seriously as it raises the possibility of atherothrombosis in patients with ESKD and malnutrition than those without malnutrition; this table shows also highly significant decrease in serum arginine in group A1 (60.5±12.3 μg/L) than group A2 (83±27 μg/L) with P> 0.05, also it shows a highly significant lower serum albumin in group A1 (3±0.3 g/L) than group A2 (3.7±0.4 g/L) and significant decrease in serum arginine level in group A1 (60.5±12.3 μg/L) than group B (83±27 μg/L) [Table 3].
[Figure 2] shows a lower level of serum arginine in group A1 (60.5 ± 12.3 μg/L) than group A2 (83±27 μg/L) and a lower level in group A2 than group B (120.5±14.6 μg/L).
[Figure 3] shows a higher percentage of platelet aggregation to ADP in group (A1) (128.7%±5.7%) over group (A2) (107.9%±5.4%) and group (B) (120.5%±14.6%).
[Figure 4] shows I-a negative correlation between serum arginine and platelet aggregation; that is describing a more liability for thrombus in patients with low arginine level.
|Figure 4: I‑Correlation between serum arginine level and platelet aggregation, II‑Correlation between serum arginine level and serum albumin, III‑Correlation between platelet aggregation and serum albumin|
Click here to view
II-positive correlation between serum albumin (as a marker of nutritional state) and serum arginine means that serum arginine decrease with malnutrition.
III-e there is negative correlation between platelets aggregation and serum albumin that reflects a more chance for thrombus formation with malnourished patients.
| Discussion|| |
Malnutrition is a major complication of ESKD. Many factors contribute to the pathogenesis of this condition, either nutritional, hormonal, or excess loss. Assessment of nutritional state can be estimated by many methods. Hypoalbuminemia is frequently seen in patients with CKD and it is one of main parameters affected by malnutrition. More than 50% of the 1937 children with ESKD identified through the United States Renal Data System for a study of hypoalbuminemia and mortality risk had serum albumin concentrations (<3.5 g/dL).  We found a significantly lower level of albumin in group A (3.3±0.5 g/dL) than group B (4.2±0.2 g/dL), also it was much lower in patients with ESKD and malnutrition group A1 (3±0.3 g/L) than group A2 (3.7±0.4 g/dL) with normal nutritional state, this finding was obtained by many studies as Locatelli et al., ,, who described that biochemical parameters are useful to determine the nutritional status in uremia, such as serum concentrations of albumin .
Several authors reported that platelet aggregation was depressed in chronic uremia,  whereas other studies provide evidence of hyperaggregability.  We found an increase in platelet aggregation state in some of our cases group A-(119%±11.9%) in comparison to control group B (81.1%±6.5%) which is in agreement with Locatelli et al.,  and Ma≥yszko et al .,  who reported increased incidence of hyperaggregability in patients with ESKD. Thus, thrombotic complications may be the predominant causes of mortality in some of those patients.
Since the total amino acid pool seems depleted in uremia, L-arginine may become an essential amino acid in this pathological state. Indeed, L-arginine metabolic pathways are directly involved in the pathophysiology of the uremic syndrome, and by-products of L-arginine metabolism, such as polyamines and urea, are potential uremic toxins.  In our results, we found a highly significant lower level of serum arginine in group A (71±45.3 μg/L) than group B (120.5±14.6 μg/L) this had been proved before by other author as Mendes-Ribeiro et al .,  also we found that level of serum L-arginine in patients with ESKD with malnutrition group A1 (60.5±12.3 μg/L) is significantly lower than those with ESKD without malnutrition group A2 (83±27 μg/L) this also had been found by da Silva et al .,  who described that there is low concentrations of plasma amino acids, including L-arginine, the precursor for NO synthesis in vascular cells in CRF, especially in malnourished patients, this results were also obtained by many other authors as Brunini et al.,  and Mendes-Ribeiro et al.,  who also described that that in patients with CRF, including those on conservative treatment and those on dialysis, continuous ambulatory peritoneal dialysis or hemodialysis, plasma levels of L-arginine are significantly reduced compared with controls. This decrease in serum level of L-arginine was explained as a part of total amino acid pool depletion,  or by a theory that urea inhibits arginine synthesis in hepatocytes, where the arginine level is extremely low to begin with, which decreases NO production  also it was explained that the arginine levels decrease and citrulline levels increase in serum from patients with renal failure, this change in arginine and citrulline has been explained by the loss of renal tubular cells which form arginine from citrulline.  In conditions of malnutrition, the additional increase in oxidative stress and deficiency in plasma L-arginine may further exacerbate the occurrence of atherosclerotic events.
Comparative studies of platelet function in malnourished and well-nourished uremic patients complemented by parallel studies of L-arginine supplementation may provide insights into the mechanisms underlying the cardiovascular alterations observed in CRF, , we found that platelet aggregation was more in group A1 (with malnutrition) (128.7%±5.7%) than A 2 (normal nutrition) (107.9%±5.4%) and that comes into agreement with da Silva et al.,  who found that in human platelets obtained from normal subjects and uremic patients have established that L-arginine is transported via system y+L, whose activity we reported is elevated two-fold in platelets taken from well-nourished patients as compared to malnourished uremic patients and thus increase platelet aggregation in those patients with ESKD with malnutrition than those with ESKD and with normal nutritional state. L-arginine transport via the high-affinity system y(+) L and NO synthesis are only stimulated in platelets from well-nourished chronic renal failure patients, leading to impaired platelet aggregation. The absence of this adaptive response in the l-arginine-NO pathway in platelets from malnourished chronic renal failure patients may account for the enhanced occurrence of thrombotic events in these patients.
In our study, we found that patients with ESKD and malnutrition group A1 have a lower level of L-arginine and a higher platelet aggregation than group A2, that is, those with ESKD and normal nutrition, this had been found by Brunini et al.,  Adams et al.,  and this relation between low level of L-arginine and high aggregation had been described also by many other authors. ,,,,,,,,
Oral administration of L-arginine inhibits human platelet aggregation through increased intraplatelet NO synthesis in healthy young adults and in hypercholesterolemic patients, and L-arginine stimulated NO production is inhibited following the administration of L-NMMA N-monomethyl-L-arginine, an endogenous arginine analogue and inhibitor of NO synthase, which increases platelet activation and adhesion.  In healthy young men, oral supplementation with L-arginine increases platelet cGMP production, impairs platelet aggregation without affecting endothelium-dependent vasodilatation, and results in platelet-specific increases in NO synthesis.  The pathological importance of platelet-derived NO is highlighted by reports that the bleeding time in rats depends on endogenous platelet-derived NO production, and that platelets obtained from patients with acute cardiac events generate significantly less NO.  Under these conditions, platelets would be more prone to aggregation.
| Conclusions|| |
We conclude that patients with ESKD have a low level of L-arginine especially those with malnutrition and that leads to increase in platelet aggregation and this may increase the possibility of thrombus formation in malnourished patients with ESKD, and we suggest the use of nutritional supplementation of L-arginine to suppress this process of atherothrombosis .
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]