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Abstract

The aim of this study is to investigate the effects of exogenous l-arginine and HDL on LDL and oxidized LDL (ox-LDL)-mediated platelet activation. Adenosine diphosphate (ADP)-activated platelets have been incubated with lipoproteins with or without l-arginine. P-selectin receptor numbers per platelet have been measured by flow cytometry. After incubation with only l-arginine (without lipoproteins), platelet nitric oxide (NO) levels and P-selectin receptor numbers significantly increased compared to the controls (P < .05). After incubation with LDL or ox-LDL, receptor numbers of P-selectin significantly increased (P < .001). However, P-selectin receptor numbers in platelets treated with l-arginine + LDL or l-arginine + ox-LDL decreased compared to the levels in platelets treated with only LDL or ox-LDL (P < .01, P < .001, respectively). Addition of HDL to l-arginine + ox-LDL caused significant reduction in P-selectin receptor numbers as in the control values (P < .001).We have concluded that l-arginine causes enhanced platelet NO levels and blocks the effects of LDL or ox-LDL on platelet P-selectin receptor numbers and HDL also strengthens this effect of l-arginine.

Keywords

nitric oxide low-density lipoprotein high density lipoprotein oxidized low-density lipoprotein platelet P-selectin

Introduction

Platelet activation and aggregation play an important role in the development of cardiovascular diseases (CAD). The interaction between plasma lipoproteins and platelets can affect platelet activation.¹ As it is known, low-density lipoprotein (LDL) is an atherogenic lipoprotein and elavated plasma LDL levels promote thrombotic events or atheriosclerotic lesion progression.² Low-density lipoprotein enhances expression of glycoprotein IIb/IIIa (GpIIb/IIIa), P-selectin, and fibrinogen binding as well as increasing platelet aggregation and trombaxane A₂ release. Increased reactive oxygen species in various pathological conditions such as CAD and diabetes give rise to oxidative processes like lipid peroxidation in LDL, which causes oxidative modification of LDL. Oxidized LDL (ox-LDL) is more effective on platelet activation than native LDL. It has been reported that ox-LDL increases platelet aggregation without an agonist.³ Oxidized-LDL stimulates platelet activation primarily by diminishing nitric oxide sythase (NOS) expression and release of nitric oxide (NO) in endothelial cells and platelets.^4,5 Thus, it can promote atherosclerotic lesion formation on the surfaces of arteries. At the same time, it not only increases foam cell formation but also increases cell proliferation in smooth muscles and platelet adhesion.^6–8 High-density lipoprotein (HDL) has anti-atherogenic effects on the platelet function. High-density lipoprotein reduces total body cholesterol by a mechanism known as reverse cholesterol transport and thereby reduces the risk of developing CAD. However, HDL has additional antithrombotic actions. It also promotes the production of the atheroprotective signalling molecule NO.⁹ Several studies showed that ox-LDL reduces NOS activity but HDL reverses ox-LDL’s effects on neutrophils.¹⁰ Extracellular l-arginine modulates intracellular NO synthesis by providing the substrate for cNOS.^11,12

Nitric oxide, previously known as an endothelium-derived relaxing factor, is a biological mediator and is produced from l-arginine by NOS. There are 3 isoforms of NOS: the endothelial type (eNOS), the neuronal type (nNOS), and the inducible type (iNOS). Endothelial type NOS and nNOS are expressed constitutively and are generally regulated by Ca²⁺/calmodulin as well as by phosphorylation. Inducible type NOS is essentially Ca²⁺-independent and normally inactive without cytokines stimulation. However, expression of iNOS results in the production of large amounts of NO.^13,14 Nitric Oxide has a short life (seconds) and is oxidized to its metabolites (nitrite and nitrate, NOx). It plays an important role in many physiological processes of cardiovascular, nervous, and immune systems.¹⁵ Nitric Oxide also causes inhibition of platelet adhesion and aggregation by increasing cyclic guanosine monophosphate (cGMP) levels.¹⁶ It is a potent platelet inhibitor and vasorelaxant.⁴

Platelets themselves synthesize NO.¹⁷ Investigations on NO production in platelets began in 1990 with a study of Randomski et al¹⁸ describing the presence of l-arginine-NO pathway, which is able to regulate collagen-induced aggregation. Then, Mehta et al¹⁹ showed the presence of eNOS and iNOS isoforms in platelets. It has been reported that platelet NO modestly inhibits platelet activation but markedly inhibits additional platelet recruitment to a growing trombus. Thus, thrombus formation is limited by endogenous NO production in platelets.^20,21

Expression of P-selectin on the platelet surface membrane is a specific and useful index of platelet activation.²² Platelets and endothelial cells possess P-selectin. P-selectin is an adhesion molecule of selectin family and plays an important proinflamatory role in interactions of leukocytes, platelets, and vascular endothelium. P-selectin is normally stored in the α-granule membranes of resting platelets. P-selectin is rapidly translocated to the plasma membrane and is expressed on the platelet surface after activation by several mediators such as thrombin, histamine, and protein kinase C.^22,23

The aim of this study was to investigate the effects of exogenous l-arginine and HDL on LDL and ox-LDL-mediated platelet activation status using P-selectin receptor numbers.

Materials and Methods

Participants

All participants were healthy adult donors. Exclusion criteria were medical history of renal, liver, and cardiovascular disease; acute-chronic infectious diseases; thrombotic disease; alcohol consumption; smoking; use of antiplatelet, anticoagulant, or lipid-lowering drugs for at least 10 days. Inclusion criteria: normolipidemic, age between 20 and 35 years, and no medical history of platelet function disorders. All participants gave written informed consent to study participation. The study protocol was approved by the Ethics Committee at the Medical Faculty of Marmara University.

Chemicals

CD62-FITC was from Beckman Coulter (Marseille, France); QIFI kit was from DAKO (Copenhagen, Denmark); TITAN GEL Lipoprotein Kit was from Helena Laboratories (Sunderland, UK; Cat No: 3045). Paraformaldehyde (PFA), phosphate buffered saline (PBS), thiobarbituric acid (TBA), apyrase, adenosine diphosphate (ADP), l-arginine, N-(1-Naphthyl) ethylenediamine dihydrochloride, sulphanilamide, sodium nitrite, and ethylenediamine tetraacetic acid (EDTA) were obtained from Sigma Company (St Louis, Missouri, USA). Other chemicals were of reagent grade obtained from Merck (Darmstadt, Germany).

Isolation of Lipoproteins and Modification of Native LDL

For the purification of lipoproteins, peripheral venous blood of normolipemic healthy volunteers (N = 9, F:M = 5:4) was collected into vacutainer tubes containing EDTA (1 mg/mL) after 12 hours fast and was incubated at room temperature for 20 minutes. Blood samples were centrifuged at 150g for 15 minutes and plasma was separated. Native lipoproteins were isolated from the plasma by discontinuous density gradient ultracentrugation at 155 000g in the density range d = 1.019 to 1.063 g/mL for LDL and d = 1.063 to 1.24 g/mL for HDL according to the modified method of Sclavons et al.²⁴ Cholesterol (CHO), total triglyceride (TG), apoprotein A-I (Apo A-I), and apoprotein B (Apo B) contents of isolated lipoprotein fractions were measured by using standard methods at Hithachi 917 autoanalyzer (Boehringer Manheim, Gmbh, Diagnostica, Germany). Lipoproteins were pooled separately and EDTA (100 µmol/L, final concentration) was added to them. When needed, they were dialyzed and used immediately. Lipoprotein concentrations were expressed in terms of their protein content. The protein concentration was determined according to the method of Bradford,²⁵ with bovine serum albumin used as the standard.

Oxidation of native LDL (nLDL) by copper ions was performed according to a commonly used protocol. Before oxidation, nLDL was dialyzed overnight at 4°C against phosphate buffer saline (PBS, 8 mmol/L NaH₂PO₄, 5 mmol/L KCl, 125 mmol/L NaCl) to remove EDTA. Oxidation of nLDL (1 mg/mL) was carried out with 10 μmol/L CuSO₄ in PBS for 48 hours at 37°C. Oxidation of nLDL was terminated by refrigeration. After dialysis, lipid peroxidation (LPO) levels were measured by the TBA method and expressed as malondialdehyde (MDA) equivalent.²⁶

The electrophoretic mobility of isolated lipoprotein fractions and ox-LDL on agarose gels (Titan gel lipoprotein system, Helena Laboratories) was visualized by staining with Fat Red 7B.

Platelet Isolation

Peripheral blood from the participants (N = 8, F:M = 4:4) was collected into 10-mL tubes containing 1 mL of 130 mmol/L trisodium citrate for a final ratio of 9:1 and 1 U/mL apyrase was added to prevent activation during isolation. Venous blood samples were obtained with a 21-gauge butterfly needle from participants. The blood samples were centrifuged at 200g for 8 minutes to obtain platelet-rich plasma (PRP). Platelet-rich plasma was centrifuged at 2500g for 15 minutes at 4°C (Hettich, Universal 32R) to obtain a platelet pellet. The platelets were washed with Tris-NaCl buffer (0.03 mol/L Tris, 0.12 mol/L NaCl, pH:7.4) containing 5 mmol/L EDTA and 1 U/mL apyrase.

Determination of NO Levels

The levels of NO in platelets were measured by using the method of Chen et al,²⁷ with minor modifications, indicated as nitrite plus nitrate (NOx) which is commonly used to monitor NO concentration. Buffer or 1 mmol/L l-arginine was added to washed platelets. Then ADP (10 µmol/L) was added to samples and incubated for 1 hour at 37°C. The platelet suspensions were sonicated. After centrifugation for 15 minutes at 2500g, each sample was incubated with 1.44 mmol/L NADPH and 20 mU nitrate reductase for 15 minutes at 37°C for the reduction of nitrate to nitrite. After centrifugation for 15 minutes at 2500g, the supernatant was used for nitrite levels. Samples were allowed to react with the Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% H₃PO₄). Nitric oxide concentration was estimated from a standard curve with sodium nitrite as the standard and results were given as nmol NOx/mg protein.

Quantitation of Platelet P-selectin Receptor Numbers

Flow cytometric analysis of P-selectin expressed on platelets was performed by a previously described method.²² Washed platelets were incubated with l-arginine and 10 μmol/L ADP for 1 hour at 37°C to investigate the effect of l-arginine on platelet P-selectin numbers. To investigate the effects of lipoproteins on NO-mediated platelet activation, 100 mL washed platelet aliquots were first incubated with 1 mmol/L l-arginine or PBS buffer for 15 minutes at 37°C. Then nLDL (100 µg/mL) or ox-LDL (100 µg/mL) was added and platelets were incubated for 30 minutes at 37°C. Thereafter, ADP (10 µmol/L) was added and incubated for 15 minutes at 37°C. In other experiments, after preincubation with l-arginine or with PBS buffer, platelets were incubated with HDL (100 µg/mL) and HDL + ox-LDL (100 µg/mL). Then, 10 µmol/L ADP was added and the samples were incubated for 15 minutes at 37°C.

Following the incubation for total 1 hour, FITC-labeled CD62-P (anti-P-selectin) was added to each tube. The tubes were incubated for 15 minutes in the dark. For fixation, PFA (%1 vol/vol) was added and then the samples were diluted with PBS. CD62-P fluorescence intensity was measured by flow cytometer.

For the estimation of unspecific and background fluorescence, an unactivated sample was stained with an isotype FITC-conjugated immonuglobulin G (IgG) control. The analysis of all samples were carried out on FACS Calibur flow cytometer (Becton Dickinson, San Jose, California, USA). Daily quality control and fluorescence standardization were checked with CaliBrite beads (Becton Dickinson). A total of 50 000 cells were counted in each tube. The results represent the means of duplicate samples. Platelet discrimination was made according to scatter properties of cells at FS/SS (Forward Scatter/Side Scatter) scattergram with logaritmic scale. The negative control cursor was set to 2% of the fluorescence of control cells on histograms. The results were expressed as percentage of positive cells and number of receptors per cell.

Calculation of Specific Antibody Binding Capacity per Platelet

Before the flow cytometric analysis of the platelet samples stained with specific CD62-P antibodies, calibration beads, which are used as reference beads for calculation of binding capacity of CD62-P were run. The calibration beads were prepared according to the kit’s procedure (QIFI kit, DAKO, Denmark) and the same voltage and gain values were used for the measurement of samples. The calibration beads were covered with known numbers of IgG molecules in increasing numbers (3100 to 623 000). Mean fluorescence intensity (MFI) values of the calibration beads were measured by flow cytometer. Mean fluorescence intensity values for each of the platelet samples were also determined. Histograms of the geometric mean fluorescence intensity of 10 000 events were recorded and used to plot a log-log graph of the mean fluorescence intensity versus the number of antibodies attached to each bead. The number of platelet- bound CD62P antibody was estimated from this graph on the basis of the geometric mean fluorescence intensity of the sample. After subtraction of nonspecific binding, the number of specifically bound antibody molecules was taken as the number of bound sites for CD62P/platelet.

Statistics

Data were expressed as mean SD. The results were analyzed by the nonparametric Mann-Whitney U-test. Comparisons of multiple groups were made using the friedman test (SPSS 11.0 Chicago, Illinois). P < .05 was considered statistically significant.

Results

Analysis of Isolated Lipoproteins

High-density lipoprotein and nLDL were isolated from the plasma of 9 different persons by discontinuous density gradient ultracentrifugation. The composition of the lipoprotein fractions was assessed by examining the TG, CHO, protein, Apo A-I and Apo B contents (Table 1). Electrophoretic mobilities and protein, CHO, and apoprotein contents of LDL and HDL that we isolated were compatible with the expected criteria (Figure 1). Our findings have shown that lipoprotein isolation was succesfully performed. Table 2 shows oxidation levels of lipoproteins. We observed that lipid peroxidation levels of ox-LDL were found significantly higher than the nLDL (P < .001). Electrophoretic mobility of oxidized LDL by copper ions clearly increased according to the nLDL (Figure 1).

Table 1.

The Composition of the Lipoprotein Fractions

	CHO (mg/dL)	TG (mg/dL)	Apo A-I (mg/dL)	Apo B (mg/dL)	Total Protein (mg/mL)
nLDL	145.6 ± 35.3	44.2 ± 11.6	3.9 ± 0.83	110.6 ± 23.7	1.89 ± 0.771
HDL	22.3 ± 4.9	10.4 ± 1.8	68.12 ± 12.6	4.04 ± 0.49	19.62 ± 5.711

Abbreviations: CHO, cholesterol; TG, triglyceride; Apo A-I, apoprotein A-I; Apo B, apoprotein B; LDL, low-density lipoprotein; nLDL, native LDL; HDL, high-density lipoprotein.

Figure 1.

Lipoprotein electrophoresis (1-2: LDL, 3-4 ox-LDL, 5-6: HDL)

Table 2.

Lipid Peroxidation (LPO) Levels of Lipoproteins

	MDA (nmol/mg protein)
nLDL	0.23 ± 0.85
HDL	0.19 ± 0.34
Ox-LDL	3.71 ± 1.24^a

Abbreviations: MDA, malondialdehyde; LDL, low density lipoprotein; nLDL, native LDL; HDL, high density lipoprotein; Ox-LDL, oxidized LDL.

^a P < .001 versus nLDL.

Platelet NO Levels

To see the effects of exogenous l-arginine on platelet NO production, platelets were incubated with l-arginine (1 mmol/L) and then NOx levels were measured. l-Arginine significantly attenuated ADP-induced NO production in platelets compared to the control group (control: 1.76 ± 0.45 nmol/mg protein, l-arginine: 2.5 ± 0.6 nmol/mg protein, P < .05, Figure 2).

Figure 2.

Effect of l-arginine on the platelet nitric oxide (NO) production (*P = .038 vs control).

Flow Cytometric Analysis of Platelet Receptor numbers on l-Arginine-Treated Platelets

Histograms of control group and platelets treated with l-arginine are shown in Figure 3A. P-selectin receptor numbers on activated (10 µmol/L ADP) platelets were analyzed after treatment with l-arginine. P-selectin receptor numbers on platelets after incubation with l-arginine significantly decreased compared to the control group (control: 4543 ± 629, l-arginine: 3470 ± 533, P < .05, Figure 3B).

Figure 3.

A, Histogram of platelet sample. CD62-P-positive platelets are in the area marked as M1. B, P-selectin receptor numbers on l-arginine-treated platelets (*P = .040 vs. control).

The Effects of Lipoproteins on Platelet P-Selectin Numbers in the Absence or Presence of l-Arginine

As shown in Figure 4, nLDL and primarly ox-LDL promoted platelet P-selectin numbers (control: 4543±629, nLDL: 6160 ± 881, ox-LDL: 7051 ± 800, P < .001). However, P-selectin receptor numbers in platelets treated with l-arginine + ox-LDL markedly decreased compared to the levels in platelets treated with ox-LDL (ox-LDL: 7051 ± 800, L-arg + ox-LDL: 5332 ± 572, P < .001). P-selectin numbers on platelets treated with l-arginine + nLDL also decreased compared to the levels in platelets treated with only nLDL (nLDL: 6160 ± 881, L-arg + nLDL: 4858 ± 540, P < .01).

Figure 4.

The effects of lipoproteins on platelet P-selectin receptor numbers in the absence or presence of l-arginine (L-arg). Washed platelets were incubated with tested compounds and then the cells were labeled with anti-CD62-FITC. Platelet P-selectin receptor numbers were determined by flowcytometry (***P < .001 vs. control, ⁺⁺ P < .01 vs. nLDL, ⁺⁺⁺ P < .001 vs. ox-LDL).

The Effects of HDL on P-Selectin Receptor Numbers in the Absence or Presence of l-arginine and ox-LDL

When the platelets were incubated with only HDL, some reduction was observed in the numbers of P-selectin; however, this reduction was not found significant compared to the control group (control: 4543 ± 629, HDL: 3894 ± 526, P > .05). However, HDL plus ox-LDL-treated platelet P-selectin numbers were significantly lower than those in the ox-LDL group (ox-LDL: 7051 ± 800, HDL + ox-LDL: 4867 ± 778, P < .001, Figure 5). At same time, addition of HDL to l-arginine + ox-LDL caused a significant reduction in P-selectin receptor numbers per platelet (ox-LDL: 7051 ± 800, L-arg + HDL + ox-LDL: 3960 ± 522, P < .001). This decrease was also significant compared to the HDL + ox-LDL-treated group (L-arg + HDL + ox-LDL: 3960 ± 522, HDL + ox-LDL: 4867 ± 778, P < .05).

Figure 5.

The effects of HDL on P-selectin receptor numbers of ox-LDL-treated platelets. Washed platelets were incubated with tested compounds and then the cells were labeled with anti-CD62 P-FITC. Platelet P-selectin receptor numbers were determined by flow cytometry (***P < .001 vs. ox-LDL).

Discussion

Platelet-derived NO plays a role as a natural antithrombotic mechanism and inhibits additional platelet recruitment to a growing thrombus.²⁸ Malinski et al²⁹ reported that activated platelets release approximately 10^-11 mol NO/min per 10⁸ platelets. In our study, we first investigated the effects of exogenous addition of l-arginine, essential amino acid and substrates of NOS on washed platelet NO production. For this aim, platelets were incubated with l-arginine (1 mmol/L).²⁷ We have shown that l-arginine at 1 mmol/L concentration is effective on platelet NO production.

In the current study, the platelet P-selectin expression was flow cytometrically measured to observe the effect of l-arginine on the platelet activation. Experimental data suggest that P-selectin of platelets is an important link between thrombosis and vascular injury.^30,31 Measurement of platelet activation is important to better recognize the role of platelets in cerebrovascular and cardiovascular diseases. Some problems such as sensitivity and low specificity may occur in the tests that measure the platelet functions such as the measurement of platelet aggregation and specific platelet proteins. However, flow cytometry has the advantage of directly analyzing individual platelets with a high degree of sensivity.³² Flow cytometric results obtained from studies using glycoprotein receptors are generally expressed as the percentage of antibody-positive cells. But, these data do not reflect the minimal variations of receptor expressions after activation. The results termed as mean fluorescence intensity are much more sensitive and can easily convert the receptor numbers per cell by using calibrated beads.²² Thus, the receptor numbers of P-selectin per platelet have been established by using standard beads in our study. Our data have shown that treatment of 1 mmol/L l-arginine significantly decreases the P-selectin receptor numbers/platelet. In a similar study by Murohara et al,³³ it was shown that P-selectin expression and protein kinase C activity increased in the feline platelets incubated by N^G-nitro-l-arginine methyl ester (L-NAME, a NOS inhibitor) and decreased by the treatment of l-arginine. In another study, it was shown that inhibition of endogenous NO production with L-NAME resulted in 56% increase in numbers of P-selectin-positive platelets.¹⁶ However, the results were given as percentage binding in those studies. The number of platelet P-selectin receptors at the baseline was found to be around ≤500 per cell.^22,31 After TRAP activation, P-selectin numbers per platelet were determined as about 3500.²² We found the receptor numbers per platelet as about 4500 in ADP-activated platelets. l-Arginine treatment reduced P-selectin receptor numbers as low as 3500 per platelet.

Hyperactivity of human blood platelets is accompanied by an enhanced risk of atherosclerosis and arterial thrombosis.³⁴ Lipoproteins, especially modified lipoproteins such as ox-LDL, can also affect platelet functions. There are several studies in the literature that separately investigated the relationship between plasma lipoproteins with platelet activation and platelet NO production with platelet activation via P-selectin expression.^35–37 However, there is not a comprehensive study that has investigated the effects of endogenous NO production on the relationship of nLDL, HDL, and ox-LDL with platelet activation.

In the in vitro studies carried out with lipoproteins, it was observed that the dose-dependent incubation of LDL with platelets can result in shape changes, aggregation, and secretion in the platelets.³⁸ Activation of LDL receptors (apoER2′ or GpIIb/IIIa) in platelets initiates different signal ways such as phosphorilation of p38^MAPK, activation of cytosolic phospholipase A₂, and formation of thrombaxane A₂, a potent platelet-activating agent. Ox-LDL is much more effective than natural LDL in platelet signal routes via several receptors (GpIIb/IIIa, LOX-1, CD36, apoER2, SR-A).^3,39,40

The effect of ox-LDL on platelet NO production proceeds in 2 ways, namely the inhibition of NOS and uptake of l-arginine. It was shown that ox-LDL reduced platelet NOS protein.²⁷ According to our findings, ox-LDL markedly increased platelet P-selectin receptor numbers about 1.5 times. Native LDL also caused a significant increase in P-selectin receptor numbers, but this increase was not as much as ox-LDL. These effects of lipoproteins were blocked by the preincubation of platelets with l-arginine in both nLDL and ox-LDL groups. In the current study, we aimed to achieve the uptake of l-arginine into platelets by 15-minute incubation with l-arginine before the final incubation of platelets with LDL and ox-LDL. Thus, we tried to prevent the blocking effects of lipoproteins on the l-arginine uptake into the cell. The earlier addition of l-arginine resulted in a meaningful decrease in the platelet activation caused by these lipoproteins.

High-density lipoprotein has a cardiovascular protective role with its endothelial and antithrombotic effects.⁹ In our previous study, we concluded that HDL has a protective role by reducing the platelet activation and oxidative stress triggering effects of LDL/ox-LDL.⁴¹ In the current study, we indicated that HDL alone also inhibited the platelet activation increase depending on ox-LDL. The effect of HDL was further fortified by the application of l-arginine with HDL. Some antithrombotic effects of HDL are realized by stimulating NO production, which is an atheroprotective signal molecule.⁹ It has been proposed that its effect on NO production is based on apo E, a minor apoprotein.⁴² However, in this study, we showed that HDL is effective on platelet activation without the addition of l-arginine. This state shows that other mechanisms apart from l-arginine pathway may be involved in the effect of HDL on platelet activation. In recent studies, it was stated that serum paraoxonase (PON) has a potent antioxidant effect and the enzyme is related to Apo-A1 and Apo-J (Clustrein) proteins of HDL.^43,44 This enzyme may also contribute to the blocking effect of HDL in the platelet activation increase resulting from ox-LDL.

As a result, it can be concluded that platelet NOS activity is sensitive to l-arginine application as it was previously pointed out in the literature. We have flow-cytometrically determined that LDL, which we purified from human plasma, and the ox-LDL increases platelet activation and preincubation with l-arginine prevents this activation. Additionally, in our flowcytometric study, we have expressed the results of CD62-P (P-selectin) as receptor numbers per platelet but not as percentage binding. We have seen that platelet l-arginine-NO pathway has a significant role in the effects of LDL and ox-LDL on platelet activation. High-density lipoprotein inhibits platelet activation, but we think that other mechanisms may also play a role concerning this effect besides l-arginine-NO pathway.

The limitation of the study is the low number of samples. Nevertheless, the small sample size of the study allows the finding of significant clinical differences.

Footnotes

This study was presented at the 2007, AACC Annual Meeting, San Diego, California, July 15-19, 2007.

The author(s) declared no conflicts of interest with respect to the authorship and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article: Supported in part by Marmara University Research Unit Grants SAG 070/060904.

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Exogenous l -Arginine and HDL Can Alter LDL and ox-LDL-Mediated Platelet Activation

Abstract

Keywords

Introduction

Materials and Methods

Participants

Chemicals

Isolation of Lipoproteins and Modification of Native LDL

Platelet Isolation

Determination of NO Levels

Quantitation of Platelet P-selectin Receptor Numbers

Calculation of Specific Antibody Binding Capacity per Platelet

Statistics

Results

Analysis of Isolated Lipoproteins

Platelet NO Levels

Flow Cytometric Analysis of Platelet Receptor numbers on l-Arginine-Treated Platelets

The Effects of Lipoproteins on Platelet P-Selectin Numbers in the Absence or Presence of l-Arginine

The Effects of HDL on P-Selectin Receptor Numbers in the Absence or Presence of l-arginine and ox-LDL

Discussion

Footnotes

References