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(Circulation. 1996;93:1740-1746.)
© 1996 American Heart Association, Inc.

Articles

Oxidized LDL Decreases L-Arginine Uptake and Nitric Oxide Synthase Protein Expression in Human Platelets

Relevance of the Effect of Oxidized LDL on Platelet Function

L.Y. Chen, MD; P. Mehta, MD; J.L. Mehta, MD, PhD

From the Departments of Medicine and Pediatrics, College of Medicine, University of Florida, and the VA Medical Center, Gainesville, Fla.

Correspondence to J.L. Mehta, MD, PhD, University of Florida College of Medicine, PO Box 100277 JHMHC, Gainesville, FL 32610-0277.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Oxidized LDL (ox-LDL) promotes vasoconstriction and platelet activation. The present study was undertaken to determine the involvement of the L-arginine–nitric oxide (NO) pathway in ox-LDL–mediated platelet activation.

Methods and Results Washed human platelets were incubated with native LDL or ox-LDL for 1 hour at 37°C followed by measurement of platelet function and indexes of the L-arginine–NO pathway. Ox-LDL but not native LDL caused a concentration-dependent increase in thrombin-induced platelet aggregation and ¹⁴C-serotonin release. These effects of ox-LDL were inhibited by pretreatment of platelets with L-arginine, the precursor of NO. Ox-LDL also caused a concentration-dependent reduction in the uptake of ³H-L-arginine by platelets. In addition, NO synthase activity, measured as conversion of ³H-L-arginine to ³H-L-citrulline, decreased on incubation of platelet cytosol with ox-LDL. Nitrite production was also reduced by treatment of platelets with ox-LDL. These effects of ox-LDL on NO synthase activity and nitrite production were reversed by pretreatment of platelets with L-arginine. Concurrent with the decrease in NO production, cytosolic cGMP was inhibited in ox-LDL–treated platelets. The inhibitory effects of ox-LDL were dependent in part on the increase of cholesterol in the platelets. Western blot analysis demonstrated 50% reduction in the expression of NO synthase protein in platelets treated with ox-LDL.

Conclusions These observations indicate that the L-arginine–NO pathway is involved in the effects of ox-LDL on platelet function and that ox-LDL stimulates platelet function primarily by diminishing NO synthase expression as well as decreasing the uptake of L-arginine.

Key Words: vasodilation • vasoconstriction • amino acids • platelets • lipoproteins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Elevated plasma level of LDL is a major risk factor in the pathogenesis of atherosclerosis. It has been proposed that native LDL must undergo oxidative modification before it can give rise to foam cells, the key component of the fatty-streak lesion of atherosclerosis, because monocyte-derived macrophages cannot take up native LDL rapidly enough to cause lipid loading.¹ Evidence has been presented for the oxidation of LDL within the blood vessels.¹ Previous studies showed that ox-LDL impairs the release of NO, a potent platelet inhibitor and vasorelaxant,² from endothelial cells.^{3 4} Impaired formation of NO in the blood vessels not only predisposes to vasoconstriction but also favors leukocyte deposition, platelet adhesion, platelet aggregation, and subsequent release of vasoconstrictor species such as serotonin and TXA₂. Deposition of leukocytes, especially T lymphocytes and macrophages, results in inflammatory and immunologic reactions that play a key role in the process of atherogenesis.⁵

Several in vitro^{6 7} and in vivo^{8 9} studies showed that ox-LDL directly causes an increase in platelet aggregation and TXA₂ release, which could contribute to intravascular thrombus formation and vasoconstriction. Fuhrman et al¹⁰ also demonstrated that activated platelets secrete a protein-like factor that stimulates ox-LDL uptake by macrophages. Thus, platelets work in close association with macrophages, the precursor of most foam cells, in the developing atherosclerotic lesion. However, the precise mechanism of ox-LDL–mediated platelet activation remains to be elucidated.

Besides endothelial cells, platelets also generate NO from L-arginine, which, by a feedback mechanism, regulates platelet aggregation by increasing cGMP accumulation in platelets.¹¹ Durante et al¹² demonstrated that NO inhibits platelet aggregation by inhibiting platelet phospholipase C, which would result in diminished TXA₂ formation. Molecular cloning studies have characterized the human cDNA encoding two distinct constitutive NO synthase enzymes in brain¹³ and endothelial¹⁴ cells. Our recent work utilizing reverse transcription–polymerase chain reaction and Southern blot analysis has shown that human platelets contain endothelial-type constitutive NO synthase.¹⁵

In view of the key role of NO in the regulation of platelet function and the adhesion of platelets and monocytes/macrophages to the vessel wall, we studied the interaction of ox-LDL and the L-arginine–NO pathway in human platelets.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Reagents
2,3,4,5-³H-L-arginine (69 Ci/mmol; 1 Ci=37 GBq; 1.0 mCi/mL) was obtained from Amersham. An RPN 2108 ECL (enhanced chemiluminescence) Western blotting analysis system was also obtained from Amersham. Mouse monoclonal anti-human endothelial NO synthase was obtained from Transduction Laboratories. A 10-kD protein ladder (10 000 to 200 000 D) was obtained from Life Technologies. A cGMP ELISA kit was obtained from Cayman Chemical Co. All other chemicals were purchased from Sigma Chemical Co..

Preparation and Characterization of Lipoproteins
Native lipoproteins (LDL: d=1.025 to 1.063 g/mL) were isolated from human plasma by discontinuous density gradient ultracentrifugation as described previously.¹⁶ Briefly, the density of plasma was adjusted to 1.006 g/mL with sodium chloride medium, and the plasma was centrifuged at 150 000g for 24 hours. VLDL and the chylomicron-rich layer were discarded. The remaining fraction, after adjusting density at 1.063 g/mL with potassium bromide medium, was centrifuged at 150 000g for 24 hours to isolate LDL from the HDL fraction. The purified LDL was dialyzed for 96 hours against PBS, degassed with N₂ and containing 0.3 mmol/L EDTA, at 4°C. LDL was stored under N₂ at 4°C, and suitable aliquots were then oxidized in the presence of 5 µmol/L CuSO₄ for 18 to 20 hours at 37°C.¹⁷ Oxidation was terminated by refrigeration. Oxidation of LDL was confirmed by the presence of TBARS, with malondialdehyde used as a standard. Protein content was determined according to the method of Bradford,¹⁸ with BSA used as the standard.

Platelet Aggregation and ¹⁴C-Serotonin Release
Washed platelets were prepared as described previously,¹⁹ incubated with ¹⁴C-serotonin (1 µCi/mL) for 1 hour at room temperature, and washed twice. Washed platelet aliquots were incubated with buffer, ox-LDL (10, 25, 50, and 100 µg protein/mL), or native LDL (100 µg protein/mL) for 1 hour at 37°C. In some experiments, washed platelets were preincubated with L-arginine (1 mmol/L) for 15 minutes before ox-LDL was added. After incubation, platelet aggregation was induced by thrombin (in subthreshold concentrations) in a dual-channel aggregometer.^{19 20} The concentration of thrombin was kept constant and repeatedly checked to ensure that platelet function did not change in each experiment. EDTA (13.4 mmol/L) was added to the washed platelet suspension 5 minutes after the onset of aggregation, and the sample was centrifuged at 800g for 15 minutes. Supernatant (175 µL) was removed for scintillation counting. An aliquot of washed platelets labeled with ¹⁴C-serotonin was saved for total counts. Static release count was expressed as described previously.¹⁹

Measurements of Cholesterol in Platelets
Washed platelets (10⁷/mL) were incubated with buffer, native LDL (50, 100, and 200 µg protein/mL), and ox-LDL (50, 100, and 200 µg protein/mL) in 1 mL Tyrode's buffer (in mmol/L: NaCl 137, KCl 2.7, MgCl₂ 1.0, CaCl₂ 1.0, NaH₂PO₄ 0.35, NaHCO₃ 11.9, glucose 5.5, pH 7.5) for 1 to 3 hours at 37°C, with gentle agitation every 30 minutes. After incubation, platelets were washed to remove the unincorporated lipoproteins. Total cholesterol in platelets was extracted and measured by previously described methods.^{21 22}

Determination of ³H-L-Arginine Uptake
Details of the methodology for determination of ³H-L-arginine uptake and constitutive NO synthase activity in platelets have been described recently.^{15 23} Uptake of ³H-L-arginine by platelets was measured by incubating washed platelets with ³H-L-arginine for 5, 15, 30, 45, and 60 minutes and was established to be maximal after 45 minutes of incubation. The effect of lipoproteins on ³H-L-arginine uptake by platelets was measured by incubating washed platelets (10⁷ cells/mL) and ox-LDL (0, 10, 25, 50, and 100 µg protein/mL) or native LDL (50 µg protein/mL) and ³H-L-arginine (7.25 nmol/L; average count 2 000 000 dpm) for 60 minutes in 1 mL NO buffer (in mmol/L: HEPES 25, NaCl 140, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1, pH 7.4) at 37°C. The reaction was stopped with 1 mL cold buffer (in mmol/L: HEPES 25, NaCl 118, KCl 4.7, KH₂PO₄ 1.18, NaHCO₃ 24.8, N-nitro-L-arginine 5, EDTA 4, pH 5.5), and each tube was centrifuged at 800g for 20 minutes at 4°C and washed twice. The platelet pellet was disrupted by adding 1 mL of 0.3 mol/L HClO₄ and neutralized with 65 µL of 3 mol/L KHCO₃. ³H-L-arginine in the disrupted platelet suspension was quantified by liquid scintillation spectroscopy. ³H-L-arginine uptake by platelets was calculated as described previously.¹⁵

To determine the specificity of ³H-L-arginine uptake by intact platelets and competitive inhibition of the uptake by unlabeled L-arginine, platelets were incubated with the same amount of ³H-L-arginine plus different concentrations of unlabeled L-arginine (0 to 10 mmol/L) for 45 minutes at 37°C. The uptake of ³H-L-arginine was measured as described above.

Determination of NO Synthase Activity in Platelet Cytosol
Purified platelets were suspended in 25 mmol/L HEPES buffer containing dithiothreitol (1 mmol/L), phenylmethylsulfonyl fluoride (0.01 mg/mL), trypsin inhibitor (0.01 mg/mL), leupeptin (0.01 mg/mL), antipain (0.01 mg/mL), chymostatin (0.01 mg/mL), and pepstatin (0.01 mg/mL) and were lysed by sonication for 30 seconds and kept on ice. The lysate was centrifuged at 10 000 rpm for 20 minutes at 4°C. Supernatant was applied to an AG50W-X8 (Na⁺ form) column to deplete endogenous L-arginine. Crude cytosol of platelets (4x10⁷ cells) was incubated with buffer, native LDL (50, 100, and 200 µg protein/mL), and ox-LDL (50, 100, and 200 µg protein/mL) in 400 µL buffer containing 25 mmol/L HEPES (pH 7.4), 1.5 mmol/L NADPH, 1 mmol/L dithiothreitol, 1 mmol/L CaCl₂, 1 mmol/L MgCl₂, 2.5 µmol/L flavin adenine dinucleotide, and 0.1 µmol/L tetrahydrobiopterin for 15 minutes at 37°C. After incubation, 100 µL ³H-L-arginine diluted with cold L-arginine (final concentration, 1 mmol/L) was added to the tubes, followed by incubation for 30 minutes at 37°C. The reaction was terminated with stop buffer (in mmol/L: HEPES 20, EDTA 2, pH 5.5) and an aliquot was applied to Dowex AG50W-X8 (Na⁺ form) columns and eluted with 4 mL of distilled water. NO synthase activity was expressed as picomoles of ³H-L-citrulline per milligram of platelet protein per minute.

Determination of Nitrite in Platelets
Nitrite production in platelets was measured by the Griess reaction.^{15 23 24} Washed platelets (10⁸/mL) were suspended in NO buffer containing 1.44 mmol/L NADPH and incubated with buffer or ox-LDL (10, 25, 50, and 100 µg protein/mL). In parallel experiments, L-arginine (1 mmol/L) was preincubated with washed platelets for 15 minutes before adding ox-LDL. The reaction was stopped by freeze-thawing the sample. After sonication, each aliquot was incubated in the presence of 20 mU of nitrate reductase for 1 hour at 37°C, thereby reducing nitrate to nitrite. After centrifugation at 30 000 rpm for 15 minutes, the supernatant was allowed to react with the Griess reagent (1% sulfanilamide/0.1% naphthylenediamine dihydrochloride/2.5% phosphoric acid) to form a chromophore; its absorption was measured subsequently at 546 nm. Nitrite concentration was determined with sodium nitrite (0.2 to 4 nmol) as the standard.

Determination of cGMP Levels in Platelets
Platelet-rich plasma (3x10⁸ platelets/mL) aliquots were incubated with buffer, ox-LDL (10, 50, and 100 µg protein/mL), or native LDL (100 µg protein/mL) for 1 hour at 37°C. Thereafter, 0.5 mL trichloroacetic acid (final concentration, 10%) was added to platelet-rich plasma aliquots. After centrifugation at 3 000 rpm for 15 minutes, trichloroacetic acid was extracted five times from the supernatant with water-saturated ether. The aqueous phase was dried under a stream of nitrogen and resuspended in 1.5 mL phosphate buffer. cGMP levels were measured by ELISA. The values of cGMP in platelet-poor plasma were subtracted, and the results were expressed as fmol/3x10⁸ platelets.

Western Blot Analysis
Washed platelets were incubated with buffer, native LDL (200 µg protein/mL), and ox-LDL (50 to 200 µg protein/mL) in platelet-poor plasma for 1 to 3 hours. After incubation, platelets were washed to remove lipoproteins and lysed with lysis buffer (1% SDS, 0.1% Triton-X 100, 10 mmol/L Tris-HCl, pH 7.4) supplemented with protease inhibitors and centrifuged at 300 000 rpm for 60 minutes at 4°C. The cytosolic protein from different platelet aliquots (10 µg per lane) was separated by 8% SDS-PAGE by use of a Bio-Rad Mini-Protean Cell, transferred to nitrocellulose filters (Amersham Life Science), and then immunoblotted with a mouse monoclonal antibody against human endothelial-type NO synthase peptide sequence 1030 to 1209 at 1:250 dilution. Mouse monoclonal antibody against rat neuronal NO synthase was used as a negative control. Anti-mouse horseradish peroxidase–conjugated antibody was used as a secondary antibody at 1:2500 dilution. The blots were detected with the enhanced chemiluminescence method (ECL Western blot kit, Amersham). Relative intensities of bands of interest were analyzed by use of an MSF-300G Scanner (Microtek Lab) and Scan Analysis software (Biosoft) and expressed as the ratio to positive control (human endothelial cell lysate). Total protein content of different platelet preparations was measured by Bradford's method.¹⁸

Cytotoxicity Determination
The potential cytotoxic effect of ox-LDL was assessed by determination of platelet aggregation. After incubation with ox-LDL (100 to 200 µg/mL), platelets were washed and thrombin-induced aggregation was performed to check platelet function.

Statistics
All data are based on at least three experiments and are expressed as mean±SE. Statistical analyses were performed by use of ANOVA followed by Scheffé's F test or Student's t tests (paired or unpaired data) as appropriate. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
The TBARS content of ox-LDL was 1.39±0.05 and that of native LDL was 0.22±0.07 nmol/100 µg protein, indicating oxidation of LDL in the ox-LDL aliquots but not in the native LDL aliquots.

Platelet Aggregation and ¹⁴C-Serotonin Release
Ox-LDL markedly enhanced thrombin-induced platelet aggregation, and the increase in aggregation was dependent on the concentration of ox-LDL in each experiment. Occasionally, ox-LDL alone, especially in high concentration, induced platelet aggregation. On the other hand, native LDL had only a minor effect on platelet aggregation. The ox-LDL–mediated platelet aggregation was totally inhibited by preincubation of platelets with L-arginine (1 mmol/L). L-Arginine (100 µmol/L) did not block the stimulatory effect of ox-LDL, although L-arginine alone at this concentration decreased platelet aggregation by 15%. Representative experiments are shown in Fig 1, and data from several experiments are summarized in Table 1.

View larger version (12K): [in this window] [in a new window]   Figure 1. Representative experiments showing stimulation of platelet aggregation when platelets were incubated with different concentrations of ox-LDL. The stimulation of platelet aggregation with 1 to 2.5 U/mL thrombin was attenuated by prior incubation of platelets with L-arginine.

View this table: [in this window] [in a new window]   Table 1. Effect of LDL and L-Arginine on Platelet Aggregation, 14C-Serotonin Release, and Nitrite Formation

Data on ¹⁴C-serotonin release are also presented in Table 1. Ox-LDL enhanced ¹⁴C-serotonin release in a concentration-dependent fashion. The highest concentration of ox-LDL used, 100 µg protein/mL, caused a threefold to fourfold increase in ¹⁴C-serotonin release. On the other hand, native LDL had no effect on ¹⁴C-serotonin release. The potentiation of serotonin release by ox-LDL was reversed by preincubation of platelets with L-arginine at 1 mmol/L but not at 100 µmol/L. The effects of ox-LDL on platelet aggregation and ¹⁴C-serotonin release were parallel except at high concentrations, when a marked increase in ¹⁴C-serotonin release was observed.

³H-L-Arginine Uptake and Conversion to ³H-L-Citrulline
Incubation of washed platelets with ³H-L-arginine (average count, 2 000 000 dpm) alone resulted in uptake of 11±1% (n=5). Uptake of ³H-L-arginine was inhibited in a concentration-dependent fashion by unlabeled L-arginine (magnitude of inhibition, 95±5% by 1 mmol/L L-arginine), which indicates competitive inhibition of ³H-L-arginine uptake.

Data on the effect of ox-LDL on uptake of ³H-L-arginine are depicted in Fig 2. Ox-LDL markedly inhibited the uptake of ³H-L-arginine by platelets, and the magnitude of inhibition was dependent on the concentration of ox-LDL. The lowest concentration of ox-LDL (10 µg protein/mL) reduced ³H-L-arginine uptake by platelets by 50%, whereas the highest concentration of ox-LDL (100 µg protein/mL) diminished the uptake by 90%. Native LDL had no effect on ³H-L-arginine uptake.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of ox-LDL and native LDL on the uptake of 3H-L-arginine in platelets. See "Methods" for methodology for determination of 3H-L-arginine uptake. Values are mean±SE (n=5).

Ox-LDL also decreased the conversion of ³H-L-arginine to ³H-L-citrulline in platelet cytosol in a concentration-dependent manner. Ox-LDL at 200 µg protein/mL reduced the reaction rate by 50% (P<.05 versus control), indicating that ox-LDL directly affects NO synthase activity in platelets (Fig 3). In contrast to the effect of ox-LDL, native LDL did not have any effect on NO synthase activity in platelet cytosol.

View larger version (44K): [in this window] [in a new window]   Figure 3. Effect of native LDL and ox-LDL on formation of 3H-L-citrulline in platelets. Whereas ox-LDL decreases 3H-L-citrulline formation, native LDL has no similar effect. Values are mean±SE (n=5).

Nitrite Production
Data on the effect of ox-LDL on platelet nitrite production are also shown in Table 1. Low concentrations of ox-LDL (10 and 25 µg protein/mL) had only a modest effect, whereas the higher concentrations of ox-LDL markedly inhibited platelet nitrite production. This effect was totally blocked by the preincubation of platelets with L-arginine. Notably, the same concentration of L-arginine also reversed the effects of ox-LDL on platelet aggregation and ¹⁴C-serotonin release in platelets stimulated by thrombin.

Platelet cGMP Levels
As more evidence for inhibition of NO production, ox-LDL 10 µg protein/mL caused a marked decrease in platelet cGMP content from 550±35 to 50±15 fmol/3x10⁸ cells per milliliter (P<.01). Higher concentrations of ox-LDL caused no additional decrease in cGMP accumulation. Notably, native LDL also resulted in a small but significant decrease in platelet cGMP levels (200±47 versus 550±35 fmol/3x10⁸ platelets per milliliter, P<.05), which indicates some oxidation of native LDL during the incubation period.

Platelet Cholesterol Content
To investigate whether ox-LDL–induced inhibition of the L-arginine–NO pathway was related to lipid uptake, platelet lipids were extracted and measured. The mean cholesterol content of control platelets was 2.7±0.2 µg/10⁷ cells. One or 3 hours of incubation of ox-LDL (50 to 200 µg protein/mL) with platelets markedly increased total cholesterol content in platelets. Three hours of incubation resulted in a slightly but not significantly greater increase of platelet cholesterol content than 1-hour incubation. Native LDL also caused a modest but insignificant increase in total cholesterol in platelets (Table 2).

View this table: [in this window] [in a new window]   Table 2. Cholesterol Concentrations in Platelets Incubated With Native LDL or Ox-LDL

Western Blot Analysis
Western blot analyses of platelet constitutive NO synthase protein were performed with mouse anti-human endothelial constitutive NO synthase monoclonal antibody. Immunoblotting consistently identified a band with an estimated molecular weight of 140 to 150 kD with human endothelial constitutive NO synthase monoclonal antibody but not with neuronal NO synthase antibody in platelets. In all analyses, NO synthase protein levels were lower in platelets treated with ox-LDL 200 µg protein/mL (0.35±0.03 versus 0.90±0.11 arbitrary units; P<.01; Fig 4). Treatment of platelets with native LDL also caused a modest reduction in NO synthase protein (0.76±0.06 versus 0.90±0.11; P<.05). One hour of incubation with ox-LDL caused slightly less of a decrease in NO synthase protein than the 3-hour period of incubation. Whereas incubation with 50 µg protein/mL of ox-LDL did not affect NO synthase protein level, incubation of platelets with 100 or 200 µg protein/mL of ox-LDL reduced NO synthase level, which indicates that the effect of ox-LDL on NO synthase protein was concentration dependent (Fig 5). It is noteworthy that total protein content of platelets was not affected by incubation with ox-LDL for 1 to 3 hours (11.8±1.4 versus 11.4±1.2 µg/µL; P=NS).

View larger version (28K): [in this window] [in a new window]   Figure 4. Western blot analysis of platelets with a mouse monoclonal antibody against human endothelial-type NO synthase from control (Lane B), ox-LDL–treated (200 µg protein/mL, Lane C), and native LDL–treated (200 µg protein/mL, Lane D) platelets. An equal amount of protein (10 µg) was loaded onto each lane. Lane A is endothelial cell cytosol. Constitutive NO synthase protein in endothelial cells has a molecular weight of 140 kD, whereas platelet constitutive NO synthase has a molecular weight of 140 to 150 kD. This Western blot analysis is representative of three separate analyses in which platelets were incubated with ox-LDL for 3 hours.

View larger version (18K): [in this window] [in a new window]   Figure 5. Western blot analysis of platelets with a mouse monoclonal antibody against human endothelial-type NO synthase from control platelets (Lane B) and from platelets treated with ox-LDL 50 µg protein/mL (Lane C), 100 µg protein/mL (Lane D), and 200 µg protein/mL (Lane E). An equal amount of protein (10 µg) was loaded onto each lane. Lane A is endothelial cell cytosol. Whereas treatment with 50 µg protein/mL ox-LDL had no effect on platelet NO synthase expression, treatment with both 100 and 200 µg protein/mL reduced NO synthase expression. In this representative experiment, platelets were incubated with ox-LDL for 1 hour.

In control experiments, CuSO₄ (up to 5 µmol/L) used for oxidation of LDL had no significant effect on platelet aggregation, ¹⁴C-serotonin release, or the L-arginine–NO pathway (data not shown). In addition, ox-LDL (up to 200 µg/mL) significantly increased washed platelet aggregation but had no significant effect on the total platelet protein content. These results suggest that the effects of ox-LDL on platelet function and NO synthase in platelets were not caused by cytotoxicity.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We confirmed previous observations^{6 7 8 9} that ox-LDL stimulates platelet activity, measured as aggregation and ¹⁴C-serotonin release. The novel findings of the present study relate to the important regulatory role of ox-LDL on the L-arginine–NO pathway in platelets.

Human platelets are unable to synthesize cholesterol but possess specific receptors for LDL,^{25 26 27} and platelet cholesterol content is regulated by plasma concentration of cholesterol. This was apparent from our observations of a marked increase in platelet cholesterol content when platelets were incubated with ox-LDL. Cholesterol content also increased after incubation of platelets with native LDL, but the increase was very modest. Ox-LDL has a predilection for uptake through the LDL receptor–independent pathway.²⁸ However, there is some question as to whether the high-affinity binding of ox-LDL to platelets per se is responsible for its effects on platelet activation.²⁹

The increase in platelet aggregation and ¹⁴C-serotonin release in response to low concentrations of ox-LDL paralleled one another, but the increase in ¹⁴C-serotonin release was greater than platelet aggregation in the presence of high concentrations of ox-LDL in the present study. Although the precise mechanism of the variable effects on aggregation and serotonin release is not known, ox-LDL has been shown to differentially affect serotonin-mediated (Gi) and ADP or thrombin-mediated (Gq) NO-dependent vascular responses.³⁰ Whether Gi and Gq signal-transduction processes are present in platelets is not clear.

Previous studies^{3 4 31} in vascular tissues suggested that ox-LDL reduces NO synthase activity. The L-arginine–NO pathway is also believed to be important in the regulation of platelet activity.^{11 15} We speculated that incubation of platelets with ox-LDL would result in a decrease in constitutive NO synthesis in platelets. On the basis of the measurement of nitrite in platelet supernatants, it became quite evident that ox-LDL, especially in high concentrations, markedly reduces nitrite formation. Bereta et al³² recently showed that LDL inhibits nitrite accumulation in mouse brain endothelial cell cultures and in a cell-free system in which sodium nitroprusside was used as a source of NO. To explore whether the mechanism underlying inhibition of nitrite generated in platelets by ox-LDL relates to the L-arginine–NO pathway, we examined modulation of ³H-L-arginine uptake and NO synthase activity by ox-LDL.

Data on ³H-L-arginine uptake by intact platelets revealed a striking concentration-dependent decrease in the presence of ox-LDL but not in the presence of native LDL. This observation suggests that ox-LDL interferes with the intracellular availability of L-arginine. Measurement of ³H-L-citrulline production showed that ox-LDL but not native LDL decreased the conversion of ³H-L-arginine in the platelet cytosol, which suggests that ox-LDL may directly destabilize NO synthase. The reduced availability of L-arginine together with decreased NO synthase activity may limit the formation of NO in platelets, resulting in diminished nitrite levels in the platelet supernatants. This concept was further confirmed by the decreased accumulation of cGMP in the cytosol of platelets incubated with ox-LDL. The inhibitory effect of ox-LDL was more pronounced on cytosolic cGMP accumulation than on NO production. Even native LDL caused a significant decrease in platelet cGMP accumulation but not on nitrite production, indicating great sensitivity of guanylate cyclase, one of the targets of NO. Interestingly, Yang et al³³ showed that ox-LDL inhibits inducible NO synthase activity in macrophages, whereas native LDL has no such effect. This observation is consistent with ours despite the difference in NO synthase isotypes in platelets and macrophages.

Work by Liao et al³⁴ showed that ox-LDL regulates endothelial NO synthase expression through a combination of early transcriptional inhibition and posttranscriptional mRNA destabilization. Although platelets are anucleated cells, these cells retain appreciable amounts of poly(A)⁺ RNA, and this RNA can be harvested.³⁵ This concept was recently confirmed in our previous studies¹⁵ that showed the presence of endothelial-type NO synthase in human platelets. To investigate whether ox-LDL alters the translation of NO synthase mRNA, we measured NO synthase levels in platelets after treatment with ox-LDL. It was repeatedly evident that ox-LDL significantly decreased the quantity of endothelial-type constitutive NO synthase in platelets, whereas total protein content of the platelets was unaffected. Notably, NO synthase level was also modestly reduced in the presence of native LDL. Together with the decreased cGMP level by native LDL, it is possible that native LDL was somewhat oxidized during the process of incubation with platelets.³⁶

Native LDL was oxidatively modified in the presence of 5 µmol/L CuSO₄ to form ox-LDL, as indicated by the TBARS measurement, which was almost 10 times higher than in the presence of unoxidized native LDL. CuSO₄ (up to 5 µmol/L) used for oxidation of LDL had no significant effect on platelet aggregation, ¹⁴C-serotonin release, or the L-arginine–NO pathway. Thus, the inhibitory effects of ox-LDL on the L-arginine–NO pathway were related to the altered composition of lipoprotein produced by the oxidization process. This speculation is supported by the observation of Yang et al,³³ in which the inhibitory effect of a fixed dose of ox-LDL on inducible NO synthase activity was greater when the degree of lipid peroxidation, measured by TBARS, was increased. Previous studies⁴ have shown that during oxidative modification of LDL, lecithin (phosphatidylcholine) is converted to lysolecithin (lysophosphatidylcholine), which results in high concentrations of lysolecithin in ox-LDL. The lysolecithin fraction in ox-LDL is responsible for inhibition of NO-dependent relaxation in the rabbit aorta.⁴ In addition, effects of other products of LDL oxidation³⁷ may also relate to the inhibition of NO-mediated relaxation by ox-LDL.³⁸

Pretreatment of platelets with exogenous L-arginine at 0.5 to 1 mmol/L concentration negated the effects of ox-LDL on platelet function as well as on nitrite (Table 1) and ³H-L-citrulline formation (data not shown). Although these concentrations of L-arginine are above the physiological concentrations of L-arginine, supraphysiological concentrations generally are needed for appropriate uptake in in vitro systems. In accordance with our observations, others³⁹ have shown restoration of vasorelaxation in atherosclerotic blood vessels after supplementation with large amounts of L-arginine.

In summary, the present study demonstrates that the major mechanism by which ox-LDL stimulates platelet activity is inhibition of NO synthase activity. Ox-LDL regulates NO synthase activity both by blocking its substrate availability and decreasing its protein levels. Other mechanisms, such as enhanced release of arachidonic acid and formation of proaggregant TXA₂ by cholesterol-rich platelets,⁴⁰ may also be operative in the phenomenon of platelet hyperactivity in hyperlipidemia.

	Selected Abbreviations and Acronyms

 

dpm = disintegrations per minute NO = nitric oxide ox-LDL = oxidized LDL TBARS = thiobarbituric acid–reactive substances TXA2 = thromboxane A2

	Acknowledgments

 
This study was supported by funds from the Division of Sponsored Research, University of Florida, Gainesville.

Received September 27, 1995; revision received October 27, 1995; accepted November 9, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Palinski W, Rosenfeld ME, Herttuala SY, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A.1989;86:1372-1376.
   
2. Moncada S, Higgs E, Palmer R. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev. 1991;43:109-142. [Medline] [Order article via Infotrieve]
   
3. Chin JH, Azhar S, Hoffman BB. Inactivation of endothelial derived relaxing factor by oxidized lipoproteins. J Clin Invest. 1992;89:10-18.
   
4. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature. 1990;344:160-162. [Medline] [Order article via Infotrieve]
   
5. Russell R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
   
6. Aviram M, Brook JG. The effect of human plasma on platelet function in familial hypercholesterolemia. Thromb Res. 1982;26:101-109. [Medline] [Order article via Infotrieve]
   
7. Carvallho ACA, Colman RW, Lees RS. Platelet function in hyperlipoproteinemia. N Engl J Med. 1974;290:434-438.
   
8. Hassall DG, Owen JS, Bruckdorfer KR. The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin. Biochem J. 1983;216:43-49. [Medline] [Order article via Infotrieve]
   
9. Koller E, Vukovich T, Doleschel W, Auerswald W. The influence of lipoproteins on ADP-induced platelet aggregation. Atherogenase. 1979;4(suppl IV):53-58.
   
10. Fuhrman B, Brook GJ, Aviram M. Activated platelets secrete a protein-like factor that stimulates oxidized-LDL receptor activity in macrophages. J Lipid Res. 1991;32:1113-1123. [Abstract]
    
11. Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3',5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood. 1981;57:946-955. [Free Full Text]
    
12. Durante W, Kroll MH, Vanhoutte PM, Schafer AI. Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C. Blood. 1992;79:110-116. [Abstract/Free Full Text]
    
13. Nakane M, Schmidt HHHW, Pollock JS, Forstermann U, Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett. 1993;316:175-180. [Medline] [Order article via Infotrieve]
    
14. Janssens SP, Shimouchi A, Quertermous T, Bloch DB, Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem. 1992;267:14519-14522. [Abstract/Free Full Text]
    
15. Mehta JL, Chen LY, Mehta P, Kone BC, Turner P. Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. J Lab Clin Med. 1995;125:370-377. [Medline] [Order article via Infotrieve]
    
16. Fisher WR. Heterogeneity of plasma low density lipoproteins manifestation of the physiologic phenomenon in man. Metabolism. 1983;32:283-291. [Medline] [Order article via Infotrieve]
    
17. Steinbrecher VP, Witztum JL, Parthasarathy S, Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Arteriosclerosis. 1987;7:135-143. [Abstract/Free Full Text]
    
18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
    
19. Chen LY, Mehta JL. Lys- and glu-plasminogen potentiate the inhibitory effect of recombinant tissue plasminogen activator on human platelet aggregation. Thromb Res. 1994;74:555-563. [Medline] [Order article via Infotrieve]
    
20. Nicolini FA, Wilson AC, Mehta P, Mehta JL. Comparative platelet inhibitory effects of human neutrophils and lymphocytes. J Lab Clin Med. 1990;116:147-152. [Medline] [Order article via Infotrieve]
    
21. Zlatski A, Zak B, Boyle AJ. A new method for the direct determination of serum cholesterol. J Lab Clin Med. 1953;41:486-492. [Medline] [Order article via Infotrieve]
    
22. Rose HG, Oklander M. Improved procedure for the extraction of lipids from human erythrocytes. J Lipid Res. 1965;6:428-431. [Abstract]
    
23. Chen LY, Mehta JL. Inhibitory effect of high density lipoprotein on platelet function is mediated by increase in nitric oxide synthase activity in platelets. Life Sci. 1994;55:1815-1821. [Medline] [Order article via Infotrieve]
    
24. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnork JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [¹⁵N]nitrate in biological fluids. Anal Biochem. 1982;126:131-138. [Medline] [Order article via Infotrieve]
    
25. Pedreño J, de Castellarnau C, Sánchez J, Gómez-Gerique J, Ordóñez-Llanos J, González-Sastre F. LDL binding sites on platelets differ from the `classical' receptor of nucleated cells. Arterioscler Thromb. 1992;12:1353-1362. [Abstract/Free Full Text]
    
26. Shmulewitz A, Brook JG, Aviram M. Native and modified low-density-lipoprotein interaction with human platelets in normal and homozygous familial-hypercholesterolemic subjects. Biochem J. 1984;224:13-20. [Medline] [Order article via Infotrieve]
    
27. Aviram M, Brook JCT. Platelet interaction with high and low density lipoproteins. Atherosclerosis. 1983;46:259-263. [Medline] [Order article via Infotrieve]
    
28. Chatterjee S. Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation. Mol Cell Biochem. 1992;111:143-147. [Medline] [Order article via Infotrieve]
    
29. Hassall DG, Bruckdorfer KR. Platelet low-density lipoprotein receptors. Biochem Soc Trans. 1985;13:1189-1190.
    
30. Tanner FC, Noll G, Boulanger CM, Lüscher TF. Oxidized low-density lipoproteins inhibit relaxations of porcine coronary arteries. Circulation. 1991;83:2012-2020. [Abstract/Free Full Text]
    
31. Plane F, Bruckdorfer KR, Kerr P, Steuer A, Jacobs M. Oxidative modification of low-density lipoproteins and the inhibition of relaxations mediated by endothelium-derived nitric oxide in rabbit aorta. Br J Pharmacol. 1992;105:216-222.[Medline] [Order article via Infotrieve]
    
32. Bereta M, Bereta J, Cohen S, Cohen MC. Low density lipoprotein inhibits accumulation of nitrites in murine brain endothelial cell cultures. Biochem Biophys Res Commun. 1992;186:315-320. [Medline] [Order article via Infotrieve]
    
33. Yang X, Cai B, Sciacca RR, Cannon PJ. Inhibition of inducible nitric oxide synthase in macrophages by oxidized low-density lipoproteins. Circ Res. 1994;74:318-328. [Abstract/Free Full Text]
    
34. Liao JK, Shin WS, Lee WY, Clark SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J Biol Chem. 1995;270:319-324. [Abstract/Free Full Text]
    
35. Roth GJ, Hickey MJ, Chung DW, Hickstein DD. Circulating human blood platelets retain appreciable amounts of poly(A)⁺ RNA. Biochem Biophys Res Commun. 1989;160:705-710. [Medline] [Order article via Infotrieve]
    
36. Aviram M, Fuhrman B, Keidar S, Maor I, Rosenblat M, Dankner G, Brook G. Platelet-modified low density lipoprotein induces macrophage cholesterol accumulation and platelet activation. J Clin Chem Clin Biochem. 1989;27:3-12. [Medline] [Order article via Infotrieve]
    
37. Esterbauer H, Jurgens G, Quehenberger O, Koller E. Autooxidation of human LDL: loss of polyunsaturated fatty acid, vitamin E and generation of aldehydes. J Lipid Res. 1987;28:495-509. [Abstract]
    
38. Gardaz JP, Py P, Suter PM, Junod AA. Effects of oleic acid-, alpha-naphthylthiourea-, and phorbol myristate-induced microvascular damage on indexes of pulmonary endothelium function in anesthetized dogs. Am Rev Respir Dis. 1988;137:1350-1355. [Medline] [Order article via Infotrieve]
    
39. Creager MA, Gallagher SJ, Girard XJ, Coleman SM, Dzau VJ, Cooke JP. L-Arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest. 1992;90:1248-1253.
    
40. Stuart MJ, Gerrard JM, White JG. Effect of cholesterol on production of thromboxane B2 by platelets in vitro. N Engl J Med. 1980;302:6-10.[Abstract]
    

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