Reversal by high-density lipoprotein of the effect of oxidized low-density lipoprotein on nitric oxide synthase protein expression in human platelets

Reversal by high-density lipoprotein of the effect of oxidized low-density lipoprotein on nitric oxide synthase protein expression in human platelets J. L. MEHTA a n d L. Y. CHEN GAINESVILLE,FLORIDA

Oxidized low-density lipoproteins (ox-LDLs) induce vasoconstriction and platelet activation, and high-density lipoproteins (HDLs) reverse these effects of ox-LDL. To determine the involvement of the L-arginine-nitric oxide (NO) pathway in the effects of lipoproteins on platelets, washed human platelets were incubated with nativeLDL, ox-LDL, or HDL plus ox-LDL. Ox-LDL, but not native LDL, potentiated thrombininduced platelet aggregation and carbon 14-1abeled serotonin release, and these effects of ox-LDL were blocked by pretreatment of plateiets with HDL Incubation of ox-LDL with platelets resulted in reduction in the uptake of tritiated L-arginine by intact platelets and in NO synthase activity in platelet lysate. These effects of ox-LDL on platelet NO synthase activity were also reversed by pretreatment of piatelets with HDL. Western blot analysis demonstrated about a 50% reduction in the expression of NO synthase protein in platelets treated with ox-LDL. Whereas HDL alone had no effect on NO synthase protein expression, it blocked the decrease in NO synthase expression caused by ox-LDL. Thus ox-LDL stimulates platelet function primarily by diminishing NO synthase expression, and this effect of ox-LDL can be blocked by pretreatment of platelets with HDL. (J LABCLIN MED 1996;127:287-95)

Abbreviations:

EDTA - e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ; HDL high-density Iipoprotein; HEPES = N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N - 2 - e t h a n e s u l p h o n i c a c i d ; LDL = l o w - d e n s i t y l i p o p r o t e i n ; L-NAME = N~-nitro-L-arginine m e t h y l ester; NADPH = r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ; N O = nitric o x i d e ; ox-LDL - o x i d i z e d l o w - d e n s i t y l i p o p r o t e i n ; TBARS = t h i o b a r b i t u r i c a c i d - r e a c t i v e s u b s t a n c e s ; TxA 2 = t h r o m b o x a n e A 2

igh plasma levels of LDLs and low levels of HDLs are major risk factors in the pathogenesis of atherosclerosis. 1 It has been proposed that native L D L undergoes oxidative modification resulting in the formation of ox-LDL before it can give rise to foam cells, the key component of the early lesion of atherosclerosis. 2 L D L can be

H

From the Department of Medicine, College of Medicine, University of Florida, and the Veterans Affairs Medical Center. Submitted for publication June 26, 1995; revisionsubmitted Oct. 11, 1995; accepted Oct. 16, 1995. Reprint requests: J. L Mehta, MD, PhD, University of Florida College of Medicine, P.O. Box 100277JHMHC, Gainesville,FL 32610-0277. 0022-2143/96 $5.00 + 0 5/1/70183

=

oxidized within the blood vessels and also in leukocytes and platelets. 2 Previous studies have shown that ox-LDL impairs the release of the potent vasodilator species NO from endothelial cells. 1'3'4 Impaired formation of NO in the blood vessels not only predisposes to vasoconstriction but also favors leukocyte deposition, platelet adhesion and aggregation, and subsequent release of the vasoconstrictor species serotonin and TxA 2. Deposition of leukocytes, especially T lymphocytes and monocytes, results in inflammatory and immunologic reactions that play a key role in the process of atherogenesis. 5 In recent years several in vitro 6'7 and in vivo8'9 studies have shown that ox-LDL directly stimulates platelet aggregation and TxA 2 release, which could contribute to intravascular thrombosis and vasocon287

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striction. Fuhrman et al. 1° have demonstrated that activated platelets secrete a protein-like factor that stimulates uptake of ox-LDL by macrophages. Thus platelets work in close association with macrophages, the precursors of most foam cells, in the developing atherosclerotic lesion. However, the precise mechanism of ox-LDL-mediated platelet activation remains to be elucidated. H D L has been shown to decrease the cytotoxic effects of ox-LDL in cultured endothelial cells. 11 H D L also reverses the inhibitory effect of ox-LDL on NO-mediated smooth muscle relaxation. 12 Schmidt et al. 13 have demonstrated that ox-LDL reduces the response of soluble guanylyl cyclase to nitrovasodilators and that this desensitization can be antagonized by H D L . Recent studies show a reduction in atherosclerotic lesions in hypercholesterolemic rabbits by the administration of a mutant of human apo A-l, which determines the H D L subclass missing in patients with premature coronary atherosclerosis. 14 This phenomenon may have its basis in the modulation of alterations in NO-mediated vasorelaxation caused by ox-LDL. Other studies suggest that platelets also generate NO from L-arginine, which, by increasing cyclic guanosine monophosphate accumulation, regulates platelet aggregation. 15 Durante et al. 16 demonstrated that N O inhibits platelet aggregation by inhibiting platelet phospholipase C, which would result in diminished TxA 2 formation. Molecular cloning studies have characterized the human c D N A encoding two distinct constitutive N O synthase enzymes in the brain 17 and in endothelial cells. ~a Our recent work with reverse transcription polymerase chain reaction and Southern analysis has revealed that human platelets contain endothelial-type constitutive NO synthase. ~9 In consideration of the key role of NO in the regulation of platelet function and adhesion of platelets and monocytes/macrophages to the vessel wall, we studied the interaction between ox-LDL and H D L with regard to the L-arginine-NO pathway in human platelets.

METHODS

Reagents. 2,3,4,5-3H-L-arginine (69 Ci/mmol; 1 Ci = 37 GBq; 1.0 mCi/ml) was obtained from Amersham, Arlington Heights, Ill. An RPN 2108 ECL Western blotting analysis system was also obtained from Amersham. Mouse monoclonal anti-human endothelial NO synthase was obtained from Transduction Laboratories, Lexington, Ky. A 10 kd protein ladder (10,000 to 200,000 daltons) was obtained from Life Technologies, Gaithersburg, Md. All

J Lab Clin Med March 1996

other chemicals were purchased from Sigma Chemical Co., St. Louis, Mo. Preparation and characterization of lipoproteins. Native lipoproteins (LDL, density = 1.025 to 1.063 gm/ml) were isolated from human plasma by discontinuous density gradient ultracentrifugation as described earlier. 2° In brief, the density of plasma was adjusted to 1.006 gm/ml with NaC1 medium, and the plasma was centrifuged at 150,000 g for 24 hours. Very-low-density lipoproteins and the chylomicron-rich layer were discarded. The remaining fraction, after density was adjusted to 1.063 gm/ml with KBr medium, was centrifuged at 150,000 g for 24 hours to isolate the LDL fraction from the HDL fraction. The purified LDL was dialyzed for 96 hours against phosphate-buffered saline solution being degassed with N2 and containing 0.3 mmol/L EDTA at 4° C. LDL was stored under N 2 at 4° C, and suitable aliquots were then oxidized in the presence of 5 i~mol/L CuSO4 for 18 to 20 hours at 37° C. 21 Oxidation was terminated by refrigeration. Oxidation of LDL was confirmed by the presence of TBARS by using malondialdehyde as standard. Protein content was determined according to the method of Bradford 22 by using bovine serum albumin as the standard. Platelet aggregation and carbon 14-1abeled serotonin release. Washed platelets were prepared as described

previously23 and were incubated with 14C-labeled serotonin (1 ixCi/ml) for 1 hour at room temperature and washed twice. Washed platelet aliquots were incubated with buffer, native LDL (100 Ixg protein per milliliter of ox-LDL; 10, 25, 50, and 100 p~g protein/ml), HDL alone (100 ~g protein/ml), or HDL plus ox-LDL for 1 hour at 37° C. After incubation, platelet aggregation was induced by thrombin (in subthreshold concentrations) in a dualchannel aggregometer,23'z4 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 washed platelet suspension at 5 minutes after the onset of aggregation, and the sample was centrifuged at 800 g for 15 minutes. Supernatant (175/xl) was removed for scintillation counting. An aliquot of washed platelets labeled with 14C-serotonin was saved for total counts. 14C-labeled serotonin release was calculated as described earlier. 23 Measurement of cholesterol in platelets. Washed platelets (107/ml) were incubated with buffer, native LDL, ox-LDL, HDL alone, or HDL plus ox-LDL in 1 ml Tyrode's buffer (composition in mmol/L: NaC1 137, KC1 2.7, MgC12 1.0, CaC12 1.0, NaH2PO 4 0.35, NaHCO3 11.9, and 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 tipoproteins. Cholesterol in platelets was measured by the method of Zlatski et al. 25 Determination of tritiated l.-orginine uptake. Details of the methods for determination of tritiated L-arginine uptake and constitutive NO synthase activity in platelets have been described earlier. 19'26 Uptake of tritiated Larginine by platelets was measured by incubating washed

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platelets with tritiated 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 tritiated L-arginine uptake by platelets was measured by incubating washed platetets (107 cells/ml) with buffer, native LDL, ox-LDL, HDL, or H D L plus ox-LDL and tritiatedL-arginine (7.25 nmol/L; average count 1 million dpm) for 60 minutes in 1 ml NO buffer (composition in mmol/L: HEPES 25, NaC1 140, KC1 5.4, CaC12 1.8, and MgC12 1, pH 7.4) at 37 ° C. The reaction was stopped with 1 ml cold buffer (composition in mmol/L: HEPES 25, NaC1 118, KC1 4.7, NI-I2PO 4 1.18 NaHCO 3 24.8, N=-nitro-L-argi nine 5, EDTA 4, pH 5.5), and each tube was centrifuged at 800 g for 20 minutes at 4 ° C and washed twice. The platelet pellet was disrupted by adding 1 ml of 0.3 mol/L H C I O 4 and neutralized with 65 Ixl of 3 mol/L KHCO 3. Tritiated L-arginine in the disrupted platelet suspension was quantified by liquid scintillation spectroscopy. Tritiated L-arginine uptake by platelets was calculated as described earlier. 19 Determination of NO synthase activity in platelet lysate.

Platelets treated with buffer, ox-LDL, HDL alone, or H D L plus ox-LDL were suspended in 25 mmol/L HEPES buffer containing 1 mmol/L dithiothreitol, 0.1% Triton X-100, 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), pepstatin A (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 a AG50W-X8 (Na + form) column to deplete endogenous L-arginine. Crude lysate of platelets (4 × 10 7 cells) was incubated with tritiated L-arginine diluted with cold Larginine (final concentration 1 mmol/L) in 400 ixl buffer containing 25 mmol/L HEPES (pH 7.4), 1.5 mmol/L NADPH, 1 mmol/L dithiothreitol, 1 mmol/L CaCla, 1 mmol/L MgC12, 2.5 ixmol/L flavin adenine dinucleotide, and 0.1 ixmol/L (6R)-BH 4 for 15 minutes at 37° C. The reaction was terminated with stop buffer (in mmol/L: HEPES 20, EDTA 2, pH 5.5) and an aliquot applied to Dowex AGSOW-X8 (Na ÷ form) columns (Bio-Rad Laboratories, Hercules, Calif.) and eluted with 4 ml of distilled water. NO synthase activity was expressed as picomoles of tritiated Lcitrulline per milligram of platelet protein per minute. Determination of nitrite in platelets. Nitrite production in platelets was measured by the Griess reaction. 19'26'27 Washed platelets (108/ml) were suspended in NO buffer containing 1.44 mmol/L NADPH and incubated with buffer, ox-LDL (50 and 100 txg protein per milliliter), HDL alone (100 ~g protein per milliliter), or H D L plus 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% H3PO4) to form a chromophore; its absorption was mea-

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sured subsequently at 546 nm. Nitrite (plus nitrate) concentration was determined with sodium nitrite as the standard. Western analysis. Washed platelets were incubated with buffer, ox-LDL, HDL alone, or H D L (200 ~g/ml) plus ox-LDL (200 ~g/ml) in platelet-poor plasma for 1 to 3 hours. After incubation, platelets were washed to remove lipoproteins and were lysed with lysis buffer (1% sodium dodecyl sulfate, 0.1% Triton X-100, 10 mmol/L Tris-HC1, pH 7.4) supplemented with protease inhibitors and centrifuged at 30,000 rpm for 60 minutes at 4 ° C. The platelet proteins from different aliquots (10 ~g per lane) were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Bio-Rad Mini-Protean Cell (Bio-Rad Laboratories, Richmond, Calif.), transferred to nitrocellulose filters (Amersham Life Sciences), and then immunoblotted with a mouse monoclonal antibody against human endothelial NO synthase peptide sequence 1030 to 1209, at 1:250 dilution. Mouse monoclonal antibody against rat neuronal NO synthase peptide sequence 1095 to 1289 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 an enhanced chemiluminesence method (ECL Western blot kit; Amersham Life Science, Buckinghamshire, England). Relative intensities of bands of interest were analyzed with an MSF-300G Scanner (Microtek Lab, Torrance, Calif.) and the Scan Analysis software (Biosoft, Cambridge, England) and expressed as the ratio to positive control (human endothelial cell lysate). Statistics. All data are based on at least three experiments and are expressed as mean -+ SEM. Statistical analyses were performed by using analysis of variance followed by Scheffh's F test or Student's t test (paired or unpaired data), as appropriate. A p value less than 0.05 was considered significant. RESULTS

T h e T B A R S content of o x - L D L was 1.39 + 0.05 nmol/100 ~g, and that of native L D L was 0.22 _+ 0.07 nmol/100 ~g, indicating oxidation of L D L in the o x - L D L aliquots but not in the native L D L aliquots. Lipoproteins and platelet cholesterol content. T h e m e a n cholesterol content of control platelets was 2.7 _+ 0.2 ixg/107 cells. O n e to 3 hours of incubation of o x - L D L m a r k e d l y increased total cholesterol content in a c o n c e n t r a t i o n - d e p e n d e n t manner. T h r e e hours of incubation resulted in a slightly greater, but not significant, increase in platelet cholesterol content than the 1-hour incubation. Native L D L also caused a small but significant increase in cholesterol content in platelets. P r e t r e a t m e n t of platelets with H D L markedly diminished the o x - L D L - i n d u c e d rise in platelet cholesterol content. T h e data are s u m m a r i z e d in Table I.

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Table I. Cholesterol concentrations in platelets incubated with lipoproteins Total cholesterol (p.gll 07 platelets) Treatment of platelets Buffer Native LDL 200 Fg/ml Ox-LDL 50 l~g/ml 100 Fg/ml 200 ;~g/ml HDL 100 Fg/ml HDL plus ox-LDL 100 i~g/ml

1 hr incubation 3 hr incubation 2.6 _+ 0,3 ND

2.7 _+ 0.2 3.1 -+ 0.1

3.9 -- 0.4* 4.0 -+ 0,1" 5.0 _+ 0.4* ND ND

4.3 4,5 7.5 2.8 2.7

_+ 0,1" -+ 0,1" _+ 0.1 *I-+ 0.4 _+ 0.25

Data from three experiments, expressed as mean _+ SEM. ND, Not done. *p < 0.01 vs buffer-treated platelets. tP < 0.05 vs native LDL-treated platelets. Sp < 0.02 vs ox-LDL-treated platelets.

Platelet aggregation and serotonin release. Ox-LDL markedly increased platelet aggregation, and the increase in aggregation was dependent on the concentration of ox-LDL in each experiment. On the other hand, native LDL had only a minor effect on platelet aggregation. The ox-LDL-mediated platelet aggregation was totally inhibited by the preincubation of platelets with HDL. HDL per se also inhibited platelet aggregation. Representative experiments are shown in Fig. 1, and data from several experiments are summarized in Table II. Data on 14C-lableled serotonin release are also presented in Table II. Ox-LDL enhanced 14C-serotonin release in a concentration-dependent fashion. The highest concentration of ox-LDL used, 100 ixg protein per milliliter, caused a threefold to fourfold increase in 14C-labeled serotonin release. In contrast, native LDL did not affect ~4C-lableled serotonin release. The potentiation of 14 C-lableled serotonin release by ox-LDL was reversed by preincubation of platelets with HDL. HDL per se also decreased 14Clanded serotonin release. Lipoproteins and uptake of tritiated L-arginine and N O

synthase activity. Data on the effect of ox-LDL on

uptake of tritiated L-arginine are depicted in Fig. 2. Ox-LDL, but not native LDL, markedly diminished the uptake of tritiated L-arginine in intact platelets, and the magnitude of inhibition was dependent on the concentration of ox-LDL. The lowest concentration of ox-LDL (25 ixg/ml) reduced tritiated L-arginine uptake in platelets by 50%, whereas the highest concentration of ox-LDL (100 txg/ml) diminished the uptake by 90%. Although H D L had no effect on the uptake of tritiated L-arginine, it markedly reduced the effect of ox-LDL on the uptake of triti-

ated L-arginine by platelets (p < 0.01 vs the effect of ox-LDL alone). Importantly, ox-LDL decreased the conversion of tritiated L-arginine to tritiated L-citrulline in platelet lysate. At 200 txg protein per milliliter concentration, ox-LDL reduced the conversion to tritiated L-citrulline by ----50%, indicating that ox-LDL directly affects NO synthase activity in platelets. In contrast to the effect of ox-LDL, native LDL did not have any significant effect either on the uptake of tritiated L-arginine in intact platelets or on NO synthase activity in platelet lysate. H D L alone enhanced (p < 0.05) NO synthase activity, and it reversed the effect of ox-LDL alone on the formation of tritiated L-citrulline. These data are summarized in Fig. 3. Data on the effect of ox-LDL on platelet nitrite (plus nitrate) production are shown in Table II. Low concentrations of ox-LDL (10 and 25 Ixg/ml) had only a modest effect (data not shown), whereas higher concentrations of ox-LDL markedly inhibited platelet nitrite production. This effect was totally blocked by preincubating platelets with HDL. Notably, the same concentration of HDL also reversed the effects of ox-LDL on platelet aggregation and serotonin release in platelets stimulated by thrombin. Lipoproteins

and platelet NO synthase expression.

Western blot analyses of platelet constitutive NO synthase protein were performed with mouse antihuman endothelial constitutive NO synthase monoclonal antibody. Immunoblotting consistently identified a band with estimated molecular weight of 140 to 150 kd with human endothelial NO synthase monoclonal antibody but not with neuronal NO synthase antibody in platelets. In all analyses, NO synthase protein level was lower in platelets treated with ox-LDL (mean density 0.35 -+ 0.03 arbitrary units vs 0.90 _+ 0.11 arbitrary units, p < 0.01). Treatment of platelets with native LDL also caused a modest reduction in NO synthase protein. Most importantly, the presence of HDL abolished the effect of ox-LDL on NO synthase expression (mean density 1.0 + 0.3 arbitrary units vs 0.35 -+ 0.03 arbitrary units, p < 0. 01). A representative Western blot is shown in Fig. 4. In control experiments, CuSO4 (up to 5 txmol/L) used for oxidation of LDL had no significant effect on platelet aggregation, 14C-lableled serotonin release, and L-arginine-NO pathway (data not shown). DISCUSSION

This study shows that ox-LDL stimulates platelet activity and that this effect of ox-LDL is mediated by

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I - __F9 =

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-rhmn'~in Fig. 1. Representative experiments showing stimulation of thrombin-induced platelet aggregation when platelets were incubated with ox-LDL (100 ~g/ml). The stimulation of platelet aggregation was attenuated by prior incubation of platelets with HDL (100 txg/mi). HDL per se decreased platelet aggregation. Concentration of thrombin was kept constant in each experiment.

Table II. Effect of lipoproteins on plafelet aggregation, ~4C-Iobeled serotonin release, and nitrite plus nitrite formation Platelet aggregation (%) Control Ox-LDL 50 r~g protein/ml !00 Fg protein/ml Native LDL (100 ixg protein/ml) HDL (100 lxg protein/ml) HDL (100 l~g protein/ml) plus Ox-LDL (100 l~g protdn/ml)

14C-labeledserotonin release (%)

Nitriteplus nitrate (nmollmg protein)

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35 ± 5* 67 _+ 1"113_+2 5±1" 26 ± 75

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_+ 1" _+ 3*1_+ 3 ± 3* _+ 45

Data from three to five experiments,expressedas mean _+SEM. AID, Net done. "p < 0.05 vs control. tP < 0.05 vs native LDL-treated platelets, Sp < 0.02 vs ox-LDL treated platelets.

important alterations in the L-arginine-NO pathway. Ox-LDL inhibits the uptake of tritiated Larginine by intact platelets, reduces NO synthase activity in platelet lysate, and markedly decreases NO synthase protein expression in these cells. Most importantly, our data show that pretreatment of platelets with H D L completely reverses these effects of ox-LDL on platelets. Incubation of platelets with ox-LDL resulted in an increase in total cholesterol in our study. Platelet cholesterol levels also increased, although modestly, during the incubation of platelets with native LDL. Pretreatment of platelets with H D L reversed the ox-LDL-mediated increase in platelet content of total cholesterol. This effect of H D L

has been previously documented to relate most likely to active transport of cholesterol out of the cell. 2s The L-arginine-NO pathway is believed to be important in the regulation of platelet activity. ~s'19 Radomski et al. 29 clearly documented stimulation of platelet aggregation by w-substituted L-arginine analogs mediated via inhibition of cyclic guanosine monophosphate accumulation. We have also shown that L-NAME potentiates the effect of a subthreshold concentration of thrombin on washed platelet aggregation and ~4C-labeled serotonin release, and this stimulatory effect of L-NAME can be overcome by an excess of L-arginine in platelets. 19 Additional studies with reverse transcription polymerase chain

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Fig. 2. Effectof native LDL, ox-LDL,HDL, and HDL plus ox-LDLon the uptake of tritiated L-arginine in intact platelets. Whereas ox-LDLdecreased uptake of tritiated L-arginine,preincubation of platelets withHDL (100 ixg/ml)attenuatedthe effectof ox-LDL(100 ixg/ml).NativeLDL (100 pog/ml)or HDL (100 txg/ml) had no effect on the uptake of tritiated L-arginine. See text for methods for determination of tritiated L-arginineuptake. Data are from five experiments,expressed as mean ± SEM.

reaction and Southern analysis have identified the presence of both constitutive (endothelial-type) and inducible forms of NO synthase enzymeJ 9 We speculated that lipoproteins may alter the L-arginine-NO pathway and result in previously described changes in platelet function. Tritiated L-arginine uptake by intact platelets fell in the presence of ox-LDL but not in the presence of native LDL. This observation suggests that ox-LDL, and perhaps an increase in platelet membrane cholesterol content, interferes with the intracellular availability of L-arginine. In addition, measurement of tritiated L-citrulline production showed that oxLDL, but not native LDL, decreases the conversion of tritiated L-arginine in the platelet cytosol, which suggests that ox-LDL affects constitutive NO synthase activity in platelets. Based on the measurement of nitrite (plus nitrate) in the platelet supernatants, it became quite evident that ox-LDL markedly reduces nitrite formation. The reduced availability of L-arginine, together with decreased NO synthase activity, may limit the formation of NO in platelets, resulting in diminished nitrite (plus nitrate) levels in the platelet supernatants. Interestingly, Yang et al. 3° found that ox-LDL inhibited inducible NO synthase activity in macrophages, whereas native LDL had no such effect. This observation is consistent with ours, despite the difference

in NO synthase isoforms in platelets and macrophages. Recent work by Liao et al. 31 shows that ox-LDL regulates endothelial NO synthase expression through a combination of early transcriptional inhibition and post-transcriptional m R N A destabilization. I t has been reported that human platelets retain appreciable amounts of poly (A) + RNA and that this RNA can be harvested. 32 To investigate whether 0x-LDL alters the translation of NO synthase mRNA, we measured NO synthase levels in platelets after treatment with LDL. It was consistently evident that ox-LDL decreases the amount of constitutive NO synthase in platelets. Notably, NO synthase level was also modestly reduced in the presence of native LDL, which is contrary to its effects on the uptake of tritiated L-arginine. It is known that platelets oxidatively modify native LDL, 33 and it is possible that native LDL was oxidized during the process of prolonged incubation with platelets. Therefore it is likely that the apparent discrepancy between the effects of native L D L on NO synthase protein expression and platelet bioactivity probably is due to the longer incubation time in Western analysis. These results indicated that ox-LDL may regulate the expression of or destabilize NO synthase in platelets. H D L clearly reversed the effect of ox-LDL on NO

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Fig. 3. Effect of native LDL, ox-LDL, HDL, and HDL plus ox-LDL on formation of tritiated L-citrulline in platelet cytosol. Whereas ox-LDL decreased tritiated L-citrulline formation, native LDL had no similar effect. HDL (100 ~g/ml) increased the formation of tritiated L-citrulline in platelet lysate and inhibited the effect of ox-LDL (200 ixg/ml). Data from five experiments, expressed as mean ___SEM.

160 KD 140 KD 80 KD

A

B

C

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E

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Fig. 4. Western analysis of platelets from control (lane B), ox-LDL-treated (200 ~g/ml, lane C), native LDL-treated (200 Ixg/ml, lane D), HDL-treated (i00 ~xg/ml,lane E), and HDL (200 ~g/mI) plus ox-LDL (200 Ixg/ml)-treated (lane F) platelets. An equal amount of protein (10 I~g) was loaded onto each lane. Lane A is endothelial cell lysate. Constitutive NO synthase protein in endothelial cells has a molecular weight of approximately 140 kd, whereas platelet constitutive NOS has a molecular weight of approximately 140 to 150 kd. This Western analysis is representative of three separate analyses.

synthase activity in platelets, measured as the conversion of tritiated L-arginine to tritiated L-citrulline and the formation of nitrite in platelets. In addition, pretreatment of platelets with H D L prevented the decrease in constitutive NO synthase protein as measured by Western blot analysis. These effects of H D L correlated with alterations in platelet cholesterol content and in platelet aggregation and

14C-labeled serotonin release. H D L by itself had no effect on the uptake of tritiated L-arginine, but it decreased platelet activity and simultaneously increased NO synthase activity (Table II). Although changes in NO synthase activity correlated with the decrease in platelet function in the presence of HDL, other effects of HDL, such as its effect on cholesterol transport z8 and inhibition of

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LDL oxidation, 34 may also relate to its overall effects on platelet function. HDL also stimulates release and stabilization of prostaglandin I2,35 which inhibits platelet function. However, platelets do not synthesize prostaglandin I2, and this mechanism may not be operative in in vitro conditions used by us. Several investigators have shown that HDL reverses the effect of ox-LDL on endothelium-dependent vascular relaxation. ~z~3 However, the alterations in the L-arginine-NO pathway by ox-LDL and their reversal by HDL shown in this study strongly suggest that this pathway is a major mechanism by which lipoproteins regulate platelet function. Native LDL was oxidatively modified in the presence of 5 ixmol/L CuSO4 to form ox-LDL, as indicated by almost 10 times higher TBARS than in the presence of native LDL. CuSO4 (up to 5 ~mol/L) used for oxidation of LDL had no significant effect on platelet aggregation, ~4C-Iabeled serotonin release, and 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., 3° 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. In summary, this study demonstrates that the major mechanism by which ox-LDL stimulates platelet activity is by reduction in the uptake of L-arginine and NO synthase activity as a result of NO synthase protein expression. HDL reverses these effects of ox-LDL on platelet function and thereby overcomes the pro-atherogenic effects of ox-LDL. REFERENCES

1. Moncada S, Higgs EA, Palmer RMJ. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43: 109-42. 2. Palinski W, Rosenfeld ME, Herttuala SY, et al. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 1989;86:1372-6. 3. Chin JH, Azhar S, Hoffman BB. Inactivation of endothelial derived relaxing factor by oxidized lipoproteins. J Clin Invest 1992;89:10-8. 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;344:160-2. 5. Russell R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-9. 6. Aviram M, Brook JG. The effect of human plasma on platelet function in familial hypercholesterolemia. Thromb Res 1982; 26:101-9. 7. Carvallho ACA, Colman RW, Lees RS. Platelet function in hyperlipoproteinemia. N Engl J Med 1974;290:434-8.

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8. Hassall DG, Owen JS, Bruckdorfer KR. The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin. Biochem J 1983;216:43-9. 9. Koller E, Vukovich T. Doleschel W, Auerswald W. The influence of lipoproteins on ADP-induced platelet aggregation. Atherogenesis 1979;4(suppl IV):53-8. 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;32:1113-23. 11. Henriksen T, Evensen SA, Carlander B. Injury to cultured endothelial cells induced by low density lipoproteins: protection by high density lipoproteins. Scand J Clin Lab Invest 1979;39:369-75. 12. Matsuda Y, Hirata K, Inoue N, et al. High density lipoproteins reverse inhibitory effect of oxidized low density lipoprotein on endothelium-dependent arterial relaxation. Circ Res 1993;72:1103-9. 13. Schmidt K, Graier WF, Kostner GM, Kukovetz WR. High density lipoprotein antagonizes the inhibitory effect of oxidized low density lipoprotein and lysolecithin on soluble guanylyl cyclase. Biochem Biophys Res Commun 1992;182: 302-8. 14. Ameli S, Hultgardh-Nilsson A, Cercek B, et al. Recombinant apolipoprotein A-1 Milano reduces intimal thickening after balloon injury in hypercholcsterolemic rabbits. Circulation 1994;90:1935-42. 15. Mellion BT, Ignarro J, 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;57:946-55. 16. Durante W, Kroll MH, Vanhoutte PM, Schafer AI. Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C. Blood 1992;79:110-6. 17. 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;316:175-80. 18. 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;267:14519-22. 19. Mehta JL, Chen LY, Kone BC, Mehta P, Turner P. Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. J LAB CLIN MED 1995;25:370-7. 20. Mehta JL, Bryant JL Jr., Mehta P. Oxidized-LDL decreases nitric oxide synthase activity in human neutrophils. Reversal of the efffect of oxidizcd-LDL by HDL and L-arginine. Biochem Pharmacol 1995;50:1181-5. 21. Steinbrecher VP, Witztum JL, Parthasarathy S, Steinberg D. Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Arteriosclerosis 1987;7: 135-43. 22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976;72:248-54. 23. 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: 555-63. 24. Nicolini FA, Wilson AC, Mehta P, Mehta JL. Comparative platelet inhibitory effects of human neutrophils and lymphocytes. J LAB CLIN MED 1990;116:147-52. 25. Zlatski A, Zak B, Boyle AJ. A new method for the direct

J Lab CIin Med Volume 127, Number 3

26.

27.

28.

29.

30.

determination of serum cholesterol. J LAB CLIN MED 1953; 41:486-92. Chert 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: 1815-21. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnork JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [lSN]nitrate in biological fluids. Ann Biochem 1982;126: 131-8. Schmitz G, Brtining T, Williamson E, Nowicka G. The role of HDL in reverse cholesterol transport and its disturbances in tangier disease and HDL deficiency with xanthomas. Eur Heart J 1990;11(suppl E):197-211. Radomski MW, Palmer RMJ, Moncada S. An L-argininenitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci USA 1990;87:5193-7. Yang X, Cai B, Sciacca RR, Cannon PJ. Inhibition of induc-

Mehta and Chen

31.

32.

33.

34.

35.

295

ible nitric oxide synthase in macrophages by oxidized lowdensity lipoproteins. Circ Res 1994;74:318-28. Liao JK, Shin WS, Lee WY, Clark SL. Oxidized low density lipoprotein decreases the expression of endothelial nitric oxide synthase expression. J Biol Chem 1995;270:319-24. Roth GJ, Hickey MJ, Chungand DW, Hickstein DD. Circulating human blood platelets retain appreciable amounts of poly(A) ÷ RNA. Biochem Biophys Res Commun 1989;160: 705-10. Aviram M, Fuhrman B, Keidar S, et al. Platelet-modified low density lipoprotein induces macrophage cholesterol accumulation and platelet activation. J Clin Chem Clin Biochem 1989;27:3-12. Parthasarathy S, Barnett J, Fong LG. High-density lipoprotein inhibits the oxidative modification of low density lipoprotein. Biochim Biophys Acta 1990;1044:275-83. Lefer AM. Prostacyclin, high density lipoproteins and myocardial ischemia. Circulation 1990;81:2013-5.