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(Circulation. 1996;94:2434-2440.)
© 1996 American Heart Association, Inc.

Articles

-Tocopherol Inhibits Aggregation of Human Platelets by a Protein Kinase C–Dependent Mechanism

Jane E. Freedman, MD; John H. Farhat, MPH; Joseph Loscalzo, MD, PhD; John F. Keaney, Jr, MD

the Whitaker Cardiovascular Institute and Evans Memorial Department of Medicine, Boston (Mass) University School of Medicine.

Correspondence to Dr John F. Keaney, Jr, Whitaker Cardiovascular Institute, Room W507, Boston University School of Medicine, 80 E Concord St, Boston, MA 02118-2394. E-mail jkeaney@acs.bu.edu.

	Abstract

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Background Epidemiological studies indicate that vitamin E (-tocopherol) exerts a beneficial effect on cardiovascular disease. The effect of vitamin E has generally been attributed to its antioxidant activity and the antioxidant protection of LDL. Distinct from its effect on LDL, vitamin E is also known to inhibit platelet aggregation and adhesion in vitro, but the mechanism(s) responsible for these observations are not known.

Methods and Results Using gel-filtered platelets derived from platelet-rich plasma treated with -tocopherol (500 µmol/L) or vehicle (0.5% ethanol), we found that inhibition of platelet aggregation by -tocopherol was closely linked to its incorporation into platelets (r=-.78; P<.02). Platelet incorporation of -tocopherol was associated with a significant reduction in platelet sensitivity to aggregation by adenosine 5'-diphosphate, arachidonic acid, and phorbol ester (PMA) by approximately 0.15-, 2-, and 100-fold, respectively. In contrast, platelets treated similarly with butylated hydroxytoluene, another potent lipid-soluble antioxidant, did not demonstrate any change in sensitivity to these agents. Platelet incorporation of -tocopherol inhibited PMA-induced stimulation of platelet protein kinase C (PKC) as determined by phosphorylation of the 47-kD PKC substrate. In 15 normal subjects, oral supplementation with -tocopherol (400 to 1200 IU/d) resulted in an increase in platelet -tocopherol content that correlated with marked inhibition of PMA-mediated platelet aggregation (r=.67; P<.01). Platelets derived from these subjects after supplementation also demonstrated apparent complete inhibition of PKC stimulation by PMA.

Conclusions These data indicate that platelet incorporation of -tocopherol at levels attained with oral supplementation is associated with inhibition of platelet aggregation through a PKC-dependent mechanism. These observations may represent one potential mechanism for the observed beneficial effect of -tocopherol in preventing the development of coronary artery disease.

Key Words: vitamins • antioxidants • platelets • platelet aggregation inhibitors • thrombosis • coronary disease

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Recent evidence suggests that dietary vitamin E may limit the clinical expression of coronary artery disease. Prospective cohort studies have shown that dietary vitamin E consumption is inversely associated with the clinical manifestations of coronary artery disease in both men¹ and women.² In patients with established coronary artery disease, vitamin E supplementation reduces the incidence of nonfatal myocardial infarction.³

-Tocopherol, the principle form of vitamin E in human plasma and LDL,⁴ inhibits the oxidation of LDL,⁵ a process that has been implicated in the development of atherosclerosis.⁶ Consequently, there has been considerable speculation that -tocopherol is antiatherogenic by virtue of its ability to inhibit LDL oxidation,^{1 2} although more recent studies do not support this contention.^{7 8} In addition to the antioxidant protection of LDL, -tocopherol has other properties that may indirectly contribute to its beneficial effects on coronary artery disease. For example, in animal models of atherosclerosis, -tocopherol prevents the development of endothelial dysfunction.^{9 10} Vascular cell incorporation of -tocopherol is also associated with enhanced resistance to oxidant-mediated injury.¹¹

In addition to its effects on vascular cells, -tocopherol is known to inhibit platelet aggregation in vitro,^{12 13 14} an effect that was initially attributed to the inhibition of lipid peroxidation.¹² However, observations that the oxidized form of -tocopherol (which is devoid of antioxidant activity) also inhibits platelet aggregation¹⁵ cast doubt on this hypothesis. In these early studies, inhibition of platelet aggregation by -tocopherol required high concentrations (1 mmol/L) and platelets derived from -tocopherol–supplemented individuals aggregated normally, suggesting that the effect of -tocopherol was not physiologically relevant.¹⁶ Subsequent studies indicated that -tocopherol inhibits platelet adhesion at physiological blood levels,¹⁷ although the mechanism(s) responsible for this effect is unknown. The effect of -tocopherol on platelet function has been highlighted recently by one clinical study demonstrating an increase in hemorrhagic strokes with daily vitamin E supplementation.¹⁸ The purpose of this study was to examine the mechanism(s) responsible for the inhibition of platelet aggregation by physiologically relevant concentrations of -tocopherol.

	Methods

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Materials
Leupeptin, phenylmethylsufonyl fluoride (PMSF), BHT, 2-mercaptoethanol, Triton X-100, apyrase, arachidonic acid, PMA, and ADP were purchased from Sigma Chemical Co. [³²P]orthophosphate was purchased from New England Nuclear. Sepharose 2B was obtained from Pharmacia Fine Chemicals. HEPES buffer solution (HBS), pH 7.35, consisted of 5.8 mmol/L sodium HEPES, 140 mmol/L NaCl, 6.1 mmol/L KCl, 2.5 mmol/L MgSO₄, 2.4 mmol/L Na₂SO₄, 59 µmol/L bovine serum albumin, and 5.6 mmol/L dextrose. -Tocopherol (R,R,R form) (a gift from Henkel Corp, Kankakee, Ill) was dissolved in 95% ethanol and the concentration confirmed by spectrophotometric measurement with the use of an extinction coefficient of 3270 mol·L^-1·cm^-1.¹⁹

Preparation of Platelet-Rich Plasma and Gel-Filtered Platelets
Venous blood was obtained from healthy subjects not receiving vitamin supplements who had not consumed platelet inhibitors for at least 7 days. All studies were performed with informed consent and in accordance with the policies of the Institutional Review Board at Boston University Medical Center. PRP, PPP, and GFP were prepared as previously described.²⁰ Platelet counts were determined with the use of a Coulter Counter (model ZM, Coulter Electronics). In some experiments, PRP was incubated with -tocopherol, BHT, or vehicle control (all dissolved in 95% ethanol and added as less than 1% [vol/vol]) and subsequently gel-filtered to separate the platelets from plasma and free -tocopherol or BHT. Adequate separation of free -tocopherol from platelets during gel filtration was confirmed by high-performance liquid chromatography (HPLC).

Plasma and Platelet -Tocopherol Content
Plasma and platelet -tocopherol content were determined by the use of HPLC with electrochemical detection as previously described.²¹ Calibration of the HPLC system was performed daily with fresh solutions of R,R,R-–tocopherol in ethanol.

Platelet Aggregation
Aggregation was induced in 0.4 mL PRP or GFP by the addition of freshly prepared arachidonic acid, PMA, or ADP. These studies were performed at 37°C at a constant stirring rate of 1200 rpm in a BioData four-chamber aggregometer, as previously described.²⁰

Preparation of Soluble and Particulate Fractions From Platelets
PRP was incubated in the presence or absence of 500 µmol/L -tocopherol for 30 minutes at room temperature and pelleted by centrifugation (800g for 15 minutes) at 25°C. Pellets were resuspended in lysis buffer as previously described.²² The solution was centrifuged at 100 000g for 60 minutes in a Beckman L-80 ultracentrifuge and the soluble fraction separated from the pellet (particulate fraction). The particulate fraction was resuspended in lysis buffer containing 0.1% Triton X-100 and homogenized by sonication at 4°C. Particulate and soluble fractions were extracted and analyzed by HPLC for -tocopherol content as described above.

Phosphorylation of Platelet Proteins
PRP was incubated with 500 µmol/L -tocopherol or an equal volume of vehicle control at room temperature for 30 minutes followed by centrifugation at 800g for 10 minutes to separate platelets from plasma proteins. The platelets were resuspended in phosphate-free HBS containing 2 units/mL apyrase and incubated for 60 minutes at room temperature with [³²P]orthophosphate (0.5 mCi/mL).²³ Free [³²P] was removed by gel filtration through Sepharose 2B. The samples (corresponding to 1x10⁸ platelets in 0.5 mL) were incubated with PMA (27 nmol/L) for 1 minute and stimulation terminated by the addition of an equal volume of 2x Laemmli sample buffer as described.²³ Samples were boiled in the presence of 10% (vol/vol) 2-mercaptoethanol and analyzed by electrophoresis on a 10% polyacrylamide gel and autoradiography. Patient platelet samples were processed exactly as above except for preincubation with -tocopherol or vehicle.

Statistics
All data are presented as mean±SEM. Paired samples were compared by a paired Student's t test. Dose-response curves were evaluated by repeated-measures ANOVA and, if significant, were further evaluated with a post hoc Newman-Keuls or Dunnett's comparison where appropriate. Correlations were evaluated with the Spearman rank order correlation. Statistical significance was accepted if the null hypothesis was rejected, with P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Plasma and Platelet -Tocopherol on Platelet Aggregation
We assessed the effect of added -tocopherol on platelet aggregation. As seen in a representative tracing (Fig 1), -tocopherol incubated with PRP led to a dose-dependent inhibition of platelet aggregation with an IC₅₀ of 450 µmol/L exogenous -tocopherol. To estimate the relative contribution of the plasma and platelet content of -tocopherol on inhibition of aggregation, PRP was incubated with 500 µmol/L -tocopherol or vehicle for 30 minutes. Each incubation was gel-filtered, producing two preparations each of PPP and GFP, one treated with -tocopherol and the other treated with vehicle. Combinations of -tocopherol–treated and vehicle-treated PPP and GFP were mixed and aggregation rapidly induced to minimize equilibration of -tocopherol between PPP and GFP. Aggregation was inhibited by 70% when both plasma and platelets were treated with -tocopherol (Fig 2, P<.05 compared with vehicle-treated PPP and GFP). Most importantly, compared with vehicle-treated PPP and GFP, -tocopherol–treated GFP mixed with vehicle-treated PPP produced a 40% reduction in platelet aggregation (P<.05). These data suggest that inhibition of platelet aggregation by -tocopherol is a consequence of its presence in platelets as well as plasma. Unfortunately, in this type of experiment it is difficult to control for redistribution of -tocopherol between plasma and platelets. Therefore, subsequent experiments were performed using gel-filtered platelets.

View larger version (21K): [in this window] [in a new window]   Figure 1. Representative tracing of -tocopherol–mediated inhibition of in vitro platelet aggregation. PRP was incubated with 0, 0.1, 0.5, or 1.0 mmol/L R,R,R–-tocopherol for 5 minutes and aggregation induced with 50 µmol/L arachidonic acid. Data are representative of 5 independent experiments.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effect of plasma and/or platelet -tocopherol content on the extent of platelet aggregation. PRP was incubated with -tocopherol (500 µmol/L) or vehicle for 30 minutes and PPP and GFP prepared from each incubation as described in "Methods." Preparations of PPP and GFP from control or -tocopherol–treated plasma were combined and aggregation induced with 50 µmol/L arachidonic acid. Data are expressed as mean±SEM and are derived from 3 experiments. *P<.05 vs untreated group.

Incorporation of -Tocopherol Into Platelets Correlates With Inhibition of Platelet Aggregation
To define the role of platelet -tocopherol in tocopherol-mediated inhibition of platelet aggregation, we characterized -tocopherol incorporation into platelets. PRP was incubated with -tocopherol over a range of times and concentrations, gel-filtered, and the platelet -tocopherol content determined. PRP incubated with -tocopherol (500 µmol/L) demonstrated a time-dependent increase in platelet -tocopherol content (Fig 3A; P<.01 by ANOVA) with essentially complete incorporation of -tocopherol within 30 minutes. The concentration-dependent incorporation of -tocopherol into platelets is shown in Fig 3B (P<.01 for effect of -tocopherol concentration by ANOVA).

View larger version (39K): [in this window] [in a new window]   Figure 3. Dose- and time-dependent incorporation of -tocopherol into platelets and its effect on aggregation. A, PRP was incubated with 500 µmol/L -tocopherol for the indicated times followed by gel filtration and determination of platelet -tocopherol content () or aggregation induced by 50 µmol/L arachidonic acid (). B, PRP was incubated with the indicated concentration of -tocopherol for 30 minutes followed by gel filtration and determination of -tocopherol content () or aggregation () as above. Data are mean±SEM and are derived from 3 experiments. *P<.05 vs time 0 or vehicle.

Using the same platelet preparations, we examined platelet aggregation in response to 50 µmol/L arachidonic acid. We found a striking parallel between the degree of -tocopherol incorporation into platelets and the extent of platelet inhibition (Fig 3, A and B). The platelet content of -tocopherol was inversely correlated with the extent of platelet aggregation in response to 50 µmol/L arachidonic acid (r=-.78; P<.02). Furthermore, we found that platelet incorporation of -tocopherol was strictly required for inhibition of platelet aggregation. In contrast to our observations with PRP (Fig 3, A and B), the treatment of platelets after gel filtration with -tocopherol (dissolved in 95% ethanol; <1% [vol/vol]) produced no platelet incorporation of -tocopherol and no inhibition of platelet aggregation in response to 50 µmol/L arachidonic acid (Fig 4).

View larger version (14K): [in this window] [in a new window]   Figure 4. -Tocopherol–mediated platelet inhibition requires platelet -tocopherol incorporation. Original tracings demonstrating arachidonic acid–induced platelet aggregation in GFP incubated directly with (A) 500 µmol/L -tocopherol or GFP derived from (B) vehicle-treated or (C) -tocopherol–treated (500 µmol/L) PRP. The platelet -tocopherol content is indicated at the right of each tracing. Data are representative of 3 independent experiments.

To investigate the subcellular site of -tocopherol incorporation into platelets, PRP was incubated in the presence of 500 µmol/L -tocopherol for 30 minutes, gel-filtered, the particulate and soluble fractions separated, and the -tocopherol content determined. The -tocopherol content of the platelet particulate fraction increased significantly from 11.1±3.3 to 26.4±8.1 pmol/10⁸ platelets with treatment (P<.05 compared with untreated particulate fraction). No significant difference was found between the -tocopherol–treated and untreated cytosolic fractions (1.5±0.3 versus 0.6±0.3 pmol/10⁸ platelets; P=NS).

Inhibition of Platelet Aggregation by -Tocopherol Is Agonist-Dependent
To determine the mechanism by which -tocopherol inhibits platelet function, aggregations were conducted using agonists that activate platelets by different mechanisms. PRP was incubated with 500 µmol/L -tocopherol or BHT (an antioxidant with potency comparable to -tocopherol²⁴ ), and platelets were gel-filtered and activated with increasing doses of ADP (0.15 to 6.1 µmol/L), arachidonic acid (10 to 100 µmol/L), or PMA (1.6 to 800 nmol/L). Treatment of PRP with BHT before gel filtration produced no significant inhibition of platelet aggregation as determined by the EC₅₀ for the dose response to ADP, arachidonic acid, and PMA (Table). Moreover, we were not able to detect incorporation of BHT into platelets using HPLC with electrochemical or UV detection. In contrast, GFP derived from -tocopherol–treated PRP demonstrated significant inhibition of aggregation induced by ADP, arachidonic acid, and PMA. The shift in the dose response to arachidonic acid and PMA, two agonists that are dependent on PKC stimulation, were much more marked than for ADP, which does not require PKC stimulation at the doses used in this assay.²⁵

View this table: [in this window] [in a new window]   Table 1. Agonist-Dependent Platelet Inhibition by -Tocopherol

Effect of -Tocopherol on Platelet Protein Kinase C Activity
Since -tocopherol is particularly effective at inhibiting platelet aggregation to PKC-dependent agonists (Table) and -tocopherol has been shown to inhibit PKC in smooth muscle cells,²⁶ we studied the effect of -tocopherol on platelet PKC activity. Stimulation of platelets with PMA induces the phosphorylation of a 47-kD platelet protein, which is a well-known substrate of PKC.²² PRP was incubated with 500 µmol/L -tocopherol or vehicle, loaded with [³²P]orthophosphate, and gel-filtered. The resulting GFP were subjected to PKC stimulation with 27 nmol/L PMA. As shown in Fig 5, vehicle-treated platelets demonstrated the expected increase in phosphorylation of the 47-kD protein, but in -tocopherol–treated platelets, phosphorylation was prevented. Quantitative densitometry revealed a 3.8-fold increase in PMA-induced phosphorylation in vehicle-treated platelets, while no detectable increase was found in the tocopherol-treated platelets. Thus, platelet incorporation of -tocopherol is associated with inhibition of PKC stimulation.

View larger version (47K): [in this window] [in a new window]   Figure 5. Effect of -tocopherol on platelet PKC-dependent protein phosphorylation. PRP was incubated with 32[P]orthophosphate in the presence of -tocopherol or vehicle, gel-filtered, stimulated with 27 nmol/L PMA for 1 minute, and the platelet proteins electrophoresed by SDS-PAGE. Lanes 1 and 2 demonstrate the phosphorylation of the 47-kD PKC substrate in control platelets before and after stimulation with PMA, respectively. Lanes 3 and 4 demonstrate phosphorylation of the 47-kD protein in platelets derived from -tocopherol–treated plasma before and after stimulation with PMA, respectively. Data are representative of 3 independent experiments.

Effect of In Vivo -Tocopherol Supplementation on Platelet Aggregation and Platelet Protein Kinase C Activity
To determine if the above-described in vitro findings with -tocopherol are relevant in vivo, 15 normal volunteers were given -tocopherol supplements (400, 800, or 1200 IU/d) for 14 days and blood was obtained at baseline and after completion of the regimen. GFP were prepared, and -tocopherol content was determined and examined for extent of aggregation induced by ADP (0.15 to 6.1 µmol/L), arachidonic acid (10 to 100 µmol/L), or PMA (1.6 to 1600 µmol/L). There was no change in the dose response to ADP with any dose of -tocopherol; the ratio of the EC₅₀ after -tocopherol supplementation to that obtained before treatment was 1.8±0.4, 1.8±0.5, and 1.7±0.2, with 400, 800, and 1200 IU/d, respectively (P=NS; Fig 6A). In contrast, -tocopherol administration at all doses significantly inhibited platelet aggregation in response to the PKC-dependent agonist PMA (ratio of EC₅₀ postsupplementation to presupplementation of 71±44, 79±47, and 64±50 for 400, 800, and 1200 IU/d -tocopherol, respectively; Fig 6A; P<.009). With respect to arachidonic acid, platelet aggregation was inhibited only with 1200 IU/d -tocopherol (ratio of EC₅₀ postsupplementation to presupplementation of 3.5±0.8; P<.01; Fig 6A). Before supplementation, platelet -tocopherol in all subjects was 38.9±7.0 pmol/10⁸ platelets and increased to 81.2±21.6, 96.0±32.9, and 160.5±70.5 pmol/10⁸ platelets after supplementation with 400, 800, and 1200 IU/d, respectively (Fig 6B; P.05 for dose effect of -tocopherol by ANOVA). Platelet -tocopherol content was correlated with inhibition of platelet aggregation in response to PMA both before supplementation (r=.59; P<.02) and after supplementation with -tocopherol (r=.67; P<.01).

View larger version (40K): [in this window] [in a new window]   Figure 6. The effect of in vivo -tocopherol supplementation on platelet aggregation (A) and platelet -tocopherol levels (B). Individuals consumed 400, 800, or 1200 IU of -tocopherol for 14 days and PRP obtained before and after supplementation was gel-filtered and aggregation induced by ADP (0.15 to 6.1 µmol/L), arachidonic acid (10 to 100 µmol/L), or PMA (1.6 to 1600 µmol/L). A, The effect of -tocopherol on platelet aggregation was reported as the fold change in concentration of agonist required to produce a 50% extent of aggregation (EC50). B, Platelets were extracted with methanol/hexane and the platelet -tocopherol content determined. Data are mean±SEM and are derived from 5 individuals in each supplementation group. *P<.05 vs presupplementation by ANOVA.

To determine the effect of oral -tocopherol supplementation on platelet PKC stimulation, phosphorylation of the 47-kD platelet PKC substrate was studied in GFP prepared from two volunteers who consumed 1200 IU/d -tocopherol. After [³²P]orthophosphate incubation and gel filtration, the platelet samples were stimulated with 27 nmol/L PMA. Normal phosphorylation of the 47-kD protein was observed in platelets before supplementation; however, PMA failed to induce phosphorylation of the 47-kD protein in the platelets obtained after -tocopherol supplementation (Fig 7). Quantitative densitometry revealed a 2.5-fold increase in PMA-induced phosphorylation before supplementation, whereas no change was found after supplementation.

View larger version (55K): [in this window] [in a new window]   Figure 7. Effect of oral -tocopherol supplementation on platelet PKC-dependent protein phosphorylation. PRP from a normal subject was incubated with 32[P]orthophosphate, gel-filtered, and stimulated with vehicle (lane 1) or 27 nmol/L PMA (lane 2) followed by SDS-PAGE and autoradiography. Lanes 3 and 4 represent vehicle and PMA stimulation of similarly treated platelets from the same subject after 14 days of oral supplementation with -tocopherol (1200 IU/d). In this individual, platelet -tocopherol content increased from 49.2 to 124.5 pmol/108 platelets. Data are representative of two independent experiments on two subjects.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The data presented here demonstrate for the first time that -tocopherol attenuates platelet aggregation by inhibiting PKC stimulation and that these effects occur with in vivo supplementation. Using PKC-dependent and -independent platelet agonists, we found that -tocopherol was most effective in inhibiting PKC-dependent platelet aggregation. In fact, platelet incorporation of -tocopherol both in vitro and in vivo was associated with apparently complete inhibition of platelet PKC stimulation as assessed by the phosphorylation of a 47-kD PKC substrate. Thus, these data indicate that -tocopherol inhibits platelet aggregation at physiologically relevant concentrations by inhibiting platelet PKC stimulation.

Inhibition of platelet aggregation by -tocopherol has been demonstrated previously. Higashi and Kikuchi²⁷ were the first to demonstrate that -tocopherol inhibited platelet aggregation using hydrogen peroxide as a platelet agonist. Subsequently, Steiner and Anastasi¹² demonstrated dose-dependent inhibition of platelet aggregation and 5-hydroxytryptamine release by -tocopherol in response to ADP, epinephrine, and collagen.

A major problem with these studies has been the supraphysiological concentrations of -tocopherol required to produce meaningful platelet inhibition (0.3 to 2.0 mmol/L).^{12 27} In the present study, we found that -tocopherol produced dose-dependent inhibition of arachidonic acid–mediated platelet aggregation with an IC₅₀ of 450 µmol/L (Fig 1), a value that greatly exceeds the range for plasma -tocopherol content in normal subjects (15 to 40 µmol/L⁴ ) or subjects taking supplemental -tocopherol (30 to 120 µmol/L^{28 29} ). However, our data suggest that these supraphysiological concentrations are required in in vitro experiments to effectively load the platelets with -tocopherol. In PRP and GFP, we found no significant inhibition of platelet aggregation with -tocopherol concentrations <500 µmol/L (Figs 1 and 3), the threshold concentration for significant incorporation of additional -tocopherol into platelets (Fig 3B). Most importantly, the platelet -tocopherol levels achieved with in vitro loading (117.6±15.3 pmol/10⁸ platelets) were comparable to the levels measured after in vivo -tocopherol supplementation (160.5±70.5 pmol/10⁸ platelets). This finding suggests that in vitro loading of platelets using supraphysiological levels of -tocopherol is a reasonable means of increasing platelet -tocopherol to levels that occur in vivo with supplementation. The increased efficiency of -tocopherol incorporation into platelets in vivo may reflect intestinal- or hepatic-dependent metabolism of -tocopherol.³⁰

Our work contains several lines of evidence that platelet incorporation of -tocopherol is critical for inhibition of platelet aggregation. First, in PRP we found that -tocopherol–treated platelets are inhibited even when removed from -tocopherol–treated plasma (Fig 2). Second, and perhaps most important, the dose- and time-dependent increases in platelet -tocopherol levels coincide with the dose and time dependency of platelet inhibition by -tocopherol (Fig 3). In fact, platelet -tocopherol content and arachidonic acid–mediated platelet aggregation were inversely correlated (r=-.78; P<.05). Finally, treatment of platelets after gel filtration with -tocopherol does not result in platelet incorporation of -tocopherol and, as a consequence, does not produce inhibition of platelet aggregation (Fig 4). The precise mechanism(s) responsible for platelet incorporation of -tocopherol is not known. It is possible that certain plasma components, such as lipoproteins or tocopherol binding proteins, are required for the incorporation of -tocopherol into platelets. In fact, Kostner and colleagues³¹ have recently described -tocopherol exchange/transfer activity in human plasma that is due to a human plasma phospholipid transfer protein.

Since the discovery that -tocopherol inhibits the aggregation of platelets, there has been considerable effort directed at elucidating the mechanism responsible for this effect. Platelet aggregation involves the consumption of oxygen,³² the production of reactive oxygen species, and the formation of lipid hydroperoxides.³³ Since treatment of platelets with -tocopherol inhibits the formation of lipid hydroperoxides during aggregation, and since hydroperoxide formation has been linked to cyclooxygenase function,³⁴ Steiner and Anastasi¹² speculated that -tocopherol inhibits platelet aggregation by preventing lipid peroxidation. Subsequent efforts at demonstrating an effect of -tocopherol on the activity of cyclooxygenase have produced conflicting results.^{35 36 37 38 39} In fact, there are data to suggest that the antioxidant activity of -tocopherol is not required for the inhibition of platelet aggregation. Tocopheryl quinone, the oxidized form of -tocopherol that is devoid of antioxidant activity, also inhibits platelet aggregation with a potency approximately equal to -tocopherol.^{15 40}

While some component of platelet inhibition by -tocopherol may be a consequence of its antioxidant activity, the data presented here also suggest an additional mechanism of -tocopherol–mediated platelet inhibition. We found -tocopherol particularly effective in inhibiting platelet aggregation in response to arachidonic acid and PMA, two agonists that are dependent on PKC stimulation (Table). In contrast, -tocopherol was less effective against ADP-induced aggregation using ADP concentrations that do not strongly stimulate PKC²⁵ (Table). This trend was particularly striking when platelets were loaded with -tocopherol in vivo via oral supplementation (Fig 6). This indirect evidence for PKC inhibition by -tocopherol was directly confirmed by platelet protein phosphorylation studies demonstrating reduced phosphorylation of the 47-kD PKC substrate in PMA-stimulated platelets loaded with -tocopherol both in vitro and in vivo (Figs 5 and 7).

Inhibition of PKC stimulation provides an attractive mechanism for the platelet inhibitory effects of -tocopherol, particularly in reference to previous studies that failed to demonstrate inhibition of platelet aggregation with in vivo -tocopherol supplementation.^{12 16} These studies used ADP (2.5 µmol/L), epinephrine (25 µmol/L), and collagen (118 µg/mL) as stimuli for aggregation, and at these concentrations, these agents are not particularly dependent on PKC stimulation to induce platelet aggregation. As such, on the basis of the data presented here, we would predict that the full antiaggregatory effects of -tocopherol would not be realized. Using a similar regimen of -tocopherol supplementation, we observed significant inhibition of platelet aggregation only with the PKC-dependent agonists arachidonic acid and PMA, but not with ADP (Fig 6A). Thus, our data are entirely consistent with previous observations.

In contrast to the aforementioned results with ex vivo platelet aggregation, oral -tocopherol supplementation with as little as 200 IU/d has been shown to significantly inhibit shear stress–induced platelet adhesion to protein-coated glass slides.¹⁷ Recently, Kroll and colleagues⁴¹ have found that shear stress–induced platelet aggregation is associated with stimulation of platelet PKC. Increased blood vessel wall shear stress stimulates von Willebrand factor–mediated platelet adhesion as well as aggregation.⁴² In light of these observations, it is attractive to speculate that the effectiveness of -tocopherol in preventing shear stress–induced platelet adhesion¹⁷ is also a consequence of its ability to inhibit PKC stimulation.

The precise mechanism through which -tocopherol inhibits PKC stimulation is not known. -Tocopherol has been shown to inhibit PKC activity in isolated enzyme preparations and cultured cells.²⁶ Boscoboinik and colleagues²⁶ demonstrated attenuation of cellular proliferation by -tocopherol in association with PKC inhibition in vascular smooth muscle cells. In a related study, inhibition of PKC activity was associated with an increase in phorbol ester binding and a decrease in PKC translocation to the membrane.⁴³ Recent observations that staurosporine, a microbial alkaloid and potent PKC inhibitor, induces similar effects in human platelets²² prompts speculation that these two compounds may inhibit PKC by similar mechanisms.

Summary
The data presented here demonstrate for the first time that -tocopherol attenuates human platelet aggregation through its incorporation into the platelet and inhibition of platelet PKC stimulation. Moreover, we have shown that -tocopherol inhibits aggregation at physiologically relevant platelet levels and that its effects on PKC are also relevant to in vivo -tocopherol supplementation. These properties of -tocopherol could partially explain its beneficial effect on coronary artery disease as well as its association with an increase in cerebral hemorrhagic risk. In addition, these data represent another potentially beneficial effect of -tocopherol that is not related to the antioxidant protection of LDL.

	Selected Abbreviations and Acronyms

 

ADP = adenosine 5'-diphosphate BHT = butylated hydroxytoluene EC50 = effective concentration required for a 50% maximal response GFP = gel-filtered platelets IC50 = inhibitory concentration required for 50% inhibition PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate PPP = platelet-poor plasma PRP = platelet-rich plasma SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis

	Acknowledgments

 
Dr Freedman is the recipient of a Fellowship Award from the American Heart Association, Massachusetts Affiliate, and Dr Keaney is the recipient of a Clinical Investigator Development Award (HL-03195) from the National Institutes of Health. Dr Loscalzo is the recipient of a Research Career Development Award (HL-02273) from the NIH. This work was supported in part by NIH grants HL-53919, HL-48743, and HL-55993, by a Merit Review Award from the Veterans Administration, and by grants from the AHA, Massachusetts Affiliate, Henkel Corporation, Fine Chemicals Division, and The Council for Tobacco Research. The authors wish to thank Stephanie Tribuna for her expert secretarial assistance.

Received April 9, 1996; revision received May 28, 1996; accepted June 11, 1996.

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