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-Tocopherol and protein kinase C inhibition enhance platelet-derived nitric oxide release¹

Departments of Pharmacology and Medicine, Georgetown University Medical Center, Washington, D.C. 20007, USA; and the
^* Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA

²Correspondence: Medicine-Dentistry Bldg., Rm. NE 403, Georgetown University Medical Center, 3900 Reservoir Road, N.W., Washington, DC 20007. E-mail: Freedmaj{at}gunet.georgetown.edu

SPECIFIC AIMS

Platelet activation is tightly regulated by products of the endothelium and platelets, including nitric oxide (NO). Although antioxidant status has been shown to alter endothelium-derived NO bioactivity and physiological levels of -tocopherol are known to inhibit platelet function, the effect of -tocopherol on platelet NO release is unknown.

PRINCIPAL FINDINGS

1. -Tocopherol inhibits platelet aggregation and increases release of platelet-derived NO
Platelets acutely loaded with -tocopherol (0–1 mM) demonstrated a dose-dependent increase in NO release in response to 5 µM ADP. Vehicle-treated platelet NO release was 1.3 ± 0.1 pmol/10⁸ platelets and increased approximately threefold to a maximum of 3.2 ± 0.1 pmol/10⁸ platelets. This change in platelet NO release was associated with a reduction in the extent of ADP-induced aggregation from 97 ± 8% to 28 ± 4% (P<0.05). To determine the physiologic relevance of these findings, we examined platelet NO release before and after five normal subjects were treated with 400 IU vitamin E daily for 14 days. This regimen increased plasma -tocopherol from 14.0 ± 3.0 µM to 20.4 ± 4.1 µM (P<0.05) and platelet -tocopherol from 42.2 ± 6.1 pmol/10⁸ platelet to 74.4 ± 18.9 pmol/10⁸ platelet, respectively, and was associated with a 50% increase in platelet NO release from 2.2 ± 0.2 pmol/10⁸ platelets to 3.3 ± 0.3 pmol/10⁸ platelets (P<0.05). Thus, -tocopherol increases aggregation-induced platelet NO release both in vitro and in vivo.

2. -Tocopherol attenuates platelet-derived superoxide release
Aggregating platelets produce superoxide, which is known to react readily with NO and reduce its bioactivity. To determine the role of superoxide in the effect of -tocopherol on platelet-derived NO, we acutely loaded platelets with -tocopherol (0–1 mM) and examined platelet-derived superoxide. The ADP-induced chemiluminescence signal from vehicle-treated platelets was 98.3 ± 35.4 arbitrary units (AU)/10⁸ platelets, and fell in a dose-dependent fashion by 84% to 16.3 ± 9.8 AU/10⁸ platelets in platelets treated with 1 mM -tocopherol for 30 min [P<0.05 vs. vehicle by one-way analysis of variance (ANOVA) with Student Newman-Keuls comparison]. In the presence of 300 IU/ml SOD, platelet superoxide release was reduced to 3.1± 1.7 (P<0.05).

Since -tocopherol inhibits platelet aggregation and platelet superoxide production is aggregation dependent, the effect of -tocopherol may be related to its inhibition of aggregation. To examine this possibility, we developed a dose-response relation between ADP-induced (0–2 µM) aggregation and platelet-derived superoxide in the presence and absence of -tocopherol (1 mM for 30 min). Platelets loaded with -tocopherol demonstrated less superoxide release independent of the level of aggregation. Thus, -tocopherol intrinsically reduces platelet-derived superoxide in response to ADP-induced aggregation.

To determine if superoxide scavenging by -tocopherol accounts for its effects on NO bioactivity, we examined aggregation-induced platelet superoxide production in the presence of -tocopherol (0–1 mM) added only at the time of the assay. We have previously demonstrated that GFP incubated with ethanolic -tocopherol do not incorporate -tocopherol. Under these conditions, we did not observe any effect of -tocopherol on ADP-induced, platelet-derived superoxide.

3. -Tocopherol influences aggregation-induced superoxide and NO independently
To determine whether the effect of -tocopherol on platelet-derived nitric oxide is a consequence of reduced superoxide, we examined the effect of -tocopherol in the presence and absence of superoxide dismutase (SOD; 300 IU/ml). In the presence of SOD, platelet-derived NO increased 72% from 1.1 ± 0.1 pmol/10⁸ platelets to 1.9 ± 0.2 pmol/10⁸ platelets (P<0.05, n=3). Despite the presence of SOD, we continued to observe a dose-dependent stimulatory effect of -tocopherol on platelet-derived NO (P<0.05 by ANOVA). To determine whether the effect of -tocopherol on platelet-derived superoxide is a consequence of increased NO production, we examined the effect of -tocopherol in the presence of 300 µM L-NAME, an inhibitor of NO synthase that blocks aggregation-induced platelet NO production by 92%. The presence of L-NAME alone produced a 20% increase in superoxide during ADP-induced aggregation. Even in the absence of NO synthesis, however, -tocopherol incorporation was associated with a significant dose-dependent reduction of aggregation-induced platelet-derived superoxide (P<0.05 by two-way ANOVA). These data indicate that -tocopherol influences aggregation-induced superoxide and NO independently.

4. -Tocopherol antioxidant activity is not necessary for its action on platelet-derived NO or superoxide
To determine whether the antioxidant activity of -tocopherol is required for its action on platelet NO and superoxide, we examined the effects of -tocopheryl acetate and -tocopheryl quinone, two forms of tocopherol devoid of antioxidant activity. Both -tocopheryl quinone and -tocopheryl acetate incubation with platelets was associated with an increase in platelet-derived NO (from 1.3±0.3 to 2.7±0.9 and 2.5±0.6 pmol/10⁸ platelets, respectively; P<0.05) and a decrease in platelet-derived superoxide (from 100±7.2 to 58±4.4 and 51±4.3 AU, respectively; P<0.05).

5. Implications of protein kinase C (PKC) inhibition for platelet-derived NO and superoxide release
We have previously demonstrated that -tocopherol inhibits platelet aggregation through a PKC-dependent mechanism and that PKC activity is an important determinant of both NO and superoxide production in vascular cells. Therefore, we examined the role of PKC in platelet-derived NO and superoxide. Chelerythrine (30 µM) produced both a 90% increase in platelet NO release from 1.1 ± 0.1 pmol/10⁸ platelets to 2.1 ± 0.2 pmol/10⁸ platelets (P<0.05 vs. control) and a 49% reduction in platelet-derived superoxide from 100 ± 15 AU/10⁸ platelets to 51 ± 7.1 AU/10⁸ platelets (P<0.05 vs. control) in response to aggregation. There was minimal additional effect of -tocopherol on NO release and a significant further decrease in superoxide production in the presence of chelerythrine (P=ns and P<0.05, respectively). Thus, the effect of tocopherol on platelet PKC stimulation is sufficient to explain its effect on NO release and may partially explain its effect on superoxide production.

6. -Tocopherol inhibits platelet endothelial nitric oxide synthase (NOS) phosphorylation
Phosphorylation of eNOS by PKC is associated with a reduction in catalytic activity in vascular endothelial cells. Therefore, we evaluated the effect of -tocopherol on PKC-dependent phosphorylation of platelet eNOS by immunoprecipitation. We found that PKC stimulation of platelets is associated with an increase in eNOS phosphorylation that was inhibited by tocopherol (Fig. 1 ).

View larger version (34K): [in this window] [in a new window]   Figure 1. -Tocopherol- and PKC-dependent phosphorylation of platelet eNOS. Platelets were incubated with 0.5 mM -tocopherol) or ethanol control for 30 min, gel-filtered, and incubated with 32P-orthophosphate. Platelets were then treated with either PMA or vehicle and lysed. Lysates were subjected to immunoprecipitation with eNOS antibody; precipitated protein was resolved on a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and evaluated by autoradiography (representative of 3 experiments).

7. -Tocopherol minimally alters platelet superoxide release in NOS3-deficient mice
Superoxide generation by eNOS has been reported under certain conditions. To determine the contribution of eNOS and -tocopherol to superoxide release in platelets, PRP from NOS3-deficient mice or control animals were incubated with 500 µM -tocopherol or vehicle control. After isolation, platelets were stimulated with PMA and superoxide was measured. As seen in Fig. 2 , platelets from NOS3-deficient mice have markedly decreased stimulation-dependent superoxide release as compared to wild-type animals. In addition, incubation with -tocopherol is associated with a nonsignificant decrease in superoxide release for both the knockout and wild-type mice (Fig. 2) .

View larger version (19K): [in this window] [in a new window]   Figure 2. -Tocopherol and superoxide release in NOS3-deficient mice. Platelet-rich plasma from NOS3-deficient or wild-type (C57BLl6) mice was incubated with 500 µM -tocopherol (white bars) or vehicle control (black bars) for 30 min, followed by gel filtration. Platelet superoxide release was measured in PMA-stimulated gel-filtered platelet (*P < 0.01, N=3).

CONCLUSIONS AND SIGNIFICANCE

In this study, we investigated the role of -tocopherol in platelet NO production. The principal finding in this study is that platelet tocopherol content is an important determinant of the balance between superoxide and NO production in aggregating platelets. We found that loading platelets with -tocopherol enhances platelet NO production and reduces superoxide release. Although supra-physiological concentrations of -tocopherol were required to alter NO release in vitro, comparable changes were also found after oral vitamin E supplementation. This is consistent with previous studies establishing that supra-physiological concentrations of -tocopherol in vitro are equivalent to levels achieved in vivo. These effects appear to have functional implications as they were associated with a dose-dependent reduction in platelet aggregation. We also found that the effect of -tocopherol on platelet NO production was not dependent on its effect on superoxide. The converse was also true, namely, that the action of -tocopherol to inhibit platelet superoxide production was not mediated by NO since it was also observed in mouse platelets lacking eNOS. Rather, we found the effect of -tocopherol was reproduced, in part, by inhibiting platelet protein kinase C stimulation with chelerythrine. This latter finding implies that -tocopherol acts through modulating phosphorylation events, an implication supported by our demonstration that aggregation-dependent eNOS phosphorylation is inhibited by -tocopherol. Thus, these findings suggest that physiological incorporation of tocopherol modulates the balance between NO and superoxide in human platelets.

The bioactivity of platelet-derived NO is intimately related to the production of superoxide. As demonstrated, platelet NO release is enhanced by the presence of superoxide dismutase, and one obvious explanation for the increase in NO in the presence of -tocopherol is the reduction in superoxide production. However, platelet NO release is enhanced by -tocopherol even in the presence of SOD at concentrations that eliminate superoxide. Thus, -tocopherol must also enhance NO release by some other mechanism than simply the limitation of superoxide. One possible mechanism is the inhibition of PKC stimulation, a known activity of -tocopherol in platelets. Protein kinase C directly phosphorylates eNOS and reduces its catalytic activity, suggesting that PKC inhibition in activated platelets should enhance NO release. In fact, platelets incubated with the PKC inhibitor chelerethyrine demonstrated a striking twofold increase in NO release and a concomitant decrease in superoxide production. This was associated with direct demonstration of eNOS phosphorylation in aggregating platelets that was inhibited by -tocopherol (Fig. 1) . These data indicate that protein kinase C stimulation plays an important role in regulating the balance between NO and superoxide production in aggregating platelets.FIGURE 3

View larger version (45K): [in this window] [in a new window]   Figure 3. No caption available.

The precise mechanism through which PKC inhibition modulates the balance between NO and superoxide in platelets is not known. Under conditions of limited cofactor availability, eNOS may generate superoxide. In the complete absence of eNOS (Fig. 2) , however, we observed a marked reduction in superoxide generation. Further incubation with -tocopherol only leads to an incremental decrease in platelet superoxide production. Taken together, these findings prompt speculation that the effects of -tocopherol in the stimulated platelet may be partially eNOS dependent. -Tocopherol may influence PKC-dependent phosphorylation of platelet eNOS, subsequently influencing the relative production of NO and superoxide. Further investigation will be required to determine if this is indeed the case.

In summary, our observations indicate that -tocopherol enhances platelet-derived NO release through a PKC-dependent mechanism that is not dependent on the antioxidant activity of -tocopherol. These data suggest that platelet PKC stimulation plays a pivotal role in the balance between platelet-derived superoxide and NO.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0360fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.00-0360fje

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