| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 1999 American Heart Association, Inc.
Thrombosis |
Vitamin E Inhibits Collagen-Induced Platelet Activation by Blunting Hydrogen Peroxide
Correspondence to Prof Pier Paolo Gazzaniga, Dipartimento di Medicina Sperimentale e Patologia, Università degli Studi di Roma "La Sapienza," Viale Regina Elena 324, 00161 Roma, Italy. E-mail gazzaniga{at}axrma.uniroma1.it
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—In this study, we investigated whether vitamin E at concentrations achievable in blood after supplementation inhibits platelet function in humans. Gel-filtered platelets were incubated 30 minutes with scalar concentrations (50 to 250 mmol/L) of vitamin E and then stimulated with collagen. Compared with controls, vitamin E inhibited collagen-induced platelet aggregation and thromboxane A2 formation in a dose-dependent manner. Furthermore, vitamin E inhibited, in a dose-dependent manner, Ca2+ mobilization and formation of inositol 1,4,5-triphosphate. Because it was previously shown that hydrogen peroxide formation mediates arachidonic acid metabolism and phospholipase C activation in collagen-induced platelet activation, we investigated whether vitamin E was able to blunt hydrogen peroxide. In experiments performed in unstimulated platelets supplemented with hydrogen peroxide and in collagen-stimulated platelets, vitamin E was able to blunt hydrogen peroxide. In 6 healthy subjects given vitamin E for 2 weeks (600 mg/d), we found a significant decrease of collagen-induced H2O2 formation, platelet aggregation, and calcium mobilization. This study demonstrated in vitro and ex vivo that vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide formation.
Key Words: antioxidants • platelet aggregation • arachidonic acid metabolism • phospholipase C enzyme • thromboxane A2
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Use of antioxidants to prevent coronary heart disease is based on the assumption that atherosclerosis progression is determined by oxidative modification of LDL, which is then taken up by macrophages of atherosclerotic plaque.1 Among antioxidants, vitamin E has been widely investigated to assess its clinical effectiveness. In healthy subjects, dietary vitamin E consumption has been associated with fewer cardiovascular events.2 3 In patients with angiographically proven coronary heart disease, 400 to 800 mg/d vitamin E significantly reduced the rate of nonfatal myocardial infarction over 2 years of follow-up.4 These data have not been confirmed by other studies, such as that of Rapola et al5 ; however, in that study, daily administration of vitamin E was only 50 mg. Despite these contradictory results, additional studies have been conducted to assess whether vitamin E has biological properties that could prevent progression of atherosclerosis.
Several in vitro and in vivo studies have analyzed whether vitamin E inhibits platelet function. Recent studies demonstrated that vitamin E supplementation in healthy subjects or patients with hypercholesterolemia diminishes platelet function, as assessed by ex vivo platelet aggregation and urinary excretion of 11-dehydro-thromboxane B2, a marker of in vivo platelet activation.6 7 Inhibition of platelet function was dependent on the daily dose of vitamin E, indicating that plasma and platelet vitamin E concentrations are crucial for the inhibition of platelet function. These data seem to contradict those of in vitro studies, in which higher concentrations of vitamin E (0.5 to 1 mmol/L) than observed in plasma after supplementation were necessary to inhibit human platelet activity.8 9 To analyze this discrepancy, we performed an in vitro study using collagen as the platelet agonist. Collagen is an important platelet agonist thought to be involved in the early stages of platelet activation during both hemostasis and thrombosis. We recently demonstrated that collagen-induced platelet aggregation is associated with a burst of hydrogen peroxide, an oxidant species that contributes to activation of platelets.10 Because vitamin E is an antioxidant, we speculated that collagen-induced platelet aggregation may represent an interesting model to further analyze the relationship between vitamin E and platelet function. We found that a concentration of vitamin E very close to that found in human plasma after supplementation is able to inhibit collagen-induced platelet activation by blunting hydrogen peroxide formation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
32Pi was from Amersham. Fura 2-AM and 2'7'-dichlorofluorescein diacetate (DCFH-DA) were from Molecular Probes, and Sepharose 2B was from Pharmacia. Collagen, type 1, was from Semmelweis. High-performance liquid chromatography (HPLC) columns (Partisil 10 SAX) were from Whatman. BSA, HEPES, acetylsalicylic acid,
-tocopherol (vitamin E), fibrinogen, inorganic
pyrophosphatase, acid/citrate/dextrose, digitonin, EGTA, EDTA, Tris,
perchloric acid, formaldehyde, indomethacin, ammonium
formate, maleic acid, creatine phosphate, and creatine phosphokinase
were from Sigma Chemical Co.
Platelet Preparation
Human blood was obtained from drug-free healthy volunteers and
anticoagulated with acid/citrate/dextrose.11
Platelet-rich plasma, obtained by centrifugation
(15 minutes at 180g), was recentrifuged (20 minutes
at 800g) to concentrate the platelets, and the pellet
was resuspended in a 0.5 vol of autologous platelet-poor plasma.
Platelet suspensions were incubated for 1 hour at 37°C with Fura
2-AM (3 mmol/L of cell suspension), DCFH-DA (40 mmol/L), or
32Pi (2 mCi/mL).
Platelets were separated from plasma proteins and from excess Fura 2-AM and 32Pi by gel filtration on Sepharose 2B using Ca2+-free Tyrode's buffer containing 0.2% BSA, 5 mmol/L glucose, and 10 mmol/L HEPES, pH 7.35. After gel filtration, the cell suspension (gel-filtered platelets [GFPs]) was adjusted to a final concentration of 2x108 cells/mL.
Because the addition of methanol to GFP suspensions in concentrations lower than 0.5% did not induce any change in GFP response to collagen in preliminary experiments, this ratio was always used to obtain final concentrations of vitamin E of 50 to 250 mmol/L. Vitamin E was added to GFP suspensions under continuous stirring for 30 minutes at 37°C before GFP stimulation with collagen (2 to 12 µg/mL). According to results of our previous study,10 the collagen concentrations that gave reproducible values varied on the basis of the test used, ie, a higher concentration of collagen was necessary to induce inositol 1,4,5-triphosphate (IP3) formation compared with platelet aggregation tests. To serve as controls, GFPs were incubated with the dilution medium in all experiments.
Flow Cytometric Analysis
Platelet-rich plasma was recentrifuged
(800g for 20 minutes) to concentrate the platelets, and
the pellet was resuspended in PBS. DCFH-DA (8 mL/mL; 5-mmol/L
concentration) was added, and after 15 minutes of incubation, the
platelet suspension was washed twice and resuspended in Tyrode's
buffer at a final concentration of 2x108
cells/mL. The platelet preparation was activated with
collagen, added with or without vitamin E, and the reaction was stopped
after 1 minute with EGTA (2 mmol/L).
All samples were analyzed on a Coulter Epics flow cytometer equipped with an argon laser (480 nm emission). The instrument was set up to measure logarithmic forward light scatter, a measure of particle size; logarithmic 90° light scatter, a measure of cell granularity; and green (dichlorofluorescein [DCF]) fluorescence (at 510 to 550 nm). Fluorescent parameters (expressed in arbitrary units [au]) were collected using 3-decade logarithmic amplification.
Platelet Aggregation
In vitro platelet aggregation was evaluated according to the
method of Born12 in a 4-sample Aggrecorder II
(Menarini) at 37°C using siliconized glass cuvets under continuous
stirring at 1000 rpm. Fibrinogen (1 mg/mL) was added before the
agonist.
Production of Thromboxane A2
Platelet activation (see above) was stopped after 3 minutes
with indomethacin (14 mmol/L). Thromboxane
A2 production was determined by using thromboxane
B2 ELISA kits (Boehringer Mannheim GmbH).
Platelet Cytosolic Ca2+ Concentrations
Calcium concentrations were measured by using the
fluorescent indicator dye Fura-2. Changes in
fluorescence were monitored with a SFM 25 fluorometer (Kontron)
set at 340 nm excitation and 510 nm emission. To convert
fluorescence measurements into Ca2+
concentrations, minimum fluorescence was determined after
addition of digitonin (50 mmol/L) in the presence of EGTA (2
mmol/L) and Tris base (20 mmol/L); maximum fluorescence
was measured by addition of excess CaCl2 (10
mmol/L). The calcium concentration was calculated using these values
and a Kd of 224 nmol/L, according to the
method of Grynkiewicz et al,13 after correction for
extracellular dye.
Phospholipase C Activation
Because activation of phospholipase C (PLC) produces
IP3 from phosphatidyl-inositol-4,5-bis-phosphate,
and because IP3 is converted within 30 to 60
seconds into inositol 1,3,4,5-tetraphosphate, which is rapidly
degraded into the more stable inositol
1,3,4-triphosphate,14 we studied inositol
1,3,4-triphosphate production 1 minute after platelet
stimulation with 12 µg/mL of collagen, which was the lowest collagen
concentration able to induce a reproducible response.
Collagen-stimulated activation of the
[32P]-labeled platelets, resuspended in
phosphate-free Tyrode's buffer, was stopped by means of perchloric
acid (0.44 N). The neutralized platelet extracts
(1x109 cells/mL) were treated overnight with
Zn2+-pyrophosphatase (20 U/mL) in the presence of
Tris-maleic buffer (0.1 mol/L, pH 6.5) and then passed on an HPLC
column eluted with a 50-minute linear gradient that used water as the
first buffer and ammonium formate (1.5 mol/L, pH 3.75) as the final
buffer. Inositol peaks were detected with a dual-channel
(3H-32P) HPLC radioactivity
detector (Flo-One A100 Radiomatic, Camberra Co) that used
[3H]-inositol 1,3,4-triphosphate as the pure
standard.14
Ex Vivo Study
In 6 healthy volunteers (3 men and 3 women; age, 30 to 48
years), collagen-induced platelet
H2O2 formation,
platelet aggregation, calcium mobilization, and vitamin E plasma
levels were measured before and after 2 weeks of oral vitamin E
supplementation (600 mg/d). Collagen-induced platelet activation
was analyzed by using the methods described above; all
experiments were performed within 10 minutes after gel filtration. The
plasma concentration of vitamin E was measured by HPLC according to the
method of Bieri et al.15 A flow rate of 2.0 mL/min was
used on an octadecilica C18 (ODS) 5 µm with a LC/233 diod array
detector (Restek Corp) set at 0.02 to 0.1 attenuation.
Statistical Analysis
Data are reported as mean±SEM. Comparisons between
variables were analyzed by Student's t test for
paired and unpaired data. Significance was set at
P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In Vitro Study
Flow Cytometric Analysis
The flow cytometric method uses the properties of DCFH-DA,16 17 18 which rapidly diffuses across cell membranes and is then trapped within the cell by a deacetylation reaction. In the presence of hydrogen peroxide, this compound is oxidized to DCF, which is highly fluorescent.19 Figure 1
shows the DCF green
fluorescence distribution of unactivated platelets
(31±2.8 au, Figure 1A). The largest increase of DCF
fluorescence occurred with 0.1 mmol/L
H2O2 (74.1±4.3 au,
P<0.01 versus control, Figure 1B). Collagen-induced
platelet activation doubled the shift of DCF fluorescence
in comparison to unstimulated platelets (65.0±7 au,
P<0.02 versus control, Figure 1C). Vitamin E inhibited the
shift of DCF fluorescence induced by collagen in a
concentration-dependent manner (with 50 µmol/L, DCF
fluorescence=43±7 au, P<0.05 versus collagen
alone, Figure 1D; with 125 µmol/L, 28.8+3 au, P<0.05
versus collagen alone, Figure 1E; and with 250 µmol/L, 24±3 au,
P<0.003 versus collagen alone, Figure 1F). To investigate
the mechanism underlying such effects, we analyzed whether
vitamin E was able to quench
H2O2 in a system containing
unactivated platelets. As shown in Figure 2, we found that vitamin E quenched up to
1x10-4 mmol/L
H2O2. The fact that
collagen induced a fluorescence shift lower than that induced
by 1x10-3 mmol/L
H2O2 might suggest
that in vitamin E-treated platelets stimulated with collagen, the
decrease of fluorescence is due to a direct interaction between
vitamin E and H2O2 produced
by platelets.
|
|
Platelet Aggregation
Inhibition of platelet aggregation by vitamin E was closely
dependent on incubation time, with >50% inhibition observed after at
least 15 minutes of incubation (Figure 3). More than 80% inhibition was
observed after 30 minutes of incubation; therefore, in the following
experiments, platelets were incubated with vitamin E for 30 minutes
before collagen was added. We found that vitamin E inhibited
collagen-induced platelet aggregation depending on the
concentration used. A 50-µmol/L concentration of vitamin E
significantly inhibited platelet aggregation; almost complete
inhibition of platelet aggregation was observed with 250
µmol/L (Figure 4). No inhibition was
observed in adenosine diphosphate, arachidonic
acid, U46619, or thrombin-activated platelets (data not
shown).
|
|
Platelet Thromboxane A2 Formation
Collagen-induced thromboxane A2 formation was
inhibited in a dose-dependent manner by vitamin E (Figure 5). A 10-µg/mL concentration of
collagen produced 67±7 ng/mL thromboxane A2, which was
reduced to 35±3 ng/mL (P<0.05), 19±10 ng/mL
(P<0.01), and 11±3 ng/mL (P<0.01) by 50, 125,
and 250 µmol/L vitamin E, respectively.
|
Changes in Intracellular Calcium Concentration
Percentages of change in intracellular calcium concentration
induced by collagen in control GFPs and GFPs treated with vitamin E (50
to 250 µmol/L) are reported in Figure 6. In samples stimulated with collagen (2
µg/mL), vitamin E inhibited intracellular calcium mobilization in a
dose-dependent manner.
|
) of calcium mobilization in
collagen-stimulated platelets in the absence and presence of scalar
doses (50 to 250 µmol/L) of vitamin E. Data are mean±SEM of 5
experiments. *P<0.05; **P<0.01.
PLC Activation
Production of 32P-labeled inositol
1,3,4-triphosphate by collagen-stimulated platelets (12 µg/mL),
added with and without vitamin E, is shown in Figure 7. Collagen-induced formation of inositol
1,3,4-triphosphate was inhibited by vitamin E in a
dose-dependent manner.
|
Ex Vivo Study
In 6 healthy subjects given vitamin E, the plasma concentration of
vitamin E rose from 19.0±3.1 to 75.0±8.4 µmol/L
(P<0.05). After vitamin E supplementation, we observed a
significant decrease of collagen-induced platelet
H2O2 formation,
platelet aggregation, and calcium mobilization
(Table).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This study showed that a concentration of vitamin E as low as 50 µmol/L is able to inhibit collagen-induced platelet activation by interfering with arachidonic acid metabolism and the PLC pathway. We recently demonstrated that during platelet aggregation by collagen, there is a burst of hydrogen peroxide that amplifies the platelet response to the agonist.10 Thus, hydrogen peroxide stimulated arachidonic acid metabolism, contributing to platelet production of thromboxane A2; in addition, hydrogen peroxide stimulated IP3 formation and Ca2+ mobilization. In this experimental model, vitamin E inhibited platelet thromboxane as well as IP3 formation and Ca2+ mobilization, suggesting that it influences both arachidonic acid metabolism and the PLC pathway. The inhibition of platelet function by vitamin E was due to its interaction with hydrogen peroxide. Thus, in experiments performed by incubating unstimulated platelets with scalar concentrations of hydrogen peroxide, vitamin E blunted hydrogen peroxide depending on the concentration of hydrogen peroxide present in the system; with a concentration of hydrogen peroxide of 1x10-4 mmol/L, 250 µmol/L vitamin E almost completely prevented the hydrogen peroxide–mediated increase in DCF fluorescence. In collagen-stimulated platelets, vitamin E blunted hydrogen peroxide formation in a dose-dependent manner. Inhibition was evident with concentrations of vitamin E as low as 50 mmol/L, because in collagen-stimulated platelets, the concentration of hydrogen peroxide is much lower than 1x10-3 mmol/L. Together, these findings indicate that vitamin E inhibits platelet function through its antioxidant properties, in particular by blocking the capacity of hydrogen peroxide to stimulate platelet arachidonic acid metabolism and the PLC pathway.
Our results differ from those of previous studies showing that concentrations of vitamin E much higher than those found in human plasma after supplementation are necessary to reduce in vitro platelet function. Recently, Freedman et al9 reported that 0.5 mmol/L vitamin E inhibits platelet aggregation by interfering with the protein kinase C pathway, with no evidence of platelet inhibition using lower concentrations. In this study, however, the relationship between vitamin E and collagen-induced platelet aggregation was not investigated.
In the same study,9 it was also demonstrated that incorporation of vitamin E into platelet membrane was time-dependent; incorporation was crucial to obtaining evident platelet inhibition. Even if we did not measure vitamin E incorporation into platelets, our finding is in agreement with the demonstration that vitamin E inhibits platelet aggregation depending on its incubation time. In this study, we observed the maximum effect on platelet inhibition by incubating platelets with vitamin E for 30 minutes.
Our finding may be of clinical relevance because it provides experimental support for 2 recent studies showing that vitamin E supplementation in humans decreases ex vivo and in vivo platelet activation.6 7 Both of these studies indicate a close relationship between plasma vitamin E levels and platelet activation (ie, higher plasma levels of vitamin E and lower platelet function). In particular, a plasma vitamin E concentration of about 50 µmol/L was associated with ex vivo and in vivo platelet inhibition, which is in accord with our data indicating that 50 µmol/L vitamin E inhibits in vitro platelet activation. This suggestion is supported by results of our ex vivo study, which showed that vitamin E significantly inhibits collagen-induced platelet activation; interestingly, after supplementation, the plasma concentration of vitamin E was very close to that shown in vitro to inhibit collagen-induced platelet activation. However, there is a discrepancy between the results of the in vitro and ex vivo studies that requires more investigation. The ex vivo study showed complete suppression of platelet H2O2 formation, but this was observed in vitro only with the addition of 250 µmol/L. However, this difference could be due to the different incubation times that were used in the in vitro and ex vivo studies.
In conclusion, these in vitro and ex vivo studies showed that vitamin E inhibits collagen-induced platelet activation. This effect is due to its interaction with hydrogen peroxide, an important mediator of collagen-induced platelet activation. This finding gives new insight into the mechanism of action of vitamin E and provides experimental support for its possible use as an antiplatelet agent.
|
Acknowledgments |
|---|
This work was partially supported by a grant from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica.
|
Footnotes |
|---|
Department of Experimental Medicine and Pathology (P.P., F.M.P., L.L., P.P.G.) and Institute of First Clinical Medicine (F.V.), University of La Sapienza, Rome, Italy.
Received July 2, 1998; accepted March 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793–795.[Medline] [Order article via Infotrieve]
2.
Stampfer MJ, Hennekens CH, Manson JE, Coldits GA,
Rosner B, Willett WC. Vitamin E consumption and the risk of
coronary disease in women. N Engl J Med. 1993;328:1444–1449.
3.
Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E,
Coldits GA, Willett WC. Vitamin E consumption and the risk of
coronary heart disease in men. N Engl J
Med. 1993;328:1450–1456.
4. Stephens NG, Parson A, Schofield PM, Kelly F, Cheeseman K, Mitchinson MJ. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study. Lancet. 1996;347:781–786.[Medline] [Order article via Infotrieve]
5.
Rapola JM, Virtamo J, Ripatti S. Randomized trial
of -tocopherol and ß-carotene supplements on
incidence of major coronary events in men with previous
myocardial infarction. Lancet. 1997;349:1715–1720.[Medline]
[Order article via Infotrieve]
6.
Davì G, Alessandrini P, Mezzetti P, Minotti G,
Bucciarelli T, Costantini F, Cipollone F, Bittolo Bon G, Ciabbattoni G,
Patrono C. In vivo formation of 8-epi-prostaglandin F2a is
increased in hypercholesterolemia.
Arterioscler Thromb Vasc Biol. 1997;17:3230–3235.
7. Calzada C, Bruckdorfer KR, Rice-Evans CA. The influence of antioxidant nutrients on platelet function in healthy volunteers. Atherosclerosis. 1997;128:97–105.[Medline] [Order article via Infotrieve]
8. Steiner M, Anastasi J. Vitamin E: an inhibitor of platelet release reaction. J Clin Invest. 1976;57:732–737.
9.
Freedman JE, J Farhat H, Loscalzo J, Keaney JF
Jr. -Tocopherol inhibits aggregation of human
platelets by a protein kinase C–dependent mechanism.
Circulation. 1996;91:2434–2440.
10.
Pignatelli P, Pulcinelli FM, Lenti L, Gazzaniga PP,
Violi F. Hydrogen peroxide is involved in collagen-induced platelet
activation. Blood. 1998;91:484–490.
11. Aster RH, Jandel JH. Platelet sequestration in man: I: methods. J Clin Invest. 1964;43:834–855.
12. Born GVR. The aggregation of blood platelets by adenosine diphosphate and its reversal. Nature. 1962;194:927–929.[Medline] [Order article via Infotrieve]
13.
Grynkiewicz G, Poenie M, Tsien RY. A new generation of
Ca2+ indicators with greatly improved
fluorescence properties. J Biol Chem. 1985;260:3440–3450.
14. Pulcinelli FM, Gazzaniga PP, Salganicoff L. Use of Zn-pyrophosphatase in the high-performance liquid chromatography analysis of cell extracts containing 32P-labelled inositol phosphates. J Chromatogr. 1992;575:51–55.[Medline] [Order article via Infotrieve]
15.
Bieri JG, Tolliver TJ, Catignani GL.
Simultaneous determination of
-tocopherol and retinol in plasma or red cells by high
pressure liquid chromatography. Am J Clin
Nutr. 1979;32:2143–2149.
16. Burrow S, Valet G. Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF). Eur J Cell Biol. 1987;43:128–133.[Medline] [Order article via Infotrieve]
17. Del Principe D, Menichelli A, De Matteis W, Di Giulio S, Giordani M, Savini I, Finazzi Agrò AF. Hydrogen peroxide is an intermediate in platelet activation cascade triggered by collagen, but not by thrombin. Thromb Res. 1991;62:365–375.[Medline] [Order article via Infotrieve]
18. Maresca M, Colao C, Leoncini G. Generation of hydrogen peroxide in resting and activated platelets. Cell Biochem Funct. 1992;10:79–85.[Medline] [Order article via Infotrieve]
19. Leoncini G, Maresca M, Colao C. Oxidative metabolism of human platelets. Biochem Int. 1991;25:647–655.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  P. Pignatelli, F. M Pulcinelli, A. Celestini, L. Lenti, A. Ghiselli, P. P. Gazzaniga, and F. Violi The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide Am. J. Clinical Nutrition, November 1, 2000; 72(5): 1150 - 1155. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||