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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1086-1096
Biological Inactivation of 5-oxo-6,8,11,14-Eicosatetraenoic
Acid by Human Platelets
By
William S. Powell,
Sylvie Gravel,
Subhash P. Khanapure, and
Joshua Rokach
From Meakins-Christie Laboratories, Department of Medicine, McGill
University, Montreal, Quebec, Canada; and the Claude Pepper Institute
and Department of Chemistry, Florida Institute of Technology,
Melbourne, FL.
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ABSTRACT |
Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)
is a potent activator of neutrophils and eosinophils. In the present
study we examined the biosynthesis and metabolism of this substance by
platelets. Although platelets contain an abundant amount of
5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the
formation of 5-oxo-ETE, they synthesize only very small amounts of this
substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless
endogenous NADPH is converted to NADP+ by
addition of phenazine methosulfate. Similarly, relatively small amounts
of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and
neutrophils, which instead formed substantial amounts of two 12-hydroxy
metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE,
which were identified by comparison with authentic chemically
synthesized compounds. These metabolites were also formed from
5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast,
unstimulated platelets converted 5-oxo-ETE principally to 5-HETE.
Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects
on neutrophil calcium levels or platelet aggregation at concentrations
as high as 10 µmol/L, but both blocked 5-oxo-ETE-induced calcium
mobilization in neutrophils with IC50 values
of 0.5 and 2.5 µmol/L, respectively. We conclude that platelets can
biologically inactivate 5-oxo-ETE. Unstimulated platelets convert
5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas
stimulated platelets convert this substance to 12-hydroxy metabolites,
which possess antagonist properties.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ARACHIDONIC ACID IS metabolized in
neutrophils primarily by the 5-lipoxygenase (5-LO) pathway, resulting
in the formation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE)
and leukotriene B4 (LTB4).1,2
5-HETE is further metabolized to the biologically active compound
5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)3 by these
cells, whereas LTB4 is converted to  -oxidation products with reduced biological activity.4-6 In contrast to
neutrophils, platelets do not possess 5-LO, but instead convert
arachidonic acid to products of the 12-lipoxygenase (12-LO) and
cyclooxygenase pathways. The major 12-LO product formed by these cells
is
12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE), whereas the major cyclooxygenase products are thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid
(12-HHTrE).7,8
Although platelets cannot synthesize 5-LO products from arachidonic
acid, they can metabolize a number of neutrophil-derived 5-LO products.
For example, platelets convert 5-HETE to
5S,12S-dihydroxy-6E,8Z,10E,14Z-6,8,10,14-eicosatetraenoic acid (5S,12S-diHETE) by the action of 12-LO.9 Platelets can also transform neutrophil-derived leukotriene A4
(LTA4) to leukotriene C4
(LTC4) because, unlike neutrophils, they contain
LTC4 synthase.10-12 Furthermore, neutrophils
can convert platelet-derived 12-HETE to its -oxidation product
12,20-diHETE13 as well as to 5S,12S-diHETE.14 Thus, the profile of products formed by mixtures of platelets and
neutrophils is considerably more complex than the sum of products formed by each of the two types of cells alone because of the formation
of additional products by transcellular metabolism.
The objective of the present study was to determine whether platelets
can synthesize and metabolize 5-oxoeicosanoids. We have shown that
various leukocytes, including neutrophils,3
eosinophils,15 monocytes, and lymphocytes16 can
convert 5-HETE to 5-oxo-ETE by the action of 5-hydroxyeicosanoid
dehydrogenase. 5-oxo-ETE is a potent chemoattractant for
eosinophils15,17,18 and neutrophils19 and
induces a variety of other responses in these cells including calcium
mobilization,19,20 adherence,21 integrin
expression,21 actin polymerization,21,22 and
degranulation.20 There is considerable evidence that its
actions are mediated by a specific G protein-linked
receptor.22-24 We report here that platelets contain 5-hydroxyeicosanoid dehydrogenase. However, in resting platelets this enzyme seems to act in the reverse direction, converting 5-oxo-ETE
to the less biologically active 5-HETE. Stimulated platelets, on the
other hand, convert 5-oxo-ETE to its 12-hydroxy metabolite, 5-oxo-12S-hydroxy-6E,8Z,10E,14Z-eicosatetraenoic
acid (5-oxo-12-HETE), which has little or no effect on neutrophils, and
can antagonize 5-oxo-ETE-induced calcium mobilization. Thus, platelets
could make an important contribution to the biological inactivation of
5-oxo-ETE.
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MATERIALS AND METHODS |
Eicosanoids.
5-HETE was synthesized by incubation of arachidonic acid (NuChek Prep
Inc, Elysian, MN) with an ammonium sulfate fraction from potatoes as
described in the literature.25 5S,12S-diHETE was prepared
by incubating arachidonic acid with porcine leukocytes in the presence
of A23187 (Calbiochem, La Jolla, CA).6 LTB4 was
prepared similarly from porcine leukocytes except that incubations were
performed in the presence of 5 µmol/L of 5,8,11,14-eicosatetraenoic acid.26 All products were extracted on octadecylsilyl
(ODS)-silica Sep-Paks (Waters Associates, Milford, MA) and purified by
high pressure liquid chromatography (HPLC).
5-oxo-ETE and its 8-trans isomer27 as well as
6-trans-LTB4 were prepared by total chemical synthesis as
described previously. 5-oxo-12S-HETE and 8-trans-5-oxo-12S-HETE were
also prepared by total chemical synthesis.28 Prostaglandin
B2 (PGB2) was purchased from Sigma Chemical Co
(St Louis, MO), whereas 12-epi-6-trans-LTB4 was obtained
from Cayman Chemicals (Ann Arbor, MI).
Preparation of human neutrophils.
Human neutrophils were prepared by treatment of whole blood with
Dextran T-500, followed by centrifugation over Ficoll-Paque and removal of remaining red blood cells by hypotonic
lysis. The cells were resuspended in calcium and magnesium-free
Dulbecco's phosphate-buffered saline (PBS) containing 137 mmol/L NaCl,
2.7 mmol/L KCl, 1.5 mmol/L KH2PO4, and 8.1 mmol/L Na2HPO4 at a pH of 7.4.
Preparation of human platelets.
Blood was treated with Dextran T-500 (final concentration, 1%)
for 45 minutes, and the resulting supernatant centrifuged
for 10 minutes at 200g at room temperature. EDTA (final
concentration, 1.54 mmol/L) was added to the supernatant, which was
centrifuged at 700g for 20 minutes at room temperature. The top
white portion of the pellet was resuspended in PBS (without
Ca++ and Mg++) at a concentration of 2 × 108 platelets/mL unless otherwise indicated.
Preparation of subcellular fractions from platelets.
Platelets (65 × 106 cells/mL) were sonicated in PBS
containing phenylmethylsulfonyl fluoride (2 mmol/L), dithiothreitol (1 mmol/L), and EDTA (1 mmol/L). The supernatant obtained by
centrifugation at 1,500g at 4°C for 10 minutes was
centrifuged at 10,000g at 4°C for 10 minutes. The
supernatant was centrifuged at 200,000g at 4°C for 60 minutes. The 10,000g and 200,000g pellets were
resuspended in PBS containing CaCl2 (1.8 mmol/L) and
MgCl2 (1 mmol/L) at concentrations equivalent to 50 × 106 and 20 × 106 cells/mL, respectively.
Analysis of eicosanoids by precolumn extraction/reversed-phase
(RP)-HPLC.
Suspensions (1 mL) of platelets, platelet-neutrophil mixtures, or
subcellular fractions derived from platelets were incubated in PBS
containing calcium (1.8 mmol/L) and magnesium (1 mmol/L) in the
presence of various eicosanoids or agonists. The incubations were
terminated by addition of methanol (0.6 mL) and cooling to 0°C.
Before analysis, the concentration of methanol in the samples was
adjusted to 15% by addition of water and PGB2 (200 ng) was added as an internal standard. Eicosanoids were quantitated by precolumn extraction/RP-HPLC29 using a Waters gradient
controller, WISP automatic injector, WAVS automated switching valve,
Model 991 diode array detector, and a Model 600 solvent delivery system (Waters Associates, Milford, MA). The mobile phase was a
linear gradient between a mixture of 70% solvent A
(water/acetonitrile/acetic acid [80:20:0.02]) and 30% solvent B
(water/acetonitrile/methanol/acetic acid [7.5:38.5:54:0.02]) and a
mixture of 15% solvent A and 85% solvent B over 50 minutes unless
otherwise indicated. The flow rate was 1 mL/minute. The stationary
phase was a column of Novapak C18 (3.9 × 150 mm; Waters). Products were quantitated by comparing the areas of
their peaks of ultraviolet (UV) absorbance at their  max
with that of the internal standard, PGB2. The extinction coefficients used were: 5-HETE (23,000), 5-oxo-ETE (20,500),
LTB4 (39,500), 5S,12S-diHETE (40,000),
5-oxo-12-hydroxyeicosanoids (40,000), and PGB2
(28,680). The identities of the products measured were confirmed by
examination of their complete UV spectra.
Mass spectrometry.
Mass spectrometry was performed using a Quattro II instrument
(Micromass, Manchester, UK) with an electrospray
interface, which was situated in the Biomedical Mass Spectrometry Unit
of McGill University (Montreal, Quebec, Canada). Before analysis, 5-oxo-ETE metabolites were dissolved in acetonitrile/methanol/water (2:1:1) containing 1.25 mmol/L ammonium acetate, and were introduced into the ion source via direct inlet.
Measurement of cytosolic calcium levels.
Neutrophils (107 cells/mL) were preincubated for 5 minutes
at 37°C in PBS and then incubated with the acetoxymethyl ester of Calcium Green-1 (1 µmol/L; Molecular Probes, Eugene, OR) for a further 30 minutes. The cells loaded with Calcium Green-1 were then
washed twice in PBS and resuspended in the same medium to obtain a
final cell concentration of 3.22 × 106 cells/mL.
Fluorescence was measured at 37°C using a Photon Technology International (Monmouth Junction, NJ) Deltascan 4000 spectrofluorometer with a temperature-controlled cuvette holder
equipped with a magnetic stirrer. The excitation and emission
wavelengths were set at 506 nm and 531 nm, respectively. Before the
addition of agonists, CaCl2 and MgCl2 in 56 µL of PBS were added to an aliquot of the cell suspension (934 µL)
to give final concentrations of 1 mmol/L of each, a final concentration
of cells of 3 × 106/mL, and a final volume of 1 mL.
Responses to agonists were measured after stabilization of the baseline
fluorescence. Fmax was determined by adding digitonin
(final concentration, 0.1%), whereas Fmin was calculated
after determination of autofluorescence as described in the
literature.30,31
Measurement of platelet aggregation.
Platelet aggregation was measured as described in the
literature.32 Washed platelets were prepared by
centrifugation of whole blood, collected in citrate, at 180g
for 15 minutes at room temperature. The supernatant was diluted with an
equal amount of citrate buffer (93 mmol/L sodium citrate, 7 mmol/L
citric acid, and 140 mmol/L dextrose) and centrifuged at 1,000g
for 10 minutes and the pellet resuspended in
Ca++/Mg++-free Hanks' Balanced Salt
Solution. Aggregation of platelets (0.5 mL; 300 × 106/mL) was measured in a Payton aggregometer
(Payton Associates, Scarborough, Ontario, Canada), after addition of
calcium and magnesium to give final concentrations of 1 mmol/L of each.
After stabilization of the baseline, 5-oxo-eicosanoids or vehicle (2.5 µL dimethyl sulfoxide [DMSO]) were added 2 minutes before addition
of the minimal maximally effective concentration of arachidonic acid (2 to 4 µmol/L), which was determined for each experiment.
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RESULTS |
Metabolism of 5-HETE by subcellular fractions from platelets.
We previously showed that 5-hydroxyeicosanoid dehydrogenase in
neutrophils was localized in the microsomal fraction of these cells.3 To determine whether this enzyme is also present in platelets, 5-HETE was incubated with subcellular fractions prepared from sonicates of these cells. Platelet sonicates were centrifuged successively at 1,500g, 10,000g, and 200,000g.
The chromatographic profiles of products formed after incubation of
5-HETE with the 1,500g supernatant fraction and the
200,000g pellet in the presence of NADP+
(1 mmol/L) are shown in Fig
1. The 1,500g supernatant fraction converted 5-HETE to both
5-oxo-ETE and 5S,12S-diHETE, indicating that it contained both
5-hydroxyeicosanoid dehydrogenase and 12-LO. In addition, two products
with retention times of 24.8 and 28.1 minutes, respectively, were
detected at 316 nm. These products had identical UV spectra with
max values at 316 nm (Fig 1A, inset). Because it seemed
likely that these two products were 12-hydroxy metabolites of
5-oxo-ETE, both 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE were prepared
by total chemical synthesis. The product with the longer retention time
(13.2 minutes) cochromatographed with 5-oxo-12-HETE, whereas that with
the shorter retention time (11.1 minutes) cochromatographed with
8-trans-5-oxo-12-HETE. The platelet-derived products had identical UV
spectra to the corresponding chemically synthesized compounds. To
provide further support for the identities of the above two products,
their retention times were compared with those of the authentic
chemically synthesized compounds using both normal phase and RP-HPLC
(Table 1). In this case, the
two 5-oxo-12-hydroxy isomers were prepared by incubation of 5-oxo-ETE
with platelets in the presence of A23187 (see below). The chemically
and biologically derived products had identical retention times under
each of the three conditions used (Table 1), and, when mixed together,
gave single peaks (data not shown). The identities of biologically prepared 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE were further confirmed
by electrospray mass spectrometry, which showed M-1 ions at m/z
333 for both compounds (data not shown).
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| Fig 1.
HPLC profiles of products formed by platelet subcellular
fractions. Platelet sonicates were centrifuged at 1,500g for 10 minutes and the supernatant centrifuged successively at 10,000g
for 10 minutes and 200,000g for 60 minutes as described in
Materials and Methods. The 1,500g supernatant (A) and the
200,000g pellet (B) fractions (equivalent to 2 × 107 platelets/mL) were incubated for 40 minutes at 37°C
with 5-HETE (2 µmol/L) in the presence of NADP+ (1 mmol/L). The products were analyzed by precolumn extraction/RP-HPLC on
a Novapak C18 column (3.9 × 300 mm) using a gradient
between water/acetonitrile/acetic acid (50:50:0.02) and
water/acetonitrile/acetic acid (40:60:0.02) over 30 minutes, followed
by isocratic elution with 60% acetonitrile for a further 10 minutes.
PGB2 (90 ng) was added as an internal standard. The inset
to (A) shows the UV spectrum of biologically synthesized 5-oxo-12-HETE,
which was identical to that of the chemically synthesized compound.
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The 5-hydroxyeicosanoid dehydrogenase activity of subcellular fractions
from platelets was estimated by adding together the amounts of all the
5-oxo-eicosanoids formed by these fractions (ie, 5-oxo-ETE + 5-oxo-12-HETE + 8-trans-5-oxo-12-HETE). Although 5-oxo-ETE was the most
abundant product in all cases, significant amounts of 12-hydroxy
products were formed by the 1,500g supernatant (Fig 1A) and
10,000g pellet fractions. The fraction containing the highest
5-hydroxyeicosanoid dehydrogenase activity was the 200,000g
pellet fraction (Fig 1B), which, in the presence of NADP+,
showed about three times the specific activity of the 1,500g supernatant (Table 2). In contrast, the 10,000g pellet fraction showed about the same activity as the 1,500g pellet, whereas
the 200,000g supernatant fraction had little
5-hydroxyeicosanoid dehydrogenase activity.
The effects of cofactors on 5-hydroxyeicosanoid dehydrogenase activity
were also investigated. In the absence of any cofactors, the
200,000g pellet fraction showed little activity (Table 2). Although addition of NAD+ resulted in markedly increased
enzyme activity, it was only about one-fifth as effective as
NADP+ in serving as a cofactor for the dehydrogenase.
Profile of eicosanoids released by mixtures of platelets and
neutrophils.
Although platelets contain 5-hydroxyeicosanoid dehydrogenase, they
would not be expected to synthesize 5-oxo-ETE or other 5-oxo-eicosanoids from endogenous substrate because they do not contain
5-LO. However, addition of platelets to neutrophils could increase the
formation of 5-oxo-eicosanoids by transcellular metabolism. To test
this hypothesis, a mixture of platelets (2 × 108/mL)
and neutrophils (5 × 106/mL) were incubated with the
calcium ionophore A23187 (5 µmol/L) and phorbol 12-myristate
13-acetate (PMA; 30 nmol/L) for 30 minutes at 37°C. Analysis of the
resulting products by RP-HPLC showed the presence of a large number of
substances derived from the two types of cells
(Fig 2). The major compounds detected by UV absorption were the platelet-derived products 12-HETE and 12-HHTrE, along with 5S,12S-diHETE, a product of transcellular metabolism. The
neutrophil-derived products 5-HETE and LTB4 and its 6-trans isomers were also detected, along with similar amounts of 5-oxo-12-HETE and its 8-trans isomer.
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| Fig 2.
HPLC profile of products formed after incubation of a
mixture of platelets and neutrophils with A23187 and PMA. Platelets (2 × 108/mL) and neutrophils (5 × 106/mL) were
incubated for 30 minutes at 37°C with A23187 (5 µmol/L) and PMA
(30 nmol/L). After addition of the internal standard (200 ng
PGB2), the products were analyzed by precolumn
extraction/RP-HPLC as described in Materials and Methods.
Abbreviations: B4, LTB4; t-B4,
6-trans-LTB4; 12e-t-B4,
12-epi-6-trans-LTB4; 5o-12h-ETE, 5-oxo-12-HETE;
8t-5o-12h-ETE, 8-trans-5-oxo-12(RS)-HETE.
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Effects of different ratios of platelets and neutrophils on
eicosanoid production.
The effects of different ratios of platelets to neutrophils on the
synthesis of 5-oxo-eicosanoids from endogenous substrate were
investigated. Stimulation of a suspension of platelets alone (200 × 106 cells/mL) with A21387 (5 µmol/L) and PMA (30 nmol/L) failed to result in the release of detectable amounts of
5-oxo-eicosanoids (Fig 3A). However,
addition of increasing numbers of neutrophils resulted in progressive
increases in the amounts of these compounds. The amounts of
5-oxo-12-hydroxy metabolites far exceeded that of 5-oxo-ETE at all
concentrations of neutrophils tested (Fig 3A).
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| Fig 3.
Effects of different concentrations of platelets and
neutrophils on the formation of 5-oxo-eicosanoids. Mixtures of
platelets and neutrophils were incubated for 30 minutes at 37°C
with A23187 (5 µmol/L) and PMA (30 nmol/L). The products were
quantitated by RP-HPLC with PGB2 as an internal standard as
shown in Fig 1. (A) Formation of 5-oxo-12-HETE ( ),
8-trans-5-oxo-12-HETE ( ), and 5-oxo-ETE ( ) by platelets (2 × 108/mL) in the presence or absence of various
concentrations of neutrophils. (B) Formation of 5-oxo-12-HETE (),
8-trans-5-oxo-12-HETE (), and 5-oxo-ETE () by neutrophils (5 × 106/mL) in the presence or absence of various
concentrations of platelets. The inset shows the formation of 5-HETE
() and LTB4 ( ) by neutrophils (5 × 106/mL) in the presence or absence of various
concentrations of platelets. The results are means ± standard error
(SE) of experiments on mixtures of platelets and neutrophils from four
different subjects.
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The effects of adding increasing numbers of platelets to a constant
number of neutrophils (5 × 106 cells/mL) were
somewhat more complicated. In the absence of platelets, the principal
5-oxo-eicosanoid formed was 5-oxo-ETE (Fig 3B), along with very small
amounts of 8-trans-5-oxo-12-HETE. The latter substance was presumably
formed by oxidation of 6-trans isomers of LTB4 by
5-hydroxyeicosanoid dehydrogenase in neutrophils to give
5-oxo-12S-hydroxy and 5-oxo-12R-hydroxy metabolites, which would not be
separated by RP-HPLC. The 5S,12S-diHETE metabolite 5-oxo-12-HETE
(retention time, 28.1 minutes) was not detected in the absence of
platelets. Addition of platelets caused dramatic increases in the
formation of both 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, but reduced
the amount of 5-oxo-ETE detected. The total amount of 5-oxo-eicosanoids
(5-oxo-ETE + 5-oxo-12-HETE + 8-trans-5-oxo-12-HETE) synthesized by a
mixture of neutrophils with the highest concentration of platelets
tested (400 × 106/mL) was 124 ± 31 pmol/mL,
compared with 29 ± 5 pmol/mL in the presence of neutrophils alone
(P < .05). Addition of platelets to neutrophils also resulted
in increased formation of 5-HETE (P < .05) (Fig 3B,
inset). In contrast, LTB4 production seemed to be somewhat
lower in the presence of platelets, but this difference was not significant.
As shown above, changes in the ratios of platelets to neutrophils
affected the distribution of products. However, the absolute numbers of
cells could also be important, as has been shown to be the case for the
synthesis of LTB433 and
20-hydroxy-LTB46 by neutrophils. To test this
hypothesis, mixtures of increasing numbers of platelets and neutrophils
at a constant ratio of 40:1 were incubated with A23187 and PMA as
described above. The amounts of the two 5-oxo-12-HETE isomers increased
nearly linearly with cell concentration, whereas the amount of
5-oxo-ETE increased only modestly. Thus at a relatively low
concentration of cells (50 × 106 platelets; 1.25 × 106 neutrophils), the total amount of 5-oxo-12-HETE
was 5.4 ± 0.5 times greater than the amount of 5-oxo-ETE. This
ratio increased markedly as the total numbers of cells were increased
so that at a high concentration of cells (400 × 106
platelets; 10 × 106 neutrophils) the total amount of
5-oxo-12-HETEs was 26.6 ± 3.0 times greater than that of 5-oxo-ETE.
This presumably reflects an increased rate of metabolism of 5-oxo-ETE
at higher concentrations of platelets.
Metabolism of 5-HETE and related compounds by intact platelets.
The increased amounts of 5-oxo-eicosanoids formed by addition of
platelets to neutrophils suggest that intact platelets can convert
5-HETE or 5S,12S-diHETE to their 5-oxo metabolites. To test this
hypothesis more directly, 5-HETE (2 µmol/L) was incubated with
platelets (200 × 106 cells/mL) for 30 minutes at
37°C. As shown in Fig 4A, platelets converted 5-HETE to only small amounts of 5-oxo-ETE. It is possible that the modest metabolism of 5-HETE could have been related to incorporation into membranes, as occurs in neutrophils,34
but this is probably not a major consideration, because most of the 5-HETE was recovered unmetabolized after incubation with unstimulated platelets (Fig 4A). It is also possible that 5-HETE may not readily be
taken up by platelets, but subsequent experiments showed that the
metabolism of this substance was markedly enhanced by activation by
thrombin or A23187 (see below). Previous studies with neutrophils had
shown that conversion of 5-HETE to 5-oxo-ETE was tightly regulated by
intracellular NADP+ levels.35 Therefore,
platelets were incubated with 5-HETE in the presence of phenazine
methosulfate (PMS; 100 µmol/L), which nonenzymatically converts NADPH
to NADP+ 36 without oxidizing
5-HETE.35 Under these conditions, the formation of
5-oxo-ETE was dramatically increased (Fig 4B). In addition to 5-HETE,
platelets also oxidized 5S,12S-diHETE in the presence of PMS to
5-oxo-12-HETE, along with a small amount of the corresponding 8-trans
isomer (Fig 4C). Both 6-trans-LTB4 and 12-epi-6-trans-LTB4 were also converted by platelets to
their 5-oxo metabolites (ie, 8-trans-5-oxo-12R-HETE and
8-trans-5-oxo-12S-HETE) which both had retention times similar to that
of chemically synthesized 8-trans-5-oxo-12S-HETE (data not shown). Only
small amounts of 5-oxo-12-hydroxy metabolites were formed from the
above compounds in the absence of PMS (data not shown). Unlike all of
the above 5S-hydroxy-6-trans eicosanoids, LTB4 was not
converted to significant amounts of products absorbing at 316 nm in the
presence of PMS (data not shown).
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| Fig 4.
HPLC profiles of 5-oxo-eicosanoids formed by platelets in
the presence and absence of phenazine methosulfate. Platelets (2 × 108/mL) were preincubated for 5 minutes at 37°C in the
absence (A) or presence (B and C) of PMS (100 µmol/L) and then
incubated for a further 30 minutes with 5-HETE (2 µmol/L; A and B) or
5S,12S-diHETE (2 µmol/L; C). The products were analyzed by precolumn
extraction/RP-HPLC as described in Materials and Methods.
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The effects of different concentrations of PMS on the formation of
5-oxo-ETE by platelets are shown in Fig 5.
As in Fig 4A, only small amounts of 5-oxo-ETE were formed in the
absence of PMS. Formation of 5-oxo-ETE seemed to be increased at
concentrations of PMS as low as 1 µmol/L, although this effect was
not statistically significant. However, all concentrations of PMS
higher than 1 µmol/L significantly and dramatically increased the
production of 5-oxo-ETE from 5-HETE by platelets. These results clearly
show that intact platelets have a high capacity to synthesize
5-oxo-ETE, even though it may not normally be realized.
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| Fig 5.
Concentration-response for the effect of PMS on the
formation of 5-oxo-ETE. Platelets (2 × 108/mL) were
preincubated for 5 minutes at 37°C in the absence or presence of
various concentrations of PMS and then incubated for a further 30 minutes with 5-HETE (2 µmol/L). The products were analyzed by
precolumn extraction/RP-HPLC as described in Materials and Methods. The
only product formed in appreciable amounts under these conditions was
5-oxo-ETE. The results are means ± SE of determinations on platelets
from three different individuals.
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Metabolism of 5-HETE by stimulated platelets.
The modest amounts of 5-oxo-eicosanoids formed after incubation of
5-HETE with platelets in the absence of PMS compared with the amounts
synthesized by stimulated mixtures of platelets and neutrophils
suggested that the synthesis of these compounds could be enhanced in
stimulated platelets. To test this hypothesis, platelets were incubated
with 5-HETE, 5S,12S-diHETE, or 5-oxo-ETE for 10 minutes in the presence
or absence of A23187 (5 µmol/L) or thrombin (1 U/mL). Unstimulated
platelets converted 5-HETE to small amounts of 5-oxo-ETE but little or
no 5-oxo-12-hydroxy-ETEs (Fig 4A), whereas platelets stimulated with
either A23187 or thrombin formed mainly the latter compounds (data not
shown). Both A23187 and thrombin stimulated the dehydrogenase-catalyzed
oxidation of 5-HETE to 5-oxo-eicosanoids (ie, 5-oxo-ETE + 5-oxo-12-HETE + 8-trans-5-oxo-12-HETE) by over twofold
(Fig 6). Similarly, these two agonists
increased the conversion of 5S,12S-diHETE to its 5-oxo metabolites by
twofold to threefold. These results show that the formation of
5-hydroxyeicosanoid dehydrogenase products by platelets is enhanced as
a result of platelet stimulation.
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| Fig 6.
Effects of platelet stimulation on the metabolism of
5-HETE, 5S,12S-diHETE, and 5-oxo-ETE. Platelets (2 × 108)
were incubated with either 5-HETE (2 µmol/L; 5h), 5S,12S-diHETE (2 µmol/L; 5,12-dh), or 5-oxo-ETE (2 µmol/L; 5o) for 10 minutes at
37°C in the absence () or presence of A23187 (5 µmol/L;  )
or thrombin (1 U/mL;  ). The products were quantitated by precolumn
extraction/RP-HPLC as described in Materials and Methods. The four
groups of bars (from left to right) show: (1) the amounts of
5-hydroxyeicosanoid dehydrogenase (dh) products (5-oxo-ETE + 5-oxo-12-HETE + 8-trans-5-oxo-12-HETE) formed from 5-HETE, (2) the
amounts of dehydrogenase products (5-oxo-12-HETE + 8-trans-5-oxo-12-HETE) formed from 5S,12S-diHETE, (3) the amounts of
5-ketoreductase (kr) products (5-HETE + 5S,12S-diHETE) formed from
5-oxo-ETE, and (4) the amounts of 12-LO products (5-oxo-12-HETE + 8-trans-5-oxo-12-HETE + 5S,12S-diHETE) formed from 5-oxo-ETE. The
values are means ± SE of determinations on platelets from four
different donors.
|
|
Metabolism of 5-oxo-ETE by platelets.
5-oxo-ETE was incubated with platelets in the presence or absence of
thrombin (1 U/mL) for various times and the products analyzed by
RP-HPLC. As shown in Fig 7A, the major
5-oxo-ETE metabolite formed by unstimulated platelets was 5-HETE, which
reached near maximal levels by 10 minutes, and then increased slowly
with time thereafter. Only small amounts of 5-oxo-12-HETE isomers were
formed by unstimulated platelets. 5S,12S-diHETE was nearly undetectable for the first 10 minutes, and then increased slowly with time. In
contrast, in the presence of thrombin, only small amounts of 5-HETE
were detected (Fig 7B), and instead, the major 5-oxo-ETE metabolites
were the cis and trans isomers of 5-oxo-12-HETE, which reached maximal
levels by 10 minutes. In this case, the trans isomers seemed to
predominate. The ratios of the cis and trans isomers varied somewhat
from one experiment to another, probably because of nonenzymatic
conversion of the cis to the trans, which occurs very easily. Little or
no 5S,12S-diHETE was formed after 3 minutes, but its concentration
gradually increased over 30 minutes.
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| Fig 7.
Time course for the formation of 5-oxo-ETE metabolites by
resting and activated platelets. Platelets (2 × 108/mL)
were incubated with 5-oxo-ETE (2 µmol/L) for various times at
37°C in the absence (A) or presence (B) of thrombin (1 U/mL).
5-HETE (), 5-oxo-12-HETE ( ; 5o-12h), and 8-trans-5-oxo-12-HETE
( ; 8t-5o-12h), and 5S,12S-diHETE (; 5S,12S) were analyzed by
precolumn extraction/RP-HPLC as described in Materials and Methods. All
values are means ± SE of determinations on platelets from four
different donors.
|
|
The effects of incubation of platelets with A23187 or thrombin for 10 minutes on the metabolism of 5-oxo-ETE by the 5-ketoreductase and 12-LO
pathways are shown in Fig 6. In agreement with Fig 7, both agents inhibit the formation of 5-ketoreductase metabolites of
5-oxo-ETE, and strongly enhance its conversion to its 12-hydroxy metabolites by 12-LO. Thus stimulated platelets metabolize 5-oxo-ETE principally via the 12-LO pathway.
Effects of 12-hydroxy metabolites of 5-oxo-ETE on calcium
mobilization in neutrophils.
Because 5-oxo-ETE is a potent stimulus of calcium mobilization in
neutrophils, we examined the effects of its 12-hydroxy metabolites on
this response. Because the 5-oxo-12-hydroxy-ETE isomers absorbed strongly above 300 nm, they interfered with the fluorescence emission of indo-1, the calcium indicator used in our previous
studies.19 We therefore used calcium green-1, which has
much higher excitation and emission wavelengths. The results we
obtained for 5-oxo-ETE with this indicator were identical with those
obtained with indo-1. In contrast to 5-oxo-ETE, which stimulates
calcium mobilization in neutrophils with an EC50
of about 4 nmol/L,23 5-oxo-12-HETE and
8-trans-5-oxo-12-HETE have little effect on cytosolic calcium levels,
even at concentrations as high as 10 µmol/L
(Fig 8). However, both of these 12-hydroxy
metabolites block the effects of 5-oxo-ETE on calcium mobilization in
neutrophils. 5-oxo-12-HETE (EC50 0.5 µmol/L) is somewhat more potent than 8-trans-5-oxo-12-HETE
(EC50 2.5 µmol/L) in this respect (Fig 8, inset).
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| Fig 8.
Effects of 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE on
cytosolic calcium levels in human neutrophils. Neutrophils (3 × 106 cells/mL) loaded with calcium green-1 were treated with
either vehicle, 5-oxo-12-HETE (10 µmol/L; 8-cis) or
8-trans-5-oxo-12-HETE (10 µmol/L; 8-trans). Approximately 2 minutes
later, 5-oxo-ETE (10 nmol/L) was added. Fluorescence was measured as
described in Materials and Methods. The inset shows the inhibitory
effects of different concentrations of 5-oxo-12-HETE () and
8-trans-5-oxo-12-HETE () on calcium mobilization induced by addition
of 5-oxo-ETE (10 nmol/L). The values are means ± SE of determinations
on platelets from three different donors.
|
|
Effects of 5-oxo-eicosanoids on platelet aggregation.
The effects of 5-oxo-ETE, 5-oxo-12-HETE, and 8-trans-5-oxo-12-HETE on
platelet aggregation were investigated. None of these 5-oxo-eicosanoids
induced aggregation of washed platelets at concentrations up to 20 µmol/L (Fig 9A). However, 5-oxo-ETE (20 µmol/L) completely prevented aggregation induced by a maximally
effective concentration (ca. 3 µmol/L) of arachidonic acid (Fig 9A).
5-oxo-12-HETE (20 µmol/L) also inhibited arachidonate-induced
aggregation, but only by about 50%. Its 8-trans isomer was
ineffective. The effects of 5-oxo-ETE and 5-oxo-12-HETE were
concentration dependent, with much smaller effects observed at
concentrations of 10 µmol/L, and little or no effect at 3 µmol/L
(Fig 9B).
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| Fig 9.
Effects of 5-oxo-eicosanoids on platelet aggregation. (A)
The effects of vehicle (veh; 2.5 µL DMSO), 5-oxo-ETE (5o),
5-oxo-12-HETE (cis), and 8-trans-5-oxo-12-HETE (tr) on platelet
aggregation induced by a maximally effective concentration of
arachidonic acid (AA; 4 µmol/L), added 2 minutes later. The ticks on
the bar below the tracings are 1 minute apart. (B)
Concentration-response curves for the effects of 5-oxo-ETE (;
5o-ETE), 5-oxo-12-HETE (; 5o-12h [cis]), and 8-trans-5-oxo-12-HETE
(; 5o-12h [tr]) on platelet aggregation induced by the lowest
maximally effective concentration of arachidonic acid (2 to 4 µmol/L). All values are means ± SE of determinations on platelets
from three different donors.
|
|
| |
DISCUSSION |
Platelets possess 5-hydroxyeicosanoid dehydrogenase activity and can
thus convert 5-HETE and related eicosanoids to their 5-oxo metabolites.
The properties of this enzyme are very similar to those of the
corresponding enzyme that we previously identified in
neutrophils.3 The enzyme is localized in the microsomal fraction of both neutrophils and platelets and requires
NADP+ as a cofactor. It can oxidize 5-HETE and other
5-hydroxyeicosanoids in which the 5-hydroxyl group is followed by a
6-trans double bond. However, it cannot oxidize LTB4, which
has a 6-cis double bond.
The presence of 5-hydroxyeicosanoid dehydrogenase in platelets raised
the possibility that these cells could convert neutrophil-derived 5-HETE to 5-oxo-ETE by transcellular metabolism. However, mixtures of
platelets and neutrophils synthesized relatively small amounts of
5-oxo-ETE, but instead formed substantial amounts of its 12-hydroxy metabolites. There are several pathways for the formation of the two
5-oxo-12-hydroxy metabolites, as shown in
Fig 10. The 8-cis isomer, which is
normally the more abundant of the two, can be formed either by
oxidation of 5S,12S-diHETE by 5-hydroxyeicosanoid dehydrogenase or
by the action of 12-LO on 5-oxo-ETE. The 8-trans isomer is probably a
mixture of 12R-hydroxy and 12S-hydroxy epimers, formed by the oxidation
of 6-trans-LTB4 or 12-epi-6-trans-LTB4 by
5-hydroxyeicosanoid dehydrogenase. Because the two
5-oxo-12-hydroxy-8-trans isomers possess only one chiral center, they
would not be separated by RP-HPLC. In agreement with this, stimulated
neutrophils alone synthesized very small amounts of
8-trans-5-oxo-12(RS)-HETE but not the corresponding 8-cis isomer (Fig
3B). It is likely that some of the 8-trans-5-oxo-12-HETE formed in the
presence of platelets was formed nonenzymatically from the 8-cis
isomer, because the latter substance is rather unstable and can
decompose to the trans form under certain conditions. We have also
detected the formation of 8-trans-5-oxo-ETE in incubations with
neutrophils,23 and it is also possible that isomerization,
either enzymatic or nonenzymatic, of the 8-cis double bond of 5-oxo-ETE
precedes the formation of the 12-hydroxy metabolite.
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| Fig 10.
Scheme for the formation of 5-oxo-12S-HETE and
8-trans-5-oxo-12(RS)-HETE by mixtures of platelets and neutrophils.
Arachidonic acid (AA) is converted to 12-HETE by 12-LO and to 5-HETE
and LTA4 by 5-LO. 12-HETE and 5-HETE can be converted to
5S,12S-diHETE by neutrophil 5-LO or platelet 12-LO, respectively,
whereas 5-HETE can also be converted to 5-oxo-ETE by
5-hydroxyeicosanoid dehydrogenase (5h-dh) in neutrophils or platelets.
5-oxo-12S-HETE can be formed either by the action of 5h-dh on
5S,12S-diHETE or the actions of 12-LO on 5-oxo-ETE. The 8-trans isomers
of 5-oxo-12-HETE can be formed by the actions of 5h-dh on
6-trans-LTB4, which is converted to 8-trans-5-oxo-12R-HETE,
and 12-epi-6-trans-LTB4, which is converted to
8-trans-5-oxo-12R-HETE.
|
|
It is clear that the total amounts of 5-oxo-eicosanoids formed by
stimulated neutrophils increased considerably as increasing numbers of
platelets were added. This is likely to be caused at least in part by
the presence of 5-hydroxyeicosanoid dehydrogenase in platelets.
However, addition of platelets to neutrophils also increased the
amounts of 5-HETE that were formed (Fig 3B, inset), and this could have
resulted in increased 5-oxo-eicosanoid formation by both neutrophils
and platelets. The stimulatory effect of platelets on neutrophil 5-HETE
formation could have been because of the utilization of
platelet-derived arachidonic acid by neutrophil 5-LO. It has been shown
that when platelets and neutrophils are coincubated and stimulated,
platelets release arachidonic acid, which is converted to 5-HETE and
other lipoxygenase products by neutrophils.14,37 Stimulated
platelets could also increase the formation of 5-LO products through
the release of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid, which,
along with other hydroperoxy fatty acids, activates this
enzyme.38 In contrast to the production of 5-HETE, the production of LTB4 was not enhanced, and was perhaps
even diminished, by the presence of platelets. The difference in the
effects of platelets on 5-HETE and LTB4 formation could
possibly be caused by the existence of different pools of 5-LO in
neutrophils, because it was previously shown that platelet-derived
arachidonic acid was used preferentially for the synthesis of 5-HETE
rather than LTB4.39 Platelets could also divert
LTA4, released by neutrophils,33 to
cysteinyl-LTs, because they possess LTC synthase
activity,10-12 as well as to lipoxins.37,40
Platelets can interact with neutrophils in multiple ways to form
eicosanoids by transcellular metabolism. They possess two enzymes that
are specific for 5-LO products, namely LTC synthase, and, as shown in
the present study, 5-hydroxyeicosanoid dehydrogenase, which
interconverts 5-HETE and 5-oxo-ETE. In addition, platelets can
metabolize a variety of 5-LO products by the 12-LO pathway. 5-HETE is
converted to 5S,12S-diHETE by this enzyme,41 whereas LTA4 is converted to lipoxins.42 As shown here,
5-oxo-ETE is also a substrate for platelet 12-LO.
To determine whether the major 5-oxo-ETE metabolites formed by
activated platelets are biologically active, their effects on calcium
mobilization in neutrophils were tested. As shown in Fig 8, neither
5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE at concentrations as high as 10 µmol/L has a significant effect on cytosolic calcium levels in
neutrophils. In contrast, concentrations of 5-oxo-ETE as low as 1 nmol/L mobilize calcium in these cells.19 Thus, addition of
a 12-hydroxyl group and replacement of the 11-cis double bond of
5-oxo-ETE with a 10-trans double bond result in a loss of potency of
over 10,000-fold. This adds support to the concept that neutrophils
possess a highly specific recognition mechanism for 5-oxo-ETE.
We19,23 and others43 have shown that relatively
minor changes in the structure of 5-oxo-ETE result in substantial
losses in biological activity. This, along with evidence implicating
the involvement of a G-protein in 5-oxo-ETE-induced responses,22-24 suggests that the actions of this substance
are mediated by its interaction with a specific G-protein coupled 7-transmembrane domain receptor.
Although both 5-oxo-12-HETE and its 8-trans isomer lack significant
agonist activity, both substances seem to have some antagonist activity, because they were able to substantially reduce
5-oxo-ETE-induced calcium mobilization. In the case of the 8-cis
derivative, the calcium response to 5-oxo-ETE was virtually abolished
at a concentration of 10 µmol/L and reduced by over 50% at a
concentration of 1 µmol/L. These results raise the possibility that
it may be feasible to develop specific antagonists to 5-oxo-ETE that
could be used to investigate its physiological role.
In contrast to its potent effects on neutrophils and eosinophils,
5-oxo-ETE had only a very modest effect on human platelets. None of the
5-oxo-eicosanoids synthesized by platelets induced platelet aggregation
at concentrations up to 20 µmol/L. However, relatively high
concentrations of 5-oxo-ETE and 5-oxo-12-HETE inhibited
arachidonate-induced aggregation. It is interesting that there was some
structural specificity for this inhibitory response, because
8-trans-5-oxo-12-HETE was ineffective. However, the high concentrations
of 5-oxo-eicosanoids required for this response would not support a
physiological role for these compounds in regulating platelet function,
and it is likely that they are more relevant to granulocytes.
It would seem unlikely that the role of 5-hydroxyeicosanoid
dehydrogenase in platelets is to synthesize 5-oxo-ETE. Although platelets possess a high capacity to synthesize this compound, as shown by the experiments with PMS shown in Figs 4 and 5, this capacity may not be realized under normal circumstances. Unstimulated platelets synthesize relatively small amounts of 5-oxo-ETE, presumably because insufficient levels of NADP+ are available in the
resting state. Thus, even if platelets were exposed to 5-HETE released
by inflammatory cells, little 5-oxo-ETE would be formed unless the
platelets were stimulated. Although increased amounts of
5-oxo-eicosanoids are formed by stimulated platelets, this also results
in the activation of 12-LO, which converts 5-oxo-ETE to its
biologically inactive 12-hydroxy metabolites, which, if present in high
concentrations, could antagonize the effects of 5-oxo-ETE. Thus it
would seem unlikely that platelets could be a significant source of
biologically active 5-oxo-ETE under physiological conditions.
The small amounts of 5-oxo-ETE formed by platelets could be caused by
the reversibility of 5-hydroxyeicosanoid dehydrogenase, which would
explain the high 5-ketoreductase activity observed in these cells (Fig
6). Thus, when unstimulated platelets are presented with either
5-oxo-ETE or 5-HETE, the equilibrium would always be on the side of
5-HETE. We have previously shown that neutrophil microsomes
stereospecifically convert 5-oxo-ETE to 5S-HETE in the presence of
NADPH.3 It is therefore possible that platelet
5-hydroxyeicosanoid dehydrogenase serves to inactivate 5-oxo-ETE by
converting it to 5-HETE, which has only about 1% of the biological
potency of the former substance.19 Thus, resting platelets
could inactivate 5-oxo-ETE by converting it to 5-HETE, whereas
stimulated platelets could inactivate it by converting it to
5-oxo-12-HETE.
In conclusion, we have shown that platelets possess 5-hydroxyeicosanoid
dehydrogenase, which displays properties similar to the neutrophil
enzyme. In spite of this, intact resting platelets form relatively
little 5-oxo-ETE and instead may use this enzyme as a 5-ketoreductase
to inactivate 5-oxo-ETE released by inflammatory cells. Activated
platelets convert 5-oxo-ETE to biologically inactive 12-hydroxy
metabolites, which at high concentrations seem to act as 5-oxo-ETE
antagonists. Thus, platelets may be a site for the biological
inactivation of 5-oxo-ETE in blood.
| |
ACKNOWLEDGMENT |
We are grateful to Dr Daniel Boismenu, Biomedical Mass Spectrometry
Unit, McGill University, for assistance with the mass spectrometry.
| |
FOOTNOTES |
Submitted May 8, 1998; accepted September 21, 1998.
Supported by the Medical Research Council of Canada (W.S.P.; grant no.
MT-6254), the Quebec Heart and Stroke Foundation (W.S.P.), the J.T.
Costello Memorial Research Fund, and the National Institutes of Health
(J.R.; grant no. DK44730). J.R. also wishes to acknowledge the National
Science Foundation for an AMX-360 NMR instrument (grant no.
CHE-90-13145).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to William S. Powell, PhD, Meakins-Christie
Laboratories, 3626 St Urbain St, Montreal, Quebec, Canada H2X 2P2;
e-mail: Bill{at}Meakins.LAN.McGill.ca.
| |
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Abstract
Introduction