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INTRODUCTION |
The protease thrombin is known to play a central role in
physiologic hemostasis. Thrombin catalyzes the formation of fibrin from
fibrinogen and amplifies the coagulation process through the
proteolytic activation of factors V, VIII, and XI. By means of the
proteolysis of specific cell membrane receptors, thrombin activates
platelets and regulates multiple cellular processes such as
proliferation and chemotaxis (1, 2). The rapid formation of thrombin
from prothrombin is thought to be mediated mainly by the prothrombinase
complex assembled on the outer leaflet of the plasma membrane of the
activated platelets. There is general agreement that under in
vivo conditions this complex mediates the large scale synthesis of
thrombin. The assembly of factors Va and Xa on the platelet surface is
most probably triggered initially by the translocation of
aminophospholipids such as phosphatidylserine (PS)1 from the inner to the
outer leaflet of the cell membrane (3). The appearance of specific
phospholipids on the platelet surface facilitates the interaction of
factor Va with the platelet cell membrane. This, in turn, enables the
binding of factors Xa and II (prothrombin), thereby allowing the
proteolysis of prothrombin to thrombin as catalyzed by factor Xa.
High local concentrations of thrombin within and around the
atherosclerotic plaque (4) suggest that the protease may also be of
importance for the development of thrombosis. Because arterial thrombosis is the major single cause of mortality in industrialized countries, the elucidation of the mechanisms causing the increased formation of thrombin is of particular interest. According to recent
evidence, coronary thrombosis leading to myocardial infarction is in
most cases induced by a thrombus that develops following the rupture of
a lipid-rich, unstable atherosclerotic plaque (5). The nature of the
lipidic material present in the unstable plaque is apparently of great
relevance for the extent of pathological activation of coagulation.
Among other components, the plaques are enriched with oxidized low
density lipoproteins (LDL). Substantial evidence obtained over the last
decade suggests a causal role for oxidized LDL in the development of
atherosclerosis (6). Recent data, moreover, support the view that
oxidized LDL may also directly promote thrombogenesis. This hypothesis
is based on results demonstrating that oxidized LDL are able to promote the initiation of coagulation by enhancing the expression of tissue factor (7, 8) and to mitigate anticoagulant mechanisms such as
thrombomodulin-dependent protein C activation (9) and the expression of tissue factor pathway inhibitor (10). In the present study, we investigated the influence of oxidized LDL on the activity of
the platelet prothrombinase complex. The modified lipoproteins were
observed to increase thrombin formation strongly, suggesting a relevant
role for oxidized LDL in the development of thrombosis. A series of
separation procedures led to the identification of oxidized
phosphatidylethanolamine (PE) as the lipoprotein component mediating
the strongest stimulation of the platelet thrombin generation.
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EXPERIMENTAL PROCEDURES |
Materials--
Egg phosphatidylcholine (PC),
1-palmitoyl,2-palmitoyl-PC (dipalmitoyl-PC),
1-palmitoyl,2-linoleoyl-PC, egg PE, 1-palmitoyl,2-linoleoyl-PE, 1-palmitoyl,2-lyso-PE (lyso-PE),
1-palmitoyl,2-linoleoyl-phosphatidylinositol (PI), sphingomyelin (from
bovine brain), 1-palmitoyl,2-lyso-PC (lyso-PC), sphingomyelin (bovine
brain), copper acetate,
-thrombin, collagen (type VIII),
myeloperoxidase, diethylenetriaminepentaacetic acid,
NaCNBH3, and factor Xa were obtained from Sigma.
Fluorescein isothiocyanate-labeled annexin V was purchased from
Alexis, and phycoerythrin-labeled anti-P-selectin antibody was from
Serotec. 4-Hydroxynonenal (HNE) was from Calbiochem. Iloprost was
kindly provided by Schering. The anti-CD36 antibody was obtained from Immunotech. 2,2'-Azobis-(2-amidinopropane hydrochloride) (AAPH) was
provided by Polysciences Inc. Factor Va and factor II were from Enzyme
Research Laboratory. Substrate S-2238 was obtained from Chromogenix.
Plasmalogen-PE was isolated from total bovine brain PE by alkaline
methanolysis as described (11).
Lipoprotein Isolation and Oxidation--
LDL was prepared from
healthy donors by ultracentrifugation (12), dialyzed against
argon-bubbled phosphate-buffered saline containing 0.3 mM
EDTA and stored under argon at 4 °C. The amount of LDL-associated
protein was determined using the Bradford procedure (13). Before the
start of oxidation, LDL was freshly dialyzed against phosphate-buffered
saline. To oxidize the lipoproteins with copper and peroxyl
radicals, the particles (0.2 mg protein/ml) were treated for different
time intervals at 37 °C with 10 µM copper acetate and
for 4 h with 4 mM AAPH, respectively. Oxidation was
terminated by the addition of butylated hydroxytoluene (BHT, 20 µM). Myeloperoxidase-mediated LDL oxidation was performed
according to a previously published protocol (14). LDL (0.5 mg
protein/ml) was treated for 1 h at 37 °C with 100 nM myeloperoxidase in the presence of 100 µM
L-tyrosine, 100 µM
diethylenetriaminepentaacetic acid, 1 mM
H2O2 suspended in 100 mM NaCl, and
20 mM Na2HPO4. After the addition
of BHT, the suspensions were passed through centricon filters, and the
lipoproteins were recovered.
Preparation of Microemulsions and Lipid Vesicles--
Following
the oxidation of the lipoproteins, the particles (50 µg of protein)
were divided into aqueous and lipid phases according to the Bligh and
Dyer procedure (15). The upper two-thirds of aqueous fractions
(containing less than 10% of the total protein contents of the whole
LDL particles) was tested directly for its effects on platelet
prothrombinase activity. The organic phase was evaporated under
N2 and dispersed as a microemulsion by three sonication
steps (3 min each, with 1-min breaks in between) at 4 °C under a
constant stream of N2. Thereafter, the microemulsions were
incubated with the platelets for analysis of the thrombin formation. To
prepare the lipid mixtures from oxidized LDL deficient in specific
lipid components, the lipid phases (from 1 mg LDL) were first
resuspended in 500 µl of CHCl3/CH3OH (2:1).
Then, 10 identical portions of the suspensions were applied in parallel onto Silica G60 plates (Merck), and the samples were developed in
CHCl3/CH3OH/H2O/NH3
(90:54:5.5:5.5). The spots on the lanes were visualized with
diphenylhexatriene spray. The following areas were scraped from the
plates: (i) the complete lane between the origin and the top; (ii)
complete lanes lacking one specific spot (which was left on the plate);
(iii) a portion of the plate to which no material had been applied
having the same length and width as the lane mentioned under (i) above
(blank). In other experiments, the individual lipid spots were
recovered and further processed as described below.
For elution of the lipids, CHCl3/CH3OH (1:4)
was added to the silica, and the procedure was repeated twice.
Subsequently, the lipids were resuspended in a biphasic system
consisting of 5 volumes of CHCl3/CH3OH (2:1)
and 2 volumes of CH3OH/H2O (1:2) at 4 °C to
remove the residual silica. After centrifugation at 4 °C, the lower
phase was saved. Dipalmitoyl-PC (100 nmol) was added to the purified
organic phase. Following evaporation, the lipids were dispersed by
sonication in a buffer composed of 140 mM NaCl, 10 mM Hepes, 5 mM KCl, 1 mM
MgCl2, 5 mM glucose (pH 7.4) (resuspension
buffer). The mixtures were subjected to two sonication steps (2 min
each, with 1-min breaks in between). The dispersions were centrifuged
for 10 min at 4 °C and 1200 × g, and the upper 80%
of the supernatant was used as vesicle solution.
Lipid vesicles were also prepared directly from either dipalmitoyl-PC
or 1-palmitoyl,2-linoleoyl-PC (95 mol % each) and enriched with
1-palmitoyl,2-linoleoyl species of PE, PI, PC (diacyl subtypes), or
freshly prepared plasmalogen-PE (all at 5 mol %). The plasmalogen PE
used had been shown previously to contain predominantly fatty acids
with two double bonds at the sn-2 position (11). The
phospholipids were dispersed in the resuspension buffer as described
above for the LDL-extracted lipids. Subsequently, the vesicle
suspensions were oxidized for 10-12 h at 37 °C with 10 µM copper acetate. The oxidation was terminated with BHT,
and the oxidized vesicles were added to the platelet suspensions in
order to study their effects on the prothrombinase activity.
Separation and Analysis of LDL Lipids following
Oxidation--
After completion of the incubation with copper
acetate, the lipids were extracted from LDL (15) using chloroform
supplemented with BHT (50 µg/ml). The LDL lipids were separated by
one-dimensional thin layer chromatography (tlc) with the solvent
CHCl3/CH3OH/H2O/NH3 (90:54:5.5:5.5). The lipid oxidation products were visualized with
iodine vapor. Although the possibility cannot be excluded that some
lipid oxidation products may not have been detected by the iodine
vapor, this method allows the visualization of a broad spectrum of
modified lipids. Fractions I (Rf value = 0.11)
and II (Rf = 0.23) comigrated with native lyso-PC and native sphingomyelin, respectively. Fraction III
(Rf = 0.34)) migrated at the same height as native
egg PC and native PI, whereas fractions IV (Rf = 0.55) and V (at the solvent front) comigrated with native egg PE and
native neutral lipids (cholesterol and triglycerides), respectively.
For quantification of the modified phospholipids present in fractions
I-IV, the corresponding spots were scraped off and their phosphate
contents determined (16). Commercially obtained phospholipids
(sphingomyelin (bovine brain), egg PC, and egg PE) were enriched in
dipalmitoyl-PC vesicles and oxidized for 12 h with copper acetate
(10 µM). After extraction, the oxididation products were
subjected to the one-dimensional tlc procedure described above. The
oxidation products of sphingomyelin migrated with native sphingomyelin,
whereas those generated by oxidation of egg PC comigrated with native
lyso-PC and native PC. Oxidation of egg PE yielded oxidation products
comigrating with lyso-PE (Rf = 0.52) and native PE.
An additional spot became visible at the solvent front following
oxidation of egg PC and egg PE. To substantiate the identity of the
modified lipids contained in the fraction migrating at an
Rf value of 0.55, this spot was eluted from the
plate using CHCl3/CH3OH (1:4). Thereafter, the
lipids were further separated by two-dimensional tlc using the solvents
CHCl3/CH3OH/CH3COOH (65:25:10)
(first direction) and
CHCl3/CH3OH/HCOOH/H2O
(65:25:8.9:1.1) (second direction). Therefore, only a single spot
became visible that could be stained with the amino group reagent
ninhydrine. Fraction IV of the oxidized LDL therefore predominantly
contained modified ethanolamine phospholipids.
Enrichment of LDL with Phospholipids--
Fresh venous blood
obtained from healthy donors was drawn into tubes containing EDTA (1 mg/ml), and plasma was prepared by centrifugation. For the enrichment
of LDL with ethanolamine phospholipids, 1 µmol of either
1-palmitoyl,2-linoleoyl-PE or plasmalogen-PE (from bovine brain) was
dissolved together with 3 µmol of egg PC in 100 µl of ethanol. For
the preparation of the control samples, 4 µmol of egg PC was
dissolved in the same volume of ethanol. The solutions were added very
slowly under stirring to 10 ml of plasma, and the suspensions were
incubated at 37 °C under argon for 6 h. The LDL particles were
isolated by ultracentrifugation and dialyzed against phosphate-buffered
saline under argon at 4 °C.
Synthesis of Aldehyde-PE Adducts--
Synthetic HNE-PE adducts
were prepared by adding 1 mol of 1-palmitoyl,2-linoleoyl-PE (dissolved
in 1 ml of diethylether) to 2 mol of HNE suspended in 1 ml of 0.75 M NaCl and 1 mM Hepes (pH 8.5) (17). The
mixture was incubated under vigorous shaking for 2 h in the
dark in the presence of argon (30 °C). Thereafter, the lipids of the
ether phase were separated by one-dimensional tlc with the solvent
CHCl3/CH3OH/NH3/H2O
(90:54:5.5:5.5; vol:vol). The PE adducts migrating at the
Rf value of 0.55 were eluted from the silica using
CHCl3/CH3OH (1:4). To assess the amount of
Schiff base adducts formed, the reaction mixture was incubated in the
absence and presence of NaCNBH3 (25 mM). The extracted lipids were separated with the solvent
CHCl3/CH3OH (9:1). The spots representing
underivatized PE and ethanolamine phospholipids with reduced Schiff
base (18) were eluted from the silica. Phosphate analysis indicated
that 9.1% of the total PE recovered had been reduced by
NaCNBH3.
Prothrombinase Activity--
Platelet-dependent
prothrombinase activity was analyzed as described previously (19). For
isolation of the platelets, blood (anticoagulated by 0.38% citrate)
from healthy volunteers was centrifuged at 330 × g for
15 min and the supernatant recovered. Platelet-rich plasma was
supplemented with iloprost (10 ng/ml) and centrifuged at
3700 × g for 10 min, and the pellet washed once at
room temperature with a buffer consisting of 138 mM NaCl, 3 mM KCl, 1 mM MgCl2, 15 mM Hepes, 5 mM glucose (pH 6.3, washing buffer). Subsequently, the supernatant was removed, and the cells were
added to the resuspension buffer. The mixture was slowly warmed up to
37 °C. The platelets were adjusted to 2 × 107/ml
and incubated in the presence of native and oxidized LDL, microemulsions, and phospholipid vesicles for 5 min at 37 °C in the
resuspension buffer. 60 µl of this suspension were given to 540 µl of resuspension buffer containing, additionally, 5 mM CaCl2, 1.1 nM factor Va, and
0.52 nM factor Xa (final concentrations). Subsequently,
factor II (dissolved in resuspension buffer, final concentration 0.43 µM) and the chromogenic substrate S-2238 were added
(final concentration 0.26 mM). The increase in absorption at 405 nm was measured and compared with standard curves obtained with
different concentrations of thrombin.
Flow Cytometry--
Washed platelets (107, isolated
as described above) were incubated for 10 min at 37 °C in 500 µl
of the resuspension buffer (supplemented with 2.5 mM
CaCl2 and 1 mg/ml bovine serum albumin) in the presence of
the oxidized LDL (50 µg protein/ml). Thereafter, the platelet
suspensions were incubated for 30 min at room temperature with 100 µl
of CellFIX (Becton Dickinson). After removal of the supernatant by
centrifugation, the fixated platelets were incubated for 10 min in
CellWash buffer (Becton Dickinson) with fluorescein isothiocyanate-labeled annexin V (150 nM). In separate
samples, the platelets were incubated in the dark with the
phycoerythrin-labeled anti-P-selectin antibody (10 µg/ml). The
suspensions of the labeled platelets were centrifuged and washed once
with CellWash (Becton Dickinson), and following the addition of 250 µl of CellWash, their mean fluorescence intensities were analyzed
using a FACSCan flow cytometer (Becton Dickinson). Platelets were
identified on the basis of their typical forward and sideward scatter characteristics.
Statistics--
All mean values given are ± S.D.
p values < 0.05 were considered significant.
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RESULTS |
Enhancement of Platelet Prothrombinase Activity by Oxidized
LDL--
In a first series of experiments, native LDL as well as
lipoprotein particles previously oxidized for 4 h with copper were added to the platelet suspensions. Although native LDL did not affect
the platelet dependent thrombin formation, the copper oxidized LDL
enhanced the synthesis of the protease by 5.5-fold (Fig.
1a). When platelets had been
incubated for 5 min with copper (10 µM) in the absence of
the lipoproteins, their prothrombinase activity remained unchanged
(1.2 ± 0.4-fold versus control). Moreover, when the
4-h incubation of LDL with copper was performed in the presence of the
hydrophobic radical scavenger BHT (50 µg/ml), again no stimulation of
the platelet-dependent thrombin formation was observed
(1.0 ± 0.2-fold versus control; means ± S.D.
from three independent experiments). Taken together, these results make
it unlikely that soluble or lipoprotein bound copper per se
contributed to the enhancement of the prothrombinase activity by copper
oxidized LDL. The platelet agonists thrombin and collagen, known
stimulators of platelet prothrombinase activity, augmented the thrombin
formation by 3.6 (thrombin)- and 6.0-fold (thrombin plus collagen)
(Fig. 1a). In separate experiments, the particles were
oxidized for 4 h with the peroxyl radical generator AAPH (4 mM). Therefore, thrombin generation was stimulated by
4.6 ± 1.2-fold (means ± S.D. from four independent
experiments). LDL oxidized for 2, 4, and 12 h with copper elevated
platelet prothrombinase activity by 3.1-, 5.7-, and 4.6-fold,
respectively, as compared with the untreated platelets (Fig.
1b). In the presence of thrombin, the LDL particles
previously treated for different time periods with copper accelerated
the prothrombinase activity by 7.4 (oxidation, 2 h)-, 10.5 (4 h)-,
and 10.9-fold (12 h), respectively. Because the 4-h oxidation of LDL
appeared to be the shortest time period inducing the maximal
stimulation of the thrombin formation, this time interval was adopted
in the subsequent experiments.

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Fig. 1.
Platelet prothrombinase activity is
stimulated by oxidized LDL. a, platelets (2 × 107/ml) were incubated for 5 min at 37 °C with native
LDL (nLDL) and oxidized LDL (oxLDL) (both at 50 µg protein/ml). Oxidation of the particles was performed by a 4-h
incubation with copper (10 µM). Platelets were also
stimulated with thrombin (T, 0.5 units/ml) and collagen
(C, 10 µg/ml). The results are expressed as fold increase
versus the prothrombinase activity of untreated platelets.
b, LDL was oxidized for the indicated time intervals
with 10 µM copper and the prothrombinase activity
analyzed in the absence (open symbols) and presence
(filled symbols) of thrombin. The mean of 4-12 independent
experiments is shown.
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The prothrombinase activity in the absence of platelets was barely
promoted by the copper oxidized LDL (0.012 ± 0.004 (oxidized LDL)
versus 0.006 ± 0.003 milliunits/ml (control)), the
value being below the one determined in the presence of the untreated platelets (0.028 ± 0.007 milliunits/ml, means ± S.D. from
three independent experiments). Thus, the presence of the platelets was
necessary to elicit substantial stimulation of the thrombin formation
by oxidized LDL. Activated platelets are known to enhance the
prothrombinase activity by exposing aminophospholipids on their
surface. To register the appearance of the aminophospholipids, we
analyzed the platelet binding of annexin-V (Fig.
2a). Native LDL did not alter
the aminophospholipid exposure. In contrast, oxidized LDL caused a
4.8-fold increase in annexin V binding compared with the untreated
platelets. Thrombin alone and oxidized LDL plus thrombin augmented the
annexin-V binding by 2.5- and 7.5-fold (Fig. 2a). In
parallel experiments, we measured the exposure of P-selectin on the
platelet surface, an activation-dependent process indicative of the degranulation of the
-granula (Fig.
2b). Compared with the untreated platelets, the modified LDL
increased the P-selectin fluorescence by 2.5-fold, whereas native LDL
was ineffective. Thrombin alone enhanced the P-selectin exposure by
2.3-fold. In the presence of thrombin plus oxidized LDL, the P-selectin
fluorescence was increased by 4.8-fold (Fig. 2b). Together,
the results demonstrate that oxidized LDL stimulates the prothrombinase
activity by promoting the formation of a procoagulant surface, the
effect being additive to the one elicited by thrombin.

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Fig. 2.
Oxidized LDL increases the aminophospholipid
exposure on the platelet surface. a, platelets (2 × 107/ml) were incubated for 5 min at 37 °C with native
LDL (nLDL) and oxidized LDL (oxLDL) (both at 50 µg protein/ml; copper-oxidized particles) as well as with thrombin
(0.5 units/ml) and oxidized LDL plus thrombin for 5 min at 37 °C.
The exposure of the aminophospholipids was estimated by determining the
binding of fluorescein isothiocyanate-labeled annexin-V. co,
untreated platelets alone. b, under identical
conditions, the P-selectin appearance was measured using
phycoerythrin-labeled anti-P-selectin antibody. The mean of 5 independent experiments is shown.
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Active Components of Oxidized LDL Promoting Thrombin
Formation--
To identify the components of oxidized LDL responsible
for the stimulation of thrombin generation, we separated the oxidized particles into aqueous and lipid phases. The addition of the aqueous phases from native and oxidized LDL to the platelets did not alter their prothrombinase activity (Fig.
3a). Because the protein
contents of the aqueous supernatants amounted to less than 10% of the
protein contents of the intact LDL particles, no conclusion can be
reached from these data regarding the potential influence of the
modified protein components of the oxidized LDL. Microemulsions
prepared from the total lipids of the oxidized particles enhanced the
synthesis of the protease by 7.6-fold. The microemulsions obtained from the native lipoproteins were without effect. The stimulation elicited by the lipid phases of the modified particles was further increased when the platelets were concomitantly activated with thrombin (data not
shown). Because the results showed that the active components were
mainly in the lipid portion of the modified LDL, we further separated
the modified lipids into five different fractions by a one-dimensional
tlc procedure. The lipids of the lanes were reisolated in a way that
only one respectively different fraction was left on the plate.
Thereafter, the fractions were mixed with dipalmitoyl-PC and tested for
their influence on the prothrombinase activity. Oxidized LDL lipids
deficient in fraction I and those lacking fraction II increased the
prothrombinase activity by 13 and 30%, respectively, as compared with
the thrombin formation in the presence of all modified lipids (Fig.
3b). The removal of fraction III resulted in a 24% decrease
of the thrombin generation. An even stronger reduction (by 44%) was
observed in the presence of the modified lipid mixture specifically
deficient in fraction IV. When fraction V was selectively removed, the
thrombin formation was comparable with the one determined in the
presence of the total oxidized LDL lipids (Fig. 3b). We
concluded from these results that the active components of the oxidized
LDL mediating the stimulation of the thrombin generation were present
mainly in fractions IV and III.

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Fig. 3.
Active components of oxidized LDL promoting
platelet prothrombinase activity, a, native and
copper-oxidized LDL particles (50 µg protein) were separated into
organic and aqueous phases (diagonally striped and
horizontally striped columns, respectively). The organic
phases were dispersed as microemulsions and then added to the platelets
for the measurement of the prothrombinase activity (performed as
described in legend to Fig. 1). The aqueous phases were added directly
to the platelets. b, the total lipid portion of the
oxidized LDL as well as lipid mixtures lacking in one specific fraction
were prepared as described under "Experimental Procedures," mixed
with dipalmitoyl-PC (100 nmol), and dispersed by sonication in the
resuspension buffer. *, p < 0.05 versus all
lipids. The mean of 4 independent experiments is shown.
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Next, we quantified the changes in the contents of the single lipid
fractions as induced by oxidation of LDL. Because control experiments
indicated that fractions I-IV contained native and modified
phospholipids (see "Experimental Procedures"), their phosphate
contents were determined. In Table I, the
phosphate contents of the single fractions are expressed as percentages of the total phosphate content of the LDL lipids. Although the percentages of fraction I were elevated by 3.4-fold, those of fraction
II were unchanged at the end of the oxidation period (Table I). The
proportion of fraction III was reduced by 23%. In addition, the
oxidation procedure elicited a 45% decrease in the percentage of
fraction IV (Table I). Because the modified lipids of fractions III and
IV emerged as likely candidates for causing the enhancement of
the thrombin formation by oxidized LDL, we isolated these fractions
from the modified lipoproteins and incorporated them into
dipalmitoyl-PC vesicles. As a control, vesicles containing all oxidized
LDL lipids were analyzed, which accelerated the prothrombinase activity
by 7.1-fold (Fig. 4a). Vesicles supplemented with fraction III of the oxidized LDL led to a
2.1-fold elevation of the prothrombinase activity. In the presence of
vesicles enriched with fraction IV of the oxidized LDL, the thrombin
formation was increased by 5.2-fold (Fig. 4a). Together, the
results demonstrated that fraction IV and, to a considerably lesser
extent, fraction III were the active components of the oxidized LDL
enhancing the formation of thrombin.
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Table I
Changes in the contents of LDL lipid fractions after oxidation with
copper
LDL particles (0.2 mg protein/ml) were oxidized for 4 h with 10 µM copper; their lipids were extracted (15) and separated
by one-dimensional tlc. The phosphate contents of the single spots were
measured. For the tentative identification of the lipids present in the
individual fractions, see "Experimental Procedures." Mean values
are given for lipoproteins from 3 different donors.
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Fig. 4.
Modified PE in oxidized LDL enhances
prothrombinase activity. a, total oxidized LDL lipids (from
100 µg of LDL protein) as well as fractions III and IV were isolated
from the oxidized LDL and mixed with dipalmitoyl-PC (100 nmol).
Subsequently, they were dispersed in the resuspension buffer and tested
for their effects on platelet prothrombinase activity. b,
LDL particles were enriched with 1-palmitoyl,2-linoleoyl-(diacyl)-PE
(DPE) and plasmalogen-PE (PPE, from bovine brain)
by incubation with egg PC vesicles (3 µmol) containing the
ethanolamine phospholipids (1 µmol each). Thereafter, the LDL content
of diacyl-PE was increased from 16 to 27 nmol/mg protein (DPE
enrichment), and the content of plasmalogen-PE was elevated from 29 to
47 nmol/mg protein (PPE enrichment). As control, the particles were
incubated with egg PC alone (4 µmol, PC). Subsequently,
the modified lipoproteins were oxidized for 4 h with copper (10 µM). Thereby the LDL concentrations of diacyl-PE
were reduced by 33% (control), 31% (DPE-enriched LDL), and 15%
(PPE-enriched LDL). The same pro-oxidant lowered the contents of
plasmalogen-PE by 73% (control), 66% (DPE-enriched LDL), and 49%
(PPE-enriched LDL). The oxidized LDL particles were then added
to the platelets for the measurement of prothrombinase activity. The
mean of 3-5 independent experiments is shown.
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To learn more about the identity of the modified lipids present in
fraction IV, vesicles supplemented with specific phospholipids were
oxidized and the oxidation products separated by tlc (see "Experimental Procedures"). Part of the products generated by oxidation of egg PE migrated at the same height as fraction IV. Moreover, fraction IV of the oxidized LDL stained positive after application of the amino group reagent ninhydrine. The same fraction contained phosphate (cfr. Table I). Together, these
findings indicated that oxidized species of PE were present in fraction IV isolated from the oxidized LDL. About 60% of the total ethanolamine phospholipids of the LDL particles is formed by plasmalogen-PE (alkenylacyl-PE), the rest being mostly diacyl-PE (20). LDL particles
were specifically enriched with 1-palmitoyl,2-linoleoyl-(diacyl)-PE and
with plasmalogen-PE (from bovine brain, containing mostly double
unsaturated fatty acids at sn2) by incubation with
egg PC vesicles containing the ethanolamine phospholipids. Therefore, the LDL contents of diacyl-PE and plasmalogen-PE were increased by 56 and 62%, respectively (see legend to Fig. 4b). Lipoproteins previously enriched with egg PC alone and thereafter oxidized with
copper led to a 4.5-fold enhancement of the thrombin generation (Fig.
4b). Oxidation of LDL with increased initial concentrations of diacyl-PE stimulated the prothrombinase activity by 9.5-fold, whereas the oxidative modification of lipoproteins containing higher
contents of plasmalogen-PE augmented the formation of the protease by
7.6-fold. The data suggested that oxidation products of diacyl-PE were
more effective stimulators of the prothrombinase activity than oxidized
plasmalogen-PE.
Oxidized Diacyl-PE as Strongest Stimulator of Thrombin
Generation--
To analyze in more detail the role of the
phospholipids for the stimulation of the thrombin generation, we
oxidized lipid vesicles consisting of defined species of the
phospholipids. Copper oxidation of vesicles consisting of the
1-palmitoyl,2-linoleoyl-(diacyl) species of PC led to a 1.7-fold
increase of the thrombin generation (Fig.
5a). Oxidation of PC vesicles
(95 mol %) containing additionally 5 mol % of
1-palmitoyl,2-linoleoyl-PI and 1-palmitoyl,2-linoleoyl-PE enhanced the
prothrombinase activity by 3.1- and 5.9-fold, respectively. Native
PC/diacyl-PE vesicles that had been incubated in the absence of copper
did not stimulate the thrombin formation. Following the oxidation of PC
vesicles supplemented with 5 mol % of plasmalogen-PE (from bovine
brain) and with 5 mol % of 1-palmitoyl,2-lyso-PE, generation of the
protease was promoted by 2.5- and 1.9-fold, respectively (Fig.
5a). When copper treatment of the PC/diacyl-PE vesicles was
performed in the presence of BHT, the vesicles were unable to enhance
the thrombin formation (not shown). As expected, oxidized vesicles made
from dipalmitoyl-PC did not alter the thrombin formation by the
platelets (Fig. 5b). However, oxidation of dipalmitoyl-PC vesicles (95 mol %) supplemented with 5 mol % of the
1-palmitoyl,2-linoleoyl species of PI and PE resulted in a 2.6- and
5.6-fold-enhancement of the prothrombinase activity. Concomitant
activation of the platelets with thrombin additively enhanced the
effect of the oxidized diacyl-PE and -PI (Fig. 5b).
The results indicated that oxidized unsaturated diacyl-PE elicits the
most effective stimulation of the platelet prothrombinase activity.
Both the ethanolamine head group and the unsaturated fatty acid at the
C-2 atom were required for the stimulation induced by the ethanolamine
phospholipid.

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Fig. 5.
Oxidation of unsaturated diacyl-PE promotes
thrombin formation. a, 95 mol % of
1-palmitoyl,2-linoleoyl-(diacyl)-PC was mixed with 5 mol % of
1-palmitoyl,2-linoleoyl species of PC, PI, and PE (DPE), as
well as with plasmalogen-PE (PPE; from bovine brain) and
1-palmitoyl,2-lyso-PE (LPE). The vesicles prepared from the
mixtures were oxidized for 10-12 h at 37 °C with 10 µM copper and the oxidized vesicles were added to the
platelet suspensions for the analysis of the prothrombinase activity.
oxPC/phospholipid, copper-oxidized vesicles;
nPC/DPE, untreated vesicles. b, vesicles were
prepared from mixtures of 95 mol % dipalmitoyl-PC and 5 mol % 1-palmitoyl,2-linoleoyl species of PI and PE (DPE). After
oxidation with copper, the vesicles were tested for their influence on
platelet-dependent thrombin formation. Light gray
columns, absence of thrombin; dark gray columns,
presence of thrombin. co, untreated platelets. The mean
value from 4-6 experiments is shown.
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Aldehydes generated by the oxidation of unsaturated fatty acids are
known to react with the free amino group of the PE head group yielding
adducts such as Schiff bases (17). To analyze whether Schiff bases
contributed to the stimulation of the prothrombinase activity, fraction
IV containing the oxidized PE was isolated from the modified LDL and
enriched in dipalmitoyl-PC vesicles. Thereafter, the vesicles were
treated with NaCNBH3, which specifically reduces the imino
group of the Schiff base. The 5.5-fold stimulation of the
prothrombinase activity elicited by the vesicles was reversed by 82%
following treatment with the reducing agent (Fig.
6a). In contrast, activation
of the thrombin formation by thrombin itself was not altered by
NaCNBH3. In further experiments, whole oxidized LDL
particles were treated with NaCNBH3. As a consequence, the
increased thrombin generation induced by the oxidized LDL was lowered
by 78% (Fig. 6b). NaCNBH3 also antagonized the
enhanced prothrombinase activity induced by oxidized PC/diacyl-PE
vesicles, whereas the augmented thrombin formation elicited by the
oxidized PC/PI vesicles was not altered by the reductant (not
shown).

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Fig. 6.
Prothrombinase activation by oxidized LDL is
prevented by NaCNBH3. a, LDL particles (0.2 mg
protein/ml) were oxidized for 4 h with copper (10 µM), and the supensions were treated subsequently for
2 h with NaCNBH3 (18 mM) or vehicle.
Fraction IV containing the modified ethanolamine phospholipids was
isolated from the lipoproteins, enriched in dipalmitoyl-PC vesicles,
and analyzed for its influence on platelet prothrombinase activity. In
further experiments, platelets (2 × 107/ml) were
stimulated with thrombin (0.5 units/ml) in the absence or presence of
NaCNBH3 (18 mM). Gray columns,
absence of NaCNBH3; diagonally striped columns,
presence of NaCNBH3. b, copper-oxidized LDL was
treated for 2 h with NaCNBH3 (18 mM) or
vehicle and thereafter added to the platelets for the analysis of the
thrombin generation. Gray columns, presence of oxidized LDL;
diagonally striped column, additional presence of
NaCNBH3. c, the enhancement of
platelet-annexin-V binding by oxidized LDL is reversed by
NaCNBH3. Gray columns, untreated platelets
(co) and platelets treated with oxidized LDL (as indicated);
diagonally striped column, presence of NaCNBH3.
The mean of 4-6 independent experiments is shown.
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In the experiments shown in Fig. 6c, annexin V binding to
the platelet cell membrane was determined in the presence of oxidized LDL treated with NaCNBH3. The reducing agent suppressed by
88% the stimulation of the aminophospholipid exposure by the modified LDL. To further substantiate the role of the PE adducts for the stimulation of the prothrombinase activity, synthetic adducts were
prepared from egg PE and HNE, a major lipid peroxidation product of
unsaturated fatty acids accumulating in the oxidized particles (21).
The synthetic PE adducts, which had been enriched in dipalmitoyl-PC
vesicles, enhanced thrombin formation by 3.3-fold (Table
II). Treatment of the synthetic adducts
with NaCNBH3 prevented stimulation of the prothrombinase
activity by 87% suggesting that Schiff bases were responsible for the
increased generation of thrombin. To investigate whether the presence
of the fatty acid at sn2 of the PE-HNE adduct was
essential for the enhancement of the thrombin generation, we
synthesized adducts between 1-palmitoyl,2-lyso-PE and HNE. The
lyso-PE-HNE adducts increased the prothrombinase activity by 3.0-fold,
the activation being lowered by 90% subsequent to the treatment
with NaCNBH3. The results suggested that the imino group
between the ethanolamine head group and the aldehyde was the major
determinant for the stimulation of the prothrombinase activity (see the
Discussion).
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Table II
Effect of aldehyde-PE reaction products on platelet prothrombinase
activity
Aldehyde-PE adducts were synthesized from HNE and egg PE as well as
from HNE and 1-palmitoyl,2-lyso-PE. The products were incorporated into
dipalmitoyl-PC vesicles. After treatment for an additional 2 h
with NaCNBH3 (18 mM) or vehicle, the vesicles were
subsequently added to the platelet suspensions. Mean values are given
from experiments on platelets from 3-4 different donors.
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Oxidation of LDL with myeloperoxidase in the presence of tyrosine and
H2O2 yields specific aldehydes that react with
PE to form Schiff bases similar to those occurring in vivo
in the atherosclerotic vessel wall (14). LDL was oxidized with
myeloperoxidase (see "Experimental Procedures"), and the spot
migrating at the same height as fraction IV (of the copper-oxidized
LDL) was isolated. Subsequently, the modified lipid fraction was
enriched in dipalmitoyl-PC vesicles and treated for 2 h with
NaCNBH3 or vehicle. The vesicles incubated with the buffer
alone enhanced the prothrombinase activity by 5.9 ± 2.4-fold.
Following treatment of the vesicles containing the lipids modified with
NaCNBH3 (18 mM, 2 h incubation), the stimulatory effect was completely abolished (0.8 ± 0.4-fold
versus untreated platelets, means ± S.D. of triplicate
determinations from two independent experiments). Accordingly, also the
enhancement of prothrombinase activity by myeloperoxidase-treated LDL
is likely to be caused by the PE-aldehyde adduct.
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DISCUSSION |
The importance of thrombin in the physiologic and pathophysiologic
activation of coagulation led us to analyze whether oxidized LDL, a
proatherogenic agent, affected the synthesis of the protease. We
observed a pronounced stimulation of platelet-dependent
thrombin formation by oxidized LDL, which was nearly equally as potent as the one elicited by the strong platelet agonists thrombin plus collagen. Oxidized LDL increased the prothrombinase activity by enhancing the exposure of aminophospholipids on the platelet surface. To characterize the active components within the oxidized LDL, the
particles were separated into aqueous and lipid phases. Although the
lipid phases increased the prothrombinase activity to an extent similar to that in total oxidized LDL, no stimulatory activity was
found when the platelets were exposed to the aqueous phases. From these
results the possibility cannot be excluded that the modified
protein components of the oxidized LDL might have contributed to the
promotion of the thrombin formation. The strong effect elicited by the
lipid phases led us to separate them into different oxidized lipid
fractions. Although several fractions were found to increase
platelet-prothrombinase activity, the most active component was
detected in the portion of the oxidized lipoproteins containing
modified ethanolamine phospholipids. We therefore decided to analyze in
more detail the oxidative modification of the ethanolamine phospholipids mediating the procoagulant response of the platelets.
Under in vivo conditions, the platelets are continuously
exposed to PE present in the plasma lipoproteins. The LDL-associated PE
is known to consist mainly of the plasmalogen and diacyl subgroups, both types of ethanolamine phospholipids being enriched particularly with unsaturated fatty acids (22). To evaluate the contributions of
oxidation products of the different PE subtypes for enhanced thrombin
formation, we oxidized phospholipid vesicles and LDL enriched with
unsaturated species of diacyl- and plasmalogen-PE. Oxidation of the
diacyl-PE-supplemented lipid carriers resulted in a more pronounced
acceleration of thrombin formation as compared with the oxidative
modification of the particles enriched with plasmalogen-PE. The
presence of the plasmalogen-specific enol ether thus prevented to some
extent the generation of the oxidation product promoting thrombin
formation. The enol ether has been shown previously to attenuate the
oxidative degradation of unsaturated fatty acids as induced by
different oxidants (11, 23-25). Oxidation of vesicles containing
unsaturated PC and PI resulted in a weaker stimulation of the
prothrombinase activity as compared with oxidation of vesicles with
unsaturated diacyl-PE. Moreover, oxidized lyso-PE barely enhanced the
prothrombinase activity. Together, the results demonstrate that only in
the presence of both the ethanolamine head group and unsaturated fatty
acids are oxidation products generated that are capable of
activating the thrombin formation.
Because the ethanolamine head group per se is insensitive
toward oxidation, whereas, in contrast, unsaturated fatty acids are
easily degraded, the latter components will be decomposed first by the
oxidative attack. Oxidation of the unsaturated fatty acids results in
the formation of aldehydes, which are known to react with the free
amino groups of lysine residues and of phospholipids. Thereby, several
types of adducts are generated including Schiff bases. When we treated
the modified ethanolamine phospholipids isolated from the oxidized LDL
with NaCNBH3, a specific reductant for Schiff bases, the
stimulatory effect of the oxidized phospholipids on prothrombinase
activity was lost. Furthermore, NaCNBH3 inhibited the
exposure of the aminophospholipids on the platelet surface as promoted
by the oxidized LDL. The reducing agent also prevented the enhancement
of the prothrombinase activity elicited by the synthetic aldehyde-PE
adducts. Taken together, the results suggest that Schiff base
PE-aldehyde adducts are the most active components of the oxidized LDL
provoking the stimulation of the prothrombinase activity.
Oxidation of vesicles made from saturated PC and unsaturated diacyl-PE
stimulated prothrombinase activity to an extent comparable to that in
the oxidized vesicles composed of unsaturated PC plus unsaturated
diacyl-PE. This finding indicated that the extent of
non-saturation of PC molecules does not play a major role for the
promotion of the activity of the platelet prothrombinase complex. Most
likely, therefore, the aldehyde partner for the adduct formation originates mainly from the PE-associated unsaturated fatty acid itself.
Thus, the condensation reaction most likely occurrs intramolecularly. The generation of the aldehyde-PE adduct results in the loss of the
positive charge of the ethanolamine moiety. Consequently, PE acquires a
net negative charge. One might assume that this modification could
enable the phospholipid to interact with CD36, a platelet cell membrane
protein with high affinity for anionic phospholipids and for oxidized
LDL (26, 27). However, because the negative charge of the modified PE
is retained after treatment with NaCNBH3, whereas the
stimulatory influence on prothrombin cleavage is lost, the enhancement
of the prothrombinase activity must be caused by other structural
components of the adduct. As the treatment with NaCNBH3
results in the selective chemical reduction of the Schiff base-specific
imino group, this moiety appears to be crucial for the stimulation of
the prothrombinase activity. Together with the
-,
-unsaturated
double bond of the aldehydes, the imino group forms a system of
conjugated double bonds. The high reactivity of the conjugated double
bonds could enable the modified PE to interact with platelet cell
membrane receptors involved in the activation of the platelets. Earlier
data already indicate that extracellular N-substituted
phosphatidylethanolamine is able to stimulate platelet aggregation and
secretion (28). Our results suggest that the platelet activation
induced by the modified PE of oxidized LDL results in the enhanced
exposure of aminophospholipids on the platelet surface, a mechanism
facilitating the assembly of the prothrombinase complex.
It is well known that aldehydes originating from the oxidation of
unsaturated fatty acids can react with lysine residues of apoprotein
B100 yielding adducts that are recognized by the classic scavenger
receptors of the A type (29). Such aldehyde-protein adducts were
recently shown to be responsible for the stimulation of macrophage
growth induced by oxidized LDL (30) and for the decreased binding of
the modified particles to aortic proteoglycans (31). However,
the protein adducts are unlikely to have contributed to a major extent
to the stimulation of platelet prothrombinase activity by oxidized LDL.
Indeed, the lipid microemulsions prepared from the oxidized LDL
enhanced the prothrombinase activity in a manner comparable to the
whole particles. Furthermore, the inhibitory effect elicited by
chemical reduction with NaCNBH3 was rather similar when the
activation of the platelets had been induced by the oxidized LDL as
compared with the one triggered by the modified PE fractions of the lipoproteins.
Our results might contribute to a more profound understanding of the
mechanisms leading to the formation of thrombi at the site of the
unstable atherosclerotic plaques. Upon rupture of the plaque, the
oxidized LDL of the vascular wall is exposed immediately to the blood
platelets. According to the results of our study, this will
strongly augment platelet prothrombinase activity. Therefore, the local
formation of thrombin is expected to be markedly increased; this will
result in the enhanced generation of fibrin with subsequent intravascular thrombus formation. In summary, we find that among several modified lipid fractions of oxidized LDL that increase platelet prothrombinase activity, oxidation products of ethanolamine phospholipids elicit the strongest stimulation. Schiff bases formed by
the condensation of the head group of ethanolamine phospholipids with
aldehydes generated by the oxidation of unsaturated fatty acids
contribute to a major extent to the enhancement of the prothrombinase activity. By promoting the large scale production of thrombin through
the prothrombinase complex, oxidized LDL is expected to implement a
strong prothrombotic stimulus under in vivo conditions.