From the Departments of Cardiovascular Biology and
§ Free Radical Biology and Aging, Oklahoma Medical Research
Foundation, the Departments of ¶ Pathology and
Biochemistry
and Molecular Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, and the ** Howard Hughes Medical Institute, Oklahoma
City 73104
Received for publication, July 6, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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Although lipid oxidation products are usually
associated with tissue injury, it is now recognized that they can also
contribute to cell activation and elicit anti-inflammatory lipid
mediators. In this study, we report that membrane phospholipid
oxidation can modulate the hemostatic balance. Oxidation of natural
phospholipids results in an increased ability of the membrane surface
to support the function of the natural anticoagulant, activated protein
C (APC), without significantly altering the ability to support thrombin generation. Lipid oxidation also potentiated the ability of protein S
to enhance APC-mediated factor Va inactivation.
Phosphatidylethanolamine, phosphatidylserine, and polyunsaturation of
the fatty acids were all required for the
oxidation-dependent enhancement of APC function. A subgroup
of thrombotic patients with anti-phospholipid antibodies specifically
blocked the oxidation-dependent enhancement of APC function. Since leukocytes are recruited and activated at the thrombus
or sites of vessel injury, our findings suggest that after the initial
thrombus formation, lipid oxidation can remodel the membrane surface
resulting in increased anticoagulant function, thereby reducing the
thrombogenicity of the thrombus or injured vessel surface.
Anti-phospholipid antibodies that block this process would therefore be
expected to contribute to thrombus growth and disease.
The central role of the membrane surface in the reactions of the
coagulation cascade has been well known for many years (1, 2). A
negative charge on the membrane is recognized as necessary for the
binding of the vitamin K-dependent enzymes and substrates through their N-terminal
Gla1 domains.
Through this binding, the local concentrations of the proteins are
markedly increased, augmenting activation rates. Binding may also
induce conformational changes in the proteins, aligning substrate
cleavage sites with the active site of the enzyme (3). The nature of
the phospholipid head group was known to play a role, and
phosphatidylserine (PS) has been considered the most important (4, 5).
For many years, it was believed the reactions of the coagulation
cascade shared similar requirements for the membrane surface. However,
most experiments were performed with the prothrombin-activating
complex, prothrombinase, with a tendency to generalize to the other
complexes. More recently, it has become apparent that the presence of
phosphatidylethanolamine (PE) potently enhanced the rate of
inactivation of factor Va by the activated protein C (APC) complex (6)
and the anticoagulant activity in plasma (7) while having little to no
effect on the prothrombinase reaction when the PS concentration was
optimal. Polyunsaturation of the phospholipids further enhanced the
activity of the APC complex (8). Subsequently, roles for PE in factor VIII binding (9, 10), tissue factor-factor VIIa activation of factor X
(11), and prothrombin activation (12, 13) were reported. In the latter
studies, the incorporation of PE into the vesicles decreased the amount
of PS required for optimal prothrombin activation (12) much as we
observed (8). Unlike the APC system, the PE stimulation could be
overcome by increased PS.
The role of oxidation in disease has gained considerable attention in
the last several years. Oxidation is believed to play a key role in the
pathogenesis of many inflammatory diseases including atherosclerosis,
reperfusion injury, and autoimmune diseases (14-17). Oxidized LDL
plays a major role in the initiation and propagation of the
atherosclerotic plaque. It can also lead to the activation of
endothelium and platelets, possibly through lysophosphatidic acid, a
product of oxidation (18). Hydrogen peroxide, a substance elaborated by
activated leukocytes, can also act as a direct cell agonist (19, 20).
Endothelial cell activation can then lead to the elaboration of other
inflammatory mediators which themselves exacerbate coagulation (21, 22)
and induce the expression of adhesion molecules, allowing the
attachment of monocytes and neutrophils (23). Endothelial cells and
leukocytes themselves can elaborate reactive oxygen species leading to
oxidation of additional surfaces (16, 24, 25). Oxidation of red blood cells results in increased exposure of both PS and PE on the membrane surface, making these cells potentially procoagulant (15).
Oxidized LDL can also be immunogenic and lead to the development of
antibodies that cross-react with cardiolipin or the surface of
endothelial cells (26, 27). In addition, apoptotic cells with
negatively charged surfaces can be immunogenic and lead to the
development of anti-phospholipid antibodies (APAs) with procoagulant activity (28). These observations led Horrko et
al. (29, 30) to determine that many APAs are directed toward
epitopes of oxidized phospholipids.
Oxidation has long been recognized as having an effect in the in
vitro coagulation assays (31), usually observed as a change in
clotting time with the age of the phospholipid preparation. In general,
the response to this observation has been to minimize oxidation by
using only freshly prepared lipid vesicles. Very recently, Weinstein
et al. (32) have reported enhanced prothrombinase activity
in response to oxidation using defined systems. Whether the hemostatic
balance is affected as a consequence of the oxidation was not directly addressed.
Clinical studies have shown that decreases of as little as 50% in the
plasma protein C concentrations increase the risk of thrombosis. There
is an even more profound effect when other relatively common risk
factors, such as factor V Leiden, are also present (33). Clinical
studies have shown that the levels of activated protein C in the
circulation correlate directly with the levels of plasma protein C
(34), most likely because the physiological protein C concentration is
below the Km value for the physiological activation
complex. Given the relationship between protein C levels and the risk
of thrombosis, it follows that mechanisms that modulate APC
anticoagulant function even 2-3-fold will have a substantial impact on
the probability of thrombus formation. In our studies of the
phospholipid requirements of the APC complex in contrast to those of
the prothrombinase complex, we noticed a dramatically different effect
of aging of the phospholipids on the two reactions. Here we report the
differential effect of lipid oxidation on these reactions and the
identification of anti-phospholipid antibody populations that
selectively inhibit the influence of oxidation.
Proteins and Reagents--
Human prothrombin (35), human APC
(36), human factor V and Va (37), the protein C-prothrombin Gla domain
chimera (PC-PtGla) (37), and the factor X activator from Russell's
viper venom (X-CP) (38) were prepared as described previously. Ancrod,
bovine serum albumin, gelatin, and all chemicals were purchased from Sigma. Protein G columns (HiTrap) were from Amersham Pharmacia Biotech;
Chelex 100 resin was from Bio-Rad, and the Spectrozyme-TH was from
American Diagnostica. All lipids were from Avanti Polar Lipids Inc.
(Birmingham, AL). Unless specified, phospholipids derived from bovine
brain were utilized, including lysoplasmologen PE. Synthetic
phospholipids included 1-palmitoyl-2-oleoyl-PS (16:0-18:1 PS),
1-palmitoyl-2-oleoylphosphatidylcholine (16:0-18:1 PC),
1-palmitoyl-2-oleoyl-PE (16:0-18:1 PE), 1,2-dilinoleoyl-PS (18:2-18:2
PS), 1,2-dilinoleoyl-PC (18:2-18:2 PC), 1,2-dilinoleoyl-PE (18:2-18:2
PE), 1-palmitoyl-2-arachidonoyl-PC (16:0-20:4 PC),
1-palmitoyl-2-arachidonoyl-PE (16:0-20:4 PE), 1-oleoyl-2-hydroxy-sn-glycero-3-PE,
1-palmitoyl-2-hyroxy-sn-glycero-3-PE, and
1-oleoyl-2-hydroxy-sn-glycero-3-PC.
Liposome Preparation--
Extruded vesicles were prepared as
described (6) using a 100-nm Nucleopore membrane. PS:PC vesicles were
20% PS, 80% PC and PE:PS:PC vesicles were 40% PE, 20% PS, 40% PC.
Lipids were mixed in the weight proportions indicated, dried under
argon, and lyophilized 4 h to remove organic solvents.
[14C]PC (Amersham Pharmacia Biotech) was included as
tracer for the determination of lipid concentrations. The lipids were
then reconstituted at 5 mg/ml under argon in 150 mM NaCl,
20 mM HEPES, pH 7.5, which had been Chelex-treated,
extensively degassed, and bubbled with argon.
Phospholipid Oxidation--
To 1 ml of liposomes (200 µg/ml)
was added 1 µl of 10 mM CuSO4 in a glass
tube. The suspension was vortexed to introduce air into the solution,
and the liposomes were incubated at 37 °C for the appropriate period
(39). Standard oxidized liposomes were composed of brain-derived
phospholipids oxidized for 20 h.
Analysis of Oxidation Products--
The extent of oxidation was
determined spectrophotometrically (40) or by 2-thiobarbituric acid
reactivity (41). 130 nmol of phospholipid as liposomes were added to 1 ml of absolute ethanol. The amount of conjugated dienes or trienes
present was determined by absorbance at 233 or 270 nm, respectively, in
a Hewlett-Packard diode array 400N spectrophotometer. An "oxidation
index" was defined as the ratio of absorbance at 233 nm to that at
215 nm, with a ratio of 0.02 corresponding to 0.1% oxidation (40).
Malondialdehyde (MDA), an end product of lipid peroxidation, was
measured by reactivity with 2-thiobarbituric acid by standard
techniques. A standard curve was constructed by acid treatment of
1,1,3,3-tetraethoxypropane (Sigma) as described (41).
Lipoxygenase Oxidation of Phospholipids--
Liposomes (2 mg/ml)
were dispersed in 100 mM
n-octyl- Immunoglobulin Purification--
Plasma or serum was
precipitated with 50% NH4SO4. The precipitate
was resuspended and dialyzed versus 150 mM NaCl,
20 mM Tris-HCl, pH 7.5. The material was applied to a
protein G column, washed, and eluted with 100 mM glycine,
pH 2.5. The eluate was immediately neutralized with 1 M
Tris-HCl, pH 9, and dialyzed versus 150 mM NaCl,
20 mM Tris-HCl, pH 7.5. Total IgG fractions were
concentrated with Millipore Ultrafree30 centrifuge concentrators. IgG
fractions were aliquoted and stored at Coagulation Assays--
Clotting times were determined by a
modified factor Xa one-stage clotting assay using the factor
X-activating enzyme from Russell's viper venom (X-CP) to activate
factor X and an ST4 coagulometer (Diagnostica Stago). The assays were
adapted to allow evaluation of the phospholipid or APC dependence or
the effect of IgG on the reactions. The buffer used was 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.1% gelatin.
In the standard assay, normal pooled plasma (50 µl) was mixed with
X-CP sufficient to obtain a 30-s clotting time in the absence of APC,
phospholipid (10 µg/ml), in the presence or absence of APC (0.2 µg/ml) in a total volume of 150 µl. When present, IgG was at the
concentrations indicated in the figure. After 1 min of incubation at
37 °C, 50 µl of 25 mM CaCl2 was added to
initiate clotting.
Determination of Thrombin Generation in Defibrinated
Plasma--
Plasma was defibrinated by the addition of Ancrod to 0.1 unit/ml citrated plasma for 10 min at 37 °C. The fibrin was removed by two centrifugations at 8000 × g for 10 min. 240 µl of defibrinated plasma was mixed with 40 µl of 200 µg/ml
PE:PS:PC vesicles, 50 µl of buffer, and 30 µl of X-CP (2.3 nM). The buffer in all cases was 100 mM NaCl,
50 mM Tris-HCl, pH 7.5, 0.5% bovine serum albumin. The
mixtures were incubated for 3 min at 37 °C. At regular intervals, thrombin generation was started by the addition of 10 µl of the above
mixture to 10 µl of 16 mM CaCl2 with or
without 2 nM APC in a "reverse" time course. This
resulted in the final concentrations of 10 µg/ml phospholipid, 100 pM X-CP, 1 nM APC (when present), and 8 mM CaCl2 in the assay. At the end of the time
course, 80 µl of 100 mM NaCl, 50 mM Tris-HCl,
pH 8, 10 mM EDTA, and 100 mM benzamidine HCl
was added. After further dilution of 1:10 in 100 mM NaCl,
50 mM Tris-HCl, pH 8, 10 mM EDTA,
Spectrozyme-TH was added, and the rate of hydrolysis measured in a
Vmax microplate reader (Molecular Devices).
Thrombin concentration was determined by comparison to a standard curve
of known concentration.
Measurement of APC and Prothrombinase Activity in Purified
Systems--
Factor Va (50 nM) was reacted with 2.5 pM APC in the presence or absence of 20 nM
protein S and 20 µg/ml oxidized or nonoxidized PE:PS:PC vesicles at
37 °C. At various times, the APC was inhibited by the addition of
benzamidine HCl to 10 mM. After dilution, the remaining
factor Va was determined by standard one-stage clotting assays using
factor V-deficient plasma (43) or by its activity in the prothrombinase
complex as described previously (6, 37).
To determine the effect of oxidation on the prothrombinase reaction
directly, reaction mixtures contained 0.2 nM factor Va, 2 nM factor Xa, 1-120 µg/ml phospholipid, and 5 mM CaCl2. After 5 min of preincubation at
37 °C, the reactions were started with the addition of prothrombin
to 1.4 µM final. After 3 min the reaction was stopped
with the addition of 10 µl 50 mM EDTA, 0.2 M
HEPES, pH 7.5, in a final volume of 100 µl. Aliquots were further
diluted in 0.15 M NaCl, 20 mM HEPES, 5 mM EDTA, pH 7.5, and the resultant thrombin was measured
using a chromogenic assay with Spectrozyme TH and a
Vmax microplate reader (Molecular Devices). All
buffers contained 1 mg/ml gelatin and 10 mg/ml bovine serum albumin.
Effect of Lipid Oxidation on APC Anticoagulant Function--
In
our recent studies we noticed that the ability of the liposomes to
support APC anticoagulant activity increased as the liposomes aged.
Little effect was seen on the ability to support clotting initiated at
the level of factor X activation using the Russell's viper venom
factor X activator, X-CP. PE is known to increase the sensitivity of
phospholipid membranes to oxidation (44). Because our liposomes now
routinely incorporate PE, we theorized that variability in our
coagulation assays with time might represent oxidation. We therefore
investigated the impact of phospholipid oxidation on the coagulation
reactions (Fig. 1). Whether or not the
liposomes contained PE, the clotting time of plasma in the absence of
APC was not affected significantly by oxidation. Oxidation of vesicles
containing only PS and PC or PE and PC (see below) did not enhance the
ability of the vesicles to support APC anticoagulant activity.
Oxidation of PE:PS:PC vesicles dramatically enhanced the anticoagulant
activity of APC, at least doubling the APC prolongation of the clotting
time.
Effect of Oxidation on the Coagulation Reactions in Purified
Systems--
To be sure that membrane oxidation was affecting the APC
complex directly and not acting through another plasma component, the
effect on factor Va inactivation by APC was investigated in a purified
system (Fig. 2). In addition, the effect
of oxidation on prothrombinase in the purified system was studied (Fig.
3). Phospholipid oxidation enhanced
factor Va inactivation in the presence or absence of protein S. Protein
S was not required for oxidation-dependent enhancement of
APC inactivation of factor Va. However, interestingly, the ability of
protein S to enhance APC-dependent factor Va inactivation
was much greater on oxidized lipids. Thus, oxidation may have a
multifaceted effect on the anticoagulant pathway. Equivalent results
were obtained whether remaining factor Va was assessed by clotting or
prothrombinase assay. In contrast to a recent report (32),
prothrombinase was not affected at any concentration of phospholipid
studied under the conditions of our study (see "Discussion").
A reagent previously found to be useful in dissecting the membrane
requirements of the APC complex is a chimeric form of the protein in
which the Gla domain of APC is replaced with that of prothrombin,
referred to as APC-PtGla. This molecule shows enhanced anticoagulant
activity in plasma relative to the native protein. However, in addition
to protein S not being required for activity, the presence of PE does
not affect its activity. We therefore investigated whether it was also
unaffected by oxidation. Oxidation of the vesicles had no effect on the
activity of the protein either in clotting assays (Fig.
4) or the purified system described
above.
The natural brain-derived phospholipids are composed of different fatty
acids of varying length and degree of unsaturation. Increasing
unsaturation increases the potential for oxidation (14, 28). In
addition, according to the supplier, the brain-derived PE contains
~50% plasmalogen forms of PE. To control these variables better, we
investigated whether synthetic phospholipids could support
oxidation-enhanced APC activity to the extent of the brain-derived material (Fig. 5). Direct comparisons of
clotting times are not straightforward, as changes in lipid composition
affect the base-line clotting times, especially in the presence of APC,
when the same clotting stimulus (concentration of X-CP) is used (8).
However, it is apparent that APC activity is not affected by oxidation of minimally unsaturated phospholipid (all components 16:0-18:1) (Fig.
5A). Oxidation of lipids containing two diunsaturated fatty acids (18:2) only marginally enhanced the APC anticoagulant response. Inclusion of arachidonate (20:4) increased the clotting time by 50%
(Fig. 5B). When oxidation was allowed to continue in these highly polyunsaturated liposomes, clotting times increased even in the
absence of APC. Light scattering was performed to determine whether
this was due to the loss of liposome structure (Fig.
6). After 20 h of copper-catalyzed
oxidation, the liposomes made with synthetic phospholipids no longer
scattered light, indicating that the liposomes were no longer intact.
Similar light-scattering experiments indicated that liposomes made with
brain-derived components remained intact. Plasmalogens, present in the
brain phospholipids and the plasma membranes of most cells, are known
to stabilize membranes against oxidation (45). Thus, it is possible
that liposomes made with the highly unsaturated synthetic phospholipids were not as effective as the brain-derived vesicles because the liposome concentration was decreasing with increasing oxidation, thereby offsetting any continued enhancement of APC function by continued oxidation.
Phospholipid Concentration Dependence of APC Activity--
A
possible function of oxidation is to alter the phospholipid
concentration dependence of the coagulation reactions. Therefore, the
effect of oxidation on phospholipid concentration dependence was
determined for PS:PC, PE:PC, and PE:PS:PC vesicles (Fig.
7). In the absence of APC, all curves
showed an initial drop in clotting time, consistent with the
phospholipid concentration dependence of the prothrombinase reaction.
With PS:PC vesicles, there was some increased anticoagulant function in
the presence of APC at relatively high phospholipid concentrations, but
this was not affected by oxidation. PE:PC vesicles mirrored the
prothrombinase curves, and oxidation had no effect. With PE:PS:PC
vesicles in the absence of oxidation, the rise in anticoagulant
function after the initial drop in clotting time was dramatic. Lipid
oxidation increased the APC anticoagulant response at all phospholipid
concentrations and also abrogated the initial decrease in clotting
time. Thus, although oxidation of these vesicles altered the
concentration dependence at low lipid concentrations, the overall
phospholipid concentration dependence of APC anticoagulant activity was
not altered in that enhanced activity was observed at all lipid
concentrations. It should be noted that the PE:PC vesicles made using
brain-derived material do not show enhanced activity with oxidation.
This implies that PE or PE plasmalogens per se are not
sufficient to result in enhanced APC anticoagulant activity.
Effect of Oxidation on Total Thrombin Generation--
A prolonged
clotting time may indicate only a lengthening of the lag period before
feedback loops lead to the explosive generation of thrombin, rather
than a decrease in the total amount of thrombin eventually formed (46,
47). These two possibilities may not have the same ultimate
physiological effect. To determine which was the case here, thrombin
generation rather than clotting time was determined (Fig.
8). APC slightly prolonged the lag time
for thrombin generation on lipids with or without oxidation. The
additional effect of oxidation was to decrease total thrombin
generation. The decrease in thrombin with time represents inhibition by
antithrombin and Preliminary Attempts to Determine the Active Species in Oxidized
Phospholipids--
The formation of conjugated dienes and trienes and
MDA was monitored as a function of time and correlated to the
anticoagulant activity of the resulting liposomes (Fig.
9). When PE was present in the vesicles,
the formation of diene and triene correlated with increased
anticoagulant function. "Unoxidized" liposomes were <1% oxidized
based on absorbance at 233 nm. Liposomes were never more than 4%
oxidized based on conjugated diene formation for optimal activity. If
PE was not present, even though increases in these parameters were
observed spectrophotometrically, no increase in function was observed.
Formation of MDA did not correlate with increased anticoagulant
function.
We used soybean lipoxygenase, which produces conjugated dienes only
(38), to determine whether formation of these structures alone was
sufficient to produce enhanced APC anticoagulant activity. At no time
was enhanced anticoagulant activity observed, even when the
concentration of conjugated dienes present was equivalent to or greater
than that observed in the oxidized phospholipids as monitored by
absorbance at 233 nm (data not shown).
Lysophospholipids represent a possible product of lipid peroxidation.
The lyso forms of oleoyl-PE, palmitoyl-PE, oleoyl-PC, or brain
plasmalogen PE were incorporated into vesicles from 1 to 25% of the
total phospholipid. No concentration of these molecules reproduced the
effect observed with copper/air oxidation of the vesicles. If anything,
incorporation of lyso-plasmalogen PE or lyso-PC slightly inhibited APC
anticoagulant activity (data not shown).
The Effect of Patient Immunoglobulin on APC
Activity--
Previously we showed that plasma or immunoglobulin
derived from at least a subset of lupus patients could inhibit APC
function selectively in a PE-dependent manner (37, 49).
Screening of IgG from several patients with thrombosis and APA or lupus
anticoagulant indicated the anticoagulant activity of APC was inhibited
more effectively on oxidized phospholipid than on nonoxidized
phospholipid (data not shown). A typical IgG titration curve for one
patient is shown in Fig. 10.
The results from the present studies begin to address several
important and unresolved clinical issues. First, the mechanism by which
new thrombi or newly injured vessels which are very thrombogenic become
less so with time, a process known as "passivation" (50, 51). The
ability of phospholipid oxidation, a time-dependent process, to enhance APC anticoagulant function would be expected to
contribute to this process. Second, although protein S is clinically established as a key component of the anticoagulant pathway with total
deficiencies resulting in massive thrombosis, protein S effects on
factor Va inactivation observed in purified systems have been
relatively modest. As seen in Fig. 2, the ability of protein S to
accelerate APC inactivation of factor Va is markedly enhanced by
phospholipid oxidation. If oxidation is indeed involved in thrombus
passivation, this may demonstrate a potentially important function for
protein S in that process. Third, patients with APAs have an increased
propensity to thrombose. Oxidized lipids are a target for at least some
of these APAs. APAs that can block the ability of the
oxidized lipid to enhance anticoagulant function may
contribute to a prothrombotic state.
Our previous studies indicated the importance of PE and
polyunsaturation in the expression of APC activity (6, 8, 49). The
studies reported here indicate yet another feature that distinguishes procoagulant from anticoagulant phospholipid surfaces. We have found
that oxidization of the phospholipid membrane further enhances the
activity of APC in the factor Va inactivation complex with little or no
effect on the prothrombinase reaction. The enhanced factor Va
inactivation results in increased anticoagulant activity. That the
effect is greater in the presence of protein S in the purified system
indicates oxidation may be impacting more than one interaction.
The present results demonstrate that lipid oxidation has a significant
effect on the ability of APC to block total thrombin generation without
significantly influencing thrombin generation in the absence of APC.
These findings exclude the possibility that the increased anticoagulant
activity in plasma is due to an increase in the lag time before
thrombin generation is initiated. It is of interest that studies of
deficiency states and inhibitors have demonstrated that the impact on
total thrombin generation is usually greater than the effect on the
clotting time. Indeed, studies of total thrombin generation were able
to demonstrate the presence of an acquired inhibitor of APC in pregnant
women and women on birth control pills that is thought to contribute to
thrombotic risk (52). Standard coagulation tests failed to detect this inhibitor.
For the oxidation to be effective, both PE and PS must be present in
addition to a significant degree of polyunsaturation. Based on the
studies with APC-PtGla, it is apparent that the oxidation effect is
mediated by features of the Gla domain of protein C that differ from
those of prothrombin. We demonstrated previously that the PE head group
specificity was also resident in the Gla domain of protein C (37). The
requirements for the assembly of the anticoagulant complex include
interaction of the enzyme, APC, and the substrate, factor Va, with the
membrane surface. Optimal factor Va binding requires the presence of
the PS head group, probably explaining the need for PS. APC is
essentially inactive in the absence of PE. PE, polyunsaturation, and
lipid oxidation all favor formation of hexagonal phase lipids (28, 53).
Based on the observations that PE, polyunsaturation, and oxidation all
favor APC function, we would presume that the oxidation is favoring
hexagonal phase lipid formation. This would be consistent with the
ability of the anti-PL antibodies to block the oxidation effect as
other investigators (54, 55) have demonstrated these antibodies bind
preferentially to hexagonal phase phospholipids and that oxidation can
enhance their binding.
We have not yet obtained a liposome composition using synthetically
produced phospholipids that is as effective as the naturally occurring
brain-derived phospholipids. This may be due to the progressive loss of
liposome structure during the oxidation process of highly unsaturated
phospholipids. This is quite possibly a function of the difficulty to
control the rate of oxidation in these preparations. When these
liposomes are oxidized in the absence of copper, greater APC activity
is observed after an extended period, but the unpredictability of this
reaction makes it impractical for detailed study.
One constituent present in the brain-derived material that is absent in
the synthetic mixtures is various plasmalogen forms of PE that may
serve to stabilize the membranes (45). Although possibly serving this
role, plasmalogens themselves are not sufficient for the effect, as
liposomes composed solely of brain-derived PC and PE, which contain the
plasmalogens, lack any enhancing activity after oxidation.
Lysophospholipid or conjugated diene derivatives of the fatty acid side
chains are also not sufficient to support the enhanced activity of
these membranes. Conjugated diene formation was a useful monitor of
oxidation. However, experiments using soybean lipoxygenase, which
produces only conjugated dienes (42), failed to produce enhanced
activity, even though the concentration of conjugated dienes formed was
equivalent to or greater than that observed in the copper-oxidized
phospholipids (data not shown). Although conjugated triene formation
appears to correlate with activity in the data presented, this was not
always the case (data not shown). Attempts to produce trienes
specifically to directly test whether their formation is sufficient
have not been successful. Only a small degree of oxidation of the
phospholipids (<4%) is required for the observed effects as monitored
by conjugated diene/triene formation. Thus, isolation and
identification of the putative moieties responsible for the activity
will require sensitive and extensive investigation. Such studies are ongoing.
Very recently, Weinstein et al. (32) reported on enhanced
prothrombinase activity in the presence of oxidized
1-stearoyl-2-arachidonoyl-PC (18:0, 20:4-PC) (SAPC). However, the
conditions used by these authors are very different from those used in
the studies reported here, making comparisons difficult. In the cited
reference, other than the SAPC, the vesicles are composed of
dimyristoyl (14:0, 14:0)-PC and/or -PS. These are very sterically
hindered nonphysiological forms of these molecules. Extremely strong
oxidation conditions (2 mM H2O2 and
200 µM CuSO4) are used relative to the mild
conditions used here (10 µM CuSO4 and air),
which will most likely yield different products. The use of EDTA at
concentrations as high as 75 mM may also result in
unexpected effects. Butylated hydroxytoluene is added to all
mixtures to stop oxidation. However, it may also be incorporated into
the vesicles used for the prothrombinase reactions and have direct
effects on observed activity. The tocopherols (and ascorbic acid to
some extent), used in some experiments to block oxidation, would be
coextracted with the SAPC and presumably reincorporated into the
vesicles used for the prothrombinase reactions. Incorporation of
tocopherols into membranes has been found to decrease membrane fluidity
(56, 57), further complicating interpretation of these experiments.
Indeed, the reversal of the known effects of unsaturated fatty acids
(in this case, SAPC whether or not it is oxidized) on prothrombinase
activity (58, 59) would argue for a direct effect of the lipophilic
antioxidants on the membrane structure, further complicating
interpretation of these experiments.
In addition to cell or platelet surfaces, plasma lipoproteins have been
found to be active in the coagulation reactions. Both Rota et
al. (60) and Moyer et al. (61) observed very low
density lipoproteins supported prothrombin generation. Rota et
al. (60) also found that copper oxidation of the low density (LDL)
population led to a marked increase in prothrombin activation. However,
oxidation of the other lipoprotein classes did not lead to enhanced
activation. Since the authors showed that the relative concentration
and composition of phospholipids did not vary significantly between the
lipoprotein populations, oxidation of the phospholipid per
se was unlikely to be the major mechanism of the enhanced
anticoagulant activity. More likely, oxidation of the protein moieties
in the LDL permitted improved display of the phospholipid present in
these particles. Griffin et al. (62) observed APC
anticoagulant function on high density lipoprotein particles but not
LDL. Neither very low density lipoproteins nor the effect of oxidation
on any of the lipoproteins was investigated.
When a coagulant stimulus is generated, leukocytes are recruited into
the forming clot (63, 64). Platelet activation occurring during this
time results in the expression of P-selectin on the platelet surface,
serving to rosette the incoming neutrophils and monocytes around the
activated platelet (65). Lipid oxidation as a result of the
leukocyte-platelet interaction leads to the evolution of lipoxins,
eicosanoids with anti-inflammatory effects including inhibition of
neutrophil chemotaxis (66, 67). Activation of the adherent leukocytes
may also enhance the anticoagulant properties of the platelet through
lipid oxidation.
Relative to phospholipid surfaces, the platelet expresses a relatively
high prothrombinase activity compared with their ability to support
factor Va inactivation (68). Despite this in vitro observation, it has been shown that APC (69), or thrombin infusion which elicits APC formation (70), can block platelet deposition on
thrombogenic surfaces, a system that has been shown previously to
involve thrombin formation. The latter observations are opposite to
what would be predicted from the in vitro experiments. We
would propose that the key difference between the in vitro
and in vivo results may be leukocyte-mediated oxidation that
helps passivise the developing thrombus. The mechanisms responsible for
this process are unknown and undoubtedly complex. Lipid oxidation
caused by the adherent leukocytes could contribute significantly to
this phenomenon that is critical for successful vascular repair. The first response to a low or moderate inflammatory stimulus would be the
development of a procoagulant surface. As leukocytes enter the clot,
oxidation of cell surfaces would begin after a time lag. The
anticoagulant response would then be promoted to prevent excessive
thrombus growth. Membrane constituents such as plasmalogens (45, 71) or
sphingomyelin (72) may help stabilize minimally oxidized surfaces to
maintain APC activity for a longer period. The red cell membrane could
also be oxidized by the infiltrating leukocytes in addition to the
platelets normally envisioned, possibly adding anticoagulant surface
despite the externalization of PS and PE that can occur on these cells
in response to oxidation (15).
The effect of oxidation on the APC complex may also further
differentiate subclasses of autoimmune antibodies. Previously we showed
that plasma or immunoglobulin derived from at least a subset of lupus
patients could inhibit APC function selectively in a
PE-dependent manner (37, 49). Although normal precautions against oxidation were taken, including storage under argon and usage
within 1 week of liposome preparation, the oxidation state of the
phospholipids used in those studies was not specifically known. In a
small sampling of patients, immunoglobulin populations have been
identified that inhibit APC anticoagulant activity on oxidized
phospholipids much more effectively than on nonoxidized liposomes. It
is not known how common this reactivity is. The diagnostic utility of
this distinction is also unknown at this time as many more patients
will need to be screened for oxidation dependence of APC inhibition.
Clinically, if lipid oxidation is involved in making clots and vascular
injury sites less thrombogenic, then APAs with specificity for oxidized
lipids may have a different physiological effect than those that are
not oxidation-dependent. Oxidation-dependent
antibodies would minimize the ability to passivise the clot or injured
surface. Such antibodies may not be significantly procoagulant in the
absence of an oxidized surface. Violi and co-workers (73, 74) have
reported that markers of clotting activation correlate with markers of
in vivo lipid peroxidation in patients with APA but not in
individuals devoid of APAs. Both markers decrease in response to antioxidants.
It might be argued that the 2-3-fold enhancement of APC anticoagulant
function and factor Va inactivation is too small an effect to be
physiologically relevant. However, decreases in protein C levels to
50% of normal are sufficient to increase thrombotic risk (75).
Furthermore, small changes in the level or function of natural
anticoagulants can have a major effect on potential thrombin
generation. Studies of Mann and colleagues (46, 47) indicate that a
"threshold effect" occurs in coagulation. Thrombin must be
generated to a level greater than that threshold for clotting to ensue.
Doubling the rate of inhibition of the procoagulant reactions can be
sufficient to keep thrombin generation below that required for
threshold, changing the balance such that normal hemostasis is maintained.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside, 200 mM sodium borate, pH 10, with or without soybean lipoxygenase type I-B (Sigma) at 5000 units/ml (42). The suspensions were vortexed to introduce air into the solutions. The mixtures were
then incubated at room temperature for 1 h. Each sample was then
dialyzed versus 150 mM NaCl, 20 mM
HEPES, pH 7.5, with three buffer changes over 2 days. Chelex 100 resin
was present in the dialysis vessels.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
Oxidation of PE containing liposomes enhances
APC anticoagulant activity. Phospholipids derived from bovine
brain were used to prepare liposomes composed of 20% PS, 80% PC
(triangles) or 40% PE, 20% PS, 40% PC
(circles) as described under "Materials and Methods."
Liposomes (200 µg/ml) were oxidized with 10 µM
CuSO4 and air up to 20 h in a "reverse time
course." Liposomes stored under argon served as "nonoxidized"
liposomes. Clotting times were measured in a one-stage assay as
described in the presence (open symbols) or absence
(closed symbols) of 0.2 µg/ml APC. Bars
represent the standard error of three experiments performed in
duplicate.
View larger version (22K):
[in a new window]
Fig. 2.
Oxidized phospholipids enhance factor Va
inactivation by APC in a purified system. Factor Va (50 nM) was reacted with APC (2.5 pM) in the
presence (open symbols) or absence (closed
symbols) of 20 nM protein S using 20 µg/ml oxidized
( and
) or nonoxidized (
and
) PE:PS:PC vesicles at
37 °C. At the times indicated, the APC was inhibited with 10 mM benzamidine HCl. After dilution, the remaining factor Va
was determined by standard one-stage clotting assay using factor
V-deficient plasma. Bars represent the standard error of
three experiments performed in duplicate.
View larger version (20K):
[in a new window]
Fig. 3.
Oxidation does not affect prothrombinase
activity in a purified system. PE:PS:PC vesicles were either not
oxidized ( ) or oxidized for 30 min (
) and 1 (
), 3 (
), 6 (
), or 20 h (
) and incorporated in the prothrombinase
reactions at 1-120 µg/ml. Thrombin generation after 3 min of
reaction at 37 °C was determined as described in the text.
View larger version (17K):
[in a new window]
Fig. 4.
The APC-PtGla chimera is unaffected by
phospholipid oxidation. Plasma clotting was initiated with X-CP as
described under "Materials and Methods," and the anticoagulant
response was measured as a function of increasing APC ( and
) or
APC-PtGla (
and
) concentration. Open symbols,
oxidized phospholipid; closed symbols, nonoxidized
phospholipid.
View larger version (18K):
[in a new window]
Fig. 5.
Synthetic phospholipids are not as effective
as brain-derived phospholipids in enhancing APC activity.
Synthetic phospholipids were used to make the liposomes as described
under "Materials and Methods." All liposomes are 40% PE:20%
PS:40% PC. A, and
. All lipids were 16:0-18:1
(palmitoyl-, oleoyl-); (
and
) PE was 18:2-18:2 (dilinoleoyl-);
(
,
) PS was 18:2-18:2. B, PS is 18:2-18:2, PE and PC
are 16:0-20:4 (palmitoyl, arachidonyl-). Open symbols are
in the presence of 0.2 µg/ml APC; closed symbols are in
the absence of APC. Graphs represent a typical experiment,
performed in duplicate.
View larger version (26K):
[in a new window]
Fig. 6.
Liposomes made with unsaturated synthetic
phospholipids are not stable to oxidation. Liposomes were prepared
from brain-derived or synthetic (18:2-18:2 PS, 16:0-20:4 PC,
16:0-20:4 PE when present) phospholipids as described under
"Materials and Methods." Aliquots of each were oxidized with 10 µM copper for 20 h as described. Light scattering at
490 nm was then measured on all samples at 200 µg/ml phospholipid.
Hatched bars, nonoxidized liposomes; solid bars,
oxidized liposomes.
View larger version (21K):
[in a new window]
Fig. 7.
Oxidation does not alter the phospholipid
concentration dependence of the clotting reactions. Clotting was
initiated in the presence (open symbols) or absence
(closed symbols) of 0.2 µg/ml APC using oxidized ( and
) or nonoxidized (
and
) brain-derived phospholipids at the
concentrations indicated. A, 20% PS:80% PC; B,
40% PE:60% PC; C, 40% PE:20% PS:40% PC. Bars
represent the standard error of two experiments performed in
duplicate.
2-macroglobulin (48). The residual
amidolytic activity is due to the fact that
2-macroglobulin does not completely inhibit thrombin
amidolytic activity.
View larger version (28K):
[in a new window]
Fig. 8.
Oxidation of phospholipid affects total
thrombin generation and not lag time. Nonoxidized (A)
or oxidized (B) phospholipid vesicles (10 µg/ml final
concentration) and X-CP (0.1 nM final concentration) were
added to Ancrod defibrinated plasma and incubated for 3 min at
37 °C. At regular time intervals, thrombin generation was initiated
by the addition of 1 volume of 16 mM CaCl2 with
(open symbols) or without (closed symbols) APC (1 nM final concentration) to develop a "reverse" time
course. At the end of the time course, the reactions were diluted
5-fold with buffer containing 10 mM EDTA, 100 mM benzamidine HCl, pH 8. After 1:10 dilution in buffer
containing 10 mM EDTA, pH 8, Spectrozyme-TH (American
Diagnostica) was added, and the rate of hydrolysis was measured.
Thrombin concentration was determined by comparison to a standard
curve of known thrombin concentration. Bars represent
the standard error of three determinations.
View larger version (14K):
[in a new window]
Fig. 9.
Time course of lipid oxidation products.
PE:PS:PC ( ) or PS:PC (
) were oxidized as described under
"Materials and Methods." At the times indicated, conjugated diene
(A), conjugated triene (B), and MDA formation
(C) were determined as described. A and
B represent the means ± S.E. of the 3 experiments
represented in Fig. 1. C, is the average of two of those
experiments. The average maximal "oxidation index" obtained at
20 h represents ~3% oxidation.
View larger version (22K):
[in a new window]
Fig. 10.
Titration of an
oxidation-dependent inhibitory APA immunoglobulin.
Normal or patient IgG was purified as described under "Materials and
Methods" and incorporated in the clotting assays at the final
concentrations indicated. Assays were performed as described in Fig. 1.
Symbols: normal Ig, dashed lines; patient Ig, solid
lines; presence of APC, open symbols; absence of APC,
closed symbols; oxidized liposomes, upside down
triangles; nonoxidized liposomes, circles. Data
represents the average clotting time ± S.E. of four experiments
performed on 4 separate days with different phospholipid
preparations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Joan T. Merrill Division of Rheumatology, St. Luke's-Roosevelt Hospital Center, New York), Dr. Armando D'Angelo (Coagulation Service and Thrombosis Research Unit, Scientific Institute H. S. Raffaele, Milan, Italy), and Dr. Philip C. Comp (Oklahoma University Health Sciences Center, Oklahoma City, OK) for supplying plasma and serum from lupus and/or thrombotic patients. The technical assistance of Tommy Norton is appreciated. We also thank Nici Barnard for preparation of the figures.
![]() |
FOOTNOTES |
---|
* This work was supported by a Specialized Center on Thrombosis NHLBI Grant P50 HL54502 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Investigator of the Howard Hughes Medical Institute and holds
the Lloyd Noble Chair in Cardiovascular Research at the Oklahoma Medical Research Foundation.
§§ To whom correspondence should be addressed: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104. Tel.: 405-271-7399; Fax: 405-271-3137; E-mail: Naomi-esmon@omrf.ouhsc.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M005931200
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ABBREVIATIONS |
---|
The abbreviations used are:
Gla, -carboxyglutamic acid;
APC, activated protein C;
PS, phosphatidylserine;
PE, phosphatidylethanolamine;
PC, phosphatidylcholine;
APA, anti-phospholipid antibody;
PC-PtGla, a
chimeric form of protein C in which the gla domain has been replaced
with that of prothrombin;
X-CP, the factor X activating factor from
Russell's viper venom;
MDA, malondialdehyde;
SAPC, 1-stearoyl-2-arachidonoyl-PC;
LDL, low density lipoprotein.
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REFERENCES |
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