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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 508-515
By
From the University Department of Clinical Biochemistry and the
Department of Haematology, Addenbrooke's Hospital, Cambridge, UK.
The importance of lipoproteins in the etiology of atherosclerosis is
well established. Evidence is now accumulating to implicate thrombin in
the pathogenesis of atherosclerosis. We have investigated whether
atherogenic lipoproteins can support thrombin generation. In the
absence of platelets or endothelial cells, both very low-density lipoprotein (VLDL) and oxidized low-density lipoprotein (LDL) support
assembly of the prothrombinase complex and generation of thrombin.
Thrombin generation (per µg of apolipoprotein) supported by VLDL was
19.4-fold greater than that supported by high-density lipoprotein
(HDL), P < .00001, and 11.7-fold greater than that supported by LDL, P < .00001. Oxidation of LDL increased
lipoprotein-supported thrombin generation 12-fold compared to
unmodified LDL, P < .0001. We have shown that the
phenomenon of lipoprotein-supported thrombin generation is mediated
predominantly by specific phospholipids and is enhanced by oxidation of
these phospholipids. The addition of vitamin E (
THERE IS INCREASING evidence to support
the hypothesis that thrombin generation may influence the development
of atherosclerosis.1-7 Thrombin generation is dependent on
assembly of the prothrombinase complex on a phospholipid
surface.8,9 Whether platelets provide the only important
phospholipid surface for prothrombinase assembly is uncertain. Thrombin
can also be generated in vitro in the presence of phospholipid bilayers
and micelles; and it is therefore possible that cell-free phospholipid
may be able to support assembly of the prothrombinase complex in vivo.
Various cell-free phospholipids appear capable of binding the
individual components of the prothrombinase complex. Liposome vesicles
containing phosphatidyl choline were able to support thrombin
generation in the presence of factor Va.10 Very low-density
lipoprotein (VLDL) has also been shown to bind
prothrombin11,12 and it has been suggested that intact VLDL
and VLDL phospholipid extracts were able to accelerate prothrombin conversion, although contamination of prothrombin by factor V may have
contributed to this process.13
It is well established that hyperlipidemia is a risk factor for
coronary heart disease (CHD) but the relationship between increased plasma lipoprotein concentrations and CHD is poorly defined.
Given the role of phospholipids in thrombin generation we hypothesized
that lipoprotein-supported thrombin generation provides a link
between increased concentrations of atherogenic lipoproteins and CHD.
There is indirect evidence supporting this notion because subjects with
hyperlipidemia have increased concentrations of markers of
thrombin generation14,15 and treatment of hyperlipidemia with lipid-lowering drugs reduced levels of these
markers14-16 without necessarily affecting platelet
function.17 Thus, there is in vitro, pathological, and
clinical evidence suggesting that cell-free plasma lipids and
lipoproteins may be capable of supporting assembly of the
prothrombinase complex and thrombin production. We have tested the
ability of lipoprotein fractions to support thrombin generation and
examined whether modification of lipoproteins by oxidation,
nonenzymatic glycosylation, or vitamin E alters this function.
Preparation of lipoprotein subclasses.
Lipoprotein preparation was undertaken as described
previously.18 Initially, experiments were undertaken in the
presence of protease inhibitors, antibiotics, and an antioxidant to
determine whether lipoprotein-supported thrombin generation was
affected by these reagents. Omission of protease inhibitors and
antioxidant did not alter lipoprotein-supported thrombin generation and
therefore subsequent experiments were undertaken immediately after
preparation of lipoproteins without addition of any of these reagents.
Preparation of each lipoprotein class was undertaken by collection of
venous blood from 12 healthy volunteers into EDTA (1.5 mg/mL of blood). Plasma was separated by centrifugation at 1,750×g for 20 minutes and the density adjusted to 1.215 g/mL by adding KBr. After
ultracentrifugation at 235,000×g for 36 hours at 4°C,
the lipoprotein fraction was removed. Purified lipoprotein fractions
were obtained by separation through a Sepharose CL-6B gel filtration
column (Sigma, St. Louis, MO), in phosphate-buffered
saline (PBS), pH 7.2. Analysis of lipoprotein fractions collected from
the gel filtration column by agarose gel electrophoresis (1% agarose,
0.4% bovine serum albumin (BSA), 50 mol/L sodium barbitone, 1.9 mol/L
EDTA, pH 8.6, at 220 V, 150 mA for 30 minutes) was undertaken to ensure
that each fraction used in the thrombin generation assay contained only
a single class of lipoprotein. Agarose gels were fixed by incubation
with 5% trichloroacetic acid and dried at 90°C for 15 minutes.
Lipoproteins were visualized by incubation with Sudan Black in 60%
ethanol. Using the above technique, pure fractions of chylomicrons,
VLDL, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were obtained. The protein content of each lipoprotein fraction was
measured by a modified Lowry method.19 Cholesterol and
triglyceride concentrations were measured by a standard enzymatic
method and apo B concentrations measured by enzyme-linked immunosorbent
assay (ELISA) with a polyclonal antibody apo B and apo B standard from The Binding Site (Birmingham, UK).20
Quantitation of lipoprotein-supported thrombin generation.
A chromogenic assay to quantify thrombin generation by lipoproteins and
platelets was developed based on established work.21,22 Prothrombin, factor V, and factor Xa purified from human plasma were
obtained from Diagnostica, Stago (Asnières, France). Each factor
was reconstituted in 0.05% human albumin in phosphate-buffered saline
(PBS), pH 7.2. Lipoprotein [5 µL of 100 µg/mL (apolipo)protein] was added to 1 µmol/L prothrombin, 1.5 nmol/L factor V, and 1 nmol/L
factor Xa (final concentrations). After incubation for 15 minutes at
37°C, 2.5 mmol/L CaCl2 in 6.25 mmol/L Tris HCl, 12.5 mmol/L NaCl, 0.0625% fatty acid-free BSA, pH 7.5 was added. After a
further incubation for 25 minutes at 37°C, 10 µL of this solution
was added to tubes containing 465 µL of buffer (50 mmol/L Tris HCl,
20 mmol/L EDTA, 0.1 mol/L NaCl, 0.5% fatty acid-free BSA at pH 7.5)
and 25 µL of 4 mmol/L S2238, a thrombin-specific chromogenic
substrate (Kabi Diagnostica, Kabi Vitrum, Stockholm, Sweden). After 4 minutes the reaction was stopped by the addition of 300 µL of 1 mol/L
citric acid. Three hundred µL of the final reaction mixture was
transferred to flat-bottomed 96-well microtitre plates (Nunc Maxisorp,
Roskilde, Denmark) and the absorbance determined at 405 nm
in an Anthos HTII, Anthos Labtec HT2 version 1.05 microtitre plate reader. The ability of each lipoprotein class to
support thrombin generation was quantified separately for individual
subjects and the results (mean ± standard error of the mean
[SEM]) were expressed as nmol/L thrombin, calculated from a standard
curve generated with Inhibition by annexin V.
Lipoproteins (5 µL of 100 µg/mL) were incubated with 5 µmol/L annexin V at 37°C for 30 minutes. The thrombin generation
assay was then undertaken as described above. Control experiments were undertaken by adding equal volumes of PBS instead of annexin V. The
small volume of PBS used did not produce a calcium precipitate when
added to the solution of coagulation factors and calcium chloride.
Quantitation of phospholipid concentration and identification of
lipoprotein phospholipids.
Phospholipid concentrations were measured using a commercial kit
(Randox Laboratories Ltd, Crumlin, Co Antrim, UK). Phospholipids were
analyzed by high performance thin-layer chromatography (HPTLC). Two
hundred µg by total weight of each lipoprotein class was dried down
under nitrogen. Lipoproteins were resuspended in 30 µL chloroform and
methanol (2:1) and analyzed by HPTLC (silica gel, 60 precoated plates;
MERCK, Darmstadt, Germany); in methyl
acetate:isopropanol:chloroform:methanol:0.25% KCl (25:25:25:10:9)
polar solvent. Commercial phospholipid standards were obtained from
Sigma. Lipids were stained with a solution containing 3% cupric
acetate and 8% phosphoric acid. Phospholipid bands were photographed
using an Eagle Eye II Still Video System (Stratagene, La Jolla,
CA). The image was exported in a TIFF file and
phospholipid bands were quantified using NIH Image 1.55 software for
Macintosh23(Apple Computer, Cupertino, CA).
Quantitation of phospholipid-supported thrombin generation.
To test each component of lipoproteins for thrombin generating
capacity, the thrombin generation assay was modified and optimized for
use in 96-well microtitre plates. Lipid or apolipoprotein was dried
onto microtitre wells (PolySorp [lipid], MaxiSorp [protein] Nunc),
blocked in 1% fatty acid-free BSA in PBS for 1 hour, and washed in
PBS. The assay was performed in the microtitre plate with the same
ratio of prothrombin, factor V, and factor Xa as described above. The
factors were incubated for 15 minutes at 37°C before adding
CaCl2. The plate was then incubated for a further 60 minutes before subsampling into the substrate solution. After 8 minutes
incubation the reaction was terminated and assessed as described above.
The results are expressed as nmol/L thrombin, calculated from a
standard curve generated with Oxidation, glycosylation, and vitamin E treatment of lipoproteins
and phospholipids.
Lipoproteins were modified by oxidation and nonenzymatic glycosylation.
Oxidation of lipoprotein fractions was undertaken by incubation with
2.5 µmol/L CuSO4 (final concentration; Sigma) at 37°C
for 17 to 20 hours.24 Lipoprotein oxidation was checked by
comparison of diene conjugates before and after incubation with
CuSO425 and by agarose gel
electrophoresis. Oxidation was stopped by addition of 0.1 mol/L EDTA
and refrigeration at 4°C. Lipoprotein fractions were also modified
by nonenzymatic glycosylation with 5 mmol/L, 20 mmol/L, 50 mmol/L, and
100 mmol/L glucose by incubation for 3 days at 37°C.26
After modification, the lipoprotein fractions were washed with PBS by
centrifugation with Centricon 30 concentrators (Amicon, Beverly, MA)
and were resuspended at 100 µg/mL protein concentration. The effect
of vitamin E on thrombin generation produced after copper oxidation of
LDL was examined. LDL from 9 of the subjects was mixed and incubated
with 850 IU vitamin E/mL [(+)- Statistics.
Lipoprotein and phospholipid-supported thrombin generation was normally
distributed. Presented data of means ±SEM and comparison of means
was undertaken by unpaired Student's t-test.
Subject Characteristics
Optimization of Thrombin Generation Assay
Thrombin Generating Capacity of Lipoproteins and Effect of Annexin V Binding Thrombin generation [per µg of (apolipo)protein] supported by VLDL was 19.4-fold greater than that supported by HDL, P < .00001 (and 11.7-fold greater than that supported by LDL, P < .00001). Levels of thrombin generation supported by LDL and HDL were not significantly different from each other. Annexin V reduced lipoprotein-supported thrombin generation and platelet-specific thrombin generation by similar amounts (Table 1) (45.7%, P = .002 and 47.4%, P = .015, respectively). Annexin V is a member of a family of Ca2+-dependent phospholipid-binding proteins. The exact prerequisites for binding are not fully established but binding is known to be strongly dependent on the presence of an sn-3 phosphate group and hydrophobic interaction with the sn-2 acyl chain of the phospholipid.30 Therefore, the reduction in lipoprotein-supported thrombin generation caused by annexin V strongly suggested that phospholipids are an important mediator of this phenomenon.
Phospholipid Composition of Lipoproteins Next, we analyzed the phospholipid components of each lipoprotein by HPTLC to determine if the presence of different phospholipids between lipoprotein classes might be responsible for the marked difference in lipoprotein-supported thrombin generation between classes. Similar percentages of each type of phospholipid were observed across lipoprotein classes. Typical values of individual phospholipids expressed as a percentage of total phospholipid extracted from the lipid core of a lipoprotein were: phosphatidyl choline, 46%; sphingomyelin, 12%; phosphatidyl serine, 5%; phosphatidyl ethanolamine, 12%; phosphatidyl inositol, 11%; and lysophosphatidyl choline, 14%. Sphingomyelin varied most in percentage content (±5%) between lipoprotein classes.Thrombin Generating Capacity of VLDL and LDL Lipoproteins Calculated With apo B100 Concentration as Denominator Because neither the total amount nor the type of phospholipid could explain the marked differences in lipoprotein-supported thrombin generation between VLDL and HDL (or LDL), we excluded the possibility that the observed differences with VLDL were attributable to different numbers of VLDL and LDL particles in the thrombin generation assay. Therefore, we determined lipoprotein-supported thrombin generation results per ng of apo B100, for VLDL and LDL, because this represented an indirect measure of the amount of thrombin generated per lipoprotein particle (VLDL and LDL contain a single apo B100 molecule per particle). VLDL-supported thrombin generation was 32-fold greater than LDL-supported thrombin generation per ng of apo B100 (P < .001). These data provided further support for the suggestion that the differences between lipoprotein classes were not an artifact resulting from the choice of denominator for expression of thrombin generation results.Thrombin Generation Supported by Phospholipids The magnitude of the difference in lipoprotein-supported thrombin generation between lipoprotein classes suggested that our findings had not occurred by chance. Confounding by amount and type of phospholipid or lipoprotein particle number was also excluded and therefore we investigated the mechanism of lipoprotein-supported thrombin generation by analyzing individual components of lipoproteins for thrombin generating capacity. Of the phospholipids present in each lipoprotein class, phosphatidyl inositol, phosphatidyl ethanolamine, and phosphatidyl serine provided the greatest support for assembly of the prothrombinase complex and thrombin generation and this phenomenon could be inhibited partially by annexin V (Table 2). Although sphingomyelin, lysophosphatidyl choline, and phosphatidyl choline were present in highest concentration in each lipoprotein class, thrombin generation supported by these phospholipids was at levels close to the limit of detection of the assay. Thrombin generation supported by sphingomyelin (685 µmol/L) was 25 nmol/L, lysophosphatidyl choline (1008 µmol/L) was 55 nmol/L, and phosphatidyl choline (654 µmol/L) was 38 nmol/L (results are means of two separate experiments). Other
components of lipoproteins including cholesterol, triglyceride, apo B,
and apo E were also examined for their capacity to support thrombin generation, but none of these components supported thrombin generation beyond the lower limit of detection of the assay. Thrombin generation supported by cholesterol (1292 µmol/L), triglyceride (1293 µmol/L), apo B (0.15 µmol/L), and apo E (0.2 µmol/L) was 25 nmol/L. A threefold increase in concentration of sphingomyelin, lysophosphatidyl choline, phosphatidyl choline, cholesterol, triglyceride, apo B, or apo
E did not significantly increase thrombin generation.
Effect of Oxidation and Nonenzymatic Glycosylation on Lipoprotein-supported Thrombin Generation The effect of modification of lipoproteins by oxidation and nonenzymatic glycosylation on lipoprotein-supported thrombin generation was then examined. Oxidation of LDL produced a 12-fold increase in lipoprotein-supported thrombin generation, compared to thrombin generation by unmodified LDL (P < .0001). No significant increase in lipoprotein-supported thrombin generation was observed with oxidation of any of the other lipoprotein classes (Table 1). LDL and VLDL lipoproteins were then studied after modification by nonenzymatic glycosylation. A range of glucose concentrations were chosen from physiological (5 mmol/L) to pathological (100 mmol/L). Incubation of VLDL and LDL with glucose concentrations over this range of concentrations did not increase lipoprotein-supported thrombin generation.Effect of Oxidation on Thrombin Generating Capacity of Specific Phospholipids In view of the effect of oxidation on LDL-supported thrombin generation, we examined the effect of oxidation of individual phospholipids on phospholipid-supported thrombin generation. Phospholipids contained within lipoproteins, identified by HPTLC, were tested before and after modification by oxidation. A variety of phospholipids were examined for their capacity to support thrombin generation. Oxidation increased phospholipid-supported thrombin generation with phosphatidyl inositol, phosphatidyl ethanolamine, and phosphatidyl serine (Table 2). Another phospholipid, phosphatidyl glycerol, that was not detected in lipoproteins by HPTLC was also tested and oxidation did not increase phosphatidyl glycerol-supported thrombin generation. Both oxidation and nonenzymatic glycosylation of apo B100, apo E, cholesterol, and triglyceride did not significantly increase thrombin generation, with thrombin levels remaining close to the lower limit of detection of the assay. The ability of both lipoproteins and phospholipids to support thrombin generation was Ca2+-dependent, and omission of any of the three coagulation factors reduced the signal to background levels confirming that all three coagulation factors are necessary for prothrombinase complex assembly.Effect of Vitamin E on Thrombin Generation Capacity of Oxidized Lipoproteins On a separate occasion, LDL from nine subjects was preincubated with vitamin E then subjected to copper oxidation. The LDL was washed and thrombin generating capacity was tested (Fig 1). In accordance with the results noted above, there was a dramatic increase in the thrombin generation potential when the LDL had been oxidized (unmodified LDL = 76 ± 12 mmol/L v oxidized LDL = 822 ± 57 nmol/L; P < .0001). The effect of copper oxidation alone on lipoprotein-supported thrombin generation was markedly reduced by preincubating LDL with vitamin E before oxidation (138 ± 47 nmol/L v 822±57; P < .0001). Thrombin generation supported by LDL that had been incubated with vitamin E only was 53 ± 2 nmol/L, whereas incubation with vitamin E without lipoprotein was below the limit of detection of the assay, suggesting that vitamin E and its solvent had little effect on baseline thrombin generation. The LDL samples were subsequently analyzed by agarose gel electrophoresis (Fig 2). Native LDL and LDL incubated with vitamin E migrated the least from the wells. In comparison, oxidized LDL migrated further down the gel. LDL that had been preincubated with vitamin E before oxidation migrated an intermediate distance. This result suggests a difference in charge between these forms of LDL with oxidation increasing net negative charge. In addition, the poorly defined bands in both the copper oxidized and copper oxidized after vitamin E preincubation fractions suggests heterogeneity within the oxidized species of LDL formed. Results obtained with the conjugate diene assay supported the observation that vitamin E diminished LDL oxidation. Thus, these results suggest that vitamin E prevents the copper-mediated increase in LDL-supported thrombin generation by decreasing LDL oxidation.
The novel and potentially important findings of our study are that atherogenic lipoproteins have the capacity to support assembly of the prothrombinase complex and to generate thrombin. Thrombin generation supported by VLDL is significantly greater than for any other unmodified lipoprotein species and oxidation specifically increases the capacity of LDL to support thrombin generation. Vitamin E is able to attenuate the increased capacity of oxidized LDL to support thrombin generation and this result suggests a unique mechanism by which vitamin E may protect against the development of CHD.
Submitted June 4, 1997;
accepted August 25, 1997.
We gratefully thank Dr P. Flynn (University of Cambridge, UK) for assistance with ultracentrifugation of plasma samples; Dr J. Tait (University of Washington, Seattle, WA) for the kind gift of annexin V; Dr S. Stone (University of Cambridge, UK) for the kind gift of a thrombin; and Dr S.H. Wild for critical reading of the manuscript.
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| Copyright © 1998 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Abstract
Introduction
-tocopherol)
markedly reduced the increase in thrombin generation observed after
oxidation of LDL (822 ± 57 v 138 ± 47 nmol/L;
P < .0001). These effects suggest that lipoproteins
are important in the production of thrombin and that vitamin E may confer protection from the detrimental effects of lipoprotein oxidation
by limiting thrombin formation. These results suggest that atherogenic
lipoproteins are linked to the development of atherosclerosis in part
by their capacity to support thrombin generation.
Abstract
