© 1998 American Heart Association, Inc.
Original Contributions |
Association of Vitamin K–Dependent Coagulation Proteins and C4b Binding Protein With Triglyceride-Rich Lipoproteins of Human Plasma
From the Department of Clinical Chemistry, University Hospital of Malmö (N.X., B.D.); and the Division of Clinical Chemistry, Department of Laboratory Medicine (A.-K.Ö.), and the Department of Medicine (Å.N.), University Hospital of Lund, Sweden.
Correspondence to Åke Nilsson, MD, PhD, Department of Medicine, University Hospital of Lund, S-221 85 Lund, Sweden.
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Abstract |
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Abstract—The triglyceride (TG) concentration in plasma is an independent risk factor for coronary heart disease. There is evidence that TG-rich lipoprotein (TGRLP), ie, chylomicrons (CMs), chylomicron remnants (CMRs), and VLDLs associate with factor VII and prothrombin and that the association enhances a platelet factor Xa–mediated prothrombin activation when the CM-prothrombin complex is exposed to platelets. In this study, we examined the association of the vitamin K–dependent coagulation factors VII, IX, X, and prothrombin, as well as the anticoagulation protein C and its cofactor protein S, in plasma lipoproteins obtained from human fasting and postprandial plasma. We also analyzed some other proteins that are related to the coagulation system but not to vitamin K–dependent proteins, including factor V, serum amyloid P component (SAP), C4b binding protein (C4BP), and thrombomodulin (TM), and as a control, Ig G. Human TGRLP (d<1.006 kg/L), LDL (d=1.006 to 1.063 kg/L), and HDL (d=1.063 to 1.210 kg/L) were separated from normal subjects both in fasting and 2 to 3 hours after the ingestion of a meal containing 100 g fat. The different coagulation proteins, SAP, C4BP, TM, and Ig G were determined by SDS-polyacrylamide gel electrophoresis combined with Western blotting, using specific polyclonal or monoclonal antibodies, and were visualized by peroxidase staining. All the vitamin K–dependent proteins associate with TGRLP in both fasting and postprandial plasma, but not with LDL or HDL. Factor V, SAP, TM, and Ig G were not found in any lipoprotein classes. C4BP, which is a regulatory protein of the classic pathway of the complement system and which binds protein S in vivo to regulate blood coagulation, was present in TGRLP, especially postprandial, but not in LDL or HDL. The amounts of prothrombin, protein S, and C4BP in postprandial TGRLP were larger than those in fasting TGRLP. Vitamin K–dependent procoagulation and anticoagulation proteins, as well as C4BP, could be associated with TGRLP in vivo. If the association enhances prothrombin activation, this effect may thus be counteracted by simultaneous binding of protein S.
Key Words: triglyceride • lipoproteins • blood coagulation • atherosclerosis
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Introduction |
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Despite more than 30 years of epidemiological and clinical research, the relations between hypertriglyceridemia, TGRLP, and CHD remain obscure and much debated.1 2 3 4 5 6 7 TGRLPs, including CMs, CMRs, and VLDLs, are a heterogeneous population of particles varying in origin, structure, and interactions with cellular receptors. More than a decade ago, Zilversmit8 advanced the hypothesis that postprandial lipoproteins are important in atherosclerosis in humans. As summarized by Patsch et al,4 this possibility was discussed also in earlier studies.9 10 11 Since there are only a few clinical studies of postprandial lipoproteins in subjects with premature CHD, this hypothesis has not yet been definitely confirmed.12 13 14 However, decreased clearance of CMs and CMRs after a fat load has been indicated to be associated with the presence of CHD in studies using semiquantitative determination of apolipoprotein B-4813 or quantification of the lipid moiety only.13 14 In an angiographic study on survivors of a myocardial infarction, Karpe et al15 demonstrated that the number of small CMRs during the postprandial phase was related to the progression of coronary artery diseases.
The vitamin K–dependent procoagulation and anticoagulation proteins have been considered to be related to arteriosclerosis and thrombosis.16 17 18 19 It has been suggested that all four vitamin K–dependent coagulation factors are related to a higher risk of ischemic heart disease. Burns et al16 analyzed factor IX and X activities, as well as concentrations of prothrombin and factor VII, and found general increases of all four vitamin K–dependent coagulation factors in high-risk CHD subjects. Increased factor VII activity has been identified as a risk factor for ischemic heart disease.17 18 20 21 22 A general increase in all four vitamin K–dependent coagulation factors could produce a hypercoagulable state much greater than that predicted for an isolated increase in factor VII. There is also a strong correlation between factor VII and plasma TG level, suggesting that there is a probable interaction of factor VII with TGRLP in vivo. Deficiencies of vitamin K–dependent anticoagulation proteins, ie, protein C and its cofactor protein S, are also related to the initiation and progression of deep venous thrombosis.23 24 25 It is still unknown whether vitamin K–dependent proteins interact with TGRLP in vivo, although prothrombin and factor Xa may bind to VLDLs in vitro, partially through a calcium-dependent association.26 27 Carvalho de Sousa et al28 found that coagulation factors VII and X could associate with isolated plasma CMs and VLDLs by using immunoenzyme assay. The associations of factor VII and factor X were stronger on CM and VLDL fractions than on LDL fraction, and no such association was found on HDLs. Furthermore, we have demonstrated that CMs bind prothrombin in vitro in a calcium-dependent manner.29 30 31 The question was therefore raised whether the TGRLPs bind vitamin K–dependent proteins and other coagulation proteins in vivo. In this study, we examined the association in vivo with human fasting and postprandial TGRLP of prothrombin, factors VII, IX, and X, protein C, and protein S and found that all these vitamin K–dependent proteins, but not SAP, factor V, TM, or Ig G, associated with the lipoproteins. We also analyzed C4BP, which is a regulatory protein of the classical pathway of the complement system and which can also bind protein S in vivo to regulate blood coagulation.32 33 34 This protein was earlier shown to bind to a soybean TG–egg phospholipid emulsion (Intralipid) and was also demonstrated in human plasma CMs (then called proline-rich protein).35 The present report confirms its association with TGRLP and shows a postprandial increase in this association.
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Methods |
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Materials
Highly purified human plasma-derived prothrombin, human coagulation factor VII, and polyclonal sheep antibody against human factor VII were obtained from Enzyme Research Laboratories. Polyclonal rabbit antibodies against human protein C, protein S, coagulation factors V, IX, and X, SAP, and Ig G were from DAKO A/S. Purified human protein C, protein S, factors V, IX, and X, C4BP, and mouse antibody against human protein S (MK 54) were available in the Department of Clinical Chemistry, University Hospital of Malmö, Sweden.36 37 Polyclonal rabbit antibody against human TM was a kind gift from Dr J. Morser, Berlex Biosciences. Rabbit anti–human prothrombin antiserum and monoclonal mouse antibodies against human factor IX (MK 13) and prothrombin (MK 10) were kind gifts from Prof Johan Stenflo, Department of Clinical Chemistry, University Hospital of Malmö, Sweden. Monoclonal mouse antibody against human factor V (MK 30) was described earlier.34 Rabbit antiserum against C4BP and monoclonal mouse antibody against C4BP (MK 104) were characterized earlier.38 39 Rainbow-colored protein molecular-weight standards of 14.3 to 220 kD and 4 to 250 kD were from Amersham Life Science and Novel Experimental Technology, respectively. Hirudin was purchased from Sigma, and PPACK from Calbiochem-Nonabiochem Co.
Volunteers and Blood Samples
Nine healthy volunteers, aged 27 to 55 years (four men and five
women; mean±SD 40.9±10.8 years), were included in the present
study, which was approved by the local ethics commission. No medication
was used within 2 weeks before the examination. Blood samples were
obtained from each volunteer after fasting overnight and 2 to 3 hours
after eating a meal containing 100 g fat in total as butter,
cheese, and cream, together with strawberry.
K3EDTA (4 mmol/L) or the mixture of hirudin
(2 U/mL blood), benzamidine (10 mmol/L), and PPACK (1
µmol/L) was used as anticoagulant. Thimerosal (sodium
ethylymercurithiosalicylate), purchased from Sigma, was immediately
added to the blood to a final concentration of 25 µmol/L to
inhibit proteolytic activity.
Lipoprotein Separation and Delipidation
TGRLPs (d<1.006 kg/L) including VLDLs, CMs, and
CMRs; LDL (d=1.006 to 1.063 kg/L); and HDL
(d=1.063 to 1.210 kg/L) were isolated by sequential
flotation.40 Briefly, 10 to 13 mL of plasma
obtained from each volunteer both in fasting and 2 to 3 hours after fat
meals were adjusted to d=1.006 kg/L with EDTA saline
(188 mmol/L NaCl in 1 mmol/L Na2EDTA,
d=1.006 kg/L) and ultracentrifuged at 37 000 rpm
for 18 hours at 10°C in a Beckman L5 to 65 ultracentrifuge
with SW 40 Ti swing-out rotor. The top layer containing TGRLP was
collected. LDL was obtained after adjusting the infranatant to
d=1.063 kg/L and ultracentrifuged at 38 000 rpm for
24 hours at 10°C. After the LDL fraction was removed and adjusted to
d=1.210 kg/L by KBr, the HDL was separated by
ultracentrifugation at 38 000 rpm for 48 hours at 10°C. Each
lipoprotein fraction was washed once by
ultracentrifugation at the designed density in the
presence of 1 mmol/L Na2EDTA. In the case of
the hirudin, benzamidine, and PPACK mixture as anticoagulant, the
lipoprotein fractions were separated as described above but without
Na2EDTA. The samples were also washed once by
buffer containing the mixture of hirudin, benzamidine, and PPACK. The
three lipoprotein fractions were delipidated with ethanol: diethyl
ether (3:1, vol/vol) at 4°C for 24 hours.41 The
delipidation procedure was repeated three times, and the protein
precipitate was washed twice with cold diethyl ether and dried under
nitrogen. The protein precipitate was resolubilized in 50 mmol/L
Tris–acetic acid buffer (pH 7.5) in presence of 0.01% Triton X-100
and then passed through 0.2-µm Millipore filters. The final
concentration of protein mass was adjusted to 1.0 mg/mL.
Electrophoresis and Immunological Determinations of
Proteins
Proteins were separated by 10% or 12% polyacrylamide
or 4% to 15% polyacrylamide gradient slab gel electrophoresis
in the presence of SDS, and Western blotting analysis was
performed according to the manufacturer's instructions, using the
Bio-Rad system (Bio-Rad Laboratories). Aliquots of delipidated samples
containing 30 µg of total protein were applied to each well of the
SDS–polyacrylamide slab gels. Purified specific proteins were
used as standards, as indicated in the figure legends.
Determination of Factor VII Activity
Factor VII activity in plasma and in different lipoprotein
fractions that had been delipidized was determined by a photometric
determination kit according to the manufacturer's protocol
(Chromogenix AB). The mixture of human normal plasma obtained from 31
volunteers was used as control plasma. The factor VII activity in the
control plasma was considered as 100%.
Proportion of Prothrombin, Factors IX and X, Protein C, Protein S,
and C4BP in Fasting and Postprandial TGRLP
The separation of the proteins and the Western blotting
analysis were carried out as described above. Different amounts
of purified specific proteins were used to make a standard curve that
was subjected to linear regression analysis after the membranes
were scanned by a Bio-Rad imaging densitometer (model GS-670) combined
with a software for image analysis system, version 2.0
(Hercules).
Chemical Determinations
The protein contents of the lipid-free lipoprotein fractions
were determined with the method of Lowry et al,42
using bovine serum albumin as the standard. The concentrations
of TG, total cholesterol, and choline-containing
phospholipids in plasma were determined by the respective enzymatic
assay methods, using Boehringer test-combination kits according
to the manufacturer's protocols.
Statistical Analysis
Values are reported as mean±SEM. Data were analyzed by
using an IBM personal computer statistical software package. One-way
ANOVA followed by unpaired Student's t test was employed
for statistical analysis. A value of P<.05 in a
two-tailed test was considered significant.
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Results |
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Plasma Lipids in Fasting and Postprandial States
As shown in Table 1
, the plasma TG
concentrations of fasting and postprandial plasma were 105.6±17.1 and
218.0±28.5 mg/dL, respectively. Total cholesterol
concentrations of the plasma were 195.3±9.9 and 197.6±8.4 mg/dL. The
choline-containing phospholipid concentrations of the samples,
determined by a phospholipase C choline oxidase kit, were 211.6±12.7
and 255.4±16.6 mg/dL.
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Association of Vitamin K–Dependent Proteins With TGRLP
As shown in Fig 1, prothrombin and
factors IX and X are found in the TGRLP in both fasting and
postprandial samples, but not in LDL and HDL fractions. The results
were the same in the determinations with polyclonal or monoclonal
antibodies (data not shown). Also, protein C and protein S are found in
the TGRLP, but not in LDL and HDL (Fig 2). Again, the results were the same when
using polyclonal or monoclonal antibodies (data not shown). No distinct
immunoreaction of factor VII in the TGRLP was observed in the Western
blotting examination, possibly due to limitation of the sensitivity of
the method (data not shown); nor was factor VII found in LDL and HDL.
We therefore also determined factor VII activity in both the plasma and
delipidized lipoprotein fractions; the factor VII activity was
increased by 30% to 35% compared with the control plasma in the
postprandial plasma. Factor VII activity was also found in TGRLP,
although it was only about 4% of the plasma factor VII activity, but
was not found in LDL and HDL. There was no obvious change of factor VII
activity between fasting and postprandial TGRLP.
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Determination of SAP, TM, Factor V, C4BP, and Ig G
No SAP, TM, factor V, or Ig G could be demonstrated in TGRLP, LDL,
or HDL (data not shown). In contrast, C4BP was found in the TGRLP (Fig 2C
), and the amount was increased in postprandial samples (Table 2). Only two of nine volunteers were
found to have C4BP in their fasting TGRLP samples, but all of the
postprandial TGRLP samples have C4BP. No positive reaction for C4BP was
found in the LDL or HDL fractions in either fasting or postprandial
samples.
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Comparison of Fasting and Postprandial States
The proportions of the vitamin K–dependent proteins and C4BP in
both fasting and postprandial TGRLP are shown in Table 2 and Fig 3. There were increases of prothrombin,
protein S, and C4BP per mass of delipidized protein in postprandial
samples compared with fasting samples, whereas factor IX, factor X, and
protein C were not increased in the TGRLP after fat meals. The
proportion of these proteins associated with the TGRLP is about 1% of
the total delipidized protein mass of the lipoproteins. There was no
statistical difference in the association of these proteins with TGRLP
by using the mixture of hirudin, benzamidine, and PPACK or the
K3EDTA (data not shown).
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Discussion |
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Factors VII, IX, X, and prothrombin, as well as protein C and its cofactor protein S, are synthesized in the liver. They show considerable sequence homology and all undergo a posttranslational vitamin K–dependent step to acquire
-carboxyglutamic acid
residues, which form medium-affinity calcium-binding
sites.43 44 The modification of glutamic acid
residues endows these proteins with the property of binding to
negatively charged phospholipid membranes at
physiological calcium
concentration.45 The calcium binding is
characterized by an initial positive
cooperativity46 and induces two conformational
transitions in the molecule.47 The first of these
is cation nonselective, whereas the second transition, necessary for
membrane binding, is specifically dependent on calcium ions. In some
hypertriglyceridemic subjects, not only
factor VII but also the other procoagulant vitamin K–dependent
proteins are increased in plasma.48 Bradley and
Gianturco27 reported that the large VLDLs that
were separated from hypertriglyceridemic
patients contained prothrombin, but not those of normal subjects; they
also demonstrated that prothrombin can bind to plasma VLDLs in vitro at
physiological concentrations of VLDLs, prothrombin,
and calcium, whereas binding to LDLs and HDLs was
negligible.26 In our previous studies, we
examined binding of prothrombin to native chyle CMs, which contain more
phospholipids and a higher proportion of phosphatidylethanolamine in
the polar surface coat49 than the particles that
have been exposed to plasma or lipoprotein lipase. We found that human
plasma-derived prothrombin can bind to rat chyle CMs with a high
affinity and in a calcium-dependent manner.31 The
data thus indicate that prothrombin binding is not due to a simple
electrostatic association but may rather be related to a conformational
change of the prothrombin during the interaction with calcium ions and
CM phospholipids.47
The data in the present study indicate that all vitamin
K–dependent procoagulation and anticoagulation proteins could
associate with TGRLP in vivo, but not with LDLs or HDLs. There was a
potential loss of proteins during a long procedure of the separation.
EDTA was used as anticoagulant during the separation of lipoproteins to
minimize the association occurring during the procedure. This factor
may cause an underestimation of the association in vivo, since any
reversible association dependent on divalent metal ions will be
disrupted. However, when we used the mixture of hirudin, benzamidine,
and PPACK instead of EDTA, the association of these proteins in the
TGRLP was not increased. Any loss of bound coagulation factors or C4BP
that may occur during the isolation of the lipoproteins was thus not
dependent on EDTA. The data indicate that the amount of prothrombin
associated with TGRLP (50±11 ng/30µg lipoprotein apoproteins:
approximately 1% of the circulation pool) should be sufficient to have
functional consequences if the association facilitates activation of
prothrombin. Such an activation could be counteracted by the
simultaneous association of protein C and its cofactor
protein S. The proportion of prothrombin, protein S, and C4BP in TGRLP
was increased after the fat meal. No such increase was seen for factors
VII, IX, or X or for protein C (Table 2). The study also showed that no
non–vitamin K–dependent proteins, including SAP, TM, factor V, and Ig
G, associated with TGRLP, indicating that the association of vitamin
K–dependent proteins and C4BP with TGRLP is specific. The mechanism of
this association needs to be examined in vitro, particularly for other
factors than prothrombin and factor Xa.26 27 31
For example, the mechanism of association of C4BP, which is not a
vitamin K–dependent protein but which can bind protein S, has not been
studied. The questions of whether C4BP interacts with TGRLP directly or
with TGRLP containing protein S and whether the association influences
the ability of C4BP to bind protein S are particularly interesting.
The functional and pathophysiological implication of the association of vitamin K–dependent proteins and C4BP with TGRLP so far remains unknown. Our earlier in vivo study in rats injected with CM–125I-prothrombin complexes50 indicated that most of the bound prothrombin is released in blood and metabolized as free prothrombin, although some is catabolized with the CMRs and a small portion remains circulating in lipoprotein-bound form for 2 hours. Zilversmit7 8 has stressed the hypothesis that during development of arteriosclerosis, TGRLP binds to arterial endothelium and to deendothelialized areas. Locally present lipoprotein lipase then initiates lipolysis. If this is so, coagulation factors binding to the lipoproteins may become activated in proximity to endothelium. This action may be facilitated if activated platelets are adhering to areas with damaged endothelium. The balance between associated prothrombin and other procoagulant factors and the protein C–protein S system may be crucial. In further studies, it is important to examine how initiation of lipolysis influences the interaction of coagulation factors with TGRLP. If TGRLP binds to lipoprotein lipase or related structures in the endothelium or structures in the damaged vessel wall, the hypothesis that coagulation factors associated with TGRLP are important in atherosclerosis development is still compatible with the observation that severe type I hyperlipoproteinemia due to lipoprotein lipase deficiency does not seem to cause premature arteriosclerosis. A recent prospective study evaluated patients with lipoprotein lipase deficiency for atherosclerosis and found that premature atherosclerosis can occur in patients with familial chylomicronemia as a result of mutations in the lipoprotein lipase gene.51 It is suggested that defective lipolysis may increase susceptibility to atherosclerosis in humans.
TGRLPs such as human VLDLs and CMs were shown to bind with a high affinity and to cause rapid, receptor-mediated lipid accumulation, creating a foam cell morphology, in murine peritoneal macrophages and P388D1 macrophages.52 53 Other studies show that human monocyte-derived macrophages also have specific high-affinity TGRLP binding sites, producing a saturable uptake of TGRLP. This uptake was apolipoprotein E and LPL independent.54 55 56 The accumulation of lipids in the cells leads to foam cell formation. Two membrane-binding activities were identified as receptor candidates for this uptake.55 The effect of bound coagulation factors on the process of foam cell formation and on the normal receptor-mediated catabolism of remnants formed from TGRLP is another interesting area for further studies. In addition, the binding of the different vitamin K–dependent factors to TGRLP in different hypertriglyceridemic states should be examined.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Swedish Medical Research Council (Nr-3969, Nr-11194, and Nr-07143), the medical faculty of the University of Lund, The Albert Påhlsson Foundation, the Nutrition Foundation of the Swedish Margarine Industry, the Crafoord Foundation, the Anna and Sven-Erik Lundgren Foundation, and the Göran Gustafsson Foundation. Polyclonal rabbit antibody against human thrombomodulin was a kind gift from Dr J. Morser, Berlex Biosciences. Rabbit anti–human prothrombin antiserum and monoclonal mouse antibodies against human factor IX (MK 13) and prothrombin (MK 10) were given by Prof Johan Stenflo, Department of Clinical Chemistry, University Hospital of Malmö, Sweden. We thank Maria Hansson for expert technical assistance.
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Footnotes |
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Presented in part at the Lipids, Membranes, and Thrombosis meeting, The Netherlands, July 10 through 13, 1996.
Received May 27, 1997; accepted September 9, 1997.
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