© 1997 American Heart Association, Inc.
Articles |
Lipoprotein(a) Isoforms Display Differences in Affinity for Plasminogen-Like Binding to Human Mononuclear Cells
From INSERM U.143 (C.K., T.S., E.A-C.) and the Cardiology Department (C.F.), Hôpital de Bicêtre, Paris, and Clinique Médicale of the CHUR de Reims (V.D.), France.
Correspondence to Dr. E. Anglés-Cano, INSERM U.143, Hôpital de Bicêtre, Bât. C. Bernard, F-94276-Cedex, Bicêtre, France. E mailangles{at}infobiogen.fr or angles{at} kb.inserm.fr
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Abstract |
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Abstract Binding of lipoprotein(a) (Lp(a)) to membrane proteins of the monocyte-macrophage cell lineage may be an important event in atheroma formation. Since Lp(a) with distinct apolipoprotein(a) (apo(a)) isoforms may show differences in their affinity with regard to fibrin binding, the existence of such a functional behavior in the interaction of apo(a) in Lp(a) with these cells was explored using the monocytic cell line THP-1. Lp(a) preparations containing small size apo(a) isoforms (Mr=450 000 to 550 000) and high molecular mass isoforms (Mr
700 000) were purified from
plasmas containing >0.35 g/L of Lp(a) obtained from subjects (n=14)
with cardiovascular atherosclerotic disease. Binding of
plasminogen to THP-1 cells was performed using the method
of radioisotopic dilution. For binding of Lp(a) to cells, the THP-1
monocytic cells were incubated with varying concentrations of the
different Lp(a) preparations; cells were then washed and the amount of
Lp(a) bound was detected with a radiolabeled polyclonal antibody
directed against apo(a). Binding due to kringle interactions with
lysine residues was calculated by subtracting from the total bound the
amount of Lp(a) bound (
10%) in the presence of 6-aminohexanoic
acid. Analysis of data with the Langmuir equation indicated
identical and independent (noninteracting) sites and allowed evaluation
of the Kd. Binding isotherms of small size
isoforms showed saturation and a high affinity
(Kd=25.8±19 nmol/L) relative to that of
plasminogen (Kd=1750±760 nmol/L). A
similar difference (Kd=17.5±7.9 nmol/L versus
Kd=600±220 nmol/L) was found when binding
experiments were performed with a fibrin surface. In contrast, binding
isotherms of the high molecular mass isoforms did not show saturation
at the highest Lp(a) concentrations used, thus indicating a lower
affinity. In conclusion, these results show that apo(a) isoforms may
display polymorphism-linked functional
heterogeneity with regard to cell binding, which may
explain the higher association with cardiovascular risk
of small size isoforms. These qualitative differences in the binding of
apo(a) isoforms to fibrin or cells may modulate the
cardiovascular risk associated with high levels of
Lp(a).
Key Words: lipoprotein(a) • apo(a) isoforms • plasminogen • monocytes
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Introduction |
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An elevated plasma concentration of the lipoprotein Lp(a) is now considered to be an independent risk factor for cerebro- and cardiovascular atherosclerotic occlusive disease. The mechanism by which Lp(a) may favor this pathologic state may be related to its particular structure, a nonactivable serine-proteinase component, apo(a), linked to the apo B-100 of an atherogenic LDL-like particle. The cDNA-derived sequence of apo(a) shows important homology (75 to 94%) with kringles 4 and 5 and the catalytic region of plasmin(ogen), including the Ser-His-Asp residues of the active site in plasmin.1 Single copies of plasminogen-like kringle 5 and the serine-proteinase region are present in apo(a), whereas the number of copies of kringle 4 varies from 10 to 40, giving rise to a series of apo(a) isoforms of variable size. In spite of this striking homology, apo(a) is resistant to activation by activators of plasminogen, a phenomenon most probably related to the Ser-Ile substitution at the Arg-Val plasminogen cleavage site to which other substitutions may also contribute.2 Therefore, competition between Lp(a) and plasminogen for binding to lysine residues of cell membrane proteins or fibrin surfaces results in decreased plasmin formation.3 4 5 In recent reports it has been proposed that apo(a) isoforms may show variability in their lysine-binding function and thereby different antifibrinolytic effects.6 7 8 Indeed, we have recently shown that Lp(a) particles containing distinct apo(a) isoforms display functional heterogeneity for fibrin binding, with the low molecular mass isoforms having the highest affinity for fibrin.9 In the present study, using the monocytic cell line THP-1 and Lp(a) purified from subjects with cardiovascular disease, we have explored the existence of such a structural/functional relationship in the interaction of apo(a) in Lp(a) with monocyte-macrophages, since binding of Lp(a) to these cells may be an important event in atheroma formation.
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Methods |
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Subjects and Blood Samples
Patients having one or several manifestations of atherosclerotic vascular occlusion and high levels of Lp(a) (>0.35 g/L) were selected among the in-patients of the Endocrinology and Metabolism Department of the Medical Clinic of the Centre Hospitalier Universitaire de Reims (Reims, France) and the Cardiology Department of the Hôpital de Bicêtre (Paris, France). Venous blood was drawn from the forearm into sterile polypropylene tubes containing 4 mmol/L of EDTA final concentration. Plasma was separated by centrifugation at 2000g for 20 minutes at +4°C and supplemented with 1 mmol/L of EDTA, 0.01% NaN3, proteolytic inhibitors (aprotinin, Trasylol, Bayer, 100 kallikrein inhibitory units (mL); D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) 1 µmol/L, D-valyl-L-phenylalanyl-L-lysine chloromethyl ketone 1 µmol/L, France Biochem, p-nitrophenyl-p'-guanidobenzoate (PNPGB) 10 µmol/L, Sigma) and an antioxidant (butylhydroxytoluene 0.05 mg/mL), final concentrations from stock solutions in water or in formamide for the two last named. The concentration of Lp(a) was determined by immunoelectrodiffusion according to Laurell,10 using a commercial kit (ImmunoFrance).
Lp(a) Purification and Characterization
Isolation of Lp(a)
Lp(a) was purified by sequential
ultracentrifugation in the density interval 1.050 to
1.100 g/mL; plasma was first adjusted to a solvent density of
1.050 g/mL with KBr and centrifuged at 134
000g for 24 hours at 10°C in a 50-Ti rotor (Beckman,
France). The floating lipoproteins (chylomicrons, VLDL, and LDL) were
removed by aspiration; the infranatant containing Lp(a) and HDL was
then adjusted with KBr to a density of 1.100 g/mL and
recentrifuged under similar conditions for 48 hours. The 1.050-
to 1.100-g/mL density fraction was removed and subjected to gel
filtration on a Biogel A-5m column (BioRad, Richmond, California)
equilibrated with 80 mmol/L of NaCl and 50
mmol/L of sodium phosphate, pH 7.4. The peak fractions
containing Lp(a) were pooled, concentrated against polyethylene-glycol
20 000 (Serva, France), and dialyzed against the same buffer containing
2 mmol/L of EDTA. The final product was conserved at
+4°C in this state until assay (less than 24 hours).
Determination of apo(a) Isoform Size
The content in apo(a) isoforms of Lp(a) preparations obtained by
sequential ultracentrifugation was evaluated as
previously described9 with minor modifications. In brief,
amounts of 1 µg of purified Lp(a) were incubated at 60°C for 30
minutes in a volume of 20 µL, 10-mmol/L of Tris buffer, pH
8.8, containing 2 mmol/L of EDTA, and 100
mmol/L of DTT; samples were then diluted 1:4 (vol/vol)
with a solution containing 4% SDS, 20% glycerol, and 0.04%
bromophenol blue and further incubated at 60°C for 15 minutes.
Amounts of 10 µL were loaded on 3.75% polyacrylamide/0.8%
agarose gels (10x12 cm), and electrophoresis was performed at 20°C
for 16 hours at 16 mA/gel constant current in 40 mmol/L of
Tris, pH 7.4, 20 mmol/L of sodium acetate, 2
mmol/L of EDTA, and 0.2% of SDS. Protein bands were
electroblotted to nitrocellulose sheets with a graphite electroblotter
system (Millipore, Bedford, Mass.) according to
Kyhse-Andersen.11 Apo(a) protein bands were localized with
a sheep antibody to human apo(a) followed by a peroxidase-conjugated
rabbit antibody to sheep IgG revealed with 4-chloro-1-naphtol. To
quantify the proportion of each isoform from a given preparation,
immunoblots were incubated overnight with a
125I-labeled polyclonal antibody directed against apo(a)
and autoradiographed on Kodak XS-films for 24 to 48 hours at -70°C.
The apo(a) bands were cut and counted in a 
-radiation counter.
Apo(a) isoforms were identified using reference plasmas containing
isoforms B, S1, S3, S4, and S4+ generously provided by Dr G. Utermann
(University of Innsbrück, Austria).
Antisera and Specific Immunoglobulins
Sheep antiserum against human apo(a) was prepared as
described.4 The IgG fraction of this antiserum was
separated by ammonium sulfate precipitation, ion-exchange
chromatography on diethylaminoethyl-Trisacryl, and
affinity chromatography on protein-A Sepharose
(Pharmacia). The purified IgG was further purified using
Sepharose-immobilized plasminogen and apo
B-100; the final antibody preparation did not cross-react with these
proteins. The affinity of this antibody for apo(a) was determined using
the method of radioisotopic dilution as follows: varying concentrations
of the purified IgG containing a constant amount of the
125I-labeled antibody were incubated for 1 hour at 22°C
with a constant concentration of different isoforms of Lp(a) bound to a
fibrin surface. Unbound antibody was removed, and bound radioactivity
was then counted in a -radiation counter. Data were fitted to the
Langmuir adsorption isotherm equation (see Analysis of Binding
Data); the mean dissociation constant thus calculated was 3
nmol/L.
Purification of Proteins
Glu-plasminogen was purified as previously
described12 from diisopropylfluorophosphate-treated
fresh-frozen human plasma by affinity chromatography on
lysine-Sepharose 4B,13 gel filtration on Ultrogel AcA 44,
and ion-exchange chromatography on
diethylaminoethyl-Trisacryl. All procedures were performed at 4°C in
the presence of aprotinin. No contaminant plasmin activity was detected
by incubating the plasminogen with the
chromogenic substrate CBS 0065 (1.5 mmol/L
final concentration) for 48 hours at 37°C. The
plasminogen preparation was considered to be more than 99%
pure and was shown to be Lp(a) free as assessed by
SDS-polyacrylamide gel electrophoresis and
immunoblotting, respectively. The concentration of the
plasminogen preparation was measured spectrophotometrically
in 50 mmol/L of sodium phosphate buffer, pH 7.4, containing
80 mmol/L of NaCl, 0.01% NaN3, and 0.01%
Tween-20 (buffer A), using E(1%, 1 cm)=16.8 at 280
nm.14
Fibrinogen was purified as previously described15 from fresh-frozen human plasma supplemented with proteinase inhibitors (100 kallikrein inhibitory units/mL of aprotinin, 2 mmol/L of diisopropyl fluorophosphate, 1 µmol/L of PPACK, 0.2 mol/L of 6-Ahx, 10 µmol/L of PNPGB, 4 mmol/L of benzamidine, and 0.5 unit/mL of Hirudin, final concentrations) by glycine precipitation according to Kazal et al16 with minor modifications. It was then chromatographed on a sepharose 6B column (Pharmacia) in 50 mmol/L of phosphate buffer, pH 7.4, containing 0.5 mol/L of NaCl, 2 mmol/L of EDTA, and all inhibitors except 6-Ahx. Additional purification was obtained by affinity chromatography on lysine-Sepharose 4B (Pharmacia), gelatin-Ultrogel (BioSepra), and organomercurial agarose (BioRad) with use of the same buffer. Fibrinogen was concentrated by precipitation with 25% ammonium sulfate, extensively dialyzed against 0.1 mol/L of phosphate buffer, pH 7.4, containing 0.3 mol/L of NaCl, separated into aliquots (12 mg/mL), and stored at -70°C. The purified fibrinogen was free of von Willebrand factor, plasminogen, fibronectin, and factor XIII as determined by an enzyme-linked immunosorbent assay specific for these proteins. The absence of plasminogen or plasmin was confirmed by incubation for 72 hours at 37°C of the fibrin-agar plates prepared with the purified products supplemented or not with a plasminogen activator. Fibrinogen was more than 98% clottable and appeared homogeneous by SDS-polyacrylamide gel electrophoresis and autoradiography of the 125I-labeled product. Protein concentration was determined by measuring the absorbance in buffer A at 280 nm using E(1%, 1 cm)=15.1 at 280 nm.17
Cell Culture
The human monocytic leukemia-derived cell line
THP-118 was grown in Dulbecco-Iscove medium supplemented
with 10% fetal calf serum, 4 mmol/L of glutamine, 100
µg/mL of penicillin, 100 units/mL of streptomycin, 0.5
mmol/L of sodium pyruvate, 0.5% essential amino acids, and
0.5% nonessential amino acids. Cells were maintained in tissue culture
flasks at 37°C in a humidified atmosphere of 5% CO2 and
95% air at a density of 1.0 to 2.0x105 cells/mL.
Preparation of Fibrin Surfaces
Fibrin surfaces were prepared as previously
described.12 19 Briefly, fibrinogen was covalently bound
to poly(vinyl chloride)-bound stable polyglutaraldehyde
derivatives. The fibrinogen monolayer was then treated with thrombin
(20 NIH U/mL), and its transformation into a fibrin surface was
verified by the disappearance of immunoreactivity with a monoclonal
antibody (Y 18) directed against the A
stretch 1-51 of human
fibrinogen.20 Plasmin-degraded fibrin surfaces were
prepared by treatment with 25 nmol/L of plasmin for 30 minutes
at 37°C in 50 mmol/L of sodium phosphate buffer, pH 7.4,
containing 80 mmol/L of NaCl, 0.01% NaN3,
0.01% Tween-20, and 2 mg/mL of bovine serum albumin
(assay-buffer). Plasmin was then eluted by incubating (three cycles of
8 hours) the degraded surface of fibrin with assay-buffer containing
0.2 mol/L of trans-4-(aminomethyl)-cyclohexane
carboxylic acid, 1 mmol/L of benzamidine, and 10
µmol/L of the plasmin inhibitor
ValPheLysCH2Cl. The degradation of fibrin by plasmin was
verified with a monoclonal antibody (FDP-14) directed against fibrin
fragment E.21
Radioiodination of Proteins
Plasminogen and the purified IgG against apo(a) were
radioiodinated with Na[125I] using the
lodogen(TM) method of Fracker and Speck,22 an iodination
time of 4 minutes at 4°C, and removal of free 125I by
molecular sieving on a PD-10 Sephadex column (Pharmacia). The specific
radioactivity obtained was 9 to 11 nCi/ng of plasminogen
and 3 nCi/ng of anti-apo(a) IgG.
Binding of Purified Lp(a) and Plasminogen to THP-1
Cells
THP-1 monocytic cells were harvested, centrifuged at
200g for 10 minutes at 22°C, washed twice with 50 mL of
Hanks' buffer containing 2 mg/mL of bovine serum
albumin, and resuspended in the same buffer at a density of
5x106 cells/mL. Varying concentrations of purified Lp(a)
were prepared in Hanks' buffer containing 4 mg/mL of bovine
serum albumin; 25 µL of each dilution were incubated with 25
µL of the cellular suspension for 1 hour at 22°C in a microtiter
plate. To remove unbound Lp(a), cells were washed three times with
Hanks' buffer containing 2 mg/mL of bovine serum
albumin, resuspended in 25 µL of dilution buffer, and probed
for 1 hour at 22°C with a known concentration of the
125I-labeled polyclonal antibody directed against apo(a).
Unbound antibody was removed by washing thrice with the same buffer,
and bound radioactivity was counted in a -radiation counter. The
mass of antibody bound was calculated by dividing the radioactivity of
each well by the specific radioactivity (dpm/mol of IgG) of the labeled
antibody.
Binding of plasminogen to THP-1 cells was performed using
the method of radioisotopic dilution. A constant amount of
125I-labeled plasminogen was added to varying
concentrations of nonlabeled plasminogen (0 to 10
µmol/L) to obtain solutions with different specific activities
(dpm/mol). A volume of 25 µL of these solutions was incubated with 25
µL of the cellular suspension for 1 hour at 22°C. Cells were then
washed, and radioactivity bound to cells was counted in a -radiation
counter and transformed into pmol of plasminogen bound,
using the following relation:
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Binding of Purified Lp(a) and Plasminogen to
Fibrin Surfaces
A volume of 25 µL of each solution of Lp(a) or
125I-labeled plasminogen prepared as described
above was loaded on fibrin surfaces containing 25 µL per well of
Hanks' buffer supplemented with 4 mg/mL of bovine serum
albumin. After a 1-hour incubation at 22°C, unbound Lp(a) or
plasminogen was removed by washing with Hanks' buffer
containing 2 mg/mL of bovine serum albumin. Surfaces
with bound radiolabeled plasminogen were cut and counted.
Surfaces containing bound Lp(a) were incubated with the
125I-labeled polyclonal antibody directed against apo(a)
and after 1 hour at 37°C, the plate was washed, cut, and counted. The
radioactivity in each well was transformed into mass of bound antibody
or plasminogen as described above.
To evaluate the effect of Lp(a) isoforms S1 and S3 on plasminogen binding, varying amounts of Lp(a) were added to a plasma containing 2 µmol/L of plasminogen and no Lp(a). These mixtures were incubated with fibrin surfaces as described above, and the amount of plasminogen bound was detected by incubating during 1 hour at 37°C a monoclonal antibody that recognized plasminogen kringle 1,23 followed by a goat antimouse IgG radiolabeled as indicated before.
Analysis of Binding Data
Binding (n) of Lp(a) and plasminogen to
THP-1 cells and fibrin surfaces and of the apo(a) antibody to fibrin
bound Lp(a) was measured as a function of ligand concentration
(C) at a given temperature (T). In such a case,
the quantity bound is a function of the equilibrium solute
concentration, as represented by the adsorption isotherm
function
 | (1) |
 | (2) |
using nonlinear
regression analysis (see Figs 2
and 3) means that the surface
consists of adsorption sites, that all adsorbed species are confined to
localized sites without any interactions between adjacent molecules,
and that the limit of adsorption with increasing concentration of
ligand corresponds to saturation of the surface with a monolayer. By
relating the number of occupied sites
(S · X=bound plasminogen or bound
anti-apo(a) antibody) to ligand concentration
(X=plasminogen or Lp(a), equation 2 enables one
to calculate the two parameters, the maximum bound
Bmax=S0, and the dissociation
constant Kd=K-1.
Bmax and Kd for
plasminogen were calculated by direct fit of raw data to
the nonlinear equation. The amount of Lp(a) adsorbed onto cells or
fibrin was, however, expressed by the mass of antibody bound using an
algebraic expression of equation 2, which describes the linear
relationship of antibody binding at low antibody concentrations with
the amount of fibrin-bound Lp(a):12
 | (3) |
 | (4) |
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, specific binding; 
, binding obtained with 0.2
mol/L of 6-Ahx; 
, total binding. Kd=940
nmol/L, Bmax=1.28 pmol of plasminogen bound.
Bars represent the error of triplicates.
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Results |
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Characteristics of the Plasmas Studied
A total of 110 plasmas were studied, and 59 had an Lp(a) concentration greater than 0.35 g/L. Fourteen of these patients had a second consultation during the duration of this study, and plasma was obtained on this occasion for the purification of Lp(a). Characteristics of these patients with regard to Lp(a) plasma concentration, apo(a) isoform phenotype, and cardiovascular involvement are given in Table 1
. The apo(a) isoforms were identified
using reference plasmas containing isoforms B, S1, S3, S4, and S4+
generously provided by Dr G. Utermann (University of Innsbrück,
Austria).
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This classification25 is based on the electrophoretic mobility of apo B-100 (Mr 500 000); isoform B has an electrophoretic migration similar to that of apo B-100, whereas isoforms S1 to S4 migrate more slowly than apo B-100. Those in intermediate positions were binned with the closest isoform type with a plus or minus sign indicating higher or lower molecular mass. Isoform S2 was assigned when migration of the lipoprotein was intermediate between isoforms S1 and S3. The sheep antibody to human apo(a) was shown to react equivalently with the different isoforms.
Among the 14 patients, 12 were heterozygous with, in most of the cases,
a predominant isoform in the zone of isoform S1; this is in agreement
with the greater frequency of these isoforms in the Caucasian
population. Patients L.j and H.p can be considered as homozygous for
isoforms S1+ and S2, respectively. In contrast, patients M.m, D.f, B.m,
and L.a have a predominant high molecular mass isoforms S3 or S4 (Fig 1).
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Binding of Plasminogen to THP-1 Cells
In Fig 2, the binding of
plasminogen to THP-1 cells is shown. The binding was dose
dependent, saturating, and specific for carboxy-terminal lysine
residues as indicated by its inhibition with the lysine analog 6-Ahx;
indeed, 90% of the binding was inhibited when the experiment was
performed in the presence of 0.2 mol/L of 6-Ahx. A dissociation
constant of 940 nmol/L and a Bmax of 1.28 pmol of
plasminogen bound /25·105 cells were
calculated applying equation 2 to raw data. The mean value obtained
from nine experiments was 1750±760 nmol/L; the corresponding
Kd on fibrin surfaces was 660±220 nmol/L
(Table 2). These values were in the same
order of magnitude, thus indicating a similar type of interaction
between plasminogen and carboxy-terminal lysine residues in
both fibrin and THP-1 cells.
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Binding of Purified Lp(a) to THP-1 Cells
The binding isotherms obtained with isoforms S1 and S3 on THP-1
cells and fibrin can be seen in Figs 3 and 4. The x axis represents the
molar concentration of Lp(a) in terms of total protein content
[apo(a)+apo B-100], assuming one molecule of each apolipoprotein
component per Lp(a) particule.26 The y axis
represents the amount of anti-apo(a) antibody bound to THP-1
cells or fibrin. The Lp(a) preparation used in the experiment
represented in Fig 3 was purified from patient L.j. The
binding was, as for plasminogen, dose dependent,
saturating, and specific. Binding due to kringle interactions was
calculated by subtracting the amount bound in the presence of 6-Ahx
acid (~10%) from the total Lp(a) bound. Only the specific binding is
represented in Fig 3. The Kd values
obtained with this Lp(a) preparation on THP-1 cells and on fibrin were
similar, 25 and 29 nmol/L, respectively.
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Fig 4 represents the binding of high molecular mass isoform S3
purified from patient M.m to cell membranes and to fibrin surfaces.
Binding of this isoform was specific as indicated by its inhibition
with 6-Ahx. However, saturation could not be reached at the highest
input concentrations of Lp(a) used. It was therefore impossible to
calculate the dissociation constants of these interactions, thus
indicating that isoform S3 has less affinity for THP-1 cells and for
fibrin than isoform S1.
Kd values obtained with plasminogen
and Lp(a) particles containing isoforms of small molecular mass are
summarized in Table 2. The Kd value (mean±SD)
obtained with different Lp(a) preparations containing a majority of
small size isoforms (S1 with/or S2) was 50-fold lower than the value
obtained for plasminogen; this difference may explain the
high ability of small size isoforms to compete efficiently with
plasminogen for binding to fibrin and THP-1 cells. In
contrast, isoforms of high molecular mass had lower affinity and a
lower ability to compete with plasminogen.
Effect of Lp(a) on the Binding of Plasminogen to
Fibrin Surfaces
In Fig 5, the effect of Lp(a)
isoforms S1 and S3 on the binding of plasminogen to a
fibrin surface is shown. The amount of plasminogen bound to
fibrin decreased with the increase in the binding of added Lp(a); at
identical molar concentrations, the effect of isoform S1 was more
pronounced (>20%) than the effect of isoform S3 (~10%). These
experiments were performed under conditions as close as possible to a
physiologic condition, the plasma/fibrin interface, and in the presence
of 2 µmol/L of plasminogen. These results are
indicative of competitive inhibition between plasminogen
and Lp(a) for the same type of binding sites.
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) were added to a plasma containing a normal
plasminogen concentration (2 µmol/L) and
undetectable Lp(a). These mixtures were incubated with fibrin surfaces,
and the amount of Lp(a) bound (right graph) was detected and expressed
as indicated in Fig 3 |
Discussion |
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The definition of a threshold risk value for Lp(a) is complicated by variations in the distribution of Lp(a) among different populations, eg, black Africans, Eskimos, and Caucasians. It has been stated, however, that a high concentration of Lp(a) is a major independent risk factor for atherosclerosis. As plasma Lp(a) concentrations are inversely related to the number of sequences encoding kringle 4, the question is to know whether short apo(a) alleles are an atherosclerotic risk factor. Indeed, it has been reported that the apo(a) allele length distribution among atherosclerotic patients differs substantially from that among healthy subjects.25 27 28 29 30 According to these data, the short apo(a) alleles contribute to atherogenesis by increasing the Lp(a) content, thus suggesting a crucial role for protein concentration rather than for apo(a) size in pathogenesis. However, attempts to establish the role of apo(a) allele length on the development of atherosclerosis have not taken into account the qualitative behavior of Lp(a) isoforms, ie, their ability to inhibit plasminogen binding to fibrin and cell surfaces, and thereby to allow fibrin deposition, and to interfere with a variety of cellular processes. Indeed, variations in repeats number in the genes may also alter protein functioning.31 Therefore, the influence of the allelic apo(a) variants on the development of cardiovascular disease may be more reliably estimated by determining the affinity of their interactions with fibrin and cell surfaces. We (E.A-C., V.D.) have previously shown that the concentration-dependent effect of Lp(a) on the inhibition of plasminogen binding is subordinated to its affinity for lysine-binding sites on fibrin.9 32 In the present work, our aim was to investigate whether a similar phenomenon is the dominant rule in the interaction of different Lp(a) particles with cells of the monocytic/macrophage lineage, since these cells may play an important role in atheroma formation. For that purpose, Lp(a) particles isolated from the plasma of subjects with cardiovascular disease and the monocytic cell line THP-1 were used. These cells constitute a well-known model of human monocytes and have been used to study the binding of plasminogen33 34 and its activators,35 as well as the formation of foam cells.36 37 Bottalico et al,38 Keesler et al,39 and Skiba and colleagues40 have studied the internalization and degradation of Lp(a) and apo(a) by macrophage foam cells and have concluded that these phenomena were mediated by a calcium-dependent cell-surface receptor induced by cholesterol loading and down-regulated by interferon-
.
Because in our studies we have used nonstimulated THP-1 cells, the
internalization and degradation of Lp(a) by this cell-surface receptor
was not considered. Also, interactions between the LDL-like particle of
Lp(a) and the apoB/E receptor or the LDL receptor reported in some
studies41 42 were not analyzed in the present
work. Indeed, data concerning the role of the LDL receptor in Lp(a)
uptake and degradation are conflicting. Moreover, Rader et
al43 have recently investigated the in vivo catabolism of
Lp(a) and LDL in patients with homozygous familial
hypercholesterolemia and have concluded that
the LDL receptor was not required for the catabolism of Lp(a).
In the present work, we were specifically concerned with cell
interactions of Lp(a) through its apo(a) component, and experiments
were designed to specifically detect plasminogen-like
binding to lysine residues of certain membrane proteins. The exact
nature of these proteins is still controversial; however, it has been
suggested that Lp(a) can interact directly with -enolase and other,
not as yet identified, proteins having carboxy-terminal lysine
residues.44 The value of the dissociation constant we have
measured for plasminogen with these cells
(Kd=1750±760 nmol/L) is in agreement
with previously reported values.33 34 35 Lp(a) particles in
sufficient amounts for binding studies were isolated from plasma (n=14)
containing more than 0.35 g/L of Lp(a). A single isoform was
detected in two cases, a predominant isoform (80 to 95%) was
present in nine other cases, and three plasmas contained two
isoforms that were small size (~S1) at nearly equal proportions. Of
note, in three cases, the main isoform in plasma was an isoform of high
molecular mass (S3 or S4).
To prevent complications in the evaluation of binding data due to
effects (oxidation, changes in conformation) of radiolabeling
procedures on the lipoprotein complex,45 our strategy was
to quantify the apo(a)-dependent interactions of Lp(a) with cell
surfaces by using a polyclonal antibody specific for human apo(a).
Indeed, the structural diversity of individual antibodies in a
polyclonal preparation prevents interference of radioiodination in the
detection of Lp(a), even if the procedure may be harmful for some
antibody subtypes. Because the affinity of the antibody for apo(a) was
independent of isoform size and, as described by equation 3, a linear
relationship of antibody binding exists at low antibody concentrations,
the mass of antibody bound becomes proportional to the amount of Lp(a)
adsorbed on cells or fibrin. The amount of Lp(a) bound thus calculated
at each Lp(a) concentration was characteristic of a dose-response curve
for Lp(a) and described a typical Langmuir isotherm saturation profile
that allowed calculation of the affinity of the different apo(a)
isoforms for cell surfaces using equation 2. The
Kd values for the interaction of Lp(a) with
THP-1 or fibrin were similar; the affinity of small size isoforms was
nearly 50-fold more important than the affinity of
plasminogen for similar surfaces
(Kd=25.8±19 versus 1750±760 nmol/L),
thus suggesting that these isoforms could efficiently compete with
plasminogen for binding to cell or fibrin surfaces.
Experimental evidence on this competitive binding was obtained (Fig 5).
Consequently, these small size isoforms may have an antifibrinolytic
effect. In contrast, isoforms of high molecular mass displayed weak
affinities with Kd values approaching the
Kd for plasminogen. Binding of the
different Lp(a) preparations to THP-1 cells was specifically inhibited
by 6-Ahx as was their binding to fibrin; in a similar fashion, the
interactions of plasminogen with both THP-1 cells and
fibrin were inhibited by 6-Ahx. These results are in agreement with
previously published data32 46 and indicate the existence
of single site interactions between domains in apo(a) or
plasminogen and the lysine residues in fibrin or THP-1
membrane proteins. The difference in affinity between
plasminogen and Lp(a) for lysine residues in fibrin
resulted in reciprocal inhibition of binding as demonstrated by
quantitative analysis.32 This type of interaction
is typical of multiple binding by two (or more) ligands that bind to
equivalent but independent sites, ie, binding is mutually
exclusive.46 Therefore, binding of either Lp(a) or
plasminogen to THP-1 cells will be the result of
interactions governed by their relative affinities and concentrations.
As plasminogen concentration (~2 µmol/L in
plasma) and affinity are relatively constant, variation in Lp(a)
concentration and apo(a) isoform type should be determinant in the
resulting fibrinolytic potential of a given subject (Fig 5).
Accordingly, Lp(a) isoforms with high affinity for THP-1 cells will
inhibit binding of plasminogen in a concentration-dependent
manner, thus explaining the concentration-dependent effect of small
apo(a) size isoforms on atherogenesis. In contrast, the contribution of
high molecular mass isoforms to this process will be less pronounced
even at high concentrations as their affinity for fibrin and THP-1
cells approaches the corresponding affinities of
plasminogen. Therefore, the plasma concentration of high
molecular mass isoforms will not considerably influence
plasminogen binding.
In conclusion, the results presented in this study demonstrate that Lp(a) isoforms display heterogeneity with regard to cell binding, with small size isoforms having the highest affinity for lysine residues of membrane proteins in THP-1 cells. These qualitative differences in the behavior of Lp(a) with regard to fibrin or cell binding may modulate the role of high levels of Lp(a) as a cardiovascular risk factor. If present in vivo, this phenomenon may explain the greater cardiovascular risk associated with small size isoforms.9 Studies aimed at determining the presence of these isoforms in atheroma plaques or at sites of vascular injury will be most useful for this purpose.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by grant 3AM002 from INSERM/CNAMTS and grant ACC-SV from the Ministère de l'Enseignement Supérieur et de la Recherche. C.K. is a recipient of a research scholarship from the latter. T.S. is a recipient of a research grant from the Sanofi Foundation. The technical assistance of Stéphane Loyau is gratefully acknowledged. We are grateful to Dr T. Lambert (Blood Bank, Hôpital de Bicêtre) for providing fresh-frozen human plasma for the purification of plasminogen and fibrinogen.
Received July 15, 1996; accepted February 28, 1997.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Mc Lean JW, Tomlinson JE, Kuang WJ, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn RM. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature.. 1987;330:132-157.[Medline] [Order article via Infotrieve]
2. Gabel BR, Koschinsky ML. Analysis of the proteolytic activity of a recombinant form of apolipoprotein(a). Biochemistry.. 1995;34:15777-15784.[Medline] [Order article via Infotrieve]
3. Harpel PC, Gordon BR, Parker TS. Plasmin catalyses binding of lipoprotein(a) to immobilized fibrinogen and fibrin. Proc Natl Acad Sci U S A 1989;86:3847-3851.
4.
Rouy D, Grailhe P, Nigon F, Chapman J, Anglés-Cano
E: Lipoprotein(a) impairs generation of plasmin by fibrin-bound
tissue-type plasminogen activator.
Arterioscler Thromb.. 1991;11:629-638.
5.
Loscalzo J, Weinfeld M, Fless GM, Scanu AM.
Lipoprotein(a), fibrin binding and plasminogen
activation. Arteriosclerosis.. 1990;10:240-245.
6.
Scanu AM, Pfaffinger D, Lee JC, Hinman J. A
single point mutation (Trp
Arg) in human apo(a) kringle 4-37
associated with a lysine binding defect in Lp(a). Biochem
Biophys Acta.. 1994;1227:41-45.[Medline]
[Order article via Infotrieve]
7. Armstrong VW, Harrach B, Robenek H, Helmhold M, Walli AK, Seidel D. Heterogeneity of human lipoprotein Lp(a): cytochemical and biochemical studies on the interaction of two Lp(a) species with the LDL receptor. J Lipid Res.. 1990;31:429-441.[Abstract]
8. Leerink CB, Duif PFCCM, Gimpel JA, Kortlandt W, Bouma BN, van Rijn HJM. Lysine-binding heterogeneity of Lp(a): consequences for fibrin binding and inhibition of plasminogen activation. Thromb Haemostas.. 1992;68:185-188.[Medline] [Order article via Infotrieve]
9.
Hervio L, Chapman J, Thillet J, Loyau S,
Anglés-Cano E. Does apolipoprotein(a)
heterogeneity influence lipoprotein(a) effects on
fibrinolysis? Blood.. 1993;82:392-397.
10. Laurell CB. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem.. 1984;15:283.
11. Kyhse-Andersen J. Electroblotting of multiple gels: a single apparatus without buffer tank for rapid transfer of protein from polyacrylamide to nitrocellulose. J Biophys Biochem Methods.. 1984;10:203-209.
12. Fleury V, Anglés-Cano E. Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines. Biochemistry.. 1991;30:7630-7638.[Medline] [Order article via Infotrieve]
13.
Deutsch D, Mertz ET. Plasminogen
purification from human plasma by affinity
chromatography. Science.. 1970;170:1095-1096.
14. Wallen B, Wiman B. Characterization of human plasminogen. II. Separation and partial characterization of different molecular forms of human plasminogen. Biochim Biophys Acta.. 1972;257:122-134.[Medline] [Order article via Infotrieve]
15. Grailhe P, Nieuwenhuizen W, Anglés-Cano E. Study of tissue-type plasminogen activator binding sites on fibrin using distinct fragments of fibrinogen. Eur J Biochem.. 1994;219:961-967.[Medline] [Order article via Infotrieve]
16. Kazal LA, Amsel S, Miller OP, Tocantins LM. The preparation and some properties of fibrinogen precipitated from human plasma by glycine. Proc Soc Exp Biol Med.. 1963;113:989-994.
17. Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Ark Kemi.. 1956;10:415-443.
18. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer.. 1980;26:171-176.[Medline] [Order article via Infotrieve]
19. Anglés-Cano E. A spectrophotometric solid-phase-fibrin-tissue plasminogen activator activity assay (SOFIA-t-PA) for high-fibrin-affinity tissue plasminogen activators. Anal Biochem.. 1986;153:201-210.[Medline] [Order article via Infotrieve]
20.
Koppert PW, Huisjsmans CMG, Nieuwenhuizen W. A
monoclonal antibody specific for human fibrinogen,
fibrinopeptide A-containing fragments and not reacting
with free fibrinopeptide A. Blood.. 1985;66:503-507.
21.
Koppert PW, Koppman J, Haverkate F, Nieuwenhuizen
W. Production and characterization of a monoclonal
antibody reactive with a specific neoantigenic determinant (comprising
Bß 54-118) in degradation products of fibrin and of
fibrinogen. Blood.. 1986;68:437-441.
22.
Fracker PM, Speck JC. Protein cell membrane
iodination with a sparingly soluble chloramide, 1, 3, 4,
6-tetrachloro-3, 6-diphenylglycoluril. Biochem
Biophys Res Commun.. 1978;80:849-857.[Medline]
[Order article via Infotrieve]
23.
Montes R, Páramo JA, Anglés-Cano E, Rocha
E. Development and clinical application of a new ELISA assay to
determine plasmin-2-antiplasmin complexes in plasma.
Br J Haematol.. 1996;92:979-985.[Medline]
[Order article via Infotrieve]
24. Adamson AW. The Langmuir Adsorption Isotherm: Physical Chemistry of Surfaces. New York, NY; John Viley & Sons, Inc., 1990:595-606.
25. Utermann G, Menzel H, Kraft H, Duba H, Kemmler H, Seitz C. Lp(a) glycoprotein phenotypes: inheritance and relation to Lp(a)-lipoprotein concentrations in plasma. J Clin Invest.. 1987;80:458-465.
26. Albers JJ, Kennedy H, Marcovina SM. Evidence that Lp(a) contains one molecule of apo(a) and one molecule of apoB: evaluation of amino acid analysis data. J Lipid Res.. 1996;37:192-196.[Abstract]
27. Sandholzer C, Boerwinkle E, Saha N, Tong MC, Utermann G. Apolipoprotein(a) phenotypes, Lp(a) concentration and plasma lipid levels in relation to coronary heart disease in a chinese population: evidence for the role of the apo(a) gene in coronary heart disease. J Clin Invest.. 1992;89:1040-1046.
28. Sandholzer C, Saha N, Kark JD, Rees A, Jaross W, Dieplinger H, Hoppichler F, Boerwinkle E, Utermann G. Apo(a) isoforms predict risk for coronary heart disease: a study in six populations. Arterioscler Thromb.. 1992;12:1214-1226.[Abstract]
29. Yiyi Z, Janjun L, Junjun W. Apolipoprotein(a) phenotypes in cardiocerebrovascular diseases. Chem Phys Lipids. 1994;67/68:293-297.
30. Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson GR, Boerwinkle E, Utermann G. Relation of serum lipoprotein(a) and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hyper cholesterolemia. N Engl J Med.. 1990;322:1494-1499.[Abstract]
31. Lancaster CA, Peat N, Duhig T, Wilson D, Taylor-Papadimitriou, Gendler SJ. Structure and expression of the human polymorphic epithelial mucin gene: an expressed VNTR unit. Biochem Biophys Res Commun.. 1990;173:1019-1029.[Medline] [Order article via Infotrieve]
32. Hervio L, Durlach V, Girard-Globa A, Anglés-Cano E. Multiple binding with identical linkage: a mechanism that explains the effect of lipoprotein(a) on fibrinolysis. Biochemistry.. 1995;34:595-606.
33.
Miles LA, Dahlberg CM, Plescia J, Felez J, Kato K, Plow
EF. Role of cell-surface lysines in plasminogen
binding to cell: identification of -enolase as a candidate of
plasminogen receptor. Biochemistry.. 1991;30:1682-1691.[Medline]
[Order article via Infotrieve]
34.
Felez J, Miles LA, Plescia J, Plow EF.
Regulation of plasminogen receptor expression on human
monocytes and monocytoid cell lines. J Cell Biol.. 1990;111:1673-1685.
35. Felez J, Miles LA, Fabregas P, Jardi M, Plow EF, Lijnen RH. Characterization of cellular binding sites and interactive regions within reactants required for enhancement of plasminogen activation by t-PA on the surface of leukocytic cells. Thromb Haemost.. 1996;76:577-584.[Medline] [Order article via Infotrieve]
36. Hassall DG. Three probes flow cytometry of a human foam-cell forming macrophages. Cytometry.. 1992;13:381-388.[Medline] [Order article via Infotrieve]
37. Banka CL, Black SA, Cheryl A, Cutiss LK. THP-1 cells form foam cells in response to coculture with lipoproteins but not with platelets. J Lipid Res.. 1991;32:35-45.[Abstract]
38.
Bottalico LA, Keesler GA, Fless GM, Tabas I.
Cholesterol loading of macrophages leads to marked
enhancement of native lipoprotein(a) and apoprotein(a) internalization
and degradation. J Biol Chem.. 1993;268:8569-8573.
39.
Keesler GA, Li YQ, Skiba PJ, Fless GM, Tabas I.
Macrophage foam cell lipoprotein(a)/apoprotein(a)
receptor-cell-surface localization, dependence of induction on new
protein synthesis, and ligand specificity. Arterioscler
Thromb.. 1994;14:1337-1348.
40.
Skiba PJ, Keesler GA, Tabas I. Interferon-
down-regulates the lipoprotein(a)/apoprotein(a) receptor activity on
macrophage foam cells—Evidence for disruption of
ligand-induced receptor recycling by interferon-gamma.
J Biol Chem.. 1994;269:23059-23067.
41. Hofmann SL, Eaton DL, Brown MS, McConathy WJ, Goldstein JL, Hammer RE. Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice. J Clin Invest.. 1990;85:1542-1547.
42.
Utermann G, Hoppichler F, Dieplinger H, Seed M,
Thompson J, Boerwinkle E. Defects in low density lipoprotein
receptor gene effects lipoprotein(a) levels: multiplicative interaction
of two gene loci associated with premature
atherosclerosis. Proc Natl Acad Sci
U S A.. 1989;86:4171-4174.
43. Rader DJ, Mann WA, Cain W, Kraft HG, Usher D, Zech LA, Hoeg JM, Davignon J, Lupien P, Grosman M, Wilson JM, Brewer HB Jr. The low density lipoprotein receptor is not required for normal catabolism of Lp(a) in humans. J Clin Invest.. 1995;95:1403-1408.
44. Miles LA, Fless GM, Scanu AM, Baynham P, Sebald MT, Skocir P, Curtiss LK, Levin EG, Hoover-Plow JL, Plow EF. Interaction of Lp(a) with plasminogen binding sites on cells. Thromb Haemost.. 1995;73:458-465.[Medline] [Order article via Infotrieve]
45. Fidge N. A review of methods and metabolic studies associated with the radioiodination of lipoproteins. Clin Chim Acta.. 1974;52:5-13.[Medline] [Order article via Infotrieve]
46. Hervio L, Girard-Globa A, Durlach V, Anglés-Cano E. The antifibrinolytic effect of lipoprotein(a) in heterozygous subjects is modulated by the relative concentration of each of the apolipoprotein(a) isoforms and their affinity for fibrin. Eur J Clin Invest.. 1996;26:411-417.[Medline] [Order article via Infotrieve]
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