Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2036-2043
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2036-2043.)
© 1997 American Heart Association, Inc.
Lipoprotein(a) Isoforms Display Differences in Affinity for Plasminogen-Like Binding to Human Mononuclear Cells
C. Kang;
V. Durlach;
T. Soulat;
C. Fournier;
;
E. Anglés-Cano
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
 |
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
 |
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.
 |
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 2000
g for 20 minutes at
+4°C
and supplemented with 1 mmol/L of EDTA, 0.01%
NaN
3, 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:
Binding of Lp(a) and plasminogen to THP-1 cells
in
the presence of 0.2 mol/L of 6-Ahx was considered as unrelated
to
interactions with lysine residues.
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) |
which is the most convenient
form in which to obtain and plot
data. Our experimental approach
was therefore to determine this
function. The simpliest functional
form for
f is based on a
specific model, the Langmuir adsorption
equation for bimolecular
phenomena at heterogeneous
interfaces,
12 24
 | (2) |
where (
S ·
X) represents the
equilibrium fraction
of plasminogen or Lp(a) bound to the
surfaces, (
S0) the total
amount of
plasminogen or Lp(a) binding sites,
K the
association
constant of the ligand/surface interaction, and
(
X) the total
input of ligand. A correct fitting of the
original raw data
(experimental points) to equation 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
B
max=
S0,
and the dissociation
constant
Kd=
K-1.
B
max 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) |
and by rearrangement
 | (4) |
where
Kd represents
the
dissociation constant of the interaction of the anti-apo(a)
antibody
(Ab) with fibrin-bound apo(a) (
Kd=3
nmol/L).
 |
Results
|
|---|
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).
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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Figure 1. Immunoblot analysis of apo(a)
isoform content in purified Lp(a) preparations isolated from single
donors. Lp(a) preparations were reduced with 100 mmol/L of
dithiotreitol for 30 minutes at 60°C and loaded on a 3.75%
polyacrylamide/0.8% agarose gel. The protein bands were
transferred to a nitrocellulose membrane, and the 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. Isoforms were identified using reference plasmas
containing isoforms B, S1, S3, S4, and S4+.
|
|
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.
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.
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.
 |
Discussion
|
|---|
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
plasminogen
33 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 colleagues
40 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
studies
41 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
al
43 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.
 |
Selected Abbreviations and Acronyms
|
|---|
| apo(a) |
= |
apolipoprotein(a) |
| IgG |
= |
immunoglobin G |
| Lp(a) |
= |
lipoprotein(a) |
| SDS |
= |
sodium dodecyl sulfate |
| 6-Ahx |
= |
6-aminohexanoic acid |
|
 |
Acknowledgments
|
|---|
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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