© 2000 American Heart Association, Inc.
Thrombosis |
Effect of Individual Plasma Lipoprotein(a) Variations In Vivo on Its Competition With Plasminogen for Fibrin and Cell Binding
An In Vitro Study Using Plasma From Children With Idiopathic Nephrotic Syndrome
From INSERM U.143, Hôpital de Bicêtre (T.S., S.L., E.A.-C.), Bicêtre, France, and the Departments of Pediatric Nephrology (V.B., C.L.) and Hematology (L.M., M.-F.H.-R., N.S.), Hôpital Robert-Débré, Paris, France.
Correspondence to Dr E. Anglés-Cano, INSERM U.143, Hémostase-Biologie Vasculaire, 84 rue du Général Leclerc, F-94276-Cedex, Bicêtre, France. E-mail angles{at}infobiogen.fr
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Abstract—Simultaneous natural changes in lipoprotein(a) [Lp(a)] and plasminogen occur in the nephrotic syndrome and offer a unique opportunity to investigate their effects on plasminogen activation under conditions fashioned in vivo. Plasminogen, Lp(a), and apolipoprotein(a) in plasma were characterized, and their competitive binding to carboxy-terminal lysine residues of fibrin and cell membrane proteins was determined in nephrotic children during a flare-up of the disease (61 cases) and after 6 weeks (33 cases) and 6 months (42 cases) of remission. Low plasminogen concentrations (median 1.34 µmol/L, range 0.39 to 1.96 µmol/L) and high Lp(a) levels (median 0.27 g/L, range 0.07 to 2.57 g/L) were detected at flare-up. These changes were associated with an increased Lp(a) binding ratio onto fibrin (3.13±0.48) and cells (1.53±0.24) compared with binding ratios of control children (1.31±0.19 and 1.05±0.07, respectively) with normal plasminogen and low Lp(a) (median 0.071 g/L). After 6 weeks and 6 months of remission, the values for net decrease in Lp(a) binding to fibrin were 1.7±0.22 (after 6 weeks) and 1.88±0.38 (after 6 months) and were correlated with low Lp(a) concentrations (median 0.2 g/L, range 0.07 to 0.8 g/L; and median 0.12 g/L, range 0.07 to 1.34 g/L) and inversely associated with increased plasminogen levels (median 1.82 µmol/L, range 1.4 to 2.1 µmol/L; and median 1.58 µmol/L, range 1.1 to 2.1 µmol/L). These studies provide the first quantitative evidence that binding of Lp(a) to lysine residues of fibrin and cell surfaces is directly related to circulating levels of both plasminogen and Lp(a) and that these glycoproteins may interact as competitive ligands for these biological surfaces in vivo. This mechanism may be of relevance to the atherothrombotic role of Lp(a), particularly in nephrotic patients.
Key Words: lipoprotein(a) • apolipoprotein(a) isoforms • binding, competitive • plasminogen inhibition • lysine binding site
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Thromboembolic events represent some of the most serious complications in patients with nephrotic syndrome.1 2 3 Several alterations, potentially related to these events, have been identified in blood coagulation factors (increased concentrations of factors V and VIII and fibrinogen) and inhibitors (decreased concentrations of antithrombin III and free protein S).4
Modifications of the fibrinolytic system are less well defined.5 6 For instance, modifications in the levels of plasminogen may be related to age, in view of the fact that low levels have been found in nephrotic children7 8 and normal or increased levels have been found in nephrotic adults.9 10 In contrast, increased concentrations of Lp(a), the lipoprotein particle containing apo(a), a glycoprotein genetically related and structurally homologous to plasminogen, have been reported in nephrotic adults9 11 12 13 and in children.14 15 Because high plasma levels of Lp(a) are now recognized as a risk factor in cerebrovascular and cardiovascular diseases,16 17 18 increased concentrations of Lp(a) may represent an added thrombotic risk in nephrotic subjects.19 20
Plasminogen and apo(a) contain triple-loop disulfide-linked structures called "kringles" and a serine-proteinase domain.21 22 Among the 5 kringles of plasminogen, kringles 1 and 4 contain a lysine-binding site, which allows binding of plasminogen to carboxy-terminal lysine residues of fibrin and cell membrane proteins. Plasminogen thus bound is transformed into plasmin, the enzyme responsible for fibrinolysis and pericellular proteolysis, after cleavage of the Arg561-Val562 peptide bond by activators.23
Apo(a) contains multiple tandem repeats of plasminogen-like
kringle 4 (61% to 75% homology) followed by a single copy of kringle
5 and of the protease domain (
94% homology).24 The
variable number of plasminogen-like kringle 4 copies in
apo(a) gives rise to isoforms of different size, which have been
identified by analysis of the protein25 and the
cDNA.26 The size of these isoforms (300 to >800 kDa) is
inversely correlated with the plasma level of Lp(a).27
Data concerning the distribution of these apo(a) isoforms in children
with renal diseases are scarce.14
Some of the plasminogen-like kringle 4 copies endow apo(a) with the ability to compete with plasminogen for binding to cells28 29 and fibrin.30 31 However, the substitution of the Arg-Val plasminogen cleavage site by Ser-Ile in apo(a) impairs the generation of plasmin-like activity by activators. Thus, the competitive binding of Lp(a) for lysine residues of fibrin and cell membrane proteins results in decreased plasmin formation and may favor the deposit of fibrin and lipids within the vascular wall.32
Because a decreased plasminogen activation may be associated with thrombosis,33 34 we thought it interesting to explore the interactions of Lp(a) with this enzyme system in nephrotic children. We hypothesized that the low levels of plasminogen and the high levels of Lp(a) induced by the nephrotic syndrome may favor binding of Lp(a) to fibrin and to human monocytes, thus inducing a decrease in fibrinolysis and pericellular proteolysis, which may contribute to the development of atherothrombosis. We undertook to explore this hypothesis in 61 children with idiopathic nephrotic syndrome. Blood samples were collected during a flare-up of the disease (n=61) and after remission at 6 weeks (n=33) and at 6 months (n=42). Taking into account the plasma concentrations of Lp(a), apo(a), and plasminogen and the functional characteristics of the apo(a) isoforms, we evaluated the pathogenicity of Lp(a) under conditions fashioned in vivo.
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Subjects and Blood Samples
Sixty-one children with idiopathic nephrotic syndrome were studied during a flare-up of the disease (stage 1), defined by heavy proteinuria and plasma albumin levels
25g/L. Thirty-three of
these children were also studied after 6 weeks of remission (stage 2),
and 42 were studied after 6 months of remission (stage 3). Children
aged <1 year were excluded from the study. All children were treated
in the Pediatric Nephrology Department of Hôpital
Robert Debré, Paris, France. The present study was approved
by the ethical committee of Bichat-Claude Bernard Medical Faculty and
by the Délégation à la Recherche Clinique, Assistance
Publique-Hôpitaux de Paris. Written informed consent was obtained
from parents and, eventually, from patients. Control samples for Lp(a)
determination were obtained from the Centre Hospitalo-Universitaire of
Rheims, Rheims, France, for 200 age-matched children (109 boys, 91
girls) who exhibited neither renal nor lipid disorders. All samples
were obtained during routine biological sampling over 36 months. Venous
blood was drawn (volume 3 mL) on 4 mmol/L EDTA and 100 kallikrein
inhibitory units (KIU)/mL aprotinin (final concentrations).
Plasma was separated from blood by centrifugation at
2000g for 20 minutes at 4°C, supplemented with
D-valyl-L-phenylalanyl-L-lysine
chloro- methyl ketone (VPL, 1 µmol/L), and stored at
-80°C.
Reagents and Buffers
Materials were purchased from the following sources: BSA was
from Eurobio; VPL and
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone (PPACK), from France-Biochem; aprotinin (Trasylol),
from Bayer Pharma; AEBSF, from Interchim; peroxidase-labeled sheep
immunoglobulins, from Dako; and nitrocellulose sheets, from Sartorius.
Others products were obtained as previously
described.31 35
Buffer A consisted of 0.05 mol/L sodium phosphate, pH 7.4, containing 0.08 mol/L NaCl, 0.01% Tween 20, and 0.01% NaN3. Assay buffer consisted of buffer A containing 2 mg BSA/mL. Mass buffer, a buffer that provided the mass action effects on the competitive nonspecific adsorption of plasminogen and Lp(a) to fibrin and cell surfaces, consisted of buffer A containing 40 mg of BSA/mL. PBS buffer consisted of 0.015 mol/L Na2HPO4 and 0.06 mol/L KH2PO4, pH 7.4, containing 0.14 mol/L NaCl.
Determination of Lp(a) and Plasminogen Concentrations
The concentrations of plasma Lp(a) and plasminogen
were determined by immunoelectrophoresis according to the method of
Laurell.36 An interassay coefficient of variation of <8%
was determined by using plasmas with a known concentration of
plasminogen and Lp(a). A commercial kit (ImmunoFrance) was
used for Lp(a), and the concentration is given in grams per liter by
reference to the kit standard. The lower limit of detectability was
0.067 g/L. For the detection of plasminogen, an assay was
developed, and the concentration was expressed in moles per liter with
the use of a home standard prepared by adding varying concentrations of
purified plasminogen to plasminogen-depleted
plasma. Electrophoresis was performed on 1% agarose gels containing a
sheep antiserum directed against human plasminogen. The
antiserum was prepared at the Institut National de la Recherche
Agronomique (Centre de Clermont-Ferrand-Theix) by immunizing the animal
with 3 subcutaneous injections of purified human
plasminogen. The agarose (Indubiose A37, Sepracor) was
dissolved in a buffer consisting of 50.8 mmol/L Tris, 27.2
mmol/L H3BO3, 1.0
mmol/L Na2-EDTA, and 0.01%
NaN3 and was mixed at 60°C with 1% and 0.4%
(final concentrations) of polyethylene glycol 6000 and the sheep
antiserum, respectively. A 2-mm-thick gel layer was poured on a
polyester film (GelBond, FMC Bioproducts), and 5-µL samples were
loaded and electrophoresed at 70 V for 2 hours in the same buffer.
After electrophoresis, the gel was washed (0.15 mol/L NaCl), dried, and
stained with an amido-black solution dye in 45% methanol and 10%
acetic acid.
Determination of Size of Plasma Apo(a) Isoforms
The apo(a) isoforms were identified as follows: plasma was
electrophoresed under reducing conditions with 3.75%
polyacrylamide/0.8% agarose gels, and protein bands were
electroblotted to a nitrocellulose membrane according to
Khyse-Andersen.37 Apo(a) bands were localized by using a
sheep antibody to human apo(a) prepared as described below, followed by
a peroxidase-conjugated rabbit antibody to sheep IgG (Dako A/S)
revealed with 4-chloro-1-naphthol. To quantify the proportion of each
isoform from a given plasma, immunoblots were incubated
overnight with the apo(a)-specific antibody, radiolabeled with
125I, and autoradiographed on Kodak XS films for
24 to 48 hours at -70°C. The apo(a) bands were cut and counted in a
gamma-radiation counter. Apo(a) isoforms were identified by using a
recombinant apo(a) standard prepared in our laboratory (INSERM U.143)
and composed of isoforms containing 10, 14, 18, 26, and 34
kringles.38 The molar concentrations of the apo(a)
isoforms were calculated by relating their molecular mass and relative
proportion in plasma with the concentration of Lp(a). Molecular mass
estimation was based on sequence24 and by assuming a
constant carbohydrate content of 23 wt%.39
Purification of Proteins and Lp(a)
Human plasminogen and fibrinogen were purified from
fresh-frozen human plasma under conditions that avoid proteolysis, as
previously described,40 41 with
modifications.42 43 Lp(a) was isolated from healthy adult
volunteers whose plasma contained >0.5 g/L Lp(a), as previously
described.35 The concentration of the purified Lp(a) was
determined by immunoelectrophoresis, and its apo(a) content was
characterized as indicated above. The final product was conserved
at 4°C in buffer A containing proteolytic inhibitors and
was used in binding experiments within 24 hours of preparation.
Cell Culture and Preparation of Cell Surfaces
The monocytic cell line THP-144 was obtained from
the American Type Culture Collection. The cells were plated in 25- and
80-cm2 Falcon plastic flasks containing
Dulbecco-Iscove supplemented with 10% FCS, 0.5% sodium pyruvate, and
1% antibiotics (streptomycin and penicillin) and cultured at 37°C in
5% CO2. The cells were recovered after 3 or 4
days of culture, washed with PBS buffer, resuspended in the same buffer
at a concentration of 1.5x106 cells per
milliliter, and immobilized on microtitration plates by
using an adaptation of the method of Stocker and
Heusser.45 We introduced the following modifications:
flexible polyvinyl chloride plates were first treated with
polyglutaraldehyde during 2 hours at 22°C, excess
glutaraldehyde was eliminated by washing the plates
with distilled water, and 80 000 cells were added into each well.
After centrifugation at 233g during 10
minutes, the cells were incubated in the plates for 1 hour at 22°C,
followed by an incubation of 10 minutes in a bath of 0.25%
polyglutaraldehyde; after 2 final washes with PBS, 100
µL per well of PBS containing 4 mg/mL BSA and 0.1% thimerosal was
added, and the plates were sealed and stored at 4°C until further
use.
Purification and Radioiodination of Immunoglobulins Directed
Against Human Apo(a)
The IgG fraction of a sheep antiserum against human apo(a)
obtained as described31 was separated by ammonium sulfate
precipitation, ion-exchange chromatography on DEAE, and
affinity chromatography on Protein-A Sepharose
(Pharmacia). The purified IgG was further immunodepleted by using
Sepharose-immobilized apoB-100 and plasminogen;
the final antibody preparation did not cross-react with these proteins
and was shown to react equivalently with different apo(a)
isoforms.46 The IgG was labeled with sodium iodide
I 125 by using the Iodogen method of Fraker and Speck,47
an iodination time of 4 minutes at 22°C, and removal of free
125I by molecular sieving on a PD-10 Sephadex
column (Pharmacia). The specific radioactivity obtained was 3 nCi/ng of
anti-apo(a) IgG.
Competitive Binding of Lp(a) and Plasminogen at
Plasma/Fibrin Interface
The experiments were performed with fibrin surfaces
prepared and characterized as previously described42 and
with plasminogen/Lp(a)-depleted plasma prepared as follows:
Human venous blood obtained from 2 healthy volunteers was drawn into
polypropylene tubes containing 4 mmol/L EDTA, 0.01%
NaN3, and inhibitors of proteolysis
(10 KIU/mL aprotinin, 1 µmol/L PPACK, 1 µmol/L VPL,
10 µmol/L p-Nitrophenyl-p'-guani dinobenzoate, and 1
mmol/L AEBSF). Plasma was then separated by
centrifugation at 2000g for 15 minutes at
4°C and was depleted in plasminogen and Lp(a) (initial
concentrations 1.5 µmol/L and <0.067 g/L, respectively) by 3
batch adsorptions on lysine-Sepharose 4B (Pharmacia). After depletion,
no plasminogen or Lp(a) antigen was detected by
immunoelectrophoresis.
Lp(a) purified as described above was added at varying concentrations to plasminogen/Lp(a)-depleted plasma supplemented with 1 or 2 µmol/L plasminogen. In parallel experiments, the plasma was also supplemented with purified fibrinogen to obtain final concentrations of 4 and 6 g/L (initial concentration 2 g/L). A volume of 50 µL of each mixture was incubated with the fibrin surfaces for 18 hours at 4°C. Unbound proteins were removed by washing, and the surface (3 wells for each concentration) was probed with the 125I-labeled antibody directed against apo(a). After 1 hour at 37°C, the surface was washed, and the radioactivity was counted and transformed into mass of antibody bound to fibrin.46 Data were fitted to the Langmuir adsorption isotherm equation, as described.42 Bound plasminogen was detected in 3 other wells by adding 50 µL per well of assay buffer containing a synthetic substrate from Diagnostica Stago selective for plasmin (CBS OO65, 1.5 mmol/L) and tissue plasminogen activator (tPA, 10 IU/mL). In this system, the activation of fibrin-bound plasminogen by tPA is detected by measuring the change in absorbance (change at 405 nm/min) produced by the release of p-nitroaniline from the synthetic substrate. Initial rates were transformed into femtomoles of plasmin bound per well as described by Fleury et al.48
Binding of Plasma Lp(a) to Fibrin and Cell Surfaces
The procedure for the binding of plasma Lp(a) to fibrin and cell
surfaces was based on previous studies31 35 and the above
competitive binding experiments. Plasma (1:2 to 1:16 in mass buffer, pH
6.8, supplemented with 2 mmol/L EDTA, 1 µmol/L VPL, and
1 µmol/L dansyl-Dansyl-L-glutamyl-glycyl-L-arginine
chloromethyl ketone) was incubated with fibrin or cell surfaces. After
18 hours at 4°C, unbound proteins were removed by washing, and the
surfaces were probed with a known concentration of the
125I-labeled polyclonal antibody directed against
human apo(a). Excess antibody was removed by washing, and the
radioactivity in the wells was counted in a gamma-radiation counter and
transformed into an Lp(a) binding ratio by relating the signal obtained
with the plasma from patients to the signal of plasma from a healthy
control with undetectable levels of Lp(a). For each plasma sample, the
value used to calculate the binding ratio was the highest signal
obtained as a function of the plasma dilutions tested. Specificity of
the binding of Lp(a) to carboxy-terminal lysine residues of fibrin and
membrane proteins of THP-1 cells was assessed as previously
described.35 42 Briefly, binding experiments were
performed either with surfaces previously treated with carboxypeptidase
B or with plasma supplemented with 0.2 mol/L 6-aminohexanoic acid
before incubation with untreated surfaces.
Statistical Analysis
Differences between groups were calculated by using a 2-tailed
paired Student t test for statistical significance. The
possible effect of various hemostasis and lipid parameters
on the competitive binding of Lp(a) and plasminogen to
fibrin and cell surfaces was analyzed by using a linear
regression model. The variability of Lp(a) and plasminogen
concentrations during the course of the nephrotic syndrome was
expressed by the percentiles of the distributions. In all cases,
significance was established at P<0.05.
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Demographic Data
Sixty-one children (37 boys, 24 girls) with idiopathic nephrotic syndrome were included in the study during a flare-up of the disease (stage 1). Median age was 4 years, ranging from 1 to 16 years. Among these 61 patients, 51 had steroid-responsive and 10 had steroid-resistant idiopathic nephrotic syndrome. Renal biopsy, performed in 16 children, showed minimal change in glomerular lesions in 10 of the children, focal segmental glomerular sclerosis in 5, and mesangial proliferation in 1.
At the time of flare-up, 17 children had received no treatment, 40 had received prednisone, and 4 had received prednisone and cyclosporine. Three children had thromboembolic complications: 2 children exhibited pulmonary thromboembolism at the time of the study, and 1 child had experienced thrombosis of the external iliac vein 15 months before the study. Thirty-five children received anticoagulant therapy with either heparin (2 children), anti–vitamin K (30 children), or a combination of both (5 children). Mean serum creatinine at flare-up was 46±40 µmol/L. Five children had transient renal insufficiency: serum creatinine levels were between 74 and 106 µmol/L in 3 children and between 208 and 284 µmol/L in 2 children. At stage 2 (6 weeks of remission, 33 children), all patients received prednisone (which was associated with cyclosporine treatment in 3 children). One child received anti–vitamin K treatment. At stage 3 (6 months of remission, 42 children), 17 children received no treatment, 11 received prednisone alone, 9 received prednisone and cyclosporine, 1 received prednisone and cyclophosphamide, and 4 received cyclosporine alone. Only 1 child received anti–vitamin K treatment.
Albumin, Lipids, and Fibrinolytic Parameters
As indicated in Table 1
, all
patients showed heavy proteinuria, hypoalbuminemia,
hypercholesterolemia, and
hypertriglyceridemia during flare-up.
Results of the measurement of proteins involved in
fibrinolysis are depicted in Table 2; fibrinogen and plasminogen
activator inhibitor type 1 (PAI-1) antigen were
markedly elevated at flare-up.
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Plasminogen and Apo(a)/Lp(a) Quantification
The plasma concentrations of plasminogen and
apo(a)/Lp(a) are indicated in Table 3.
The molar concentration of apo(a) was calculated by using the
proportion of each isoform in plasma and concentrations of Lp(a)
0.067 g/L, the detection limit of the immunoelectrophoretic assay. An
Lp(a) concentration <0.067 g/L was detected in 29% of patients at
diagnosis (n=18 of 61), in 39% at 6 weeks of remission (n=13 of 33),
and 45% at 6 months of remission (n=19 of 42). Opposite modifications
in the levels of plasminogen and apo(a)/Lp(a) were
observed. The concentration of plasminogen (1.34
µmol/L) at flare-up was significantly lower (P<0.0001)
than the levels at 6 weeks (1.82 µmol/L) and at 6 months
(1.58 µmol/L) of remission. In contrast, at flare-up the levels
of apo(a) (median 255 µmol/L) and Lp(a) (median 0.27 g/L) were
markedly elevated compared with levels at 6 weeks (0.167 µmol/L
and 0.2 g/L, respectively) and at 6 months (0.234 µmol/L and
0.121 g/L, respectively) of remission. In 28 of these patients, a
plasminogen concentration <1.3 µmol/L was detected.
Although plasminogen levels were normal at remission, the
concentrations of apo(a)/Lp(a) were still higher at 6 months of
remission compared with levels previously reported for
children14 15 and by reference to an age-matched
control population of children composed of 109 boys and 91 girls.
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Apo(a) Phenotyping
To simplify the analysis and taking as a basis a
mean isoform size of 22 kringles, which discriminates apo(a) isoforms
expressing a greater risk for coronary heart
disease,49 we classified the apo(a) isoforms of nephrotic
children into 2 groups: small molecular size isoforms (range 16 to 21
kringles) and high molecular size isoforms (range 23 to 30 kringles).
Apo(a) isoforms could not be identified in 23 plasma samples with Lp(a)
concentrations 0.067 g/L. Six patients were apparently homozygous and
had single isoforms (16 kringles, 1 patient; 17 kringles, 2 patients;
and 18 kringles, 3 patients), whereas all others (32 patients) were
heterozygous and had a low molecular mass isoform (mean size 16±1
kringles) and a high molecular mass isoform (mean size 26±2 kringles)
as shown in Figure 1. In agreement with
the known inverse relation between Lp(a) concentration and apo(a)
isoform size, the mean concentration of low molecular mass isoforms
(0.23±0.27 µmol/L) was higher than the mean concentration of
high molecular mass isoforms (0.16±0.22 µmol/L). Individual
modifications in the relative concentration of the apo(a) isoforms in
heterozygous subjects were observed with the decrease in Lp(a)
concentrations after remission. In some cases, the observed decrease in
Lp(a) did not allow detection of the apo(a) isoform band of high
molecular mass with our phenotyping system (Figure 1).
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Competitive Binding of Plasma Lp(a) to Fibrin and Cell
Surfaces
Competition between plasminogen and Lp(a) for binding
to fibrin has been previously demonstrated in purified
systems.50 In the present study, the binding assays
were performed in plasma. Therefore, we sought to rule out potential
confounding plasma factors that might influence the assay. For that
purpose, the competitive binding of plasminogen and Lp(a)
separated from the plasma of volunteer adults was studied at the
plasma/fibrin interface by using a system consisting of a fibrin
surface and plasma depleted in plasminogen and Lp(a). Data
shown in Figure 2 correspond to an
Lp(a) containing an 18-kringle apo(a) isoform and are
representative of data obtained with different Lp(a)
preparations (n=12). The amount of Lp(a) bound to fibrin was a function
of the input concentration added to plasma and decreased as the
concentration of plasminogen was incremented from 0 to 1
and 2 µmol/L (Figure 2, main graph). In a similar
fashion, the amount of plasminogen bound at these
concentrations decreased as a function of the plasma
Lp(a)/plasminogen molar concentrations (Figure 2, inset). Lp(a) and plasminogen thus detected were
specifically bound to carboxy-terminal lysine residues of fibrin and
cell surfaces, as indicated by the absence of binding to surfaces
treated with carboxypeptidase B and by its inhibition with the lysine
analogue 6-aminohexanoic acid. These results indicate that binding of
Lp(a) and plasminogen to fibrin at the interface with
plasma complies with mechanisms similar to those described in purified
systems; ie, the amount bound is a function of their plasma
concentration and of their affinities for fibrin. Of note, the amount
of Lp(a) and plasminogen bound to fibrin was not affected
by fibrinogen at concentrations similar to those found in nephrotic
plasmas (data not shown). On the basis of these results, the
competitive binding behavior of Lp(a) and plasminogen was
explored at fibrin and cell interfaces with plasmas from nephrotic
children during a flare-up (n=61) and after 6 weeks (n=33) and 6 months
(n=42) of remission. The plasmas were incubated with the surfaces at
various dilutions to mimic different ligand/surface interactions, as
indicated in Methods. The amount of Lp(a) bound that was measured with
an antibody to apo(a) was chosen as the end point of the assay and was
expressed by a patient/control binding ratio that relates the
respective binding values. The control plasma used had no detectable
Lp(a), as assessed by immunoelectrophoresis and phenotyping. The value
used to calculate the binding ratio was the highest amount of
fibrin-bound Lp(a) detected as a function of the plasma dilution (1:8
in most patients). The results are expressed in Table 4. Mean binding ratios obtained with both
fibrin (3.13±0.48) and cell (1.53±0.24) surfaces at flare-up were
significantly higher (P<0.0001) than the mean values
obtained at 6 weeks (1.7±0.22 and 1.16±0.09) and at 6 months
(1.88±0.38 and 1.22±0.08) of remission. Values obtained at 6 months
of remission approximated the mean normal values obtained with plasmas
from 14 age-matched children (fibrin 1.31±0.19; cells 1.05±0.07) and
the cut-off Lp(a) fibrin binding ratio (mean+2 SD 1.74) established in
a healthy adult population (n=108; E.A.-C. et al, unpublished data,
1999). Figure 3 clearly shows that the
decrease in the Lp(a) binding ratio (Figure 3A) that evolves
with the nephrotic syndrome from the clinical active phase (stage 1) to
remission at 6 weeks (stage 2) and at 6 months (stage 3) was directly
linked to the increase in plasma plasminogen levels and to
the decrease in apo(a) concentration (Figure 3B). The relation
between the increased concentration of apo(a) and the decreased
concentration of plasminogen with the binding ratio is
clearly shown in Figures 4 and 5. Binding ratios for each apo(a) isoform
were calculated by using their relative concentrations in plasma and
affinity values, ie, the dissociation constant
(Kd), calculated with equivalent Lp(a)
isoforms purified from the plasma of healthy adults. Typical binding
isotherms used to calculate binding affinities are shown in Figure 2 for an 18-kringle Lp(a) isoform
(Kd 12 nmol/L). In Figure 4, the
amount of each apo(a) isoform bound to fibrin (panel A) and THP-1 cells
(panel B) is plotted against the apo(a) isoform/plasminogen
molar concentration ratio. An increase in the amount of apo(a) bound is
observed as a function of the apo(a) isoform/plasminogen
molar ratio, thus indicating that the binding of Lp(a) to the fibrin
and cell surfaces was directly related to the increase in the
concentration of Lp(a) and inversely related to plasminogen
concentrations. Further evidence of the inverse relation between
plasminogen concentrations and the amount of apo(a) bound
could be clearly shown at concentrations of plasminogen
<1.3 µmol/L (Figure 5). In contrast, the Lp(a) binding
data thus obtained did not correlate with any of the lipid and
hemostatic variables, including serum albumin
(P=0.20), cholesterol (P=0.17),
triglycerides (P=0.25), tPA (P=0.13),
PAI-1 (P=0.21), and fibrinogen (P=0.21)
concentrations.
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) and in the presence of
plasminogen at 1 (•) and 2 (
) µmol/L. Bound
Lp(a) was detected with a radiolabeled sheep antibody specific for
apo(a). Radioactivity bound was then transformed into femtomoles of
antibody bound to fibrin. In separate wells, bound
plasminogen was activated with tPA and detected
with a chromogenic substrate selective for plasmin; the
initial velocity of the reaction was transformed into mass of
plasmin(ogen) as indicated.
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, large isoforms. Binding ratios for each apo(a) isoform
were calculated as indicated in Methods. The molar concentrations of
the apo(a) isoforms were calculated by relating their molecular mass
and relative proportion in plasma with the concentration of
Lp(a).
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Thromboembolic complications increase the risk of morbidity and mortality in patients with nephrotic syndrome. Vascular occlusions (pulmonary thromboembolism and arterial or venous thrombosis) have been observed in both adults and children.1 3 The reported incidence is lower in children despite more marked coagulation abnormalities.2 However, the possibility of clinically silent thrombosis in children cannot be ruled out.1 These conditions result, most probably, from the conjunction of hypoalbuminemia and hyperlipemia with several changes in blood coagulation, fibrinolysis, and platelet functions.5 51 Marked lipid abnormalities, such as elevation of lipoproteins containing apo B, ie, VLDL, LDL, and Lp(a), have been previously reported52 and were confirmed by results of the present studies. Nephrotic children have significantly higher plasma concentrations of Lp(a) than do healthy children14 15 53 54 55 56 ; these high levels of Lp(a) are an abnormality currently considered to be an independent risk factor for the development of cardiovascular disease in the general population.18 The mechanism by which Lp(a) may favor the atherothrombotic process may be related to the lysine-binding properties of apo(a).32 57 It has been proposed that Lp(a) may inhibit the binding of plasminogen to cells and fibrin by a competitive mechanism and, thereby, interfere with the fibrinolytic process.28 29 31 Furthermore, we have previously shown that in subjects heterozygous for the apo(a) trait, the influence of the various apo(a) isoforms on fibrinolysis depends on their affinity for fibrin and on their concentrations relative to each other and to plasminogen.50 According to these data, high levels of Lp(a) and low plasminogen concentrations, conditions that were present in a majority of the nephrotic children included in the present study, may promote the deleterious effect of Lp(a). Indeed, congenital hypoplasminogenemia and high plasma levels of Lp(a) were recently associated with thrombosis.58 In addition, it has recently been demonstrated that plasminogen deficiency greatly accelerates the formation of intimal lesions in transgenic hypercholesterolemic apo E–deficient mice.59 Furthermore, it has been shown that modification of apo(a) lysine-binding sites reduces atherosclerosis in transgenic mice.32 The acquired decrease in plasminogen and the elevated levels of Lp(a) observed in our patients may thus be considered a predisposing condition to thrombosis and atherosclerosis. Therefore, the plasma Lp(a)/plasminogen modifications documented at a flare-up of the nephrotic syndrome and their normalization with remission constitute a model for the study of common interactions between these proteins and biological surfaces. This natural model offers a unique opportunity to disclose the potential atherothrombotic role of Lp(a) under exceptionally large changes in the concentrations of 2 genetically linked homologous glycoproteins: apo(a) and plasminogen. The present study was therefore restricted to experimental procedures performed with native plasma to preserve the in vivo molecular interactions between plasminogen and Lp(a) induced by the nephrotic syndrome and to determine their effects on fibrin and cell binding in vitro. The experimental setup was designed to functionally isolate the Lp(a)/plasminogen competition for carboxy-terminal lysine residues of fibrin and cell membrane proteins and to preserve the original plasma environment.
One of the most important features of the present study was the evolution of Lp(a) binding to fibrin and cells with disease activity. The increased binding of Lp(a) to fibrin and cell surfaces observed during a flare-up of the nephrotic syndrome was significantly correlated with the apo(a)/plasminogen molar concentration ratio, thus indicating that it was directly related to the concentration of Lp(a) and inversely proportional to the level of plasminogen. Of note, other hemostatic and lipid parameters that also showed large variations were not correlated with the binding of Lp(a). Indeed, high fibrinogen in plasma from nephrotic adults has been shown to have no influence on plasminogen activation.5 Furthermore, the current in vitro experiments that used whole plasma failed to show any effect of plasma proteins, including high fibrinogen concentrations, on the competitive binding of Lp(a) and plasminogen. This binding was mainly due to the effect of apo(a) isoforms of low molecular mass and was accentuated at low plasminogen concentrations. Of note, 2 of the nephrotic children in the present study and 2 of 20 nephrotic children reported by Garnotel et al15 developed thrombosis and had high levels of apo(a) isoforms of low molecular mass. The decrease in the binding of Lp(a) to fibrin and cell surfaces at 6 weeks and at 6 months of remission, which was simultaneous with the normalization of plasminogen and the decrease in Lp(a) levels, confirmed this hypothesis and indicated clearly that the binding was a consequence of the important modifications in the plasma levels of Lp(a) and plasminogen. On the other hand, the increased levels of PAI-1 observed during flare-up may efficiently neutralize tPA available for fibrinolysis and thereby accentuate the antifibrinolytic effect of Lp(a).
Another important feature of the present study was the observed differences in the proportion of each isoform with disease activity. The decrease in the concentration of Lp(a) from the acute phase to remission produced a concomitant decrease in the relative proportion of the apo(a) isoforms, which in some cases could not be detected with the phenotyping system used. This phenomenon was more frequently observed with isoforms of high molecular mass, which also presented the lowest initial concentration, thus suggesting a difference in protein synthesis rather than a selective decrease in one of the apo(a) isoforms. However, the underlying mechanism responsible for the increased Lp(a) concentrations in nephrotic patients remains obscure. Lp(a) may behave as an acute phase reactant,60 and its plasma concentration may be modified by the immunosuppressive treatment used in these patients. Steroid treatment was reported to have no effect on Lp(a) levels, but it has been suggested that cyclosporin may affect it.61 However, contradictory results have been recently published.62 63 In the present study, the effect of corticosteroid and cyclosporin treatment could not be separately evaluated because a majority of patients were under treatment with these drugs, and the levels of Lp(a) in the 17 children without treatment at flare-up were not significantly different from the levels in treated children.
Although the plasminogen levels were raised to normal values in all cases at remission, an effect most probably related to decreased proteinuria, the levels of Lp(a) remained relatively high even at 6 months of remission. Similar findings were reported in a series of 20 nephrotic children.15 It is difficult to ascertain whether these Lp(a) levels represent the original concentration of patients or whether they are a persistent manifestation of the disease. However, it has been reported that hyperlipidemia persists indefinitely, even when the patients enter remission.64
Altogether, these data provide evidence that changes in plasma plasminogen and Lp(a) occurring in vivo affect the competitive binding of these proteins to fibrin and monocytic cells in vitro. The potential in vivo relevance of these findings cannot be extrapolated from these in vitro experiments. However, the present study may provide new insight into our understanding of the role of Lp(a) in the pathophysiology of atherothrombosis.
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Acknowledgments |
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This study was conducted at INSERM U.143 under the support of the Institut National de la Santé et de la Recherche Médicale to Dr Anglés-Cano and grant No. CRC940189 from Assistance Publique-Hôpitaux de Paris to Dr Loirat. We gratefully acknowledge V. Durlach (Centre Hospitalo-Universitaire of Rheims, Rheims, France) for his continuous support with samples for Lp(a) binding studies in adults. We are indebted to R. Garnotel and P. Gillery (CHU of Rheims, Rheims, France) for their collaboration with the control population of children for Lp(a) testing. We thank D. Durand (I.N.R.A., Centre de Clermont-Ferrand-Theix, Clermont-Ferrand-Theix, France) for preparing the immune sera against plasminogen.
Received May 5, 1999; accepted July 14, 1999.
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