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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2036-2043

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2036-2043.)
© 1997 American Heart Association, Inc.

Articles

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 ({approx}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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 *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 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 {gamma}-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 {gamma}-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{alpha} 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 {gamma}-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 {gamma}-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 2Up using nonlinear regression analysis (see Figs 2Down and 3Down) 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 2Up 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 2Up, 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).



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  		Figure 2. Binding isotherm of plasminogen to the monocytic cell line THP-1. Varying concentrations of plasminogen were mixed with a constant concentration of 125I-radiolabeled plasminogen and incubated with 25·105 cells. After elimination of unbound plasminogen by washing, radioactivity was counted in a {gamma}-counter and transformed into the amount of plasminogen bound to cells. {bullet}, specific binding; {blacksquare}, binding obtained with 0.2 mol/L of 6-Ahx; {diamond}, total binding. Kd=940 nmol/L, Bmax=1.28 pmol of plasminogen bound. Bars represent the error of triplicates.



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  		Figure 3. Binding isotherm of low molecular mass apo(a) isoform S1 (patient L.j) to the human monocytic cell line THP-1 (left), and to a fibrin surface (right). Varying concentrations of purified preparations of Lp(a) were incubated with 25·105 cells. After three washes, Lp(a) bound to the surfaces was detected with a 125I-radiolabeled polyclonal antibody directed against human apo(a). Radioactivity bound was then transformed into fmol of antibody bound to cells or to the fibrin surface. Data were fitted to the Langmuir adsorption isotherm equation 2Up. The x axis represents the molar concentration of Lp(a) in terms of total protein content [apo(a)+apo B-100]. The y axis represents the mass of antibody bound, a quantity proportional to the amount of Lp(a) adsorbed onto fibrin or THP-1 cells, as described by equation 3Up. Bars represent the error of duplicates. To simplify the plot, only the specific binding, calculated as indicated in Methods, is shown in the graphs. The Kd calculated with equation 2Up was 25 nmol/L for THP-1 cells and 29 nmol/L for fibrin.


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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 1Down. 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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  		Table 1. Characteristics of the Subjects Studied

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 1Down).



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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 2Up, 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 2Up 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 2Down). 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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  		Table 2. Binding of Lp(a) and Plasminogen to THP-1 Cells and Fibrin Surfaces

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 3Up and 4Down. 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 3Up 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 3Up. 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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  		Figure 4. Binding isotherm of high molecular mass apo(a) isoform S3 (patient M.m) to the human monocytic cell line THP-1 (left), and to a fibrin surface (right). The experiments were performed as indicated in Fig 3Up. The x and y axes were as indicated in Fig 3Up. {blacksquare}, binding obtained with 0.2 mol/L of 6-Ahx; {diamond}, total binding. Error bars represent the error of duplicates. As saturation could not be reached with the highest input concentrations of Lp(a), it was impossible to calculate the dissociation constants of these interactions.

Fig 4Up 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 2Up. 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 5Down, 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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  		Figure 5. Inhibition of the binding of plasminogen to fibrin by Lp(a). Varying amounts of Lp(a) isoform S1 ({bullet}) or S3 ({circ}) 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 3Up. The left graph shows the decrease in plasminogen bound detected as indicated in Methods; bound plasminogen represented as a percentage of the total amount bound in the absence of Lp(a) is plotted against the concentration of Lp(a) added. Experimental points represent the mean of duplicates.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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-{gamma}. 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 {alpha}-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 3Up, 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 2Up. 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 5Up). 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 5Up). 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.


*    References
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 *References
 

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