Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents
Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1232-1238
Published online before print May 9, 2002, doi: 10.1161/01.ATV.0000021144.87870.C8
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
22/7/1232    most recent
01.ATV.0000021144.87870.C8v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, C.
Right arrow Articles by Anglés-Cano, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, C.
Right arrow Articles by Anglés-Cano, E.
Related Collections
Right arrow Risk Factors
Right arrow Fibrinolysis
Right arrow Fibrinogen/fibrin
Right arrow Thrombosis risk factors
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1232.)
© 2002 American Heart Association, Inc.

Thrombosis

Lp(a) Particles Mold Fibrin-Binding Properties of Apo(a) in Size-Dependent Manner

A Study With Different-Length Recombinant Apo(a), Native Lp(a), and Monoclonal Antibody

Chantal Kang*; Miguel Dominguez*; Stéphane Loyau; Toshiyuki Miyata; Vincent Durlach; Edouard Anglés-Cano

From INSERM U460 (C.K., M.D, S.L., E.A.-C.), Faculté de Médecine Xavier Bichat, Paris, France; the Laboratory of Thrombosis Research (T.M.), National Cardiovascular Center Research Institute, Osaka, Japan; and Service Endocrinologie-Diabetologie (V.D.), Centre Hospitalo-Universitaire de Reims, Reims, France.

Correspondence to Dr E. Anglés-Cano, INSERM U460, UFR de Médecine Xavier-Bichat, 16 rue Henri Huchard–BP 416, F-75870-Cedex 18, Paris, France.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Objective— Small-sized apolipoprotein(a) [apo(a)] isoforms with high antifibrinolytic activity are frequently found in cardiovascular diseases, suggesting a role for apo(a) size in atherothrombosis. To test this hypothesis, we sought to characterize the lysine (fibrin)-binding function of isolated apo(a) of variable sizes.

Methods and Results— Recombinant apo(a) [r-apo(a)] preparations consisting of 10 to 34 kringles and a monoclonal antibody that neutralizes the lysine-binding function were produced and used in parallel with lipoprotein(a) [Lp(a)] particles isolated from plasma in fibrin-binding studies. All r-apo(a) preparations displayed similar affinity and specificity for lysine residues on fibrin regardless of size (Kd 3.6±0.3 nmol/L) and inhibited the binding of plasminogen with a similar intensity (IC50 16.8±5.4 nmol/L). In contrast, native Lp(a) particles displayed fibrin affinities that were in inverse relationship with the apo(a) kringle number. Thus, a 15-kringle apo(a) separated from Lp(a) and a 34-kringle r-apo(a) displayed an affinity for fibrin that was higher than that in the corresponding particles (Kd 2.5 versus 10.5 nmol/L and Kd 3.8 versus 541 nmol/L, respectively). However, fibrin-binding specificity of the r-apo(a) preparations and the Lp(a) particles was efficiently neutralized (IC50 0.07 and 4 nmol/L) by a monoclonal antibody directed against the lysine-binding function of kringle IV-10.

Conclusions— Our data indicate that fibrin binding is an intrinsic property of apo(a) modulated by the composite structure of the Lp(a) particle.


Key Words: lipoprotein(a) • apolipoprotein(a) • plasminogen • fibrin surfaces • fibrin affinity


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
High plasma levels of the Lp(a) are now recognized as a risk factor in cerebrovascular and cardiovascular diseases.1 However, the mechanism by which Lp(a) may favor the atherogenic and thrombogenic processes is not, as yet, clearly understood. Its composite structure, consisting of an inactive serine proteinase, apo(a), which is disulfide-linked to apoB-100 of an atherogenic LDL-like particle,2 may explain this phenomenon in part. Apo(a) is a highly glycosylated protein that shares a high degree of sequence identity (75% to 94%) with plasminogen, the precursor of the fibrinolytic enzyme plasmin.3,4 Apo(a) and plasminogen are derived from a common ancestral gene and are constituted by disulfide-bridged structures of 80 to 90 amino acid residues called kringle (K) domains and a catalytic domain. Plasminogen consists of 5 different kringle domains, 2 of which, K1 and K4, bear a lysine-binding site (LBS) that displays affinity for lysine residues exposed on fibrin and cell membrane proteins. Binding and activation of plasminogen through these interactions leads to fibrinolysis and pericellular proteolysis. Apo(a) contains a copy of plasminogen-like K5 and multiple tandem repeats of a kringle that shares 61% to 75% sequence identity with K4, hereafter designated K-IV. The K-IV repeats of apo(a) differ by specific point mutations or deletions and have been classified as 10 different types.5 Apo(a) K-IV type 2 has no functional LBS, and its variable number gives rise to a series of apo(a) isoforms containing a single copy of the other 9 K-IV types.6 Sequence comparison and molecular modeling5,7 have shown that an LBS similar to that of plasminogen K4 is present in K-IV type 10. A slightly modified LBS that is present in K-IV copies (type 5 to type 8) and appears to be masked in the intact Lp(a) particle may display lysine (fibrin) binding.8,9 Kringle copies with a functional LBS may endow apo(a) with lysine-binding capabilities similar to those of plasminogen. Indeed, it has been recently shown that apo(a) accumulation in the vessel wall of fibrin(ogen)-deficient apo(a) transgenic mice is importantly reduced.10 Lp(a) particles containing distinct apo(a) isoforms may show variability in their lysine-binding function11–13 and display functional heterogeneity for fibrin14,15 and cell16 binding. Furthermore, several studies have found a correlation between the prevalence of low-molecular-mass isoforms and coronary artery disease17,18 or advanced stenotic atherosclerosis.19 In the present study, we have further explored the existence of such a structural/functional relationship in the interaction of apo(a) with fibrin by using recombinant apo(a) [r-apo(a)] isoforms containing a variable number of K-IV type 2 repeats and a monoclonal antibody (mAb) that neutralizes the LBS function.20 The results of the present study show that in contrast to native Lp(a) particles, all r-apo(a)s display similar affinity and specificity for lysine residues regardless of size, thus suggesting that the affinity of apo(a) for fibrin is conditioned by the composite structure of the Lp(a) particle.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Production of r-Apo(a)
The plasmids pCMV-A10, -A14, -A18, -A22, -A26, -A30, and -A34 were obtained as previously described21 and were stably transfected by electroporation into the adenovirus-transformed human embryonic kidney cell line 293.22 The culture medium containing the different r-apo(a)s, produced and harvested as described,23 was supplemented with proteinase inhibitors (20 kallikrein inhibitory units/mL aprotinin, 0.5 mmol/L AEBSF, 2 mmol/L EDTA, and 0.01% [wt/vol] NaN3, final concentrations) and stored at -80°C until use.

Purification and Amino-Terminal Sequence Analysis of r-Apo(a)
The r-apo(a)s were immunopurified on a Sepharose 4B–immobilized mAb directed against native apo(a) (mAb a3, kindly provided by L. Sorell, Center for Genetic Engineering, Havana, Cuba).24 The r-apo(a) was eluted with 20 mmol/L glycine-HCl, pH 2.0, containing 0.1 mol/L NaCl, and fractions were collected into a volume of 0.1 mmol/L Tris, pH 10, sufficient to neutralize the pH in each fraction. Fractions containing the r-apo(a) were pooled, concentrated on dried polyethylene glycol 20 000 (Serva), and dialyzed against buffer A (50 mmol/L sodium phosphate, pH 7.4, 80 mmol/L NaCl, 0.01% [wt/vol] Tween 20, and 0.01% [wt/vol] NaN3) containing 2 mmol/L EDTA. The concentration of the r-apo(a) was determined according to the method of Lowry et al,25 with purified human plasminogen used as a standard. Electrophoretic analysis (SDS/6% acrylamide or 3.75% polyacrylamide/0.8% agarose gels) of the r-apo(a) preparations was performed as previously described.15,23 Proteins were electroblotted onto polyvinylidene difluoride sheets with a graphite electroblotter system (Millipore) according to the method of Kyhse-Andersen.26 Apo(a) protein bands were stained with Coomassie brilliant blue and submitted to microsequencing with the use of a microsequencer (model 473A, Applied Biosystem) equipped with a model 610A data analysis system.

Purification of Native Lp(a) From Plasma and Isolation of Apo(a)
Volunteer blood donors were selected among individuals attending the outpatient clinic and from the medical staff of Hôpital Robert-Debré (Université de Reims, Reims, France) for their high Lp(a) plasma concentration (>=0.5 g/L) and apo(a) phenotype. Venous blood was drawn from the forearm into sterile polypropylene tubes containing 4 mmol/L EDTA (final concentration). Plasma was separated by centrifugation at 2000g for 20 minutes at 4°C and supplemented with proteinase inhibitors and an antioxidant (butyl hydroxytoluene, 0.05 mg/mL). Lp(a) was purified by sequential ultracentrifugation and molecular sieving as previously described.16 Dissociation of apo(a) 15 kringles from the purified Lp(a) of a homozygous subject [plasma Lp(a), 1.2 g/L] was performed under mild reductive conditions essentially as described by Edelstein et al.27 After reduction, the solution was adjusted to a solvent density of 1.150 g/mL with KBr and centrifuged at 134 000g for 24 hours at 10°C in a 50-Ti rotor (Beckman). The bottom fraction (extensively dialyzed against buffer A) was shown to contain free apo(a) by SDS-PAGE and Western blotting and was conserved at 4°C until assay (<24 hours).

Purification and Radioiodination of Proteins
Plasminogen was purified from fresh-frozen human plasma as previously described,28 with modifications.29 The plasminogen preparation was considered to be >99% pure and was shown to be Lp(a) free, as assessed by SDS-PAGE and autoradiography of the 125I-labeled product and by amino terminal analysis. Plasminogen concentration was calculated by measuring the absorbance at 280 nm with a molecular weight of 93 000 and


A sheep antiserum directed against human apo(a) was prepared at the Institut National de la Recherche Agronomique (Center de Clermont-Ferrand-Theix) by immunizing the animal with 3 subcutaneous injections of purified r-apo(a) A10. The IgG fraction of this antiserum was separated by ammonium sulfate precipitation, ion-exchange chromatography on diethylaminoethyl-Tris-acryl, and affinity chromatography on protein A–Sepharose (Pharmacia). The purified IgG was further immunodepleted by using Sepharose-immobilized plasminogen, and its apparent affinity for the different r-apo(a) was determined.

The monoclonal IgG1 A10.2 recognizing K-IV type 10 of r-apo(a) was obtained, cultured, and purified on protein A–Sepharose as previously described.20 Concentration of the purified IgG1 was determined by the procedure of Lowry et al.25

Plasminogen and the purified anti-apo(a) IgG were radioiodinated with sodium 125I with the use of the Iodogen method of Fracker and Speck,31 an iodination time of 4 minutes at 4°C, and the removal of free 125I by molecular sieving on a PD-10 Sephadex column (Pharmacia). The specific radioactivity obtained was 9 to 11 nCi/ng plasminogen and {approx}3 nCi/ng anti-apo(a) IgG.

Fibrin-Binding Studies
Fibrin surfaces were prepared and immunocharacterized as previously described.29 Varying concentrations of purified Lp(a) or r-apo(a) preparations and isolated 15-kringle apo(a) in buffer A supplemented with 40 mg/mL BSA were incubated with fibrin for 2 hours at 22°C. Unbound proteins were removed by 3 washes with buffer A supplemented with 2 mg/mL BSA. The surfaces were then probed with a known concentration of the 125I-labeled polyclonal antibody directed against apo(a) in buffer A supplemented with 40 mg/mL BSA. After 3 washes with buffer A supplemented with 2 mg/mL BSA, bound radioactivity was counted in a {gamma}-radiation counter and transformed into mass of antibody bound by using the specific radioactivity (dpm/mole) of the labeled IgG. Binding of ligands to fibrin in the presence of 0.2 mol/L of 6-amino-hexanoic acid was considered to be unrelated to interactions with lysine residues.

Inhibition of the Fibrin-Binding Function of r-Apo(a) and Lp(a) With a mAb
Experiments aimed at determining the fibrin-binding function of r-apo(a) and plasma Lp(a) were performed with mAb A10.2. This antibody is specifically directed against the LBS of kringle 4 in plasminogen and K-IV type 10 in apo(a)20 and does not recognize nonfunctional LBSs. Experiments were performed as indicated above except that the r-apo(a) was incubated with varying concentrations of mAb A10.2 or an unrelated mAb (7D4 directed against plasminogen activator inhibitor-1, kindly provided by P.J. Declerck, Catholic University of Leuven, Leuven, Belgium) for 30 minutes at room temperature before incubation with the fibrin surfaces.

For Lp(a) in plasma, the procedure was as described previously.32 Briefly, plasma was supplemented with protease inhibitors and incubated with varying concentrations of mAb A10.2. The samples were then incubated (50 µL per well) with fibrin for 1 hour at 37°C; the plates were washed and probed with a polyclonal antibody directed against apo(a). A peroxidase-labeled rabbit anti-sheep IgG was used as secondary antibody, and a 1 mg/mL solution of 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid) was used as a substrate for color development. The absorbance was measured in a microplate counter at 405 nm. The amount of r-apo(a) or Lp(a) bound to fibrin was calculated by using the linear regression of the relationship between the concentration of a r-apo(a) reference and the change in absorbance at 405 nm (A405/min).

Competitive Inhibition of Plasminogen Binding to Fibrin
Solutions containing varying amounts (0 to 200 nmol/L) of r-apo(a) and a constant amount ({approx}4 nmol/L) of radiolabeled plasminogen were prepared in buffer A supplemented with 40 mg/mL BSA and incubated for 2 hours at 22°C with the fibrin surface (50 µL per well). In control experiments, similar amounts of LDL were added to plasminogen and incubated with fibrin. After elimination of the supernatant, the plate was washed, and the radioactivity bound to the surface was counted in a {gamma}-radiation counter. In the absence of competitors, this amount was considered to be 100% plasminogen bound.

Analysis of Binding Data
Original raw data were fitted to the Langmuir adsorption equation for bimolecular interactions at heterogeneous interfaces29,33: Down


where S · X is the equilibrium fraction of r-apo(a) bound to the surfaces, S0 is the total amount of r-apo(a) binding sites, K is the association constant of the ligand/surface interaction, and X is the total input of ligand. By relating the number of occupied sites [S · X=bound anti-apo(a) antibody] to ligand concentration [X=r-apo(a)], Equation 1 allows calculation of the maximum bound, Bmax=S0, and the dissociation constant, Kd=K-1. The amount of r-apo(a) bound to cells or fibrin was expressed by the mass of antibody bound by using an algebraic expression from Equation 1 that describes the linear relationship of antibody binding at low antibody concentrations with the amount of Lp(a) bound to fibrin34: Down



*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Production, Isolation, and Amino-Terminal Characterization of r-Apo(a) Isoforms
Stable transfected embryonic human kidney cells, line 293, were committed to express r-apo(a) isoforms differing by their variable number of K-IV type 2 repeats (from 0 to 24). All other K-IV types (K-IV type 1 and K-IV type 3 to K-IV type 10) and the serine-protease region were encoded by a common cDNA expression plasmid fragment and were present in single identical copies in all r-apo(a) isoforms (Figure 1). Conditions chosen for development of the transfected cells allowed for the harvesting of culture medium containing high concentrations of intact unmodified r-apo(a) as detected by immunoblot analysis of agarose-acrylamide gels (Figure 2A). The immunopurified r-apo(a) migrated as single protein bands in Coomassie blue–stained 6% acrylamide gels (Figure 2B). The sequence of 10 N-terminal amino acid residues of the purified r-apo(a) was similar to the deduced N-terminal sequence of human apo(a).3,4



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Schematic representation and relative mobility of r-apo(a) isoforms. The domain structure of r-apo(a) isoforms encoded by pCMV-A10 to pCMV-A34 plasmids and relative migration of the expressed proteins (mixture of culture supernatants from transfected human embryo kidney cells) is shown. Electrophoresis was performed on 1.5% agarose gel, followed by immunoblotting, as indicated in Figure 2. Isoform A10 contains single copies of all K-IV types but K-IV type 2. The A14 to A34 isoforms contain a variable number, from 4 (A14) to 24 (A34), of K-IV type 2 copies; all other kringle structures are present in single copies.



View larger version (93K):
[in this window]
[in a new window]
  		Figure 2. SDS-electrophoresis and immunoblot analysis of purified r-apo(a) isoforms. r-Apo(a) isoforms were purified as described in Methods, reduced with 100 mmol/L dithiothreitol for 30 minutes at 60°C, and electrophoresed in the presence of SDS as indicated. A, r-Apo(a) protein bands were transferred from a 3.75% polyacrylamide/0.8% agarose gel to a nitrocellulose membrane and were immunolocalized with a sheep antibody to r-apo(a) A10, followed by a peroxidase-conjugated rabbit antibody to sheep IgG, revealed with 4-chloro-1-naphthol. A less efficient transfer was observed for isoforms A30 and A34. B, Coomassie brilliant blue staining of r-apo(a) isoforms electrophoresed on a 6% acrylamide gel is shown. Numbers at the top of each figure indicated the total number of kringles of each r-apo(a) isoform. P indicates a pool containing the indicated purified r-apo(a).

Variable Size Apo(a) Recombinants, but Not Lp(a)s, Bind to Fibrin With a Similar Affinity
The use of solid-phase fibrin as a surface acceptor for r-apo(a), native Lp(a), and plasminogen has been previously demonstrated.15,29,35 Binding isotherms were obtained by incubation of r-apo(a) and native apo(a) or Lp(a) with fibrin surfaces. Binding to these surfaces was quantified with a 125I-labeled polyclonal IgG raised against r-apo(a) A10. The affinity of the purified IgG for the different r-apo(a)s was in the same order of magnitude (Kd 47±15 nmol/L) as measured by using surface plasmon resonance (data not shown). Therefore, the amount of 125I-labeled IgG bound was assumed to be proportional to the amount of apo(a) bound according to Equation 2. All the isoforms tested (A10 to A34) showed dose dependence, saturation, and specificity for carboxy-terminal lysine residues of fibrin as indicated by its inhibition with 6-amino-hexanoic acid. Fitting of the data obeys the simple Langmuir equation (Equation 1) for adsorption at interfaces,33 as indicated by typical binding isotherms for isolated r-apo(a) A30 and the corresponding Lp(a) reported in Figure 3. The calculated dissociation constant (Kd) values for the different r-apo(a)s were similar (3.6±0.3 nmol/L), regardless of their size (Table). In contrast, a clear inverse relationship between apo(a) size and affinity for fibrin could be documented for Lp(a) particles, in agreement with previous published data.15,34 These results suggest that the LBS function of apo(a) is modified by the composite structure of the Lp(a) particle, as previously suggested by Klezovitch et al.8 Indeed, the Kd (2.5 nmol/L) of a 15-kringle apo(a) separated from native Lp(a) was similar to the mean Kd (3.4 nmol/L) of the closest size r-apo(a) A14, thus indicating that the mild reductive conditions we used to separate apo(a) from native Lp(a) did not disturb its lysine-binding function. In contrast, the corresponding native Lp(a) particle displayed a 4-fold higher Kd (10.5 nmol/L), indicative of lower affinity. The highest differences in affinity were found between r-apo(a) A30 (Kd 3.6 nmol/L) and r-apo(a) A34 (Kd 2.1 nmol/L) and their corresponding native Lp(a) particles (Table).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Isotherm of the binding to fibrin of r-apo(a) and Lp(a). Varying concentrations of purified r-apo(a) A30 and Lp(a) particles containing a similar-sized apo(a) isoform were incubated with the fibrin surface. After 3 washes, the fibrin surface was probed with a 125I-radiolabeled polyclonal antibody directed against human apo(a). Radioactivity bound was then transformed into femtomoles of antibody bound to fibrin. The x-axis represents the concentrations of r-apo(a) (nmol/L) and Lp(a) (g/L) that were incubated with fibrin. The y-axis represents the mass of antibody bound, a quantity proportional to the amount of r-apo(a) A30 adsorbed onto fibrin, as described by Equation 2. Data were fitted to the Langmuir adsorption equation. The binding isotherm of Lp(a) is represented in the main figure, with Kd of 309 nmol/L, calculated by using molecular masses of 512 and 540 kDa for 30-kringle apo(a) and apoB-100, respectively. The binding of r-apo(a) A30 is shown in the inset (Kd 2.8 nmol/L). Bars represent the error of triplicates.


View this table:
[in this window]
[in a new window]
  		Table 1. Affinity of r-apo(a) and Native Lp(a) for Fibrin

All r-apo(a)s inhibited the binding of plasminogen to fibrin with a similar intensity (Figure 4). The amount of plasminogen bound to fibrin decreased as a function of the input concentration of r-apo(a) that was added, and it attained 50% (IC50) at 16.8±5.4 nmol/L These results are indicative of competitive inhibition between plasminogen and r-apo(a) for the same type of binding sites. At identical molar concentrations, the size of the different r-apo(a) preparations had no influence on the competitive binding.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Inhibition of the binding of plasminogen to fibrin by r-apo(a) of different sizes. Varying amounts (0 to 200 nmol/L) of the different r-apo(a)s were incubated with a constant amount ({approx}4 nmol/L) of 125I-plasminogen onto the fibrin surface, and bound radioactivity was counted in a {gamma}-counter. Bound plasminogen, represented as a percentage of the total amount bound in the absence of r-apo(a), is plotted against the concentration of r-apo(a) that was added. Bars represent the error of duplicates. To simplify the figure, only the effect of r-apo(a) A10 ({circ}) and r-apo(a) A18 (•) is shown. The mean±SD IC50 value for all r-apo(a)s tested was 16.8±5.4 nmol/L.

Lp(a) and r-Apo(a) Fibrin-Binding Diversity Is Equally Neutralized by LBS-Blocking mAb
Binding of the different-sized r-apo(a) preparations and of native plasma Lp(a) particles was efficiently inhibited by a mAb directed against the LBS of K-IV type 10.20 Inhibition of the binding was dose dependent (Figure 5), and an IC50 of 0.074 nmol/L was calculated for r-apo(a) from data displayed in the inset to Figure 5. The IC50 values for plasma, shown in Figure 5, were 3.9 nmol/L for the inhibition of a plasma Lp(a) containing a 10-kringle apo(a) isoform and 4.2 nmol/L for an Lp(a) containing 19- and 28-kringle apo(a) isoforms. These IC50 values were higher than those obtained with the isolated r-apo(a) and most probably reflect competition/consumption by kringle 4 of plasma plasminogen, which is also recognized by mAb A10.2.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 5. Inhibition of the binding of plasma Lp(a) and r-apo(a) A10 to fibrin by mAb A10.2. In the main graph, plasma samples from 2 subjects [1.2 g/L Lp(a), 10-kringle apo(a) isoform ({circ}); 2.2 g/L Lp(a), 19- and 28-kringle apo(a) isoforms ({blacksquare})] were incubated with varying concentrations of mAb A10.2 (0 to 545 nmol/L, by protein assay25 and with Mr 150 000). The inset shows a fixed concentration (1 nmol/L) of r-apo(a) A10 (•) incubated with varying concentrations (0 to 2 nmol/L) of mAb A10.2. The solutions were then incubated with fibrin. Unbound proteins were discarded, and bound r-apo(a) or Lp(a) was detected with a polyclonal antibody directed against r-apo(a). The concentration of mAb that inhibits 50% (IC50) of the binding of r-apo(a) (inset) and plasma Lp(a) (main graph) to fibrin was calculated from raw data by using a modified hyperbolic decay equation derived from the original Langmuir equation. The IC50 calculated for r-apo(a) was 0.074 nmol/L, whereas the corresponding value for plasma Lp(a) was 3.9 ({circ}) and 4.2 nmol/L ({blacksquare}). An unrelated mAb (7D4) directed against plasminogen activator inhibitor (PAI)-1, which was used as control at identical concentrations, produced no modification on the binding of either r-apo(a) or Lp(a) to fibrin. To simplify the graphs, only the effect of mAb A10.2 is shown.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Definition of a threshold risk value for Lp(a) is complicated by variations in the distribution of Lp(a) among different populations.36 However, most studies have accepted an Lp(a) concentration >30 mg/dL as a major independent risk factor for atherosclerosis.1 The circulating concentration of Lp(a) is mainly regulated by the apo(a) gene, which varies in size as a function of the number of repetitive sequences encoding K-IV type 2. The smaller this hypervariable region, the higher the plasma concentration of Lp(a) will be. Thus, short apo(a) alleles may favor atherogenesis by increasing the concentration of Lp(a). In agreement with this known inverse relationship between the size of apo(a) isoforms and plasma Lp(a) concentration, a series of studies have shown a prevalence of small-sized apo(a) isoforms in coronary heart disease patients17,18,37-40 or in those with advanced stenotic atherosclerosis.19 However, it remains to be determined whether short apo(a) alleles are, per se, an atherosclerotic risk factor.41 The possibility that apo(a) phenotypes may have different functional properties regarding fibrin and cell binding has been explored; an apo(a) size–related variability in affinity has previously been reported with the use of native Lp(a) particles.14,16,34 Because the main structural difference between apo(a) isoforms almost certainly reflects a variable number of K-IV type 2 repeats, the present study was aimed at determining their effect on fibrin binding. We sought to explore this phenomenon by comparing binding to fibrin of different-sized r-apo(a) and Lp(a) particles. The r-apo(a)s differed solely by the number of copies of K-IV type 2 and were similar regarding lysine-binding kringle structures. Binding of the purified r-apo(a) and Lp(a) particles to fibrin inhibited plasminogen binding and was specific for lysine residues. Of note, LDL particles were shown to be unable to modify plasminogen binding to fibrin. Furthermore, binding to fibrin of r-apo(a) and Lp(a) in plasma was neutralized with a mAb that specifically recognizes the fibrin-binding function of apo(a) K-IV type 10.20 It has been reported that regions constituting K-IV type 5 to K-IV type 88 and K-V-protease42 may also be involved in fibrin binding. These regions are present in all the r-apo(a) that we have tested, and it is possible that mAb A10.2 may also neutralize the corresponding LBS.

The dissociation constant of the different r-apo(a) for fibrin was in the same order of magnitude (3.6±0.3 nmol/L) as the value previously determined with a radiolabeled r-apo(a) containing 17 K-IV copies.35 These values are {approx}150- to 250-fold lower than the Kd value that we have determined for plasminogen in previous reports (0.5 to 1 µmol/L)16,29 and in the present study (0.9 µmol/L). These differences in Kd values explain the ability of r-apo(a) to compete efficiently with plasminogen for binding to fibrin. Most interesting was the finding of quantitative differences in affinity between the r-apo(a) and Lp(a) preparations tested. The functional heterogeneity of native Lp(a) particles is in contrast to the homogeneity in Kd values found for all the r-apo(a)s tested and for the isolated native 15-kringle apo(a). Thus, the purified Lp(a) particles displayed lower affinities for fibrin than did the corresponding r-apo(a)s. Of greater interest was the observation that the change in affinity for the 15-kringle apo(a) isolated from native Lp(a) was moderate (4-fold) compared with the important difference in affinity (85-fold) observed between the r-apo(a) A30 and the corresponding Lp(a) particle. The study of other apo(a) phenotypes derived from reduced Lp(a) particles is hampered by the risk of severely affecting the LBS structure and function by sulfhydryl groups.43

Our data suggest that the fibrin affinity of small-sized apo(a) isoforms is preserved in Lp(a), whereas the affinity for fibrin of high-molecular-mass isoforms (>22 kringles) is reduced in a size-dependent manner in the Lp(a) particle. We hypothesize, in agreement with Klezovitch et al,8 that interactions of apo(a) kringles with components of the Lp(a) particle may contribute to the observed changes in fibrin affinity. Indeed, Ernst et al9 have shown that binding to lysine-Sepharose of apo(a) incorporated into an LDL particle was strongly decreased. These modifications in affinity may be related to conformational changes. As a matter of fact, the extended shape of free r-apo(a)44 in solution is in contrast to the natural roughly spherical shape of Lp(a), where apo(a) is closely associated with the surface, as recently visualized by Weisel et al.45 On the other hand, homocysteine has been shown to induce a concentration-dependent increase in its affinity for fibrin46 and a concomitant release of free apo(a) from reduced Lp(a). According to our present results, the free apo(a) released by homocysteine may contribute to the observed increase in the fibrin affinity of Lp(a) through the "multiple binding with identical linkage mechanism."34

Taken altogether, the results presented in the present study demonstrate that in contrast to native Lp(a) particles, all r-apo(a)s tested display a size-independent similar affinity and specificity for lysine residues on fibrin. Therefore, affinity for fibrin appears to be an intrinsic property of apo(a) that is not dependent on kringle number, except for apo(a) isoforms constitutive of an Lp(a) particle.


*    Acknowledgments
 
This work was supported by grant ACC-SV9 from the Ministère de la Recherche (France) and grant Adrienne et Pierre Sommer from the Fondation de France. We are grateful to Dr H.-J. Müller (Roche, Penzberg) for hosting C.K. in his laboratory during preparation of recombinant apo(a) plasmids made available to us.


*    Footnotes
 
*These authors contributed equally to the present study. Back

Received March 6, 2002; accepted April 1, 2002.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Danesh J, Collins R, Peto R. Lipoprotein(a) and coronary heart disease. Circulation. 2000; 102: 1082–1085.[Abstract/Free Full Text]
  2. Utermann G, Weber W. Protein composition of Lp(a) lipoprotein from human plasma. FEBS Lett. 1983; 154: 357–361.[CrossRef][Medline] [Order article via Infotrieve]
  3. McLean JW, Tomlinson JE, Kuang WJ, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn RM. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987; 330: 132–137.[CrossRef][Medline] [Order article via Infotrieve]
  4. Eaton DL, Fless GM, Kohr WJ, Xu QT, Miller CG, Lawn RM, Scanu AM. Partial amino acid sequence of apolipoprotein(a) shows that it homologous to plasminogen. Pro Natl Acad Sci U S A. 1987; 84: 3224–3228.[Abstract/Free Full Text]
  5. Guevara J, Knapp RD, Honda S, Northrup SR, Morrisett JD. A structural assessment of the apo(a) protein of human lipoprotein(a). Proteins. 1992; 12: 188–199.[CrossRef][Medline] [Order article via Infotrieve]
  6. Lackner C, Cohen JC, Hobbs HH. Molecular definition of the extreme size polymorphism in apolipoprotein(a). Hum Mol Genet. 1993; 2: 933–940.[Abstract/Free Full Text]
  7. Guevara J, Jan YJ, Knapp R, Tulinsky A, Morrisett JD. Comparison of ligand-binding sites of modeled apo(a) kringle-like sequences in human apolipoprotein(a). Arterioscler Thromb. 1993; 13: 758–770.[Abstract/Free Full Text]
  8. Klezovitch O, Edelstein C, Scanu AM. Evidence that the fibrinogen binding domain of apo(a) is outside the lysine binding site of kringle IV-10: a study involving naturally occurring lysine binding defective lipoprotein phenotypes. J Clin Invest. 1996; 98: 185–191.[Medline] [Order article via Infotrieve]
  9. Ernst A, Helmhold M, Brunner C, Pethö-Schramm A, Armstrong VW, Müller HJ. Identification of two functionally distinct lysine-binding sites in kringle 37 and in kringles 32–36 of human apolipoprotein(a). J Biol Chem. 1995; 270: 6227–6234.[Abstract/Free Full Text]
  10. Lou XJ, Boonmark NW, Horrigan FT, Degen JL, Lawn RM. Fibrinogen deficiency reduces vascular accumulation of apolipoprotein(a) and development of atherosclerosis in apolipoprotein(a) transgenic mice. Proc Natl Acad Sci U S A. 1998; 95: 12591–12595.[Abstract/Free Full Text]
  11. Scanu AM, Pfaffinger D, Lee JC, Hinman J. A single point mutation (Trp72->Arg) in human apo(a) kringle 4–37 associated with a lysine binding defect in Lp(a). Biochem Biophys Acta. 1994; 1227: 41–45.[Medline] [Order article via Infotrieve]
  12. Armstrong VW, Harrach NB, Robenek NH, Helmhold NM, Walli NA, Seidel D. Heterogeneity of human lipoprotein(a) Lp(a): cytochemical and biochemical studies on the interaction of two Lp(a) species with the LDL receptor. J Lipid Res. 1990; 31: 429–441.[Abstract]
  13. Leerink CB, Duif PFCCM, Gimpel JA, Kortland W, Bouma B, van Rijn HJM. Lysine-binding heterogeneity of Lp(a): consequences for fibrin binding and inhibition of plasminogen activation. Thromb Haemost. 1992; 68: 185–188.[Medline] [Order article via Infotrieve]
  14. Fless GM, Snyder ML. Polymorphic forms of Lp(a) with different structural and functional properties: cold-induced self-association and binding to fibrin and lysine-Sepharose. Chem Phys Lipids. 1994; 67/68: 69–79.
  15. Hervio L, Chapman J, Thillet J, Loyau S, Anglés-Cano E. Does apolipoprotein(a) heterogeneity influence lipoprotein(a) effects on fibrinolysis? Blood. 1993; 82: 392–397.[Abstract/Free Full Text]
  16. Kang C, Durlach V, Soulat T, Fournier C, Anglés-Cano E. Lipoprotein(a) isoforms display differences in affinity for plasminogen-like binding to human mononuclear cells. Arterioscler Thromb Vasc Biol. 1997; 17: 2036–2043.[Abstract/Free Full Text]
  17. Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson GR, Boerwinkle E, Utermann G. Relation of serum lipoprotein(a) and apolipoprotein(a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. N Engl J Med. 1990; 322: 1494–1499.[Abstract]
  18. Sandholzer C, Boerwinkle E, Saha N, Tong MC, Utermann G. Apolipoprotein(a) phenotypes, Lp(a) concentration and plasma lipid levels in relation to coronary heart disease in a Chinese population: evidence for the role of apo(a) gene in coronary heart disease. J Clin Invest. 1992; 89: 1040–1046.
  19. Kronenberg F, Kronenberg MF, Kiechl S, Trenkwalder E, Santer P, Oberhollenzer F, Egger G, Utermann G, Willeit J. Role of lipoprotein(a) and apolipoprotein(a) phenotype in atherogenesis: prospective results from the Bruneck study. Circulation. 1999; 100: 1154–1160.[Abstract/Free Full Text]
  20. Dominguez M, Rojas G, Loyau S, Bazurco M, Sorell L, Anglés-Cano E. Kringles of the plasminogen–prothrombin gene family share conformational epitopes with recombinant apolipoprotein (a): specificity of the fibrin-binding site. Biochim Biophys Acta. 2001; 1548: 72–80.[CrossRef][Medline] [Order article via Infotrieve]
  21. Brunner C, Lobentanz EM, Pethö-Schramm A, Ernst A, Kang C, Dieplinger H, Müller HJ, Utermann G. The number of identical kringle IV repeats in apolipoprotein(a) affects its processing and secretion by HepG2 cells. J Biol Chem. 1996; 271: 32403–32410.[Abstract/Free Full Text]
  22. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977; 36: 59–77.[Abstract/Free Full Text]
  23. Anglés-Cano E, Loyau S, Cardoso-Saldana G, Couderc R, Gillery P. A novel kringle-4 number based recombinant apo[a] standard for human apo[a] phenotyping. J Lipid Res. 1999; 40: 354–359.[Abstract/Free Full Text]
  24. Sorell L, Rojas G, Rodriguez M, Ramos C, Torres L, Torres MB. A sandwich ELISA based on anti-apo(a) and anti-apo B monoclonal antibodies for lipoprotein(a) measurement. Clin Chim Acta. 1995; 236: 59–70.[CrossRef][Medline] [Order article via Infotrieve]
  25. Lowry OH, Rosenbrough NJ, Farr AR, Randall R. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265–275.[Free Full Text]
  26. Kyhse-Andersen J. Electroblotting of multiple gels: a single apparatus without buffer tank for rapid transfer of protein from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984; 10: 203–209.[CrossRef][Medline] [Order article via Infotrieve]
  27. Edelstein C, Mandala M, Pfaffinger D, Scanu A. Determinants of lipoprotein(a) assembly: a study of wild-type and mutant apolipoprotein(a) phenotypes isolated from human and rhesus monkey lipoprotein(a) under mild reductive conditions. Biochemistry. 1995; 34: 16483–16492.[CrossRef][Medline] [Order article via Infotrieve]
  28. Deutsch D, Mertz ET. Plasminogen purification from human plasma by affinity chromatography. Science. 1970; 170: 1095–1096.[Abstract/Free Full Text]
  29. Fleury V, Anglés-Cano E. Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines. Biochemistry. 1991; 30: 7630–7638.[CrossRef][Medline] [Order article via Infotrieve]
  30. Wallen B, Wiman B. Characterization of human plasminogen, II: separation and partial characterization of different molecular forms of human plasminogen. Biochem Biophys Acta. 1972; 257: 122–134.[Medline] [Order article via Infotrieve]
  31. Fracker PM, Speck JC. Protein cell membrane iodination with a sparingly soluble chloramide, 1,2,4,6-tetrachloro-3{alpha}, 6{alpha}-diphenylglycoluril. Biochem Biophys Res Commun. 1978; 80: 849–857.[CrossRef][Medline] [Order article via Infotrieve]
  32. Soulat T, Loyau S, Baudouin V, Maisonneuve L, Hurtaud Roux MF, Schlegel N, Loirat C, Anglés-Cano E. 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. Arterioscler Thromb Vasc Biol. 2000; 20: 575–584.[Abstract/Free Full Text]
  33. Adamson AW. Physical Chemistry of Surfaces. 5th ed. New York, NY: Wiley-Interscience Publication; 1992.
  34. Hervio L, Durlach V, Girard-Globa A, Anglés-Cano E. Multiple binding with identical linkage: a mechanism that explains the effects of lipoprotein(a) on fibrinolysis. Biochemistry. 1995; 34: 13353–13358.[CrossRef][Medline] [Order article via Infotrieve]
  35. Rouy D, Koschinsky ML, Fleury V, Chapman J, Anglés-Cano E. Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments. Biochemistry. 1992; 31: 6333–6339.[CrossRef][Medline] [Order article via Infotrieve]
  36. Cobbaert C, Kesteloot H. Serum lipoprotein(a) levels in racially different populations. Am J Epidemiol. 1992; 136: 441–449.[Abstract/Free Full Text]
  37. Sandholzer C, Saha N, Kark JD, Rees A, Jaross W, Dieplinger H, Hoppichler F, Boerwinkle E, Utermann G. Apo(a) isoforms predict risk for coronary heart disease: a study in six populations. Arterioscler Thromb. 1992; 12: 1214–1226.[Abstract]
  38. Wild SH, Fortmann SP, Marcovina SM. A prospective case-control study of lipoprotein(a) levels and apo(a) size and risk of coronary heart disease in Standford Five-City Project participants. Arterioscler Thromb Vasc Biol. 1997; 17: 239–245.[Abstract/Free Full Text]
  39. Kraft HG, Lingenhel A, Köchl S, Hoppichler F, Kronenberg F, Abe A, Mühlberger V, Schönitzer D, Utermann G. Apolipoprotein(a) kringle IV repeat number predicts risk for coronary heart disease. Arterioscler Thromb Vasc Biol. 1996; 16: 713–719.[Abstract/Free Full Text]
  40. Klausen C, Sjol A, Hansen PS, Gerdes LU, Moller L, Lemming L, Schroll M, Faergeman O. Apolipoprotein(a) isoforms and coronary heart disease in men: a nested case-control study. Atherosclerosis. 1997; 132: 77–84.[CrossRef][Medline] [Order article via Infotrieve]
  41. Marcovina SM, Koschinsky ML. Lipoprotein(a) concentration and apolipoprotein(a) size: a synergistic role in advanced atherosclerosis? Circulation. 1999; 100: 1151–1153.[Free Full Text]
  42. Xue S, Madison EL, Miles LA. The kringle V-protease domain is a fibrinogen binding region within apo(a). Thromb Haemost. 2001; 86: 1229–1237.[Medline] [Order article via Infotrieve]
  43. Leerink CB, van Ham ADFJ, Heeres A, Duif PFCCM, Bouma BN, van Rijn HJM. Sulfhydryl compounds influence immunoreactivity, structure and functional aspects of lipoprotein(a). Thromb Res. 1994; 74: 219–232.[CrossRef][Medline] [Order article via Infotrieve]
  44. Phillips ML, Lembertas AV, Schumaker VN, Lawn RM, Shire SJ, Zioncheck TF. Physical properties of recombinant apolipoprotein(a) and its association with LDL to form an Lp(a)-like complex. Biochemistry. 1993; 32: 3722–3728.[CrossRef][Medline] [Order article via Infotrieve]
  45. Weisel JW, Nagaswami C, Woodhead JL, Higazi AA-R, Cain WJ, Marcovina SM, Koschinsky ML, Cines DB, Bdeir K. The structure of lipoprotein(a) and ligand-induced conformational changes. Biochemistry. 2001; 40: 10424–10435.[CrossRef][Medline] [Order article via Infotrieve]
  46. Harpel PC, Chang VT, Borth W. Homocysteine and other sulfhydryl compounds enhance the binding of lipoprotein(a) to fibrin: a potential biochemical link between thrombosis, atherosclerosis, and sulfhydryl compound metabolism. Proc Natl Acad Sci U S A. 1992; 89: 10193–10197.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CirculationHome page
R. Guerra, Z. Yu, S. Marcovina, R. Peshock, J. C. Cohen, and H. H. Hobbs
Lipoprotein(a) and Apolipoprotein(a) Isoforms: No Association With Coronary Artery Calcification in The Dallas Heart Study
Circulation, March 29, 2005; 111(12): 1471 - 1479.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Tsurupa, B. Ho-Tin-Noe, E. Angles-Cano, and L. Medved
Identification and Characterization of Novel Lysine-independent Apolipoprotein(a)-binding Sites in Fibrin(ogen) {alpha}C-domains
J. Biol. Chem., September 26, 2003; 278(39): 37154 - 37159.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. A. Hancock, M. B. Boffa, S. M. Marcovina, M. E. Nesheim, and M. L. Koschinsky
Inhibition of Plasminogen Activation by Lipoprotein(a): CRITICAL DOMAINS IN APOLIPOPROTEIN(a) AND MECHANISM OF INHIBITION ON FIBRIN AND DEGRADED FIBRIN SURFACES
J. Biol. Chem., June 20, 2003; 278(26): 23260 - 23269.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
22/7/1232    most recent
01.ATV.0000021144.87870.C8v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, C.
Right arrow Articles by Anglés-Cano, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, C.
Right arrow Articles by Anglés-Cano, E.
Related Collections
Right arrow Risk Factors
Right arrow Fibrinolysis
Right arrow Fibrinogen/fibrin
Right arrow Thrombosis risk factors

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2002 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.