Interaction of lipoprotein (a) with the extracellular matrix

Fibdnolysis & Proteolysis(1998) 12 (2), 79-87 © HarcourtBrace & Co. Ltd 1998

Interaction of lipoprotein (a) with the e x t r a c e l l u l a r matrix L. A. Miles, ~ M. T. Sebald, 1 G. M. Fless, 3 A. M. Scanu, 3,4 L. K. Curtiss,t2 E. F. Plow, 5 J. L. H o o v e r - P l o w 5 Departments of 1Vascular Biology (VB-1) and 2Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA 2Departments of 3Medicine and 4Department of Biochemistry and Molecular Biology, University of Chicago, 5841 S. Maryland Avenue, Box 231, Chicago, Illinois 60637, USA 5Joseph J. Jacobs Center for Thrombosis and Vascular Biology (FF2), Department of Molecular Cardiology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

Summary Differences in the interactions of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) with extracellular matrices may account for their different pathogenic effects and localization within atherosclerotic lesions. Accordingly, we have compared the binding of Lp(a), plasminogen (which is highly homologous in structure to the apolipoprotein(a) [ape(a)] moiety of Lp(a)), and LDL with the extracellular matrix using Matrigel as a model. Under conditions where radiolabeled Lp(a) bound to the matrix, LDL binding was not observed. However, at higher LDL input concentrations, binding of this lipoprotein was detected, suggesting a lower affinity interaction of LDL than Lp(a) with the matrix. Binding of Lp(a) and plasminogen was time dependent, specific, saturable and reversible. Lp(a) exhibited a higher affinity for Matrigel (K d = 12-130 nM) than plasminogen (K~ = 0.86 gM); but Lp(a) had fewer binding sites in the matrix than plasminogen. Both Lp(a) and plasminogen binding were inhibited by lysine analogs, implicating the lysine binding sites associated with their kringle structures in the interactions. In addition, Lp(a) binding was inhibited by certain other amino acids, proline and alanine, and LDL. We conclude that the interaction of Lp(a) with the extracellular matrix involves multiple recognition specificities that synergize to achieve a higher affinity interaction than observed for LDL and plasminogen. These differences may lead to different pathogenetic mechanisms for these lipoprotein particles.

INTRODUCTION Central to the atherogenic effects of lipoproteins is their interaction and retention within extracellular matrices} The deposition of lipoprotein particles into matrices may be followed by their uptake and metabolism by macrophages and smooth muscle cells and by their oxidative modification to form deleterious products} These processes are believed to be key steps in the contribution of low density lipoprotein (LDL) to the formation of atherosclerotic lesions. LDL appears to bind with relatively low affinity to the glycosaminoglycans and proteoglycans within extracellular matrices.3 Elevated levels of lipoprotein(a) (Lp(a)) are associated with an increased risk of cardiovascular disease. 4-~ Received: 29 July 1997 Accepted after revision: 26 February 1998 Correspondence to: Jane L. Hoover-Plow, Tel: +1216 445 8207; Fax: +1216 445 8204; E-mail: hooverj@cesmtp, ccf. org

Proatherothrombotic Lp(a) is similar in structure to LDL, with an hydrophobic lipid core in which the amphipathic apoprotein B is embedded, but is distinguished by the presence of an additional apoprotein, apoprotein(a) or [apo(a)].7 Because Lp(a) and LDL are independent risk factors for atherosderotic diseases, ~ apo(a) must impart unique properties to the Lp(a) particle. Consistent with this premise, several functions and properties have been ascribed to Lp(a) which are not shared with LDL.s-~ Notably, Lp(a) and LDL show differential localizations within atherosclerotic lesions,~Z~3which suggests distinct mechanisms of interaction with extracellular matrices. Apo(a) is remarkably similar in structure to plasminogen. Both apo(a) and plasminogen contain mukiple kringles followed by a protease domain.14,~ Kringles are triple-looped disulfide motifs of 80-90 amino acids. Certain kringles within both Lp(a) and plasminogen are functional lysine binding sites (LBS). These LBS mediate binding of both Lp(a) and plasminogen to certain cells ~6-~9and proteins, 2°-25 and these interactions can be inhibited by lysine analogs, such as e-aminocaproic acid

79

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Miles et al

(EACA). Indeed, some of the properties of Lp(a), which distinguish it from LDL, are shared with plasminogen and are inhibited by EACA.~9,26-28 The protease domain of apo(a) contains the amino acids of the active catalytic triad of plasmin, but lacks the activating cleavage site so that proteolytic activity is not generated in apo(a) by the plasminogen activators.15 In this study, we compared the interactions of Lp(a), LDL and plasminogen with the extracellular matrix. Using Matrigel, the basement membrane-like extracellular matrix from murine sacroma cells, distinct interactions of LDL and Lp(a) were observed. These differences may account for the unique localization and pathogenetic contribution of Lp(a) to cardiovascular diseases. MATERIALS AND METHODS Proteins and lipoproteins

Unless otherwise specified, a single band Lp(a) species having an apo(a) isoform of 281 000 Mr (F isoform) was purified from human plasma by flotation centrifngation and affinity chromatography on lysine-Sepharose as describedY 4 In certain experiments, a second apo(a) isoform was used. This isoform, with a Mr -- 520 000, was isolated by the same procedure. By both Coomassie blue staining of polyacrylamide gels run in the presence of sodium dodecyl sulfate and plasminogen activation assays, plasmin(ogen) contamination of the Lp(a) preparations was _<1%. Lp(a) -free LDL and HDL were prepared as described 29 and their concentrations were determined using a modified Lowry assay with bovine serum albumin (BSA) as standard. 3° Lp(a) and Lp(a) -free LDL were radioiodinated using a modified iodine monochloride method. 28,31For Lp(a), the labeling mixture contained 0.2 M EACA to protect its LBS during the iodinationY Unbound iodine was removed by gel filtration on a PD 10 column and dialysis into 0.15M NaC1, 0.01% Na 2 EDTA, 0.01% NAN3, pH Z4. Specific activities of 0.42 + 0.16 ~Ci/~tg and 0.42 + 0.08 ~tCi/~g were obtained for Lp(a) and LDL, respectively, during the course of these studies. The concentrations of radiolabeled proteins were determined by determining the TCA precipitable counts before and after dialysis. Glu-plasminogen was purified by affinity chromatography on lysine-Sepharose 32 as described previously. 33 The concentration of plasminogen was determined spectrophotometrically at 280 nm using an extinction coefficient of 16.8. 34 Plasminogen was radiolabeled using a modified chloramine:F procedure 3~to a specific activity of 0.58 + 0.2 ~tCi/~g. Extensive measures were followed in the handling of the lipoproteins to minimize oxidation. Exposure to air and light was minimized; samples were mixed by gentle inversion and not vortexed; and EDTA (0.01%) was present during storage. Possible oxidation was monitored at 234 nm to Fibrinolysis & Proteolysis (1998) 12(2), 79-87

measure conjugated diene formation. Each Lp(a) and LDL preparation was used within 3 months, and iodinated lipoproteins were used within 3 weeks. Binding of ligands to extracellular matrix

To measure the ligand binding to an extracellular matrix, wells of 96-well flexible flat bottom microtiter plates (No. 3912, Falcon, Becton Dickinson, Bedford, MA, USA) were coated with Matrigel (Collaborative Biomedical Products, Becton Dickinson). The Matrigel was thawed at 4°C, and the wells were coated at 4°C typically using 500 ~g]ml diluted in 0.01 M sodium phosphate, 0.15 M NaC1, pH Z3 (phosphate buffered saline, PBS). After 18 h at 4°C, the wells were washed, and then postcoated with 3% ovalburain (Sigma, St Louis, MO, USA) in PBS for 1 h at 22°C. The wells were washed 3 times with 200 ~1 0.05% Tween 80 in PBS. Radiolabeled ligands were diluted in PBS conraining 0.1% Tween 80 and 100 units/ml Trasylol (FBA Pharmaceuticals, New York, NY, USA) and incubated in triplicate in a volume of 100 ~zlfor 2 h at 22°C. Wells were washed six times with 200 ~1 PBS containing 0.05% Tween 80. The microtiter plates were inverted, tapped to remove excess liquid and dried. Individual wells were cut out and counted in an Iso-Data gamma counter (IsoData, Inc., Palatine, IL, USA). The fmoles of ligand bound were calculated based on the specific activities of the radiolabeled ligands. Analyses and statistics Data

are reported

as mean

+ standard

deviation.

Dissociation constants, Ka; the maximum number of binding sites, Bmax; and the concentration of inhibitor producing 50% inhibition of binding (ICs0) were derived from Scatchard plots of binding studies in which varying concentrations of nonlabeled Lp(a), plasminogen or other inhibitors were added with a constant concentration of radiolabeled Lp(a) or plasminogen (15 nM). Data from Scatchard analyses were analyzed using the LIGAND computer program? 6 RESULTS

Matrigel, produced by Engelbreth-Holm-Swarm mouse sarcoma cells,3z has been used as a model basement membrane-like extracellular matrix in many studies (e.g.38-41). This material provides a matrix source that can be compared among laboratories. Its major components are typical matrix constituents including laminin, collagen IV, heparin sulfate proteoglycans, entactin and nidogen as well as certain growth factors. Initially we tested whether radiolabeled Lp(a) or plasminogen, each added at a 15 nM concentration, could interact with this matrix. © Harcourt Brace & Co. Ltd 1998

Interaction of lipoprotein (a) with the extracellular matrix

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Table 1 Interaction of Lp(a) and LDL with Matrigel* Lipoprotein Concentration added (nM)

Lp(a) bound (fmol)

LDL bound (fmol)

15 50 150

11.3±4.5 33.5±7.5 71.0±26.2

1,8±1.4 7,7±4.4 26.7±16.0

*Microtiter plates were coated with 500 #g/ml Matrigel. Binding of the lipoproteins was measured at 120 rain at 22°C, and interaction with the wells in the absence of Matrigel was subtracted.

The influence of Matrigel coating concentrations on binding is shown in Figure 1. Both 125I-Lp(a) and ~25I-plasminogen bound to the extracellular matrix. Their binding increased with increasing Matrigel concentration, and plateaux were attained at Matrigel concentrations above 500 gg/ml. However, the extent of plasminogen binding was substantially greater than for Lp(a). Similar results were obtained with four different lots of Matrigel. In contrast to the binding of Lp(a) and plasminogen, no specific binding of 125I-LDL (15 nM) to the matrix could be demonstrated. The extent of binding of LDL to the non-coated microtiter well was similar to that observed at Matrigel concentrations between 25 and 1000 ~tg/ml. However, as summarized in Table 1, when higher © Harcourt Brace & Co. Ltd 1998

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Fig. 2 Effects of LDL and Lp(a) on 12SI-Lp(a)binding to Matrigel. l~SI-Lp(a) (15 nM) was incubated with Matrigel in the presence of the indicated concentration of unlabeled Lp(a) (open circles) or unlabeled LDL (closed circles) diluted into Tris buffered saline. Specific binding was determined by subtracting counts bound to wells in the absence of Matrigel [1.71 fmoles 12~l-Lp(a)]. In the absence of competitor, 3.02 ± 0.68 fmoles 12SI-Lp(a)were bound/well.

concentrations of a25I-LDLwere added (50 or 150 nM) to the matrix-coated wells, specific binding could be detected. Nonetheless, the extent of LDL binding remained low compared to Lp(a). If the lipoprotein properties of Lp(a) contribute to its binding to the matrix, then LDL, despite its low affinity for Matrigel, might still inhibit Lp(a) binding. This possibility is supported by the data shown in Figure 2 where LDL inhibited 125I-Lp(a) binding. LDL was less potent than non-labeled Lp(a) in inhibiting 125I-Lp(a)binding; its ICso was - 6-fold greater than Lp(a). Nonetheless, LDL completely inhibited 125I-Lp(a) binding at high concentrations. These data are consistent with previously reported weak interactions of LDL with matrices. 42-45 Accordingly, in subsequent detailed analyses, we focused primarily upon the binding of Lp(a) and plasminogen to the matrix. The kinetics of the interaction of these ligands with the Matrigel is shown in Figure 3. An apparent steady state binding of ~SI-Lp(a) was achieved by 90 rain (Fig. 3A). ~25I-plasminogen binding exhibited a similar time course (Fig. 3B). Since binding of each radiolabeled ligand was inhibited by excess non-labeled ligand, Lp(a) and plasminogen must each bind to a limited number of sites within the matrix. The specificity of the interactions of ~25I-Lp(a) and ~2~I-plasminogen with the matrix was analyzed further. Non-labeled Lp(a) inhibited the binding of both ~25I-Lp(a) and ~25I-plasminogen in a dose-dependent manner (Fig. 4A). For both radiolabeled ligands, the extent of inhibition exceeded 84% at 1.9 gM unlabeled Lp(a). This level of non-specific binding, 16%, was similar to the level of Fibrinolysis & Proteolysis (1998) 12(2), 79-87

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Time (min) Fig. 3 Time dependence of Lp(a) and plasminogen binding to Matrigel. (A) Microtiter wells were either uncoated (open squares) or coated with 500 #g/ml Matrigel and incubated with ~2SI-Lp(a)(15 nM) in the presence of PBS (closed circles), or 1 #M unlabeled Lp(a) (open circles) for the indicated times. Specific binding (closed squares) was determined by subtracting counts bound in the presence of unlabeled Lp(a) from counts bound in the presence of PBS. (B) Microtiter wells were either uncoated (open squares) or coated with 500 #g/ml Matrigel and incubated with ~251-plasminogen (15 nM) in the presence of either PBS (closed circles) or 10 p.M unlabeled plasminogen (open circles) for the indicated times. Specific binding (closed squares) was determined by subtracting counts bound in the presence of unlabeled plasminogen from counts bound in the presence of PBS (total binding). Values are mean _+SD of triplicate measurements.

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binding in the absence (coated with ovalbumin only) of Matrigel (24 + 6%). The inhibitory effects of non-labeled Lp(a) on 12SI-Lp(a) and 12SI-plasminogen binding was specific; unrelated molecules - ovalbumin, RNase and lysozyme - when present at 10 pM, gave < 8% inhibition of binding of either ~25I-Lp(a) or ~25I-plasminogen. The concentration of non-labeled Lp(a) producing 500/0 inhibition of its own binding or that of radiolabeled plasminogen was very similar, approximately 0.23 pM. For comparison, unlabeled plasminogen (10 pM) inhibited the total binding to 11.6 + 2%, similar to the level of ~25I-plasminogen binding to the wells in the absence of Matrigel (13.8 + 5%) and the concentration of nonlabeled plasminogen required to inhibit ~25I-plasminogen binding by 50% was 0.4 p_M (data not shown). Binding isotherms were constructed for Lp(a) and gave evidence of saturability (Fig. 4B). To determine if it was appropriate to derive affinity constants from the data in

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Fig. 4 (A) Effect of unlabeled Lp(a) on ~2~l-plasminogenand 12SI-Lp(a)binding to Matrigel. Increasing concentrations of unlabeled Lp(a) were mixed with either 12~l-Lp(a) (15 nM) (closed circles) or ~2~l-plasminogen(15 nM) (open circles) and incubated for 90 min with Matrigel as described in Materials and Methods. Per cent saturable binding is shown and was calculated by subtracting cpm bound in the absence of Matfigel from counts bound in the presence of competitor divided by counts bound in the presence of PBS from counts bound in the absence of Matrigel. In this experiment, counts bound in the absence of Matrigel represented 10% and 15% of total binding of ~2~l-plasminogenand la~l-Lp(a), respectively. (B) Data from an inhibition curve of unlabeled Lp(a) inhibiting 1251-Lp(a)binding to Matrigel was transformed to a binding isotherm using the LIGAND program. (C) Data from (B) were subjected to a non-linear curve fitting analysis using the LIGAND program, and the resulting Scatchard plot is shown.

p l a s m i n o g e n to t h e m a t r i x was reversible. 125I-Lp(a) or ~25I-plasminogen was b o u n d to Matrigel for 90 min. The u n b o u n d l i g a n d was r e m o v e d , a n d 200 m M EACA was © Harcourt Brace & Co. Ltd 1998

Interaction of lipoprotein (a) with the extracellular matrix

added. In the next 90 min, 64% of the 125I-Lp(a) bound to the matrix was displaced. When E.4C.4 was added simultaneously with 125I-Lp(a) for 180 min, 88% of the binding was inhibited. A similar resuk was obtained with plasminogen. Under the same experimental conditions, EACA displaced 63% of the ~25I-plasminogen bound to Matrigel. With evidence of extensive reversibility, the binding of Lp(a) to the matrix was subjected to Scatchard analyses..4 representative plot is shown in Figure 4C. The data could be fitted to a straight line, suggesting that the matrix provided a single class of binding sites for Lp(a) with respect to affinity. A Ka of 0.22 gM and Bmax of 24.8 fmoles were calculated for the experiment shown in Figure 4C. From five experiments, the average Ka value was 0.13 + 0.08 pM, with Lp(a) from the same donor (a single apo(a) isoform, F (Mr = 281 000)). Similar analyses for plasminogen gave a Ka of 0.86 + 0.4 gM (n = 3). Under the conditions of analysis, the maximum number of matrix binding sites, B.... was 23.1 + 15.9 fmoles (n = 5) and 2.1 + 2.8 pmoles (n = 3) for Lp(a) and plasminogen, respectively. For comparison, the binding parameters were determined for a second Lp(a) isoform, (Mr 520 000) Lp(a). ,4 Ka of 12 + 0.4 nM and B of 15.2 + 2.6 fmole (n = 2) was determined, Therefore, the B appears to be similar for different isoforms while the affinities of distinct isoforms for this matrix may be different. Because the LBS within apo(a) and plasminogen participate in their interactions with cells ~6-~9and a variety of other proteins/9-2~ we also tested whether the lysine analog, E.4CA could affect the binding of these ligands to Matrigel. At a high concentration (200 raM), E,4C.4 inhibited the binding of both ligands by more than 85% (3-5 experiments). This inhibition suggests that the kringleassociated lysine binding sites are involved in the binding of both ligands to the matrix. The role of the LBS was further explored by comparing the effects of varying concentrations of lysine analogs on binding of Lp(a) and plasminogen to Matrigel. Lysine and E,4C.4 inhibited the interactions of both ligands in a dose-dependent manner with E.4C,4 being approximately 50-fold more potent than lysine for both radiolabeled ligands (Fig. 5). Previous studies have shown that certain Lp(a) interactions can be inhibited by proline. 46-48 Accordingly, we tested the effects of proline, as well as other amino acids, on the interactions of Lp(a) and plasminogen with the matrix. Both proline and alanine inhibited the interaction of Lp(a) with the matrix; when these amino acids were present at concentrations of 50 raM, ~25I-Lp(a) binding was inhibited by > 90o/0 (Fig. 5_4). Glycine produced no inhibition of ~25I-Lp(a) binding at this concentration. These amino acids were considerably less effective in inhibiting plasminogen binding (Fig. 5B). To effectively interact with the LBS of Lp(a) or plasminogen, lysines © Harcourt Brace & Co. Ltd 1998

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Amino Acid Concentration (mM) Fig. 5 Effect of lysine, EACA and hydrophobic amino acids on Lp(a) and plasminogen binding to Matrigel. 12SI-Lp(a)(15 nM) (A), or ~2Sl-plasminogen(15 nM) (B) were incubated with the indicated concentrations of lysine (closed circles), EACA (open circles), proline (closed squares), alanine (open squares), glycine (closed triangles) or buffer. The 100% binding (buffer only) was defined as the counts bound in the presence of PBS minus the counts bound to the wells in the absence of Matrigel. ~2SI-Lp(a)and ~251plasminogenbound to the wells without Matrigel was 4.4 fmoles and 12.1 fmoles, respectively.

Table 2 Effect of proline-containing peptides on Lp(a) binding to matrigel Peptide

None Proline Gly-Pro Gly-Pro-Gly-Gly Glycine

Concentration (mM)

Inhibition of Lp(a) binding (%)

10 100 10 100 10 100 10 100

0.0 58.3 98.3 58.7 90.0 61.0 88.4 3.0 8.4

Microtiter wells were coated with 500 gg/ml Matrigel, and binding of 12SI-Lp(a)(15 nM) was measured after 120 min at 22°C. Binding in the absence of Matrigel was subti'acted from that in its presence and used to calculate % inhibition.

must be present in a carboxy-terminal position within proteins or peptides. However, the results shown in Table 2, in which a series of prolyl-containing peptides were tested, indicate that the position of the proline did not determine inhibitory capacity: peptides with a carboxy or an internal proline were equally effective as proline in blocking 125I-Lp(a) binding to the matrix. DISCUSSION

In this study, we have examined the interaction of Lp(a) with the extracellular matrix using Matrigel as a Fibrinolysis & Proteolysis (1998) 12(2), 79-87

84

Miles et al

model. 49-53 Matrigel was selected because it is a reproducible matrix that affords comparison of results between laboratories and is not subject to differences in cell culture and matrix harvesting condition. In addition, since transgenic mice overexpressing Lp(a) have now been developed, results of our studies should be directly relevant to testing hypotheses regarding development of atherosclerosis in these transgenic mice. This is the first study to identify clear differences in the mechanisms by which Lp(a) and LDL interact with the matrix which could be a basis for differences in the pathogenetic effects of high levels of these two lipoproteins as well as their differential localization in vessel walls. Since the affinity of Lp(a) was higher than that of LDL for Matrigel, we focused on the interaction of Lp(a) with the matrix. A time-dependent and saturable interaction was demonstrated. Binding of radiolabeled Lp(a) to the Matrigel reached a steady state within 90 min at 22°C, and this interaction was inhibited by non-labeled Lp(a) but not by a variety of unrelated proteins. The Lp(a) interaction, like that of plasminogen, was mostly reversible. Upon Scatchard analysis, the binding could be fit to a single class of binding sites with an apparent Kd of 0.13 ~M. This Kd value is similar to that estimated for the binding of the same Lp(a) isoform to cells, ~9 and slightly lower than the estimated Kd of plasminogen for the matrix. The Kd of the $1 isoform was lower than the F isoform studied in detail, and suggest that isoform size will be an important parameter to investigate in future studies. The small isoforms, as utilized in this study, are associated with increased risk of coronary heart disease, 54 and two recent studies found an effect of isoform size on binding of Lp(a) to THP-1 cells,55 and its binding to the LDL receptor and the LDL receptor-related protein. 56 Although fluctuations in the Kd and Bm~ were observed for Lp(a) and plasminogen, the number of Lp(a) binding sites was at least an order of magnitude lower than for plasminogen. Despite the use of human Lp(a) with a matrix derived from mice, the relationship between the number of Lp(a) and plasminogen binding sites is similar to that which exists on cells. 19 Because non-labeled Lp(a) completely blocked plasminogen binding to the matrix as well as to cells, the lipoprotein must preclude access of plasminogen to its additional binding sites. As potential explanations, one molecule of Lp(a) may bind to more than one site simultaneously; Lp(a) may sterically interfere with plasminogen binding to these sites without interacting directly with them; or Lp(a) may interact with plasminogen and prevent plasminogen binding to these sites. This latter interaction has been proposed ~7 but we have been unable to detect specific binding of I2~I-Lp(a) to immobilized plasminogen or binding of ~25I-plasminogen to immobilized Lp(a) (Plow and Hoover-Plow, unpublished observations). Fibrinolysis & Proteolysis (1998) 12(2), 79-87

Under conditions where Lp(a) binding to the matrix could be demonstrated, LDL binding was not observed. However, when LDL was present at a 3- to 10-fold higher concentration than Lp(a), direct binding of LDL was detected. These observations suggest that LDL can interact weakly with Matrigel. Consistent with this interpretation, higher concentrations of LDL were required to inhibit Lp(a) binding to the Matrigel. With 125I-Lp(a) as the ligand, the ICs0 for LDL was 0.31 ~Vl compared to 0.055 gM for non-labeled Lp(a). In separate studies (not shown) using 125I-LDLat 50 nM as a ligand, the ICs0 for unlabeled LDL was 0.33 ~VI and for unlabeled Lp(a) 0.035gM. Together with the direct binding comparisons of radiolabeled Lp(a) and LDL, these data suggest that LDL interacts with the matrix, but with lower affmity than Lp(a). The interaction of Lp(a) with Matrigel is complex. Because plasminogen, EACA and lysine inhibited the binding of Lp(a) to the matrix, the LBS of Lp(a) are clearly implicated in mediating binding. Since > 50% of plasminogen binding to the Matrigel was inhibited in the presence of 50 ~tM EACA, its interaction with the matrix probably involves its high affinity lysine binding site, 5s which is present in kringle 1Y Although Lp(a) does not contain a kringle 1 homolog, its kringles appear to function similarly to the high affinity LBS in plasminogen in this interaction. A high affinity LBS also is involved in Lp(a) binding to cells. 19At least two functional LBS have been demonstrated in apo(a)f ° and engagement of multiple lower affinity LBS could impart apparent high affinity LBS function to Lp(a). Nevertheless, the interaction of Lp(a) with matrix is not simply mediated by its LBS because LDL inhibited J25I-Lp(a) binding. This inhibition may arise from competition for hydrophobic binding sites within the matrix and may depend upon the lipid moieties of the lipoproteins, or may depend upon the apolipoprotein B-100 present in both lipoprotein particles. Trieu et al6~ reported that Lp(a) binds to a proline-Sepharose column and suggested that proline recognition could mediate binding of Lp(a) to subendothelial cell matrices which contain proline-rich collagen. We showed that proline, as well as alanine, inhibited Lp(a) binding to Matrigel. These amino acids may interact with the LBS or could define an additional and potentially hydrophobic component of Lp(a) binding to the matrix. Interestingly, the positioning of proline within a peptide did not influence its inhibitory capacity, whereas a free carboxylate group was required for high affinity binding of lysine to the LBS of Lp(a) and plasminogen. Taken together, our data suggest that multiple recognition specificities are involved in mediating Lp(a) binding to the matrix. Nevertheless, the LBS mediated component of binding appears to exhibit the highest affinity and is unique to Lp(a) as contrasted to the other lipoproteins. © Harcourt Brace & Co. Ltd 1998

Interaction of lipoprotein (a) with the extracellular matrix

A l t h o u g h several studies h a v e e x a m i n e d t h e b i n d i n g of Lp(a) or its c o m p o n e n t s to i n d i v i d u a l m a t r i x proteins, litfie i n f o r m a t i o n is available o n t h e i n t e r a c t i o n of Lp(a) w i t h a c o m p l e x matrix. 62-64 Recently, K r a m e r - G u t h et al45 r e p o r t e d t h a t Lp(a), b u t n o t LDL, b o u n d to t h e extracellular m a t r i x of m e s a n g i a l cells, a n d P e k e l h a r i n g et a165 f o u n d t h a t b o t h Lp(a) a n d LDL i n h i b i t e d p l a s m i n o g e n b i n d i n g to s u b e n d o t h e l i a l cell matrices, b u t Lp(a) was a b e t t e r c o m petitor. O u r d a t a are consistent w i t h b o t h of t h e s e reports. The ability of Lp(a) to interact w i t h t h e extracellular m a t r i x m a y h a v e two p r o a t h e r o t h r o m b o g e n i c effects. First, its r e t e n t i o n in t h e vessel wall m a y e v o k e f o r m a t i o n of atherosclerotic lesions b y lipid m e d i a t e d m e c h a n i s m s . F u r t h e r m o r e , a d d i t i o n a l m e c h a n i s m s of Lp(a), as o p p o s e d to LDL, b i n d i n g to t h e m a t r i x m a y a c c e n t u a t e t h e s e processes. Second, c o m p e t i t i o n b e t w e e n Lp(a) a n d plasm i n o g e n for m a t r i x b i n d i n g sites m a y l e a d to local inhibition of t h e proteolytic activity of p l a s m i n at t h e s e sites. 8,9,11 Such i n h i b i t i o n c o u l d result in i n c r e a s e d d e p o s i t i o n of fibrinogen/fibrin w i t h i n d e v e l o p i n g atherosclerotic lesions, 6~ or suppress g r o w t h factor a c t i v a t o r s F Either of t h e s e m e c h a n i s m w o u l d c o n t r i b u t e to t h e d e v e l o p m e n t of atherosclerosis in i n d i v i d u a l s w i t h h i g h Lp(a) levels.

ACKNOWLEDGMENTS We t h a n k Jos6 Y. Santiago for p r e p a r i n g Lp(a) a n d LDL. This w o r k was s u p p o r t e d b y N I H g r a n t s HL-18577, HL38272, HL-45934 a n d HL-50398. This w o r k was carried o u t d u r i n g t h e t e n u r e of a n Established I n v e s t i g a t o r s h i p Award from t h e A m e r i c a n H e a r t A s s o c i a t i o n a n d Smith Kline B e e c h a m to L.A.M. Blood d r a w i n g at t h e Scripps Research Institute was p e r f o r m e d in its General Clinical Research Center, s u p p o r t e d b y g r a n t No. M 01 RR008333. This is p u b l i c a t i o n n u m b e r 8805-VB f r o m The Scripps Research Institute.

REFERR[NCES 1. Simionescu M, Simionescu N. Proatherosclerotic events: Pathobiochemical changes occurring in the arterial wall before monocyte migration. F.ASEBJ 1993; 7: 1359-1366. 2. Nordestgaard BG. The vascular endothelial barrier-selective retention of lipoproteins. Curr Opin Lipidol 1996; 7: 269-273. 3. Camejo G, Hurt-Camejo E, Olsson U, Bondjers G. Proteoglycans and lipoproteins in atherosclerosis. Curr Opin Lipidol 1993; 4: 385-391. 4. Bostom .AG,Gagnon DR, Cupples .Aet al..A prospective investigation of elevated lipoprotein(a) detected by electrophoresis and cardiovascular disease in women. Circulation 1994; 90: 1688-1695. 5. Terres W, Tatsis E, Pfalzer B, Bell FU, Beisiegel U, Harnm CW. Rapid angiographic progression of coronary artery disease in patients with elevated lipoprotein(a). Circulation 1995; 91 : 948-950.

6. Groves P, Rees .A, Bishop A et al. Apolipoprotein(a) concentrations and susceptibility to coronary artery disease in © Harcourt Brace & Co. Ltd 1998

85

patients with peripheral vascular disease. Br Heart J 1993; 69: 26-30. Z Fless GM, Rolih CA, Scanu AM. Heterogeneity of human plasma lipoprotein(a): isolation and characterization of the lipoprotein subspecies and their apoproteins. I Biol Chem 1984; 259: 11470-11478. 8. Miles LA, Plow EF. Lp(a): an interloper into the fibrinolytic system? Thromb Haemost 1990; 63: 331-335. 9. Howard GC, Pizzo SV. Biology of disease: lipoprotein(a) and its role in atherothrombotic disease. Lab Invest 1993; 69: 373-386. 10. Scanu AM, Edelstein C. Kringle-dependent structural and functional polymorphism of apolipoprotein(a). Biochim Biophys Acta 1995; 1256: 1-12. 11. Harpel PC, Hermann A, Zhang XX, Osffeld I, Borth W. Lipoprotein (a) plasmin modulation, and atherogenesis. Thromb Haemost 1995; 74: 382-386. 12. Cushing GL, Gaubatz JW, Nava ML et al. Quantitation and localization of apolipoproteins(a) and B in coronary artery bypass vein grafts resected at reoperation. Arteriosclerosis 1989; 593: 603. 13. Rath M, Niendorf A, Reblin T, Dietel M, Krebber H-J, Beisiegel U. Detection and quantification of lipoprotein(a) in the arterial wall of 107 coronary bypass patients. Arteriosclerosis 1989; 9: 579-592. 14. Eaton DL, Fless GM, Kohr WJ et al. Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. Proc Nail Acad Sci USA 1987; 84: 3224-3228. 15. McLean JW, Tomlinson JE, Kuang W-I et al. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature 1987; 330: 132-13Z 16. Scanu .AM,Miles LA, Pfaffinger D et al. Rhesus monkey Lp(a) binds to lysine-Sepharose and U937 monocytoid cells less efficiently than human Lp(a). Evidence for a dominant role of kringle 43z.J Clin Invest 1993; 91: 283-291. 1Z Ezratty .A, Simon DI, Loscalzo I. Lipoprotein(a) binds to human platelets and attenuates plasminogen binding and activation. Biochemistry 1993; 32: 4628-4633. 18. Plow EF, Herren T, Redlitz .A, Miles LA, Hoover-Plow JL. The cell biology of the plasminogen system. FASEBJ 1995; 9: 939-945. 19. Miles L.A,Fless GM, Scanu AM et al. Interaction of Lp(a) with plasminogen binding sites on cells. Thromb Haemost 1995; 73: 458-465. 20. Wiman B, Lijnen HR, Collen D. On the specific interaction between the Iysine-binding sites in plasmin and complementary sites in alpha-2-antiplasmin and in fibrinogen. Biochim Biophys -Acta 1979; 579: 142-154. 21. Lucas M.A,Fretto LJ, McKee P.A.The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem 1983; 258: 4249-4256. 22. Kluft C, Jie .AFH,Los P, deWit E, Havekes L. Functional analogy between lipoprotein(a) and plasminogen in the binding to the kringle 4 binding protein, tetranectin. Biochem Biophys Res Commun 1989; 161: 427-433. 23. Harpel PC, Gordon BR, Parker TS. Plasmin catalyzes binding of lipoprotein(a) to immobilized fibrinogen and fibrin. Proc Natl Acad Sci USA 1989; 86: 3847-3851. 24. Fleury V, Angles-Cano E. Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines. Biochemistry 1991; 30: 7630-7638. 25. Leerink CB, Duif PFCCM, Gimpel J-A,Kortlandt W, Bouma BN, van Rijn HJM. Lysine-binding heterogeneity of Lp(a): consequences for fibrin binding and inhibition of plasminogen activation. Thromb Haemost 1992; 68: 185-188. Fibrinolysis & Proteolysis (1998) 12(2), 79-87

86

Miles et al

26. Edelberg JM, Conzalez-Gronow M, Pizzo SV. Lipoprotein(a) inhibits streptokinase-mediated activation of human plasminogen. Biochemistry 1989; 28: 2370-2374. 27. Edelberg JM, Gonzalez-Gronow M, Pizzo SV. Lipoprotein(a) inhibition of plasminogen activation by tissue-type plasminogen activator. Thromb Res 1990; 57: 155-162. 28. Hoover-Plow JL, Miles L_A,Fless GM, Scanu AM, Plow EE Comparison of the lysine binding functions of lipoprotein(a) and plasminogen. Biochemistry 1993; 32: 13681-1368 Z 29. Snyder ML, Polacek D, Scanu AM, Fless GM. Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte derived macrophages. J Biol Chem 1992; 267: 339-346. 30. Curtiss LK, Edgington TS. Immunochemical heterogeneity of human plasma high density lipoproteins. J Biol Chem 1985; 260: 2982-2993. 31. Shepherd J, Bedford DK, Morgan HG. Radiodination of human low density lipoprotein: a comparison of four methods. Clin Chim Acta 1976; 66: 97-109. 32. Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096. 33. Miles LA, Dahlberg CM, Plow EF. The cell-binding domains of plasminogen and their function in plasma. J Biol Chem 1988; 263:11928-11934. 34. Wallen P, Wiman B. Characterization of human plasminogen II. Separation and partial characterization of different molecular forms of human plasminogen. Biochim Biophys Acta 1972; 257: 122-134. 35. Miles LA, Plow EF. Binding and activation of plasminogen on the platelet surface. J Biol Chem 1985; 260:4303-4311. 36. Munson PJ, Rodbard D. Ligand a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 1980; 107: 220-239. 37. Orkin RW, Gehron P, McGoodwin EB, Martin GR, Valantine T, Swarm R. A murine tumor producing matrix of basement membrane. J Exp Med 1977; 145: 204-220. 38. Albini .4, Iwamoto Y, Kleinnlan HK, Matrin GR, Aaronson SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 1987; 47: 3239-3245. 39. Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, Laurie GW, Martin GR. Basement membrane complexes with biological activity. Biochemistry 1986; 25:312-318. 40. Matrisian LM. Metalloproteinases and their inhibitors in matrix remodelling. Trends Genet 1990; 6: 121-125. 41. Schnaper WH, Grant DS, Stetler-Stevenson WG et al. Type IV collagenases and TIMPs modulate endothelial cell morphogenesis in vitro. J Cell Physiol 1993; 156: 235-246. 42. Iverius P-H. The interaction between human plasma lipoproteins and connective tissues glycosaminoglycans. J Biol Chem 1972; 247: 2606-2613. 43. Saxena U, Klein MG, Vanni TM, Goldberg IJ. Lipoprotein lipase increases low density lipoprotein retention by subendothelial cell matrix. J Clin Invest 1992; 89: 373-380. 44. McConathy WJ. Interactions of Lp(a) with subendothelial cell matrix. In: Stein O, Eisenberg S, Stein Y (eds). Proceedings of the Ninth International Symposium on Atherosclerosis, 1991:119. 45. Kramer-Guth A, Greiber S, Pavenstadt H, Quaschning T, Winkler K, Schollmeyer P, Wanner C. Interaction of native and oxidized lipoprotein(a) with human mesangial cells and matrix. Kidney Int 1996; 49: 1250-1261. 46. Edelstein C, Mandala M, Pfaffinger D, Scanu AM. Determinants of lipoprotein(a) assembly: A study of wild-type and mutant

Fibrinolysis & Proteolysis (1998) 12(2), 79-87

4Z

48. 49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

apolipoprotein(a) phenotypes isolated from human and rhesus monkey lipoprotein(a) under mild reductive conditions. Biochemistry 1995; 34: 16483-16492. Gabel BR, May LF, Marcovina SM, Koschinsky ML. Lipoprotein(a) assembly: Quantitative assessment of the role of apo(a) kringle IV types 2-10 in particle formation. Arterioscler Thromb Vasc Biol 1996; 16: 1559-1567. Trieu VII, McConathy WJ. A two-step model for lipoprotein(a) formation. J Biol Chem 1995; 270:15471-15474. Niemann MA, Baggott JE, Miller EJ. Binding of SPAAT,the 44residue C-terminal peptide of alpha 1-antitrypsin, to proteins of the extracellular matrix. J Cell Biochem 1997; 66: 346-35Z Caceres J, Brandan E. Interaction between Alzheimer's disease beta A4 precursor protein (APP) and the extracellular matrix: evidence for the participation of heparan sulfate proteoglycans. J Cell Biochem 1997; 65: 145-158. Malinda KM, Goldstein AL, Kleinman HK. Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells. FASEBJ 1997; 11:474-481. Yamagishi S, Yonekura H, Yamamoto Yet al. Advanced glycation end products-driven angiogenesis in vitro. Induction of the growth and tube formation of human microvascular endothelial ceils through autocrine vascular endothelial growth factor. J Biol Chem 1997; 272: 8723-8730. Holst-Hansen C, Johannessen B, Hoyer-Hansen G, Romer J, Ellis V, Brunner N. Urokinase-type plasminogen activation in three human breast cancer cell lines correlates with their in vitro invasiveness. Clin Exp Metastasis 1996; 14: 297-307. Klausen IC, Sjol A, Hansen PS et al. Apolipoprotein(a) isoforms and coronary heart disease in men: a nested case-control study. Atherosclerosis 1997; 132: 77-84. Kang C, Durlach V, Soulat T, Fournier C, Angl6s-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. M~irz W, Beckmann A, Scharnagl H et al. Heterogeneous lipoprotein (a) size isoforms differ by their interaction with the low density lipoprotein receptor and the low density lipoprotein receptor-related protein/%-macroglobulin receptor. FEBS Lett 1993; 325: 271-275. Sangrar W, Gabel BR, Boffa MB et al. The solution phase interaction between apolipoprotein(a) and plasminogen inhibits the binding of plasminogen to a plasmin-modified fibrinogen surface. Biochemistry 1997; 36: 10353-10363. Markus G, De Pasquale JL, Wissler FC. Quantitative determination of the binding of epsilon-aminocaproic acid to native plasminogen. J Biol Chem 1982; 253: 727-732. Lerch PG, Rickli EE. Studies on the chemical nature of lysinebinding sites and on their localization in human plasminogen. Biochim Biophys Acta 1980; 625: 374-378. Ernst A, Helmhold M, Brunner C, Peth6-Schramm A, Armstrong VW, Mfiller H-J. Identification of two functionally distinct lysine-binding sites in kringle 37 and in ktingles 32-36 of human apolipoprotein(a). J Biol Chem 1995; 270: 6227-6234. Trieu VN, Zioncheck TF, Lawn RM, McConathy WJ. Interaction of apo-lipoprotein(a) with apolipoprotein B- containing lipoproteins. J Biol Chem 1991; 256: 4778-4782. Salonen E-M, Jauhiainen M, Zardi L, Vaheri .4, Ehnholm C. Lipoprotein(a) binds to fibronectin and has serine proteinase activity capable of cleaving it. EMBO J 1989; 8: 4035-4040. Van der Hoek YY, Sangrar W, C6t6 GP, Kastelein JJP, Koschinsky ML. Binding of recombinant apolipoprotein(a) to extracellular matrix proteins. Arterioscler Thromb 1994; 14:1792-1798.

© Harcourt Brace & Co. Ltd 1998

Interaction of lipoprotein (a) with the extracellular matrix

64. Edelstein C, Italia JA, Klezovitch O, Scanu AM. Functional and metabolic differences between elastase-generated fragments of human lipoprotein[a] and apolipoprotein[a]. J Lipid Res 1996; 37: 1786-1801. 65. Pekelharing HLM, Kleinveld HA, Duif PFCCM, Bouma BN, van Rijn HJM. Effect of lipoprotein(a) and LDL on plasminogen binding to extracellular matrix and a matrix-dependent plasminogen activation by tissue plasminogen activator. Thromb Haemost 1996; 75: 497-502.

© Harcourt Brace & Co. Ltd 1998

87

66. Ernst E. Fibrinogen: an important risk factor for atherothrombotic diseases. Ann Med 1994; 26:15-22. 67. Lawn RM, Pearle AD, Kunz LL, Rubin EM, Reckless J, Metcalfe JC, Grainger DJ. Feedback mechanism of focal vascular lesion formation in transgenic apolipoprotein(a) mice. J Biol Chem 1996; 217: 31367-31371.

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