Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:656-664
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:656-664.)
© 1996 American Heart Association, Inc.
A Quantitative Immunoassay for the Lysine-Binding Function of Lipoprotein(a)
Application to Recombinant Apo(a) and Lipoprotein(a) in Plasma
Jane L. Hoover-Plow;
Nataya Boonmark;
Pamela Skocir;
Richard Lawn;
Edward F. Plow
From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology,
Cleveland Clinic Foundation, Cleveland, Ohio, and the Department of
Cardiovascular Medicine (N.B., R.L.), Stanford University, Stanford, Calif.
Correspondence to Jane Hoover-Plow, Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology/FF20, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195.
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Abstract
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Abstract Apo(a), the unique apoprotein of lipoprotein(a)
(Lp[a]),
can express lysine-binding site(s) (LBS). However, the
LBS activity
of Lp(a) is variable, and this
heterogeneity may influence its
pathogenetic
properties. An LBS-Lp(a) immunoassay has been developed
to
quantitatively assess the LBS function of Lp(a). Lp(a) within
a sample
is captured with an immobilized monoclonal antibody
specific
for apo(a), and the captured Lp(a) is reacted with an antibody
specific
for functional LBS. The binding of this LBS-specific antibody
is
then quantified by using an alkaline phosphataseconjugated
disclosing
antibody. The critical LBS-specific antibody was raised to
kringle
4 of plasminogen. When applied to plasma samples,
the LBS activity
of Lp(a) ranged from 0% to 100% of an isolated
reference Lp(a);
the signal corresponded to the percent retention of
Lp(a) on
a lysine-Sepharose column but did not correlate well with
total
Lp(a) levels in plasma. Mutation of residues in the putative
LBS
in the carboxy-terminal kringle 4 repeat (K4-37) in an
eight-kringle
apo(a) construct resulted in marked but not complete
loss of
activity in the LBS-Lp(a) immunoassay. These data suggest that
this
kringle is the major but not the sole source of LBS activity
in
apo(a). The LBS-Lp(a) immunoassay should prove to be a useful
tool in
establishing the role of the LBS in the pathogenicity
of Lp(a).
Key Words: lipoprotein(a) lysine-binding site recombinant apo(a) functional immunoassay
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Introduction
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Lp(a) is similar to LDL
with respect to lipid composition and
the presence of an apoB
moiety.
1 2 Distinguishing Lp(a) from
LDL is the presence
of apo(a) within Lp(a). The primary structure
of apo(a) strongly
resembles plasminogen.
3
Plasminogen contains
five kringle domains, each composed of
80 to 90 amino acids
organized into a triple-looped structure by
three internal disulfide
bonds.
4 The homology of the
kringles of apo(a) with plasminogen,
especially K4 and K5,
is extremely high. Human apo(a) contains
multiple and variable
numbers of plasminogen K4-like kringles
and one copy of
K5.
5
The kringles of plasminogen, including K4 and K5, function
as LBS. The LBS of plasminogen recognize lysines, primarily
carboxy-terminal lysines.6 7 This activity is
responsible for the binding of plasminogen to
lysine-Sepharose and, more importantly, for the interaction of
plasminogen and plasmin with substrates,8 9
inhibitors,10 and cellular
receptors.11 12 Lp(a) also binds to
lysine-Sepharose,3 several cell types, including
hepatocytes,13 14 monocytes,15 16 17
and endothelial cells,18 19 and
extracellular matrices20 21 and matrix
proteins.22 23 These interactions can be inhibited, at
least in part, by lysine and lysine analogues.18 19 21 In
addition, Lp(a) can also interfere with certain functions of
plasminogen that are mediated by the
LBS.15 19 24 25 26 These data indicate that one or more of
the kringles of apo(a) has LBS activity. On the basis of the crystal
structure of K4 of plasminogen,27 the LBS is
composed of a trough lined by three aromatic residues, flanked on one
end by two anionic (Asp, Asp) residues and at the other end by two
cationic (Lys, Arg) residues. The sequence of apo(a) shows that its
final kringle 4 repeat, K4-37 (nomenclature of McLean et
al5 ), has the appropriate residues to form an LBS. This
prediction has been experimentally supported by using mutated
recombinant K4-3728 and by showing that naturally
occurring apo(a) with a single amino acid substitution in K4-37 lacks
LBS function.29 30 On the basis of their amino acid
sequences, other apo(a) kringles are predicted to be lower-affinity
LBS, to be nonfunctional, or to have a relaxed
specificity.31 32
The pathogenicity of Lp(a) as a risk factor for
cardiovascular disease may depend on its
LBS,26 33 34 which imparts unique functions to Lp(a) not
shared with LDL, including a potential to interfere with
fibrinolysis. However, the LBS function of Lp(a) varies
within the population.30 35 36 This variability is due at
least in part to amino acid polymorphisms in
K4-37,37 38 but other heterogeneities in Lp(a)
structure,1 such as the lipid and carbohydrate composition
or the overall organization of the particle, may also influence LBS
function.
The current approach to measure the LBS function of Lp(a) is to use
lysine-Sepharose chromatography.30 35 36
This approach is cumbersome, requires relatively large samples, and
does not discriminate graded variations in LBS activity. Accordingly,
the purpose of the present study was to develop a less cumbersome
and more accurate assay to quantify the LBS function of Lp(a). An
immunoassay that can assess the LBS function of Lp(a) in a small amount
of plasma has been developed that can quantify the effects of
enzymatic, chemical, and mutational modifications of Lp(a) on LBS.
Ultimately, this assay will permit an examination of the relationship
of size, concentration, and LBS activity of Lp(a) in plasma samples
from patients with atherosclerotic cardiovascular
disease.
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Methods
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Materials
Reference Lp(a), LDL, and Glu-plasminogen are
routinely isolated
from donor plasma.
11 16 Plasma samples
containing Lp(a) with
amino acid substitutions within
K4-37
30 were kindly provided
by Dr Angelo Scanu,
University of Chicago, Chicago, Ill. Monoclonal
antibodies against LDL
apoB-100 were generous gifts from Dr
Linda Curtiss, Department of
Immunology, Scripps Research Institute,
La Jolla, Calif, and apo(a)
monoclonal antibodies, a generous
gift of Dr Gunther Fless, Department
of Medicine, University
of Chicago. Lp(a) polyclonal antibodies from
sheep were purchased
from Immuno AG, and the MACRA Lp(a) kit was from
Strategic Diagnostics;
tissue-type
plasminogen activator (Activase) was from
Genentech;
high-molecular-weight urokinase, crystalline BSA,
and
DL-lysine
were from Calbiochem; EACA and gelatin (type
B from bovine skin
endotoxin) were from Sigma Chemical Co; aprotinin
was from FBA
Pharmaceuticals; and Tween 20 was from Fisher. All
chemicals
were of reagent grade.
Construction of Apo(a) cDNA Expression Plasmid
The expression plasmid pCMha8, containing eight K4-like domains
as well as the K5-like and protease-like domains, was constructed
from the apo(a) cDNA expression plasmid pRK5ha17, which has been
described by Koschinsky et al.39 Briefly, plasmid pRK5ha17
was partially digested with HindIII, which cleaves in 12
positions (1113, 1455, 1797, 2139, 2823, 3165, 3507, 3849, 4191, 4533,
and 4875) in the apo(a) cDNA sequence and position 7890 in the 3'
untranslated region.5 The resulting fragments, which
eliminated most repeated kringles but retained the K4-32'
untranslated region, were isolated and relegated. After transformation,
a clone that eliminated nine repeated kringles was isolated. The
resulting construct, which encoded the 5' untranslated region, the
signal sequence, the first two K4-like repeats, and a fusion kringle
containing 149 bp of the third K4-like domain and 341 bp of K4-32,
followed by the K4-33K4-37, K5-like, and protease-like domain and
67 bp of the 3' untranslated sequence, was confirmed by DNA
sequencing.
Site Direct Mutagenesis
Substitution mutants were prepared by using a unique
site-elimination mutagenesis kit (Pharmacia Biotech Inc) according
to the manufacturer's suggestions. Briefly,
5'GAATCCAGCGGCCCCTTGG3', a complementary
oligonucleotide (Lysmuta) consisting of 3-bp mutant
sequences that changed amino acid Asp at positions 55 and 57 in K4-37
of the r-apo(a) to Ala and Ala, was synthesized to eliminate the
two negatively charged residues of the lysine-binding
pocket.40 The mutation sequences also introduced a unique
Not I recognition site. This primer was annealed to
heat-denatured plasmid pCMha8 together with a second
oligonucleotide (Xhomuta),
5'GGTTCTATCCATTGAATTCTAGATCTCGTCGACCCTG3', which eliminates a unique
Xho I site from the vector backbone. The annealed primers
were extended and ligated with T4 polymerase and
T4 ligase. After digestion with Xho I to
linearize wild-type plasmids, the mixture was used to transform
repair-deficient Escherichia coli strain BMH71-18MutS.
Plasmid DNA prepared from a culture of the transformed cells was
subjected to a second round of Xho I digestion and
transformation to become further enriched with mutated plasmids.
Mutants were identified by Not I digestion and confirmed by
DNA sequencing. The resulting plasmid was designated pCMha8Lysmuta.
Cell Culture and Transfection
The 293 cells (human embryonic kidney41 ) were
cultured in 60-mm dishes with Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. Transfection experiments were
performed by using lipofectin (Life Technologies, Inc). Plasmids pCMha8
and pCMha8Lysmuta were purified by using ion-exchange columns
(Qiagen Inc). The 293 cells were seeded at 0.75x106
cells per 60-mm dish, and transfection was performed when the cells
attained 40% to 60% confluence. For each dish, 10 µg of either
plasmid DNA was mixed with 1.5 mL Opti-MEM medium (GIBCO/BRL) in a
polystyrene tube, and 1.5 mL Opti-MEM containing 70 µg lipofectin
(GIBCO/BRL) was added. The lipofectin/DNA complexes were allowed to
form for 15 minutes at room temperature and added to the dishes after
washing the monolayers twice with Puck's saline A (GIBCO/BRL). After 5
hours, the medium was replaced by 5 mL Opti-MEM, and incubation was
continued for 24 hours. The medium was harvested, adjusted to 1 mmol/L
phenylmethylsulfonyl fluoride, centrifuged for 10
minutes at 4000g to remove cell debris, and concentrated
from 50 to 1 mL by using Centriprep 100 concentrator (Amicon
Inc). The concentrated medium was analyzed by using reducing
sodium dodecyl sulfatepolyacrylamide gel
electrophoresis and subsequent immunoblotting. The
concentrated medium containing the r-apo(a) was added directly to
the LBS-Lp(a) assay. The concentrations of r-apo(a) were
determined by using the MACRA Lp(a) assay kit.
LBS-Lp(a) Assay
To identify an antibody specific for LBS, 96-well microtiter
plates were coated with 20 µg/mL Lp(a) or plasminogen in
PBS (150 mmol/L NaCl and 10 mmol/L phosphate buffer, pH 7.4) and
incubated overnight at 4°C. Plates were washed four times with buffer
I (PBS, 1 g/L BSA, 0.2 g/L sodium azide, 0.5 g/L Tween 20, and 10 U/L
aprotinin) and coated with 30 g/L gelatin in PBS at 200 µL/well for
60 minutes at 23°C. Plates were incubated at 37°C to melt the
gelatin, which was removed by aspiration. A 100-µL aliquot of a
candidate antibody was added to wells at increasing dilutions in the
presence or absence of 200 mmol/L EACA and incubated for 2 hours at
37°C. Plates were washed four times with 200 µL per well of buffer
I. To each well was added 100 µL alkaline phosphataseconjugated
secondary antibody (raised against mouse or rabbit IgG) that was
diluted in 100 mmol/L Tris, 100 mmol/L NaCl, 5 mmol/L
MgCl2, and 0.5 g/L Tween 20, pH 7.5, and incubated
for 1 hour at 37°C. Plates were washed four times with 200 µL
buffer I, and 100 µL pnitrophenyl phosphate at 1 mg/mL
in buffer (in mmol/L: Tris-HCl 100, NaCl 100, and MgCl2 5,
pH 9.5) was added to each well. The reaction was stopped with 1 mmol/L
NaOH after 10 minutes at room temperature, and the absorbance of
individual wells was read at 405 nm by using a spectrophotometer.
The antibody ultimately identified as detecting the LBS of Lp(a) was
raised in rabbits against the K4 of
plasminogen.42 The anti-K4 antibodies were
immunopurified by applying the initial antiserum to a
plasminogen-Sepharose column (4 mg
plasminogen/mL) and eluting the desired antibody with 200
mmol/L EACA.43 The immunopurified antibody was then used
in the assays at 0.06 to 0.6 mg/L.
To identify the Lp(a) capture antibody, candidate antibodies were
absorbed onto microtiter wells overnight at 4°C. The wells were
washed four times with buffer I and coated with 30 g/L gelatin as
described above. Various dilutions of reference Lp(a), negative and
positive Lp(a) serum diluted in PBS, purified kringle-containing
proteins diluted in PBS with 3 g/L BSA, and medium containing
r-apo(a) were added to the antibody-coated wells and incubated
for 1 hour at 37°C. The wells were then washed four times with buffer
I, and the LBS antibody identified above was added at the selected
dilution in 100 µL. After 2 hours at 37°C the plates were washed
four times with buffer I, and the secondary disclosing antibody was
added as described above.
Quantification of Plasma Lp(a)
The concentration of Lp(a) in plasma samples was determined by
using an established method44 45 or a commercial assay
[MACRA Lp(a) kit] using the kit standards and the isolated reference
Lp(a) standard. Both assays are specific for Lp(a) in the presence of
plasminogen, high triglyceride levels or high
cholesterol, and Lp(a) quantification is not affected by
apo(a) size.44 46 The specificity of the apo(a) antibodies
used in these assays has been described.44 45
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Results
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Antisera Selection and Assay Development
The initial objective of these studies was to develop an
immunoassay
to measure the LBS activity of Lp(a). The critical reagent
for
such an assay is an antibody that reports on the functional
status
of the LBS. To identify the appropriate reagent, the
capacity of EACA,
a lysine analogue that interacts with LBS
with high affinity, to
interfere with the binding of candidate
antibodies to
immobilized Lp(a) was examined. A total of 10
antibodies,
including both polyclonal antisera and monoclonal
antibodies, were
screened for LBS specificity. Of the five antibodies
to apo(a)/Lp(a)
tested, all reacted with Lp(a) (Table 1

). Of
these, the
binding of one Lp(a) antibody was affected by EACA,
but even this
reagent exhibited a substantial reaction with
Lp(a) in the presence of
the lysine analogue. The two apoB monoclonal
antibodies tested both
reacted with immobilized Lp(a), but their
binding was
minimally influenced by EACA. The two antisera to
the kringle regions
of plasminogen did cross-react with Lp(a).
The
antiserum to the K1-3 (the elastase degradation product
I)
region of plasminogen showed a strong cross-reaction
with
Lp(a), but this interaction was not inhibited by EACA. In
contrast,
an antiserum raised to the K4 region of
plasminogen, the elastase
degradation product II,
not only reacted well with Lp(a), but
its binding was substantially
reduced in the presence of EACA
(Table 1

); the reactivity of this
antiserum with plasminogen
in an EACA-sensitive interaction
is consistent with a previous
study.
42 Table 1

also shows the result obtained when anti-K4
was immunopurified. For
this purpose, the antiserum was passed
over a
plasminogen-Sepharose column, and a subpopulation of
bound
antibody was eluted with EACA. The EACA sensitivity of
the
immunopurified antibody was accentuated, and its reactivity
with Lp(a)
was fully blocked by EACA. This immunopurified anti-K4
was employed in
subsequent studies.
The reactivity of the immunopurified anti-K4 with the LBS of Lp(a) was
evaluated further. The binding of varying concentrations of this
antibody to immobilized Lp(a) and plasminogen
in the presence or absence of EACA is shown in Fig 1
. In
the absence of EACA, the antibody reacted in a dose-dependent
manner with both immobilized Lp(a) and
plasminogen. EACA effectively blocked the binding of all
antibody concentrations to both target antigens.

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Figure 1. Line graph shows interaction of anti-K4 with Lp(a)
and plasminogen (PLG). Varying concentrations of the
immunopurified antibody (see "Methods") were reacted with Lp(a)
or plasminogen in the absence or presence of 200 mmol/L
EACA. The Lp(a) and plasminogen were
immobilized on microtiter plates at 2 and 22 pmol/well,
respectively (2 µg/well). Binding was detected with an alkaline
phosphataseconjugated second antibody.
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While the above data indicate that anti-K4 reacts only with unoccupied
LBS, this antiserum does not discriminate between the LBS of Lp(a) from
plasminogen or other potential LBS-containing proteins. To
establish specificity for the LBS of Lp(a), monoclonal antibodies to
apo(a) were used to selectively capture Lp(a). For this purpose, the
anti-apo(a)-coated microtiter plates provided in commercially
available assay kits (MACRA, Strategic Diagnostics,
Apo-Tek, PerImmune) for quantifying Lp(a) were employed. The plates
from two different suppliers gave similar results. Both capture
antibodies from these two kits have been
characterized45 46 and are reported not to bind to
plasminogen, LDL, VLDL, or HDL. In addition, neither
triglycerides, hemoglobin, nor bilirubin interfere with the
Lp(a) quantification in these assays. The capture antibody is reported
to be insensitive to size polymorphism. Lp(a) or other
kringle-containing proteins were added to these plates followed by
anti-K4 and then the disclosing reagents. In this configuration, Lp(a)
(6.7 mg/L protein) gave a positive signal (absorbance=1.1), but
plasminogen, at its plasma concentrations of 200 mg/L
(30 000-fold above the test Lp[a] concentration), reacted poorly
(absorbance<0.2). Other kringle-containing proteins at their
approximate plasma concentrations, urokinase (100 ng/L) and
tissue-type plasminogen activator (100
ng/L), also gave minimal signals (absorbance<0.2). Furthermore, the
LBS activity of the isolated Lp(a) was not altered when assayed in the
presence of various plasma proteins (Table 2
). The
absorbance for the LBS activity of the Lp(a) plus the other proteins
was 97% to 108% of the reference Lp(a) alone.
Further evidence for the appropriate specificity of the assay with
anti-apo(a) as a capturing reagent and anti-K4 as an LBS reporter
is provided in the analysis shown in Fig 2
. When
a constant concentration of isolated Lp(a) was added to various
dilutions of an Lp(a)-negative plasma, the same signal was obtained at
all plasma dilutions. A number of plasma samples, including
hyperlipidemic plasma, have been tested in a similar
manner (Table 3
). The LBS activity of isolated Lp(a) (20
µg/mL) added in varying dilutions to either normolipidemic or
hyperlipidemic plasma was similar (coefficient of
variation=8%). Thus, the naturally occurring plasma proteins with
kringles, including plasminogen, did not interfere with the
assay. EACA blocked binding at all dilutions, indicating that the LBS
of the captured Lp(a) must be unoccupied for reaction with the anti-K4.
In addition, these data also demonstrate the feasibility of measuring
the LBS status of Lp(a) within plasma samples.
The final configuration and format of the LBS-Lp(a) immunoassay used to
quantify the LBS function of Lp(a) are summarized in Fig 3
. Step 1 entails a 1-hour incubation of the
Lp(a)-containing sample, such as plasma, in microtiter wells coated
with anti-apo(a) as a capturing antibody. Step 2 involves a 2-hour
incubation with the immunopurified anti-K4. In step 3 the disclosing
antibody is added, and after 1 hour the colorimetric
reaction is developed by the addition of substrate.

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Figure 3. Top, Format for the LBS-Lp(a) immunoassay. Step 1:
Lp(a) is captured with an immobilized monoclonal antibody
specific for apo(a); step 2: immunopurified K4 antibody is added to the
captured Lp(a); step 3: binding of the antibody is quantified with an
alkaline phosphatase disclosing antibody (anti-rabbit IgG). Bottom,
Line graph shows dose response of the reference standard Lp(a) in the
LBS-Lp(a) immunoassay. The Lp(a) was a single isoform of
Mr=0.96x106 that was
introduced into the assay at 1-21 nmol/L (1-20 mg/L).
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By using this configuration, varying concentrations of a
single-isoform Lp(a) were added to the assay. This particular Lp(a)
is completely retained on lysine-Sepharose columns.1 47 A
dose-response curve was obtained with evidence of saturation at
Lp(a) concentrations >5 mg/L (Fig 3
, bottom). In subsequent
experiments, this Lp(a) was used as a reference standard. The LBS
activity within unknown samples was quantified relative to this
reference Lp(a) as: percent reference Lp(a)=[absorbance of unknown (5
mg/L)x100]÷absorbance reference standard (5 mg/L). The dilution of
the test sample required to obtain 5 mg/L Lp(a) was determined by using
the MACRA Lp(a) assay kit. Specific applications of the LBS-Lp(a)
immunoassay using this protocol are described below.
LBS Activity of Purified Lp(a)
Two Lp(a) preparations from different donors were introduced into
the LBS-Lp(a) immunoassay at 5 µg/mL in the presence of varying
concentrations of lysine and EACA (Fig 4
). The two
lysine analogues produced similar dose-dependent inhibition of
antibody binding to the two Lp(a) isoforms (Fig 4
). The average
IC50 values for the two donors were 0.5 and 3.7 mmol/L for
EACA and lysine, respectively. These values are in excellent agreement
with those obtained for these compounds in an earlier lysine-bead
assay47 (1.3 and 3.8 mmol/L for EACA and lysine,
respectively). Thus, the LBS-Lp(a) immunoassay appears to accurately
report on the relative affinity of the LBS for lysine analogs.

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Figure 4. Line graphs show effect of lysine analogues on the
LBS-Lp(a) immunoassay. Lp(a) preparations from two different donors
were introduced into the LBS-Lp(a) in the presence of varying
concentrations of lysine (LYS) or EACA. Each Lp(a) preparation is a
different single isoform: A,
Mr=1.04x106; B,
Mr=1.13x106.
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LBS Function of Lp(a) in Plasma
The LBS-Lp(a) immunoassay was used to assess plasma samples
containing Lp(a) with established defects in LBS activity, ie, the
Lp(a) isolated from these plasma samples showed minimal retention on
lysine-Sepharose columns. The three samples used, kindly provided by Dr
Angelo Scanu, included two plasma samples with Lp(a) particles having
the Trp
Arg substitution at position 72 of K4-37, a substitution
known to markedly diminish the LBS function of Lp(a).29 30
The LBS activity of these plasma samples was quantified relative to the
reference Lp(a) as described above (Table 4
). The two
patients with the Trp
Arg substitution showed extremely low activity
in the LBS-Lp(a) immunoassay [0% and 5.7% of the reference standard
Lp(a)]. Rhesus monkey Lp(a), which also has the Trp
Arg mutation at
K4-37, has low binding to lysine-Sepharose columns30 and
lysine-bead assays47 and low LBS activity (13%) as
well. Another plasma sample known to have low reactivity with
lysine-Sepharose (8%) but lacking the Trp
Arg mutation also
exhibited low LBS-Lp(a) activity (6.7%). As discussed by Scanu et
al,30 the sample with low LBS activity but lacking the
Trp
Arg mutation may have another mutation of one or more amino acids
critical for LBS activity.48 In a wild-type plasma
sample prepared in identical fashion, another patient yielded an
LBS-Lp(a) activity of 98.6%, indicating that other Lp(a) particles in
plasma could approach the functional activity of the reference Lp(a)
standard.
Next, the relationship between LBS activity and total Lp(a) levels was
assessed in a panel of 29 plasma samples (Fig 5
).
LBS-Lp(a) activities extended from undetectable levels (0%) to levels
similar to and slightly exceeding the reference standard (defined as
100%). The relationship between LBS activity and the Lp(a)
concentration in this group of samples was not well correlated; the
correlation coefficient (R2) was only .476.
Thus, the LBS-Lp(a) immunoassay reported on a distinct property of
Lp(a).

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Figure 5. Plasma samples were diluted to 5 mg/L on the basis
of reactivity in a commercial Lp(a) assay. LBS activity was determined
in the LBS-Lp(a) immunoassay. The signal obtained for the reference
standard Lp(a) in the assay was assigned a value of 100%; other
values are expressed as a percentage of the reference standard.
Scatterplot shows the total Lp(a) protein level versus the LBS activity
measured in the LBS-Lp(a) immunoassay.
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r-Apo(a)
To test the prediction that K4-37 contains the most prominent LBS
of apo(a), this site was destroyed by changing the two key anionic
residues of the LBS (Asp 55 and 57) to alanine by in vitro mutagenesis.
The wild-type and the mutated K4-37 were expressed in an
r-apo(a) with eight K4-like domains followed by a single
K5 and a protease domain; Western blotting of the wild-type and
mutant r-apo(a) was performed (Fig 6
). The two
r-apo(a) reacted similarly with the apo(a) antibody used and were
of similar electrophoretic mobility. The culture media containing the
wild-type and mutated r-apo(a) were concentrated and
introduced into the LBS-Lp(a) assay in the presence or absence of EACA
(Fig 7
). As controls, conditioned medium from 293 cells
after 24 hours and concentrated (fourfold) conditioned medium were
tested for interference in the LBS assay. Adding 5 µg/mL of the
reference Lp(a) to these samples at varying dilutions (from zero to
1:243) did not change the LBS activity of the Lp(a). The coefficient of
variation was within the experimental error (medium, 6%; conditioned
medium, 6%; and concentrated conditioned medium, 3%). Nonspecific
binding determined in the presence of 100 mmol/L EACA was subtracted.
In the absence of EACA, the signal obtained with wild-type
r-apo(a) was 1.9-fold higher than with the mutant
r-apo(a). At 1 mmol/L EACA, the signal obtained with the
wild-type r-apo(a) was reduced by 63%. The signal obtained
with mutant r-apo(a) was reduced by 41%. Thus, LBS function was
substantially but not fully abolished in the mutant r-apo(a).


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Figure 6. Top, Structures of the mutant and wild-type
r-apo(a). The recombinant apoproteins were composed of eight
repeats, one K5, and a protease domain. The mutant contained alanine
substitutions for two aspartic acids in the last K4 repeat, which
corresponds to K4-37 in the nomenclature of McLean et al.5
Lower left, Immunoblot analysis of the
wild-type and mutant r-apo(a). Following transient
transfection of 293 cells with wild-type (pCMha8) and mutant
(pCMha8Lysmuta) apo(a) expression plasmids, culture medium was
concentrated and analyzed by sodium dodecyl
sulfatepolyacrylamide gel electrophoresis under reducing
conditions and subsequently immunoblotted by using an
antibody specific for apo(a). Wild-type and mutant
r-apo(a) migrate to the same position, corresponding to
Mr=2x105.
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Discussion
|
|---|
In this study, an immunoassay that reports on the functional
status
of the LBS of Lp(a) has been developed and applied to assess
the
LBS function of apo(a) and Lp(a). The critical reagent in
the
development of the LBS-Lp(a) immunoassay is anti-K4, which
only reacted
with functionally available LBS. This antibody,
elicited to K4 of
plasminogen, reacts with the functional LBS
of K4 but not
with K1-3 of plasminogen
42 ; and this same
requirement
for functional LBS applies for its reactivity with Lp(a).
To
establish the specificity of the assay for the LBS of Lp(a),
an
initial immunocapture step with a specific anti-apo(a) was
employed.
Failure of Lp(a) to react in the LBS-Lp(a) immunoassay arose
either
from occupancy of the LBS [lysine analogues blocked reactivity
of
Lp(a)] or ablation of LBS function by LBS modifications [mutations
in
the LBS of Lp(a)]. It is noteworthy that addition of Lp(a) to
normolipidemic,
hyperlipidemic, or hemolyzed plasma did
not block the reactivity
of Lp(a) in the assay. This observation does
not exclude the
possibility that certain plasma samples may contain
substances,
eg, tetranectin,
25 that interact with the LBS,
but it does
indicate that the LBS of Lp(a) can be available and
reactive
in a plasma milieu. Ultimately the assay of plasma samples at
several
dilutions to assign an LBS activity may be desirable to
minimize
potential interfering factors.
When varying concentrations of lysine or EACA were introduced into the
LBS-Lp(a) immunoassay, they produced a dose-dependent inhibition of
signal. The concentrations of lysine and EACA required for 50%
inhibition in the LBS-Lp(a) immunoassay were very similar to those
required47 for blocking the binding of isolated Lp(a) to
lysine-Sepharose beads. This similarity suggests that the LBS-Lp(a)
immunoassay can be used to determine the relative affinity of the LBS
of Lp(a) for lysine analogues. Blockade of the LBS of Lp(a) may be a
useful therapeutic approach to limiting the pathogenetic effects of
Lp(a). The LBS-Lp(a) immunoassay should provide a useful approach for
screening various lysine-LBS inhibitors for potency and
determining their consistency among Lp(a) samples from
different patients. The LBS-Lp(a) immunoassay should also provide a
method for screening the effects of modifications of the Lp(a) particle
on LBS activity. While several studies49 50 51 indicate that
the apo(a) portion of Lp(a) extends away from the apoB-100 and lipid
core, apoB is closely associated with the lipid core. The apo(a) is
attached to apoB via a disulfide linkage at K4-36.52 The
K4-37 kringle is located close to the kringle, as are K4-32 to K4-35,
which, according to Ernst et al,53 may also have LBS
activity and be important for the assembly of the Lp(a) particle. These
kringles near the attachment point of apoB to apo(a) may have
variable LBS activity in different Lp(a) particles depending on the
size and content of the lipid core or modification of the apoproteins.
Detergent53 or low salt concentration29
influences LBS activity, supporting the potential for variable LBS
function. Taken together, our data indicate that K4-37 is the primary
determinant of the LBS activity of Lp(a). However, other factors, such
as the exposure of secondary LBS in other kringles, changes in the
organization of the lipoprotein particle, or the association of plasma
proteins, may influence LBS function. It appears that the LBS-Lp(a)
assay can faithfully report on such variations in the LBS function of
Lp(a).
The LBS-Lp(a) immunoassay was applied to a small panel of plasma
samples. LBS activity ranged from 0% to 100% of the reference Lp(a).
The binding of Lp(a) from different individuals to a lysine-Sepharose
column varies from 40% to 80%, 17% to 91%, and 30% to
70%.35 36 54 The explanation for these ranges of LBS
activity could be one or more of the following. Mutations in K4-37 may
reduce or abolish LBS activity; other kringles with or without
mutations may have some LBS function53 ; in vivo
modifications (eg, oxidation,55
reduction,54 56 sialyation/desialyation,57 or
lipid composition and degradation58 ) of Lp(a) may
influence LBS activity; and kringle number may influence the
accessibility of some kringles, ie, Lp(a) size may influence LBS
activity.59 Limited correlation was found in the samples
studied between the Lp(a) concentrations in these plasmas and LBS
activity. Thus, the LBS-Lp(a) immunoassay may report on a distinct
property of Lp(a) independent of its plasma concentration. Nearly 20
retrospective and prospective studies have confirmed that Lp(a) is a
major risk factor for coronary heart disease and
stroke.60 61 62 63 Karmansky et al36 compared two
groups of patients, one with moderate and one with severe
coronary artery disease, and found that the LBS activity of
Lp(a) was nearly threefold higher in the severe coronary artery
disease patients. Not all subjects with high Lp(a) levels have high LBS
activity (Table 1
), just as size is not an absolute predictor of
concentration.64 65 A good test for the usefulness of the
LBS-Lp(a) assay would be to analyze a population in which high
a Lp(a) level is not a risk factor compared with a population in which
high Lp(a) is a risk factor. Studies are now under way to determine the
relationship between the signal in the LBS-Lp(a) immunoassay and
pathogenesis in such well-characterized panels of clinical
specimens. One interesting possibility is that the LBS-Lp(a)
immunoassay will help to identify those patients with elevated Lp(a)
levels who are at particularly high risk for the development of
coronary disease. As a cautionary caveat, the signal
obtained with plasma samples in the LBS-Lp(a) immunoassay is critically
dependent on the input concentration of Lp(a). The
heterogeneity among individual Lp(a) samples can
influence their quantification in the initial Lp(a)
assays.66 Thus, further development and use of the
LBS-Lp(a) immunoassay must be closely coordinated with development of
reliable assays for Lp(a) quantification.
The LBS-Lp(a) immunoassay was used to characterize the LBS activity of
a wild-type and mutant r-apo(a). On the basis of initial
sequencing, wild-type K4-37 was predicted to have a functional LBS.
This supposition has been supported by molecular
modeling,29 31 by characterization of Lp(a) particles
containing naturally occurring substitutions within this LBS, and by
expression and analyses of this recombinant kringle. The mutant
r-apo(a) contained two substitutions at the critical
aspartic acid residues in the LBS of K4-37 and should have diminished
LBS activity.48 Consistent with these predictions,
the mutant r-apo(a) showed a significant decrease in reactivity in
the LBS-Lp(a) immunoassay relative to wild-type r-apo(a).
Nevertheless, the LBS-Lp(a) activity of the mutant r-apo(a) was not
fully lost. Assembly of apo(a) into LDL requires LBS activity and is
inhibited by EACA.49 67 68 While our study was in
progress, Ernst et al53 reported that r-apo(a) may
contain two LBSs, one associated with K4-37 and a second within the
K4-32K4-36 region, and suggested that this second LBS is not
expressed within the intact Lp(a) particle. Edelstein et
al69 have shown that apo(a) from rhesus monkeys binds LDL
particles to form Lp(a); this incorporation is EACA-sensitive.
Nevertheless, rhesus monkey Lp(a), which has a nonfunctional LBS in
K4-37 due to a point substitution,29 does not have an
exposed LBS. Thus, the existence of two LBS in apo(a), with only the
one in K4-37 being available in the intact human Lp(a) particle,
provides one possible explanation for our data and is
consistent with recent reports. Transgenic mice that express
human apo(a) or Lp(a) are more susceptible to diet-induced
atherosclerosis than control animals.70
The in vivo effects of mutant r-apo(a), either as free apo(a) or
incorporated into Lp(a) particles, should be very informative in terms
of assessing the contributions of the two potential LBS (K4-37 and
K4-32K4-36) to the pathogenesis of Lp(a). The analyses of
these mice to determine whether Lp(a) particles are formed, and, if so,
whether the particles have LBS function, should resolve the
contributions of the two LBS to Lp(a) assembly.
 |
Selected Abbreviations and Acronyms
|
|---|
| BSA |
= |
bovine serum albumin |
| EACA |
= |
-aminocaproic acid |
| LBS |
= |
lysine-binding site(s) |
| Lp(a) |
= |
lipoprotein(a) |
| PBS |
= |
phosphate-buffered saline |
| r-apo(a) |
= |
recombinant apo(a) |
|
 |
Acknowledgments
|
|---|
This work was supported in part by National Institutes of Health
grant
HL 18577 and the American Heart Association, Northeast Ohio
Affiliate.
We wish to thank Brian Lowry for excellent technical
assistance
and Jane Rein and Gene Lazuta for preparation of the
manuscript.
Received June 23, 1995;
accepted December 19, 1995.
 |
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T. Huby, V. Afzal, C. Doucet, R. M. Lawn, E. L. Gong, M. J. Chapman, J. Thillet, and E. M. Rubin
Regulation of the Expression of the Apolipoprotein(a) Gene: Evidence for a Regulatory Role of the 5' Distal Apolipoprotein(a) Transcription Control Region Enhancer in Yeast Artificial Chromosome Transgenic Mice
Arterioscler. Thromb. Vasc. Biol.,
September 1, 2003;
23(9):
1633 - 1639.
[Abstract]
[Full Text]
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M. Ogorelkova, H. G. Kraft, C. Ehnholm, and G. Utermann
Single nucleotide polymorphisms in exons of the apo(a) kringles IV types 6 to 10 domain affect Lp(a) plasma concentrations and have different patterns in Africans and Caucasians
Hum. Mol. Genet.,
April 1, 2001;
10(8):
815 - 824.
[Abstract]
[Full Text]
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R. M. Lawn, K. Schwartz, and L. Patthy
Convergent evolution of apolipoprotein(a) in primates and hedgehog
PNAS,
October 28, 1997;
94(22):
11992 - 11997.
[Abstract]
[Full Text]
[PDF]
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