 |
INTRODUCTION |
Following the original observation by Oida et al.
(1), several reports have demonstrated the presence of
immunoreactive apo(a)1 in
human urine. Particularly significant were the studies by Mooser
et al. (2, 3) who by combining chemical and immunochemical techniques provided evidence that urinary apo(a) was represented by
fragments derived from its N-terminal region. When, following purification, these urinary fragments were injected intravenously into
mice, they were excreted in the urine without an apparent change in
size. The same authors also reported the presence in the plasma of
apo(a) fragments unattached to apoB100. Upon their intravenous
injection into mice, these fragments were rapidly excreted in the urine
as smaller sized components (3). Taken together, the results were
interpreted to indicate that the apo(a) fragments in human plasma are
derived from Lp(a)/apo(a) and are in turn the source of the apo(a)
fragments in the urine. Unanswered in those studies was the underlying
cause of the apo(a) fragmentation as well as the site or sites for its
occurrence. We reported previously that limited in vitro
proteolysis of apo(a), by either pancreatic or leukocyte elastase, is
attended by the cleavage at the Ile3520-Leu3521
bond located in the linker domain between kringles IV-4 and IV-5 (4,
5). This cleavage generates two main fragments, one representing the
N-terminal domain, F1, and the other the C-terminal domain, F2. When
these two fragments were separately injected intravenously into mice,
F1 but not F2 was rapidly excreted in the urine (4). This observation
prompted us to examine whether the cleavage site on apo(a) was limited
to serine proteases such as pancreatic and leukocyte or neutrophil
elastase (NE) or could also apply to other inflammatory enzymes. Among
them we considered matrix matalloproteinases (MMPs), which are
metallo-dependent enzymes involved in the homeostasis of
the extracellular matrix and also shown to play an important role in
the atherosclerotic process (6-13). In this work, we directed our
attention to macrophage metalloelastase, also referred to as MMP-12,
which was first identified as an elastolytic metalloproteinase secreted
by inflammatory macrophages (14) and structurally defined (15, 16).
This enzyme has a broad substrate specificity for matrix macromolecules
such as fibronectin, laminin, vitronectin, and proteoglycans (17). In the current study, we now show that MMP-12 cleaves apo(a) in
vitro and also provide evidence that, in the mouse strain used, it
is involved in the generation of F1 in vivo.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Human leukocyte elastase (EC 3.4.21.37) was
purchased from Sigma. Antiserum to purified preparations of apo(a),
Lp(a), and LDL were raised in the rabbit and affinity purified as
described previously (18). Anti-Lp(a) were shown to be devoid of
immunoreactivity to LDL and plasminogen; anti-LDL was unreactive to apo(a).
Studies in Mice--
Mice deficient in NE (NE
/
)
or MMP-12 (MMP-12
/
) in a pure 129/Svj background were
generated by targeted disruption of either the NE or MMP-12 gene in RW4
embryonic stem cells as described previously (19, 20). All mice were
housed in individual cages under normal light. The evening before the
experiment, the animals were given a 10% sucrose solution to drink
ad libitum in place of water. The following morning the mice
were anesthetized with Metafane prior to the injection into the tail
vein of 25-250 µg of either Lp(a), apo(a), or fragment F1 in a
volume of 100 µl. The mice were then placed in metabolic cages, given
access to standard lab chow and 10% sucrose solution to drink. Blood
samples were withdrawn from the orbital vein into heparinized
hematocrit tubes at the specified time points and immediately iced.
Urine was collected at 0-3, 3-5, and 5-24 h. ELISA quantitation,
sensitive to < 0.0015 mg/dl of apo(a), was performed on the urine
samples to determine the levels of apo(a)-reacting material. These
results indicated to what degree the sample was to be concentrated for electrophoretic detection. The latter was estimated to be > 0.03 mg/dl. The urine was concentrated in Amicon Centriprep filters.
Human Subjects--
The subjects used for the preparation of
Lp(a) and LDL were healthy donors with plasma Lp(a) protein levels in
the range of 15-43 mg/dl determined as described previously (4).
Plasmapheresis was performed in the Blood Bank of the University of
Chicago. All of the subjects used in the study gave a written informed consent. The steps for Lp(a) isolation were carried out immediately after blood drawing using the procedure outlined below. We used an
additional five subjects for studying the apo(a) fragments in their
plasma and urine. These were also healthy subjects with a known
phenotype and genotype. Their plasma Lp(a) protein levels varied
between 1 and 12 mg/dl, and the apo(a) size isoforms varied between 350 and 550 kDa. In each subject, 4-h urine samples were collected,
centrifuged for 15 min at 3000 rpm, and either used fresh or frozen
immediately at
80 °C. Before use, they were concentrated 200-fold
in microconcentrators (Amicon Corp. Beverly, MA). All of the subjects
used in the study gave a written informed consent.
Preparation of Human Lp(a) and LDL--
To prevent lipoprotein
degradation, the plasma obtained by plasmapheresis was adjusted with
0.15% EDTA, 0.01% NaN3, 10,000 units/liter aprotinin, and
1 mM phenylmethylsulfonyl fluoride. Lp(a) were isolated by
sequential ultracentrifugation and lysine-Sepharose chromatography as
described previously (21). The purity of the product was assessed by
mobility on precast 1% agarose gels (Ciba-Corning, Palo Alto, CA) and
Western blots of SDS-PAGE, utilizing anti-Lp(a) and anti-apoB100. The
LDL preparations used in this study were isolated at density of
~1.030-1.050 g/ml by sequential flotation as described previously
(22). To prevent lipoprotein degradation, the plasma obtained by
plasmapheresis was adjusted with 0.15% EDTA, 0.01% NaN3,
10,000 units/liter aprotinin and 1 mM phenylmethylsulfonyl fluoride.
Phenotyping and Genotyping of Apo(a)--
Apo(a) phenotyping was
performed on isolated apo(a) samples by SDS-PAGE followed by
immunoblotting using anti-Lp(a) (23). The mobility of the individual
apo(a) bands was compared with isolated apo(a) isoforms of known
molecular weights (21). For apo(a) genotyping, DNA plugs were prepared
from blood mononuclear cells and subsequently fractionated by pulsed
field gel electrophoresis, and the blots were developed with an
apo(a)-specific probe essentially as described earlier (24).
Isolation of Apo(a) from Lp(a)--
Apo(a) was isolated from
Lp(a) essentially as described previously (23). Briefly, Lp(a), 1 mg/ml
protein, was incubated with dithioerythritol at a final concentration
of 1.5-2 mM.
-amino caproic acid to a final
concentration of 100 mM was then added, and the mixture was
incubated at room temperature for 1 h under nitrogen gas. After
dialysis against 10 mMPO4 buffer, an equal volume
of 60% sucrose in the same buffer was added and the resulting mixture
placed into a TLA 100.3 titanium rotor and spun in a table top TL100
ultracentrifuge at 10 °C, 412,160 × g for 18 h. After centrifugation, the top 0.5-ml fraction contained LDL free of apo(a), [Lp(a
)], and unreacted Lp(a). The bottom 1.0 ml fraction contained free apo(a) in pure form. The latter was stored in the sucrose solution at
80 °C.
Isolation of Apo(a) Fragments F1 and F2--
These fragments
were the products of the limited proteolysis of apo(a) by purified
human leukocyte elastase essentially as described previously (5).
Briefly, the enzyme (1 unit = 1 nm p-nitrophenol/s from
N-1-Boc-L-Ala p-nitrophenol ester)
was diluted 1000-fold in 50 mM Tris-HCl, 100 mM
NaCl, pH 8.0. One microliter of the diluted enzyme was incubated per
7.5 µg of Lp(a) or LDL protein or 15 µg of apo(a) at 37 °C for
30 min. The reaction was terminated with 5 mM diisopropyl
fluorophosphate for 20 min at 22 °C. After digestion, the fragments
were purified by lysine-Sepharose affinity chromatography (4).
Electrophoretic Methods--
SDS-PAGE (4 and 4-12%
polyacrylamide) was performed on a Novex system (Novex, San Diego, CA)
for 1.5 h at constant voltage (120 V) at 22 °C. The samples
were prepared by heating at 95 °C for 5 min in sample buffer which
consisted of 94 mM phosphate buffer, pH 7.0, 1% SDS and 2 M urea with or without 3%
-ME. Immediately after
electrophoresis, the gels were placed onto Immobilon-P sheets (Millipore Corp.) that were previously wetted with a buffer containing 48 mM Tris, 39 mM glycine, pH 8.9. Blotting was
performed on a horizontal semi-dry electroblot apparatus (Amersham
Pharmacia Biotech, Inc.) at 0.8-1 mA/cm2 for 45 min at
22 °C.
Immunoblotting--
After electroblotting, the Immobilon-P
sheets were blocked in PBS containing 5% non-fat dry powdered milk and
0.3% Tween 20 followed by incubation with anti-apo(a) or anti-apoB100
antibody. The blots were washed and incubated with anti-rabbit
horseradish peroxidase-labeled IgG. Subsequently, the blots were
developed with the ECL Western Detection Reagent (Amersham Pharmacia
Biotech) according to the instructions of the manufacturer.
Amino-terminal Sequence Analyses--
Apo(a) digests (30-50
µg) were electrophoresed under reducing conditions as outlined above.
After electrophoresis, the gels were electroblotted onto Immobilon PSQ
sequence grade membranes (Millipore Corp.) as described above in the
immunoblotting section. The blots were rinsed in distilled water,
stained with Coomassie Blue R250 (0.025% in 40% methanol), and
destained with 40% methanol. Reduction with DTT and alkylation with
iodoacetamide was performed directly on the PSQ membrane which was then
subjected to automated Edman degradation on an Applied Biosystems 477A
unit using procedures recommended by the manufacturer. All sequencing
procedures were carried out in the core laboratory of the
Macromolecular and Structural Analysis Facility at the University of Kentucky.
Lipoprotein and Apolipoprotein Analyses--
Lp(a) and LDL
protein were quantitated by a sandwich ELISA essentially as described
previously (18) except that anti-Lp(a) IgG was used as the capture
antibody and anti-apoB100 IgG conjugated to alkaline phosphatase as the
detection antibody. For the ELISA quantitation of apo(a), anti-apo(a)
IgG conjugated to alkaline phosphatase was used as the detection
antibody. Subsequently, an extinction coefficient (
278 = 1.31 ml mg
1 cm
1) was established for apo(a)
in the 30% sucrose solution. Protein determinations were performed by
the Bio-Rad DC Protein Assay.
Proteolytic Cleavage of Lp(a)/apo(a) by Purified Recombinant
MMP-12--
Mouse recombinant MMP-12 was expressed in
Escherichia coli and purified as described previously (15).
The active enzyme was incubated with human Lp(a)/apo(a) (final
concentration, 0.2 mg/ml apo(a)) at varying weight ratios of protein to
enzyme (25, 50, and 100:1, P:E) at 37 °C in 50 mM
Tris-HCl, pH 7.5, 0.15 M NaCl, 10 mM
CaCl2, 0.05% Brij 35, 0.02% NaN3. After
18 h of incubation, samples were removed, EDTA (final
concentration 50 mM) was added to stop the reaction, and
SDS-PAGE was performed under unreduced and reduced conditions.
 |
RESULTS |
Studies on the in Vitro Proteolytic Cleavage of Lp(a)/apo(a) by a
Purified Preparation of MMP-12--
The ability of MMP-12 to hydrolyze
Lp(a) was assessed at varying weight ratios of apo(a) to enzyme using
anti-apo(a) and anti-apoB100 immunoblots of 4% SDS-PAGE. Fig.
1A shows the digestion
patterns of Lp(a) after an 18-h incubation at 37 °C, under unreduced
conditions, probed with anti-apo(a) (left) or anti-apoB100
antibodies (right). The level of digestion of apo(a) and
apoB100 increased as a function of enzyme concentration (Fig.
1A, lanes 3 to 5, left and
right panels). Notably, in the left
panel, bands marked with an arrow decreased in
intensity as the enzyme concentration increased. Three other bands of
major intensity were also present (left panel), one of which
exhibited a mobility corresponding to F1. Immunostaining of the same
gels with anti-apoB100 (right panel) showed that a large
proportion of the products of apoB100 hydrolysis in the Lp(a) particle
comigrated with bands corresponding to the apo(a) immunostained ones in
the left panel. On the reduced gels, there were two major
bands that migrated at an apparent mass of 220 and 170 kDa (Fig.
1B, lanes 2-4, left panel). The mobilities of these bands corresponded to fragments F1 and F2 previously reported (4). The band corresponding to F1 migrated with a slower mobility on
reduced gels than under unreduced conditions, likely because of the
effect of the reducing agent on the conformation of the fragment.
Immunostaining of the reduced gels with anti-apoB100 (Fig. 1B,
right panel) showed that the gel pattern was essentially the same
as observed in the unreduced gel (Fig. 1A, right panel). When apo(a) was digested with MMP-12, the pattern was the same as shown
in Fig. 1B left panel for Lp(a) under reduced conditions. The results suggest that apo(a) in Lp(a) was cleaved at a single site
producing two major fragments, F1 and F2, the latter covalently linked
to truncated forms of apoB100.

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Fig. 1.
Proteolytic cleavage of Lp(a) by MMP-12.
A, pattern of anti-apo(a) and anti-apoB100 immunostained 4%
SDS-PAGE (unreduced) of Lp(a) before and after incubation with MMP-12
for 18 h at 37 °C at various apo(a) to enzyme weight ratios.
Left panel, lanes 1 and 2, undigested Lp(a) and
purified F1, respectively; lanes 3-5, digests at
protein:enzyme weight ratios of 100, 50, and 25:1, respectively.
Right panel, lanes 1 and 2, Lp(a) and LDL;
lanes 3-5, same as in left panel. B,
electrophoretic pattern under reduced conditions of Lp(a) before and
after incubation with MMP-12. Left panel, lane 1, Lp(a);
lanes 2-4, digests at protein to enzyme weight ratios of
100, 50, and 25:1. Right panel, lane 1, LDL; lanes
2-4, same as in left panel.
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The partial amino-terminal sequences of F1 and F2 obtained by MMP-12
digestion of Lp(a) and apo(a) are shown in Fig.
2. By aligning these sequences with those
of apo(a), using the program CLUSTAL W, we located the cleavage site at
the Asn3518-Val3519 bond in the linker region
between kringles IV-4 and IV-5. These fragments correspond to the N-
and C-terminal domain of apo(a) containing kringles IV-1 to IV-4
(N-terminal), and IV-5 through IV-10, kringle V, and the protease
region (C-terminal).

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Fig. 2.
Amino acid sequence alignments of fragments
F1 and F2 obtained from MMP-12 digests of apo(a). The alignment
shown was constructed according to the program ClustalW using the
apo(a) sequence from McLean et al. (25). Apo(a) is composed
of repeats of KIV numbered 1-10, one KV, and a
protease domain (P). The first 10 amino acids obtained by
sequencing F1 were aligned to the amino acid sequence of the mature
apo(a). The amino acid sequence of F2 began with valine at position
3519. The MMP-12 cleavage site was determined to be in the linker
region between KIV-4 and KIV-5. The dashed lines refer to
undetermined amino acids, and the dotted lines are
continuation of sequences upstream and downstream of the protein
sequences.
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Because apoB100 was also digested by MMP-12, it was of interest to
determine whether the fragments remained on the surface of the Lp(a)
particle or formed separate lipoprotein species, each consisting of
various truncated forms of apoB100 disulfide linked to F2. To this end,
MMP-12-digested Lp(a) was electrophoretically separated on native
nondenatured polyacrylamide gels that were immunostained with
anti-apo(a) and anti-apoB100 (Fig. 3).
Digested Lp(a) exhibited two bands (lane 4), one of which
also stained with anti-apoB100 (lane 8). The band with the
faster mobility in lane 4 corresponded to F1. In turn, when
LDL was digested with MMP-12, the pattern was the same as for
undigested LDL (compare lanes 5 and 6).

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Fig. 3.
Native electrophoretic gels of Lp(a) before
and after digestion with MMP-12. Lp(a) was treated with MMP-12 for
18 h at 37 °C at an apo(a) to enzyme weight ratio of 100:1. The
digest was then separated on a 4% native polyacrylamide gel and
immunostained with anti-apo(a) and anti-apoB100. Lanes 1 and
2, apo(a) and F1; lanes 3 and 4, Lp(a)
before and after digestion; lanes 5 and 6, LDL
before and after digestion; lanes 7 and 8, Lp(a)
before and after digestion.
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Taken together, we interpreted these results to show that 1) MMP-12
hydrolyzes both apo(a) and apoB100 components of Lp(a); 2) the
hydrolytic products consist of apo(a) fragment F1 and a miniLp(a)
particle in which the C-terminal region of apo(a), fragment F2, is
covalently linked to truncated forms of apoB100; and 3) the apoB100
fragments remain on the surface of the particle, whereas the N-terminal
fragment of apo(a), F1, is released.
Studies in Mouse Plasma--
We examined, on anti-apo(a)
immunostained blots of reduced 4% SDS-PAGE, plasma samples from mice
bled 3 h after intravenous injection of either Lp(a), apo(a), or
F1. The electrophoretic pattern of the plasma samples after injection
of apo(a) into NE
/
and control mice (Fig.
4A, lanes 2 and
3) contained bands corresponding to full-length apo(a) and
fragments thereof, one migrating in the position of F1, indicating that
a fragmentation process had occurred in vivo. In contrast,
the 3-h plasma sample of MMP-12
/
mice exhibited a major
band corresponding to full-length apo(a) along with some minor bands of
low immunostaining intensity but none with the mobility of F1
(lane 4). The electrophoretic patterns, under reduced
conditions, of the plasma from mice injected with Lp(a) (data not
shown) were comparable with those shown in Fig. 4A,
lanes 2 to 4.

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Fig. 4.
Western blot analysis of mouse plasma and
urine after injection of apo(a) and F1 as well as normal human plasma
and urine. A, in these studies, apo(a) and F1 were
injected into the mice, and plasma was collected 3 h after
injection. The plasma samples were reduced and analyzed by anti-apo(a)
immunostaining of 4% SDS-PAGE. Lane 1, apo(a) before
injection; lanes 2-4, plasma from NE / ,
control, and MMP-12 / mice, respectively, after
injection of apo(a); lane 5, F1 before injection;
lanes 6-8, plasma from NE / , control, and
MMP-12 / mice, respectively, after injection of F1;
lane Hu, fresh human plasma (1 µl). B, mice
were injected with apo(a) and F1, and urine was collected 24 h
after injection. The urine samples were concentrated, reduced, and
analyzed by apo(a) immunostaining of 4-12% gradient SDS-PAGE. The
urine samples from NE / and control mice were
concentrated 7-fold and those from MMP-12 / mice
concentrated 21-fold. In each case, an aliquot of 30 µl of the
concentrated sample was analyzed. Lane 1, apo(a) before
injection; lanes 2-4, urine from NE / ,
control, and MMP-12 / mice, respectively, after
injection of apo(a); lane 5, F1 before injection;
lanes 6-8, urine from NE / , control, and
MMP-12 / mice, respectively, after injection of F1;
lane Hu, human urine collected from the same patient as in
panel A and concentrated 200-fold.
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The banding pattern of plasma after injection of F1 (Fig.
4A, lanes 6-8) was similar among all of the mice
and resembled the injected material (lane 5). All the
patterns contained a major band at 220 kDa and other bands of minor
immunostaining intensity in the range of 200-135 kDa. The
electrophoretic pattern of the plasma of all the mice remained
unaltered at each time point after injection, although the intensity of
these bands, including the major F1 band, decreased with time (data not shown).
Studies in Mouse Urine--
We examined the urine of mice,
collected 24 h after injection of either Lp(a), apo(a) or F1, by
anti-apo(a) immunostained blots of reduced 4-12% gradient SDS-PAGE.
The urinary patterns of apo(a) after injection into NE
/
and control mice were comparable (Fig. 4B, lanes
2 and 3) and showed a broad spectrum of
bands including those migrating at 110, 81, 57, and 33 kDa. In
contrast, the urine from MMP-12
/
mice was devoid of
bands (lane 4), even after concentrating the urine 3-fold
more than that of either the NE
/
or control mice. In
turn, the electrophoretic pattern of the urine from all of the mice
injected with F1, contained fragments (lanes 6-8) that were
smaller in size than the injected material (lane 5) and
similar to those observed in NE
/
and control mice
injected with full-length apo(a) (lanes 2 and 3).
To rule out artifactual contributions to the formation of fragments,
the collected urine samples from injected mice were incubated at
37 °C overnight and compared with freshly collected urine kept on
ice. Western blot analysis showed no formation of new fragments. In
addition, no new fragments were observed when the urine of a control
uninjected mouse was incubated with intact Lp(a) or its derivatives.
These results provide evidence that F1 is the component of apo(a)
targeted for renal excretion and that in our mouse model, this fragment
is generated by the action of MMP-12 on apo(a).
Studies in Human Plasma and Urine--
All subjects studied
exhibited similar results in terms of electrophoretic band patterns. A
representative fresh plasma sample is shown in Fig. 4A,
lane Hu. In addition to intact apo(a), there were at least
three other bands, one of which corresponded to the mobility of F1. In
terms of urine, the pattern of apo(a) fragments (Fig. 4B,
lane Hu) was comparable with those of controls and
NE
/
mice injected with apo(a) or Lp(a) and to all mice
injected with fragment F1.
 |
DISCUSSION |
Our studies have shown that, in vitro, MMP-12 cleaves
human apo(a) at the Asn3518-Val3519 bond
located in the linker between kringles IV-4 and IV-5. This bond is
immediately upstream of the Ile3520-Leu3521
bond that we previously showed to be the site of cleavage by either
pancreatic or leukocyte elastase (4, 5). The reason for the high
substrate specificity by the elastases and MMP-12 is unclear. One
plausible explanation is that the linker between kringles IV-4 and
IV-5, contrary to the other linkers of apo(a), is unglycosylated as
predicted on the basis of sequence analysis (25). It is of note that
the proximity of the bond sites cleaved by NE and MMP-12, results in
the generation of two main fragments of a nearly equal size.
One of the interesting aspects of our current studies was the
observation that Lp(a)/apo(a)-injected MMP-12
/
,
contrary to the NE
/
mice, had virtually no
immunodetectable fragments in their plasma and none in their urine.
Yet, the MMP-12
/
mice were able to excrete in the urine
intravenously injected F1. These observations when coupled with the
finding that MMP-12 was able to cleave in vitro, apo(a),
strongly suggest that in the mouse, this enzyme was responsible for the
generation of F1 from injected human apo(a). The results also suggest
that the kidney has a recognition site for free F1 but not when it is a component of a full-length apo(a). The reason for the selective recognition of F1 by the kidney is unclear. Both size (about 200 kDa)
and high degree of glycosylation (about 30% by weight) of F1 speak
against a simple filtration process as suggested by Mooser et
al. (3). A receptor mechanism is a more likely possibility. However, our knowledge about receptor(s) that specifically recognize Lp(a)/apo(a) is not firmly established. An involvement by the VLDL
receptor in the recognition of apo(a) has been recently suggested (26).
Based on the current findings, it would be of interest to determine
whether F1 is a ligand for this receptor. It is important to note that,
in all of the animals injected with either Lp(a)/apo(a) or F1, the
urinary fragments, if present, were comparable in size and smaller than
those in the plasma, suggesting that in our mouse strain neither NE nor
MMP-12 was involved in the second step degradation by the kidney
perhaps by additional MMPs. In this regard MMP-2 and 9 have been
reported in the renal tissue (27, 28).
A question that was not specifically addressed in the current studies
was the site(s) of the first step of apo(a) fragmentation generating
F1. The plasma appears to be an unlikely source based on the findings
by Mooser et al. (2, 3) and our own (4, 5) showing that
neither mouse nor human plasma has the capacity to cleave, in
vitro, Lp(a)/apo(a). Moreover, any MMP-12 activity in the plasma
would be neutralized by circulating tissue inhibitors of MMP, at least
under physiological conditions. A more likely possibility is that the
apo(a) fragments are generated at tissue sites by the action of MMPs
secreted by macrophages unopposed by local tissue inhibitors of MMP.
Because F1 and F2 are generated in vitro by the action of
both NE (5) and MMP-12 on apo(a), one has to ask why only F1 was
present in the plasma of the NE
/
and control mice
injected intravenously with apo(a). This may be because of the fact
that F2 but not F1 binds to matrix macromolecules such as fibrinogen
and fibronectin (4) and as exemplified by our recent findings on the
proteoglycan decorin (29). On the other hand, F1, for which no ligand
has been identified thus far (30), once formed at tissue sites would be
free to diffuse and return to the blood stream by a reverse flow process.
The current studies invite the question as to whether the results on
the murine models may apply to man. It is of interest to note that
mouse MMP-12 has a high degree of homology with its human counterpart
(16). Moreover, the apo(a) fragments that are spontaneously present in
the plasma of human subjects are of a similar size as those found in
the plasma of mice injected with human apo(a) (2-4) and also as shown
by the current results in Fig. 4. In addition, the urine of human
subjects has been shown previously (2-4) and in this study to contain
apo(a) immunoreactive fragments which are smaller in size than those in
the plasma, suggesting that the human kidney is involved in the second
step of degradation of apo(a). However, despite these similarities more
direct documentation is needed to establish that MMP-12, or other MMPs,
are involved in the fragmentation of apo(a) in human subjects.
In summary, our results have shown that MMP-12 cleaves apo(a) in the
linker region between kringles IV-4 and IV-5 and also provide evidence
from mouse models that such a cleavage may account for the generation
in the plasma of F1, which is subsequently degraded by the kidney. The
emerging notion that the generation of F1 may be controlled by the
activity of MMP-12, suggests that the activity of this enzyme may play
an important role in the pathobiology of Lp(a).