© 1998 American Heart Association, Inc.
Original Contribution |
Factor XIIIa Cross-links Lipoprotein(a) With Fibrinogen and Is Present in Human Atherosclerotic Lesions
From the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.
Correspondence to Dr Anne M. Romanic, Department of Cardiovascular Pharmacology, UW2510, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, King of Prussia, PA 19406. E-mail anne_romanic-1{at}sbphrd.com
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Abstract—During the development of atherosclerotic lesions, lipoprotein(a) [Lp(a)], a highly atherogenic lipoprotein, accumulates within fibrin clots attached to blood vessel walls. As Lp(a) accumulates within the fibrin clot with time, fatty streaks are formed that develop into occlusive atherosclerotic plaques. It is not understood, however, which mechanisms are involved in the binding of Lp(a) to fibrin and, hence, the stable incorporation of Lp(a) into the fibrin clot. The results of the present study demonstrate that factor XIIIa, a transglutaminase that catalyzes the formation of amide bonds between endo-
-glutaminyl and endo-
-lysyl
residues of proteins, is capable of cross-linking Lp(a) to fibrinogen,
the soluble precursor of fibrin. Biochemical assays were conducted to
demonstrate that factor XIIIa cross-links Lp(a) with fibrinogen in a
time- and concentration-dependent manner. Additionally,
immunohistochemical studies revealed that factor XIII protein
expression colocalizes with Lp(a) expression in human atherosclerotic
plaques. It is proposed that factor XIIIa–mediated cross-linking of
Lp(a) to fibrin effectively increases the local concentration of Lp(a)
within a fibrin clot. The accumulation of Lp(a) within the blood vessel
promotes an antifibrinolytic environment, foam cell formation, the
generation of a fatty streak, and an increase in smooth muscle cell
content, all of which may contribute to the pathogenesis of
atherosclerosis.
Key Words: atherosclerosis • fibrin • fibrinogen • lipoprotein • lesion
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Lipoprotein(a) [Lp(a)] is a highly atherogenic lipoprotein complex formed of apolipoprotein(a) [apo(a)] that is disulfide-bound to the apolipoprotein B-100 moiety of LDL. It has been reported that Lp(a) binds fibrin,1 which becomes deposited within the blood vessel wall in fibrin clots. Furthermore, it has been suggested that Lp(a) accumulates within the vessel wall and has the potential to contribute to the development of occlusive atherosclerotic plaques with time.2 Individuals who develop occlusive atherosclerosis suffer from coronary heart disease, and an elevated plasma Lp(a) level has been shown to be an independent risk factor for the development of premature coronary heart disease.3 Other vascular diseases associated with elevated plasma Lp(a) include restenosis and stroke.4 5 The deposition of Lp(a) within the fibrin clot is believed to be a contributing factor in atherogenesis.4 5 However, the actual mechanism by which Lp(a) binds fibrin and becomes stably incorporated into a fibrin clot remains unknown.
Factor XIII (EC 2.3.2.13) is a transglutaminase that catalyzes
the final step in the coagulation cascade.6 7
This enzyme exists as a zymogen and is present in 2 molecular
forms. The tissue form of factor XIII, identified with platelets,
macrophages, and the placenta, is a dimer that is composed of 2
identical A chains (A2). The plasma form is a
tetramer consisting of 2 A subunits and 2 B subunits
(A2B2). Activation of
factor XIII to factor XIIIa is mediated by thrombin and is initiated by
proteolytic removal of an amino-terminal propeptide followed by a
calcium-dependent conformational change that exposes a cysteine residue
in the active site. Activation also leads to dissociation of the
A2 and B2 subunits of
plasma factor XIII, at which point the tissue and plasma forms of
factor XIIIa are identical. Factor XIIIa, also referred to as plasma
transglutaminase, fibrinoligase, and fibrin-stabilizing factor, is a
calcium-dependent thiol enzyme that catalyzes the formation of amide
bonds between endo--glutaminyl and endo--lysyl residues of
proteins.
Factor XIIIa is noted primarily for its participation in the
coagulation cascade, where it covalently cross-links fibrin monomers
and converts soft fibrin clots into hard
clots.6 7 Factor XIIIa is also capable of
cross-linking 
2-antiplasmin and extracellular
matrix proteins such as fibronectin, vitronectin, and
collagen to fibrin, thus rendering the clot more resistant to
lysis and anchoring the clot to the blood vessel wall, respectively.
Furthermore, factor XIIIa has been demonstrated to cross-link primary
amines to the apo(a) moiety of Lp(a),8 suggesting
a role for factor XIIIa in atherosclerosis. Also,
elevated plasma levels of factor XIIIa have been observed in patients
with obliterative
atherosclerosis.9 To examine the
possibility that factor XIIIa plays a role in Lp(a) deposition into
fibrin clots in the development of atherosclerosis, we
conducted studies to directly demonstrate that factor XIIIa is capable
of cross-linking Lp(a) to fibrinogen, the soluble precursor of fibrin.
Additionally, immunohistochemistry directly demonstrated that factor
XIII and Lp(a) expression was identified in human atherosclerotic
lesions.
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Factor XIIIa–Mediated Cross-linking of Lp(a) With Fibrinogen: Determination by Immunoprecipitation With Anti-Fibrinogen Antibody Followed by Western Blotting With Lp(a) Antibody
Factor XIIIa was incubated with Lp(a) and fibrinogen in solution and then analyzed for Lp(a)-fibrinogen cross-linking over a time course ranging from 1 to 6 hours (n=5 per time point). In brief, purified human fibrinogen (Calbiochem) at a final concentration of 100 µg/mL was incubated with purified human Lp(a) (Sigma Chemical Co) at a final concentration of 500 µg/mL [based on the protein content of Lp(a)] in the presence of purified human factor XIIIa (Enzyme Research Laboratories) at 30 U/mL. The approximate molecular weight of Lp(a) was reported by the manufacturer to be 1 000 000, in agreement with a previous report,10 and was purified to homogeneity from fresh, nonfrozen human plasma pooled from several hundred donors. Since the Lp(a) was purified from plasma obtained from multiple donors, it can be assumed that the Lp(a) used for these studies is a mixture containing the various apo(a) isoforms and is representative of the general population.10 The chemical composition of the Lp(a) was reported to be
31% protein and 69% lipid, based on
earlier studies.11 It should be noted that the
concentrations of Lp(a) and factor XIIIa used for these studies are
within the physiological range detected in human
plasma. Also, since fibrin will spontaneously form a soft clot and
potentially trap Lp(a) within it to yield a false-positive result,
fibrinogen, the soluble precursor of fibrin, was used for these
studies. The reactions were conducted at 37°C in a buffer consisting
of 40 mmol/L Tris, 0.15 mol/L NaCl, 5 mmol/L DTT, and 10
mmol/L CaCl2, pH 8.3. Factor XIII was
preactivated to factor XIIIa immediately before each experiment
by incubating factor XIII with thrombin at 3 U/mL in 40 mmol/L
Tris and 0.15 mol/L NaCl, pH 8.3, for 1 hour at room temperature. The
activation procedure was stopped by adding hirudin at 100 U/mL (Sigma)
to inhibit thrombin. At the end of each time point, the cross-linking
reaction was terminated by adding EDTA to a final concentration of
25 mmol/L. As controls, factor XIIIa was either omitted from the
reaction (n=5) or 10 mmol/L EDTA was added in the presence of
factor XIIIa to inhibit factor XIIIa activity (n=5). As additional
controls, unactivated factor XIII was analyzed in the
reaction (n=3), and thrombin plus hirudin, in the absence of factor
XIII, was analyzed (n=2). Additional studies were conducted to
study the degree of cross-linking to fibrinogen when increasing amounts
of Lp(a) were added to the reaction (n=3 per concentration). Lp(a) was
incubated with fibrinogen in the presence or absence of factor XIIIa as
described above; however, the final concentrations of Lp(a) were
adjusted to range from 200 to 800 µg/mL [based on the protein
content of Lp(a)].
Covalently cross-linked complexes that formed between fibrinogen
and Lp(a) were isolated by immunoprecipitation. A polyclonal rabbit
anti-human fibrinogen antibody directed against the -, ß-, and
-chains of human fibrinogen (Calbiochem) was added to the reaction
mixture to a final concentration of 5 µg/mL and then incubated at
4°C for 18 hours while being rotated. Protein A–Sepharose beads (20
mg/mL, Pharmacia) were then added to the samples and incubated for an
additional 4 hours at 4°C while being rotated. The Lp(a)-fibrinogen
complexes, bound by the beads, were then subjected to a series of
washes with 1%, 0.5%, and 0.05% Triton X-100 in PBS. The beads were
pelleted between each wash by centrifugation at
14 000g, and the final pellet was resuspended in Laemmli
buffer containing 62.5 mmol/L Tris, pH 6.8, 2% SDS, 5% glycerol,
0.7 mol/L 2-mercaptoethanol, and 0.025% bromophenol blue and then
heated at 100°C for 3 minutes. To identify the presence of Lp(a)
within the immunoprecipitated complexes, samples were analyzed
by Western blotting with the use of an antibody to Lp(a). In brief, the
samples (30 µL each, 60% of the pelleted material) were subjected
to electrophoresis through a 4% to 20% polyacrylamide
gradient gel (Bio-Rad)12 and then transferred to
a nitrocellulose membrane by using a Bio-Rad semidry transfer
apparatus according to the manufacturer's instructions.
Unoccupied binding sites were blocked overnight at 4°C with 5%
nonfat powdered milk in a 0.1 mol/L Tris-HCl buffer, pH 8.0, containing
1.5 mol/L NaCl and 0.5% Triton X-100 (TBST buffer). A polyclonal sheep
anti-human Lp(a) primary antibody that recognizes the apo(a) component
of Lp(a) (Enzyme Research Laboratories), diluted in TBST to 10 µg/mL,
was then added to the membrane and allowed to incubate for 1 hour at
25°C. The membrane was washed 3 times for 20 minutes each with TBST
and then incubated for 30 minutes with a donkey anti-sheep IgG
secondary antibody conjugated to horseradish peroxidase (Sigma) diluted
1:5000 in TBST. The membrane was washed as detailed above, and the blot
was developed with the enhanced chemiluminescence method (Amersham)
according to the manufacturer's instructions. The intensity levels of
each band relative to background were determined and quantified with a
Molecular Dynamics densitometer.
Factor XIIIa–Mediated Cross-linking of Lp(a) With Fibrinogen:
Determination by ELISA-Based Assay
Microtiter ELISA plates (No. 25801, Corning) were coated with
purified human fibrinogen (American Diagnostica) at a
concentration of 80 µg/mL, 100 µL per well, for 40 minutes at room
temperature. Unoccupied binding sites were then blocked for 1 hour with
1% BSA in buffer A (40 mmol/L Tris and 0.15 mol/L NaCl, pH 8.3).
Lp(a) (Sigma) was added to the wells to a final concentration of 250,
375, or 500 µg/mL [based on the protein content of Lp(a)] in buffer
A containing 10 mmol/L CaCl2 and 5
mmol/L DTT (n=6). Factor XIIIa (Enzyme Research Laboratories),
preactivated with thrombin as described above, was then added
to the wells for a final activity of 30 U/mL. The reactions were
allowed to proceed for 2 hours at room temperature and then stopped by
the addition of EDTA to a final concentration of 15 mmol/L. The
wells were then washed 4 times with ELISA wash solution consisting of
0.002 mol/L imidazole-buffered saline (pH 7.4) and 0.02% Tween 20
(Kirkegaard and Perry).
To determine whether factor XIIIa had cross-linked Lp(a) to the immobilized fibrinogen, a sheep anti-Lp(a) antibody (Enzyme Research Laboratories), diluted to 10 µg/mL in ELISA wash solution, was added to the wells and allowed to incubate for 18 hours at 4°C. The wells were washed as described above, and then a biotinylated anti-sheep IgG antibody (Vector Laboratories), diluted to 10 µg/mL in ELISA wash solution, was added and allowed to incubate for 1 hour at room temperature. The wells were washed as described above, after which time 150 µL of streptavidin–ß-galactosidase (GIBCO/BRL), diluted to 0.35 µg/mL in ELISA wash solution, was added for 30 minutes at room temperature. The wells were washed once with ELISA wash solution, and then 150 µL of p-nitrophenyl-ß-D-galactopyranoside (Sigma) at 1 mg/mL was added. After 30 minutes of incubation at room temperature, the reaction was stopped, and the resulting color product was enhanced by adding 20 µL of 1N NaOH to each well. Absorbance was then read at a wavelength of 405 nm on a SPECTRAmax 250 microplate spectrophotometer (Molecular Devices).
Immunohistochemistry
Sections of human coronary arteries with moderate to
advanced atherosclerotic lesions were obtained from the
cardiovascular tissue bank at the University of
Washington, Seattle. The tissue samples (n=3) were obtained from
diseased hearts of cardiac transplant recipients. Tissues were
collected within 30 to 60 minutes after cardiac excision and placed
into neutral buffered formalin. After overnight fixation, the samples
were routinely processed and paraffin-embedded for histology.
Hematoxylin and eosin–stained sections were reviewed and used to
select samples for immunohistochemistry. The ages of the donors, all
male, were 51, 54, and 63 years. Sections were prepared for
immunoperoxidase staining by using the Vectastain ABC kit (Vector
Laboratories) according to the manufacturer's instructions. In brief,
endogenous peroxidase was quenched with 0.3%
H2O2 in methanol for 30
minutes. Nonspecific immunoglobulin binding sites were blocked with
normal rabbit serum for 1 hour, and then the sections were incubated
with a sheep anti–factor XIII primary antibody (2.5 µg/mL, Enzyme
Research Laboratories) or a sheep anti-Lp(a) primary antibody generated
against the apo(a) moiety of Lp(a) (2.5 µg/mL, Enzyme Research
Laboratories) for 1 hour at room temperature. As a control, serial
sections were incubated with sheep IgG (2.5 µg/mL, Sigma) instead of
the primary antibody. The sections were then incubated for 30 minutes
with a biotinylated rabbit anti-sheep IgG secondary antibody (7.5
µg/mL, Vector Laboratories) followed by 30 minutes of incubation with
the Vectastain Elite ABC reagent solution. Immunoglobulin complexes
were visualized on incubation with 3,3'-diaminobenzidine (Vector
Laboratories) at 0.5 mg/mL in 50 mmol/L Tris-HCl, pH 7.4, and 3%
H2O2. Sections were washed,
counterstained with Gill's hematoxylin, cleared, mounted with
Aquamount (Polysciences), and then examined by light
microscopy.
Statistics
Data were expressed as mean±SEM. For statistical
analysis of factor XIIIa–mediated cross-linking of Lp(a) with
fibrinogen as determined by the ELISA-based method, the t
test for unpaired data was used. Statistical significance was accepted
when P<0.05.
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Results |
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Factor XIIIa Is Capable of Cross-linking Lp(a) With Fibrinogen
To determine whether factor XIIIa cross-links Lp(a) with fibrinogen, Lp(a) and fibrinogen were incubated in solution with or without factor XIIIa (n=5). Complexes that formed between Lp(a) and fibrinogen were then immunoprecipitated with an antibody directed against fibrinogen. Those complexes were subjected to electrophoresis and then analyzed for the incorporation of Lp(a) by Western blotting with an antibody directed against Lp(a). The results demonstrated that in the presence of factor XIIIa, increasing amounts of Lp(a) became cross-linked with fibrinogen over a time course ranging from 1 to 6 hours (Figure 1
). After 6
hours, the degree of cross-linking between Lp(a) and fibrinogen when
factor XIIIa was present had increased 10.82±2.33-fold (Figure 1, bottom). When factor XIIIa was omitted from the reaction, only a
negligible amount of Lp(a) was detected in the immunoprecipitated
material, which was comparable to the amounts detected at the zero time
points (Figure 1, top and bottom). Also, when unactivated
factor XIII was added to the reaction (n=3), only a small amount of
Lp(a) was detected in the immunoprecipitated material, indicative of a
nonspecific factor XIIIa–independent interaction between Lp(a) and
fibrinogen (Figure 1, top). Similar results were generated when EDTA
was added to the reaction to inhibit factor XIIIa (data not shown, n=5)
and when only thrombin plus hirudin was added (data not shown, n=2). In
additional experiments, Lp(a) was incubated at concentrations ranging
from 200 to 800 µg/mL [based on the protein content of Lp(a)] with
fibrinogen and factor XIIIa for 6 hours (n=3). The results showed that
Lp(a) became cross-linked to fibrinogen in a concentration-dependent
fashion (Figure 2). When factor XIIIa was
not included in the reaction, Lp(a) was essentially undetected (Figure 2). These results indicate that factor XIIIa mediates cross-linking
between Lp(a) and fibrinogen. Also, with increasing amounts of Lp(a)
added to the reaction, there appeared to be complexes formed of varying
molecular weight. This was evident as a "tail" that existed beneath
each band that had been detected by Western blotting (Figure 2, top).
With increasing amounts of Lp(a) present, the complexes appeared to
be larger; hence, the "tails" became shorter. These results suggest
that there are multiple Lp(a) binding sites on each fibrinogen
molecule.
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Factor XIIIa–mediated cross-linking of Lp(a) to fibrinogen was
also analyzed by an ELISA-based assay (n=6). The results
demonstrated that during a 2-hour incubation period in the presence of
factor XIIIa, Lp(a) became cross-linked to the fibrinogen
immobilized on the plate in a concentration-dependent
manner (Figure 3). It should be noted,
though, that in the absence of factor XIIIa, a measurable amount of
Lp(a) nonspecifically adhered to the fibrinogen-coated plate (Figure 3), and this background signal modestly increased with time.
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Factor XIII and Lp(a) Expression Is Detected in Human
Atherosclerotic Lesions
To determine whether factor XIII and Lp(a) were expressed within
an atherosclerotic lesion, immunohistochemistry was performed. The
expression of factor XIII and Lp(a) was investigated in serial sections
prepared from human coronary arteries containing
atherosclerotic lesions (n=3). The results showed that factor XIII and
Lp(a) were expressed in a similar region within the atherosclerotic
lesions (Figure 4), predominantly in the
core of the lesions. Notably, the intensity of the immunoreactive
product was greater for the Lp(a)-immunostained samples
than for the factor XIII–immunostained samples (Figure 4A and 4B, respectively). Also, additional Lp(a) staining was detected in
some regions devoid of factor XIII immunoreactivity and vice versa. A
serial section that was incubated with sheep IgG instead of factor XIII
or Lp(a) primary antibodies was negative (Figure 4C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the development of atherosclerotic lesions, Lp(a) becomes deposited into fibrin clots within the blood vessel wall and has the potential to contribute to atherogenesis.1 2 Factor XIIIa, a transglutaminase that catalyzes the formation of amide bonds between the endo-
-glutaminyl and endo--lysyl residues of proteins, plays
a major role in the cross-linking of fibrin monomers for the generation
of fibrin clots and arterial
thrombi.6 9 It has also been shown that factor
XIIIa is able to cross-link primary amines to
Lp(a).8 Since the mechanism by which Lp(a)
actually becomes incorporated into the fibrin clot is poorly
understood, we propose the hypothesis that factor XIIIa is capable of
catalyzing covalent -glutamyl--lysine bonds between Lp(a) and
fibrin and contributes to the development of atherosclerotic lesions.
Our results have demonstrated for the first time that in vitro, factor
XIIIa is indeed able to catalyze the formation of complexes containing
Lp(a) and fibrinogen. Over time, increasing amounts of Lp(a)
become complexed with fibrinogen. Additionally, Lp(a) formed complexes
with fibrinogen in a concentration-dependent manner, possibly with
multiple Lp(a) molecules bound to each fibrinogen molecule.
Furthermore, we have demonstrated that factor XIII and Lp(a) expression
is present in human atherosclerotic lesions. The pathophysiological role of Lp(a) in the development of vascular diseases such as atherosclerosis, myocardial ischemia, restenosis, and stroke may lie in the ability of Lp(a) to (1) become deposited within fibrin clots on vessel walls, (2) interfere with fibrinolysis, (3) enhance cholesterol accumulation in macrophages to result in foam cell formation, and (4) promote smooth muscle cell proliferation and migration. For example, the incorporation of Lp(a) onto the vessel wall potentiates the thrombotic events that occur in the development of atherosclerotic lesions.2 13 The apo(a) moiety of Lp(a) has been shown to have high homology with plasminogen,14 15 the zymogen precursor of the fibrinolytic enzyme plasmin. Owing to the high homology between the apo(a) component of Lp(a) and plasminogen, it has been demonstrated that Lp(a) competes with plasminogen for binding to endothelial cells and monocytes.2 16 17 These studies suggest that in pathological conditions such as atherosclerosis, in which there is an increased amount of circulating Lp(a), Lp(a) prevents plasmin(ogen) from binding to the vessel wall and interferes with fibrinolysis of the thrombotic lesion.2 13
Furthermore, modified Lp(a) has been demonstrated to become bound18 and internalized19 by macrophages to generate lipid-laden foam cells. In addition to promoting foam cell formation and the generation of fatty streaks, Lp(a) has been demonstrated to induce smooth muscle cell proliferation20 and migration.21 Increased smooth muscle cell content is characteristic of atherosclerotic as well as restenotic lesions. Investigators have also shown that the effects of Lp(a) on smooth muscle cells are due to the fact that Lp(a) inhibits plasmin-mediated activation of transforming growth factor-ß, a potent inhibitor of smooth muscle cell growth and migration.22 23 24
In conclusion, we propose that factor XIIIa–mediated cross-linking of Lp(a) to fibrin effectively increases the local concentration of Lp(a) within a fibrin clot that is sequestered within the vessel wall. The accumulation of Lp(a) within the vessel wall promotes an antifibrinloytic environment, foam cell formation, and an increase in smooth muscle cell content, all of which contribute to the pathogenesis of atherosclerosis.
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Acknowledgments |
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The authors would like to thank Drs Daniel Veber and Robert Marquis for their helpful discussions in conducting this work. We would also like to thank Drs Kevin O'Brien and Charles Alpers for their work in maintaining the cardiovascular tissue bank at the University of Washington, Seattle.
Received December 12, 1997; accepted April 15, 1998.
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R. Klose, F. Fresser, S. Kochl, W. Parson, A. Kapetanopoulos, J. Fruchart-Najib, G. Baier, and G. Utermann Mapping of a Minimal Apolipoprotein(a) Interaction Motif Conserved in Fibrin(ogen) beta - and gamma -Chains J. Biol. Chem., December 1, 2000; 275(49): 38206 - 38212. [Abstract] [Full Text] [PDF]
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