(Received for publication, February 19, 1997, and in revised form, April 1, 1997)
From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Elevated plasma fibrinogen levels are a major
risk factor for thrombosis. This report shows two mechanisms by which
fibrinogen can affect the fibrinolysis rate in vitro and
thus may lead to thrombosis. First, the lysis rate of fibrin decreases
as the initial concentration of fibrinogen increases. Second, a minor
variant form of fibrinogen decreases the rate of fibrinolysis. This
variant, A/
fibrinogen, has one altered
chain and is known
to bind to factor XIII zymogen. In a fibrinolysis assay containing
purified thrombin, fibrinogen, tissue-type plasminogen activator, and
plasminogen, clots from
A/
A and
A/
fibrinogen lysed at
similar rates. However, when factor XIII was added, slower lysis was
seen in
A/
fibrin clots when compared with
A/
A fibrin
clots. A D-dimer agglutination assay showed that the
A/
clots
were more highly cross-linked than the
A/
A clots. The lysis rates
of
A/
clots were similar to
A/
A clots in the presence of
N-ethylmaleimide, a specific inhibitor of factor XIIIa. The
A/
fibrin clots made in the presence of factor XIII showed
increased proteolytic resistance to both plasmin and trypsin. Clots
made from afibrinogenemic plasma reconstituted with
A/
fibrinogen also showed significant resistance to lysis compared with
A/
A fibrinogen. These data demonstrate
A/
fibrin is
resistant to fibrinolysis, possibly as a result of concentrating factor
XIII on the clot. The total fibrinogen concentration and the amount of
A/
fibrinogen increase clot stability in vitro and
thus may contribute independently to the risk of thrombosis in
humans.
Fibrinogen is a 340,000-Da dimeric glycoprotein composed of three
polypeptide chains, (Mr = 65,000),
(Mr = 56,000), and
(Mr = 47,000) linked by disulfide bonds.
Fibrinogen is converted to fibrin through limited proteolysis by
thrombin, which exposes polymerization sites in fibrinogen (1). The
fibrin monomers spontaneously associate with each other to form the
weblike fibrin clot (2). A plasma transglutaminase, factor XIII,
strengthens the fibrin clot by forming covalent bonds between adjacent
fibrin monomers (3).
Plasma factor XIII is a 320,000-Da tetrameric protein composed of two
polypeptide a chains (Mr = 83,000)
and two polypeptide b chains (Mr = 80,000) (4). Factor XIII normally circulates as an inactive proenzyme
until it is activated by thrombin cleavage of a 4000-Da activation
peptide from each a subunit, which is followed by the
dissociation of the b subunits. Activated factor XIII, or
XIIIa, catalyzes the formation of -glutamyl-
-lysine bonds between
polypeptide chains in fibrin (5). These cross-links strengthen the
fibrin clot (6) and increase its resistance to lysis (7-9).
Recent evidence has revealed an association between a variant form of
fibrinogen and factor XIII that may play a role in modulating the
stability of a fibrin clot. This fibrinogen variant, referred to as
A/
fibrinogen, peak II fibrinogen (10),
A-
B fibrinogen (11), or F
50, 57.5 (12), contains an altered
chain
termed
(13),
B (14), or
57.5 (12), and comprises
approximately 7-15% of the total fibrinogen found in plasma (10). The
chain arises from alternative processing of the
chain mRNA
(15, 16) that leads to the translation of a polypeptide with a 20-amino
acid sequence substituted for the carboxyl-terminal four amino acids of
the
chain. The physiological function of the
chain remains
unclear; however, recent studies have shown that the
chain binds
directly to the zymogen form of factor XIII (17). This suggests that
the
chain of fibrinogen may serve as a carrier for factor XIII to
increase the local concentration of factor XIII at the fibrin clot.
Elevated fibrinogen levels are a major risk factor for thrombosis.
Several mechanisms have been proposed to explain the correlation between fibrinogen levels and thrombosis (18, 19). Fibrinogen may
contribute to thrombosis by its role in atherosclerotic plaque formation. Fibrinogen binds to platelet membranes through glycoprotein IIb-IIIa (integrin IIb
3) and thereby
mediates platelet aggregation (20, 21). Fibrin degradation products
stimulate the proliferation of vascular smooth muscle cells (22, 23),
which may contribute to narrowing of blood vessels during
atherosclerosis. In addition, once fibrinogen is incorporated into the
atherosclerotic plaque, it binds low density lipoprotein and sequesters
more fibrinogen (18), thus enhancing plaque formation. Another
mechanism by which fibrinogen may lead to thrombotic disorders is by
increasing blood viscosity. Increased blood viscosity favors the
hypercoagulable state (24). Furthermore, increased fibrinogen levels
lead to increased fibrin deposition (25-27). However, the causal role
of these potential factors in thrombosis is still unclear.
The present report demonstrates that increases in total fibrinogen
concentration result in increased clot stability toward fibrinolysis
in vitro, suggesting a mechanistic explanation of how
elevated fibrinogen levels per se may cause an increased
risk of thrombosis. In addition, clots made from A/
fibrinogen
are more stable to fibrinolysis in vitro than clots made
from
A/
A fibrinogen. Taken together, these findings suggest that
both the total amount of fibrinogen and the amount of
A/
fibrinogen in plasma may be independent risk factors for
thrombosis.
Plasminogen-free human plasma fibrinogen (Calbiochem)
was dissolved in 39 mM Tris-PO4, pH 8.6, containing 5 mM -aminocaproic acid
(EACA)1 and 0.2 mM
phenylmethylsulfonyl fluoride (PMSF) and dialyzed exhaustively into the
same buffer at 4 °C. Insoluble residue was removed by centrifugation
at 10,000 × g for 30 min at 4 °C. The
A/
A and
A/
forms of fibrinogen were separated using DEAE-cellulose as
described previously (28). Briefly, the fibrinogen solution was
adsorbed to a column of DEAE-cellulose (6 × 20 cm) and eluted with a 1200-ml exponential gradient generated in a 600-ml constant volume mixing chamber from the starting buffer (39 mM
Tris-PO4, pH 8.6, 5 mM EACA, 0.2 mM
PMSF) to the final buffer (193 mM Tris-PO4, pH
4.6, 5 mM EACA, 0.2 mM PMSF). The absorbance
was monitored at 280 nm, and 11-ml fractions were collected. The
elution profile showed two peaks;
A/
A fibrinogen composed the
first peak and
A/
fibrinogen composed the second smaller peak
(data not shown).
In some experiments, A/
A and
A/
fibrinogen were further
purified using a
glycine-L-proline-L-arginine-L-proline-L-cysteine (GPRPC)-agarose affinity resin (29). Briefly, the resin was prepared by
reacting 10 mg of
glycine-L-proline-L-arginine-L-proline-L-cysteine peptide (Howard Hughes Medical Institute Biopolymer Laboratory, Seattle, WA) with 10 ml of 5-thio-2-nitrobenzoate-agarose (Pierce) according to the manufacturer's protocol. The dialyzed
A/
A or
A/
fibrinogen pool from DEAE-cellulose was adsorbed to a column (3 ml) of GPRPC-agarose, washed with 100 mM NaCl, 50 mM Tris-PO4, pH 7.8, 5 mM EACA, 0.2 mM PMSF, and then washed with 2 M NaBr, 50 mM Tris-PO4, pH 7.8, 5 mM EACA, 0.2 mM PMSF. The fibrinogen was eluted in 1-ml fractions with 2 M NaBr, 20 mM citrate, pH 5.3, 5 mM
EACA, 0.2 mM PMSF and immediately neutralized with 0.02 volume of 2 M Tris-HCl, pH 8.0. The fibrinogen fractions
were dialyzed into 137 mM NaCl, 2.7 mM KCl, 10 mM HEPES, pH 7.4, 1 mM CaCl2 and
stored at
70 °C.
Microtiter
plate fibrinolysis assays were carried out as described previously by
Jones and Meunier (30) using 96-well assay plates (Corning 25-880-96).
Fibrinogen and Lys-plasminogen (Calbiochem) were added to an interim
mixing plate containing assay buffer (0.1 M NaCl, 30 mM NaHCO3, 4 mM KCl, 1 mM CaCl2, 1 mM
Na2HPO4, 0.3 mM MgCl2,
0.4 mM MgSO4, 10 mM HEPES, pH 7.4, 0.01% Polysorbate 80). A separate assay plate contained -thrombin
(a generous gift from Dr. Walter J. Kisiel, University of New Mexico)
and tissue plasminogen activator (Calbiochem) in assay buffer. The
fibrinogen/plasminogen solution was then dispensed from the interim
plate into the assay plate wells containing thrombin and tissue
plasminogen activator. The final concentrations of reagents were 1.25 mg/ml fibrinogen (unless stated otherwise), 30 µg/ml Lys-plasminogen,
16 ng/ml tissue plasminogen activator, and 13.2 NIH units/ml thrombin
in a total volume of 100 µl. For assays containing factor XIII (a generous gift from Drs. Michael W. Mosesson and David A. Meh, University of Wisconsin Medical School, Milwaukee Clinical Campus), factor XIII was added to a final concentration of either 10 or 100 µg/ml to the wells in the interim plate containing the
plasminogen/fibrinogen mixture. In some assays 1 mM
N-ethylmaleimide was also added to the interim plate. The
turbidity of the clot was measured at room temperature every 6 min at
405 nm. The optical density was converted to percent lysis as
follows,
![]() |
(Eq. 1) |
A D-dimer agglutination assay kit (Sigma) was
used to measure D-dimer content in lysed fibrin clots. Clots were
formed from A/
A or
A/
fibrinogen using 1.0 mg/ml
fibrinogen, 13.2 NIH units/ml
-thrombin, 16 ng/ml tissue-type
plasminogen activator, 30 µg/ml Lys-plasminogen, and 10 µg/ml
factor XIII and allowed to lyse completely. After lysis, 50 µl of the
lysed clots were serially diluted in the manufacturer's proprietary
buffered saline solution, pH 7.3, containing 0.02% sodium azide. 20 µl of the diluted sample were then mixed with 20 µl of a suspension
of latex beads coated with mouse monoclonal antibody (MA-8D3) specific for the fibrin D-dimer on the test card. The test card was rocked back
and forth, and agglutination was scored after 3 min on duplicate samples.
A/
A or
A/
fibrinogen and factor XIII were added to a 96-well assay plate
containing assay buffer (0.1 M NaCl, 30 mM
NaHCO3, 4 mM KCl, 1 mM
CaCl2, 1 mM Na2HPO4,
0.3 mM MgCl2, 0.4 mM
MgSO4, 10 mM HEPES, pH 7.4, 0.01% Polysorbate
80), and
-thrombin was added to the wells to initiate clotting. The
final concentrations of the reagents were 1.0 mg/ml fibrinogen, 10 µg/ml factor XIII, and 13.2 NIH units/ml
-thrombin in a total
volume of 100 µl. The clots were incubated at room temperature until
maximum turbidity was reached. When clotting was complete, 50 µl of
11.7 µg/ml trypsin were added to the clots. The turbidity of the
clots was measured every 5 min at 405 nm. The optical density was
converted to percent lysis as described above.
The A/
A and
A/
forms of fibrinogen were iodinated with Na125I (DuPont NEN)
according to the method of Fraker and Speck (31) to a specific activity
of 85,000-120,000 cpm/ng as described previously (29). The plasma
fibrinolysis assays were carried out using the method of Wun and
Capuano (32). Briefly, the iodinated
A/
A or
A/
fibrinogen
in 137 mM NaCl, 2.7 mM KCl, 1 mM
CaCl2, 10 mM HEPES, pH 7.4, was added 1:1 (v/v)
to fibrinogen-deficient human plasma (George King Biomedical) at a
final concentration of 1.25 mg/ml on ice. (The plasma contained less
than 0.1 mg/ml residual fibrinogen.) Clotting of the samples was
initiated with 1 NIH unit/ml
-thrombin and the clots were incubated
at 37 °C in the presence of 0.02% NaN3. Samples were
taken daily for 7 days and centrifuged for 5 min in a microcentrifuge
to pellet the insoluble clot. 20-µl aliquots of the supernatant were
counted in a gamma counter to measure the soluble
125I-fibrin degradation products. The percentage of clot
lysis was calculated using the following equation and plotted
versus time.
![]() |
(Eq. 2) |
Since high plasma fibrinogen levels predispose
individuals to a greater risk of thrombosis, we hypothesized that clots
made from high concentrations of fibrinogen may be more resistant to fibrinolysis. To test this hypothesis, fibrinolysis assays were performed using purified components, including fibrinogen, thrombin, tissue-type plasminogen activator, and plasminogen (30). Concentrations of fibrinogen from 1 to 4 mg/ml were used, and clotting was initiated by the addition of thrombin. The calculated lysis
t1/2 for clots formed from 1, 2, 3, and 4 mg/ml
total fibrinogen concentrations was 18.7 ± 0.1, 30.4 ± 0.7, 55.7 ± 0.6, and 70.3 ± 0.3 min, respectively (Fig.
1). The fibrinolysis half-times were essentially linear with respect to the initial fibrinogen concentrations (Fig. 1, inset). Similar results were seen in the absence of factor
XIII (data not shown). These results are consistent with other
published data (30) and demonstrate that fibrin clots made at high
fibrinogen concentrations are more resistant to lysis, suggesting a
mechanistic explanation for the role of fibrinogen as a risk factor for
thrombosis (33).
A second
potential risk factor for thrombosis is the amount of A/
fibrinogen in plasma. This form of fibrinogen has been shown to act as
a carrier protein for factor XIII by binding directly to factor XIII
via the
chain (17). We hypothesized that this binding may serve to
concentrate factor XIII locally at the surface of the growing fibrin
clot, thereby resulting in a more highly cross-linked and stabilized
clot. Fibrinolysis assays were therefore carried out using purified
A/
A or
A/
fibrinogen. As seen in Fig. 2,
both the rate of clotting and the rate of lysis were significantly
decreased in
A/
fibrin clots. Clots made from
A/
fibrinogen in the presence of factor XIII clotted more slowly and
subsequently lysed more slowly (Fig. 2, inset). Clots formed from
A/
A fibrinogen in the presence of physiological factor XIII
concentrations (10 µg/ml) showed a lysis t1/2 of
72 ± 0 min, whereas the clots formed from
A/
fibrinogen
had a t1/2 of 179 ± 4 min. Clot stability was
enhanced further in the presence of supraphysiological concentrations
of factor XIII. In experiments where the factor XIII concentration was
increased to 100 µg/ml, the
A/
A fibrinogen clots showed a lysis
t1/2 of 99 ± 1 min, while the clots formed
from
A/
fibrinogen had a t1/2 of 207 ± 8 min. These data suggest that factor XIII increases the clotting time as well as decreases the fibrinolysis rates of
A/
fibrin. In the
absence of added factor XIII, fibrin clots made from
A/
A and
A/
fibrinogen clotted and lysed at similar rates, with a lysis
t1/2 of 50 ± 1 min for
A/
A fibrinogen
and 54 ± 1 min for
A/
fibrinogen (Fig. 3),
demonstrating that the resistance of
A/
fibrin to fibrinolysis
is dependent on the presence of factor XIII. Furthermore, when factor
XIIIa was inhibited as it was formed during clotting by the prior
addition of N-ethylmaleimide, the fibrinolysis rates of
A/
fibrin clots were similar to clots made without factor XIII
(data not shown). These data show that active factor XIII is required
for the decreased fibrinolysis rate of the
A/
fibrin.
A D-dimer agglutination assay was performed to determine if the
A/
fibrin clots were more highly cross-linked than the
A/
A
fibrin clots. As seen in Table I, the lysed
A/
fibrin clots showed positive agglutination down to a dilution of 1:300, whereas the
A/
A fibrin clots showed positive agglutination down to a dilution of only 1:128. These data show that the
A/
fibrin clots are more highly cross-linked, possibly because of high local concentrations of factor XIII at the clot surface due to binding to the
chain.
|
It should be noted that these assays were carried out with A/
A
and
A/
fibrinogen that had been separated on DEAE-cellulose and
further purified by GPRPC-agarose chromatography. Factor XIII co-purifies with
A/
fibrinogen on DEAE-cellulose (17),
presumably by binding directly to the
chain in
A/
fibrinogen. We have verified that factor XIII co-purifies with
A/
fibrinogen on DEAE-cellulose, but is depleted from
A/
fibrinogen purified further on GPRPC-agarose (Fig. 4).
In experiments using fibrinogen purified only on DEAE-cellulose, the
A/
fibrin clots were resistant to lysis in the absence of added
factor XIII (Fig. 5), presumably due to the
contaminating factor XIII.
To ensure that the increased lysis resistance was not due to factor
XIII affecting the fibrinolytic system directly, A/
A and
A/
fibrin clots were formed with thrombin and then digested with
trypsin. The
A/
A fibrin clots had a lysis t1/2
of 5.7 ± 0.2 min, while the
A/
clots had a
t1/2 of 39.1 ± 1.8 min (Fig.
6). These data suggest that the increased fibrinolytic
resistance of the
A/
fibrin clots is due primarily to
proteolytic resistance, although inhibition of the fibrinolytic system
cannot be ruled out entirely.
Fibrinolysis assays were also carried out in whole
plasma (32) to ensure that the effect of A/
fibrinogen on clot
lysis was not an artifact of the purified fibrinolysis assay.
A/
A and
A/
fibrinogens were iodinated and added to
fibrinogen-deficient plasma obtained from an afibrinogenemic
individual. Clotting was initiated by adding thrombin, and samples were
assayed daily for 7 days to quantitate the amount of soluble fibrin
degradation products (Fig. 7). After 7 days, the clots
formed from
A/
A fibrinogen showed complete lysis, with a lysis
half-time of 4.3 ± 0.1 days, whereas clots formed from
A/
fibrinogen exhibited no detectable lysis. These results demonstrate
that
A/
fibrin clots generated in whole plasma, in which a
number of potentially confounding factors are present (particularly
protease inhibitors), are also resistant to fibrinolysis.
Fibrinolytic therapy is a widely used tool in the treatment of thrombosis. Fibrinolytic agents such as streptokinase or tissue-type plasminogen activator are used to activate the fibrinolytic system by converting the protease zymogen plasminogen to active plasmin, which then cleaves the fibrin clot into soluble components. Unfortunately, the administration of these agents often produces secondary hemorrhagic events that may be life-threatening. The development of new fibrinolytic agents with greater clot specificity is an area of intense study, but less attention has been focused on the factors that determine clot stability. If clot stability could be reduced, then lower amounts of these fibrinolytic agents could be used, thus reducing the chance of adverse side effects.
Increased fibrinogen levels are a major risk factor for cardiovascular
disease (18, 19). There are many potential mechanisms to explain how
high fibrinogen levels may contribute to the disease process. One
possible role is that high fibrinogen levels produce a hypercoagulable
state that favors thrombosis. It is known that the plasma levels of
fibrinogen have a direct influence on the amount of fibrin formed in
clots (25-27). The present study suggests two additional mechanisms
through which high fibrinogen levels may lead to thrombosis. In
agreement with previously published data, the results show that
fibrinolysis rates in vitro vary inversely with fibrinogen
concentration (33). This suggests that in hyperfibrinogenemia, there
may not only be a greater propensity for a hypercoagulable state, but
the clots that result will also be more resistant to lysis. In
addition, the present study shows that A/
fibrinogen affects the
stability of fibrin clots formed in vitro, possibly by
increasing the local concentration of factor XIII at the clot. Fibrinolysis assays using purified components revealed that clots formed with
A/
fibrinogen show significantly decreased
fibrinolysis compared with
A/
A fibrinogen, but only in the
presence of active factor XIII.
A/
fibrin clots are also more
highly cross-linked than the
A/
A fibrin clots, as assessed with a
D-dimer agglutination assay. The resistance of
A/
fibrin clots
to fibrinolysis was verified in reconstituted whole plasma. These
results suggest that
A/
fibrinogen levels in plasma may modulate
clot stability by affecting fibrinolysis rates.
The physiological function of A/
fibrinogen has been elusive for
many years. It is clear that the
chain displays much less binding
to the platelet fibrinogen receptor, glycoprotein IIb-IIIa (integrin
IIb
3) (11, 29, 34), but this negative role does not explain the raison d'être for
A/
fibrinogen. The present studies suggest that the physiological role of
A/
fibrinogen may be to increase clot stability by localizing
factor XIII at the fibrin clot (Fig. 8). In this
scenario, unactivated factor XIII is delivered to the growing fibrin
clot by
A/
fibrinogen. Through the action of thrombin,
fibrinogen (including
A/
fibrinogen) is converted to fibrin.
Factor XIII that is bound to the newly formed
A/
fibrin is
subsequently activated to XIIIa, since fibrin acts as a positive
modulator in factor XIII activation by thrombin (3). Thrombin cleavage
dissociates the active a2 dimer from the
b subunits of factor XIII, thereby allowing the free
a2 dimer to catalyze the cross-linking of the
fibrin clot. It is not clear at the present time whether the
b subunits remain bound to the
chain or dissociate with
the a2 subunits after thrombin activation. Thus,
the delivery of factor XIII to the fibrin clot by
A/
fibrinogen
could serve to increase the local concentration of factor XIIIa and
result in a more highly stabilized clot.
The concentration of A/
fibrinogen in plasma may therefore be a
risk factor for thrombosis, independent of the total plasma fibrinogen
concentration. The molecular mechanisms that affect the ratio of
A
versus
mRNA transcription are unknown, although the
liver expresses both mRNAs, while other tissues express only the
A mRNA (35). It is clear that the two mRNAs arise by
alternative processing of the original transcript (15, 16); it is
possible that the relative amounts of the mRNA pools or their
relative translation rates in liver vary among individuals, giving rise to different amounts of
A/
A and
A/
fibrinogen. There are presently no data available regarding the variability of
A/
fibrinogen levels in human populations. Epidemiologic studies to
address this issue are therefore currently underway.
We wish to thank Dr. Michael W. Mosesson and
Dr. Kevin R. Siebenlist for many helpful discussions, Dr. David A. Meh
for factor XIII, and Dr. Walter J. Kisiel for -thrombin.