From the ¶ Cancer Biology Research Group,
Department of Biochemistry and Molecular Biology, and
§ Department of Pathology, University of Calgary, Calgary,
Alberta T2N 4N1, Canada
Received for publication, February 14, 2001, and in revised form, April 20, 2001
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ABSTRACT |
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In a previous report we showed that
plasmin-dependent lysis of a fibrin polymer, produced from
purified components, was totally blocked if annexin II heterotetramer
(AIIt) was present during fibrin polymer formation. Here, we show that
AIIt inhibits fibrin clot lysis by stimulation of plasmin
autodegradation, which results in a loss of plasmin activity.
Furthermore, the C-terminal lysine residues of its p11 subunit play an
essential role in the inhibition of fibrin clot lysis by AIIt. We also
found that AIIt binds to fibrin with a Kd of 436 nM and a stoichiometry of about 0.28 mol of AIIt/mol of
fibrin monomer. The binding of AIIt to fibrin was not dependent on the
C-terminal lysines of the p11 subunit. Furthermore, in the presence of
plasminogen, the binding of AIIt to fibrin was increased to about 1.3 mol of AIIt/mol of fibrin monomer, suggesting that AIIt and plasminogen
do not compete for identical sites on fibrin. Immunohistochemical
identification of p36 and p11 subunits of AIIt in a pathological clot
provides important evidence for its role as a physiological
fibrinolytic regulator. These results suggest that AIIt may play a key
role in the regulation of plasmin activity on the fibrin clot surface.
The formation of a fibrin polymer from its soluble precursor,
fibrinogen, results from the hydrolytic catalysis of fibrinogen by
thrombin, the terminal proteolytic enzyme in the coagulation cascade
(1-3). The (patho-) physiological existence of fibrin is linked to its
homeostatic roles, such as temporary matrix formation in wound healing
and the formation of a hemostatic plug. Accumulation of fibrin is a
hallmark of a variety of diseases, such as cancer and arteriosclerosis
(4). The polymerization of the fibrin monomer and the degradation of
the fibrin polymer are physiologically balanced by the coagulation and
fibrinolysis systems, respectively (5-9). Dissolution of a fibrin clot
is mainly conducted by plasmin, the terminal enzyme of the fibrinolytic
cascade. Plasmin, an 85-kDa serine protease, is involved in a variety
of physiological and pathological processes, including fibrinolysis,
wound healing, tissue remodeling, embryogenesis, and the invasion and
spread of transformed tumor cells (5, 8, 10-16). Active plasmin is
produced from two serial activation processes. First,
[Glu]plasminogen is converted to the more reactive [Lys]plasminogen
by plasmin itself through proteolytic removal of the N-terminal 77 amino acids. Second, plasmin also activates the plasminogen activators, tissue-type plasminogen activator
(tPA)1 and urokinase-type
plasminogen activator, which, in turn, convert [Lys]- or
[Glu]plasminogen to plasmin (13).
It is well established that fibrin stimulates the rate of
tPA-dependent plasminogen activation by at least two orders
of magnitude due to the localization of plasminogen and tPA to the
fibrin surface via their lysine-binding kringle domains (17, 18). Other
proteins, such as the histidine-proline rich glycoproteins or certain
extracellular matrix proteins that interact with the kringle domains of
plasminogen, have also been shown to stimulate plasminogen activation
(19-21). Typically, these interactions involve the binding of
the kringle domains with the C-terminal lysine residues of the
plasminogen-binding protein. Hajjar's group recently reported that the
Ca2+-binding protein, annexin II, stimulated the
tPA-dependent formation of plasmin from [Glu]plasminogen
or [Lys]plasminogen 20-fold or 14-fold, respectively, in
vitro. They also reported that plasminogen activation was
inhibited on human umbilical vein endothelial cell surfaces after
transfection of these cells with antisense oligonucleotides directed
against annexin II mRNA, suggesting that annexin II may function as
a fibrinolytic receptor for plasminogen on the surface of endothelial
cells (23-27).
Annexin II was originally described as an intracellular
Ca2+- and phospholipid-binding protein, and subsequent
studies suggested that this protein could be involved in regulating
membrane trafficking events such as exocytosis or endocytosis (28).
Surprisingly, annexin II does not contain a C-terminal lysine residue,
and the mechanism by which this protein interacted with plasminogen was unclear. Annexin II also exists in cells as a heterotetramer, called
annexin II heterotetramer (AIIt), which consists of two annexin II
molecules referred to as the p36 subunit and two molecules of an 11-kDa
regulatory subunit referred to as the p11 light chain. Importantly, p11
contains two lysine residues at its C terminus. Ninety to ninety-five
percent of the total cellular annexin II is present in the
heterotetrameric form in many cells such as Madin-Darby canine kidney
cells, bovine intestinal epithelial cells, and calf pulmonary arterial
endothelial cells (29, 30).
Recently, it was established that AIIt binds tPA, plasminogen, and
plasmin and stimulates tPA-dependent plasminogen activation about 300-fold in vitro (31). This activation was mediated
by binding of plasminogen kringle domains to the C-terminal lysine residue of the p11 subunit, suggesting that the p11 subunit
facilitates the majority of the AIIt-mediated stimulation of
tPA-dependent plasminogen activation. Furthermore,
immunofluorescence and coimmunoprecipitation experiments established
the colocalization of p11 and annexin II on the surface of human
umbilical vein endothelial cells (31), BT20 human breast carcinoma
cells, U87 human glioblastoma cells (32, 33), and fibrosarcoma
cells.2 Taken together, these
data provided evidence that AIIt played a key role in plasminogen
activation on the cell surface.
We recently reported that AIIt inhibited the fibrinolytic activity of
plasmin (34). These experiments were conducted using fibrin clots
generated from purified components in a cell-free system (34). We also
demonstrated that AIIt associated with the fibrin clot in
vitro (34). In a separate series of experiments, we showed that
preincubation of AIIt and plasmin resulted in the stimulation of the
autocatalytic digestion of the plasmin heavy and light chains,
resulting in a decrease in plasmin activity (31). It was therefore
unclear from our results if the antifibrinolytic activity of AIIt was
due to its stimulation of plasmin autodegradation or due to its ability
to associate with fibrin thereby sterically hindering the interaction
of plasmin with fibrin.
In the present report, we demonstrate that the antifibrinolytic
activity of AIIt is due to its stimulation of plasmin autodegradation by a mechanism that requires the C-terminal lysine residues of the p11 subunit.
Reagents--
Bovine intestinal mucosa heparin (3 kDa, 50 USP
units/mg),
Annexin II tetramer (AIIt) was prepared from bovine lung as previously
described (36). Recombinant annexin II monomer and p11 subunits were
expressed in Escherichia coli as described previously (37,
38). Purification of recombinant annexin II from bacterial lysates
involved chromatography on hydroxyapatite (Bio-Rad), affinity chromatography on heparin-Sepharose, and gel permeation chromatography on Superose 12. Purification of recombinant p11 involved ion-exchange chromatography on Fast-Q and Fast-S and gel permeation chromatography on Superose 12. Recombinant AIIt was formed by mixing equimolar annexin
II and p11 followed by gel permeation chromatography on Superose 12. All proteins were stored at Mutagenesis of Annexin II--
The bacterial expression vector
(pDS10) encoding the cDNA of human total calcium mutant
annexin II was kindly provided by Dr. Volker Gerke. This vector has all
five Ca2+-binding sites of annexin II inactivated and has
the mutation A65E in the annexin II sequence to provide an epitope for
antibody recognition (39). This site was reverted back to the wild-type sequence (E65A) before bacterial transformation (40). The mutant annexin II cDNA sequence was excised from the pDS10 vector using PCR (40). The PCR reaction resulted in a blunt-ended cDNA encoding the exact amino acid sequence of annexin II and therefore did not
encode for the six extra amino acids as previously reported. A
BamHI site was added to the 3'-end using PCR with the
appropriate primers. This cDNA sequence was then ligated into an
NdeI-cut, Klenow-filled-in, BamHI-digested
pAED4.91 vector. These vectors were then transformed into E. coli BL21(DE3) and grown as previously described (37).
Production of the p11 Subunit Deletion Mutant--
The cDNA
of the p11 subunit deletion mutant (lacking the two C-terminal lysine
residues) was produced by PCR mutagenesis as previously described (38).
The cDNA expression construct, pAED4.91-p11del KK was
confirmed by DNA sequencing to lack Lys-95 and Lys-96. The expression
of the p11del KK in BL21(DE3) E. coli and the
subsequent purification were performed as previously described
(37).
Fibrin Polymer Lysis Assay--
Fibrin polymer lysis experiments
were performed in a volume of 400 µl at 25 °C using a Beckman DU
640 spectrophotometer to monitor changes in turbidity at 450 nm (34).
Initially, fibrin clots were formed by incubation of 8 µM
fibrinogen with 0.83 NIH units/ml thrombin in buffer A (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 5 mM CaCl2) at room temperature. After turbidity
had reached a stable level, 100 µl of buffer A containing 42 nM plasmin and test substances such as glycerol, Radioiodination of Protein--
AIIt and [Glu]plasminogen were
radioiodinated as previously described (41). Briefly, either AIIt or
plasminogen (10 µM) was incubated for 3 min at room
temperature with 37 MBq (= 1 mCi) of Na125I and three
IODO-BEADs (Pierce Chemical Co.) in phosphate-buffered saline. Free
I125 and protein were separated using a PD-10 desalting
column (Sephadex G-25, Amersham Pharmacia Biotech), equilibrated, and
eluted with buffer A. The specific activity of the protein preparations
ranged from 1000 to 2000 cpm/pmol of protein.
Plasmin Autodegradation during Fibrinolysis--
Fibrin clots
were formed as described above for the fibrin polymer lysis assay.
After turbidity had reached a stable level, fibrin clot lysis was
triggered by gently layering 200 nM radioiodinated plasminogen and 25 nM tPA with or without 1 µM AIIt. After 12 h of incubation at 25 °C, 10 µl of each reaction was subjected to SDS-PAGE, and the pattern of
plasminogen or plasmin degradation was detected by autoradiography. The
autoradiographs were quantitated by densitometric analysis using a
Hewlett Packard ScanJet 4c flatbed scanner and Adobe Photoshop 5.5.
Fibrin Binding Assay--
Fibrin polymerization was initiated by
addition of thrombin (5 NIH units/ml) to a series of Eppendorf tubes,
preheated to 37 °C, containing 5 µM fibrinogen, 4.2 nM 125I-labeled AIIt, and various
concentrations of unlabeled AIIt in buffer A. To examine the binding of
AIIt to fibrin in the presence of plasminogen, fibrin was formed by
incubation of thrombin (5 NIH units/ml), 5 µM fibrinogen,
4.2 nM 125I-labeled AIIt, 2 µM
unlabeled AIIt, and various concentrations of plasminogen in the
absence or presence of 10 mM Plasminogen Activation Assay on Fibrin Surface--
Fibrin
polymers were formed in 24-well plates by incubation of 8 µM fibrinogen with 0.83 NIH unit/ml thrombin in buffer A as described above. The kinetics of tPA-mediated plasminogen activation in the absence or presence of AIIt on fibrin was determined by measuring amidolytic activity of the plasmin generated during activation of plasminogen. The reaction was performed with the substrate
H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide (Spectrozyme 251) at a final concentration of 100 µM, and
in buffer A containing 2 nM tPA in the absence or presence
of 1 µM AIIt. Alternatively, AIIt-stimulated plasmin
formation on the fibrin clot was examined in the presence of various
concentrations of AIIt. The reaction was initiated by the addition of
0.1 µM [Glu]plasminogen and was monitored at 405 nm in
a PerkinElmer Life Sciences HTS 7000 Bioassay reader.
Data Analysis--
Initial rates of plasmin generation were
calculated using linear regression analysis of plots of
A405 nm versus time squared
as previously outlined (43). Linear regression was performed with the computer program SigmaPlot (Jandel Scientific). For time course data, the data were fit to the equation,
A405 nm = B + K*t2, where K is the rate
constant for the acceleration of p-NA generation and
B is the y intercept. This equation describes the
rate of p-nitroanilide (p-NA) production from a
mixture of tPA, plasminogen, and spectrozyme 251. Under our
experimental conditions, K was proportional to the initial
rate of plasmin formation from plasminogen. Typically, the initial
rates of plasmin generation were reported in units of
A405 nm/min2 × 10 Immunohistochemistry--
A lung tissue specimen was obtained
from an autopsy of a patient who had suffered a pulmonary
thromboembolism. The lung tissue was fixed overnight in 4%
phosphate-buffered formaldehyde solution, processed, and embedded in
paraffin. Three-micron-thick sections were cut from the paraffin blocks
and placed on coated glass slides. The sections were deparaffinized and
rehydrated. No antigen retrieval technique was necessary. Sections were
stained using a Ventana Nexes immunostainer with a Ventana DAB
detection kit. Antibody was applied manually at an appropriate time in
the cycle. Anti-p11 monoclonal antibody was diluted with Dako antibody
diluting buffer to a working concentration of 1:40, whereas the
anti-annexin II monoclonal antibody was diluted to a working dilution
of 1:25. Antibodies were incubated with samples for 30 min. The slides were counterstained with Ventana hematoxylin and bluing agent. Coverslips were placed over the slides, which were then examined using
a Zeiss microscope fitted with a SPOT digital camera.
Miscellaneous Techniques--
Nonlinear least-squares fitting
was performed using SigmaPlot (Jandel Scientific). Results are
representative of a minimum of three experiments. AIIt, fibrinogen, and
[Glu]plasminogen concentrations were determined
spectrophotometrically (A280) using the
extinction coefficients (Emg/ml) 0.68, 1.51, and 1.68, respectively.
Effects of Glycerol on the AIIt-mediated Inhibition of Plasmin
Fibrinolytic Activity--
We previously reported that the
plasmin-dependent lysis of a fibrin polymer, produced from
purified components, was totally blocked if AIIt was present during
fibrin polymer formation (34). It was unclear from these studies if, in
the presence of fibrin, AIIt inhibited plasmin activity directly or
indirectly inhibited plasmin activity by promoting plasmin
autodegradation. We therefore used an in vitro fibrin
polymer lysis assay to address this question. In this assay, fibrin was
formed from fibrinogen by thrombin and fibrin polymer dissolution was
initiated by addition of plasmin in the presence or absence of AIIt.
Fibrin clot polymerization and subsequent lysis were measured with a
simple turbidity assay (A450).
As shown in Fig. 1, the two respective
processes of fibrin polymerization (clotting) and fibrinolysis are
indicated by an initial rapid increase in turbidity to a plateau value
(polymer formation) and, after addition of plasmin to the fibrin
polymer, a subsequent return to the value of nonpolymerized material
(fibrinolysis). Lysis of the fibrin clot was completely abolished in
the presence of AIIt. Interestingly, the AIIt-mediated inhibition of
plasmin fibrinolytic activity was blocked by glycerol in a
dose-dependent manner (Fig. 1). Glycerol has been shown to
inhibit plasmin autodegradation but not plasmin-dependent
clot lysis (44). Our data therefore suggested that AIIt blocked fibrin
clot lysis by accelerating plasmin autodegradation, thereby reducing
the availability of plasmin for fibrin clot lysis.
Effects of AIIt on Plasmin Degradation during Fibrinolysis--
It
has been documented that plasmin autodegradation results in a loss in
plasmin activity and degradation of the plasmin heavy (A) and light (B)
chains. To establish if AIIt blocked fibrin clot lysis via stimulation
of plasmin autodegradation, we analyzed the plasmin reaction products
during fibrinolysis. Fibrinogen was incubated with thrombin, and, after
30 min, a stable fibrin clot was formed. Radioiodinated plasminogen was
added to the clot, and fibrinolysis was initiated by addition of tPA.
After 12 h, the reaction products were analyzed by SDS-PAGE. As
shown in Fig. 2, in the presence of
fibrin, tPA converted plasminogen into plasmin as illustrated by the
appearance of the 55-kDa A chain and 25-kDa B chain of plasmin. In
contrast, when AIIt was added to the reaction, degradation of both the
plasmin A (H-Pm) and B (L-Pm) chain was apparent
(Fig. 2). Densitometric quantitation of the autoradiogram showed that
11.8% of the A chain and 43.6% of the B chain of plasmin remained
after incubation with 1 µM AIIt. Furthermore, in the presence of 5 µM AIIt, only 5.4% of the A chain and
35.1% of the B chain of plasmin remained (Fig. 2, inset).
These results therefore establish that the inhibition of fibrin clot
lysis by AIIt is due to its acceleration of plasmin
autodegradation.
Domains of AIIt Responsible for the Inhibition of
Plasmin-dependent Fibrin Clot Lysis--
AIIt is a
Ca2+-binding protein, and binding of Ca2+ to
the p36 subunit regulates many of its biological activities (28, 46). The p36 subunit of AIIt contains five established
Ca2+-binding sites. We utilized a recombinant AIIt mutant
to establish if the Ca2+-binding sites of AIIt played a
role in the inhibition of fibrin clot lysis. TCM AIIt, which has all
five Ca2+-binding sites of the p36 subunit inactivated,
significantly blocked plasmin-dependent fibrin clot lysis
in a similar fashion to wild-type AIIt (Fig.
3A). This suggested that the
five Ca2+-binding sites in the p36 subunit of AIIt do not
play a role in the inhibitory effect of AIIt on fibrin clot lysis.
AIIt binds plasminogen via the C-terminal lysine residues of its p11
subunit (38). Removal of the C-terminal lysines blocks the ability of
AIIt to accelerate tPA-dependent plasminogen conversion to
plasmin (38). To examine if the C-terminal lysines of the p11 subunit
of AIIt participate in the antifibrinolytic activity of AIIt, we
carried out plasmin-dependent fibrin clot lysis in the
presence of recombinant mutant AIIt (AIIt(K-K) Fibrin Binding Properties of AIIt--
We previously demonstrated
that AIIt pelleted with a fibrin clot (34). This result prompted us to
characterize the binding of AIIt to the fibrin polymer. A fibrin
polymer was formed by incubation of fibrinogen and thrombin with
radioiodinated AIIt. The fibrin clot was centrifuged, and the AIIt
binding to the fibrin clot was quantitated. Fig.
4 shows the binding isotherm for the binding of AIIt to fibrin. AIIt bound fibrin polymer in a specific and
saturable manner with a Kd of 269.4 ± 60.0 nM (mean ± S.D., n = 3). The binding
approached a plateau at about 1.6 µM AIIt, which
corresponded to about 0.28 mol of AIIt/mol of fibrin monomer. Scatchard
analysis (Fig. 4, inset) established that the binding of
AIIt to fibrin was mediated by a single, moderate affinity-binding site. The low stoichiometric binding of AIIt to fibrin could be due to
steric hindrance. For example, it is possible that the binding sites in
the dense areas of the clot may not be accessible to AIIt.
It was previously reported that heparin binds AIIt (47) and that it
inhibits the association of AIIt with the fibrin polymer (34). As shown
in Fig. 5A, heparin blocks the
binding of AIIt to fibrin polymer. Inhibition of AIIt binding to fibrin
by heparin was dose-dependent, and half-maximal inhibition
occurred at 1.1 ± 0.2 µM heparin (mean ± S.D., n = 3).
To determine if the Ca2+-binding sites of the p36 subunit
or the C-terminal lysines of the p11 subunit of AIIt played a role in
fibrin binding, we investigated the ability of the AIIt mutants, TCM
and AIIt(K-K) Effects of Plasminogen on AIIt Binding to the
Fibrin Polymer--
Plasminogen exists at micromolar concentration in
blood. Because plasminogen binds to both fibrin and AIIt, we examined
the binding of AIIt to the fibrin clot in the presence of plasminogen (Fig. 6). The binding of AIIt to fibrin
polymer was increased by addition of plasminogen in a
dose-dependent manner and approached a plateau at about 1 µM plasminogen. Half-maximal binding was obtained at
~200 nM plasminogen (Fig. 6, filled circles).
This binding event was also examined in the presence of a lysine
analogue,
The interaction of AIIt with the fibrin polymer may therefore
physiologically occur in at least one of two ways; its intrinsic binding ability (independent of the C-terminal lysine as shown in Fig.
5C), or through plasminogen-mediated additional binding capacity, which is governed by its p11 C-terminal lysine residues.
Acceleration of Plasminogen Activation by AIIt on Fibrin
Surface--
AIIt has been reported to stimulate both the
tPA-dependent conversion of plasminogen to plasmin as well
as plasmin autodigestion in vitro. Although our data shows
that, in the presence of fibrin stimulation of plasmin autodigestion is
the predominant reaction, we could not rule out the possibility that
AIIt might also stimulate tPA-dependent plasmin formation.
Fig. 7A shows that fibrin
accelerated tPA-dependent plasmin formation 7.8-fold. This
fibrin-accelerated tPA-dependent plasmin formation was
increased further 6.4 times by AIIt (Fig. 7A,
inset). Analysis of the dose dependence of the stimulation
of tPA-dependent plasmin formation by AIIt showed that
about 0.58 ± 0.10 µM AIIt was required for
half-maximal stimulation of plasmin formation (Fig. 7B).
This concentration range of AIIt was similar to that required for
half-maximal inhibition of plasmin-dependent fibrin polymer
lysis. Collectively, our data suggest that AIIt may support the
turnover of plasmin at the fibrin clot surface by stimulating both
plasmin formation and destruction, although under our experimental
conditions the latter reaction predominates.
Immunohistochemical Identification of AIIt in a Physiological Blood
Clot--
AIIt or annexin II has been shown by several groups to
localize to the extracellular surface of variety of cells (31-33).
However, whether or not AIIt associates with fibrin under physiological conditions has not been established. Accordingly, lung tissue obtained
from a patient with a pulmonary thromboembolism was stained for annexin
II and p11 (Fig. 8). The results show
that both annexin II and p11 localize at contact areas of the fibrin
clot and cell components. Thus, the association of AIIt with fibrin
observed in vitro appears to be of physiological
relevance.
The formation of a fibrin polymer subsequent to blood vessel
injury not only conserves blood constituents but also provides temporary matrices for blood vessel growth. However, the accumulation of a fibrin clot due to unbalanced hemostasis may lead to a variety of
pathological disorders, including inflammation, arteriosclerosis, or
stroke. During the formation of a solid blood clot, plasmin-mediated fibrinolysis is triggered by the actions of blood vessel endothelial cells growing on the fibrin substratum during the wound healing process. This process relies on the spatial and temporal regulation of
plasmin activity within the fibrin clots.
Recently, annexin II, the 36-kDa subunit of AIIt, was shown to function
as a receptor for both plasminogen and tPA on the surface of
endothelial cells (22). Furthermore, it has recently been shown that
AIIt, but not annexin II, inhibits plasmin-dependent fibrinolysis (34) as well as accelerates both tPA-dependent plasminogen activation (31) and subsequent plasmin autodegradation (45). In this report, we demonstrate that AIIt can bind to the fibrin
clot in vitro and associates with the fibrin clot in
vivo. Furthermore, we show that AIIt inhibits
plasmin-dependent fibrin clot lysis by stimulation of
plasmin autodegradation in vitro.
The ability of AIIt to inhibit the fibrinolytic activity of plasmin
severely diminishes the concentration of active plasmin that is
normally targeted for fibrin clot dissolution. This notion is supported
by the finding that glycerol, an inhibitor of the plasmin
autodegradation, inhibits the antifibrinolytic activity of AIIt (Fig.
1). The inhibition of plasmin-dependent fibrinolysis by
AIIt also corresponded with plasmin heavy and light chain degradation (Fig. 2), suggesting a relationship between the antifibrinolytic activity of AIIt and AIIt-stimulated plasmin autodegradation. Although
the stimulation of plasmin breakdown is the predominant reaction
observed under our experimental condition, we have also shown that, in
the presence of the fibrin clot, AIIt stimulates tPA-dependent plasmin formation. This suggests that the
physiological role of AIIt may be to provide a transient pulse of
plasmin activity on the fibrin surface.
We also examined two AIIt mutants to determine the potential role of
the Ca2+-binding sites of the p36 subunit of AIIt or of the
C-terminal lysine residues of p11 subunit of AIIt in its
antifibrinolytic activity (Fig. 3). We first examined a mutant AIIt in
which all five Ca2+-binding sites of the p36 subunit were
inactivated (TCM AIIt). This mutant was chosen because Ca2+
is an essential cofactor in the coagulation cascade, and it was, therefore, reasonable to suspect that the Ca2+-binding
sites of AIIt might play a role in its inhibition of fibrin clot lysis.
However, as observed in Fig. 3A, the TCM AIIt also inhibited
plasmin-dependent fibrinolysis. Thus, the
Ca2+-binding sites of AIIt do not play a direct role in
mediating its antifibrinolytic activity.
The second mutational analysis of AIIt involved the removal of the
C-terminal lysine residues of the p11 subunit
(AIIt(K-K) The profound effect of fibrin on the regulatory properties of AIIt led
us to examine in detail the interaction of AIIt with fibrin. First we
found that AIIt bound fibrin with a Kd of 269.4 ± 60.0 nM (S.D., n = 1) (Fig. 4).
Scatchard analysis reveals that a single saturable binding site is
involved in this interaction (Fig. 4, inset). The low
stoichiometry (0.28 mol of AIIt/mol of fibrin monomer) probably
indicates that all of the AIIt binding sites in the artificial fibrin
polymer formed in test tubes are not accessible to AIIt. Second, we
found that the interaction of AIIt with fibrin was inhibited by heparin
with an IC50 of 1.1 ± 0.2 µM (S.D.,
n = 1). The heparin binding region is located at
residues 306-313 of the p36 subunit and may, therefore, be involved in
the interaction of AIIt with fibrin (Fig. 5A). Third, using
the TCM AIIt and the AIIt(K-K) The binding of these components to the p11 subunit of AIIt localizes
tPA, plasminogen, plasmin, and AIIt to the fibrin surface. It is
therefore reasonable to assume that the mechanism by which the
C-terminal lysines of the p11 subunit of AIIt stimulates plasmin autodegradation is due to the spatial localization of plasmin to the
C-terminal lysine binding sites of AIIt thus resulting in a localized
concentration of plasmin at these sites. It is also possible that the
interaction of plasmin with the C-terminal lysines of the p11 subunit
of AIIt may induce a conformational change in plasmin resulting in
stimulation of plasmin autodegradation.
Our results suggest that the inhibition of
plasmin-dependent fibrinolytic activity by AIIt requires
both the interaction of AIIt with the plasminogen activation system
(enzyme system) and with the fibrin polymer (substrate components).
These binding events appear to localize tPA, plasminogen, plasmin, and
AIIt to the fibrin surface. The demonstration of AIIt-stimulated
tPA-dependent plasminogen activation on the fibrin surface
suggests that AIIt may transiently increase plasmin activity on the
fibrin surface (Fig. 7). It is clear that, in the presence of fibrin,
AIIt stimulates both plasmin formation and destruction. However, under
our experimental conditions, the AIIt-mediated destruction of plasmin
predominates. We cannot, however, rule out the possibility that other
regulators of AIIt may be present at or in the vicinity of the fibrin
clot. These putative regulators could influence whether or not plasmin formation or destruction is the predominant reaction stimulated by
AIIt.
The above results, along with previous findings, form the basis for a
model of the mechanism of plasmin regulation by AIIt (Fig.
9). AIIt bound to fibrin is capable of
accelerating the tPA-dependent formation of plasmin from
plasminogen bound either to fibrin or AIIt itself. The ability of AIIt
to bind fibrin, plasminogen, and tPA allows recruitment of plasminogen
and tPA to the fibrin surface and thus spatially regulates plasmin
production. Moreover, AIIt also accelerates the autodegradation of
plasmin subsequent to its activation. The result is a tight regulation of plasmin activity on the fibrin surface. It is possible that the dual
role of AIIt in plasmin formation and destruction provides the fibrin
surface with a transient pulse of proteolytic activity. This allows for
dissolution of the fibrin clot and prevents constitutive activation of
plasminogen, which is also detrimental to hemostatic balance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminocaproic acid (
-ACA), and thrombin (2300 NIH
units/mg) were obtained from Sigma Chemical Co. Human recombinant tPA
was obtained from Genentech, was purified by chromatography on
benzamidine-Sepharose (35), and was an 80-90% single chain as
determined by SDS-PAGE. [Glu]plasminogen, [Lys]plasminogen,
plasmin, the amidolytic plasmin specific substrate, Spectrozyme 251 (H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide), and the tPA amidolytic substrate Spectrozyme 444 (methyl-D-cyclohexatyrosyl-glycyl-arginine paranitroaniline
acetate) were obtained from American Diagnostica. Fibrinogen was
obtained from Sigma and further purified by chromatography on Superose
12 (Amersham Pharmacia Biotech) to remove contaminating plasminogen.
70 °C in 40 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM EGTA, and
150 mM NaCl. Anti-p11 and annexin II monoclonal antibodies
were purchased from Transduction Laboratories.
-ACA,
or other proteins were gently layered on top of the fibrin polymer
reaction mixture, and turbidity was measured as described previously
(34).
-ACA (42). Fibrin clots
were incubated for 1 h at 37 °C and pelleted by centrifugation at 10,000 × g for 30 min at 4 °C. The clots were
washed with buffer A three times, and AIIt bound to the fibrin clot was
calculated from radioactivity of the fibrin pellets. Radioactivity of
the pelleted clots in the thrombin-untreated tubes was subtracted from
that of the pellet in the thrombin-treated tubes. Nonspecific binding
was typically less than 10% of total binding.
3.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Glycerol blocks the inhibition of
plasmin-dependent fibrin polymer lysis by AIIt.
Thrombin (0.83 NIH units/ml) and fibrinogen (8 µM) were
incubated at 25 °C in buffer A containing 5 mM
CaCl2, 50 mM Tris, pH 7.5, and 100 mM NaCl. After fibrin polymerization was complete (30 min),
100 µl of buffer A alone (filled squares) or buffer A
containing 1 µM plasmin and the following additions were
layered on top of the fibrin polymer; 25% glycerol (filled
circles); 25% glycerol and 1 µM AIIt (open
circles); 50% glycerol and 1 µM AIIt (filled
triangles); 1 µM AIIt (open triangles).
Fibrinolysis was measured as described under "Experimental
Procedures."
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Fig. 2.
Effects of AIIt on plasmin degradation during
fibrinolysis. Thrombin (0.83 NIH units/ml) and fibrinogen (8 µM) were incubated at 25 °C in buffer A. After fibrin
polymerization was complete (30 min), fibrin polymer lysis was
initiated by the addition of 100 nM radioiodinated
plasminogen and 2 nM tPA as described under "Experimental
Procedures." Reactions were conducted in the presence of 1 µM (+) and 5 µM AIIt (++), respectively.
After a 12-h incubation at 25 °C, 10 µl of each reaction mixture
containing fibrin clot was dissolved with SDS sample buffer and
subjected to SDS-PAGE, and the gel was vacuum-dried. Upper
panel, pattern of plasminogen or plasmin degradation was detected
by autoradiography. Lower panel, the band intensity of the
autoradiogram was quantitated using a Hewlett Packard ScanJet 4c
flatbed scanner and Adobe Photoshop 5.5. The lanes of the
autoradiogram were aligned with a densitometric histogram.
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Fig. 3.
Role of the Ca2+-binding sites of
the p36 subunit and C-terminal lysines of the p11 subunit of AIIt in
the inhibition of plasmin fibrinolytic activity. A,
effects of Ca2+-binding site deletion mutant AIIt (TCM) on
plasmin-dependent fibrin polymer lysis. Thrombin (0.83 NIH
units/ml) and fibrinogen (8 µM) were incubated at
25 °C in buffer A. After fibrin polymerization was complete (30 min), 100 µl of buffer A without (open squares) or buffer
A containing 1 µM plasmin was layered on top of the
fibrin polymer in the absence (open circles) or presence of
2 µM wild-type AIIt (filled triangles), 2 µM TCM AIIt (open triangles), 5 µM TCM AIIt (filled squares). Fibrinolysis was
measured as described under "Experimental Procedures."
B, effects of p11 C terminus K-K deletion mutant AIIt
(AIIt(K-K) ) on plasmin-dependent fibrin
polymer lysis. Thrombin (0.83 NIH units/ml) and fibrinogen (8 µM) were incubated at 25 °C in buffer A. After fibrin
polymerization was complete (30 min), 100 µl of buffer A without
(filled circles) or buffer A containing 1 µM
plasmin was layered on top of the fibrin polymer in the absence
(open circles) or presence of 5 µM wild-type
AIIt (open squares), 5 µM
AIIt(K-K)
(open triangles) or 10 µM AIIt(K-K)
(filled squares).
Fibrinolysis was measured as described under "Experimental
Procedures."
) whose two
p11 C-terminal lysine residues were deleted (Fig. 3B). The
results show that, unlike the wild-type and TCM AIIt, AIIt(K-K)
did not block plasmin-dependent
fibrin clot lysis. Therefore, these data suggested that the C-terminal
lysine residues of AIIt play an important role in the inhibitory effect
of AIIt on fibrin clot lysis.
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Fig. 4.
AIIt binding to fibrin. AIIt was
radioiodinated as described "Experimental Procedures." Fibrin
polymerization was initiated by addition of thrombin (5 NIH units/ml)
to a series of Eppendorf tubes, preheated to 37 °C, which contained
648 nM fibrinogen, 4.2 nM
125I-labeled AIIt, and various concentrations of
non-labeled AIIt in buffer A. After incubation at 37 °C for 1 h, fibrin clots were pelleted by centrifugation at 10,000 rpm for 30 min at 4 °C. After three washes with buffer A, AIIt bound to the
fibrin clot was calculated from radioactivity of clot.
Inset, Scatchard plot analysis of the binding
isotherm.
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Fig. 5.
Analysis of the interaction of AIIt with
fibrin. Fibrin was polymerized by incubation of 648 nM
fibrinogen with thrombin (5 NIH units/ml) in the presence of 4.2 nM 125I-labeled AIIt and various concentrations
of heparin (A), TCM AIIt (B), and
AIIt(K-K) (C). The binding was assessed as
described under "Experimental Procedures."
, to dissociate radiolabeled wild-type AIIt
from the fibrin clot. It was therefore expected that the recombinant
mutant AIIt would compete with the radiolabeled wild-type AIIt, unless
the mutation rendered the protein unable to bind to fibrin. Fig. 5,
B and C, shows that both TCM AIIt and
AIIt(K-K)
competitively inhibited wild-type AIIt binding
to fibrin polymer with an IC50 values of about 260 or 330 nM, respectively. Therefore, neither the
Ca2+-binding sites of AIIt nor the C-terminal lysine
residues of the p11 subunit of AIIt mediate the binding of AIIt to the
fibrin polymer.
-ACA, which inhibits the interaction between AIIt and
plasminogen. The addition of 10 mM
-ACA blocked the
increased AIIt binding to fibrin polymer mediated by plasminogen (Fig.
6, open circles). This suggests that the p11 C-terminal
lysine residues of AIIt are involved in plasminogen-facilitated binding
of AIIt to fibrin. This data also implies that binding of plasminogen
to fibrin may influence the interaction of AIIt with fibrin
polymer.
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Fig. 6.
Effects of plasminogen on AIIt binding to
fibrin in the absence or presence of
-ACA. Fibrin was polymerized by incubation of
648 nM fibrinogen with thrombin (5 NIH units/ml) in buffer
A containing 4.2 nM 125I-labeled AIIt, 2 µM unlabeled AIIt, and various concentrations of
plasminogen in the absence (filled circles) or presence
(open circles) of 10 mM
-ACA. The binding was
assessed as described under "Experimental Procedures."
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Fig. 7.
Stimulation of tPA-dependent
plasminogen activation by AIIt on fibrin surface. A,
time course of AIIt-dependent stimulation of plasmin
formation on the fibrin clot. After a stable fibrin polymer was formed,
tPA-dependent plasminogen (Pg) activation was
examined in the absence (open circles) or presence
(triangles) of 1 µM AIIt as described under
"Experimental Procedures." Potential plasmin contamination in the
plasminogen preparation was also measured (closed circles).
Inset, the rate constant for the plasminogen activation was
calculated from the slope of curve fitting of the data according to the equation
A405 nm = B + K*t2, where K is the slope
and B is the y
intercept.t-PA-dependent plasminogen activation in the
absence of fibrin was also examined as described above, and the rate of
activation was compared with that obtained in the presence of fibrin.
B, dose dependence of AIIt-stimulated plasmin formation on
the fibrin clot. Initial rates of tPA-dependent Pg
activation were determined in the presence of various concentrations of
AIIt as described under "Experimental Procedures."
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Fig. 8.
Immunohistochemical staining of p36 and p11
in a pathological blood clot. Human lung tissue was
processed for immunohistochemical staining as described under
"Experimental Procedures." The presence of the fibrin clots in the
lung tissue is indicated by arrows and immunostaining of the
p36 (A) and p11 (B) subunits of AIIt are
indicated by arrowheads.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
). We have previously shown that removal of
these key residues disables the ability of AIIt to stimulate
tPA-dependent plasminogen activation (38). These residues
are believed to be involved in the binding of kringle domains of
plasminogen, plasmin, and tPA. As shown in Fig. 3B, this
mutant AIIt does not inhibit plasmin-dependent fibrin clot
lysis. This suggests that the C-terminal lysine residues of the p11
subunit of AIIt participate in both the stimulation of plasmin
formation and destruction by AIIt. In the absence of fibrin the former
reaction is favored, whereas, in the presence of fibrin, the latter
reaction is favored.
mutants, we found that the
binding of AIIt to fibrin does not involve the Ca2+-binding
sites of the p36 subunit nor the C-terminal lysine residue of the p11
subunit (Fig. 5, B and C). Fourth, we found that
in the presence of plasminogen the binding of AIIt to fibrin was actually increased (Fig. 6). The simplest explanation for this observation was that AIIt bound to fibrin and to fibrin-associated plasminogen. This was an important finding, because it ruled out the
possibility that AIIt and plasminogen competed for similar sites on the
fibrin polymer. Fifth, we demonstrated the localization of AIIt to a
thrombus in vivo. This established that under physiological conditions AIIt associated with fibrin (Fig. 8).
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Fig. 9.
Hypothetical scheme for regulation of plasmin
activity on the fibrin surface. AIIt forms a complex with fibrin,
plasminogen, and tPA and initially accelerates
tPA-dependent plasminogen activation on the fibrin surface.
Subsequently, AIIt accelerates plasmin autodegradation resulting in a
loss of plasmin fibrinolytic activity.
In summary, the present report establishes that the ability of AIIt to
inhibit plasmin-dependent fibrin clot lysis is due to its
acceleration of plasmin autocatalytic activity and therefore the
destruction of plasmin. Additionally, AIIt accelerates
tPA-dependent plasmin formation, although under our
experimental conditions plasmin destruction is the predominant reaction
stimulated by AIIt. Furthermore, AIIt and plasminogen form a complex on
the fibrin polymer, in vitro. The data also establish for
the first time that AIIt is present on the surface of a fibrin clot
in vivo. These novel findings suggest that AIIt may play a
central role in regulation of plasmin activity on the fibrin surface.
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FOOTNOTES |
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* This work was supported by a grant from the Alberta Heart and Stroke Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.:
403-220-3022; Fax: 403-283-4841; E-mail:
waisman@acs.ucalgary.ca.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M101426200
2 K.-S. Choi et al., manuscript in preparation..
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ABBREVIATIONS |
---|
The abbreviations used are:
tPA, tissue-type
plasminogen activator;
AIIt, annexin II heterotetramer;
-ACA,
-aminocaproic acid;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
p-NA, p-nitroanilide.
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REFERENCES |
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