1 Canadian Blood Services, R&D Department, 1800 Alta Vista Drive, Ottawa, ON
K1G 4J5, Canada
2 Department of Biochemistry, Microbiology, and Immunology, University of
Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada
3 Department of Biochemistry, Queens University, Room A212 Botterell Hall,
Kingston, ON K7L 3N6, Canada
4 Department of Pathology and Laboratory Medicine, University of British
Columbia, 2211 Wesbrook Mall, Room GF-114, Vancouver, BC V6T 2B5, Canada
* Author for correspondence (e-mail: ed.pryzdial{at}bloodservices.ca)
Accepted 20 February 2003
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Summary |
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Key words: Annexin 2, Thrombin, Endothelial cell, Fibrinolysis, Coagulation
![]() |
Introduction |
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Differences in the Ca2+ requirements for aPL binding of A2m and
A2t dictate their intracellular distribution. The ability of A2t to bind aPL
at intracellular Ca2+ concentrations localizes it to the inner
leaflet of the plasma membrane, whereas A2m is found largely in the cytosol
(Courtneidge et al., 1983;
Osborn et al., 1988
;
Thiel et al., 1992
). An
interesting property of A2 cellular distribution is that it has no secretory
signal, but has nevertheless been identified on the surface of various cell
types (Chung and Erickson,
1994
; Hajjar et al.,
1994
; Kassam et al.,
1998
; Wright et al.,
1995
). The mechanism by which A2 is shuttled to the cell surface
is not known, but might involve localization to caveolae
(Sagot et al., 1997
;
Stahlhut et al., 2000
).
The presence and availability of A2 on the cell surface plays a crucial
role in many of its proposed functions. Of relevance to the current study, A2
has been identified as an endothelial cell-surface co-receptor for the
fibrinolysis proteins plasminogen and tissue plasminogen activator (tPA)
(Hajjar et al., 1994). It has
been reported that both A2m and A2t can function as cofactors in the
tPA-dependent proteolytic activation of plasminogen to plasmin
(Cesarman et al., 1994
;
Liu et al., 1995
), which
proteolytically solubilizes fibrin clots for subsequent clearance. In vivo
evidence for an involvement of A2 in fibrinolysis
(Rand, 2000
) has been
demonstrated in acute promyelocytic leukemia, where overexpression of
endothelial A2 correlated with accelerated fibrinolysis and bleeding
(Menell et al., 1999
).
The enzyme directly responsible for fibrin clot production, thrombin, is
also a potent cell modulator. Thrombin-mediated activation of cell-surface
receptors, including those on endothelial cells, is pleiotropic and results
in: increased cytosolic Ca2+, stimulation of various second
messenger systems, kinase activation, induction of mitosis, and the flux of
ions in and out of the cell (Bartha et al.,
1989; Brock and Capasso,
1989
; Garcia et al.,
1995
; Magazine et al.,
1996
; Molino et al.,
1997
; Pollock et al.,
1988
; Schini et al.,
1989
; Sugama and Malik,
1992
; Tesfamariam et al.,
1993
). Thrombin receptors belong to the protease-activated
receptor (PAR) family of seven-transmembrane-domain, G-protein-linked,
cell-surface receptors (reviewed by Brass
and Molino, 1997
; Hou et al.,
1998
; Jamieson,
1997
). To date, three members of the PAR family, PAR-1
(Rasmussen et al., 1991
;
Vu et al., 1991
), PAR-3
(Ishihara et al., 1997
) and
PAR-4 (Kahn et al., 1998
),
have been identified as thrombin sensitive. The mechanism of PAR activation is
by proteolytic generation of a new N-terminus
(Vu et al., 1991
), which acts
as a `tethered' receptor ligand. Peptides whose sequences are identical to the
first six amino acids of the newly revealed PAR N-terminus have been shown to
activate the receptor without a proteolytic step
(Garcia et al., 1993
;
Muramatsu et al., 1992
;
Tiruppathi et al., 1992
).
To ensure that the sequential initiation of coagulation and then of fibrinolysis is stringent, feedback communication mechanisms between these pathways have evolved. In the current study, we addressed the hypothesis that an additional mode of communication could be facilitated by the cell-modulating capabilities of thrombin, which might affect activation of plasminogen on human umbilical vein endothelial cells (HUVECs) by altering the availability of cell-surface A2 and p11. The data revealed that thrombin or the analogous thrombin receptor-activating peptide (TRAP) selective for PAR-1 enhances both A2 and p11 on the HUVEC surface, which correlated with increased plasminogen binding and tPA-mediated plasmin generation. The regulation of A2 accessibility on the HUVEC surface is thus a novel variable to be considered in hemostasis.
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Materials and Methods |
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Antibodies
Several primary antibodies specific for A2m, p11 or vimentin were evaluated
to maximize the signal-to-background ratio for cell-surface or intracellular
immunofluorescence microscopy, western blot and plasminogen cell-binding
analyses. For A2m detection, three different antibodies were found to be best
suited for different applications. mAb anti-A2m IgG2a (Oncogene, Cambridge,
MA), mAb anti-A2m IgG1 (Transduction Laboratories, San Jose, CA) and mAb
anti-A2m IgG1 (Zymed, San Fransisco, CA) were used for cell-surface
immunofluorescence microscopy, intracellular immunofluorescence microscopy and
western blotting, respectively. mAb anti-p11 IgG1 (Transduction Laboratories)
was used for both intracellular immunofluorescence microscopy and western blot
analyses. The polyclonal rabbit anti-p11 IgG used in plasminogen-binding
experiments was a kind gift from D. Waisman (University of Calgary). This
antibody was raised to a synthetic peptide corresponding to residues 21-38 of
p11 linked to keyhole limpet hemocyanin and affinity purified using the same
peptide bound to Sepharose. Polyclonal goat anti-vimentin serum (Sigma) or mAb
anti-vimentin IgG1 (Sigma) were used for surface or intracellular detection of
antigen by immunofluorescence microscopy, respectively. Species-, subclass-
and (where applicable) isotype-matched non-immune negative control antibodies
were purchased from Sigma and used at an identical concentration as the
specific primary antibodies in every experiment involving antibodies. The
secondary antibodies used in immunofluorescence microscopy experiments were
fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat IgG (Sigma),
FITC-conjugated goat anti-rabbit IgG (Sigma) and Cy3-conjugated
donkey anti-mouse IgG (Jackson ImmunoResearch, Westgrove, PA). For western
blots, purified A2m and p11 provided by D. Waisman were used as positive
controls, and mAb anti-ß-actin (Sigma) was used to confirm that
comparable amounts of cell extract were loaded in each electrophoresis lane.
HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) was used as the
secondary antibody in all western blotting experiments.
Cell culture
HUVECs were isolated from fresh umbilical cords as previously described
(Dudani et al., 1991;
Jaffe et al., 1973
). Isolated
cells were propagated in complete IMDM, consisting of Iscove's Modified
Dulbecco's Media (IMDM) supplemented with 5% fetal bovine serum, 2 mM
L-glutamine, 20 µg/ml gentamycin (all from GibcoBRL, Burlington, ON), 59
µg/ml heparin (Sigma) and 10 µg/ml endothelial cell growth supplement
(Calbiochem, Mississauga, ON). All experiments were performed on cells between
passage one and five, with no observable differences within this range.
Thrombin and TRAP treatment of cells
HUVECs were grown to confluence on gelatin-coated 22x22 mm glass
coverslips or multiwell tissue culture plates. Monolayers were washed once
with IMDM/BSA (IMDM supplemented with 1 mg/ml BSA and 2 mM L-glutamine), and
treated with the indicated concentrations of -thrombin (Haematologic
Technologies, Burlington, VT) or TRAP in IMDM/BSA for 5 minutes at 37°C.
The cells were then washed once with complete IMDM supplemented with 2 mM
Ca2+ (IMDM/Ca2+), and further incubated in
IMDM/Ca2+ for 1 hour at 37°C. In the absence of the additional
hour of incubation, cells were prone to detachment.
Surface A2 and p11 detection
Fluorescence microscopy
To detect cell-surface A2 exclusively, thrombin- or TRAP-treated cells were
washed once with complete IMDM, and simultaneously incubated with a purified
mAb against A2m (Oncogene; 1.33 µg/ml) and polyclonal goat anti-vimentin
serum ([protein]=0.20 mg/ml) for 30 minutes at room temperature (RT). Vimentin
is an intracellular cytoskeletal component and was used as a marker for
inadvertent permeabilization of cells when surface antigens were being probed.
After washing with complete IMDM, cells were incubated with
Cy3-conjugated donkey anti-mouse IgG (1.25 µg/ml) and
FITC-conjugated rabbit anti-goat IgG (1.66 µg/ml) for 30 minutes at RT.
Stained cells were washed extensively and fixed with 4% paraformaldehyde and
0.25% gluteraldehyde in HBS. After fixation, nuclei were stained with DAPI (25
ng/ml) and the cells were washed with PBS and then water prior to mounting on
glass slides with 5 µl of mounting media [0.1% phenylenediamine (Sigma),
50% glycerol in PBS].
To visualize the distribution of A2 relative to submembranous cytoskeletal elements, thrombin- or TRAP-treated HUVECs were fixed with 4% paraformaldehyde and 0.25% gluteraldehyde in HBS before immunofluorescent microscopy, as described (Traverso, 2000), to detect A2, vimentin and nuclei simultaneously.
All micrographs were obtained using a Zeiss Axioplan 2 epifluorescent microscope fitted with a Sony 3 CCD color video camera (model DXC-950P). Images were captured by Northern Eclipse (Empix, Mississauga, ON, Canada) software. For all immunofluorescence experiments, individual micrographs presented for comparison were conducted on the same day and subjected to identical image acquisition parameters to optimize observable fluorescence intensity differences between treated and mock-treated cells.
Cell-surface biotinylation
After three washes with HBS, the surface proteins of thrombin- or
TRAP-treated HUVECs were biotinylated by incubating the cells for 30 minutes
at RT with 0.5 mg/ml of the hydrophilic probe, sulfo-NHS-Biotin (Pierce,
Brockville, ON). The washing was repeated, and the cellular proteins were
solubilized using Triton X-100 lysis buffer (40 mM TRIS, 150 mM NaCl, 3%
Triton X-100, 1 mM EGTA and 0.2 mM PMSF). Surface-labeled proteins were
isolated by incubation with avidin-conjugated Sepharose (Sigma) overnight at
4°C. After three washes, the surface proteins bound to the beads were
released by the addition of Laemmli sample buffer containing 12.5 mg/ml DTT
for 2 minutes at 95°C. To compare the effects of thrombin or TRAP
treatment on the amount of surface A2 that is labeled, the affinity-depleted
surface proteins were separated by SDS-PAGE (15% polyacrylamide), transferred
to polyvinyl difluoride (PVDF) membranes, and probed with anti-A2 mAb (Zymed;
0.05 ng/ml). After washing, the membranes were incubated with
peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch; 0.09
ng/ml), and the bands were detected by chemiluminesce (ECL; Pierce).
The identification of biotinylated surface p11 required an overnight incubation of solubilized cell supernatants at 4°C with non-immune mouse IgG1 (6.7 µg/ml) and then 2 hours at RT with protein A/G Sepharose to reduce nonspecific antibody-protein interactions. After centrifugation, p11 was depleted from clarified supernatants by incubation with anti-p11 mAb (Transduction Laboratories; 6.7 µg/ml) for 2 hours at RT followed by 2 hours with protein A/G Sepharose. As a control for specificity, identically treated cells were incubated with non-immune mIgG1 (6.7 µg/ml). The samples were then subjected to electrophoresis as above for A2. The biotinylated proteins were visualized by probing with horseradish peroxidase conjugated avidin (HRP-avidin) (Sigma; 33.3 ng/ml), followed by ECL detection. To confirm the identity of the observed bands, the blot was subsequently reprobed with anti-p11 (0.05 ng/ml).
Intracellular A2 and p11 detection
Fluorescence microscopy
To investigate effects on intracellular A2 and p11, thrombin or
TRAP-treated HUVECs were simultaneously fixed and permeabilized with 4%
paraformaldehyde, 0.25% gluteraldehyde and 0.2% Triton X-100 prior to
incubation with polyclonal goat anti-vimentin serum, anti-A2m mAb
(Transduction Laboratories), or anti-p11 mAb (Transduction Laboratories), all
at 2.5 µg/ml. The primary antibodies were probed with
Cy3-conjugated donkey anti-mouse IgG or FITC-conjugated rabbit
anti-goat IgG both at 1.25 µg/ml. Following DAPI treatment, the cells were
mounted and visualized as described above.
Western blots
After treatment with thrombin or TRAP, HUVECs grown in tissue culture
plates were washed twice with HBS and lysed with Laemmli sample buffer. The
proteins were separated by SDS-PAGE under reducing conditions and analyzed by
western blotting. A2m and p11 were stained as described for the surface
detection of these proteins. As before, the membranes were reprobed with
anti-actin mAb to ascertain whether constant amounts of cell extract were
electrophoresed.
Plasminogen cell binding and activation
Fluorescence microscopy
To visualize the effects of thrombin or TRAP on plasminogen binding to
HUVECs, cells were washed with HBS/BSA and incubated with F-Plg (150 nM) for
30 minutes at 37°C in the dark. After three washes with HBS/BSA, the cells
were fixed with 4% paraformaldehyde and 0.25% gluteraldehyde in HBS. The
nuclear DNA was then stained with DAPI, and the cells were mounted. A
requirement for p11 in F-Pg binding to cells was investigated by including
polyclonal rabbit anti-p11 antibody (16 µg/ml). As a control for antigen
specificity in these experiments, the effects of an identical amount of
non-immune rabbit IgG was also evaluated.
Avidin blots
To confirm the F-Pg experiments, after washing once with HBS/BSA, thrombin-
or TRAP-treated cells were incubated with B-Plg (150 nM) in HBS/BSA for 30
minutes at 37°C. Following the removal of unbound B-Pg by washing, the
clarified cell extracts were subjected to SDS-PAGE (10%). Biotinylated surface
p11 was detected as above with avidin-HRP. In each case, actin was used as a
control for consistency in the amount of cell extract loaded per lane.
Identical experiments were conducted with either unlabeled Glu-plasminogen
(1.5 µM) or -ACA (3 mM) being added to the B-Plg solutions as
competitive inhibitors.
Chromogenic assay
HUVECs grown to confluence in 48-well culture plates were treated with 8 nM
thrombin or 10 µM TRAP as described above. After recovery in
IMDM/Ca2+ for 30 minutes at 37°C, the cells were further
incubated with 5 nM recombinant tPA in HBS/BSA for 20 minutes. Glu- or
Lys-plasminogen (150 nM) was then added to the reaction mixture. Aliquots were
removed at various time points, and the amount of plasmin generated was
measured by cleavage of the chromogenic substrate S-2251. To control for
cellular tPA production or the presence of plasminogen in the culture media,
the experiment was repeated without exogenous tPA, plasminogen, or both. The
experiment was furthermore performed in the presence of the protease inhibitor
aprotinin [100 KIU (Kallikrein inactivator units)], to confirm that the
chromogenic activity observed was attenuated by a known plasmin inhibitor.
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Results |
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|
The finding that staining unfixed, native HUVECs must be conducted to
ensure the exclusive detection of surface A2 by immunofluorescence microscopy,
excluded using the p11 antibodies we evaluated. These gave an unacceptable
signal-to-background ratio when cells were not fixed. The exposure of HUVEC
surface p11 was consequently probed by chemically modifying native cells with
a hydrophilic amine-reactive derivative of biotin that cannot cross the plasma
membrane into the cell, as previously described
(Kassam et al., 1998). These
experiments confirmed that HUVEC surface A2m was increased by pretreatment of
the cells with thrombin or TRAP (Fig.
2). Furthermore, p11 was increased compared with mock-treated
cells, suggesting that A2t may be shuttled to the HUVEC surface after cell
stimulation by thrombin or TRAP. The identity of the biotinylated proteins was
determined antigenically and by electrophoretic migration compared with
purified A2 or p11 (data not shown).
|
Thrombin or TRAP enhance total HUVEC p11
As shown in Fig. 3A, the
effects of thrombin or TRAP on intracellular amounts of A2 and p11 were
evaluated by immunofluorescence microscopy of fixed and
detergent-permeabilized HUVECs. When HUVECs were pretreated with thrombin or
TRAP, the detection of p11 antigen was enhanced compared with mock-treated
cells. By contrast, the amount of A2 detected was not changed. No obvious
difference in the distribution of intracellular A2 was observed by this
method. Identically treated cells were stained for vimentin to ensure all
cells were equally permeabilized, and that thrombin or TRAP did not affect the
extent of permeabilization. The expected spindle pattern for vimentin was seen
for each cell. In addition, the corresponding isotype controls for thrombin-
or TRAP-treated cells were negative (data not shown), indicating that the
observed A2, p11 and vimentin staining required the respective specific
primary antibody. Visualization of nuclei with DAPI confirmed that a similar
number of cells per field were being evaluated.
|
To corroborate the effects of thrombin or TRAP on intracellular A2 or p11 observed by immunofluorescence microscopy, western blot analyses were conducted (Fig. 3B). This experiment also showed that both stimuli enhanced the amount of intracellular p11, while A2 levels remained unchanged. The PVDF membranes were subsequently reprobed for the cytoskeletal protein ß-actin, confirming that the same amount of cell extract was being loaded in each lane.
Thrombin or TRAP enhance submembranous A2
To determine if A2 colocalizes with submembranous cytoskeletal patches as
previously described for annexin I
(Traverso et al., 1998),
HUVECs were fixed with paraformaldehyde and gluteraldehyde in the absence of
detergent. These `pre-fixed' cells were simultaneously stained for A2,
vimentin and nuclear material, and evaluated by fluorescence microscopy.
Fig. 4 demonstrates that this
fixing protocol results in an obviously different distribution of vimentin and
A2 compared with the surface staining of unfixed cells
(Fig. 1). Here, patches of
vimentin were observed to colocalize with A2, which is highlighted by
overlaying the micrographs (Fig.
4). The absence of typical vimentin spindles seen for
detergent-permeabilized cells (Fig.
3) demonstrated that typical permeabilization did not occur and
was consistent with the previous well-established conclusion that these
represent submembranous domains (Traverso
et al., 1998
). After treatment with thrombin or TRAP, the number
and extent of these locales of vimentin and A2 were increased
(Fig. 4). Isotype controls
conducted on identically treated cells confirmed that all staining observed
was dependent on specific antibody-antigen interactions (data not shown).
|
Enhancement of plasminogen binding to thrombin- or TRAP-treated
HUVECs
A2 has been shown to accelerate plasmin generation by functioning as a
cell-surface coreceptor for plasminogen and tPA
(Hajjar et al., 1994;
Kassam et al., 1998
). To
determine whether thrombin- or TRAP-induced enhancement of surface A2 and/or
p11 could play a role in plasmin generation, we studied their effects on the
binding of fluorescein (F-Plg)- or biotin (B-Plg)-labeled recombinant
S741C-plasminogen. After thrombin or TRAP treatment of HUVECs
(Fig. 5A), F-Pg binding
followed by fluorescence microscopy was enhanced several fold compared with
the mock-treated cells. To correlate the increase in F-Plg binding to p11, a
rabbit anti-p11 polyclonal antibody was able to inhibit completely the
enhancement of F-Plg binding observed. At comparable concentrations of
non-immune rabbit IgG, or other p11 or A2m antibodies, F-Pg binding to the
treated cells was not affected (data not shown). Staining of nuclei with DAPI
demonstrated that similar numbers of cells were being evaluated in each field.
Since these inhibition experiments were performed using the conditions we
established for detection of surface antigens on HUVECs, they further support
the presence of p11 on the stimulated HUVEC surface.
|
To confirm the F-Plg fluorescence microscopy experiments, binding of B-Plg
to cells treated with thrombin or TRAP was evaluated by HRP-avidin blots
(Fig. 5B). We observed that
either thrombin or TRAP enhanced B-Plg binding to HUVECs compared with
mock-treated cells. The addition of -ACA, which inhibits the interaction
of plasminogen with essential C-terminal lysines on known receptors
(Miles et al., 1991
), or a
tenfold excess of unlabeled purified Glu-plasminogen completely inhibited
B-Plg binding (data not shown). Reprobing the PVDF membrane for ß-actin
served as a loading control and verified that comparable amounts of cell
extract were subjected to electrophoresis.
Plasmin generation on HUVECs is enhanced by thrombin
Having established that thrombin-mediated stimulation of HUVECs can enhance
plasminogen binding, we investigated whether the effects translated to
increased plasmin generation on the HUVEC surface. tPA-mediated activation of
the Lys (Fig. 6A) or Glu forms
of plasminogen to plasmin and was measured using a chromogenic substrate
specific for plasmin (Fig. 6B).
These experiments demonstrated a significant acceleration of plasmin
generation on the HUVEC surface due to prior treatment with thrombin. The
omission of tPA or plasminogen prevented generation of chromogenic activity,
indicating that the cells were not secreting detectable amounts of tPA, and
that plasmin generation was dependent solely on exogenous tPA and plasminogen
(data not shown). The addition of aprotinin, an effective inhibitor of
plasmin, blocked all chromogenic activity observed (data not shown), a further
indicator that the assay was specific for plasmin generation.
|
![]() |
Discussion |
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Using identical conditions that were found by immunofluorescence microscopy
to ensure no inadvertant permeabilization of cells, surface biotinylation of
HUVECs confirmed that thrombin treatment resulted in an increase to the amount
of A2 on the HUVEC surface. Furthermore, this method enabled us to conclude
that HUVEC surface p11 was concomitantly increased due to thrombin-mediated
effects. Since A2 is required to anchor p11 to the cell membrane
(Zobiack et al., 2001), our
observation of enhanced p11 implies that the tetrameric form of A2 (i.e. A2t)
is being increased on the cell surface by thrombin.
Thrombin triggers transmembrane signaling through proteolytic stimulation
of the PAR family of cell-surface receptors
(Coughlin, 2001). To determine
whether PAR-1 participates in the effects of thrombin that we observed on A2
surface exposure, a well-characterized peptide corresponding exclusively to
the PAR-1 tethered receptor ligand, SFLLRN (TRAP), was used to treat HUVECs
instead of thrombin. By immunofluorescence or surface biotinylation of
TRAP-treated cells, we found an enhancement of A2 and p11 on the HUVEC
surface, demonstrating that at least PAR-1 is involved.
To determine whether thrombin-mediated translocation of A2 and p11 to the cell surface was concurrent with antigenic changes inside of cells, immunofluorescence microscopy of fixed and permeabilized cells and western analysis of total cell extracts were conducted. These experiments revealed that, whereas thrombin has no effect on the amount of A2, p11 is increased significantly. This finding suggests that levels of intracellular p11 might control the extent of transmembrane trafficking.
Neither A2 nor p11 contain secretory signals, therefore the finding of
either on the cell surface is intriguing since the mechanism by which they are
trafficked to the cell surface is not known and cannot involve the typical
endoplasmic reticulum-dependent pathway
(Siever and Erickson, 1997).
The observations presented here document the only known trigger for
redistribution of A2 and p11 to the HUVEC exterior and provide insight into
the mechanism since thrombin induces signaling through G proteins. Like
thrombin, the effects of nicotine (Zheng
et al., 1997
) and nerve growth factor
(Rakhit et al., 2001
) on
certain cells have been suggested to involve functions of G proteins.
Combining the current thrombin studies with reports that: (1) nicotine
stimulation of chromaffin cells causes translocation of A2 from the cytosol to
caveolar domains (Sagot et al.,
1997
); (2) A2 has been identified as a component of caveolae
(Stahlhut et al., 2000
;
Harder and Gerke, 1994
;
Stan et al., 1997
); (3)
cell-surface A2 has been colocalized with caveolin
(van der Goot, 1997
); and (4)
nerve growth factor induces exposure of A2 on the neurite surface
(Jacovina et al., 2001
), we
propose that A2 transport to the cell surface may involve G-protein-linked
control of caveolae.
To add credence to the hypothesis that submembranous structures may be
involved in the transmembrane trafficking of A2, we utilized a cell-fixation
method in the absence of detergent that previously enabled identification by
electron and immunofluorescence microscopy of distinct regions proposed to
occur at junctions between the cytoskeleton and the plasma membrane
(Traverso et al., 1998). This
fixation method showed that thrombin stimulation enhances the HUVEC
submembranous A2, which colocalized with the intermediate cytoskeletal
component vimentin. The thrombin-mediated increase in intracellular p11 we
observed might in part explain the elevation of submembranous A2 by
facilitating A2t formation and consequent shuttling to the submembrane, as
demonstrated recently for HepG2 cells
(Zobiack et al., 2001
). The
probability that submembranous A2t would be preferentially translocated to the
extracellular surface compared with cytosolic A2m helps to rationalize the
previous conclusion that A2t represents much of the cell-surface A2
(Kassam et al., 1998
).
A2 is recognized as an endothelial cell-surface coreceptor for tPA and
plasminogen, and a cofactor in the tPA-dependent conversion of plasminogen to
plasmin. Although the ability of A2 to bind plasminogen is well documented,
there is controversy in the literature over whether A2m
(Cesarman et al., 1994;
Hajjar et al., 1994
;
Hajjar et al., 1998
;
Kang et al., 1999
) or A2t
(Choi et al., 1998
;
Liu et al., 1995
) is the
functional form on the HUVEC surface. Our observations that both A2 and p11
antigen were increased on the HUVEC surface after thrombin treatment led us to
test whether this effect correlated to enhanced fibrinolytic cell-surface
activity. As predicted, greater binding of fluorescein- or biotin-labeled
plasminogen to the cell surface was observed after thrombin or TRAP-mediated
HUVEC stimulation. Like previous experiments with purified A2
(Cesarman et al., 1994
;
Kang et al., 1999
), we found
-ACA inhibited plasminogen binding to HUVECs. Because thrombin might
enhance numerous putative plasminogen receptors on the cell surface,
immuno-inhibition was conducted. Our observation that polyclonal anti-p11
antibody and not non-immune control antibody inhibited the thrombin- or
TRAP-mediated increase in F-Plg binding confirmed the importance of p11.
Although we were unable to identify an antibody specific for A2 that was
inhibitory (data not shown), we cannot exclude the possible simultaneous
direct involvement of A2m or the A2 subunit of A2t. However, a role for p11 is
strongly supported to explain our additional finding that thrombin
pretreatment of HUVECs enhances tPA-dependent plasmin generation on the cell
surface.
Cumulatively, the data presented here are consistent with a model where
stimulation of HUVECs by thrombin enhances the accessibility of A2 and p11 on
the cell surface. Once on the surface of the cell, at least the p11 subunit of
A2t functions as a plasminogen receptor to accelerate plasmin generation by
tPA. Thus, a novel feedback regulatory step in hemostasis is indicated that
links thrombin, the biological mediator of coagulation, to enhanced cellular
production of the fibrinolytic enzyme, plasmin. Cell-surface A2 has been
suggested to have importance in several processes, such as tenascein C-binding
(Chung and Erickson, 1994),
tumor invasion (Mai et al.,
2000
) and cytomegalovirus infection
(Raynor et al., 1999
;
Wright et al., 1995
).
Therefore, the finding that exposure of cell-surface A2 can be induced by
thrombin might have implications in areas additional to fibrinolysis.
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Acknowledgments |
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References |
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---|
Ali, S. M., Geisow, M. J. and Burgoyne, R. D. (1989). A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells. Nature 340,313 -315.[CrossRef][Medline]
Bartha, K., Muller-Peddinghaus, R. and van Rooijen, L. A. A. (1989). Bradykinin and thrombin effects on polyphosphoinositide hydrolysis and prostacyclin production in endothelial cells. Biochem. J. 263,149 -155.[Medline]
Brass, L. F. and Molino, M. (1997). Protease-activated G protein-coupled receptors on human platelets and endothelial cells. Thromb. Haemost. 78,234 -241.[Medline]
Brock, T. A. and Capasso, E. L. (1989).
GTPS increases thrombin-mediated inositol triphosphate accumulation in
permeabilized human endothelial cells. Am. Rev. Respir.
Dis. 140,1121
-1125.[Medline]
Cesarman, G. M., Guevara, C. A. and Hajjar, K. A.
(1994). An endothelial cell receptor for plasminogen/tissue
plasminogen activator: Annexin II-mediated enhancement of tPA-dependent
plasminogen activation. J. Biol. Chem.
269,21198
-21203.
Choi, K. S., Ghuman, J., Kassam, G., Kang, H. M., Fitzpatrick, S. L. and Waisman, D. M. (1998). Annexin II tetramer inhibits plasmin-dependent fibrinolysis. Biochemistry 37,648 -655.[CrossRef][Medline]
Chung, C. Y. and Erickson, H. P. (1994). Cell surface annexin II is a high affinity receptor for tenascin-C. J. Cell Biol. 126,539 -548.[Abstract]
Coughlin, S. R. (2001). Protease-activated receptors in vascular biology. Thromb. Haemost. 86,298 -307.[Medline]
Courtneidge, S., Ralston, R., Alitalo, K. and Bishop, J. M. (1983). Subcellular localization of an abundant substrate (p36) for tyrosine-specific protein kinases. Mol. Cell. Biol. 3,340 -350.[Medline]
Drust, D. S. and Creutz, C. E. (1988). Aggregation of chromaffin granules by calpactin at micromolar levels of calcium. Nature 331,88 -91.[CrossRef][Medline]
Dudani, A. K., Hashemi, S., Aye, M. T. and Ganz, P. R. (1991). Identification of an endothelial cell surface protein that binds plasminogen. Mol.Cell. Biochem. 108,133 -139.[Medline]
Erikson, E., Tomasiewicz, H. G. and Erikson, R. L. (1984). Biochemical chracteriszation of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein. Mol. Cell. Biol. 4,77 -85[Medline]
Garcia, J. G. N., Patterson, C., Bahler, C., Aschner, J., Hart, C. M. and English, D. (1993). Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet-derived growth factor mRNA expression in cultured endothelium. J. Cell Physiol. 156,541 -549.[Medline]
Garcia, J. G. N., Pavalko, F. M. and Patterson, C. E. (1995). Vascular endothelial cell activation and permeability responces to thrombin. Blood Coagul. Fibrinolysis 6, 609-626.[Medline]
Gerke, V. and Weber, K. (1984). Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders: calcium-dependent binding to non-erythroid spectrin and F-actin. EMBO J. 3, 227-233.[Abstract]
Gerke, V. and Moss, S. E. (1997). Annexins and membrane dynamics. Biochim. Biophys. Acta 1357,129 -154.[Medline]
Glenney, J. (1986). Phospholipid dependent
Ca2+ binding by the 36 kDa tyrosine kinase substrate (calpactin)
and its 33-kDa core. J. Biol. Chem.
261,7247
-7252.
Glenney, J. R., Boudreau, M., Galyean, R., Hunter, T. and Tack,
B. (1986). Association of the S-100-related calpactin I light
chain with the NH2-terminal tail of the 36-kDa heavy chain. J.
Biol. Chem. 261,10485
-10488.
Hajjar, K. A., Guevara, C. A., Lev, E., Dowling, K. and Chacko,
J. (1996). Interaction of the fibrinolytic receptor, annexin
II, with the endothelial cell surface: Essential role of endonexin repeat 2.
J. Biol. Chem. 271,21652
-21659.
Hajjar, K. A., Mauri, L., Jacovina, A. T., Zhong, F., Mirza, U.
A., Padovan, J. C. and Chait, B. T. (1998). Tissue
plasminogen activator binding to the annexin II tail domain. Direct modulation
by homocysteine. J. Biol. Chem.
273,9987
-9993.
Hajjar, K. A., Jacovina, A. T. and Chacko, J.
(1994). An endothelial cell receptor for plasminogen/tissue
plasminogen activator: identity with annexin II. J. Biol.
Chem. 269,21191
-21197.
Harder, T. and Gerke, V. (1994). The annexin II2p112 complex is the major protein component of the triton X-100-insoluble low-density fraction prepared from MDCK cells in the presence of calcium. Biochim. Biophys. Acta 1223,375 -382.[CrossRef][Medline]
Horrevoets, A. J. G., Pannekoek, H. and Nesheim, M. E.
(1997). Production and characterization of recombinant human
plasminogen (S741C-fluorescein): A novel approach to study zymogen activation
without generation of active protease. J. Biol. Chem.
272,2176
-2182.
Hou, L., Howells, G. L., Kapas, S. and Macey, M. G. (1998). The protease-activated receptors and their cellular expression and function in blood related cells. Br. J. Haematol. 101,1 -9.[CrossRef][Medline]
Ishihara, H., Connolly, A. J., Zeng, D., Kahn, M. L., Zheng, Y. W., Timmons, C., Tram, T. and Coughlin, S. R. (1997). Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386,502 -506.[CrossRef][Medline]
Jacovina, A. T., Zhong, F., Khazanova, E., Lev, E., Deora, A. B.
and Hajjar, K. A. (2001). Neuritogenesis and the nerve growth
factor-induced differentiation of PC-12 cells requires annexin II-mediated
plasmin generation. J. Biol. Chem.
276,49350
-49358.
Jaffe, E. A., Nachman, R. I., Becker, C. G. and Minick, C. R. (1973). Culture of human endothelial cells derived from human umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest. 52,2745 -2756.[Medline]
Jamieson, G. A. (1997). Pathophysiology of platelet thrombin receptors. Thromb. Haemost. 78,242 -246.[Medline]
Johnsson, N., Vandekerckhove, J., Van-Damme, J. and Weber, K. (1986). Binding sites for calcium, lipid, and p11 on p36, the substrate of retrobiral tyrosine-specific protein kinases. FEBS Lett. 198,361 -364.[CrossRef][Medline]
Johnsson, N., Marriott, G. and Weber, K. (1988). p36, the major cytoplasmic substrate of src tyrosine protein kinase, binds to its p11 regulatory subunit via a short amino-terminal amphiphatic helix. EMBO J. 7,2435 -2442.[Abstract]
Kahn, M. L., Zhen, Y. W., Huang, W., Bigornia, V., Zeng, D., Moff, S., Farese, R. V. J., Tam, C. and Coughlin, S. R. (1998). A dual thrombin receptor for platelet activation. Science 394,690 -694.[CrossRef]
Kang, H. M., Choi, K. S., Kassam, G., Fitzpatrick, S. L., Kwon, M. and Waisman, D. M. (1999). Role of annexin II tetramer in plasminogen activation. Trends Cardiovasc. Med. 9, 92-102.[CrossRef][Medline]
Kang, H. M., Kassam, G., Jarvis, S. E., Fitzpatrick, S. L. and Waisman, D. M. (1997). Characterization of human recombinant annexin II tetramer purified from bacteria: Role of N-terminal acetylation. Biochemistry 36,2041 -2050.[CrossRef][Medline]
Kassam, G., Choi, K. S., Ghuman, J., Kang, H. M., Fitzpatrick,
S. L., Zackson, T., Zackson, S., Toba, M., Shinomiya, A. and Waisman, D.
M. (1998). The role of annexin II tetramer in the activation
of plasminogen. J. Biol. Chem.
273,4790
-4799.
Kim, J. and Hajjar, K. A. (2002). Annexin II: a plasminogen-plasminogen activator co-receptor. Front. Biosci. 7,d341 -d348.[Medline]
Liu, L., Fisher, A. B. and Zimmerman, U. J. (1995). Lung annexin II promotes fusion of iolated lamellar bodies with liposomes. Biochim. Biophys. Acta 1259,166 -172.[Medline]
Magazine, H. I., King, J. M. and Srivastava, K. D. (1996). Protease activated receptors modulate aortic vascular tone. Int. J. Cardiol. 53,S75 -S80.[CrossRef][Medline]
Mai, J., Waisman, D. M. and Sloane, B. F. (2000). Cell surface complex of cathepsin B/annexin II tetramer in malignant progression. Biochim. Biophys. Acta 1477,215 -230.[Medline]
Menell, J. S., Cesarman, G. M., Jacovina, A. T., McLaughlin, M.
A., Lev, E. A. and Hajjar, K. A. (1999). Annexin II and
bleeding in acute promyelocytic leukemia. N. Engl. J.
Med. 340,994
-1004.
Miles, L. A., Dahlberg, C. M., Plescia, J., Felez, J., Kato, K. and Plow, E. F. (1991). Role of cell-surface lysines in plasminogen binding to cells: Identification of alpha-enolase as a candidate plasminogen receptor. Biochemistry 30,1682 -1691.[Medline]
Molino, M., Woolkalis, M. J., Reavy-Catewell, J., Pratico, D.,
Andrade-Gordon, P., Barnathan, E. S. and Brass, L. F. (1997).
Endothelial cell thrombin receptors and PAR-2: Two protease-activated
receptors located in a single cellular environment. J. Biol.
Chem. 272,11133
-11141.
Muramatsu, I., Laniyonu, A., Moore, G. J. and Hollenberg, M. D. (1992). Vascular actions of thrombin receptor peptide. Can. J. Physiol. Pharmacol. 70,996 -1003.[Medline]
Osborn, M., Johnsson, N., Wehland, J. and Weber, K. (1988). The submembranous location of p11 and its interaction with the p36 substrate of pp60 src kinase in situ. Exp. Cell Res. 175,81 -96.[Medline]
Pollock, W. K., Wreggett, K. A. and Irvine, R. F. (1988). Inositol phosphate production and Ca2+ mobilization in human umbilical-vein endothelial cells stimulated by thrombin and histamine. Biochem. J. 256,371 -376.[Medline]
Powell, M. A. and Glenney, J. R., Jr (1987). Regulation of calpactin I phospholipid binding by calpactin I light chain binding and phosphorylation by p60v-src. Biochem. J. 247,321 -328.[Medline]
Rakhit, S., Pyne, S. and Pytela, R. (2001).
Nerve growth factor stimulation of p42/p44 mitogen-activated protein kinase in
PC12 cells: role of G(i/o), G protein-coupled receptor kinase 2, beta-arrestin
I, and endocytic processing. Mol. Pharmacol.
60, 63-70.
Rand, J. H. (2000). The annexinopathies: a new category of diseases. Biochim. Biophys. 1489,169 -173.
Rasmussen, U. B., Vouret-Craviari, V., Jallat, S., Schlesinger,
Y., Pages, G., Pavirani, A., Lecocq, J.-P., Pouysségur, J. and van
Obberghen-Schilling, E. (1991). cDNA cloning and expression
of a hamster -thrombin receptor coupled to Ca2+
mobilization. FEBS Lett.
288,123
-128.[CrossRef][Medline]
Raynor, C. M., Wright, J. F., Waisman, D. M. and Pryzdial, E. L. G. (1999). Annexin II enhances cytomegalovirus binding and fusion to phospholipid membranes. Biochemistry 38,5089 -5095.[CrossRef][Medline]
Regnouf, F., Sagot, I., Delouche, B., Devilliers, G., Cartaud,
J., Henry, J.-P. and Pradel, L.-A. (1995). `In vitro'
phosphorylation of annexin II tetramer by protein kinase C: Comparative
properties of the unphosphoryated and phosphorylated annexin II on the
aggregation and fusion of chromaffin granule membranes. J. Biol.
Chem. 270,27143
-27150.
Sagot, I., Regnouf, F., Henry, J. P. and Pradel, L. A. (1997). Translocation of cytosolic annexin 2 to a triton-insoluble membrane subdomain upon nicotine stimulation of chromaffin cultured cells. FEBS Lett. 410,229 -234.[CrossRef][Medline]
Schini, V. B., Hendrickson, H., Heublein, D. M., Burnett, J. C. J. and Vanhoutte, P. M. (1989). Thrombin enhances the release of endothelin from cultured porcine aortic endothelial cells. Eur. J. Pharmacol. 165,333 -334.[CrossRef][Medline]
Siever, D. A. and Erickson, H. P. (1997). Extracellular annexin II. Int. J. Biochem. Cell Biol. 29,1219 -1223.[CrossRef][Medline]
Stahlhut, M., Sandvig, K. and van Deurs, B. (2000). Caveolae: Uniform structure with multiple functions in signalling, cell growth, and cancer. Exp. Cell Res. 261,111 -118.[CrossRef][Medline]
Stan, R. V., Roberts, W. G., Predescu, D., Ihida, K., Saucan, L., Ghitescu, L. and Palade, G. E. (1997). Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae). Mol. Biol. Cell 8,595 -605.[Abstract]
Sugama, Y. and Malik, A. B. (1992). Thrombin receptor-14 amino acid peptide mediates endothelial hyperadhesivity and neutrophil adhesion by P-selectin dependent mechanism. Circ. Res. 71,1015 -1019.[Abstract]
Swairjo, M. A. and Seaton, B. A. (1994). Annexin structure and membrane interactions: a molecular perspective. Annu. Rev. Biophys. Biomol. Struct. 23,193 -213.[CrossRef][Medline]
Tesfamariam, B., Allen, G. T., Normandin, A. and Antonaccio, M.
J. (1993). Involvement of the `tethered ligand' receptor in
thrombin-induced endothelium-mediated relaxations. Am. J. Physiol.
Heart Circ. Physiol. 265,H1744
-H1749.
Thiel, C., Osborn, M. and Gerke, V. (1992). The
tight association of the tyrosine kinase substrate annexin II with the
submembranous cytoskeleton depends on intact p11- and Ca(2+)-binding sites.
J. Cell Sci. 103,733
-742.
Tiruppathi, C., Lum, H., Andersen, T. T., Fenton, J. W. I. and
Malik, A. B. (1992). Thrombin receptor 14 amino acid
peptide binds to endothelial cells and stimulates calcium transients.
Am. J. Physiol. Lung Mol. Physiol.
263,L595
-L601.
Traverso, V., Morris, J. F., Flower, R. D. and Buckingham,
J. (1998). Lipocortin 1 (annexin 1) in patches associated
with the membrane of a lung adrenocarcinoma cell line and in the cell
cytoplasm. J. Cell Sci.
111,1405
-1418.
van der Goot, F. G. (1997). Separation of early steps in endocytic membrane transport. Electrophoresis 18,2689 -2693.[Medline]
Vu, T.-K., Hung, D. T., Wheaton, V. I. and Coughlin, S. R. (1991). Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64,1057 -1068.[Medline]
Waisman, D. M. (1995). Annexin II tetramer: structure and function. Mol. Cell. Biochem. 149/150,301 -322.
Wright, J. F., Kurosky, A., Pryzdial, E. L. G. and Wasi, S. (1995). Host cellular protein annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts. J. Virol. 69,4784 -4791.[Abstract]
Zheng, J., Zhang, P. and Hexum, T. D. (1997).
Neuropeptide Y inhibits chromaffin cell nicotinic receptor-stimulated tyrosine
hydroxylase activity through a receptor-linked G protein-mediated process.
Mol. Pharmacol. 52,1027
-1033.
Zobiack, N., Gerke, V. and Rescher, U. (2001). Complex formation and submembranous localization of annexin 2 and S100A10 in live HepG2 cells. FEBS Lett. 500,137 -140.[CrossRef][Medline]