© 1995 American Heart Association, Inc.
Articles |
Thrombin Decreases the Urokinase Receptor and Surface-Localized Fibrinolysis in Cultured Endothelial Cells
From the Division of Cardiovascular Disease, Department of Medicine, University of Alabama, Birmingham.
Correspondence to Francois M. Booyse, PhD, Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, 809 BBRB, 845 19th St S, Birmingham, AL 35294-2170.
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Abstract The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (<5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluorophosphate–tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase–polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5x104 cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C–treated human aortic cultures yielded
10- and
12-fold more 55-kD Mr and 6-fold more
35-kD Mr 35S-labeled u-PAR forms
than control cultures, respectively. The u-PAR antigen forms
(Mr, 54 kD) and the
glycosyl-phosphatidylinositol–anchored protein CD59
(Mr, 20 kD) were also simultaneously
identified by immunoprecipitation in the media of thrombin-treated
cultures. This suggests that thrombin may release u-PAR and decrease
u-PA ligand binding through a common pathway involving phospholipase C.
These results establish a novel interrelation between thrombin and EC
fibrinolysis and suggest that thrombin may also have an additional
regulatory role in the net expression of surface-localized EC
fibrinolytic activity.
Key Words: thrombin • endothelial urokinase receptor • fibrinolysis • plasmin • ligand binding
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Introduction |
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Maintaining a balance between coagulation and fibrinolytic systems preserves normal hemostasis. Endothelial cells (ECs) play a major role in maintaining this balance by synthesizing both coagulation and fibrinolytic proteins and by providing a surface for the assembly, interaction, and activation of these proteins. ECs are able to achieve this high degree of regulation through the expression of surface binding sites or receptors for fibrinolytic and coagulant proteins. The urokinase-type plasminogen activator receptor (u-PAR) is a key component of the EC regulation of fibrinolysis, which specifically localizes and enhances u-PA plasminogen activation on the EC surface.1 The u-PAR is a 55-kD Mr glycoprotein that is reduced to 35 kD after deglycosylation and is attached to the plasma membrane by a unique glycosyl-phosphatidylinositol anchor. The u-PAR expression can in turn be influenced by a variety of agents. Phorbol myristate acetate increases u-PAR synthesis in smooth muscle cells (SMCs) and U937 cells.2 3 4 Forskolin increases u-PAR expression in human umbilical vein endothelial cells (HUVECs) through a cAMP-mediated process.5 Cytokines,6 eg, interferon and tumor necrosis factor, and growth factors, eg, basic fibroblast growth factor,7 have been shown to increase u-PAR expression in cultured human monocytes and HUVECs, respectively.
Thrombin has profound effects on the EC-mediated regulation of coagulation and fibrinolysis. Thrombin promotes clot formation by stimulating EC production of platelet activating factor8 and thromboplastin9 and by inducing EC release of factor VIII.10 Conversely, thrombin also has important anticoagulant properties. It induces EC release of prostacyclin11 and nitric oxide12 13 14 and interacts with EC-bound thrombomodulin to promote the activation of protein C, an inhibitor of factors VIII and V.15 The regulatory role of thrombin in EC fibrinolysis has also been the subject of intense interest. Thrombin has been shown to induce the secretion of tissue-type plasminogen activator (TPA),16 u-PA,17 18 and plasminogen activator inhibitor–1(PAI-1)19 from ECs. Recent reports have also shown that thrombin can increase the expression of u-PAR in various SMC lines.2 Surprisingly, only a few studies describe the influence of thrombin on EC-related u-PAR expression.20 Given the significant role that u-PAR may have in regulating EC surface-localized fibrinolysis, it would be important to define the specific effects of thrombin on u-PAR and surface-localized fibrinolytic activity in ECs. In these studies, we describe the effects of thrombin on u-PA ligand binding and expression of u-PAR activity and mRNA levels in different cultured EC types.
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Materials
The binding domain peptide for single-chain urokinase-type PA (scu-PA) (residues 4 through 43) was provided by Dr J. Henken and A. Mazar (Abbott Laboratories). tcu-PA was obtained from The Green Cross Corp; monoclonal antibody to u-PAR from American Diagnostica, Inc; monoclonal antibody to CD59 from Serotec; cyanogen bromide (CNBr)–activated Sepharose CL-4B and Sephadex G-25 column (PD-10) from Pharmacia, Inc; Glu-plasminogen (Glu-Pmg) from Enzyme Research Products, Inc; collagenase (type I, CLS) from Boehringer Mannheim Biochemicals; fetal bovine serum (FBS) from Intergen Corp; human thrombin, heparin (porcine intestinal mucosa), hirudin, diisopropylfluorophosphate (DFP), phospholipase C (phosphatidylinositol specific, PI-PLC), and bovine serum albumin (BSA) from Sigma Chemical Co; chromogenic substrates for plasmin (S-2251) and thrombin (S-2238) from Chromogenix; human fibrinogen from KabiVitrum; aprotinin (Trasylol) from Mobay Corp; Na125I (specific activity, 14.0 mCi/µg) and L-[35S]methionine (specific activity, >1000 Ci/mmol) from Amersham Corp; [35S]methionine-cystine (specific activity, 1150 Ci/mmol) and methionine-cystine–free RPMI from ICN Biomedicals, Inc; p-aminobenzamidine–agarose and Iodo-Beads from Pierce Chemical Co; acetylated LDL labeled with 1,1'-dioctadecyl-1-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI-Ac-LDL) from Biomedical Technologies, Inc; TRIzol Reagent (total RNA isolation reagent) and Medium-199 (M199) from GIBCO BRL; M-MLV reverse transcriptase (RT) and random primer from Promega, Inc; and Gene Amp PCR Core Reagents Kit from Perkin Elmer. Thrombin receptor activation peptide SFLLRNP and inactive control peptide FSLLRNP were kindly provided by Dr Davell H. Carney, University of Texas Medical Branch at Galveston, Department of Human Biological Chemistry and Genetics.
Cell Culture
HUVECs, human aortic ECs (HAECs), and human saphenous vein ECs
(HSVECs) were obtained from fresh cords, thoracic aortas (3 to 5 in.)
from human organ donors (Alabama Organ Center), and surgical specimens
left over after completion of coronary artery bypass surgery,
respectively, by mild collagenase treatment.21 All human
EC types were seeded into human fibronectin-coated plastic Petri dishes
(9.6 cm2) and grown to confluence in complete culture
medium consisting of M199, 0.025 mol/L HEPES buffer, pH 7.4, 0.002
mol/L fresh L-glutamine, 100 U/mL penicillin, 100 µg/mL
streptomycin, 10% heat-deactivated FBS, 90 µg/mL heparin, and 50
µg/mL EC growth factor, as we described previously for
HUVECs.1 Cultures were refed every 48 hours with complete
medium and maintained in a 95% air–5% CO2 humidified
atmosphere. Porcine aortic ECs (PAECs) were obtained from fresh
slaughterhouse aortas by mild collegenase treatment as we described
previously and cultured in the same medium as described above for
HUVECs, minus heparin and EC growth factor. All experiments were
carried out with postconfluent (2 to 3 days after reaching their stable
confluency density), pooled first- or second-passage cultured ECs
(HUVECs, three to five cords; HAECs, two aortas; HSVECs, three to four
veins; and PAECs, four to six aortas) to minimize individual vessel
variability.
Ligand binding studies and mRNA analysis were carried out with confluent cultures grown in 24-well multiwell plastic plates (2 cm2 per well) and fibrinolytic activity assays with confluent cultures in 96-well multiwell plastic plates (0.33 cm2 per well). Cells were counted with phase microscopy and a 1.0x1.0-mm counting reticle.
Cultured cells were characterized as ECs by their content of immunologically identifiable von Willebrand factor antigen (immunofluorescence staining), uptake of the fluorescent probe DiI-Ac-LDL,22 and their typical monolayer "cobblestone" tight-packing morphology.22 23 24 25 26 27 28 29
Treatment of tcu-PA and Thrombin With DFP
Purified tcu-PA (10 to 20 µg) or thrombin (10 to 20 µg) in
0.2 mL of phosphate-buffered saline (PBS) was treated with DFP (0.01
mol/L, final) at 4°C for 6 hours. The DFP–tcu-PA was then passed
through a p-aminobenzamidine–agarose column to remove any
remaining active tcu-PA, and the absence of active tcu-PA in the
follow-through solution containing the DFP–tcu-PA was confirmed by the
absence of measurable fibrinolytic activity with the chromogenic
plasmin substrate S-2251. The absence of active thrombin in the
DFP-thrombin was confirmed by use of the chromogenic thrombin substrate
S-2238.
Iodination of tcu-PA, DFP–tcu-PA, and Glu-Pmg
Purified high-molecular-weight (54 kD) tcu-PA or DFP–tcu-PA (20
to 50 µg) in 0.2 mL of Dulbecco's PBS (DPBS) and purified human
Glu-Pmg (100 µg) in 0.25 mL of DPBS were iodinated with 250 to 300
µCi of Na125I by the Iodo-Bead method
(Pierce).30 The reaction was terminated by removal of the
Iodo-Beads from the sample, and the free iodine was removed by
gel-filtration chromatography with a Sephadex G-25 column. Specific
activities of the 125I-labeled proteins were determined at
1.5 to 2.0x106 cpm/µg and 1.6 to
2.0x106 cpm/µg for plasminogen activators and
Glu-Pmg, respectively.
Treatment of Cultured EC Types With Thrombin, DFP-Thrombin, and
Thrombin Receptor Activation Peptide
Confluent cultured ECs were washed three times with warm
(37°C) DPBS containing 1% BSA (DPBS-BSA) and treated with thrombin
or DFP-thrombin (0.1 to 8 U/mL), thrombin receptor activation peptide
SFLLRNP, or inactive control peptide FSLLRNP (50 to 200 µmol/L) at
37°C for various times (0 to 30 minutes). Pretreated cultures were
then washed (three times) with DPBS-BSA and used immediately for
125I-labeled tcu-PA or 125I-labeled DFP–tcu-PA
ligand binding, tcu-PA activity, and fibrinolytic assays or were refed
complete culture medium for subsequent 125I-labeled tcu-PA
ligand binding assays and RT–polymerase chain reaction (RT-PCR)
analysis (see below).
Binding of 125I-Labeled tcu-PA and DFP–tcu-PA to
Cultured ECs
The binding of 125I-labeled tcu-PA to cultured ECs
was carried out in 24-well multiwell plates, as we previously
described.1 Briefly, confluent cultured EC monolayers were
washed (three times) with DPBS-BSA. Various concentrations or
saturation levels (2 nmol/L) of 125I-labeled tcu-PA were
added and incubated at 4°C for various time periods (0 to 30 minutes)
with continuous gentle shaking. To determine the nonspecific binding of
125I-labeled ligand, a 50-fold molar excess of unlabeled
tcu-PA was added simultaneously with the 125I-labeled
ligand to parallel wells. Supernatants from individual wells were
removed, and the cell monolayers washed (five times) with DPBS-BSA to
remove unbound 125I-labeled ligand. Cell monolayers in
individual wells were then solubilized with 1% sodium dodecyl sulfate
(SDS) (wt/vol), 0.5 mol/L NaOH, and 0.01 mol/L EDTA,1 and
cell-associated radioactivity was determined by a gamma counter.
Specific binding was calculated by subtracting the nonspecific
cell-associated radioactivity from its respective total cell-associated
radioactivity. These specific binding data were analyzed with the
LIGAND program of Munson and Rodbard21 for
estimating ligand binding parameters for a one-model system. This
program provided estimates of the dissociation constant
(Kd) and the number of binding sites per cell
(Bmax) for each ligand.
125I-labeled DFP–tcu-PA ligand binding studies were carried out as described for 125I-labeled tcu-PA ligand binding above to determine whether the observed decrease in binding of active 125I-labeled tcu-PA ligand to thrombin-treated ECs might be due to the rapid formation of tcu-PA-PAI-1 complexes rather than the actual decrease in u-PAR binding sites.
Studies were also carried out with acid-treated control after thrombin-treated PAEC cultures by use of 0.1 mol/L glycine buffer (pH 2.2) for 3 minutes at 4°C to remove bound u-PA ligand2 to determine whether occupancy of u-PAR binding sites by thrombin-induced, endogenously released u-PA might contribute to the observed decrease in binding of 125I-labeled tcu-PA.
To determine whether tcu-PA may directly cleave the u-PAR ligand binding domain from the cell surface, PAEC cultures were washed with DPBS-BSA, one culture was preincubated with buffer (control), and two cultures were incubated with tcu-PA (2 nmol/L) at 37°C. After various incubation times (0 to 30 minutes), cultures were washed (three times) with DPBS-BSA; the control and one of the tcu-PA–treated cultures were treated with acid. 125I-labeled tcu-PA ligand binding was then carried out with the two acid-treated and one remaining tcu-PA–treated cultures, as described above.
Ligand Affinity Isolation of the
[35S]Methionine-Labeled u-PAR
Confluent cultured PAECs (three T-25 flasks) were washed with
DPBS containing 0.25% BSA (DPBS-BSA), incubated with methionine-free
M199 containing 5% FBS for 12 hours and [35S]methionine
(333 µCi per flask), and then added to the medium. After incubation
for 16 hours, the media from each of the three flasks were aspirated
and the cell monolayers were washed (three times) with DPBS-BSA. The
washed cultures in DPBS-BSA were then incubated with thrombin (2 U/mL),
phospholipase C (1.5 U/mL), or DPBS-BSA for only 30 minutes, and the
[35S]methionine-labeled u-PAR was isolated by affinity
chromatography, as we previously described in detail.1 The
medium containing [35S]methionine-labeled proteins from
each flask was individually collected, Aprotinin (300 KIU/mL) and EDTA
(0.01 mol/L, final) were immediately added, and centrifugation was
carried out at 1500g for 5 minutes at 4°C to remove intact
cells and cellular debris. Each of the
[35S]methionine-labeled supernatants (3 mL) was then
preadsorbed onto Sepharose CL-4B (precolumn, 1x5 cm) to remove
proteins that nonspecifically bound to the Sepharose CL-4B. Each
flow-through volume was collected, and then affinity was adsorbed onto
three separate immobilized DFP–tcu-PA–Sepharose CL-4B columns (1x3
cm) at 4°C. The columns were washed with 50 mL of 0.1 mol/L phosphate
buffer, pH 7.0, containing 0.5 mol/L NaCl, 0.01% Triton X-100, and 50
KIU/mL aprotinin, followed by 50 mL of the same buffer containing only
0.01% Triton X-100. Elution of the
[35S]methionine-labeled u-PAR proteins was carried out
with 1x10-6 mol/L of the specific scu-PA
N-terminal binding domain peptide (residues 4 through 43) in
0.1 mol/L phosphate buffer, pH 7.0, containing 0.5 mol/L NaCl and
0.01% Triton X-100. The [35S]methionine-labeled
u-PAR–containing elution fractions were pooled and concentrated to
about 0.25 mL with a Centricon-10 concentrating unit (Amicon). Each of
the concentrated [35S]methionine-labeled
u-PAR–containing fractions was further analyzed by SDS–polyacrylamide
gel electrophoresis (PAGE)–autoradiography as described above, under
reduced conditions (5% 2-mercaptoethanol), to identify the positions
of radiolabeled 55- and 35-kD Mr bands of the
single-chain glycosylated and nonglycosylated u-PAR forms,
respectively. The individual [35S]methionine-labeled 55-
and 35-kD Mr bands were cut out, and the
radioactivity content of each band was determined with a gamma
counter.
The DFP–tcu-PA Sepharose CL-4B was prepared by coupling 1 mg DFP–tcu-PA to 2 mL swollen CNBr-activated Sepharose CL-4B in 0.1 mol/L NaHCO3 buffer, pH 8.3, containing 0.5 mol/L NaCl, as we described previously.1
Immunoprecipitation31 of
[35S]Methionine-Cystine–Labeled u-PAR and CD59
Confluent cultured HSVECs (four T-75 flasks) incubated in
methionine-cystine–free RPMI medium for 2 hours were refed
[35S]methionie-cystine–containing RPMI culture medium
for 12 hours followed by gentle washing (three times) with DPBS-BSA.
Washed cultures were then incubated in the presence of thrombin (4
U/mL) or control buffer for 30 minutes at 37°C, and the conditioned
medium from each flask was separated and concentrated to 100 µL
total. Immunoprecipitation of radiolabeled u-PAR and CD59 antigens from
the concentrated, conditioned medium of thrombin- and control
buffer–treated cells was carried out in separate 1.5-mL Eppendorfs
with rabbit anti-human u-PAR antibody or rat anti-human CD59 antibody,
respectively, in solution A (50 mmol/L Tris, pH 7.4, 190 mmol/L NaCl, 6
mmol/L EDTA, 2.5% Triton X-100, 0.02% NaN3, and
100 U/mL aprotinin) at 4°C overnight. Subsequently, each sample was
rotated for 2 hours at room temperature with the addition of 20 µL of
a protein A–Sepharose solution (0.1 g protein A–Sepharose in 700 µL
solution A). The solution A–antigen-antibody complexes formed were
then separated out by centrifugation and washed (three times) with
solution A plus 0.2% SDS. After the last wash, the solution
A–antigen-antibody complexes were resuspended in 50 µL 2x Laemmli
reducing sample buffer, boiled for 5 minutes, and subjected to
SDS-PAGE. The positions of proteins were determined by phosphorimaging
autoradiography with a Molecular Dynamics Series 425F PhosphorImager in
combination with IMAGEQUANT software (Molecular
Dynamics).
Measurement of Plasminogen Activator Activity by Fibrin
Autography
The plasminogen activator activity forms secreted by cultured
PAEC monolayers, after treatment with thrombin (2 U/mL) or DPBS-BSA
(control), were analyzed by SDS-PAGE–fibrin autography as described by
Erickson et al.32 Postconfluent PAEC cultures were washed
(two times) with DPBS-BSA and thrombin or DPBS-BSA added to the
cultures in DPBS-BSA at 37°C. After various incubation times (0, 15,
30, 60, 120, and 240 seconds), samples (25 µL) were rapidly removed
from thrombin-treated and control cultures and immediately mixed with
an equal volume of buffer containing 2% SDS and 0.065 mol/L Tris-HCl,
pH 6.8. Electrophoresis was performed according to
Laemmili33 with 1.5x82x74-mm slab gels consisting of
upper stacking gels of 4% acrylamide and lower resolving gels of 8%
acrylamide. After electrophoresis, the polyacrylamide gels were washed
with PBS containing 2.5% Triton X-100 and overlaid with fibrin gels in
the absence or presence of anti–TPA and anti–u-PA IgG, as
described.32 Additional controls included the presence of
54-kD Mr tcu-PA and 68-kD
Mr TPA standards in separate lanes. Gels were
incubated at 37°C for 3 hours, and the tcu-PA or TPA type was
identified by the formation of lytic zones in the fibrin gels in the
absence or presence of specific anti-PA IgGs.
Fibrinolytic (Plasmin) Activity Assay
Plasmin formation was measured by the direct conversion of
single-chain 125I-labeled Glu-Pmg to two-chain
125I-labeled plasmin after SDS-PAGE under reduced
conditions, according to Mussoni et al,34 as modified in
this laboratory with live cultured ECs. Postconfluent cultured PAECs in
96-well plates were washed (three times) with 0.01 mol/L HEPES, 0.1
mol/L sodium acetate, pH 7.4, containing 1% BSA (buffer A). Thrombin
(2 U/mL) or buffer A (control) was added (in triplicate) to individual
wells and incubated at 37°C for 30 minutes. Thrombin-treated and
control PAEC cultures were washed (three times) with warm (37°C)
buffer A and equilibrated with buffer A (50 µL per well) at 4°C for
20 minutes. A saturating concentration of tcu-PA (2 nmol/L) was added
to each well, and cultures were incubated at 4°C for 30 minutes and
then washed (five times) with cold buffer A containing 100 KIU/mL
aprotinin. 125I-labeled Glu-Pmg (150 nmol/L) in buffer A
containing 1000 KIU/mL aprotinin (40 µL) was added to each well and
incubated at 4°C for 15 minutes. Culture plates were placed in a
37°C water bath to initiate the tcu-PA–mediated conversion of
125I-labeled Glu-Pmg to 125I-labeled plasmin.
Reactions were stopped at various times (0, 8, 16, and 24 minutes) by
the rapid addition of 40 µL hot (56°C) solubilizing buffer (4%
SDS, 10% glycerol, 0.2 mol/L Tris-HCl, pH 6.8). The total contents of
each well were analyzed by 0.1% SDS-PAGE, as described above. The
amounts of 125I-labeled heavy-
(Mr, 65 kD) and light-
(Mr, 20 kD) chain plasmin thus generated
were quantified by measurement of the radioactivity content in each
band by phosphorimaging autoradiography with a Molecular Dynamics
Series 425F PhosphorImager in combination with IMAGEQUANT
software. The radioactivity content in each band was then converted to
plasmin by comparison of the radioactivity content of these individual
bands with the radioactivity content of plasmin
125I-labeled heavy- (Mr, 65
kD) and light- (Mr, 20 kD) chain derived
from varying known amounts of fully converted 125I-labeled
Glu-Pmg. 125I-labeled Glu-Pmg (1.0 µg) in buffer A
containing 1000 KIU/mL aprotinin (minus BSA) was fully converted to
plasmin by incubation with tcu-PA (2 IU/mL) at 37°C for 60
minutes.
Measurement of u-PAR mRNA Level by RT-PCR
Cultured HAECs (in 24-well multiplates) were washed (three
times) with 1% BSA–DPBS and incubated (in duplicate) with thrombin (2
U/mL) at 37°C for 30 minutes. Thrombin-treated cultures were washed
(three times) with 1% BSA–DPBS and subsequently incubated with
complete 10% serum-containing medium at 37°C for various times (0,
0.5, 1, 2, 4, 8, 16, and 24 hours). The total cytoplasmic RNA was
extracted from each of these HAEC cultures with TRIzol Reagent. RT was
carried out with M-MLV reverse transcriptase, followed by 29 cycles of
PCR, according to the manufacturer's instructions, with specific
primer pairs for u-PAR (upstream, 5'-CTGCGGTGCATGCAG-3' and downstream,
5'-CACAACCTCGGTAAG-3') and an internal standard, glyceraldehyde
phosphate dehydrogenase (GAPDH) (upstream,
5'-CCACCCATGGCAAATTCCATGGCA-3' and downstream,
5'-TCTAGACGGCAGGTCAGGTCCACC-3'). Cycling parameters were 90 seconds at
94°C, 90 seconds at 55°C, and 60 seconds at 72°C. PCR products
were analyzed on a 1.2% agarose–ethidium bromide gel. The gels were
photographed, and the intensity of each band of the u-PAR mRNA was
quantified by laser densitometric scanning and expressed as a relative
ratio of the GAPDH mRNA band intensity in each
lane.35 36 37 38 39
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Effect of Thrombin on 125I-Labeled tcu-PA Binding to Cultured ECs
Thrombin caused a significant overall decrease (0.82 to 0.37x105 sites per cell,
60% to 70%) in both
125I-labeled tcu-PA and 125I-labeled
DFP–tcu-PA ligand binding to cultured PAECs. The initial decrease in
125I-labeled tcu-PA ligand binding was rapid (<5 minutes),
representing 40% to 50% of the total reduction, followed
by a gradual decrease that reached a maximum by 30 to 40 minutes
(Fig 1
). This thrombin-induced decrease in
125I-labeled tcu-PA ligand binding (sites per cell) was
dose-dependent (0.1 to 8 U/mL), with the most significant effect
obtained with 1 U/mL (Fig 2). No significant effects
were observed at thrombin concentrations >4 U/mL. However, DFP-treated
thrombin had no effects on 125I-labeled tcu-PA ligand
binding in cultured PAECs (Fig 2). The thrombin receptor activation
peptide SFLLRNP but not control peptide FSLLRNP showed a similar but
reduced (20%) decrease in the specific binding of
125I-labeled tcu-PA to u-PAR (Fig 3).
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) or thrombin (
, 
, 2 U/mL) at
37°C for various times (0, 5, 10, 20, and 30 minutes).
125I-labeled tcu-PA ligand binding (
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The decrease in thrombin-induced 125I-labeled tcu-PA ligand
binding in cultured PAECs was reversible. Thrombin-treated (30 minutes)
PAEC cultures incubated in complete 10% FBS-containing culture medium
resulted in the time-dependent recovery (6 to 24 hours) of
125I-labeled tcu-PA ligand binding (Fig 4).
After incubation for 12 hours, the number of 125I-labeled
tcu-PA binding sites per cell had returned to essentially normal
control levels (0.35 to 0.83x105 sites per cell). In each
experiment, however, 125I-labeled tcu-PA ligand binding
sites per cell were overexpressed by 30% to 35% (1.25 versus
0.83x105 sites per cell) after 24 hours of incubation in
thrombin versus control PAEC cultures (Fig 4). Typically, the increased
125I-labeled tcu-PA ligand binding sites per cell in
thrombin-treated PAEC cultures returned to essentially normal control
levels after 36 hours of incubation.
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Cultured ECs derived from different human vascular beds showed
considerable variation in the number of 125I-labeled tcu-PA
ligand binding sites per cell (Fig 5). Similar but less
significant effects of thrombin, however, were also observed with
cultured human ECs, including HUVECs, HSVECs, and HAECs, compared with
cultured PAECs. Incubation of cultured human ECs with thrombin (2 U/mL)
for 30 minutes at 37°C decreased 125I-labeled tcu-PA
ligand binding in cultured HSVECs by 25% (from
2.02±0.13x105 to 1.51±0.01x105 sites per
cell), HUVECs by 21% (from 1.25±0.07x105 to
0.99±0.10x105 sites per cell), and HAECs by 44% (from
0.969x105 to 0.546x105 sites per cell),
compared with 66% in cultured PAECs (from 0.872±0.05x105
to 0.296±0.08x105 sites per cell) (Fig 5).
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Further Scatchard analysis of the specific 125I-labeled
tcu-PA ligand binding data with cultured PAECs confirmed that thrombin
decreased Bmax but had no apparent effect on the
Kd of 125I-labeled tcu-PA ligand
binding for u-PAR. The Kd for
125I-labeled tcu-PA ligand binding to cultured PAECs was
determined at 1.31±0.78 nmol/L for thrombin-treated and 1.22±0.73
nmol/L for control cultures (Fig 6).
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) or buffer (
Thrombin-Induced Release of u-PA Activity From Cultured PAECs
Fibrin autography was used to determine whether the
thrombin-induced decrease in cultured EC 125I-labeled
tcu-PA ligand binding might be due to increased u-PAR occupancy by
endogenously released u-PA ligand. Analysis of the DPBS-BSA media
from PAEC cultures treated with thrombin for various time periods (0,
15, 30, 60, 120, and 240 seconds) by SDS-PAGE followed by fibrin
autography in the absence or presence of specific anti–TPA IgG
indicated the very rapid appearance (15 seconds) of a lytic zone
corresponding to the 54-kD Mr tcu-PA standard
(Fig 7). This plasminogen activator activity form was
further identified immunologically as u-PA by complete inhibition of
activity by specific anti–u-PA IgG but not anti-TPA IgG. Although
tcu-PA lytic zones were readily identifiable at 15, 30, 60, and 120
seconds, this activity rapidly decreased with time and was no longer
visible after 240 seconds. The disappearance of tcu-PA from the media
suggested that the released tcu-PA may be rapidly binding to u-PAR,
resulting in the decreased availability of binding sites for subsequent
125I-labeled tcu-PA ligand binding.
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To further examine whether thrombin-induced, endogenously released
tcu-PA may subsequently decrease the availability of u-PAR binding
sites, cultures were acid-treated before the 125I-labeled
tcu-PA ligand binding assays. Acid treatment did not affect or restore
125I-labeled tcu-PA ligand binding to control levels in
thrombin-treated cultures (Fig 8). These acid treatment
results suggested that occupancy of u-PAR binding sites by
thrombin-induced, endogenously released tcu-PA did not appear to
account for or contribute to the observed decrease in
125I-labeled tcu-PA ligand binding in thrombin-treated PAEC
cultures.
|
Cleavage of u-PAR by Thrombin
Incubation of cultured PAECs with tcu-PA for 30 minutes with and
without acid treatment did not affect 125I-labeled tcu-PA
ligand binding, indicating that tcu-PA did not appear to cleave and
release the u-PAR ligand binding domain from the cell surface, as was
described for U937 cells.40 These results suggest that
thrombin-induced, endogenously released tcu-PA does not decrease
125I-labeled tcu-PA ligand binding through cleavage of the
u-PAR ligand binding domain during the 30-minute time course of this
reaction.
PI-PLC has been shown to cleave the glycolipid anchor of u-PAR in U937
cells.2 These studies were carried out to determine
whether thrombin might decrease u-PAR levels and hence
125I-labeled tcu-PA ligand binding through a similar
anchorage cleavage mechanism. Metabolically 35S-labeled 55-
and 35-kD Mr u-PAR forms were isolated from the
buffer media of PAEC cultures treated with buffer, thrombin, or PI-PLC
by ligand affinity adsorption and specific elution with the
N-terminal u-PA binding domain peptide. Comparative
analysis of the radioactivity content of each of the isolated
35S-labeled 55-kD Mr u-PAR bands
after SDS-PAGE indicated that brief PI-PLC and thrombin treatment of
cell monolayers released 12- and 9.8-fold more cleaved
35S-labeled 55-kd Mr u-PAR,
respectively, than the control buffer treatment. The
Table summarizes results from a typical isolation
experiment. Similar results were obtained in three different isolation
experiments. Analysis of the isolated, nonglycosylated
35S-labeled 35-kD Mr u-PAR bands
from PI-PLC– and thrombin-treated monolayers also indicated 6.4-
and 5.9-fold more release of cleaved 35S-labeled 35-kD
Mr u-PAR, respectively, than the control buffer
treatment (Table). Similar results were obtained with three separate
independent 35S-labeled 55- and 35-kD
Mr u-PAR isolation experiments. These data
suggest that thrombin may act directly or indirectly through activation
of endogenous PI-PLC to decrease 125I-labeled tcu-PA ligand
binding in cultured PAECs by increased cleavage and release of
surface-localized u-PAR.
|
To further explore this potential hypothesis, CD59, a
glycosyl-phosphatidylinositol–anchored membrane glycoprotein cleaved
by PI-PLC,41 was chosen to serve as a control protein for
subsequent thrombin-mediated experiments. Thrombin- and control
buffer–treated ECs were incubated with
[35S]methionine-cystine medium for 12 hours, and
the conditioned medium was analyzed by immunoprecipitation and
phosphorimaging autoradiography for radiolabeled u-PAR and CD59
antigen. Only thrombin-treated medium contained radiolabeled u-PAR and
CD59 antigen (Fig 9). This result also suggests that
thrombin may act directly or indirectly through activation of
endogenous PI-PLC to release surface-localized u-PAR and CD59.
|
Effect of Thrombin on u-PAR mRNA Level in Cultured ECs
Relative u-PAR mRNA levels were measured by RT-PCR analysis of
the RNA extracted from HAEC cultures at various times (0 to 24 hours)
after thrombin treatment. Expression of the intensity ratios of u-PAR
mRNA to GAPDH mRNA demonstrated a rapid (<1 hour) increase in
the u-PAR mRNA level that peaked at 8 to 12 hours and appeared to
decrease with time (Fig 10). The thrombin-induced,
rapid increase in u-PAR mRNA was coincident with the rapid increase and
recovery of 125I-labeled tcu-PA ligand binding activity
(Fig 4
), suggesting the thrombin induction of new u-PAR synthesis and
expression on the cell surface.
|
Effect of Thrombin on Surface-Localized Plasmin Generation in
Cultured PAECs
To determine whether the decrease in 125I-labeled
tcu-PA ligand binding activity, induced by thrombin, corresponded with
a concomitant decrease in surface-localized fibrinolytic activity, the
generation of plasmin was measured in PAEC cultures by direct
activation of 125I-labeled Glu-Pmg. Measurement of the
specific u-PAR–bound tcu-PA–mediated generation of
125I-labeled 20-kD Mr plasmin light
chain after a 30-minute thrombin treatment showed a rapid (<1 minute),
time-dependent (0 to 16 minute) decrease (50% to 60%) compared
with the buffer control (125±8 to 62±18 fmol/16 min) in the net
expression of cultured PAEC surface-localized fibrinolytic activity
(Fig 11).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
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These studies have demonstrated that thrombin will rapidly (<5 minutes) decrease the binding of 125I-labeled tcu-PA to cultured ECs, which results in a significant and concomitant decrease in surface-localized fibrinolytic activity. This decrease in ligand binding and fibrinolytic activity is associated with a rapid release of u-PAR from the cell surface. The thrombin-induced decrease in 125I-labeled tcu-PA ligand binding is reversible, returns to control levels within 12 hours of incubation under standard culture conditions, and is preceded by the rapid (<1 hour) expression of u-PAR mRNA levels.
Although TPA is generally considered the major physiological
plasminogen activator in thrombolysis, the additional contributory role
of u-PAR–bound u-PA should not be undermined or overlooked as an
equally important plasminogen activator in EC fibrinolysis and
thrombolysis. Recent studies clearly suggested a pivotal role for u-PAR
in the surface localization and regulation of cellular
fibrinolysis.1 u-PARs are widely distributed on many cell
types.42 43 44 45 The 55- to 60-kD Mr
u-PAR has been isolated from U937 cells46 47 and the cDNA
cloned and sequenced.48 A 46- to 55-kD
Mr u-PAR with identical properties has been
isolated and characterized from cultured HUVECs.1 The
u-PAR consists of three internal repeats (90 amino acid residues
each) or domains with the high-affinity ligand binding domain located
in domain 1 (N-terminal residues 1 through 87;
Mr, 16 kD), which is attached to the
plasma membrane by a glycosyl-phosphatidylinositol anchor at the
C-terminal portion of the molecule.48
In these studies, we have demonstrated that thrombin will induce the rapid release of u-PA from cultured PAECs. This endogenously released u-PA may decrease subsequent ligand binding during thrombin treatment by receptor occupancy. Acid treatment has been shown to dissociate receptor-bound u-PA and would be expected to restore u-PA ligand binding to control levels if occupancy by endogenously released u-PA is responsible for the reduction in 125I-labeled tcu-PA ligand binding. Because acid treatment of thrombin-pretreated PAEC cultures did not restore u-PA ligand binding to control levels, we concluded that this reaction did not contribute significantly to the observed decrease in 125I-labeled tcu-PA ligand binding.
Alternatively, endogenous u-PA released by thrombin may decrease subsequent ligand binding by the cleavage of u-PAR as described by Hoyer-Hansen et al.40 These investigators demonstrated the time-dependent (1 to 20 hours) and dose-dependent (1 to 100 nmol/L) cleavage of purified U937-derived u-PAR between domains 1 and 2 to release the ligand binding domain. Their studies with U937 cells incubated with a monoclonal antibody to u-PA suggested that u-PA may also cleave the u-PAR on the cell surface. In our experiments, however, incubation of cultured PAECs with saturating amounts of tcu-PA (2 nmol/L) for 5 to 30 minutes followed by removal of any bound tcu-PA by acid treatment and subsequent 125I-labeled tcu-PA ligand binding assays did not decrease 125I-labeled tcu-PA ligand binding. This inability to demonstrate a rapid (<5 minute) tcu-PA–induced decrease in 125I-labeled tcu-PA ligand binding strongly suggested that tcu-PA did not appear to cleave the u-PAR in cultured PAECs during the relatively short incubation with thrombin.
Interestingly, DFP-treated thrombin has no effect on the binding of 125I-labeled tcu-PA to cultured ECs. This result is consistent with the newly published data describing the novel activation of thrombin of its own receptor. Thrombin, through the proteolytic cleavage of the NH2-terminal portion of the receptor, generates a new tethered ligand that then binds to and activates the receptor. Thus, for thrombin to affect u-PAR through a receptor-mediated event, its activation domain needs to remain intact. Given that the activation domain of thrombin is needed to achieve the noted decrease in 125I-labeled tcu-PA ligand binding, one might reason that thrombin simply cleaves the u-PAR off the cell surface. The results of the activation peptide experiments argue against this simple mechanism. Because the activation peptide does not contain a serine protease portion and results in a similar decrease in 125I-labeled tcu-PA ligand binding to thrombin, one may reason that the activation of the thrombin receptor is needed to produce the observed decrease in ligand binding. These peptide studies therefore strongly suggest that activation of the thrombin receptor is required to decrease u-PAR on ECs but that thrombin does not directly cleave the u-PAR.
Miles et al20 showed that preincubation with thrombin for
16 hours will increase 125I-labeled tcu-PA binding (17%)
to cultured HUVECs. The results reported here indicate that thrombin
will rapidly (<5 minutes) decrease 125I-labeled tcu-PA
binding by 40% to 50% in cultured PAECs. However, this effect is
reversible, with recovery of binding to normal levels being achieved
within 12 hours of incubation under standard culture conditions. It is
important to note that the recovery in binding is also typically
associated with a gradual overexpression of u-PA binding sites per
cell, reaching a maximum of 30% to 35% after 24 hours. These
results demonstrate that thrombin will induce a previously undescribed
early, rapid decrease in u-PA ligand binding followed by a later
increase in u-PA ligand binding. These latter results are consistent
with the thrombin-induced increase in 125I-labeled tcu-PA
binding reported by Miles et al.20 A similar, late (14 to
16 hours) fivefold increase in 125I-labeled tcu-PA ligand
binding was demonstrated in cultured bovine aortic SMCs incubated with
thrombin.2
Thrombin rapidly decreased the Bmax for tcu-PA
in each of the different cultured EC types studied, including PAECs,
HAECs, HSVCs, and HUVECs. The decrease in Bmax
varied from 21% to 66% and appeared to reflect differences between
the arterial (aortic) and venous (saphenous and umbilical) origins of
the ECs rather than interspecies differences. Early and rapid
thrombin-induced effects on Bmax were more
pronounced in cultured arterial versus venous ECs. Although the
Bmax was 1.3- to 2.2-fold higher in cultured
venous ECs, the effects of thrombin were not as striking in these
cultured systems. The variation in thrombin-induced effects on EC u-PAR
levels and activity in cultured ECs derived from different vascular
beds is consistent with previously described differences in the levels
and types of PAs produced by different cultured EC
types.49
Thrombin will initiate a cascade of transmembrane signals and events
that will eventually activate a combination of PI-PLC50 51 52 53 54
and protein kinase C50 55 56 pathways that may provide a
plausible mechanism for the early thrombin-induced decrease in the
Bmax for tcu-PA and the later expression of
u-PAR activity and mRNA levels. Activation of PI-PLC by thrombin and
the ensuing cellular events occur rapidly (<30
seconds),50 well within the time frame of the
thrombin-induced decrease in the Bmax for
tcu-PA. Thrombin-activated PI-PLC may release u-PAR from the EC surface
by a mechanism similar to the action of PI-PLC on CD59. It is well
known that CD59 can be released from the surface of ECs through a
PI-PLC–mediated cleavage of the glycosyl-phosphatidylinositol
membrane anchor of CD59.41 Because the u-PAR's
C-terminal portion is also attached to the plasma membrane
by a glycosyl phosphatidylinositol anchor,48 it is
possible that thrombin can release u-PAR by activation of PI-PLC, which
then proceeds to cleave the u-PAR glycosyl phosphatidylinositol anchor.
The detection of u-PAR and CD59 antigen in the cultured medium of ECs
pretreated with thrombin or PI-PLC (Fig 9 and the Table) is compatible
with such a mechanism. However, these results do not completely
preclude the possibility that cleavage within the amino acid portion of
u-PAR may be achieved by some other undefined, thrombin-released
proteolytic enzyme. Because thrombin also activates protein kinase C,
which has been reported to inhibit PI-PLC activity50 and
induce u-PAR synthesis,5 these combined cellular events
may contribute to and account for the eventual recovery of tcu-PA
binding after thrombin exposure. The coincident thrombin-induced
increase in steady-state u-PAR mRNA levels occurred rapidly within 30
minutes and reached a maximum in 10 to 12 hours, consistent with the
rapid increase in u-PAR mRNA induced by forskolin and PMA reported by
Langer et al5 in cultured HUVECs.
A possible alternative mechanism that should be considered to explain the thrombin-induced decrease in EC u-PAR is that thrombin may cause the rapid release or shedding of microvesicles from the EC surface. Thrombin-induced release of microvesicles from the surface of platelets57 58 and erythrocytes59 was recently described. Microvesicles released from platelets by thrombin were shown to contain procoagulant activity,57 glycoprotein IIb/IIIa, and ß-1 integrins.58 It is therefore conceivable that microvesicles released from the EC surface may also contain membrane components, including u-PAR. However, the thrombin-induced release of microvesicles from the EC surface has not yet been clearly demonstrated or established, although it has been shown that human ECs exposed to peroxide release large sealed membrane vesicles.60
In U937 cells, PAI-1 can bind to u-PA on the u-PAR, resulting in the
internalization of the u-PAR–u-PA–PAI-1 complex.61
Thrombin can induce the release of u-PA and PAI-1 from the ECs, so it
is plausible that a similar complex-internalization mechanism may have
contributed to the decrease in u-PA ligand binding seen in these
studies. This effect, however, is not described in ECs, and the time
frame of the observed reduction in 125I-tcu-PA binding in
these studies does not support this possibility. The internalization of
the u-PAR–u-PA–PAI-1 complex in U937 cells took 3 hours to reach the
maximum level, while our studies demonstrated a rapid, significant,
thrombin-induced reduction in 125I-tcu-PA binding within 30
minutes. In addition, ligand binding with 125I-labeled
DFP–tcu-PA also decreased in thrombin-treated EC cultures (Fig 1).
Because DFP–tcu-PA cannot bind PAI-1, the formation of a
u-PAR–u-PA–PAI-1 complex for internalization is less likely. Further
studies are necessary to determine whether the PAI-1–mediated
internalization of u-PAR may additionally contribute to or be
responsible for the observed thrombin-induced decrease in u-PAR and
u-PA binding described in these experiments.
Thrombin treatment (16 to 20 hours) was previously shown to increase u-PAR expression and plasmin generation in cultured bovine SMCs.2 In these studies, we have demonstrated that thrombin will rapidly (<5 minutes) decrease u-PAR–bound tcu-PA–mediated fibrinolysis on the EC surface. To facilitate and stabilize early fibrin formation and clot stabilization, it may be advantagous to be able to acutely downregulate EC fibrinolysis, as we described in these studies. Finally, these studies have identified and described a novel new interrelation between the coagulation and fibrinolytic systems and suggest that thrombin may also be an important regulator of EC fibrinolysis.
|
Acknowledgments |
|---|
This work was supported in part by National Institutes of Health grants HL-17667 and HL-49764 and Institutional National Research Service Award T32 HL-07703. Drs Li, Benza, and Koons were supported in part by the Institutional National Research Service Award during the course of this study. Dr Varma was a recipient of an American Heart Association Fellowship Award, Alabama Affiliate. We would like to thank the labor and delivery staff of AMI Brookwood Medical Center in Birmingham, Ala, for providing the umbilical cords used for the isolation of HUVECs used in these studies. We would also like to thank Drs A.D. Pacifico and W.L. Holman in the Division of Cardiothoracic Surgery, University of Alabama, Birmingham, for providing saphenous veins and the Alabama Organ Center, Birmingham, for providing aortas for these studies.
Received March 4, 1994; accepted December 20, 1994.
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  G. C. Auld, H. Ritchie, L. A. Robbie, and N. A. Booth Thrombin Upregulates Tissue Transglutaminase in Endothelial Cells: A Potential Role for Tissue Transglutaminase in Stability of Atherosclerotic Plaque Arterioscler. Thromb. Vasc. Biol., October 1, 2001; 21(10): 1689 - 1694. [Abstract] [Full Text] [PDF]
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E. M. Tabengwa, R. L. Benza, H. E. Grenett, and F. M. Booyse Hypertriglyceridemic VLDL Downregulates Tissue Plasminogen Activator Gene Transcription Through cis-Repressive Region(s) in the Tissue Plasminogen Activator Promoter in Cultured Human Endothelial Cells Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1675 - 1681. [Abstract] [Full Text] [PDF]
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H. E. Grenett, R. L. Benza, G. M. Fless, X.-N. Li, G. C. Davis, and F. M. Booyse Genotype-Specific Transcriptional Regulation of PAI-1 Gene by Insulin, Hypertriglyceridemic VLDL, and Lp(a) in Transfected, Cultured Human Endothelial Cells Arterioscler. Thromb. Vasc. Biol., November 1, 1998; 18(11): 1803 - 1809. [Abstract] [Full Text] [PDF]
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 S. Rota, N. A. McWilliam, T. P. Baglin, and C. D. Byrne Atherogenic Lipoproteins Support Assembly of the Prothrombinase Complex and Thrombin Generation: Modulation by Oxidation and Vitamin E Blood, January 15, 1998; 91(2): 508 - 515. [Abstract] [Full Text] [PDF]
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X.-N. Li, H. E. Grenett, R. L. Benza, S. Demissie, S. L. Brown, E. M. Tabengwa, S. H. Gianturco, W. A. Bradley, G. M. Fless, and F. M. Booyse Genotype-Specific Transcriptional Regulation of PAI-1 Expression by Hypertriglyceridemic VLDL and Lp(a) in Cultured Human Endothelial Cells Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 3215 - 3223. [Abstract] [Full Text]
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E. M. Conway, S. Pollefeyt, D. Collen, and M. Steiner-Mosonyi The Amino Terminal Lectin-Like Domain of Thrombomodulin Is Required for Constitutive Endocytosis Blood, January 15, 1997; 89(2): 652 - 661. [Abstract] [Full Text] [PDF]
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