Site-specific thrombin receptor antibodies inhibit
Ca2+ signaling and increased
endothelial permeability
Lan T.
Nguyen1,
Hazel
Lum2,
Chinnaswamy
Tiruppathì2, and
Asrar
B.
Malik2
2 Department of Pharmacology,
College of Medicine, The University of Illinois, and
1 Department of Pharmacology,
Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612
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ABSTRACT |
Thrombin
receptor is activated by thrombin-mediated cleavage of the receptor's
NH2 terminus between Arg-41 and
Ser-42, generating a new NH2
terminus that functions as a "tethered ligand" by binding to
sites on the receptor. We prepared antibodies (Abs) directed against
specific receptor domains to study the tethered ligand-receptor interactions required for signaling the increase in endothelial permeability to albumin. We used polyclonal Abs directed against the
peptide sequences corresponding to the extracellular
NH2 terminus [residues
70-99 (AbDD) and 1-160 (AbEE)] and extracellular loops 1 and 2 [residues 161-178 (AbL1) and 244-265
(AbL2)] of the seven-transmembrane thrombin receptor. Receptor
activation was determined by measuring changes in cytosolic
Ca2+ concentration
([Ca2+]i)
in human dermal microvascular endothelial cells (HMEC) loaded with
Ca2+-sensitive fura
2-acetoxymethyl ester dye. The transendothelial 125I-labeled albumin clearance
rate (a measure of endothelial permeability) was determined across the
confluent HMEC monolayers. AbEE (300 µg/ml), directed against the
entire extracellular NH2-terminal extension, inhibited the thrombin-induced increases in
[Ca2+]i
and the endothelial 125I-albumin
clearance rate (>90% reduction in both responses). AbDD (300 µg/ml), directed against a sequence within the
NH2-terminal extension, inhibited
70% of the thrombin-induced increase in
[Ca2+]i
and 60% of the increased
125I-albumin clearance rate. AbL2
(300 µg/ml) inhibited these responses by 70 and 80%, respectively.
However, AbL1 (300 µg/ml) had no effect on either response. We
conclude that NH2-terminal
extension and loop 2 are critical sites for thrombin receptor
activation in endothelial cells and thus lead to increased
[Ca2+]i
and transendothelial permeability to albumin.
intracellular calcium concentration; tethered ligand
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INTRODUCTION |
THROMBIN RECEPTOR activation in vascular smooth muscle
cells, platelets, and endothelial cells plays a vital role in the
induction of mitogenesis, hemostasis, and inflammation (6). The
activation of the endothelial thrombin receptor also increases
endothelial permeability to albumin (8, 15), an important early step in
the induction of the inflammatory response. The thrombin receptor, consisting of seven transmembrane domains (i.e., the extracellular NH2 terminus, 3 extracellular
loops, 3 intracellular loops, and a COOH terminus), is coupled to
heterotrimeric GTP-binding proteins (25). The receptor is activated
after thrombin proteolytically cleaves the peptide bond linking Arg-41
and Ser-42 of the receptor's NH2
terminus. This new NH2 terminus
functions as a "tethered ligand" that binds to specific sites on
the thrombin receptor and activates the receptor as depicted in Fig.
1 (25). The observation that a
14-residue synthetic peptide (SFLLRNPNDKYEPF; TRP-14) corresponding to
the NH2 terminus of the tethered
ligand mimics most of thrombin's actions supports this model of
receptor activation (7, 18, 23, 25).
Bahou et al. (2) showed that antibodies (Abs) directed against a
20-amino acid peptide (residues 34-52) spanning the thrombin cleavage site of the NH2 terminus
prevented the thrombin-induced increase in cytosolic
Ca2+ concentration
([Ca2+]i)
in human umbilical vein endothelial cells but did not affect the
TRP-14-induced rise in
[Ca2+]i.
However, an Ab directed against the entire
NH2-terminal extension plus the
first transmembrane domain (residues 1-160) abolished both
thrombin- and TRP-14-induced responses, suggesting that the NH2 terminus contains the critical
recognition sequences required for receptor activation (2). Gerszten et
al. (9) studied receptor binding domains by making chimeras of the
NH2 terminus of the
Xenopus thrombin receptor coupled to
the extracellular loop domains of the human thrombin receptor. They
concluded that both the
NH2-terminal exodomain and
extracellular loop 2 (residues 244-265) were required for receptor
activation (9).
Thrombin increases endothelial permeability by activating the thrombin
receptor and stimulating protein kinase C (PKC) (16, 17) and
Ca2+-signaling pathways (7, 14,
15). However, we (13) and others (2, 23, 24) have observed
that the synthetic tethered ligands do not reproduce all the actions of
thrombin, suggesting presence of multiple thrombin receptors and
signaling pathways. The goals of the present study were to use
site-specific Abs to determine the thrombin receptor domains
responsible for receptor activation in endothelial cells and to address
whether inhibiting receptor activation by this means would influence
the increase in endothelial permeability. We used polyclonal Abs
directed against peptide sequences corresponding to the postulated
binding sites of thrombin on the receptor's extracellular
NH2 terminus (residues 70-99
and 1-160) and of the tethered ligand on loop 1 (residues 161-178) and loop 2 (residues 244-265).

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Fig. 1.
Model of thrombin receptor activation.
A: receptor in the unactivated state.
Extracellular NH2 terminus
contains the cleavage site for thrombin and the 3 extracellular loops
(L1, L2, and L3). B: after thrombin
cleavage of the NH2 terminus, the
generated "tethered ligand" interacts with the receptor's
extracellular loop domains, leading to receptor activation. The
"?" indicates the uncertainty about binding sites of the
NH2 terminus with domains of the
receptor.
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MATERIALS AND METHODS |
Materials.
Endothelial cell culture medium MCDB 131, fetal bovine serum (FBS),
Hanks' balanced salt solution (HBSS), and Dulbecco's modified Eagle's medium (DMEM) were obtained from GIBCO Laboratories (Grand Island, NY). Trichloroacetic acid (TCA),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), bovine serum albumin (BSA), ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA),
m-maleimidobenzoyl-N-hydroxysuccinimide ester, Freund's adjuvant, hydrocortisone, endothelial growth
supplement, type II calf gelatin, fibronectin, protein
A-Sepharose, acetonitrile, trifluoroacetic acid, and
ionomycin were purchased from Sigma Chemical (St. Louis, MO). The other
materials were as follows: 125I
(Amersham, Arlington Heights, IL), fura 2-acetoxymethyl ester (AM;
Molecular Probes, Eugene, OR), tris(hydroxymethyl)aminomethane (Tris) · HCl and glycine (Bio-Rad, Hercules, CA),
polycarbonate micropore filters (Nuclepore, Pleasanton, CA), and
plastic cylinders (Adaps, Dedham, MA). Human
-thrombin
(Enzyme Research Laboratories, South Bend, IN) purified from human
plasma had an activity of 5,245 U/ml.
Endothelial cell culture.
The human dermal microvessel endothelial cell line (HMEC) was used (1).
These cells are endothelial cells in origin because they exhibited
typical cobblestone morphology when grown in monolayer culture and
presence of von Willebrand factor and angiotensin converting enzyme
(1). HMEC were grown in complete medium consisting of MCDB 131 supplemented with 5% FBS, 0.10% hydrocortisone, and 0.01%
endothelial growth factor. The cells were passaged every 7-8 days
at confluency as visualized by phase microscopy and were used at
passages 20-30.
Transendothelial 125I-labeled albumin
clearance rate.
We determined the diffusive flux of
125I-labeled albumin across
endothelial monolayers as the index of endothelial permeability (5).
The system consisted of abluminal and luminal compartments separated by
a gelatin- and fibronectin-coated polycarbonate microporous filter (13 mm diameter, 10 µm thickness, 0.8 µm pore diameter), which was
glued onto the base of a 9-mm (ID) plastic cylinder. The filters were
sterilized under ultraviolet light for 24 h, seeded with HMEC
(105 cells/filter), and grown for
3-4 days until confluent. The luminal compartment, which contained
500 µl of DMEM with 0.5% BSA, 20 mM HEPES, and ~5 µCi/ml
125I-albumin, was fitted with a
styrofoam ring and "floated" in the abluminal medium to equalize
the hydrostatic pressure difference. The abluminal compartment
contained 25 ml of the same DMEM, which was kept at 37°C and
continuously stirred to ensure rapid mixing. Thrombin was added to the
luminal chamber at final concentrations that took into account both
luminal and abluminal volumes of DMEM. Abluminal samples of 400 µl
were obtained at 5-min intervals for 60 min and counted for
radioactivity in a gamma counter (Packard Instruments, Downers Grove,
IL). At the end of the experiment, 12% TCA precipitation was used to
determine percentage of free 125I,
which was used to correct for the clearance of
125I-albumin. Clearance values are
reported as the clearance rate of
125I-albumin determined as the
volume from luminal chamber cleared into the abluminal chamber per min
(µl/min) based on weighted least-squares nonlinear regression
analysis (BMDP Statistical Software, Berkeley, CA). Transendothelial
permeability of endothelial monolayers
(Pec) was
calculated using the following equation: 1/Pec = 1/(Pec+f)
1/Pf,
where Pec+f is
the permeability of monolayer plus filter (calculated from clearance
rates) and Pf is
the permeability of filter alone [determined to be 3.9 × 10
5 cm/s on the basis of a
filter surface area of 0.635 cm2
(5)].
For these experiments, the confluent HMECs plated on the prepared
filters were washed with DMEM (without FBS, but containing 0.5% BSA),
pretreated with the thrombin receptor peptide Abs at room temperature
for 30 min, and floated onto the permeability setup for clearance
determinations.
[Ca2+]i
measurements.
HMEC were seeded at 5 × 104
cells/glass coverslip (25 mm diameter) and grown for 2-3 days
until confluent for
[Ca2+]i
determinations as described previously (11, 14). The cells were loaded
with 2 µM fura 2-AM for 60 min at room temperature. After the cells
were loaded, they were washed two times with HBSS and pretreated with
thrombin receptor peptide Abs for 30 min at room temperature. The cells
were then placed into a Sykes-Moore chamber (Bellco, Vineland, NJ) and
positioned onto the stage of an inverted Nikon Diaphot microscope that
was coupled to a Deltascan microspectrofluorometric system
[Photon Technology International (PTI), Princeton, NJ]. An
optically isolated group of three to four cells was alternately excited
at wavelengths of 340 and 380 nm, and the emitted light was collected
with a photomultiplier at 510 nm. Background autofluorescence (in the
absence of fura 2) was determined at the beginning of each day's
experiment and was automatically subtracted while data were collected.
At the end of each experiment, the fluorescence of
Ca2+-saturated fura 2 was obtained
by adding 10 µM ionomycin, and the fluorescence of free fura 2 was
obtained by adding 0.1 M EGTA. Fluorescence ratios of excitation
wavelengths 340 and 380 nm and [Ca2+]i
values were computed with PTI software that was based on a dissociation
constant of 306 nM, which corrects for measurements made at room
temperature (21).
Thrombin receptor peptide synthesis.
Thrombin receptor peptides corresponding to potential binding domains
for the tethered ligand (Table 1) were
synthesized on an Applied Systems (Foster City, CA) automated peptide
synthesizer (model 430 A), using Fmoc chemistry with FastMoc protocol
as suggested by the manufacturer. All reactive Fmoc amino acids were
protected on the side chains, and rink resin was used for amide at the
COOH terminus. The products were obtained by cleavage and deprotection with 95% trifluoroacetic acid plus the appropriate scavengers. The
peptides were purified to homogeneity by preparative reverse-phase high-performance liquid chromatography (HPLC; C18 column, using a
gradient of 0-60% acetonitrile and 0.1% trifluoroacetic acid). Each peptide was checked and verified by its single peak in the analytical HPLC chromatogram, amino acid composition, and mass spectrum
and by NH2-terminal sequencing.
Ab production and purification.
Polyclonal antisera were raised against thrombin receptor peptides in
rabbits. Peptides were conjugated to keyhole limpet hemocyanin using
the heterobifunctional coupling reagent
m-maleimidobenzoyl-N-hydroxysuccinimide ester. After conjugation, 300-500 µg of peptide in 250 µl were mixed with an equal volume of complete Freund's adjuvant and injected intramuscularly at four different sites into the rabbit. Blood was
collected before injection to obtain preimmune serum from each rabbit.
Booster injections with incomplete adjuvant were made at 2-wk intervals
for 4 wk.
Abs were affinity purified using protein A-Sepharose column. The column
was filled with 2 ml of protein A-Sepharose and washed with 0.1 M
Tris · HCl (pH 8.0). The Ab solution was passed
through the protein A bead column two times, and the column was washed with 30-40 ml of 0.1 M Tris · HCl (pH 8.0). The
column was eluted stepwise with 0.1 M glycine-HCl (pH 3.0). Five 2.5-ml
fractions of the eluate were collected in conical tubes containing 250 µl of 1.0 M Tris · HCl (pH 8.0). The tubes were
gently mixed to bring the pH back to neutral. The protein-containing
fractions were identified by absorbance at 280 nm (1 optical density
unit = 0.8 mg/ml). The eluate containing the protein was
placed in a dialysis bag and allowed to dialyze overnight at 40°C
in 2 liters phosphate-buffered saline (PBS). Protein
concentration was determined by the Lowry method. We generated Abs to
peptides corresponding to NH2
terminus and extracellular loops 1 and 2. Attempts at preparing Abs to loop 3 were unsuccessful due to the lack of antigenicity of that receptor region.
Fluorescence-activated cell sorter analysis.
Confluent monolayers of HMEC-1 were washed, incubated in serum-free
medium for 2 h, and nonenzymatically dissociated with cell dissociation
medium (Sigma Chemical). The cell suspension was incubated with 20%
horse serum in PBS containing 0.1%
NaN3 on ice for 30 min to block
nonspecific binding and then washed two times using PBS, and 5 × 105 cells were incubated for 1 h
on ice with 1 mg/ml preimmune control immunoglobulin G (IgG) or 1 mg/ml
AbL1, AbL2, or AbDD in 3% horse serum-0.1%
NaN3-PBS. After incubation with
primary Ab, the cells were washed two times and incubated with
fluorescein isothiocyanate-labeled goat anti-rabbit IgG (Sigma
Chemical) at a dilution of 1 mg/ml for 30 min on ice. The cells were
then washed two times in horse serum-NaN3-PBS and once in PBS,
fixed in 1% paraformaldehyde, and analyzed by a Coulter
fluorescence-activated cell sorter (FACS) analyzer (Flow Cytometry
Laboratory, University of Illinois, Chicago, IL).
Statistics.
Statistical analysis for transendothelial clearance of
125I-albumin was carried out using
the analysis of variance test, with level of significance set at
P < 0.05. All experiments were
performed three to four times with four to six monolayers per
experiment. A multiple-range test for comparisons with a single control
(Dunnett's test) was used for analysis of the
[Ca2+]i
responses.
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RESULTS |
Thrombin increases albumin permeability and
[Ca2+]i
in HMEC.
Activation of confluent monolayers of HMEC with human
-thrombin
(1-8 nM) resulted in dose-dependent increases in transendothelial 125I-albumin clearance rates (Fig.
2). Stimulation with 8 nM thrombin caused
an approximately fourfold increase in the clearance rate over control
values, similar to the increase seen with 6 nM thrombin (Lum,
unpublished observations). Stimulation of HMEC with 0.5 and 1 nM
thrombin increased
[Ca2+]i
to 1.9 ± 0.3 and 1.75 ± 0.1 µM, respectively (Table
2), suggesting that these
thrombin concentrations elicited the maximal rise in [Ca2+]i.

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Fig. 2.
Effects of different concentrations of thrombin (0, 1, 4, and 8 nM) on
clearance rate of 125I-labeled
albumin across confluent monolayers of human dermal microvascular
endothelial cells (HMEC). Values are means ± SE in µl/min; each
group contained 5-7 monolayers (n = 2 separate experiments). * Difference from control group
(P < 0.01).
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Effects of thrombin receptor Abs on increase in permeability.
The purified thrombin receptor Abs used in this study are listed in
Table 1. We have previously shown by immunoblotting that AbL1 (against
residues 161-178) specifically binds to the thrombin receptor of
endothelial cells (26). In the present study, FACS analysis showed that
AbL1, AbL2, and AbDD caused a similar right shift in relative log
fluorescence compared with preimmune IgG (Fig.
3), indicating the binding of the Abs to
the HMEC thrombin receptor.

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Fig. 3.
Flow cytometric analysis of binding of anti-thrombin receptor
antibodies (Abs) AbL1 (B), AbDD
(C), and AbL2
(D) to cell surface of HMEC. See
MATERIALS AND METHODS for details
[binding of AbEE to endothelial thrombin receptor has been
described by Bahou et al. (2)].
A shows effects of preimmune
immunoglobulin G (IgG).
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The involvement of thrombin receptor extracellular loops 1 and 2 in the
regulation of endothelial permeability was studied using AbL1 and AbL2.
HMEC were pretreated with 300 µg/ml AbL2 and stimulated with 8 nM
thrombin, a concentration shown to produce a maximal increase in
transendothelial 125I-albumin
clearance rate. AbL2 reduced the thrombin-induced increase in clearance
rate by ~80% (Fig.
4B). In
contrast, pretreatment with AbL1 had no significant effect on the
thrombin-induced increase in permeability (Fig.
4A). In a control experiment,
pretreatment with 300 µg/ml preimmune IgG for 30 min did not inhibit
the thrombin-induced increase in clearance rate (Fig. 4). IgG alone did
not alter the 125I-albumin
baseline clearance rate (data not shown).

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Fig. 4.
Effects of anti-thrombin receptor AbL1 (300 µg/ml,
A) and AbL2 (300 µg/ml,
B) on thrombin-induced increases in
transendothelial clearance rate of
125I-albumin across HMEC
monolayers. Cells were pretreated with Abs or IgG for 30 min and
stimulated with 8 nM -thrombin ( -Thr). Values are means ± SE;
each group contained 5-6 monolayers
(n = 3 separate experiments).
* Difference compared with control group
(P < 0.01).
+ Difference compared with
-Thr group (P < 0.01).
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We then studied the effects of Abs directed against the extracellular
NH2 terminus in mediating the
thrombin-induced increase in permeability. AbEE (300 µg/ml) (directed
against the entire NH2 terminus)
prevented the thrombin-induced increase in clearance rate (Fig.
5). However, pretreatment with AbDD
(directed against residues 70-99 of the
NH2-terminal extension) resulted
only in ~50% inhibition of the thrombin-induced permeability
response (Fig. 6).

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Fig. 5.
Effects of anti-thrombin receptor AbEE on -Thr-induced increase in
the clearance rate of 125I-albumin
in HMEC monolayers. Cells were pretreated with 300 µg/ml AbEE or
preimmune IgG for 30 min and stimulated with 8 nM -Thr. Values are
means ± SE; each group contained 5-6 monolayers
(n = 3 separate determinations).
* Difference compared with control group
(P < 0.01).
+ Difference compared with
-Thr group (P < 0.01).
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Fig. 6.
Effects of anti-thrombin receptor AbDD on -Thr-induced increase in
the clearance rate of 125I-albumin
in HMEC monolayers. Cells were pretreated with 300 µg/ml AbDD or
preimmune IgG for 30 min and stimulated with 8 nM -Thr. Values are
means ± SE; each group contained 5-6 monolayers
(n = 3 separate determinations).
* Difference compared with control group
(P < 0.01).
+ Difference compared with
-Thr group (P < 0.01).
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Effects of thrombin receptor Abs on increase in
[Ca2+]i.
We investigated the effects of thrombin receptor Abs on the
thrombin-induced increase in
[Ca2+]i
(Table 2). HMEC were pretreated with 300 µg/ml AbL2 and stimulated with 0.5 or 1.0 nM thrombin. AbL2 reduced the thrombin-induced increases in
[Ca2+]i
by ~70% at either concentration of thrombin (Table 2 and Fig. 7). Control preimmune IgG pretreatment did
not significantly affect the thrombin-induced increase in
[Ca2+]i
(Fig. 7). In contrast to AbL2, AbL1 did not significantly inhibit the
increase in
[Ca2+]i
in response to 0.5 nM thrombin (Table 2 and Fig.
8).

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Fig. 7.
Effects of anti-thrombin receptor AbL2 on -Thr-induced increase in
cytosolic Ca2+ concentration
([Ca2+]i)
in HMEC. Cells were pretreated with 300 µg/ml AbL2 or IgG for 30 min
and stimulated with 1.0 nM -Thr. Control indicates absence of Ab
pretreatment. Short arrow indicates time of -Thr addition; long
arrows indicate the groups. Results are representative of 5 separate
determinations.
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Fig. 8.
Effects of anti-thrombin receptor AbL1 on -Thr-induced increase in
[Ca2+]i
in HMEC. Cells were pretreated with 300 µg/ml AbL1 for 30 min and
stimulated with 0.5 nM -Thr. Control indicates absence of Ab
pretreatment. Short arrow indicates time of -Thr addition; long
arrows indicate groups. Results are representative of 4 separate determinations.
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Pretreatment of HMEC with AbEE (300 µg/ml) resulted in >90%
inhibition of the increase in
[Ca2+]i
in response to 0.5 or 1.0 nM thrombin (Table 2 and Fig.
9A). AbDD inhibited the increase in
[Ca2+]i
in response to 1 nM thrombin by 64% and to 0.5 nM thrombin by 74%
(Table 2 and Fig. 9B).

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Fig. 9.
Effects of anti-thrombin receptor AbEE
(A) and AbDD
(B) on the -Thr-induced increase
in
[Ca2+]i
in HMEC. Cells were pretreated with 300 µg/ml AbEE or AbDD for 30 min
and stimulated with 1 nM -Thr. Control indicates absence of Ab
pretreatment. Short arrows indicate time of -Thr addition. Results
are representative of 3 and 5 separate determinations, respectively.
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DISCUSSION |
Thrombin receptor activation involves the cleavage of its extracellular
extension (between Arg-41 and Ser-42) by thrombin, thereby generating a
new NH2 terminus beginning at
Ser-42 (25). The newly formed NH2
terminus functions as a tethered ligand that binds to sites on the
receptor to elicit receptor activation (Fig. 1). The goals of this
study were to use receptor site-specific Abs to investigate the
thrombin receptor domains in endothelial cells regulating receptor
activation and to determine whether the Abs capable of blocking
receptor activation would prevent the thrombin-induced increase in
endothelial permeability.
We used HMEC, in which we showed that the basal permeability values of
HMEC monolayers were in the range for cells that we have studied
previously (bovine pulmonary microvessel endothelial cells; Ref. 22).
Basal transendothelial
125I-albumin clearance rates in
both cell types ranged from 0.03 to 0.06 µl/min. The calculated basal
endothelial permeability of albumin was 0.13 × 10
5 cm/s (Table
3), a value 10-fold lower
than the value of 1.2 × 10
5 cm/s for bovine
pulmonary artery endothelial cells (5). Activation of HMEC by thrombin
resulted in permeability increases of two- to fourfold over baseline,
which is typical of the response observed in other endothelial cell
types (8, 13, 16).
We observed that the Ab directed against peptide sequences
corresponding to the extracellular loop 2, but not the anti-loop 1 Ab,
reduced by ~70% the thrombin-induced increases in
[Ca2+]i
and endothelial permeability. This finding suggests that the binding of
the Ab to loop 2 and the resulting steric hindrance may have prevented
the activation of the thrombin receptor. Therefore, loop 2 contains the
important recognition sequence for receptor activation that is
responsible for increasing endothelial permeability. The control IgG
and AbL1 had no effect on these responses; thus the results of the AbL2
cannot be ascribed to nonspecific effects of the Ab directed against
loop 2. The importance of the loop 2 domain in mediating thrombin
receptor activation is consistent with the work of Gerszten et al. (9)
who investigated the thrombin receptor binding domains by making
chimeras of human with Xenopus thrombin receptors and studied the activation of these modified receptors using the human thrombin receptor peptide SFLLRN. They found
that the construct consisting of the human loop 2 sequence had a 50%
effective concentration (EC50)
value 30-fold lower than the construct containing human loop 1 sequences (9), indicating the importance of loop 2 in activation of the
receptor.
Although the present data indicate that loop 2 is a critical domain
regulating thrombin receptor activation in endothelial cells, the
results also indicate that the effect of AbL2 in inhibiting the
thrombin-induced Ca2+ and
permeability responses was partial. This incomplete inhibition was not
due to the low Ab-to-receptor ratio, since a lower concentration of 200 µg/ml AbL2 showed a similar degree of inhibition as an Ab
concentration of 300 µg/ml (data not shown). The effect also cannot
be explained by activation of additional endothelial receptors such as
the proteinase-activated receptor (PAR-2) (19) because AbEE directed
against the entire NH2 terminus of
the thrombin receptor fully abrogated the responses. A logical
explanation for this finding is that the
NH2 terminus may interact with
other receptor sites besides loop 2 that also participate in activation of the thrombin receptor.
Because both a distal domain (residues 76-93) of the
NH2 terminus and the extracellular
loop 2 have been proposed to be important sites required for thrombin
receptor activation (9), we studied the effect of AbDD directed against
residues 70-99 of the NH2 terminus. This Ab reduced the thrombin-induced increases in
permeability and
[Ca2+]i
by 60-70%, respectively, consistent with chimera experiments (9).
The finding supports the hypothesis that cooperativity between this
NH2 domain with other receptor
sites (i.e., loop 2) is required for full receptor activation. The
thrombin receptor chimera containing the entire
NH2 terminus showed a 10-fold
lower EC50 to stimulation by the
thrombin receptor peptide than the construct containing a partial
NH2 terminus of residues up to 75 (9). The EC50 was lowered by
100-fold in the chimera containing both the entire human
NH2 terminus plus loop 2 (9).
These observations support our findings obtained in endothelial cells
using site-specific Abs that the
NH2 terminus and loop 2 are both
required for mediation of thrombin receptor activation and
the thrombininduced increase in endothelial cell permeability.
We showed that AbEE (directed against residues 1-160 of the
NH2 terminus) was more effective
than AbDD (directed against residues 70-99) in preventing the
thrombin-induced increases in
[Ca2+]i
and endothelial permeability. AbDD inhibited 60-70% of the thrombin-induced responses, whereas AbEE fully prevented both responses. On the basis of the model for thrombin receptor activation, AbEE may block the interaction of the
NH2 terminus at multiple sites of
the thrombin receptor (2, 9), since it is directed against the entire
extramembranous NH2 region of the
receptor including the thrombin cleavage site. In contrast, the less
effective inhibition of AbDD observed may be attributed to the fact
that this Ab is directed against a portion of the
NH2 terminus that excludes the
thrombin cleavage site.
Interestingly, the recent report of a second human thrombin receptor
(PAR-2) (4, 12) suggests that the permeability response may be subject
to regulation by activation of two distinct thrombin receptors. Both
PAR-2 and the first cloned thrombin receptor (PAR-1) mediate
Ca2+ transients and
phosphoinositide hydrolysis and appear to rely on the
fibrinogen-binding exosite for receptor recognition; however, they are
different in their tethered ligand sequences and cleavage sites (12).
Although PAR-2 is distributed in a wide range of tissues, its
occurrence in the endothelium is not clear and its role in contributing
to loss of endothelial barrier is not known.
The present study provides convincing evidence that the
thrombin-induced increase in endothelial permeability is the
consequence of thrombin receptor activation and requires the generation
of signaling molecules. The results argue against a nonspecific
proteolytic action of thrombin as being responsible for increasing
endothelial permeability. The thrombin-induced increase in endothelial
permeability is dependent in part on the increase in
[Ca2+]i
(15), the onset of which is rapid and transient, and precedes the onset
of the increase in permeability (14). The significance for a
Ca2+ requirement may be dependent
on its function as a cofactor for activation of
Ca2+-dependent PKC, a critical
kinase regulating the increase in endothelial permeability (3, 17, 22).
The rise in
[Ca2+]i
may also contribute to the force generated by the endothelial actin-myosin contractile system, which induces endothelial contraction leading to increased endothelial permeability (10, 20).
In summary, we have shown, using site-specific Abs, that domain
70-99 of the NH2 terminus and
the extracellular loop 2 of the thrombin receptor are important
determinants of thrombin receptor activation and the increase in
endothelial permeability. The mechanism of the thrombin-induced
increase in permeability may involve cleavage of the extracellular
extension between Arg-41 and Ser-42, creating a new
NH2 terminus beginning at Ser-42.
Residues 70-99 may undergo a conformational change, enabling the
NH2 terminus to bind to extracellular loop 2 and signaling the increase in permeability. The
present data suggest that increased endothelial permeability involves
in part the interaction of the NH2
terminus with loop 2 of the thrombin receptor. Moreover, the present
data suggest a novel means of preventing the thrombin-induced increase
in vascular endothelial permeability using Abs directed against
specific domains of the PAR-1 thrombin receptor.
 |
ACKNOWLEDGEMENTS |
We wish to thank Dr. Wadie F. Bahou for a generous gift of Ab
(1-160).
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-27016, HL-46350, and HL-45638 and by the American Heart
Association of Metropolitan Chicago.
Address for reprint requests: A. B. Malik, Dept. of Pharmacology,
College of Medicine, Univ. of Illinois at Chicago, 835 S. Wolcott Ave.,
M/C 868, Chicago, IL 60612.
Received 25 April 1997; accepted in final form 21 July 1997.
 |
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