(Received for publication, September 25, 1995; and in revised form, November 9, 1995)
From the
Receptor-mediated assembly of blood proteases on vascular cells
maintains the hemostatic balance and initiates intracellular signal
transduction. Effector cell protease receptor-1 (EPR-1) is an
62-kDa vascular cell membrane receptor for the clotting protease
factor Xa, participating in thrombin formation and lymphocyte
activation. Here, recombinant EPR-1 fragments were engineered in the
frame of intercellular adhesion molecule-1, transfected in mammalian
cells, and analyzed for antibody recognition and ligand binding.
Chimeric transfectants containing the EPR-1 sequence
Met
-Arg
bound the immunosuppressive
anti-EPR-1 monoclonal antibody (mAb) 2E1. In contrast, transfected
cells expressing the EPR-1 sequence Pro
-Ala
were recognized by the functionally inhibitory anti-EPR-1 mAbs
9D4 and B6, bound
I-factor Xa in a reaction
quantitatively indistinguishable from that of wild-type EPR-1
transfectants, and promoted factor Xa concentration-dependent
prothrombin activation in the absence of exogenous factor V/Va.
Chimeric transfectants expressing the COOH terminus end of the EPR-1
extracellular domain (Ala
-Glu
) did
not bind anti-EPR-1 mAbs and did not associate with factor Xa.
Mutagenesis of Asn
or Lys
in the EPR-1
ligand recognition domain abolished factor Xa binding by 80 ±
5.5 and 96 ± 4%, respectively, while mutation of
Lys
, Gly
, Asn
, and
Asn
was without effect. A synthetic peptide duplicating
the EPR-1 sequence S
PGKPGNQNSKNEPP
dose
dependently inhibited factor V/Va-independent thrombin generation of
resting endothelium (IC
1 µM), while
the adjacent EPR-1 sequence P
PKKRERERSSHCYP
was ineffective. These findings demonstrate that EPR-1 contains two
spatially distinct functional domains implicated in lymphocyte
activation (Met
-Arg
) or factor Xa
binding and prothrombin activation
(Pro
-Ala
). These interacting
sequences may provide a novel potential target for inhibition of factor
Xa-dependent vascular cell responses.
Assembly of coagulation and anticoagulation pathways occurs on vascular cells through the regulated ligand recognition of membrane protease receptors(1, 2) . Belonging to structurally different and evolutionarily unrelated gene superfamilies, cell-surface receptors for thrombin(3) , urokinase(4) , protein S(5) , or factor Xa (6) provide a controlled microenvironment for limited proteolytic activation of the clotting and fibrinolytic cascades(1, 2) . Recent studies have also underscored the participation of protease receptors in pleiotropic mechanisms of vascular cell signal transduction, including transcription of activation-dependent genes(7) , generation of intracellular second messengers(3) , modulation of immune inflammatory responses(8) , and cell proliferation(5, 9) . Aberrations of protease-dependent signaling pathways may play a primary pathogenetic role in the establishment and progression of the atherothrombotic disease (9, 10) as well as in neoplastic transformation and tumor cell dissemination(5, 11) .
Binding of factor Xa to leukocytes (12) or endothelial cells ()is contributed by effector cell protease receptor-1
(EPR-1), (
)a 337-amino acid single membrane-spanning
glycoprotein lacking significant homologies to other protease receptors
and expressed in a cell-type specific fashion by alternative mRNA
splicing(13) . Occupancy of EPR-1 with factor Xa participates
in factor V/Va-independent mechanisms of prothrombin
activation(6) , generates intermediate product(s) of factor IX
activation(14) , or contributes an accessory pathway of
lymphocyte costimulation(8) .
To gain insights into the potential pathophysiological relevance of EPR-1-factor Xa interaction in hemostasis and vascular cell signal transduction, we sought to identify the structural requirements implicated in receptor-ligand recognition. Using a eukaryotic expression strategy of EPR-1 chimeric constructs combined with site-directed mutagenesis and synthetic peptidyl mimicry, we have identified two spatially distinct regions in the EPR-1 extracellular domain separately involved in lymphocyte activation mechanisms or factor Xa binding and prothrombin activation.
Figure 1:
Design and characterization of
EPR-1-ICAM-1 chimeras. A, schematic diagram of EPR-1 chimeras.
EPR-1 sequences G2 (Met-Arg
), A1
(Pro
-Ala
), and G3
(Ala
-Glu
) were amplified by PCR and
directionally inserted in the unique NcoI and NarI
sites in the extracellular domain of ICAM-1. B, in vitro translation of EPR-1 chimeras. EPR-1-ICAM-1 chimeric constructs G2 (lane 1), A1 (lane 2), and G3 (lane 3) or
control pcDNA3 vector alone (lane 4) was translated using the
reticulocyte lysate method in the presence of
[
S]methionine, separated by electrophoresis on a
10% SDS-polyacrylamide linear gel, and visualized by autoradiography.
Relative molecular weight markers are shown on the left. TM, transmembrane domain.
For prothrombin
activation studies, wild-type CHO cells or CHO cells transfected with
full-length EPR-1 cDNA or the A1 or G3 chimera were seeded in 48-well
plates at 4 10
cells/well 24 h prior to the
experiment. Cells were incubated in phenol red-free RPMI 1640 medium
(Cellgro, Mediatech, Washington, D. C.) in the presence of increasing
concentrations of factor Xa (0.1-1 µg/ml), 1.25 mM CaCl
, and 20 µg/ml prothrombin for 5 min at 22
°C. Thrombin generated under the various conditions tested was
quantitated at 22 °C immediately after addition of a 50 µg/ml
concentration of the thrombin-sensitive chromogenic substrate S-2238
(Chromogenix, Mölndal, Sweden) by absorbance at a
405-nm wavelength on a ThermoMax microplate reader (Molecular Devices,
Menlo Park, CA). Background hydrolysis of S-2238 in the presence of
factor Xa alone (8-14%) was subtracted from the absorbance
determined under the various experimental conditions tested. Absorbance
units were converted to units of thrombin/milliliter using a standard
curve constructed with 2-fold serial increasing concentrations of
bovine thrombin (Calbiochem) from 0.015 to 2 units/ml. The A1 sequence
in EPR-1 (Pro
-Ala
) was duplicated by
two partially overlapping synthetic peptides, AG1
(S
PGKPGNQNSKNEPP
) and AG2
(P
PKKRERERSSHCYP
). Authenticity and purity
of the EPR-1 peptides were confirmed by amino acid composition and mass
spectrometry. For peptide competition experiments, human umbilical vein
endothelial cells (HUVEC) were seeded in 96-well tissue culture plates
and grown to confluency for 2-4 days prior to the assay. Two
µg/ml factor Xa was separately preincubated with increasing
concentrations (0.1-25 µM) of peptide AG1 or AG2 for
5 min at 22 °C before addition to HUVEC monolayers and
determination of prothrombin activation by S-2238 hydrolysis as
described above. Functional characterization of EPR-1 expression on
HUVEC by immunoblotting, Northern hybridization, and factor
V/Va-independent binding of
I-factor Xa will be reported
elsewhere in detail.
Figure 2: Epitope mapping of anti-EPR-1 mAbs. CHO cells were transfected with full-length ICAM-1 cDNA, with the various EPR-1-ICAM-1 chimeras, or with control vector pcDNA3 by electroporation. Forty-eight h after transfection, cells were analyzed by flow cytometry for reactivity with anti-ICAM-1 mAb 6E6, with the second generation anti-EPR-1 mAb 2E1, or with the first generation of functionally blocking anti-EPR-1 mAb 9D4 or B6. Horizontal and vertical axes measure fluorescence intensity on a 4-log scale and the cell number, respectively. WT, wild-type.
Figure 3:
I-Factor Xa binding to EPR-1
chimeras. CHO cells transiently transfected with control vector pcDNA3,
wild-type EPR-1 cDNA, or the indicated EPR-1-ICAM-1 chimeras were
incubated with increasing concentrations (0.2-2 µg/ml) of
I-factor Xa in the presence of 2.5 mM CaCl
for 15 min at 22 °C. Nonspecific binding was quantitated in
the presence of a 50-fold molar excess of unlabeled factor Xa added at
the start of the incubation and was subtracted from the total to
calculate net specific binding. Data are the means ± S.E. of at
least three independent experiments. WT,
wild-type.
The role of chimeric EPR-1
sequences in thrombin formation was investigated. In agreement with
previous observations(6) , wild-type EPR-1 transfectants
promoted prothrombin activation in a factor Xa concentration-dependent
manner, with the generation of 0.55 ± 0.06 units of
thrombin/ml for saturating concentrations of factor Xa of 1 µg/ml (Fig. 4). Consistent with their inability to bind
I-factor Xa (Fig. 3), control CHO cells
transfected with pcDNA3 vector alone did not significantly promote
prothrombin activation at comparable concentrations of factor Xa added (Fig. 4). Under these experimental conditions, A1 chimeric
transfectants generated increasing amounts of thrombin in a factor Xa
concentration-dependent reaction quantitatively indistinguishable from
the response observed with wild-type EPR-1 transfectants (Fig. 4). In contrast, G3 transfectants did not generate
thrombin under the same experimental conditions (data not shown).
Figure 4:
Prothrombin activation by EPR-1 chimeras.
CHO cells transfected with control vector pcDNA3 or EPR-1 constructs
were incubated with the indicated increasing concentrations of factor
Xa in the presence of 20 µg/ml prothrombin and 1.2 mM CaCl for 5 min at 22 °C. Thrombin generation under
the various conditions tested was quantitated by hydrolysis of the
thrombin-sensitive chromogenic substrate S-2238 at a 405-nm wavelength
and converted to units of thrombin/milliliter using a standard curve
constructed with serial increasing concentrations (0.015-2
units/ml) of bovine thrombin. Data are the means ± S.E. of three
independent experiments. WT,
wild-type.
Figure 5:
Assignment of A1 critical residues
implicated in factor Xa binding. A, CHO cells transfected with
wild-type (WT) EPR-1 cDNA or the indicated single-residue
mutagenized A1 chimeras (Pro-Ala
)
were incubated with 0.5 µg/ml
I-factor Xa in the
presence of 2.5 mM CaCl
for 15 min at 22 °C
before quantitation of specific binding as described for Fig. 3.
Data are the means ± S.E. of three independent experiments. B, shown is the secondary structure prediction of the
wild-type sequence and the Lys
Ile A1 mutant.
Secondary structure prediction of the wild-type EPR-1 sequence
Pro
-Ala
(solid line) and the
mutant Lys
Ile (broken line) was
generated with the algorithm of Chou-Fasman using the Plotstructure
program in a Genetics Computer Group software package. Hydrophilicity
and hydrophobicity according to Kyte-Doolittle (KD) are
indicated.
Figure 6:
Effect of A1-derived peptides on HUVEC
prothrombin activation. The experimental conditions are essentially as
described for Fig. 4, except that factor Xa was preincubated
with the indicated increasing concentrations of the partially
overlapping A1-derived synthetic peptide AG1
(SPGKPGNQNSKNEPP
) or AG2
(P
PKKRERERSSHCYP
) for 5 min at 22 °C
before addition of each incubation mixture to HUVEC monolayers and
determination of thrombin formation by S-2238 hydrolysis as described
for Fig. 4. Thrombin generation by HUVEC monolayers in the
absence of peptide was 0.33 ± 0.03 units/ml. Data are the means
± S.E. of three independent
experiments.
In this study, we have identified the ligand-binding site and functional epitope requirements of a novel vascular cell receptor for the clotting protease factor Xa, designated EPR-1(6) . Cellular receptors for coagulation and fibrinolytic proteases have recently emerged as a novel class of modulators of the hemostatic balance as well as potent signal-transducing molecules in vascular and nonvascular cell types. In addition to the pleiotropic cellular functions mediated by thrombin (18) and transduced by a G-protein-coupled seven-transmembrane domain receptor(3) , binding of urokinase to its receptor (4) has been implicated in the modulation of macrophage gene expression(19) , lymphocyte activation(20) , intercellular adhesion (21) , and monocyte chemotaxis(22) . Furthermore, the association of the anticoagulant protein S with the recently characterized Tyro-3 tyrosine kinase receptor (5) has been implicated in signal transduction and vascular smooth muscle cell proliferation(5, 9) . As a fourth vascular cell protease receptor for factor Xa(6) , EPR-1 participates in thrombin formation (12) and generation of intermediate products of factor IX activation (14) as well as in lymphocyte activation mechanisms(8) .
The first
functional EPR-1 domain identified here comprised the amino-terminal
sequence Met-Arg
and contained the
epitope of the second generation of anti-EPR-1 mAbs, i.e. mAb
2E1(6) . As judged from the partial overlap between this region
and the sequence of a mAb 2E1-immunoreactive cDNA clone isolated from a
phage T cell expression library(6) , the minimal epitope
recognized by this class of anti-EPR-1 mAbs is predicted to be included
between Ala
and Arg
. Characterized by high
hydrophilicity and antigenic index by Kyte-Doolittle analysis, this
region is predicted by Chou-Fasman and Robson-Garnier algorithms to be
structurally organized in two reverse turn structures, localized in a
highly surface-exposed loop in the molecule. The importance of the mAb
2E1 epitope in EPR-1 is underscored by the profound immunosuppressive
properties of this mAb, in vitro and in vivo. In
these experiments and consistent with a more general role of EPR-1 and
other protease receptors in lymphocyte activation
mechanisms(8) , mAb 2E1 completely inhibited lymphocyte
proliferation (IC
0.1 µg/ml), suppressed
cytokine release, and blocked immunoglobulin production and B cell
lymphomagenesis in a human severe combined immunodeficiency mouse
model.
The identification of this immunoregulatory epitope
on EPR-1 may facilitate the rational design of advanced derivatives of
mAb 2E1 or of synthetic peptidyl antagonists targeted at manipulating
the immune response in vivo.
The second functional domain
of EPR-1 identified here corresponded to the sequence
Pro-Arg
and contained a
receptor-binding site for factor Xa. As judged by
I-factor Xa binding parameters of
Pro
-Arg
chimeric transfectants versus wild-type EPR-1 transfectants, most, if not all, of the
EPR-1-factor Xa interaction can be recapitulated by this recognition
sequence. Consistent with this scheme,
Pro
-Arg
transfectants mediated
prothrombin activation in a factor Xa concentration-dependent manner
and were recognized by the first generation of anti-EPR-1 mAbs B6 and
9D4, previously characterized for their ability to block factor Xa
association with leukocytes (12) or endothelium.
The specificity of this recognition was also independently
substantiated by the inability of other expressed extracellular regions
in EPR-1, i.e. Ala
-Glu
, to
bind factor Xa, to participate in prothrombin activation, or to
associate with the functionally inhibitory mAb 9D4 or B6. Molecular
dissection of the EPR-1 ligand binding sequence
Pro
-Arg
by single-amino acid
mutagenesis identified Asn
and Lys
as
crucial residues involved in factor Xa recognition. Interestingly, the
Lys
Ile substitution, which completely abolished
EPR-1-factor Xa interaction, was apparently associated with a profound
structural change in this interacting domain, with disruption of a
discrete reverse turn structure, as tentatively predicted by the
Chou-Fasman algorithm of wild-type versus mutated EPR-1
sequences. Altogether, these data suggest that EPR-1-factor Xa
interaction depends on the integrity of a discrete ligand-binding
groove. As compared with the recognition of other protease receptors,
this model is reminiscent of the ligand binding requirements of the
urokinase receptor (23) as opposed to the proteolytic pathway
of receptor activation mediated by thrombin (3) .
The
identification of the EPR-1 sequence
Pro-Arg
as a novel cellular binding
site for factor Xa may have important pathophysiological implications
for vascular cell assembly of proteolytic activity and thrombin
formation. Consistent with the expression and function of EPR-1 on
endothelium,
a synthetic peptide duplicating the
interacting motif S
PGKPGNQNSKNEPP
dose-dependently inhibited thrombin formation on these cells in the
absence of factor V/Va. That prothrombin activation on HUVEC could not
be solely mediated by released factor V/Va (24) was suggested
earlier by the absence of factor V/Va on normal unperturbed endothelium (25) and/or by the lack of factor V transcript in these
cells(26) . Moreover, cellular receptors for factor Xa,
distinct from factor V/Va, were identified on HUVEC (27, 28) and implicated in ligand processing (29) and intracellular signal transduction with release of
endothelial cell mitogens(30) . The data of synthetic peptidyl
mimicry reported here suggest that under experimental conditions of
unperturbed endothelium (25) and in the absence of detectable
membrane expression of factor V/Va,
most of the prothrombin
activation potential of these cells may be recapitulated by the EPR-1
recognition of factor Xa and competitively inhibited by antagonists of
the Pro
-Arg
sequence, i.e. the AG1 peptide. This leads to the speculation that, although
considerably less catalytically efficient than the prothrombinase
complex coordinated by factor V/Va(31) , the EPR-1-factor Xa
interaction may function as a low-level thrombin-generating system in
the vasculature, thus providing an essential ``first signal''
for clotting(1) , thrombomodulin-dependent anticoagulation
pathways(32) , or protease-dependent vascular cell signal
transduction pathways.
In summary, we have identified two spatially distinct structural domains in the factor Xa receptor (EPR-1) that are separately committed to the dual function of this receptor in the modulation of leukocyte activation (8) and factor Xa assembly on vascular cells. Elucidation of the complementary EPR-1 interacting sequence(s) in factor Xa will provide new insights into the potential pathophysiological role of this pathway in protease-dependent hemostatic and vascular cell signaling processes.