Factor Xa binding to annexin 2 mediates signal transduction via protease-activated receptor 1

Article (PDF Available)inCirculation Research 102(4):457-64 · March 2008with23 Reads
DOI: 10.1161/CIRCRESAHA.107.167759 · Source: PubMed
Abstract
The serine protease zymogen factor X is converted to its catalytically active form factor Xa by the binary complex of factor VIIa bound to its cell surface receptor tissue factor (TF) or by the intrinsic Xase complex, which consists of active factors VIII (VIIIa), IX (IXa), factor X, and Ca2+. Factor Xa has procoagulant activity by conversion of prothrombin to thrombin and also induces signal transduction, either alone or in the ternary TF:VIIa:factor Xa coagulation initiation complex. Factor Xa cleaves and activates protease activated receptor (PAR)1 or -2, but factor Xa signaling efficiency varies among cell types. We show here that annexin 2 acts as a receptor for factor Xa on the surface of human umbilical vein endothelial cells and that annexin 2 facilitates factor Xa activation of PAR-1 but does not enhance coagulant function of factor Xa. Overexpression of TF abolishes annexin 2 dependence on factor Xa signaling and diminishes binding to cell surface annexin 2, whereas selectively abolishing TF promotes the annexin 2/factor Xa interaction. We propose that annexin 2 serves to regulate factor Xa signaling specifically in the absence of cell surface TF and may thus play physiological or pathological roles when factor Xa is generated in a TF-depleted environment.
ISSN: 1524-4571
Copyright © 2008 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online
TX 72514
Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas,
DOI: 10.1161/CIRCRESAHA.107.167759
2008;102;457-464; originally published online Jan 3, 2008; Circ. Res.
Mackman, Wolfram Ruf and Thomas S. Edgington
Gourab Bhattacharjee, Jasimuddin Ahamed, Rafal Pawlinski, Cheng Liu, Nigel
Protease-Activated Receptor 1
Factor Xa Binding to Annexin 2 Mediates Signal Transduction via
http://circres.ahajournals.org/cgi/content/full/102/4/457
located on the World Wide Web at:
The online version of this article, along with updated information and services, is
http://www.lww.com/reprints
Reprints: Information about reprints can be found online at
journalpermissions@lww.com
410-528-8550. E-mail:
Fax:Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050.
Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters
http://circres.ahajournals.org/subscriptions/
Subscriptions: Information about subscribing to Circulation Research is online at
at ROCKEFELLER UNIVERSITY LIBRARY on June 11, 2008 circres.ahajournals.orgDownloaded from
Factor Xa Binding to Annexin 2 Mediates Signal
Transduction via Protease-Activated Receptor 1
Gourab Bhattacharjee, Jasimuddin Ahamed, Rafal Pawlinski, Cheng Liu,
Nigel Mackman, Wolfram Ruf, Thomas S. Edgington
Abstract—The serine protease zymogen factor X is converted to its catalytically active form factor Xa by the binary complex
of factor VIIa bound to its cell surface receptor tissue factor (TF) or by the intrinsic Xase complex, which consists of active
factors VIII (VIIIa), IX (IXa), factor X, and Ca
2
. Factor Xa has procoagulant activity by conversion of prothrombin to
thrombin and also induces signal transduction, either alone or in the ternary TF:VIIa:factor Xa coagulation initiation complex.
Factor Xa cleaves and activates protease activated receptor (PAR)1 or -2, but factor Xa signaling efficiency varies among cell
types. We show here that annexin 2 acts as a receptor for factor Xa on the surface of human umbilical vein endothelial cells
and that annexin 2 facilitates factor Xa activation of PAR-1 but does not enhance coagulant function of factor Xa.
Overexpression of TF abolishes annexin 2 dependence on factor Xa signaling and diminishes binding to cell surface annexin
2, whereas selectively abolishing TF promotes the annexin 2/factor Xa interaction. We propose that annexin 2 serves to
regulate factor Xa signaling specifically in the absence of cell surface TF and may thus play physiological or pathological roles
when factor Xa is generated in a TF-depleted environment. (Circ Res. 2008;102:457-464.)
Key Words: annexin 2
endothelial cells
factor Xa
signal transduction
tissue factor
T
he serine protease zymogen factor X is converted to
catalytically active protease factor Xa on binding to the
binary complex of the cell surface receptor tissue factor (TF)
with its protease ligand factor VIIa (VIIa)
1
or by the intrinsic
pathway. The transient TF:VIIa:factor Xa ternary complex is
the target for physiological inhibitory control by TF pathway
inhibitor.
2
On dissociation from TF:VIIa and association with
its cofactor factor Va, factor Xa proteolytically converts
prothrombin to thrombin, which in turn leads to fibrin
formation, fibrin deposition, and thrombus formation (re-
viewed elsewhere
3
). The intact form of factor Xa, Xa
, can
be cleaved by autoproteolysis, resulting in the excision of a
4-kDa peptide and the formation of Xa
.
4,5
Factor Xa
and
Xa
behave identically as enzymes,
6
although recent work
has shown that further cleavage by plasmin results in modi-
fications that eliminate clotting and amidolytic activity.
7
In addition to promoting coagulation, coagulation pro-
teases induce signal transduction through the activation of G
protein– coupled protease-activated receptors (PARs).
8 –12
Thrombin signaling has been extensively characterized and
occurs via cell type–specific combinations of PAR-1, PAR-3,
and PAR-4.
13–16
TF-dependent coagulation complexes also
directly activate PARs.
2,9,17
TF:VIIa activates PAR-2
18
and
the ternary TF:VIIa:factor Xa complex signals by factor
Xa– dependent activation of either PAR-1 or PAR-2.
8,12,19
However, factor Xa– dependent signaling independent of the
TF initiation complex has also been demonstrated in certain
cell types, albeit with lesser efficiency.
2
In this study, we characterize a previously unrecognized cell
surface factor Xa receptor function for annexin 2. The annexins
are a family of phospholipid-binding membrane proteins that
were originally identified as mediators of cellular responses to
changes in intracellular Ca
2
levels.
20
Annexin 2 dimerizes with
p11 to form a heterotetramer.
21
Annexin 2 binds plasminogen
and stimulates its activation by tissue plasminogen activator,
22
with the p11 subunit believed to be responsible for the direct
interaction with tissue plasminogen activator, plasmin, and
plasminogen.
23
The interaction with p11 is dependent on
C-terminal lysine residues, and this interaction is sensitive to
inhibition by -amino caproic acid (EACA).
24
We here demonstrate that factor Xa binds to annexin 2
specifically and annexin 2 regulates factor Xa–mediated
signal transduction independent of procoagulant activity. Our
findings support the conclusion that annexin 2 serves as a
previously unrecognized cell surface receptor for factor Xa to
enable signal transduction in the absence of cell surface TF.
Materials and Methods
Mice
Generation of annexin 2
/
and TF
flox/flox
mice has been described
previously.
25,26
TF
flox/flox
mice were crossed with LysM Cre mice to
generate TF
flox/flox
LysM Cre
mice lacking TF in myeloid cells. All
Original received January 3, 2007; first resubmission received March 5, 2007; second resubmission received November 14, 2007; revised second
resubmission received December 11, 2007; accepted December 13, 2007.
From the Department of Immunology, The Scripps Research Institute, La Jolla, Calif.
Correspondence to Gourab Bhattacharjee, PhD, The Scripps Research Institute, SP-258, 10550 N Torrey Pines Rd, La Jolla, CA 92037. E-mail
gourab@scripps.edu
© 2008 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.107.167759
457
at ROCKEFELLER UNIVERSITY LIBRARY on June 11, 2008 circres.ahajournals.orgDownloaded from
mouse experiments were carried out under approved care and use
protocols.
Materials
Factor Xa, Xa
, Xa-EGR, Gla domainless factor Xa [Xa(-)Gla],
factor Va, thrombin, and mouse monoclonal antibody against the
heavy chain of factors X and Xa were from Hematologic Technol-
ogies (Essex Junction, Vt). Rabbit polyclonal antibody which binds
both factors X and Xa (anti-X/Xa) was prepared as previously
described.
27
Plasminogen was purified as previously described.
28
Angiostatin was from Calbiochem (San Diego, Calif). Mouse mono-
clonal antibodies against annexins 1 and 2 and p11 were from BD
Biosciences (San Jose, Calif). Glutathione S-transferase–fused re-
combinant annexin 2, annexin 1, and p11 were from Abnova (Taipei,
Taiwan). Fluorescein isothiocyanate and Texas red– conjugated im-
munoglobulins (IgGs) were from Vector Labs (Burlingame, Calif).
Nap5 was from Corvas Inc (San Diego, Calif). EACA and other
chemicals were from Sigma (St Louis, Mo), unless otherwise
specified. Mouse monoclonal PAR-1 antibodies, WEDI and
ATAP-2, and rabbit polyclonal PAR-2 antibody have been described
previously.
19,29
Mass Spectrometry
Matrix-assisted laser desorption/ionization (MALDI) mass spectro-
metric experiments were performed as described previously.
30
Cell Culture and Adenoviral Transduction
Human umbilical vein endothelial cells (HUVECs) were from
Clonetics (San Diego, Calif) and grown according to the instructions
of the supplier. Ad5 serotype vectors expressing full-length human
TF have been described previously in detail.
11,31
HUVECs grown to
50% to 60% confluence were transduced with TF (50 particles per
cell) for 3.5 hours and grown for 48 hours.
Cell Surface Binding, Immunoprecipitation, and
Western Blotting
Factors X and Xa were incubated with HUVECs for 4 hours at 4°C
at increasing concentrations, followed by extensive washing to
remove unbound protein and quantitation via Western blotting.
HUVECs were grown to confluence in 6-well plates and then
incubated with factor Xa (200 nmol/L) alone or in the presence of
Nap5 (1
mol/L), hirudin (100 nmol/L), EDTA (1 mmol/L), or
EACA (10 to 50 mmol/L) for 4 to 6 hours at 4°C in HBS (25 mmol/L
Hepes, 0.9% saline, pH 7.5) supplemented with 5 mmol/L CaCl
2
and
0.02% Tween 20. Cells were then extensively washed and solubi-
lized with 50 mmol/L n-octyl-
-D-glucopyranoside, followed by
incubation with anti-X/Xa coupled to Dynabeads (Invitrogen, Carls-
bad, Calif) for 12 to 16 hours at 4°C. Beads were then extensively
washed and resuspended in 2 SDS reducing buffer followed by
PAGE and Western blotting with anti-X/Xa or monoclonal antibod-
ies against annexins 1 and 2, p11, and IgG as control. Quantification
was performed using Scion Image (NIH, Bethesda Md) within the
linear range of the detection apparatus. Plasma membrane purifi-
cation was performed using the Qproteome plasma membrane
purification kit (Qiagen, Valencia, Calif). Factor Xa binding and
annexin 2 coimmunoprecipitation were also assayed in thioglyc-
ollate-elicited peritoneal macrophages isolated from TF
flox/flox
LysM
Cre
and TF
flox/flox
/LysM Cre
mice.
Protein/Protein Binding Assay
Glutathione S-transferase–fused recombinant annexin 2, annexin 1,
and p11 were coated on to Reacti-Bind glutathione-coated plates
(Pierce, Rockford, Ill) according to the protocol of the manufacturer.
Increasing concentrations of factor Xa (10 to 400 nmol/L) was
incubated on the plates for up to 4 hours at 4°C in HBS supplemented
with 5 mmol/L CaCl
2
and 0.02% Tween 20, followed by extensive
washing with ice-cold HBS to remove unbound factor Xa. The
remaining annexin 2 bound factor Xa was quantitated with the
chromogenic substrate Spectrozyme FXa (American Diagnostics,
Greenwich, Conn). Absorbance (405 nm) was converted to factor Xa
(picomoles bound) using an established standard curve.
Immunofluorescence
For confocal imaging, nonpermeabilized HUVECs, grown to con-
fluence on cover slips, were incubated with factor Xa as described
above but in the absence of Tween 20 to prevent permeabilization.
Cells were fixed in 10% formalin in HBS for 15 minutes at 4°C,
followed by incubation with rabbit anti-X/Xa (10
g/mL) in com-
bination with mouse monoclonal anti–annexin 2 (10
g/mL) for 1 to
2 hours at 4°C. Cells were then washed extensively and incubated
with Texas red–conjugated anti-rabbit IgG (1:200) and fluorescein
isothiocyanate– conjugated anti-mouse IgG (1:200) for 1 hour at 4°C,
followed by extensive washing and mounting in Vectashield with
4,6-diamidino-2-phenylindole (Vector Labs). Cells were visualized
by confocal microscopy using a Bio-Rad MRC1024 laser scanning
confocal microscope followed by quantification with Image Pro Plus
(Media Cybernetics, Silver Spring, Md). Control experiments were
performed using secondary antibodies in the absence of primary. TF
was detected in transduced cells using the 9C3 monoclonal anti-
body
32
(5
g/mL) directly conjugated to Texas red.
Extracellular Signal-Regulated Kinase
1/2 Phosphorylation
TF-transduced and nontransduced HUVECs were incubated in
serum-free conditions (medium 199 supplemented with 2 mmol/L
L-glutamine and 1 mmol/L CaCl
2
) for 5 hours at 37°C. Cells were
then incubated with factor Xa (50 nmol/L) for 5 minutes, followed
by lysis in 2 SDS reducing buffer for Western blot analysis using
phosphospecific extracellular signal-regulated kinase (ERK)1/2 and
non–phospho-ERK1/2 rabbit polyclonal antibodies (Cell Signaling,
Danvers, Mass) as described previously. To assay the effect of
annexin 2 on factor Xa–induced ERK phosphorylation, anti–annexin
2 (20
g/mL) was preincubated for 15 minutes before the addition of
factor Xa. Control experiments were performed with mouse mono-
clonal antibodies against PAR-1 (50
g/mL WEDI and 25
g/mL
ATAP-2) and rabbit polyclonal antibody against PAR-2 (200
g/
mL). All experiments were performed in the presence of hirudin to
exclude thrombin effects.
Thrombin Generation Assay
HUVECs grown to confluence in 12-well polystyrene plates were
preincubated with anti–annexin 2 (20
g/mL) for 15 minutes,
followed by addition of factor Xa or Xa
(50 pmol/L), factor Va
(400 pmol/L), prothrombin (1.3
mol/L) for 10 minutes at 37°C,
followed by addition of S-2238 Chromogenix thrombin substrate
from Diapharma (West Chester, Ohio), followed by kinetic assay at
405 nm. Absorbance was converted to units per milliliter thrombin
using an established standard curve.
Cell-Based Factor Xa Generation Assay
Cells grown to confluence in 12-well polystyrene plates were
incubated in the presence of 20
g/mL anti–annexin 2 or anti–
annexin 1 for up to 4 hours at 37°C. Cells were then washed twice
with Hepes (10 mmol/L) buffered saline supplemented with
5 mmol/L CaCl
2
. Factors VIIa (10 nmol/L) and X (50 nmol/L) were
then added. Reactions were stopped at selected time points by adding
100 mmol/L EDTA to the sample and the activity of the generated
factor Xa in the medium was quantitated with the chromogenic
substrate Spectrozyme FXa. Absorbance (405 nm) was converted to
factor Xa generated (picomoles per minute) using an established
standard curve.
Single-Stage Clotting Assay
Peritoneal macrophages were isolated from thioglycollate injected
wild-type and annexin 2 knockout mice and allowed to adhere in
6-well polystyrene plates for 3 hours (37°C). Nonadherent cells were
removed by extensive washing and the remaining, adherent macro-
phages incubated overnight at 37°C. Untreated and lipopolysaccha-
ride-treated (1
g/mL, 4 hours, 37°C) cells were washed with HBS
458 Circulation Research February 29, 2008
at ROCKEFELLER UNIVERSITY LIBRARY on June 11, 2008 circres.ahajournals.orgDownloaded from
and left intact or solubilized in 15 mmol/L n-octyl-
-D-
glucopyranoside. Time to clot was measured using a START4
Coagulation Analyzer (Diagnostica Stago, Parsippany, NJ).
Data Analysis
All data are representative of a minimum of 4 experiments performed
in triplicate and are shown as meansSD.
Results
Factor Xa Binds to Endothelial Cells and
Coprecipitates With Annexin 2
Factors X and Xa were incubated with HUVECs at 4°C to
determine whether there was a unique cell surface receptor
responsible for the observed binding of factor Xa. Factor Xa
bound to HUVECs, whereas factor X binding was negligible
(Figure 1A). To identify the putative receptor for factor Xa,
factor Xa bound to HUVECs was immunoprecipitated using
an anti-X/Xa antibody, followed by nonreducing SDS-PAGE,
resulting in multiple bands that were excised and subjected to
MALDI-TOF mass spectroscopy. Peptides corresponding to
both factor Xa and immunoglobulin heavy and light chains
were identified in addition to a peptide that represented
annexin 2 (Figure 1B). Annexin 2 coprecipitation was con-
firmed by Western blotting (Figure 1C). Factor Xa immuno-
precipitated from endothelial cells was primarily Xa
(Figure
1C), but the ratio of Xa
to Xa
ranged from 1:1 to 1:2 in
the unbound fraction, and there was no conversion of un-
bound factor X to factor Xa (Figure 1C). Active site blocked
factor Xa with Glu-Gly-Arg (EGR)-chloromethyl ketone
exhibited reduced binding relative to active factor Xa, possi-
bly attributable to incomplete processing