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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4206-4215
RhoA and the Function of Platelet Integrin
 IIb 3
By
Lijun Leng,
Hirokazu Kashiwagi,
Xiang-Dong Ren, and
Sanford J. Shattil
From the Departments of Vascular Biology and Molecular and
Experimental Medicine, The Scripps Research Institute, La Jolla, CA.
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ABSTRACT |
Integrins respond to "inside-out" signals, which enable them
to bind adhesive ligands, and ligand binding initiates
"outside-in" signals that mediate anchorage-dependent cellular
responses. RhoA is a GTPase that regulates certain actin
rearrangements and transcriptional events. It has also been implicated
in integrin signaling, but the exact relationship is not understood. To
examine this further, platelets were incubated with C3 exoenzyme to
adenine diphosphate (ADP)-ribosylate and inactivate RhoA,
and the function of integrin  IIb 3 was
studied. Despite inactivation of  90% of RhoA, platelets exhibited
normal inside-out signaling, as monitored by agonist-induced binding of
a fibrinogen-mimetic anti-IIb3 antibody
and normal fibrinogen-dependent aggregation. On the other hand, RhoA
inactivation decreased the adhesion of agonist-stimulated platelets to
fibrinogen (P < .04) and the formation of vinculin-rich focal
adhesions in platelets that did adhere (P < .001). These
effects were selective because fibrin clot retraction, a response also
dependent on IIb3 and actin
contractility, was unaffected by C3, as was the content of F-actin in
resting or agonist-stimulated platelets. Similar results were obtained
in a Chinese hamster ovary (CHO) cell model system of
IIb3: C3 exoenzyme (or overexpression of
dominant-negative N19RhoA) failed to influence integrin activation
state, but it blocked the formation of focal adhesions in cells spread
on fibrinogen. These studies establish that RhoA plays a highly
selective role in IIb3 signaling, and
they identify a subset of responses to integrin ligation that may be
uniquely dependent on the actin rearrangements regulated by this
GTPase.
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INTRODUCTION |
INTEGRINS ARE heterodimeric
receptors that mediate cell-cell and cell-extracellular matrix
interactions. Their function is tightly controlled through cellular
regulation of ligand availability and receptor number and function. Of
these variables, regulation of receptor function is responsible for
acute changes in platelet adhesiveness during hemostasis, leukocyte
transendothelial migration during inflammation, and vascular cell
migration during wound healing.1-3 Such rapid integrin
regulation, referred to as inside-out signaling, can be due to
modulation of receptor conformation and affinity or modulation of
receptor avidity. The latter may be promoted by integrin lateral
diffusion and clustering and involves the actin
cytoskeleton.1,4,5 The relative contributions of affinity
and avidity modulation to the regulation of ligand binding appear to
vary with the integrin and the cell type.
Ligand binding to integrins triggers outside-in signaling, a
coordinated biochemical and mechanical response that affects anchorage-dependent cell growth, differentiation, survival, and motility.6-8 Recently, members of the Rho family of
GTPases, among them Cdc42, Rac, and Rho, have also been shown to
regulate cytoskeletal organization and gene
transcription.9-11 Accordingly, there has been great
interest in how the functions of integrins and Rho-GTPases are
interrelated. Circumstantial evidence has implicated the Rho family of
GTPases, and Rho in particular, in inside-out and outside-in signaling
through integrins.
For example, treatment of leukocytes with C3 exoenzyme Clostridial
toxin, which specifically adenine diphosphate (ADP)-ribosylates and
inactivates Rho,12 blocks agonist-induced cell aggregation and adhesion, events that require activation of and ligand binding to
1 or 2 integrins.13,14
Furthermore, in fibroblasts, overexpression of constitutively-active
variants of Cdc42, Rac, and Rho or activation of the wild-type proteins
by guanine nucleotide exchange factors triggers assembly of
"integrin complexes" visible by immunofluorescence microscopy. In
contrast, overexpression of dominant-negative variants or
microinjection of Rho-inactivating toxins inhibits integrin complex
assembly in response to growth factors.15,16 These results
have suggested that Rho family GTPases regulate integrin affinity
and/or avidity.
The integrin complexes regulated by Rho family members in fibroblasts
and certain other cell types are present within discrete actin-based
protrusive and adhesive structures: filopodia in the case of Cdc42,
lamellipodia in the case of Rac, and focal adhesions in the case of
Rho.10,17 The clustering of integrins into these structures
may be essential for some of the cellular responses to integrin
ligation. For example, inhibitors of actin polymerization or
actin-myosin contractility block not only the formation of Rho-mediated
stress fibers and focal adhesions, but also integrin-dependent tyrosine
phosphorylation of FAK and paxillin.18,19 Thus, Rho and
related GTPases may modulate signaling responses triggered through
integrins.
The present studies were performed to systematically evaluate whether
and how RhoA affects integrin signaling in a primary cell, the blood
platelet. This system was chosen for several reasons. RhoA is the sole
substrate for C3 exoenzyme in platelets,20 and these cells
have been shown to contain a Rho guanine nucleotide dissociation
inhibitor, guanine nucleotide exhange factors, and Rho
effectors.9,21 Furthermore, affinity modulation of the platelet-specific integrin, IIb3, is
required for fibrinogen binding and platelet aggregation,1
and C3 exoenzyme has been reported to partially inhibit
thrombin-induced platelet aggregation.22 In addition,
platelet aggregation is associated with tyrosine phosphorylation of FAK
and translocation of RhoA to the Triton-insoluble cytoskeleton.23-25 Finally, platelet spreading on a
fibrinogen matrix is dependent on IIb3
and is associated with the formation of focal adhesions and actin
cables, structures characteristically regulated by
Rho.26,27
Here we have monitored multiple facets of
IIb3 function using an experimental
system in which the vast majority of RhoA in intact platelets has been
inactivated by C3 exoenzyme. Parallel studies were conducted in a
Chinese hamster ovary (CHO) cell model system that was developed to
study IIb3 function. The results establish that RhoA is not involved in regulating the ligand binding function of IIb3. Rather, RhoA is
involved in a subset of outside-in signaling responses that may be
uniquely dependent on the specific actin rearrangements controlled by
this GTPase.
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MATERIALS AND METHODS |
Reagents.
ADP, phorbol myristate acetate (PMA),1 prostaglandin
E1 (PGE1), apyrase, and hirudin
were from Sigma Chemical Co (St Louis, MO). -Thrombin was from
Organon Teknika (Durham, NC). Thrombin receptor-activating peptide
(SFFLRN, referred to here as TRAP) was from Peninsula Laboratories
(Belmont, CA). (32P)NAD was from Amersham Life
Science, Inc (Arlington Heights, IL). Integrilin, a function-blocking
cyclic peptide selective for IIb3, was a
gift from Dr David Phillips (Cor Therapeutics, South San Francisco,
CA). Monoclonal antivinculin antibody was from Sigma, monoclonal
anti-RhoA antibody was from Santa Cruz Biotechnology, Inc (Santa Cruz,
CA), fluorescein isothiocyanate (FITC) goat antimouse IgG was from
Biosource International (Camarillo, CA), and rhodamine-phalloidin,
phallacidin, bodipy-phallacidin, and BCECF, AM were from Molecular
Probes, Inc (Eugene, OR).
Dr Mark Ginsberg (Scripps Research Institute) provided CHO cell lines
expressing IIb3, mammalian expression
vectors encoding Tac- 5 or constitutively-active
V12H-Ras, and anti-LIBS6, an IIb3
activating antibody. Mammalian expression vectors encoding wild-type
RhoA and dominant-negative N19RhoA and a pGEX vector encoding
glutathione S-transferase (GST)/C3 exoenzyme were provided by Drs Gary Bokoch, Mark Renshaw, and Martin Schwartz
(Scripps).28 Recombinant C3 exoenzyme was cleaved from GST
with -thrombin and sequentially gel-purified over
glutathione-agarose, p-amino benzamidine-agarose, and Q-Sepharose
(Sigma). After extensive dialysis against C3 "vehicle
buffer" (150 mmol/L NaCl, 20 mmol/L HEPES, pH 7.35), C3 exoenzyme
was concentrated to 1 mg/mL and stored at
 70°C.
Platelet preparation and ADP-ribosylation of RhoA by C3 exoenzyme.
Fresh acid-citrate-dextrose (ACD)-anticoagulated blood
was obtained from drug-free normal donors, and platelet-rich plasma was
obtained.29 For some experiments, an additional aliquot of
blood was anticoagulated with 0.38% sodium citrate to prepare citrated
platelet-poor plasma. Platelet-rich plasma was supplemented with 1 µmol/L PGE1 and 1 U/mL apyrase and centrifuged for 15 minutes at 1,600 rpm at room temperature in a Sorvall GLC-2B centrifuge (Wilmington, DE). After gentle resuspension in a buffer containing 145 mmol/L NaCl; 20 mmol/L PIPES, pH 6.5; 1 µmol/L PGE1; and
1 U/mL apyrase; the platelets were sedimented again and resuspended in
an "incubation buffer" containing 137 mmol/L NaCl; 2.7 mmol/L KCl; 1 mmol/L MgCl2; 5.6 mmol/L glucose; 1 mg/mL bovine
serum albumin (BSA); 3.3 mmol/L NaH2PO4; and 20 mmol/L HEPES, pH 7.4. Platelets were then incubated at 1.4 × 109/mL for up to 4 hours at 37°C in the presence of 10 U/mL apyrase, 10 U/mL hirudin, and C3 exoenzyme or vehicle buffer.
Preliminary experiments established that hirudin had no adverse effect
on platelet function, but its presence was necessary to inactivate traces of thrombin variably contaminating C3 exoenzyme preparations.
ADP-ribosylation of Rho was determined as described by Morii et
al,22 except that platelets were lysed at 4°C in
radioimmunoprecipitation (RIPA) buffer (1% Triton X-100;
1% sodium deoxycholate; 0.1% sodium dodecyl sulfate (SDS); 158 mmol/L
NaCl; 10 mmol/L Tris, pH 7.4; 1 mmol/L Na2EGTA; 0.5 mmol/L
leupeptin; 0.25 mg/mL 4-(2-aminoethyl)-benzenesulfonyl fluoride
hydrochloride; 5 µg/mL aprotinin). To exclude
significant carry-over of C3 exoenzyme from the initial incubation,
control platelets were supplemented with an appropriate amount of C3
immediately before washing and lysis.
Measurements of affinity modulation of
IIb3 and platelet aggregation.
After the incubation with C3 exoenzyme or vehicle buffer, platelets
were diluted to 1.4 × 108/mL with incubation buffer
and incubated for a further 15 minutes at room temperature with an
agonist (ADP, PMA, or TRAP) and 40 µg/mL FITC-PAC1. PAC1 is a
fibrinogen-mimetic, IIb3-specific monoclonal antibody that binds to platelets in an activation-dependent and Arg-Gly-Asp (RGD)-inhibitable manner.30
PAC1 binding to platelets was quantitated in a FACSCalibur flow
cytometer (Becton-Dickinson Immunocytometry Systems, San Jose,
CA).29 To study platelet aggregation responses to agonists,
C3-treated or control platelets were diluted to 1.4 × 108/mL in citrated platelet-poor plasma as a source of
fibrinogen and then stirred at 1,000 rpm for 2 to 3 minutes in an
aggregometer (Chrono-Log, Broomall, PA). In some experiments,
aggregation was also evaluated by counting the remaining single
platelets in an electronic particle counter (Coulter Z1, Coulter Corp,
Hialeah, FL).31
Measurement of IIb3-dependent platelet
adhesion.
After 3.5 hours incubation of platelets with C3 exoenzyme or vehicle
buffer, platelets were labeled with 12 µmol/L BCECF, AM in dimethyl
sulfoxide (DMSO) (0.4% final volume) for 30 minutes at 37°C. They
were diluted to 4 × 108 cells/mL in incubation
buffer, and platelet adhesion was initiated by adding 75-µL aliquots
to fibrinogen-coated microtiter wells in the presence or absence of 30 or 100 nmol/L PMA. After 30 minutes at room temperature, nonadherent
platelets were removed by aspiration, wells were washed twice with
phosphate-buffered saline (PBS), and adherent platelets were
quantitated in a fluorescence microplate reader at 485/530 nm. Adhesion
was expressed as a percentage of input platelets and calculated from a
standard curve derived by adding known numbers of labeled platelets to
microtiter wells and measuring fluorescence.
Measurements of platelet F-actin content.
F-actin content was analyzed by flow cytometry with
bodipy-phallacidin.32 Specifically, after an initial
30-second incubation of unstirred platelets at 37°C in the presence
of 0.1 µmol/L PGE1 or 10 or 100 µmol/L TRAP, platelets
were fixed with 4 volumes of 2.6% glutaraldehyde in 5.3 mmol/L EDTA
for 2 hours at 37°C. After washing twice with PBS, the platelets
were resuspended to half their initial volume and incubated at
37°C either with 3.3 µmol/L bodipy-phallacidin or
bodipy-phallacidin in the presence of a 300-fold molar excess of
unlabeled phallacidin. After 30 minutes, the platelets were washed
twice with PBS and platelet fluorescence was analyzed in the FL1
channel of the flow cytometer. F-actin content was expressed in
terms of specific bodipy-phallacidin fluorescence, which was defined as
that competed for by the excess of unlabeled phallacidin, and it ranged
from 88% to 91% of total fluorescence.
Clot retraction was measured essentially as described by Schoenwaelder
et al.33 After incubation with C3 exoenzyme or vehicle buffer, varying numbers of platelets were added to a constant volume of
citrated platelet-rich plasma in siliconized glass tubes. Clotting was
initiated at 37°C by addition of 2 mmol/L CaCl2 and 8 U/mL -thrombin, and the extent of clot retraction was assessed 90 minutes later by measuring the volume of fluid not incorporated into
the clot.
Immunofluorescence microscopy of platelets.
Glass coverslips were coated with 100 µg/mL fibrinogen for 2 hours at
37°C and then washed with phosphate-buffered saline. After incubation
with C3 exoenzyme or vehicle buffer, 1.4 × 107
platelets in 0.5 mL of incubation buffer were added to the
fibrinogen-coated coverslips for 60 minutes at 37°C. Nonadherent
platelets were washed away and adherent cells were stained with
monoclonal antivinculin antibody, FITC antimouse IgG, and
rhodamine-phalloidin.27 Platelet spreading, vinculin-rich
focal adhesions, and F-actin were analyzed with an MRC 1024 Bio-Rad
laser scanning confocal imaging system attached to a Leitz Diaplan
microscope (Leitz, Wetzlar, Germany).
Integrin signaling in CHO cells.
The effects of C3 exoenzyme on IIb3
function were studied in CHO cells stably-expressing either wild-type
IIb3 (A5 cells) or a
constitutively-active IIb3 chimera
(IIb6A/31),
which contains the extracellular and transmembrane domains of
IIb and 3 and the cytoplasmic domains of
6A and 1.34 Cells were
cultured in the presence of vehicle buffer or 5 to 20 µg/mL of C3
exoenzyme. After 24 hours, the cultures were supplemented again with
C3, and 24 hours later they were processed for studies of PAC1 binding and fluorescence microscopy.34,35 In other experiments,
IIb6A/31 CHO cells were transfected with hemaglutinin-tagged plasmid constructs encoding either dominant-negative N19RhoA or constitutively-active V12H-Ras along with a plasmid encoding a marker protein,
Tac-5. Forty-eight hours later, recombinant protein
expression was assessed by Western blotting and
IIb3 affinity was measured in
transfectants by flow cytometry using PAC1.35 As described
previously,35 PAC1 binding to CHO cells was expressed as an
activation index, where nonspecific binding in the presence of 10 µmol/L Integrilin was assigned a value of zero, and maximal specific
binding in the presence of a saturating concentration of activating
antibody anti-LIBS6 was assigned a value of 100.
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RESULTS |
ADP-ribosylation of RhoA in intact platelets by C3 exoenzyme.
C3 exoenzyme was selected as a potentially useful tool to study
RhoA-IIb3 relationships because it
specifically inactivates Rho12 and its sole substrate in
platelets is RhoA.20 To determine whether the
bacterially-expressed preparation of recombinant C3 exoenzyme was
functional, studies were performed first with CHO cells that stably
express IIb3 (A5 cells).34
Incubation of these cells for 48 hours with C3 exoenzyme resulted in a
dose-dependent ADP-ribosylation of Rho, as determined by the subsequent
inability of C3 exoenzyme to (32P)ADP-ribosylate Rho in
cell lysates. Approximately one half of the Rho had been
ADP-ribosylated and inactivated by 5 µg/mL of C3 exoenzyme and 90%
by 20 µg/mL of C3 (Fig 1). Cells cultured with C3 and then allowed to spread on a fibrinogen matrix exhibited a
marked reduction in focal adhesions and actin stress fibers (Fig 2).
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| Fig 1.
ADP-ribosylation of Rho in A5 CHO cells by C3 exoenzyme.
In (A) A5 CHO cells were cultured for 24 hours in the presence of vehicle buffer or the indicated amounts of C3 exoenzyme. The cultures were then supplemented again with the same amounts of C3, and 24 hours
later the cells were washed and lysates were subjected to
(32P)ADP-ribosylation assay as described in Materials
and Methods. The arrow points to the 32P-labeled Rho band.
Note that the cells that had been cultured with C3 showed a subsequent
decrease in incorporation of 32P, indicating that the Rho
in these cells had become ADP-ribosylated during culture. In (B) the
solid bars represent the means ± SEM of three independent
experiments. The number above each bar represents the ribosylation
response relative to that observed in the "No C3" control
samples, which was arbitrarily assigned a value of 100%.
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| Fig 2.
Effect of C3 exoenzyme on focal adhesions and stress
fibers in A5 CHO cells adherent to fibrinogen. A5 CHO cells were
cultured as in Fig 1 in the presence of vehicle buffer (A and C) or 20 µg/mL of C3 exoenzyme (B and D). The cells were then resuspended in
Dulbecco's modified Essential medium and incubated over
fibrinogen-coated coverslips for 60 minutes at 37°C. After washing
away nonadherent cells, adherent cells were fixed, permeabilized, and
stained for vinculin (A and B) or F-actin (C and D). Vinculin-positive
focal adhesions and actin stress fibers were plentiful in the control cells, but not in the cells treated with C3 exoenzyme. The results are
representative of three experiments. Bar = 5 µm.
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Next, to establish whether C3 exoenzyme would enter platelets and
ADP-ribosylate RhoA in an experimentally useful time frame, washed
platelets were incubated at 37°C for up to 4 hours in the presence
of C3 or an equivalent volume of vehicle buffer. Preliminary studies
indicated that 4 hours was the longest time that platelets could be
incubated in this buffer system without an unacceptable loss of
responsiveness. Incubation of platelets with C3 exoenzyme caused a
time- and dose-dependent ADP-ribosylation of RhoA such that at 400 µg/mL of the toxin, approximately 90% of the GTPase had become
inactivated (Fig 3). On the other hand, C3
treatment did not affect the platelet content of RhoA, as determined by immunoprecipitation and immunoblot analysis (not shown). These results
indicate that efficient RhoA blockade can be achieved in platelets and
CHO cells under the appropriate experimental conditions.
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| Fig 3.
ADP-ribosylation of RhoA in platelets by C3 exoenzyme. In
(A) washed platelets were incubated for 4 hours at 37°C in the
presence of vehicle buffer ("No C3") or C3 exoenzyme. The
platelets were then washed and subjected to
(32P)ADP-ribosylation assay as described in Materials
and Methods. In (B) the solid bars represent the means ± SEM of three
experiments.
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Effect of RhoA inactivation on the ligand binding function of
IIb3.
A fibrinogen-mimetic anti-IIb3 monoclonal
antibody (PAC1) was used to determine the effect of C3 exoenzyme on the
activation state of IIb3. After 4 hours
of incubation with the vehicle buffer as a control, platelets exhibited
the expected marked increase in PAC1 binding in response to ADP or
TRAP, agonists that engage G protein-linked receptors and initiate
inside-out signaling (Fig 4).36,37 In three separate experiments, a 4-hour incubation with 200 or 400 µg/mL of C3 exoenzyme caused ADP ribosylation of
66.0% ± 10.3% (standard error of mean [SEM]) and
88.0% ± 5.1% of the RhoA, respectively. However, this treatment
had no effect on PAC1 binding induced by either submaximal or maximal
concentrations of ADP or TRAP (Fig 4). Moreover, C3 had no effect on
PAC1 binding induced by PMA, which initiates activation of
IIb3 at the level of protein kinase C
(Fig 4).
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| Fig 4.
Effect of RhoA inactivation by C3 exoenzyme on
agonist-induced affinity modulation of platelet
IIb3. Washed platelets were incubated
with vehicle buffer ("No C3") or C3 exoenzyme as in Fig 3. Cells
were then diluted with incubation buffer and the affinity state of
IIb3 was assessed by flow cytometry using FITC-PAC1. PAC1 binding was expressed as a percentage, 100% being arbitrarily assigned to the maximal response for the "No C3"
control sample that had been paired with the experimental sample
containing 200 µg/mL of C3 exoenzyme. Data represent the means ± SEM of three experiments.
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To determine if the inability of C3 exoenzyme to influence the
IIb3 activation process was unique to the
cellular context of the platelet, the effect of C3 on
IIb3 in CHO cells was examined. In the A5
CHO cell line, IIb3 is normally in a
low-affinity state. In contrast, in CHO cells expressing the chimera,
IIb6A/31, the integrin is in constitutive, energy-dependent high-affinity state.34 Integrin affinity was monitored by the binding of
PAC1, which was expressed as an activation index (see Materials and Methods). In two separate experiments, incubation of either cell line
for 48 hours with 20 µg/mL of C3 exoenzyme had no effect on PAC1
binding, despite the fact that 90% of the RhoA had been inactivated
(activation indices: A5 cells without C3 exoenzyme, 18; A5 cells with
C3, 14;
IIb6A/31
cells without C3, 42;
IIb6A/31 cells with C3, 42). To validate this finding in another way,
IIb6A/31 cells were transiently-transfected with dominant-negative N19RhoA. Western blotting indicated that N19RhoA was expressed to levels about
fivefold greater than endogenous RhoA, but it had no effect on PAC1
binding to these cells. In contrast, and as reported
previously,38 a constitutively-active H-Ras mutant
(V12H-Ras) inhibited PAC1 binding to these cells (not shown). Taken
together with the platelet experiments, these results strongly suggest
that RhoA is dispensable for inside-out signaling reactions that
modulate integrin affinity.
Because activation of IIb3 leads to
ligand binding and platelet aggregation, the effect of C3 exoenzyme on
fibrinogen-dependent platelet aggregation was studied. After incubation
with 400 µg/mL of C3 exoenzyme, platelets were diluted with citrated
plasma as a source of fibrinogen and then stirred in an aggregometer.
Platelets that had been incubated for 4 hours with vehicle buffer
underwent normal primary aggregation in response to TRAP or a
combination of TRAP and epinephrine (Fig
5). However, secondary aggregation was impaired due to the prolonged
incubation. C3 exoenzyme had no effect on primary aggregation, despite
ADP-ribosylation of an average of 92.6% ± 0.7% of the RhoA (Fig
5). C3 also had no effect when aggregation was monitored by a single
platelet counting method. Thus, RhoA is not required for ligand binding
to IIb3 or for postligand binding events
responsible for primary platelet aggregation.
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| Fig 5.
Effect of RhoA inactivation by C3 exoenzyme on platelet
aggregation. Washed platelets were incubated for 4 hours at 37°C
with vehicle buffer ("No C3") or 400 µg/mL of C3 ("C3").
Then citrated platelet-poor plasma was added back, and platelet
aggregation was measured in stirred samples by aggregometry. In this
system, light transmittance is assigned a value of 0% in
nonaggregated, platelet-replete samples and 100% in platelet-free
samples. Vertical bars indicate a 20% aggregation response. With this
technique, primary aggregation is generally < 50% and secondary
aggregation > 50%. (A and B) Show tracings from two separate
experiments representative of four experiments.
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Effect of RhoA inactivation on outside-in signaling through
IIb3.
C3-treated platelets were tested for their ability to undergo
outside-in signaling responses. Although the initial adhesion of
platelets to immobilized fibrinogen does not require platelet activation, the subsequent cytoskeletal reorganization and platelet spreading do.26,39 Some of these activation signals are
probably triggered through IIb3, although
additional signals emanating from agonist receptors are required for
full platelet spreading and adhesion.26,40 Therefore, the
effect of C3 exoenzyme on platelet adhesion to fibrinogen was studied,
both in the absence and presence of an exogenous agonist, PMA. In this
series of experiments, 400 µg/mL of C3 exoenzyme caused
ADP-ribosylation of 64.2% ± 5.6% of the RhoA. However, it had no
effect on the adhesion of unstimulated platelets
(Fig 6). As expected, stimulation with 30 or 100 nmol/L PMA increased the number of adherent platelets (Fig 6).
Microscopic inspection showed that this was due to an increase in
adhesion of single platelets in four of five experiments; however, in
one experiment, small platelet aggregates were observed around adherent platelets. C3 exoenzyme caused a modest 25% decrease in the adhesion of PMA-stimulated platelets, and this decrease was statistically significant (P < .04) (Fig 6). These results indicate that
RhoA is not required for the adhesion of unstimulated platelets to fibrinogen, but it may facilitate the adhesion of activated platelets to this substrate.
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| Fig 6.
Effect of RhoA inactivation by C3 exoenzyme on platelet
adhesion to immobilized fibrinogen. Washed platelets were incubated for
4 hours at 37°C with vehicle buffer ("No C3") or 400 µg/mL of C3. Then platelets were labeled with BCECF as a fluorescent marker,
washed, incubated in fibrinogen-coated microtitre wells for 30 minutes
at room temperature, and adhesion was quantitated as described in
Materials and Methods. Adhesion is expressed as the percentage of added
platelets that adhered. Data represent the means ± SEM of five
experiments.
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Platelets that spread on fibrinogen display membrane-based, ventral
structures reminiscent of focal adhesions, which can be identified by
their staining with antibodies to vinculin and by their intersection
with F-actin cables stained with rhodamine-phalloidin.27 To
assess the role of RhoA in the formation of these structures, C3-treated and control platelets were allowed to attach to
fibrinogen-coated cover slips for 60 minutes in the presence of 100 nmol/L PMA, the latter added to enhance spreading. Although about one
half of the adherent platelets became fully-spread in each case, there were marked differences between the C3-treated and control platelets in
the number of focal adhesions and the extent of actin cables (Figs 7 and
8). For example, 48.0% of spread control
platelets contained 2 focal adhesions, and 33.0% contained no focal
adhesions. In contrast, only 18.6% of C3-treated spread platelets
contained 2 focal adhesions, and 66.0% contained no focal adhesions
(Fig 8). These differences between control and C3-treated platelets were statistically significant (P < .001), and they establish that RhoA is involved in the integrin-dependent formation or
stabilization of focal adhesions in platelets.
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| Fig 7.
Focal adhesions and actin cables in fibrinogen-adherent
platelets. Washed platelets were incubated for 4 hours at 37°C in the absence (A through E) or presence (F through J) of 400 µg/mL C3
exoenzyme. They were then diluted in incubation buffer and placed over
fibrinogen-coated coverslips for 60 minutes at 37°C in the presence
of 100 nmol/L PMA to enhance spreading. Vinculin was stained with a
specific monoclonal antibody and FITC antimouse IgG and F-actin was
stained with rhodamine phalloidin. Single views of individual spread
platelets were obtained by confocal microscopy. The five platelet pairs
shown (eg, A and F, B and G, etc) are from five separate platelet
donors and are representative of predominant morphologies. Arrows point
to some of the green-staining focal adhesions, which were scored
positive on the basis of heavy focal vinculin staining, as reported
previously.27 Arrowheads point to some of the red-staining
actin cables often seen to connect the focal adhesions. Note that focal
adhesions and actin cables were prominent in control platelets, but not
in platelets treated with C3 exoenzyme. Bar = 5 µm.
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| Fig 8.
Effect of RhoA inactivation by C3 exoenzyme on focal
adhesion formation in fibrinogen-adherent platelets. Washed platelets were incubated for 4 hours at 37°C with vehicle buffer ("No
C3") or 400 µg/mL of C3. Cells were incubated over
fibrinogen-coated coverslips and adherent cells processed for
fluorescence microscopy as in Fig 7. Five hundred control and
C3-treated platelets were scored as being spread or unspread, the
latter defined as rounded and  5 µm in diameter. Also, 500 spread
platelets in each sample were scored for focal adhesions as illustrated
in Fig 7. Data represent means ± SEM of four experiments. C3 caused
ADP-ribosylation of 64.4% ± 8.0% of the RhoA in this series of
experiments.
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Effect of RhoA inactivation on clot retraction and platelet F-actin
content.
Clot retraction is dependent on the interaction of fibrin with
IIb3 and platelet
contractility.41,42 Because RhoA has been implicated in the
contractility of actin stress fibers, it is possible that these
structures function in clot retraction.18,19 However, there
was no difference in the extent of thrombin-induced clot retraction
between control platelets (Fig 9,  ) and
C3-treated platelets (Fig 9,  ), despite the fact that the C3 caused
ADP-ribosylation of 93.7% ± 2.0% of the RhoA in this series of
experiments. In contrast, cytochalasin D, an inhibitor of actin
polymerization, markedly inhibited clot retraction (Fig 9,  v  ). These results indicate that the platelet events
required for clot retraction are independent of RhoA.
View larger version (19K):
[in this window]
[in a new window]
| Fig 9.
Effect of RhoA inactivation by C3 exoenzyme on fibrin
clot retraction. Washed platelets were incubated for 4 hours at
37°C with vehicle buffer ("No C3") or 400 µg/mL of C3.
Alternatively, platelets were incubated for 3 hours, 50 minutes without
additive, and then with DMSO or 10 µmol/L cytochalasin D (Cyto D) for
10 minutes. Then platelets were added to citrated plasma in siliconized glass tubes, and clotting was initiated with 8 U/mL of thrombin and 2 mmol/L CaCl2. After 90 minutes at 37°C, clot retraction was quantitated as described in Materials and Methods. Data represent the means of three experiments. Error bars have been omitted for clarity.
|
|
Recently, Hartwig et al43 showed that Rac, but not RhoA, is
involved in stimulus-induced actin polymerization in suspended, permeabilized platelets. Consistent with those results, but using intact platelets and bodipy-phallacidin to evaluate F-actin content by
flow cytometry, we found that inactivation of 68.5% ± 8.4% of the
RhoA by C3 exoenzyme had no effect on basal F-actin content or on the
increase in F-actin stimulated by TRAP
(Fig 10).
View larger version (23K):
[in this window]
[in a new window]
| Fig 10.
Effect of RhoA inactivation by C3 exoenzyme on platelet
F-actin content. Washed platelets were incubated for 4 hours at
37°C with vehicle buffer ("No C3") or 400 µg/mL of C3.
Platelets were then diluted with incubation buffer and stirred for 30 seconds at 37°C in the presence of PGI2 or TRAP, after
which they were fixed and stained with bodipy-phallacidin for
quantitation of F-actin as described in Materials and Methods. F-actin
content was taken as the mean specific bodipy-phallacidin fluorescence expressed as a percentage of the "No C3" control sample incubated with PGI2. Data represent means ± SEM of three
experiments.
|
|
| |
DISCUSSION |
Potential relationships between RhoA and integrins were evaluated here
using the platelet as a model system, the adhesive and signaling
functions of integrin IIb3 as endpoints,
and the inactivation of RhoA by C3 exoenzyme as an experimental tool. When washed, intact platelets were incubated with 400 µg/mL of C3
exoenzyme for 4 hours, the vast majority of the RhoA in the cells
became ADP-ribosylated and inactivated. Although prolonged platelet
incubation itself caused a loss of the labile secondary aggregation
response, this system enabled us to systematically examine many of the
adhesive and signaling functions of IIb3. The major conclusions from this work are that: (1) more than 90% of
the RhoA in platelets is dispensable for inside-out signaling reactions
that are responsible for affinity modulation of
IIb3 and for initiation of platelet
aggregation; (2) on the other hand, RhoA is involved in discrete
outside-in signaling responses in fibrinogen-adherent platelets, most
prominently the formation of focal adhesions; (3) however, in suspended
platelets, RhoA does not appear to be a major regulator of F-actin
content or of thrombin-induced clot retraction, and in
fibrinogen-adherent platelets, RhoA does not regulate
IIb3-dependent cell spreading. Taken
together, the present studies establish that RhoA plays a highly
selective role in IIb3 function, implying
that integrin- and RhoA-regulated signaling pathways intersect, but
they are not one and the same.
RhoA and inside-out signaling in platelets.
Morii et al22 showed previously that incubation of
platelets for 2 hours with 5 to 20 µg/mL of C3 exoenzyme inhibited
thrombin-induced platelet aggregation, despite the fact that less than
25% of the RhoA in the platelets had become ADP-ribosylated. They
speculated that the ribosylated RhoA might function as a
dominant-negative inhibitor of inside-out signaling. In the present
studies, we were able to inactivate 90% of the RhoA by incubating
the platelets with higher concentrations of C3 for 4 hours. However,
RhoA inactivation had no effect on either agonist-induced affinity
modulation of IIb3 or primary platelet
aggregation. These results were supported by experiments in CHO cells,
which showed that PAC1 binding to an energy-dependent,
constitutively-active IIb3 chimera was unaffected by C3 exoenzyme or by overexpression of a dominant-negative Rho mutant. In contrast, PAC1 binding to this chimera was inhibited by
constitutively-active V12H-Ras, as reported previously.38 Based on the entire collection of data, we conclude that RhoA is not
involved in affinity modulation of IIb3.
This "negative" conclusion has important implications for models
of inside-out signaling in platelets and perhaps other cells.
Fibrinogen (or PAC1) binding to platelets is initiated primarily by
changes in IIb3 conformation that
increase receptor affinity for ligands.44,45 Subsequent
changes in the platelet cytoskeleton may increase receptor avidity so
as to promote irreversible ligand binding and perhaps secondary
platelet aggregation.1,45,46 Given its demonstrated effects
on the cytoskeleton, it is conceivable that RhoA may help to regulate
the formation of large, irreversible platelet aggregates, perhaps
accounting for the previously observed effect of C3 exoenzyme on
platelet aggregation.22 On the other hand, the former
studies were potentially confounded by the use of Tris-containing
buffers, which can be detrimental to platelet function. In the present
study, we could not evaluate secondary platelet aggregation because
this response was degraded by the long incubation times required to
adequately incorporate C3 into the cells. Therefore, further studies
will be required to determine the exact role of RhoA in secondary
platelet aggregation.
C3 exoenzyme has been shown to inhibit 1 and
2 integrin-dependent adhesion events in PMA-stimulated
leukocytes: aggregation of JY T-cells,13 adhesion of L1/2 B
cells to immobilized VCAM-1,14 and adhesion
of neutrophils to fibrinogen.14 These findings are
consistent with Rho involvement either in leukocyte integrin affinity
modulation, avidity modulation, or other postligand binding events.
However, even in leukocytes, it is not necessary to postulate an effect
of Rho on integrin affinity. In contrast to platelets, where a change
in receptor affinity is the dominant mode of integrin activation,
integrin clustering and avidity modulation may be major mechanisms in
leukocytes.4,47 Because Rho is involved in reorganizing
F-actin and promoting integrin clustering,16,18 it is not
too surprising, therefore, to find that C3 exoenzyme impairs the
initial aggregation of leukocytes, but not platelets. In
agonist-stimulated platelets, the initial reversible phase of
fibrinogen or PAC1 binding is relatively insensitive to inhibition by
cytochalasins, providing additional evidence that initial activation of
IIb3 does not require major cytoskeletal
changes.48-50
RhoA and outside-in signaling in platelets.
A number of platelet responses are presumed to be dependent, at least
in part, on signaling events triggered by ligand binding to
IIb3. These include actin rearrangements
to form filopodia and focal adhesions,27,51 aggregation,
spreading on vascular matrices,52 clot
retraction,33,53 and with some agonists, exocytotic
secretion and vesiculation.54 Full responses generally require collaboration between signals generated through integrins and
more traditional agonist receptors. Identification of specific roles
for RhoA downstream of IIb3 is
complicated because RhoA may also function downstream of platelet
agonists,24 and agonist and integrin pathways may converge
at several levels. Nonetheless, the present study shows that RhoA
inactivation partially inhibited the adhesion of PMA-stimulated
platelets to fibrinogen. Thus, cytoskeletal reorganization controlled
by RhoA may be required for IIb3
clustering or other postligand binding events so that adherent
platelets can withstand the shear forces to which they are exposed in
vitro during washing and possibly in vivo during encounters with the
walls of damaged blood vessels.55,56
The most dramatic effect of C3 exoenzyme in fibrinogen-adherent
platelets was on the number of focal adhesions (Fig 8). In addition to
organization of stress fibers and focal adhesions, Rho has been
implicated in cytokinesis and actin polymerization beneath certain
plasma membranes. Each of these responses may be mediated by a specific
subset of Rho effectors.9,57,58 In fibroblasts, stress
fibers and focal adhesions are thought to be regulated by
ROCK-1 and ROCK-2, serine-threonine kinases that induce
actin-myosin contractility by phosphorylating myosin light
chain59,60 and myosin phosphatase.61 Integrin
clustering within these focal adhesions may actually be secondary to
the formation and contraction of stress fibers.18,19
Because platelets contain many of the same Rho effectors and downstream
targets, the same may be true for these cells.
In fibroblasts, experiments with bacterial toxins that either
inactivate or stimulate Rho family GTPases have established a causative
link between Rho and tyrosine phosphorylation of the focal adhesion
proteins, pp125FAK, paxillin, and
p130Cas.62-65 In platelets, FAK activation
requires both integrin ligation and actin polymerization and it occurs
concomitantly with full aggregation or spreading23,40
conditions in which actin rearrangements by RhoA have already occurred.
Although tyrosine phosphorylation was not studied in C3-treated
platelets, one function of RhoA in these cells may be to promote an
appropriate actin-based microenvironment for FAK activation and certain
other biochemical events in outside-in signaling. One such event may be
activation of a subpopulation of phosphatidylinositol
3-kinase.25,66,67
C3 exoenzyme did not influence the content of F-actin in unstimulated
or TRAP-stimulated platelets maintained in suspension, as determined by
specific binding of bodipy-phallacidin (Fig 10). It is possible that
the amount of RhoA inactivated by C3 in these particular experiments
(68%) was insufficient to obtain an effect or that RhoA plays a more
prominent role in actin polymerization in adherent platelets.
Alternatively, regulation of actin filament content in platelets may be
largely independent of RhoA. Indeed, Hartwig et al43 used
detergent-permeabilized platelets to show that Rac, but not Rho,
regulates uncapping and assembly of actin filaments in platelets
stimulated by TRAP. Another prominent cytoskeletal process that was
unaffected by C3 exoenzyme was fibrin clot retraction, even though 94%
of the RhoA had become inactivated. The molecular events that regulate
clot retraction are poorly understood, but binding of fibrinogen/fibrin
to IIb3 and platelet contractility are
clearly required, as evidenced by the effects of
IIb3 blockade or inhibition of actin
polymerization by cytochalasin D (Fig 9). Electron micrographic
analyses of fibrin clots retracted under isometric conditions have
shown platelet pseudopods containing actin filaments oriented along
fibrin strands, which in turn, are oriented in the direction of
tension.41,42 Apparently, Rho is not required for these
particular protrusive and force-generating events.
Studies of cultured fibroblasts indicate that outside-in signaling
through integrins may help to regulate Rho. For example, cell adhesion
via integrins stimulates the production of phosphatidylinositol 4,5-bisphosphate, as does the stimulation of cell lysates by
GTP-Rho.68 Also, cell adhesion to fibronectin leads to a
slow increase in actin stress fibers and focal adhesions, and this
Rho-dependent response can be accelerated by addition of an RGD
peptide.65 Thus, cell adhesion through integrins may
stimulate GTP-loading of Rho or inhibit Rho-GTP hydrolysis in a manner
that complements these same responses to other plasma membrane
receptors.9,10 Whether integrin
IIb3 regulates RhoA in platelets remains
to be determined.
| |
FOOTNOTES |
Submitted October 30, 1997;
accepted January 29, 1998.
Supported by Grants No. HL56595 and HL57900 from the National
Institutes of Health, Bethesda, MD, and by a fellowship to
H.K. from the Banyu-Merck Foundation (Tokyo, Japan).
L.L. and H.K. contributed equally to this work.
Address reprint requests to Sanford J. Shattil, MD,
Department of Vascular Biology, The Scripps Research Institute, 10550 N
Torrey Pines Rd, VB-5, La Jolla, CA 92037.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs Gary Bokoch, Mark Ginsberg, David
Phillips, Mark Renshaw, and Martin Schwartz for providing DNA
constructs, cell lines, and reagents; and to Zheng Luo for technical
assistance.
| |
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Abstract
Introduction
Abstract