* Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 14195 Berlin, Germany; and Division of Biology, California Institute of Technology, Pasadena, California 91125
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Abstract |
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Platelets respond to various stimuli with
rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the
-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or
thrombin receptors. In contrast to thrombin, the TXA2
mimetic U46619 led to the selective activation of G12
and G13 in G
q-deficient platelets indicating that these
G proteins mediate TXA2 receptor-induced shape
change. TXA2 receptor-mediated activation of G12/G13
resulted in tyrosine phosphorylation of pp72syk and
stimulation of pp60c-src as well as in phosphorylation of
myosin light chain (MLC) in G
q-deficient platelets.
Both MLC phosphorylation and shape change induced
through G12/G13 in the absence of G
q were inhibited
by the C3 exoenzyme from Clostridium botulinum, by
the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that
G12/G13 couple receptors to tyrosine kinases as well as
to the Rho/Rho-kinase-mediated regulation of MLC
phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of
MLC phosphorylation participates in receptor-induced platelet shape change.
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Introduction |
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THE functional responses of platelets to various full platelet activators are well characterized and include secretion of granular contents, platelet aggregation, and platelet shape change. Platelets are discoid in their resting state and upon activation by most stimuli rapidly change into a spheroid shape and extrude pseudopodia. This shape change is one of the earliest effects detectable in response to various platelet stimuli. Platelet shape change is believed to be a prerequisite for full platelet activation including degranulation and aggregation. The activation of platelets is responsible for primary hemostasis and underlies various pathological situations such as unstable angina pectoris, myocardial infarction, or cerebrovascular diseases.
Platelet shape change results from a rapid reorganization of the cytoskeleton including formation of new actin
filaments, disappearance of the marginal band of microtubules, and centralization of granules (Siess, 1989; Wurzinger, 1990
; Fox, 1993
; Morgenstern, 1997
). Signal transduction mechanisms regulating platelet shape change are
ill defined. An involvement of tyrosine phosphorylation events, myosin light chain phosphorylation and polyphosphoinositide-induced actin polymerization have been suggested (Daniel et al., 1984
; Hartwig et al., 1995
; Negrescu
et al., 1995
; Maeda et al., 1995
). Several reports have demonstrated that, in contrast to full platelet activation, induction of platelet shape change does not require elevation of
the free cytosolic Ca2+ concentration (Rink et al., 1982
;
Simpson et al., 1986
; Ohkubo et al., 1996
). Consistent with
that, we recently demonstrated that incubation of G
q-deficient platelets with various stimuli failed to induce
phospholipase C activation and [Ca2+]i elevation as well
as aggregation and degranulation. Platelet shape change,
however, could still be elicited (Offermanns et al., 1997b
).
Platelets do not contain G
11, a close homologue of G
q
(Milligan et al., 1993
; Johnson et al., 1996
; Offermanns et al.,
1997b
).
The effect of full platelet stimuli like thromboxane A2
(TXA2)1 and thrombin are mediated through G protein-
coupled receptors which have been shown to activate Gq,
Gi, G12, and G13 (Shenker et al., 1991; Hung et al., 1992
;
Offermanns et al., 1994
; Ushikubi et al., 1994
). G proteins
are heterotrimers which are defined by their
-subunits.
According to structural and functional similarities, G protein
-subunits are grouped into four families, G
q, G
i, G
12, and G
s (Simon et al., 1991
). Although Gq-mediated
activation of phospholipase C
-isoforms appears to play a
central and essential role in agonist-induced platelet aggregation and secretion, Gi-type G proteins are involved
in the inhibitory regulation of platelet adenylyl cyclase
(Brass et al., 1997
, 1988
; Offermanns et al., 1997b
). The
role of the G12 family members, G12 and G13, in the regulation of platelet function is unclear. The
-subunits of both
G proteins appear to be involved in the regulation of cell growth and cell movement (Dhanasekaran and Dermott,
1996
; Offermanns et al., 1997a
). Since G12/G13-coupled receptors appear to also activate Gq family members it has
been difficult to selectively study the cellular signaling
processes regulated by receptor-mediated activation of
G12/G13. Most knowledge about the signaling pathways influenced by G12/G13 results from the use of constitutively
active forms of G
12 and G
13. Either mutant has been
shown to cause Na+/H+ exchanger activation, stimulation
of phospholipase D, cell transformation, and formation of
actin stress fibers through the small molecular weight GTPase Rho (Buhl et al., 1995
; Hooley et al., 1996
; Fromm et al.,
1997
; Plonk et al., 1998
). The effector directly regulated by
G
12 and G
13 has been elusive. However, the guanine nucleotide exchange factor (GEF) for Rho, p115RhoGEF,
has been shown to interact with G
12 and G
13 and represents a candidate effector (Kozasa et al., 1998
; Hart et al.,
1998
).
To study the possible role of G12/G13 in the platelet
shape change response we took advantage of platelets
from Gq-deficient mice. In this report, we demonstrate
that selective activation of G12/G13 is sufficient to induce
the platelet shape change reaction, and we provide evidence that this involves Rho/Rho-kinase-mediated phosphorylation of the myosin light chain.
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Materials and Methods |
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Materials
U46619 was from Cayman Chemical, thrombin, histone (subgroup f2b),
monoclonal anti-myosin light chain (MLC) antibody, and fluorescein
isothiocyanate (FITC)-phalloidin were from Sigma. Sp-5,6-DCl-cBIMPS
and 8-pCPT-cGMP were from Biolog. Y-27632 was kindly provided by
Yoshitomi Pharmaceutical Industries, Clostridium botulinum C3-exoenzyme was a donation from I. Just and K. Aktories (both from University
of Freiburg, Freiburg, Germany) or was purchased from Upstate Biotechnologies, anti-pp72syk antibodies as well as anti-phosphotyrosine antibodies were from Santa Cruz Biotechnology, and anti-pp60v-src antibodies
were from Oncogene. Antisera against G protein -subunits have been
described (Offermanns et al., 1994
; Laugwitz et al., 1994
).
Platelet Preparation and Aggregation
Whole blood was collected from normal and Gq-deficient mice anesthetized with pentobarbital by puncturing the inferior vena cava with heparinized syringes at a final concentration of 25 U heparin/ml blood. The
blood from three or four G
q-deficient mice and wild-type mice was
pooled for each platelet aggregation experiment. Blood was diluted with
half the volume of Hepes-Tyrode-buffer (134 mM NaCl, 0.34 mM
Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM Hepes, 5 mM glucose,
1 mM MgCl2, pH 7.3), and platelet rich plasma (PRP) was obtained by
centrifugation for 7.5 min at 250 g. Thereafter, prostacyclin at a final concentration of 300 nM was added to the PRP, and platelets were pelleted
by centrifugation at 1,200 g for 5 min. The platelet pellet was resuspended
in Hepes-Tyrode buffer and incubated for 30 min at 37°C. Platelet suspension was adjusted to 300,000 platelets per microliter with Hepes-Tyrode buffer. Optical aggregation experiments were conducted in a four-channel aggregometer (model Aggrecorder II PA-3220; Kyoto Daiichi Kagaku). Preincubation in Hepes-Tyrode buffer without and with cGMP and cAMP analogues and Y-27632 was performed for 20 min at room temperature. Immediately before the aggregation experiments, platelets were preincubated for 1 min at 37°C in Hepes-Tyrode buffer containing 1 mM
CaCl2.
Photolabeling of Membrane Proteins and
Immunoprecipitation of G-subunits
Platelet membranes were prepared and photolabeled as described (Offermanns et al., 1994). In brief, cell membranes (50-100 µg of protein per assay tube) were incubated at 30°C in a buffer containing 0.1 mM EDTA,
10 mM MgCl2, 30 mM NaCl, 1 mM benzamidine, and 50 mM Hepes-NaOH, pH 7.4. After 3 min of preincubation in the absence and presence
of receptor agonist, samples were incubated for another 15 min with 10-20
nM [
-32P]GTP azidoanilide (130 kBq per tube). [
-32P]GTP azidoanilide
was synthesized and purified as described (Offermanns et al., 1991
). For
photolabeling of Gi
-subunits, 5 µM GDP was present in the incubation
buffer. Samples were washed, dissolved in labeling buffer, and then irradiated as described (Offermanns et al., 1994
). Photolabeled membranes
were pelleted and proteins were predenatured in SDS. Solubilized membranes were preabsorbed with protein A-Sepharose beads, and immunoprecipitation was done as described (Laugwitz et al., 1994
).
SDS-PAGE and Immunoblotting
SDS-PAGE of photolabeled proteins was performed on 10% (wt/vol)
acrylamide gels. Photolabeled membrane proteins were visualized by autoradiography of the dried gels. Blotting of membrane proteins separated
by SDS-PAGE, processing of immunoblots, and detection of immunoreactive proteins by chemiluminescence procedure (Amersham) has been
described (Laugwitz et al., 1994).
Determination of Cellular cAMP Levels
Platelets (108 per tube) were preincubated for 15 min with 300 µM 3-isobutyl-1-methylxanthine and 20 µM 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) and incubated for 20 min in the absence and
presence of receptor agonists. The reaction was stopped by the addition of
300 µl of ice-cold 10% (wt/vol) trichloracetic acid. Samples were kept for
10 min on ice, and 180 µl of 1 M Tris, pH 9.8, was added to neutralize the
sample. Cyclic AMP was determined by the competitive-binding assay
(Gilman and Murad, 1974). In brief, samples were incubated for 2 h with 2 pmol of [8-3H]cAMP (925 Gbq/mmol; Amersham) and 62.5 µg of cAMP-dependent protein kinase purified from porcine heart (Sigma) in a final volume of 200 µl at 4°C. Then, 4% (wt/vol) charcoal in 5 mM EDTA and
50 mM Tris-HCl, pH 7.5, was added, and samples were immediately centrifuged for 2 min at 12,000 g. Supernatants were counted in a liquid scintillation counter, and the amount of cAMP in the test sample was calculated as described (Gilman and Murad, 1974
).
Determination of Tyrosine Phosphorylation
Isolated platelets (1-2 × 107 platelets per tube) were incubated in 40 µl Hepes-Tyrode buffer at 37°C as indicated. Reactions were stopped by addition of 20 µl of 3× sample buffer containing a final concentration of 1 mM Na3VO4. Heated samples were separated by SDS-PAGE on 10% gels. Immunoblotted proteins were analyzed for phosphotyrosine with an antiphosphotyrosine antibody.
Immunoprecipitation and Immune Complex Kinase Assay
For immunoprecipitation of tyrosine kinases pp72syk and pp60c-src, platelet
suspensions (0.4-1 × 109 platelets) were incubated in the absence or presence of 5 µM U46619 for the indicated time periods, and platelets were
lysed by addition of an equal volume of ice-cold 2× radioimmunoprecipitation assay (RIPA) buffer (final concentration: 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Hepes/NaOH, pH 7.4, 3 mM EDTA, 3 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µg/ml leupeptin). After
incubation for 20 min on ice, samples were centrifuged for 20 min at
15,000 g at 4°C, and incubated with 5 µg agarose conjugates of rabbit polyclonal anti-pp72syk IgG or 8 µl of agarose-conjugated mouse monoclonal
anti-pp60v-src IgG1 for 2 h at 4°C. Immunoprecipitates were collected by
centrifugation at 15,000 g for 10 min at 4°C and were washed twice with
1× RIPA buffer, once with 1% Triton X-100, 0.3% SDS, 600 mM NaCl,
and 50 mM Tris-HCl, pH 7.4, and once with 300 mM NaCl, 10 mM
EDTA, 100 mM Tris-HCl, pH 7.4. For detection of pp72syk phosphorylation, precipitated proteins were eluted with 40 µl of 1× SDS sample
buffer and separated by 10% polyacrylamide gels. Tyrosine phosphorylation of pp72syk and pp72syk protein were analyzed by immunoblotting. The
anti-pp60c-src immunoprecipitates were divided into two aliquots; one was
analyzed by anti-pp60c-src immunoblotting, and the other was subjected to
in vitro kinase assay. To examine in vitro kinase activity, precipitates were incubated for 5 min at 25°C in kinase buffer containing 25 mM Hepes/
NaOH, pH 7.4, 10 mM MnCl2, 1 µM ATP (7 µCi of [-32P]ATP/tube), and
0.25 mg/ml histone. Reaction was terminated by addition of 2× sample
buffer, and samples were subjected to SDS-PAGE. Phosphorylation of
histone was analyzed by autoradiography of dried gels.
Scanning Electron Microscopy
Isolated platelets were preincubated under the indicated conditions. Thereafter, platelets were incubated in the absence or presence of 1 U/ml thrombin or 5 µM U46619 for 5 s at 37°C and then fixed for 10 min with 3% paraformaldehyde, 3.75% glutaraldehyde, 0.06 mM cacodylate buffer, and 3.4 mM CaCl2. The fixed platelets were suction filtered onto polycarbonate filters (0.45 µm; Nucleopore) which had been preincubated with 10 µg/ml polylysine. Filters were washed three times with 0.9% NaCl and dehydrated stepwise in aqueous ethanol. After exchange of ethanol for hexadimethyldisilazane, samples were air-dried and sputtered with gold. Scanning electron microscopy was carried out on a Zeiss-Gemini instrument using a beam voltage of 5 kV.
MLC Phosphorylation
MLC phosphorylation was determined as described (Daniel and Sellers,
1992). Isolated platelets (1-2 × 107 platelets per tube) were incubated in
30 µl Hepes-Tyrode buffer at 37°C as indicated. Reactions were stopped
by addition of 30 µl of 40% (vol/vol) perchloric acid. Precipitated samples
were kept on ice for 20-30 min. After centrifugation (10 min at 15,000 g at
4°C) pellets were washed twice with acetone containing 10 mM DTT. 30 µl
of SDS sample buffer was added to dried samples, and proteins were solubilized by sonication for 30 min. Separation of proteins on urea/glycin gels
was done as described (Daniel and Sellers, 1992
), and MLC was detected
after immunoblotting with an anti-MLC antibody.
Determination of F-actin Content
For actin filament content measurements, platelets (108) were incubated as indicated and fixed in 2% paraformaldehyde for 30 min at 37°C. Fixed platelets were permeabilized with 0.1% Triton X-100, incubated with 10 µM fluorescein isothiocyanate (FITC)-phalloidin (Sigma) for 30 min at room temperature and were then washed. Bound FITC-phalloidin was quantified using a fluorescence spectrophotometer (Perkin-Elmer) (excitation at 495 nm; emission at 519 nm).
ADP Ribosylation of Platelet Lysates by C3 Exoenzyme
Washed platelets were incubated with the indicated concentrations of C3
exoenzyme in Hepes-Tyrode buffer. Platelets were lysed by addition of an
equal volume of lysis buffer (1.5% Triton X-100, 0.8% DOC, 0.2% SDS,
145 mM NaCl, 20 mM Hepes, pH 7.4, 3 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin, 5 µg/ml aprotinin). ADP-ribosylation
using [32P]NAD was performed as described (Morii et al., 1992), and ribosylated samples were separated on 12% polyacrylamide gels.
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Results |
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We have recently shown that Gq-deficient platelets do
not aggregate and secrete their granule contents in response to various stimuli indicating that Gq-mediated activation of phospholipase C represents the central early signal transduction process leading to full platelet activation.
However, Gq-deficient platelets were still able to undergo
ligand-induced platelet shape change. This suggests that G
proteins other than Gq mediate the platelet shape change
response. Shape change induced by the TXA2 analogue
U46619 could be observed in G
q-deficient platelets by
scanning electron microscopy of single cells (Fig. 1, A-D)
as well as by measuring the light transmission of a platelet
suspension (Fig. 2). Shape change induced by U46619 in
G
q-deficient platelets and wild-type platelets was blocked
by the cAMP analogue Sp-5,6-DCl-cBIMPS but not by the
cGMP analogue 8-pCPT-cGMP, whereas both cyclic nucleotides blocked aggregation in wild-type platelets (Fig.
2, A and B). Similar results were observed with thrombin-activated wild-type and G
q-deficient platelets (data not
shown). Preincubation of platelets with the recently described Rho-kinase inhibitor Y-27632 (Uehata et al.,
1997
) blocked U46619-induced shape change both in wild-type and G
q-deficient platelets (Fig. 1, E-H and Fig. 2, C
and D). To assess the role of Rho in agonist-induced platelet shape change we preincubated platelets for 2 h with
50 µg/ml C3 exoenzyme which ADP ribosylates and inactivates the small GTPase Rho (Morii et al., 1992
). This C3
exoenzyme concentration and preincubation time resulted
in ADP-ribosylation of 70-75% of endogenous Rho as determined by the inability of C3 exoenzyme to [32P]ADP-ribosylate Rho in subsequently prepared cell lysates (Fig. 3). Longer preincubation times and higher C3 exoenzyme concentrations further increased the ADP-ribosylated fraction of Rho (Fig. 3), but resulted in preactivation of platelets (data not shown). C3-pretreated platelets
showed markedly reduced shape change in response to U46619 with only partial spheration and occasional filopodia formation (Fig. 1, I-L and Fig. 2 E).
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Since platelet shape change including protrusion of
filopodia and lamellipodia is accompanied by actin polymerization (Siess, 1989; Wurzinger, 1990
; Fox, 1993
) we
measured F-actin content in wild-type and G
q-deficient
platelets (Fig. 4). U46619 induced an increase in F-actin
content of both, wild-type and G
q-deficient platelets,
which could be completely blocked by Y-27632. Reduction of the amount of functional Rho by pretreatment with 50 µg/ml C3 exoenzyme for 2 h markedly reduced the effect
of U46619 in wild-type and G
q-deficient platelets.
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To identify the G proteins mediating receptor-induced
platelet shape change we studied the coupling of TXA2
and thrombin receptors to heterotrimeric G proteins in
wild-type and Gq-deficient mouse platelets. Receptors
for both, thrombin and TXA2, have been shown to be able
to couple to members of the Gq, G12, and Gi families (Shenker et al., 1991
; Hung et al., 1992
; Offermanns et al.,
1994
; Ushikubi et al., 1994
). In membranes of human
platelets, receptors activated by thrombin couple to Gq,
G12, G13, and Gi, whereas TXA2 receptors only activate
Gq, G12, and G13 (Offermanns et al., 1994
; Brass et al.,
1997
). Photolabeling of receptor-activated G proteins in
mouse platelet membranes and subsequent immunoprecipitation of individual G protein
-subunits showed that
in wild-type mouse platelets, activated TXA2 and thrombin receptors couple to Gq, G12, and G13, whereas Gi was
only activated through the thrombin receptor (Fig. 5 A).
Similarly, in membranes from G
q-deficient platelets, only
G12 and G13 were activated through the TXA2 receptor, whereas activated thrombin receptors coupled to G12, G13,
and Gi (Fig. 5 B and data not shown). Coupling of thrombin receptors to Gi in murine platelets corresponded with
the ability of thrombin to decrease cAMP levels in wild-type as well as in G
q-deficient platelets, whereas activation of TXA2 receptors had no effect on adenylyl cyclase
activity in wild-type or G
q-deficient platelets (data not
shown). These data clearly demonstrate that in G
q-deficient platelets thrombin-receptors couple to G12, G13, and
Gi, whereas only G12 and G13 are activated through TXA2
receptors. Consequently, effects which can still be induced
by TXA2 receptor agonists in G
q-deficient platelets like
the shape change response are mediated by G12 and/or
G13. TXA2-activated G
q-deficient platelets therefore represent a model to study of G12/G13-mediated signaling processes.
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Agonist-induced platelet activation results in tyrosine
phosphorylation of multiple proteins (Ferrell and Martin,
1988; Nakamura and Yamamura, 1989
; Golden and Brugge,
1989
). Phosphorylation of these proteins occurs in three
temporal phases which have been experimentally distinguished. Early tyrosine phosphorylation occurs by an integrin-independent mechanism, whereas the second and
third wave of tyrosine phosphorylation depends on the aggregation of platelets through binding of fibrinogen to
IIb
3-integrin (glycoprotein IIb-IIIa) (Clark et al., 1994b
).
In G
q-deficient platelets that do not aggregate in response to thrombin or U46619, only a subset of proteins
became tyrosine phosphorylated upon exposure of platelets to both stimuli compared with wild-type platelets (Fig.
6, A and B). Most prominently, a rapid tyrosine phosphorylation of a protein of ~72 kD could be observed in G
q-deficient platelets activated with thrombin and U46619. In
contrast, several proteins with relative molecular masses
of 40 and 95-130 kD which were tyrosine phosphorylated in wild-type platelets did not show increased tyrosine
phosphorylation in activated G
q-deficient platelets (Fig.
6, A and B).
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Platelets contain several tyrosine kinases (Dhar and
Shukla, 1993; Clark et al., 1994b
; Jackson et al., 1996
)
among which pp72syk and pp60c-src are rapidly activated after stimulation of platelets in an aggregation-independent manner (Wong et al., 1992
; Taniguchi et al., 1993
; Maeda
et al., 1993
; Clark and Brugge, 1993
; Clark et al., 1994a
).
To test whether the 72-kD protein that was tyrosine phosphorylated in response to U46619 and thrombin in G
q-deficient platelets represented pp72syk, we immunoprecipitated pp72syk from lysates of platelets exposed to U46619.
Anti-phosphotyrosine immunoblots of pp72syk immunoprecipitates demonstrated increased tyrosine phosphorylation of pp72syk in response to U46619 in G
q-deficient
platelets as well as in wild-type platelets (Fig. 6 C). Autophosphorylation of pp72syk on tyrosine has been demonstrated to increase its enzymatic activity (Taniguchi et al.,
1993
; Clark et al., 1994a
; Chacko et al., 1994
; Fujii et al.,
1994
). Fig. 6 C shows that incubation of wild-type and
G
q-deficient platelets with U46619 also resulted in a
rapid increase in the activity of pp60c-src. Increases in tyrosine kinase activity could be observed within 10 s after
addition of U46619 and were not affected by pretreatment of platelets with Y-27632 or C3 exoenzyme (data not
shown). These data indicate that TXA2 receptor-mediated
activation of G12/G13 leads to rapid activation of the tyrosine kinases pp72syk and pp60c-src in mouse platelets.
MLC phosphorylation has been suggested to be involved in early processes during platelet activation (Daniel et al., 1984). To test whether TXA2 receptor-G12/G13-
mediated signaling in G
q-deficient platelets resulted in
MLC phosphorylation, we activated platelets with U46619
for different times and separated phosphorylated and unphosphorylated MLC on urea/glycin polyacrylamide gels.
Separated proteins were blotted onto nitrocellulose filters
and MLC was detected using a specific antiserum. Fig. 7 A
shows that U46619 caused phosphorylation of the total detectable pool of MLC in wild-type platelets within 10 s. Interestingly, a rapid and apparently complete phosphorylation of MLC was also observed in G
q-deficient platelets
activated by U46619. Chelation of extracellular Ca2+ by
EGTA or preincubation of platelets with various tyrosine
kinase inhibitors had no effect on U46619-induced MLC
phosphorylation in wild-type and G
q-deficient platelets
(data not shown). Although the cAMP analogue Sp-5,6-DCl-cBIMPS completely inhibited MLC phosphorylation in wild-type and G
q-deficient platelets the cGMP analogue
8-pCPT-cGMP was without effect (Fig. 7, B and C). In
smooth muscle cells and fibroblasts, the phosphorylation
state of MLC has been shown to be under dual control
of the Ca2+/calmodulin-activated myosin light chain kinase (MLCK) as well as of myosin-phosphatase (Somlyo
and Somlyo, 1994
; Burridge and Chrzanowska-Wodnicka,
1996
). Myosin-phosphatase has been demonstrated to be
regulated by Rho/Rho-kinase (Kimura et al., 1996
; Narumiya et al., 1997
). Since U46619-induced platelet shape
change was blocked by the Rho-kinase inhibitor Y-27632
and was greatly inhibited after reduction of the amount of
active Rho by C3 exoenzyme (Figs. 1 and 2) we tested the
effect of C3 exoenzyme and Y-27632 on U46619-induced phosphorylation of MLC. Fig. 7, D-F shows that Y-27632
blocked and C3 exoenzyme markedly inhibited U46619-induced MLC-phosphorylation in wild-type as well as in
G
q-deficient platelets. Incomplete inhibition of MLC
phosphorylation by C3 exoenzyme was most likely due to
incomplete inactivation of Rho by C3 exoenzyme (see
Fig. 3). Y-27632 exerted its inhibitory effect on receptor-induced MLC phosphorylation with higher potency in
G
q-deficient platelets compared with wild-type platelets
(Fig. 7, D and E). Similarly, the effect of C3 exoenzyme
appeared to be more pronounced in the absence of G
q
(Fig. 7 F). These data indicate that activation of G12/G13
through the TXA2 receptor results in MLC phosphorylation and that this process involves Rho/Rho-kinase. The
data also provide further evidence for the concept that
MLC phosphorylation underlies platelet shape change.
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Discussion |
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Full platelet activators like TXA2 and thrombin function
through G protein-coupled receptors which activate Gq,
G12, G13, and Gi type G proteins (Shenker et al., 1991;
Hung et al., 1992
; Ushikubi et al., 1994
; Offermanns et al.,
1994
). G
11, a close homologue of G
q and coexpressed
with G
q in most cells, is not present in platelets (Milligan
et al., 1993
; Johnson et al., 1996
; Offermanns et al., 1997b
).
In G
q-deficient platelets, the TXA2 mimetic U46619 and
thrombin fail to induce platelet aggregation and degranulation. This is accompanied by a lack of phospholipase C
activation and Ca2+ mobilization after TXA2 and thrombin receptor activation supporting the concept that Gq-mediated phospholipase C activation represents the main
signaling process leading to full platelet activation (Offermanns et al., 1997b
). Lack of G
q-mediated phospholipase C activation did not interfere with the ability of U46619
and thrombin to induce platelet shape change as shown by
scanning electron microscopy of activated G
q-deficient
platelets (see Fig. 1) and measurement of light transmission through a suspension of G
q-deficient platelets (see
Fig. 2) (Offermanns et al., 1997b
). Thus, induction of
platelet shape change through receptors of different platelet stimuli is mediated by G proteins other than Gq, and
G
q-deficient platelets provide a good model to study the
mechanisms underlying receptor-induced shape change independently of secondary processes involving secretion
and aggregation.
To identify the G proteins mediating platelet shape
change we studied the coupling of TXA2 and thrombin receptors to G12 family members and Gi-type G proteins.
Studies in human platelets have provided evidence that
thrombin receptors but not TXA2 receptors couple to Gi-type G proteins resulting in an inhibition of adenylyl cyclase (Aktories and Jakobs, 1984; Houslay et al., 1986
;
Brass et al., 1988
; Offermanns et al., 1994
). Similarly, in membranes from wild-type and G
q-deficient mouse platelets,
thrombin increased incorporation of GTP-azidoanilide
into Gi, whereas U46619 was without effect (see Fig. 5).
Only thrombin was able to decrease cAMP-levels in wild-type and G
q-deficient platelets (data not shown). The
fact that thrombin but not TXA2-receptors couple to Gi
in mouse platelets clearly demonstrates that Gi-mediated
processes do not play a significant role in the regulation of
platelet shape change. Both activated TXA2 (see Fig. 5)
and thrombin receptors (data not shown), coupled to G12
and G13 in wild-type and G
q-deficient platelets. Thus, in
G
q-deficient platelets, the only G proteins found to be
activated through TXA2 receptors were G12 and G13. We
therefore conclude that G12 and/or G13 are the mediators
of ligand-induced platelet shape change and that platelet
shape change induced through TXA2 receptors in G
q-deficient platelets can be regarded as a G12/G13-regulated
physiological cellular function.
The signaling mechanisms regulating receptor-dependent platelet shape change are incompletely understood.
Elevation of the cytosolic Ca2+ concentration is necessary
for full platelet activation including granule secretion and
aggregation. However, there is good evidence that elevation of [Ca2+]i alone is not sufficient to induce platelet
shape change and that agonists can induce shape change
without an increase in phospholipase C activity and without an increase in [Ca2+]i (Simpson et al., 1986; Negrescu
et al., 1995
; Ohkubo et al., 1996
; Offermanns et al., 1997b
).
Tyrosine phosphorylation of various proteins has been
associated with receptor-mediated induction of platelet
shape change since this occurs rapidly in a Ca2+- and
IIb
3-integrin-independent manner (Clark et al., 1994b
; Negrescu and Siess, 1996
). The mechanism of early receptor-induced tyrosine phosphorylation is not known. Tyrosine kinases like pp72syk and pp60c-src, which are rapidly
activated in a partially
IIb
3-integrin-independent manner, may be involved (Clark et al., 1994b
; Presek and Martinson, 1997
), and pp72syk has been implicated in the platelet shape change response in porcine platelets (Maeda et al.,
1995
). However, there is clear evidence that activation of
pp72syk alone is not sufficient for induction of shape change
(Negrescu and Siess, 1996
). We show here that TXA2-receptor-mediated activation of G12/G13 leads to tyrosine
phosphorylation of pp72syk and activation of pp60c-src (see
Fig. 6) supporting the concept that these tyrosine kinases are involved in early platelet activation. These data also
indicate that G proteins of the G12-family can regulate tyrosine kinases. The mechanism of this regulation remains unknown.
MLC phosphorylation has been implicated in the regulation of cytoskeletal reorganization during platelet shape
change (Daniel et al., 1984; Nachmias et al., 1985
). Phosphorylated myosin interacts mainly with central actin filaments in platelets, and the forming myosin-actin complex
has been suggested to be involved in the granule centralization process (Fox and Phillips, 1982
; Stark et al., 1991
;
Fox, 1993
). The phosphorylation state of MLC is under
dual control of MLCK and myosin-phosphatase. It is well established that increase in [Ca2+]i activates the Ca2+/
calmodulin-dependent MLCK. MLC phosphorylation by
MLCK leads to actin-myosin interaction resulting in actin-stimulated ATPase activity of smooth muscle and nonmuscle myosin (Somlyo and Somlyo, 1994
; Kohama et al.,
1996
). Recently, it has been shown that upstream regulation of myosin phosphatase occurs independently of the
cytosolic free calcium concentration through phosphorylation and inactivation of its regulatory subunit by Rho-kinase, a specific target of the small GTPase Rho (Kimura et al., 1996
; Narumiya et al., 1997
). In addition,
Rho-kinase can directly phosphorylate MLC in vitro (Amano et al., 1996
). There is increasing evidence that
Rho/Rho-kinase-mediated MLC phosphorylation is involved in contractile responses in various cell types like
vascular smooth muscle cells (Uehata et al., 1997
), fibroblasts (Chihara et al., 1997
), neuroblastoma cells (Amano et al., 1998
; Hirose et al., 1998
), astrocytoma cells (Majumdar et al., 1998
), or endothelial cells (Essler et al.,
1998
). It is, however, unclear how the Rho-mediated pathway is regulated through receptors.
The TXA2 mimetic U46619 caused a rapid phosphorylation of MLC in wild-type and Gq-deficient platelets (see
Fig. 7). Since U46619 does not lead to elevation of [Ca2+]i
in the absence of G
q (Offermanns et al., 1997b
) and since Rho-kinase inhibitor Y-27632 and C3 exoenzyme inhibited
U46619-induced MLC phosphorylation in G
q-deficient
platelets, we conclude that a Rho/Rho-kinase-mediated
pathway regulating MLC phosphorylation operates in
platelets. Consistent with this, the Rho-kinase p160ROCK has been shown to be phosphorylated upon activation of
human platelets in an
IIb
3-integrin-independent way
(Fujita et al., 1997
). In addition, Rho and Rho-kinase can
be coimmunoprecipitated with the myosin-binding subunit
of myosin phosphatase from human platelets, and treatment of platelets with a TXA2-mimetic leads to rapid phosphorylation and inactivation of myosin phosphatase
(Nakai et al., 1997
). Conflicting data exist with regard to
the role of Rho in early platelet activation as determined
by C3 exoenzyme treatment. This is most likely due to the
difficulties associated with the length of incubation and
the high concentration of C3 exoenzyme required to inactivate a sufficient fraction of Rho. Although partial inactivation of the RhoA pool in human platelets by C3 exoenzyme has been shown to inhibit platelet activation (Morii
et al., 1992
), a recent report showed that ADP ribosylation of ~90% of Rho in human platelets did not affect inside-out signaling of integrin
IIb
3, ligand-induced aggregation
and F-actin content (Leng et al., 1998
). Our data clearly
support a role of Rho in early platelet activation.
In wild-type platelets in which U46619 induces an elevation of [Ca2+]i and most likely leads to Ca2+/calmodulin-MLCK-mediated MLC phosphorylation, Rho-kinase
blocker Y-27632 and C3 exoenzyme also inhibited MLC
phosphorylation induced by U46619. Interestingly, both
agents appeared to be less potent in wild-type platelets
than in Gq-deficient platelets (see Fig. 7, D-F). This suggests that both, Ca2+-mediated activation of MLCK and
inhibition of myosin phosphatase through Rho/Rho-kinase
may synergistically increase MLC phosphorylation in activated wild-type platelets. In contrast, receptor-mediated MLC phosphorylation in G
q-deficient platelets depends
on the Ca2+-independent, Rho-mediated pathway. Since
shape change could be inhibited by the C3 exoenzyme as
well as by Y-27632 in G
q-deficient platelets (see Figs. 1
and 2) we suggest that Rho/Rho-kinase-mediated MLC
phosphorylation is involved in TXA2 receptor-induced
platelet shape change.
Cyclic nucleotides like cAMP and cGMP mediate physiological inhibition of platelet activation through activation
of cAMP- and cGMP-dependent kinases. Although analogues of both cyclic nucleotides can block full platelet
activation, only cAMP analogues inhibit platelet shape
change (Matsuoka et al., 1989; Menshikov et al., 1993
).
Similarly, we observed that the cAMP analogue Sp-5,6-DCl-cBIMPS but not the cGMP analogue 8-pCPT-cGMP
inhibited TXA2 receptor-G12/G13-mediated shape change
and MLC phosphorylation in G
q-deficient platelets (see
Figs. 2 and 7). Inhibition of MLC phosphorylation by Sp-5,6-DCl-cBIMPS but not by 8-pCPT-cGMP suggests that
the Rho/Rho-kinase-mediated signaling cascade may be
inhibited by the cAMP-dependent pathway. A similar
role of cAMP was suggested for the inhibition of Rho/ Rho-kinase-mediated neurite remodeling and morphology change in epithelial-like cells (Hirose et al., 1998
; Dong
et al., 1998
).
Rho has been shown to be regulated by the activated
-subunits of G12 and G13 (Buhl et al., 1995
; Gohla et al.,
1998
; Kozasa et al., 1998
). Since G12 and G13 are the only
G proteins activated through TXA2 receptors in G
q-deficient platelets and since TXA2 receptor-mediated MLC
phosphorylation in G
q-deficient platelets was inhibited
by C3 exoenzyme and Rho-kinase inhibitor Y-27632 we
suggest that TXA2 receptor-induced G12/G13 activation results in MLC phosphorylation through Rho-mediated activation of Rho-kinase. Activated Rho-kinase may phosphorylate MLC directly or act through phosphorylation
and inhibition of myosin phosphatase. Additional, synergistic regulation of MLC phosphorylation in wild-type
platelets occurs through Gq-mediated activation of MLCK.
The mechanism by which G12/G13 activate Rho remains to
be elucidated. Epidermal growth factor tyrosine kinase
has recently been involved in the G
13-induced Rho-dependent actin stress fiber formation in fibroblasts (Gohla et
al., 1998
). However, various tyrosine kinase inhibitors were unable to block TXA2 receptor-induced, G12/G13-mediated MLC phosphorylation in G
q-deficient platelets
(data not shown). This suggests that in platelets, G12/G13-induced Rho activation is not mediated by receptor- or
nonreceptor-tyrosine kinases. Another possibility is that
regulation of Rho by G12/G13 is mediated by a Rho-specific GEF. Genetic evidence in Drosophila showed that the Drosophila RhoGEF, DRhoGEF2, functions downstream of the Drosophila G12/G13 homologue concertina
(Barrett et al., 1997
), and it has recently been shown that
the related mammalian RhoGEF, p115 RhoGEF, can directly link G
13 to the regulation of Rho (Hart et al., 1998
;
Kozasa et al., 1998
).
Using Gq-deficient platelets which do not aggregate
and secrete but undergo shape change in response to various stimuli, we show that activation of G12/G13 is sufficient
to induce platelet shape change. Thus, different G protein-mediated signaling pathways appear to be specifically
involved in the regulation of distinct processes during receptor-induced platelet activation. Although Gq is necessary for full platelet activation including aggregation and
secretion, activation of Gi may counteract anti-aggregatory influences through inhibition of adenylyl cyclase, and
G12/G13 appear to be centrally involved in the platelet
shape change response. Our data also indicate that G12/G13
can link receptors to tyrosine kinases as well as to Rho/
Rho-kinase-mediated regulation of MLC phosphorylation, and we provide evidence that the latter pathway participates in the receptor-mediated induction of platelet
shape change.
![]() |
Footnotes |
---|
Address correspondence to S. Offermanns, Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany. Tel.: (49) 30-8445-1835. Fax: (49) 30-8445-1818. E-mail: stoff{at}zedat.fu-berlin.de
Received for publication 24 September 1998 and in revised form 19 January 1999.
We thank Yoshitomi Pharmaceutical Industries, Ltd. for kindly providing us with Y-27632.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 366) and the Fonds der Chemischen Industrie.
![]() |
Abbreviations used in this paper |
---|
MLC, myosin light chain; MLCK, myosin light chain kinase; GEF, guanine nucleotide exchange factor; TXA2, thromboxane A2.
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