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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3408-3417
Agonist-Induced Regulation of Myosin Phosphatase Activity in Human
Platelets Through Activation of Rho-Kinase
By
Yoshinori Suzuki,
Masatoshi Yamamoto,
Hideo Wada,
Masaaki Ito,
Takeshi Nakano,
Yasuharu Sasaki,
Shuh Narumiya,
Hiroshi Shiku, and
Masakatsu Nishikawa
From the 2nd and the 1st Departments of Internal Medicine, Mie
University School of Medicine, Tsu, Mie, Japan; Frontier 21, Life
Science Research Center, Asahi Chemical Industry Co, Ltd, Fuji,
Shizuoka; and the Department of Pharmacology, Kyoto University Faculty
of Medicine, Kyoto, Japan.
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ABSTRACT |
Human platelets contained about 15 times lower amounts of Rho-kinase
than Ca2+/calmodulin-dependent myosin light chain (MLC)
kinase. Anti-myosin-binding subunit (MBS) antibody
coimmunoprecipitated Rho-kinase of human platelets, and addition of
GTP S-RhoA stimulated phosphorylation of the 130-kD MBS of myosin
phosphatase and consequently inactivated myosin phosphatase. Two kinds
of selective Rho-kinase inhibitors, HA1077 and Y-27632, reduced both
GTPS-RhoA-dependent MBS phosphorylation and inactivation of the
phosphatase activity. Activation of human platelets with thrombin, a
stable thromboxane A2 analog STA2, epinephrine,
and serotonin resulted in an increase in MBS phosphorylation, and the
agonist-induced MBS phosphorylation was prevented by pretreatment with
the respective receptor antagonist. HA1077 and Y-27632 inhibited MBS
phosphorylation in platelets stimulated with these agonists. These
compounds also blocked agonist-induced inactivation of myosin phosphatase in intact platelets. In addition, HA1077 and Y-27632 inhibited 20-kD MLC phosphorylation at Ser19 and ATP
secretion of platelets stimulated with STA2, thrombin (0.05 U/mL), and simultaneous addition of serotonin and epinephrine, whereas
these compounds did not affect MLC phosphorylation or ATP secretion
when platelets were stimulated with more than 0.1 U/mL
thrombin. Thus, activation of Rho-kinase and the resultant phosphorylation of MBS is likely to be the common pathway for platelet
activation induced by various agonists. These results also suggest that
Rho-kinase-mediated MLC phosphorylation contributes to a greater
extent to the platelet secretion induced by relatively weak agonists.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLATELETS CAN BE activated by a number of
agonists, including thrombin, thromboxane A2, epinephrine,
and serotonin.1,2 An agonist may be classified as either
strong or weak, depending on whether it causes full activation,
including the release reaction. Thrombin is the most potent agonist for
human platelets, whereas serotonin and epinephrine are weak agonists.
All these agonist receptors are linked to heterotrimeric G proteins,
and the earliest event after receptor-ligand interaction is the
activation of phospholipase C, which hydrolyzes phosphatidylinositol
4,5-bisphosphate to generate inositol 1,4,5-triphosphate
(IP3) and 1,2-diacylglyceride.1,2 IP3 is the primary intracellular stimulus for
Ca2+ mobilization from intracellular storage sites. An
increase in Ca2+ leads to phosphorylation of the 20-kD
light chain of myosin (MLC) at Ser19 via the
Ca2+/calmodulin-dependent MLC kinase: this phosphorylation
increases an actomyosin contractile response that is involved in
platelet shape change and secretion.3-5 The level of MLC
phosphorylation is regulated not only positively by MLC kinase, but
also negatively by a myosin phosphatase. We have shown that myosin
phosphatase from human platelets is composed of a 38-kD protein
phosphatase 1 catalytic subunit, 130-kD myosin-binding subunit
(MBS), and 20-kD regulatory subunit,6 as is the case for
smooth muscle phosphatase.7-9
There is increasing evidence that a number of small G
proteins are involved in signal transduction pathways at the plasma membrane.10 The small G protein Rho and its target
Rho-kinase are implicated in physiological functions associated with
actin-myosin filaments such as shape change, cell mortility, secretion,
and smooth muscle contraction.11-14 A recent
study15 with botulinum C3 exoenzyme has suggested that RhoA
is involved in discrete outside-in signaling responses in
fibrinogen-adherent platelets, most prominently the formation of focal
adhesion, although RhoA does not appear to be involved in either
agonist-induced affinity modulation of integrin
 IIb 3 or in primary aggregation. Trimeric
G-protein-coupled receptors appear to be the major upstream pathway
for Rho activation.11 Rho-kinase, when activated by
GTP-RhoA, phosphorylates MBS and thereby inhibits the catalytic
activity of smooth muscle myosin phosphatase,16 so that MLC
phosphorylation is increased, an event that induces the consequent
contraction of smooth muscle at a constant Ca2+
concentration (referred to as Ca2+
sensitization).11 In permeabilized platelets, the
Ca2+ sensitivity of serotonin secretion can be enhanced by
GTPS, without activation of phospholipase C.17 We have
recently shown that platelet MBS is an in vitro substrate for
Rho-kinase and that phosphorylation of MBS decreases the activity of
platelet myosin phosphatase.6 Moreover, treatment of intact
platelets with a stable thromboxane A2 analog
STA2 led to an increase in MBS phosphorylation and a
decrease in the activity of myosin phosphatase.6 In
addition, a recent report has shown that Rho-kinase directly phosphorylates the 20-kD MLC in vitro at the site that is
phosphorylated by MLC kinase, which causes activation of myosin
ATPase.18 Thus, MLC phosphorylation at Ser19
induced by various stimuli in intact cells may be mediated by not only
Ca2+/calmodulin-dependent MLC kinase, but also by
Rho-kinase. A pyridine derivative Y-27632 and an
isoquinolinesulfonamide derivative HA1077 have recently been shown to
be relatively selective inhibitors of Rho-kinase.19 Y-27632
not only inhibited the Ca2+ sensitivity of vascular smooth
muscle, but also reduced high blood pressure in laboratory
animals.19 HA1077 inhibited both vascular contractions and
MLC phosphorylation in response to a variety of agents20
and has been clinically used in Japan in the treatment of the cerebral
vasospasm after subarachnoid hemorrhage.21 Using these
inhibitors, we asked if Rho-kinase-induced MBS phosphorylation occurs
in intact platelets in response to other agonists such as thrombin,
serotonin, and epinephrine, as is the case for STA2. In
addition, we also investigated whether RhoA-mediated MLC
phosphorylation contributes to platelet secretion induced by these agonists.
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MATERIALS AND METHODS |
Materials.
GST-RhoA was expressed and purified using a glutathione-Sepharose
column.13 HA1077 [1-(5-isoquinolinesulphonyl)
homopiperazine hydrochloride] was generously provided by Asahi
Chemical Industry Co, Ltd (Tokyo, Japan). Y-27632
[(+)-(R)-trans-4(1-aminoethyl)-N-(4-pynidyl) cyclohexanecarboxamide dihydrochloride, monohydrate] was a kind gift
from Yoshitomi Pharmaceutical Industries Ltd (Osaka, Japan). Other drugs and suppliers were as follows: M-1
[(±)-3-dimethylamino 1-(o-(m-methoxypheneytyl)
phenoxyl)-2-propanol] from Mitsubishi Kasei Corp,
(Yokahama, Japan) STA2 [9, 11-epithio-11,
12-methano-thromboxane A2] and ONO-3708 [(9, 11), (11, 12)-dideoxa-9, 11-dimetylmethano-11, 12-methano-13,
14-dihydro-13-aza-14-oxo-15-cyclopentyl-16, 17, 18, 19, 20-pentanor-15-epi-thromboxane A2] from Ono Pharmaceutical Co, Ltd (Osaka, Japan); KT5926 [(8R*, 9S*,
11S*)-( )-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2, 3, 9, 10-tetrahydro-8, 11-epoxy, 1H, 8H, 11H-2,
7b, 11a-triazadibenzo[a, g]cycloocta[cde]trinden-1-one] from Kyowa Medex
Co, Ltd (Tokyo, Japan); acetoxymethyl ester of
5,5'-dimethyl-bis-(-o-aminophenoxy)-ethane-N,N,N,N,-tetraacetic acid (BAPTA-AM) from Dojindo Laboratories (Kumamoto, Japan); W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride] from Seikagaku Corp (Tokyo, Japan); yohimbine from Wako Pure Chemical Industries (Osaka, Japan); and GTPS from Boehringer Mannheim GmbH
(Mannheim, Germany). [-32P]ATP (111  Bq/mmol) was
from NEN Life Science Products, Inc (Boston, MA).
[32P]orthophosphate was from ICN Pharmaceuticals, Inc
(Costa Mesa, CA).
Measurement of Rho-kinase and MLC kinase in human platelets.
Amounts of Rho-kinase and MLC kinase in human platelets were estimated
by immunoblot analysis using polyclonal antibodies specific against
human platelet MLC kinase and Rho-kinase. Antisera against MLC kinase
and Rho-kinase were obtained by immunizing rabbits with the purified
MLC kinase from human platelets22 and with the synthesized
fragment of Rho-kinase,13 respectively, and used after
purification. Quantitative estimation of the levels of MLC kinase and
Rho-kinase in human platelets was performed densitometrically by
scanning the immunoreactive band after immediately photographing the
visualized band.23 The signal was then compared with that
of known amounts of the purified platelet MLC kinase or purified
recombinant Rho-kinase.
Immunoprecipitation of platelet myosin phosphatase.
Immunoprecipitation of platelet myosin phosphatase was performed as
described,6 with a few modifications. Platelets (800 µL)
were dissolved in the lysis buffer (1% Nonidet P-40, 20 mmol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl, 4 mmol/L EDTA, 4 mmol/L
phenylmethylsulfonyl fluoride [PMSF], 200 µg/mL leupeptin, 2 mmol/L
sodium orthovanadate). The lysate was centrifuged at 15,000 × g for 15 minutes. The soluble fraction precleared with Protein
A Sepharose CL-4B (Pharmacia Biotech AB, Uppsala, Sweden) was incubated
with 10 µL of anti-MBS antibody at 4°C for 1 hour. The immune
complex was precipitated by adding protein A-Sepharose CL-4B,
incubating for an additional 1 hour at 4°C, and then washing the
beads three times with the lysis buffer. The immune complex contains
three components of myosin phosphatase, namely MBS, PP1 catalytic
subunit, and the 20-kD subunit.6 We used these preparations
to detect the phosphorylation and for assay of phosphatase activity.
In vitro MBS phosphorylation by Rho-kinase.
Immunoprecipitate with anti-MBS antibody from human platelets was used
for in vitro MBS phosphorylation by Rho-kinase. The kinase reaction for
Rho-kinase was performed in 50 µL of the reaction mixture (20 mmol/L
Tris-HCl, pH 7.5, 2 mmol/L EDTA, 6 mmol/L MgCl2, 1 mmol/L
dithiothreitol, 0.5 µmol/L okadaic acid, 25 µL of the immunoprecipitate, 5 µL of a dilution of drug, 10 µmol/L
[-P32] ATP) and 1 µmol/L GTPS·GST-RhoA or 1 µmol/L GDP·GST-RhoA.16,18 The kinase reaction was
initiated by adding the ATP solution. Reactions were performed at
30°C for 30 seconds and terminated by the addition of sodium
dodecyl sulfate (SDS) sample buffer. The samples were
subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and MBS
phosphorylation was analyzed by autoradiography. To analyze the
phosphatase activities, the kinase reaction was performed in 50 µL of
the reaction mixture (20 mmol/L Tris-HCl, pH 7.5, 2 mmol/L EDTA,
6 mmol/L MgCl2, 1 mmol/L dithiothreitol, 25 µL
of the immunoprecipitate, 5 µL of a dilution of drug, and 10 µmol/L
ATP) at 30°C for 30 seconds. The reaction was terminated by adding
10 mmol/L EDTA, and phosphatase activity was measured immediately.
Preparation of human platelet suspension and measurements of ATP
secretion.
Venous blood was freshly drawn from a healthy donor who had not taken
any drugs for at least 2 weeks previously. Platelets (109/mL) were finally resuspended in a modified
Tyrode-HEPES buffer that contained a final concentration of 0.14 mol/L
NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 0.1%
D-glucose, 3.75 mmol/L NaH2PO4, and 15 mmol/L HEPES, pH 7.5. ATP secretion was measured using a
Lumi-aggregometer (Chrono-Log Corp, Havertown, PA), as
described.3,24 The standard platelet reaction mixtures
consisted of 400 µL platelet suspension, 50 µL of Chrono-Lumi
luciferase luciferin reagent, 50 µL of a dilution of drug (or
saline), and an aggregating agent. Washed platelet suspension
preincubated with the drug at 37°C for 3 minutes was activated by
various agonists, under conditions of nonstirring. Epinephrine and/or
serotonin was reacted in the Tyrode-HEPES buffer containing 1 mmol/L
CaCl2.
Measurement of the phosphorylation of myosin phosphatase in intact
platelets.
Washed platelets (800 µL) prelabeled with 18.5 MBq/mL
[32P]orthophosphate (at 30°C for 60 minutes) were
stimulated with the agonists, including thrombin, STA2, and
epinephrine/serotonin. The reaction was terminated by adding one third
volume of ×4 lysis buffer with a final concentration of 1 µmol/L calyculin A (Wako Pure Chemicals). Myosin phosphatase was
precipitated by immunoprecipitation using an anti-MBS antibody, as
mentioned above. Phosphoproteins were separated by SDS-PAGE. The gel
was stained with Coomassie Blue, dried, and subjected to
autoradiography. The radiolabeled bands were visualized using a Bio
Imaging Analyzer BAS2000 (Fuji Photo Film Co, Ltd, Tokyo, Japan) and
then exposed to Kodak X-Omat AR film (Eastman Kodak, Co, Rochester,
NY) with an intensifying screen at 80°C.
Quantitative estimation of the level of phosphorylation was performed
densitometrically, using a Molecular Dynamics Scanning Densitometer
(Sunnyvale, CA) in conjugation with the ImageQuant program run on a
Dell Personal Computer (Austin, TX) by scanning the radioactive
bands.23 The area of an individual peak was measured above
background in densitometric tracing and was estimated as an arbitrary unit.
Measurement of myosin phosphatase activity in intact platelets.
Activities of myosin phosphatase were determined using
[32P]-phosphorylated myosin light chain from chicken
gizzards as a substrate.6 Myosin phosphatase was
precipitated by immunoprecipitation using anti-MBS antibody from
platelets activated by 1 µmol/L STA2. PP1 activity of the
sample was measured in a reaction mixture (50 µL) containing 5 µL
of immunoprecipitate, 20 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl, 0.1 mmol/L EGTA, 2 mmol/L sodium orthovanadate, 10 nmol/L okadaic
acid, a heat stable phosphatase inhibitor-2, 4 mmol/L PMSF, and 200 µg/mL leupeptin. PP1 activity can be taken as the activity that is
sensitive to the inhibitor-223 and was regarded as myosin
phosphatase activity.6
Detection of 20-kD MLC monophosphorylated at Ser19 in
intact platelets.
The monoclonal antibody specific against monophosphorylated 20-kD MLC
at Ser19 was prepared as described elsewhere.25
Washed platelets preincubated with various concentrations of HA1077 or
Y-27632 in the presence or absence of 7.5 µmol/L BAPTA-AM for 3 minutes at 37°C were stimulated with thrombin, STA2, or
serotonin plus epinephrine for 30 seconds, without stirring.
Precipitates treated with 10% trichloroacetic acid from platelets were
subjected to SDS-PAGE to assay the 20-kD MLC phosphorylation at
Ser19 and to glycerol-PAGE to determine the extent of 20-kD
MLC phosphorylation. Immunoblot analysis with the
antimonophosphorylated MLC antibody was performed to detect the
phosphorylation of 20-kD MLC. The extent of 20-kD MLC phosphorylation
was expressed as percentages of 20-kD MLC in the monophosphorylated
form.20,25,26
| |
RESULTS |
Levels of Rho-kinase and MLC kinase in human platelets.
We determined the amount of Rho-kinase and MLC kinase in whole
platelets by immunoblot analysis, using purified recombinant Rho-kinase
or the MLC kinase purified from human platelets,22 respectively, as standards. The amount of Rho-kinase in whole platelets
was 1.11 ± 0.325 ng/107 platelets (0.047 ± 0.014 µmol/L; mean ± SD; n = 4). The amount of platelet MLC kinase was
10.4 ± 2.05 ng/107 platelets (0.695 ± 0.137 µmol/L; n = 3). The amount of platelet Rho-kinase was about 15 times
lower than that of MLC kinase.
Effects of Rho-kinase inhibitors on in vitro phosphorylation of
platelet MBS.
Rho-kinase was detected by immunoblot analysis in the anti-MBS
immunoprecipitate (Fig 1A), as we
reported.6 We did not detect protein kinase N in the
anti-MBS immunoprecipitates, although activated Rho does interact with
protein kinase N and Rho-kinase and stimulates their kinase
activities.12-14,27,28 We then asked if phosphorylation of
MBS by Rho-kinase was GTP-RhoA dependent, determined using the anti-MBS
immunoprecipitates. RhoA has GDP-bound inactive and GTP-bound active
forms that are interconvertible by GDP-GTP exchange and GTPase
reactions.29,30 We made use of GTPS (a nonhydrolyzable
GTP analog) for activation of RhoA and thereby stimulation of
Rho-kinase activity. As shown in Fig 1B, phosphorylation of
MBS was prominent in the presence of GTPS-RhoA, whereas GDP-RhoA had
a much weaker effect. Phosphorylation was nil in the absence of Rho.
These findings indicate that the phosphorylation of MBS in the
immunoprecipitate is completely dependent on GTPS-RhoA. To confirm
whether the MBS phosphorylation induced by GTPS-RhoA is catalyzed by
Rho-kinase, we examined effects of Rho-kinase inhibitors. MBS
phosphorylation was inhibited by HA1077 or Y-27632 in a dose-dependent
manner (Fig 1C). IC50 values of HA1077 and Y-27632 for the
inhibition of MBS phosphorylation were 10 and 0.3 µmol/L,
respectively. We then investigated whether the inhibition of MBS
phosphorylation by HA1077 and Y-27632 was associated with the
phosphatase activity, tested using anti-MBS immunoprecipitates (Fig
1D). Incubation with GTPS-RhoA produced a significant decrease in
the phosphatase activity of anti-MBS immunoprecipitates and the
addition of HA1077 or Y-27632 restored the phosphatase activity. HA1077 and Y-27632 did not affect the phosphatase activity of protein
phosphatase 1, 2A, 2B, or 2C (data not shown). GTPS-RhoA-dependent phosphorylation of MBS was not inhibited by receptor antagonists such
as ONO-3708 (thromboxane A2 receptor
antagonist31), yohimbine (2 receptor
antagonist32), and M-1 (S2-serotonergic
receptor antagonist33), the MLC kinase inhibitor
KT5926,34 or the calmodulin antagonist W-73
(Fig 1E).
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| Fig 1.
In vitro phosphorylation of platelet MBS and
inactivation of myosin phosphatase by Rho-kinase. (A)
Coimmunoprecipitation of Rho-kinase with platelet MBS.
Immunoprecipitates with anti-MBS antibodies were immunoblotted with
antibodies against MBS (left) and Rho-kinase (right). IP,
immunoprecipitation antibodies used; IB, immunoblotting antibodies
used; Ig, cross-reacted Ig. (B) In vitro phosphorylation of platelet
MBS. MBS immunoprecipitates from platelet lysates were incubated
without RhoA, with GDP-RhoA, and with GTPS-RhoA for 30 seconds, as
described in Materials and Methods. Protein phosphorylation was
analyzed by SDS-PAGE, followed by autoradiography. (C) Inhibitory
effect of HA1077 and Y-27632 on GTPS-RhoA-dependent phosphorylation
of platelet MBS. Different concentrations of HA1077 (left panel) or
Y-27632 (right panel) were included in the reaction mixture, and MBS
phosphorylation was analyzed, as described in Materials and Methods.
Results were expressed as the percentage of the value, without the
addition of compounds. The results are representative of three
independent experiments. (D) Effects of HA1077 and Y-27632 on the
activity of myosin phosphatase derived from MBS immunoprecipitates in
the presence of GTPS-RhoA. MBS immunoprecipitates were incubated
with 20 µmol/L HA1077 or 10 µmol/L Y-27632 in the presence of
GTPS-RhoA for 30 seconds, and the activity of myosin phosphatase was
determined immediately. The value shows the mean ± SE from three
experiments. *P < .05 (E) Effects of various compounds on
GTPS-RhoA-dependent phosphorylation of platelet MBS.
Immunoprecipitates with anti-MBS antibody from platelet lysates were
incubated for 30 seconds with 10 nmol/L M-1, 10 nmol/L yohimbine, 100 nmol/L ONO-3708, 100 nmol/L KT5926, or 50 µmol/L W-7 in the presence
of GTPS-RhoA, as described in Materials and Methods. Protein
phosphorylation was analyzed by SDS-PAGE, followed by autoradiography.
Similar results were obtained in three other experiments.
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Effects of Rho-kinase inhibitors on agonist-induced phosphorylation
of MBS in intact platelets.
We reported that treatment of intact platelets with STA2
led to an increase in phosphorylation of MBS and to a decrease in the
activity of myosin phosphatase.6 To determine if
phosphorylation of MBS occurs in platelets stimulated by agonists other
than STA2, we examined immunoprecipitates using anti-MBS
antibody of [32P]Pi-labeled platelets, before and after
stimulation with STA2, thrombin, serotonin, and
epinephrine. As shown in Fig 2A, a
phosphorylated band at 130-kD was detected in the immunoprecipitates,
and the level of MBS phosphorylation increased rapidly for up to 1 minute after exposure to each agonist, in nonstirred platelets. Amounts of precipitated MBS remained unchanged after stimulation (data not
shown). Another phosphorylated protein with a molecular weight of
120-kD was detected in the case of either thrombin or STA2; the function was not investigated. To determine whether agonist-induced phosphorylation of MBS is catalyzed by Rho-kinase, we studied the
effects of the Rho-kinase inhibitors, HA1077 and Y-27632, on
STA2-induced MBS phosphorylation in intact platelets. Both HA1077 and Y-27632 produced a dose-dependent inhibition of
STA2-induced phosphorylation of MBS (Fig 2B). We then
examined effects of these Rho-kinase inhibitors on
STA2-induced inactivation of myosin phosphatase in intact
platelets (Fig 2C). Pretreatment of intact platelets with HA1077 or
Y-27632 blocked the inactivation of myosin phosphatase induced by
STA2. These data support the idea that Rho-kinase
activation is involved in agonist-induced MBS phosphorylation with the
resultant inactivation of myosin phosphatase in human platelets. These
soluble agonists have been shown to activate respective
G-protein-coupled receptors in platelets,1,2 which leads
to cytoskeletal rearrangements, granule secretion, fibrinogen receptor
activation, and aggregation. The signaling events that evoke these
receptors are unclear, but there is general agreement that low
molecular weight GTP-binding proteins play a central role. We therefore
determined whether Rho-kinase activation is linked to
G-protein-coupled receptors, and for this we used respective receptor
antagonists, including ONO-3708, M-1, and yohimbine. Pretreatment of
intact platelets with these antagonists inhibited the MBS
phosphorylation induced by respective agonists, as shown in Fig 2D. The
S2-serotonergic receptor antagonist M-1, at concentrations
(10 nmol/L) blocking the aggregatory effect of simultaneous addition of
serotonin and collagen,33 inhibited the secretory response
of platelets evoked by both serotonin and epinephrine. The finding of
complete inhibition by M-1 of synergic responses of serotonin and
epinephrine is in accordance with previous observations by other
investigators who used the S2-serotonergic receptor
antagonist ketanserin.35,36 M-1 also inhibited the MBS
phosphorylation induced by the simultaneous addition of serotonin and
epinephrine (Fig 2D). Both HA1077 and Y-27632 abolished MBS
phosphorylation induced by simultaneous addition of serotonin and
epinephrine (Fig 2D) and by 0.1 U/mL thrombin (Fig 2E). These results
suggest that activation of Rho-kinase and the resultant phosphorylation
of MBS is likely to be the common pathway for the platelet activation
induced by various agonists.
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| Fig 2.
Agonist-induced phosphorylation of MBS and the
inactivation of myosin phosphatase in intact platelets. (A)
Agonist-induced phosphorylation of MBS in intact platelets.
[32P]Pi-labeled platelets were stimulated for the
indicated time with either 1 µmol/L STA2, 0.1 U/mL
thrombin, 1 µmol/L serotonin, or 1 µmol/L epinephrine. *An unknown
protein of 120 kD. (B) Inhibition by HA1077 and Y-27632 of
STA2-induced phosphorylation of MBS.
[32P]Pi-labeled platelets incubated for 5 minutes with
various concentrations of HA1077 (left panel) or Y-27632 (right panel)
were stimulated for 30 seconds with 1 µmol/L STA2,
without stirring. Results were expressed as the percentage of the value
without the addition of compounds. Similar results were obtained in
three other experiments, using different donor platelets. (C) Effects
of HA1077 and Y-27632 on STA2-induced inactivation of
myosin phosphatase in intact platelets. Human platelets incubated with
saline (control;  ), 20 µmol/L HA1077 ( ), or 10 µmol/L Y-27632
( ) for 5 minutes were activated with 1 µmol/L STA2
without stirring. MBS was immunoprecipitated with anti-MBS antibody and
the activity of myosin phosphatase was determined immediately, as
described in Materials and Methods. Results are expressed as the
percentage of the value, without the addition of compounds. Data
represent the mean ± SE of four experiments. *P < .05, **P < .01 (D) The inhibitory effect of receptor
antagonists on agonist-induced phosphorylation of MBS in intact
platelets. [32P]Pi-labeled platelets were pretreated with
0.1 µmol/L ONO-3708, 10 nmol/L M-1, 10 nmol/L yohimbine, 10 µmol/L
Y-27632, or 20 µmol/L HA1077 for 5 minutes at 37°C and were
activated with 1 µmol/L STA2, 1 µmol/L serotonin, or 1 µmol/L epinephrine for 30 seconds, without stirring. Similar results
were obtained in three other experiments, using different donor
platelets. (E) Inhibition by HA1077 and Y-27632 of thrombin-induced
phosphorylation of MBS. [32P]Pi-labeled platelets
incubated for 5 minutes with 20 µmol/L HA1077 or 10 µmol/L Y-27632
were stimulated for 30 seconds with 0.1 U/mL thrombin without
stirring. Similar results were obtained in three other experiments,
using different donor platelets.
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Effects on agonist-induced platelet secretion and phosphorylation of
20-kD MLC by two kinds of Rho-kinase inhibitors, HA1077 and Y-27632.
Serotonin and epinephrine are weak agonists that by themselves activate
platelets with only weak potency and to a limited degree. These
agonists by themselves did not induce secretion and aggregation of
washed human platelets, unlike thrombin and STA2, but, when
combined, ATP secretion occurs to some extent, under conditions of no
stirring. As shown in Fig 3A, ATP secretion was more extensive with 0.1 U/mL thrombin, was to a lesser
extent with 0.05 U/mL thrombin or 1 µmol/L STA2, and was
least with the simultaneous addition of 1 µmol/L epinephrine and 1 µmol/L serotonin. We then examined the effects of two kinds of
Rho-kinase inhibitors, HA1077 and Y-27632, on platelet secretion
stimulated with various agonists such as thrombin, STA2 and
a combination of serotonin with epinephrine. HA1077 and Y-27632
inhibited dose-dependently the ATP secretion induced by
STA2, a low level (0.05 U/mL) of thrombin, and a
simultaneous addition of serotonin and epinephrine (Fig 3A).
IC50 values producing a 50% inhibition of ATP secretion of
HA1077 and Y-27632 for STA2-induced secretion were 5.48 ± 1.80 and 11.2 ± 1.92 µmol/L, respectively. IC50
values of HA1077 and Y-27632 for secretion induced by a low level (0.05 U/mL) of thrombin were 8.75 ± 3.25 and 7.55 ± 1.45, respectively. IC50 values of HA1077 and Y-27632 for
secretion induced by a combination of serotonin plus epinephrine were
1.67 ± 0.440 and 4.83 ± 1.09 µmol/L, respectively. On the
other hand, these compounds did not inhibit a high level (0.1 U/mL) of thrombin-induced platelet secretion. We then examined effects of HA1077 and Y-27632 on agonist-induced phosphorylation of
20-kD MLC in intact platelets (Fig 3B). MLC phosphorylation was
analyzed by immunoblot analysis using an antibody specific for the
20-kD MLC phosphorylated at Ser19, the objective being to
exclude MLC phosphorylation at other sites (eg, by protein kinase
C37). The extent of MLC phosphorylation at
Ser19 depended on strength of the agonist: most prominent
phosphorylation (75%) with 0.1 U/mL thrombin, a lesser extent (53%)
with 0.05 U/mL thrombin, a following extent (40%) with 1 µmol/L
STA2, and to the least extent (22%) with the simultaneous
addition of 1 µmol/L epinephrine and 1 µmol/L serotonin.
The extent of ATP secretion by these agonists appeared to parallel the
extent of MLC phosphorylation at Ser19. HA1077 and Y-27632
inhibited dose-dependently the phosphorylation of 20-kD MLC stimulated
with a low level (0.05 U/mL) of thrombin, STA2 and the
simultaneous addition of serotonin and epinephrine. These compounds did
not inhibit a high level (0.1 U/mL) of thrombin-induced phosphorylation
of 20-kD MLC. In the presence of a higher concentration of thrombin
(>0.1 U/mL), neither platelet secretion nor 20-kD MLC phosphorylation
was affected by these inhibitors (data not shown). Thrombin (0.1 U/mL)
stimulation of platelets is associated with a dramatic increase in
intracellular Ca2+ concentration.38,39 To
further clarify the role of Rho-kinase in mediating MLC phosphorylation
in the case of 0.1 U/mL thrombin stimulation, we examined the effect of
intracellular Ca2+ chelator BAPTA-AM on inhibition by
Rho-kinase inhibitors of thrombin-induced MLC phosphorylation. As shown
in Fig 4, thrombin (0.1 U/mL)-induced increase in MLC phosphorylation was inhibited by preincubation with
BAPTA-AM, and subsequent treatment with HA1077 or Y-27632 resulted in
further inhibition of the thrombin-induced MLC phosphorylation.
View larger version (35K):
[in this window]
[in a new window]
| Fig 3.
Effects of HA1077 and Y-27632 on agonist-induced ATP
secretion of human platelets and MLC phosphorylation at
Ser19 in intact platelets. (A) Effects of HA1077 and
Y-27632 on agonist-induced ATP secretion. Human platelets incubated for
3 minutes in the aggregometer with various concentrations of HA1077
(left panel) or Y-27632 (right panel) were stimulated with 0.1U/mL
thrombin ( ), 0.05 U/mL thrombin ( ), 1 µmol/L STA2
(), or a mixture of 1 µmol/L serotonin and 1 µmol/L
epinephrine ( ) for 30 seconds, without stirring. Control levels of
ATP secretion induced by 0.1 U/mL thrombin, 0.05 U/mL thrombin,
STA2, and a mixture of serotonin and epinephrine were 354 ± 23.1, 184 ± 15.5, 173 ± 13.1, and 39.6 ± 2.58 pmol/108 platelets (n = 3), respectively. The values
represent the average of three experiments (SD <10%). (B) Effects of
HA1077 and Y-27632 on agonist-induced MLC phosphorylation at
Ser19 in intact platelets. Washed platelets preincubated
with various concentrations of HA1077 (left panel) or Y-27632 (right
panel) were stimulated with 0.1 U/mL thrombin (), 0.05 U/mL thrombin
(), 1 µmol/L STA2 (), or a mixture of 1 µmol/L serotonin and 1 µmol/L epinephrine (), without stirring.
The extent of 20-kD MLC phosphorylation was expressed as the percentage
of 20-kD MLC in the monophosphorylated form. The values represent the
average of three experiments (SD <10%)
|
|
View larger version (17K):
[in this window]
[in a new window]
| Fig 4.
Effect of BAPTA-AM on the inhibition by HA1077 and
Y-27632 on thrombin (0.1 U/mL)-induced MLC phosphorylation in intact
platelets. Platelets pretreated with vehicle () or 7.5 µmol/L
BAPTA-AM () for 3 minutes were treated with 20 µmol/L HA1077 or 10 µmol/L Y-27632 for 5 minutes. Platelets were stimulated with
0.1 U/mL thrombin for 20 seconds without stirring, and MLC
phosphorylation was analyzed, as described for Fig 3. Data
represent the mean ± SE of three experiments. *P < .05, **P < .01.
|
|
| |
DISCUSSION |
HA1077 and Y-27632 were reported to be relatively selective inhibitors
of Rho-kinase, compared with their inhibition of MLC kinase and protein
kinase C.19 Y-27632 inhibits Rho-kinase in vitro with a Ki
value of 0.14 µmol/L, a value about 185 times lower than that for
protein kinase C (Ki = 26 µmol/L), and it does not significantly
inhibit MLC kinase (Ki > 250 µmol/L). HA1077 is also a more potent
inhibitor of Rho-kinase (Ki = 0.33 µmol/L) than against protein
kinase C (Ki = 7.7 µmol/L) and MLC kinase (Ki = 170 µmol/L). We show here that MBS was phosphorylated in a
GTPS-RhoA-dependent manner and that HA1077 and Y-27632 inhibited, dose-dependently, GTPS-RhoA-induced MBS phosphorylation, determined using anti-MBS immunoprecipitates as sources of platelet MBS and Rho-kinase. However, GTPS-RhoA-dependent MBS phosphorylation was
not inhibited by the agonist receptor antagonist, the calmodulin antagonist W-7, or the MLC kinase inhibitor KT5926. Moreover, HA1077
and Y-27632 also inhibited GTPS-RhoA-induced inactivation of myosin
phosphatase activity in anti-MBS immunoprecipitates. These results
suggest that HA1077 and Y-27632 inhibited in platelets both
Rho-kinase-induced MBS phosphorylation and the resultant attenuation
of myosin phosphatase activity.
Phosphorylation of MBS occurred in intact platelets in response to
agonists such as thrombin, STA2, epinephrine, and
serotonin. Two kinds of Rho-kinase inhibitors produced a significant
inhibition of MBS phosphorylation induced by not only a combination of
the weak agonists serotonin and epinephrine, but also by 0.1 U/mL of, a
strong agonist, thrombin in intact platelets. HA1077 and Y-27632
inhibited STA2-induced MBS phosphorylation with similar potency (IC50 values: 2 µmol/L with HA1077 and
8 µmol/L with Y-27632), whereas the IC50 value for HA1077
(10 µmol/L) in inhibiting GTPS-RhoA-dependent MBS phosphorylation
was higher than that for Y-27632 (0.3 µmol/L). The concentrations of
both compounds required to affect MBS phosphorylation and ATP secretion
in intact platelets were similar to those in other
reports.19,20 One possible explanation of discrepancy found
in a cell-free system vis-à-vis intact platelets would be the
difference in penetration of the compound through the platelet membrane, and this aspect is now being investigated. The
agonist-induced MBS phosphorylation was also inhibited by receptor
antagonists such as an S2-serotonergic receptor antagonist
M-1, a thromboxane A2 receptor antagonist ONO-3708, and a
2-adrenergic receptor antagonist yohimbine. These data
indicate that MBS phosphorylation appears to be downstream of the
agonist-receptor interaction, although the molecular mechanisms of
signal transduction between the trimeric G-protein-coupled
receptor and RhoA remain to be identified. In addition, HA1077 and
Y-27632 significantly prevented the STA2-induced decrease
in the myosin phosphatase activity in intact platelets. These results
suggest that the Rho-kinase-induced MBS phosphorylation pathway and
the resultant inactivation of myosin phosphatase are the common pathway
in platelet activation stimulated by a variety of agonists. Rho-kinase
and myosin phosphatase have been shown to regulate phosphorylation of
-adducin, a membrane cytoskeletal protein that participates in
assembly of the spectrin-actin network.40 Therefore,
Rho-kinase and myosin phosphatase may be involved in cell functions in
addition to the regulation of the contractile cytoskeleton.
The extent of platelet ATP secretion depended on strength of the
agonist; a high level (0.1U/mL) of thrombin > a low level (0.05 U/mL)
of thrombin  STA2 > simultaneous stimulation of
epinephrine and serotonin. Moreover, the extent of secretion also
appeared to parallel the extent of MLC phosphorylation at
Ser19. RhoA appears to regulate MLC phosphorylation via two
pathways: one is the direct phosphorylation of myosin at
Ser19 by Rho-kinase,18 and the second is
inactivation of myosin phosphatase through MBS phosphorylation by
Rho-kinase.16 Both Rho-kinase-mediated pathways might be
important for an increase in MLC phosphorylation, because both pathways
(as well as the MLC kinase pathway) result in increased phosphorylation
of Ser19 in the MLC and therefore activate myosin ATPase
activity. Another target for Rho, protein kinase N did not
phosphorylate MBS nor the 20-kD MLC at Ser19 (data not
shown). Rho-kinase inhibitors should have an inhibitory effect on both
of these Rho-kinase-mediated pathways in intact platelets but not MLC
kinase phosphorylation. Uehata et al19 reported that
Y-27632 inhibits the contraction of vascular and tracheal smooth
muscles induced by various agonists such as phenylephrine, histamine,
serotonin, endothelin, and a thromboxane agonist, U-46619. Interestingly, HA1077 and Y-27632 inhibited MLC phosphorylation at
Ser19 and ATP secretion of human platelets induced by a low
level (0.05 U/mL) of thrombin, STA2, and the simultaneous
stimulation with epinephrine and serotonin, whereas these compounds
affected neither MLC phosphorylation at Ser19 nor ATP
secretion stimulated by a high level (0.1 U/mL) of
thrombin. Increases in intracellular Ca2+
concentration in platelets have been observed in response to agonists
such as thrombin, STA2, and a simultaneous addition of serotonin and epinephrine, determined using the fluorescent
Ca2+ probe quin-2 and fura-2.38,39 However, the
magnitude of the peak and the duration of the Ca2+ levels
depend on the strength of the agonist. After thrombin stimulation (0.1 U/mL), the intracellular Ca2+ level increased from about
100 nmol/L to greater than 1,000 nmol/L in human
platelets.38,39 STA2 treatment led to an
increase in the intracellular Ca2+ level, but the peak
level (~200 to 300 nmol/L) was lower than that seen with thrombin
stimulation.41 Although our data suggest that either
epinephrine or serotonin by itself can activate Rho-kinase and
phosphorylate MBS in intact platelets, these weak agonists do not
induce aggregation or ATP secretion in washed platelets suspensions.35 A simultaneous stimulation with serotonin
and epinephrine induced to a certain extent intracellular
Ca2+ mobilization and ATP secretion. The peak level of
intracellular Ca2+ by the simultaneous stimulation was
about 150 to 200 nmol/L,35 which is is
substantially lower than that induced with thrombin (0.1 U/mL)
stimulation. We have shown that human platelets contain approximately
15 times lower amounts of Rho-kinase than MLC kinase, on a molar basis.
In addition, a previous report has shown that the molecular activity of
Rho-kinase for MLC is about three times lower than that of MLC
kinase,18 although the apparent Km value (0.91 µmol/L) of
Rho-kinase for the MLC is lower than that (52 µmol/L) of MLC kinase.
Therefore, the Ca2+/calmodulin-dependent MLC kinase is
thought to be the primary regulator for MLC phosphorylation at
Ser19 in human platelets. Collectively, our data suggest
the possibility that relatively high levels (>0.1 U/mL) of thrombin
that lead to rapid mobilization of intracellular Ca2+
induce phosphorylation of the 20-kD MLC by MLC kinase and thereby trigger ATP secretion. Under these circumstances, the contribution of
Rho-kinase-mediated MLC phosphorylation appears to be minimal, even
though Rho-kinase-induced MBS phosphorylation is evident. This
hypothesis is supported by the experiments with BAPTA-AM-preincubated platelets showing that chelation of cytoplasmic Ca2+ made
apparent the inhibition by HA1077 and Y-27632 of 0.1 U/mL thrombin-induced MLC phosphorylation, presumably due to less
activation of Ca2+-dependent MLC kinase. On the other
hand, our pharmacological studies suggested that Rho-kinase mediated
MLC phosphorylation (both directly and indirectly through MBS
phosphorylation), in addition to MLC kinase-dependent phosphorylation,
may be necessary for platelet secretion induced by relatively weak
agonist, STA2, and a lower level (<0.05 U/mL) of
thrombin, all of which presumably cause a submaximal elevation of
intracellular Ca2+ concentration.42,43
Platelet-activating factor and thrombin have been shown to induce MLC
phosphorylation in intact platelets, even when cytoplasmic Ca2+ concentration remains at, or close to, resting
levels44; the observation suggests the existence of
Ca2+-independent pathway that might synergize with
Ca2+ to produce MLC phosphorylation. In permeabilized
platelets, GTPS and phorbol ester significantly enhance the
Ca2+ sensitivity of serotonin secretion, without detectable
phosphatidylinositol hydrolysis.17,45 However, the
augmentation of MLC phosphorylation by GTPS at submaximal
Ca2+ concentration was slight, therefore the dominant
factor regulating MLC phosphorylation appears to be the
Ca2+ concentration in permeabilized
platelets.17 These findings are consistent with the
hypothesis that Rho-kinase activated MLC phosphorylation is involved in
mediating platelet secretion induced by relatively weak stimuli,
although Ca2+/calmodulin-dependent MLC kinase is thought to
be the essential regulator for MLC phosphorylation at
Ser19. Additional elements, such as protein kinase
C,17 also appear to be involved in the GTPS-induced
increase in Ca2+ sensitivity that accompanies secretion. In
conclusion, Rho-kinase-mediated MLC phosphorylation is involved in
agonist-induced secretion in human platelets at submaximal
Ca2+ concentrations, in synergy with the
Ca2+-dependent MLC phosphorylation, possibly with the
cooperation of other signaling systems such as protein kinase C.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr R.S. Adelstein (NIH) for helpful
discussions, to E. Imai for technical assistance, and to M. Ohara for
helpful comments.
| |
FOOTNOTES |
Submitted November 2, 1998; accepted January 11, 1999.
Supported in part by grants for research from the Ministry of
Education, Science, Sports and Culture of Japan and by grants from the
Mie Medical Research Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Masakatsu Nishikawa, MD, PhD, The 2nd
Department of Internal Medicine, Mie University School of Medicine,
2-174 Edobashi, Tsu, Mie 514-8507, Japan; e-mail:
nisikawa{at}clin.medic.mie-u.ac.jp.
| |
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[Abstract]
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E. Butt, S. Gambaryan, N. Gottfert, A. Galler, K. Marcus, and H. E. Meyer
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[Abstract]
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S. M. Schoenwaelder, S. C. Hughan, K. Boniface, S. Fernando, M. Holdsworth, P. E. Thompson, H. H. Salem, and S. P. Jackson
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[Abstract]
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Y. Watanabe, M. Ito, Y. Kataoka, H. Wada, M. Koyama, J. Feng, H. Shiku, and M. Nishikawa
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Blood,
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[Abstract]
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G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh
Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System
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[Abstract]
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G. P. v. N. Amerongen, S. v. Delft, M. A. Vermeer, J. G. Collard, and V. W. M. van Hinsbergh
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[Abstract]
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T. Ishizaki, M. Uehata, I. Tamechika, J. Keel, K. Nonomura, M. Maekawa, and S. Narumiya
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[Abstract]
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A. P Somlyo and A. V Somlyo
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J. Feng, M. Ito, K. Ichikawa, N. Isaka, M. Nishikawa, D. J. Hartshorne, and T. Nakano
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION