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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1665-1672
Dichotomous Regulation of Myosin Phosphorylation and Shape Change
by Rho-Kinase and Calcium in Intact Human Platelets
By
Markus Bauer,
Michaela Retzer,
Jonathan I. Wilde,
Petra Maschberger,
Markus Essler,
Martin Aepfelbacher,
Steve P. Watson, and
Wolfgang Siess
Institut für Prophylaxe und Epidemiologie der
Kreislaufkrankheiten, Klinikum Innenstadt, Universität
München, München, Germany; the Department of Pharmacology,
Oxford University, Oxford, UK; and Max von Pettenkofer-Institut,
Universität München, München, Germany.
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ABSTRACT |
Both Rho-kinase and the Ca2+/calmodulin-dependent
myosin light chain (MLC) kinase increase the phosphorylation of MLC. We
show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by
U46619, shape change and MLC phosphorylation in human platelets proceed
through a pathway that does not involve an increase in cytosolic
Ca2+. Under these conditions, Y-27632, a specific
Rho-kinase inhibitor, prevented shape change and reduced the
stimulation of MLC-phosphorylation. In contrast, Y-27632 barely
affected shape change and MLC-phosphorylation by adenosine diphosphate
(ADP), collagen-related peptide, and ionomycin that were associated
with an increase in cytosolic Ca2+ and inhibited by
BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates
Rho, inhibited preferentially the shape change induced by YFLLRNP
compared with ADP and ionomycin. The results indicate that the
Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation
and platelet shape change by low concentrations of certain G
protein-coupled platelet receptors, independent of an increase in
cytosolic Ca2+. Our study defines 2 alternate pathways,
Rho/Rho-kinase and Ca2+/calmodulin-regulated MLC-kinase,
that lead independently of each other through stimulation of
MLC-phosphorylation to the same physiological response in human
platelets (ie, shape change).
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PLATELET SHAPE CHANGE is the earliest
response induced by physiological agonists. It precedes platelet
spreading, aggregation, and secretion, and is characterized
morphologically by spheration and contraction of the cell, cytoskeletal
rearrangements, folding of the surface membrane, and formation of
pseudopods.1,2 The dense tubular system of resting
platelets is filled with Ca2+, and activated platelets show
a large Ca2+-influx through the plasma membrane and
pronounced mobilization of Ca2+ from intracellular stores.
However, some stimuli such as thrombin, the thrombin receptor ligand
YFLLRNP, or the thromboxane receptor agonists U46619 or U44069 induce
shape change through a pathway that is apparently independent of an
increase in cytosolic Ca2+.3-7 Thus, upon
activation of certain receptors, platelets undergo a contractile
response that is not regulated by cytosolic Ca2+.
A crucial event in triggering shape change is the stimulation of myosin
light chain (MLC) phosphorylation.8 Phosphorylated myosin
develops actin-activated adenosine triphosphatase (ATPase) activity, interacts with the cytoskeleton, and assembles into filaments properties that may lead to the cytoskeletal rearrangements, the folding of the surface membrane, and the contractile wave centralizing the secretory granules observed during shape
change.1,2 MLC-phosphorylation is regulated in nonmuscle
cells by a Ca2+/calmodulin-dependent MLC-kinase and, as
recent studies show, by Rho-kinase, which is activated by the small
guanosine triphosphate (GTP)-binding protein RhoA.
Rho-kinase directly phosphorylates MLC and phosphorylates the
130-kD myosin-binding subunit (MBS) of myosin phosphatase,
thereby inhibiting the catalytic subunit of the enzyme leading to a
further increase of MLC-phosphorylation.9,10 Indeed, recent
studies show that RhoA, Rho-kinase, and MBS form a complex in
platelets, and that in stimulated platelets and in other cells,
Rho-kinase phosphorylates MBS, thereby decreasing the activity of
myosin phosphatase.10-12 Furthermore, expression of
constitutively active Rho-kinase increased the level of
MLC-phosphorylation in fibroblasts, and introduction of active
Rho-kinase into the cytosol of permeabilized vascular smooth muscle
cells provoked MLC-phosphorylation and contraction in the presence and
absence of cytosolic Ca2+.13,14
The role of Rho-kinase in platelet function and the contribution of
Rho-kinase to the kinetic and degree of MLC-phosphorylation in intact
cells stimulated by physiological agonists are not known. By using a
newly available, specific inhibitor of Rho-kinase, Y-27632,15 we found that Rho-kinase through stimulation of
MLC-phosphorylation and inhibition of MLC-dephosphorylation plays a
central role in mediating the pathway of platelet shape change that is
independent of an increase in cytosolic Ca2+. The
Rho-kinase-dependent pathway was stimulated by the activation of
thrombin and thromboxane receptors as opposed to the
Ca2+-/MLC kinase-dependent pathway that was stimulated by
adenosine diphosphate (ADP) and collagen receptor activation.
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MATERIALS AND METHODS |
Reagents.
Y-27632 was a gift from Dr Akiko Yoshimura (Yoshitomi Pharmaceutical
Industries, Saitama, Japan). Collagen-related-peptide (CRP) was a gift
of Dr Michael Barnes (Department of Biochemistry, Cambridge, UK).
YFLLRNP peptide was obtained from Bachem Biochemica (Heidelberg,
Germany). Human thrombin (catalogue no. T 7009) was from Sigma (St
Louis, MO). Recombinant C3-exoenzyme was produced as
described.12 All other materials were obtained from sources described previously.6,16-18
Isolation of human platelets, measurement of platelet shape change,
and cytosolic [Ca2+].
Human platelets were treated with acetylsalicylate, isolated by
centrifugation in the presence of apyrase as described, and resuspended
in a buffer (pH 7.4) containing 20 mmol/L HEPES, 138 mmol/L NaCl, 2.9 mmol/L KCl, 1 mmol/L MgCl2, and 0.6 adenosine diphosphatase
(ADPase) U/mL apyrase.6 Some experiments with CRP and thrombin were also performed in the presence of indomethacin (10 µmol/L) and EGTA (1 mmol/L) in the resuspension
buffer.17 Both methods gave similar results. Platelet shape
change was measured by recording the light transmission in an
aggregometer as described6 at a cell density of (2 to 4) × 108/mL. Y-27632 and BAPTA-AM were added at the
given concentrations 30 minutes before stimulation followed by
incubation at 37°C. EGTA (2 mmol/L) was added 2 minutes before the
addition of stimulus. In experiments using C3 exoenzyme, platelets were
resuspended in buffer exactly as described and incubated with C3
exoenzyme (400 µg/mL) for 4 hours at 37°C.19 This
method has been shown to ADP-ribosylate and inactivate approximately
90% of RhoA.19 After incubation with C3 exoenzyme or
vehicle, platelets were diluted to 2 × 108/mL for
measurement of shape change.
For measurement of the cytosolic
Ca2+-concentration, washed platelets were
loaded with 4 µmol/L Fura2-AM (Sigma) as described5 and
resuspended at a cell density of 2 × 108/mL.
Ca2+ was measured in a thermostatically controlled
(37°C) chamber while stirring using a Delta-Scan-1 double-beam
fluorescence spectrophotometer (Photon Technology International, South
Brunswick, NJ) or an LS 5OB spectrofluorimeter
(Perkin-Elmer, Beaconsfield, UK). Fluorescence measurement
and calculation of cytosolic Ca2+-concentrations were
performed as described.5
Fluorescence microscopy of F-actin-stained platelets.
Samples of washed platelets were taken from platelet suspensions in the
aggregometer and fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 minutes at room temperature. The fixed platelets were centrifuged on coverslips coated with poly-L-lysine (Sigma no.
6282) at 250g for 5 minutes at room temperature. After washing 3 times with PBS, platelets were incubated with 0.2% Triton X-100 in
PBS for 10 minutes at room temperature, and, after further washing,
stained for F-actin by incubation with 8 U/mL rhodamine phalloidin
(Molecular Probes, Eugene, OR) for 1 hour at 37°C. After washing,
the coverslips were mounted on glass slides with mounting medium
(Immuno Fluore Mounting Medium; ICN Biochemicals, Inc, Aurora, OH).
Fluorescence microscopy was performed using a Leika DM IRB confocal
fluorescence microscope and Leika software TCSNT version 1.5.451 (Leika, Bensheim, Germany).
32P-labeling of human platelets and measurement of
protein phosphorylation.
Platelets were isolated and incubated with 1 to 5 mCi/mL
H332PO4 as described previously
except that albumin (0.3 mg/mL) was used instead of 10% plasma during
labeling.18 Preincubation with inhibitors and measurement
of shape change were performed as described above. Portions of 50 µL
were taken at the given time points, transferred to an equal amount of
sample buffer (final concentration: 62.5 mmol/L Tris, 3% sodium
dodecyl sulfate [SDS], 5% glycerol, 5% 2-mercaptoethanol, 1 mmol/L
EDTA; pH 6.8), and boiled for 5 minutes. 32P-labeled
proteins were separated by SDS/polyacrylamide-gel electrophoresis, stained with Coomassie Blue, and dried as described
previously.18 Autoradiography was performed by exposure of
the dried gels to Kodak X-OMAT film (Eastman Kodak, Rochester, NY) for
16 to 24 hours. The bands of phosphorylated MLC were analyzed by
densitometry using a JX-325 film scanner (Sharp, Hamburg, Germany) and
ImageMaster-1D software (Pharmacia, Uppsala, Sweden). The level of MLC
phosphorylation of resting platelets was subtracted, and
the results of the individual experiments were expressed as percent of
maximal MLC phosphorylation induced by the respective agonists.
Analysis of results.
Results are shown as mean ± SD of individual experiments from
different blood donors.
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RESULTS |
We studied various platelet stimuli that mediate their action through
distinct receptors, and chose concentrations that induced a similar
level of platelet shape change. Beside several stimuli that activate G
protein-coupled receptors, we used CRP as potent agonist of the
collagen activatory receptor GP VI.20-22 By measuring in
parallel in the same preparation of Fura2-loaded platelets cytosolic
Ca2+ concentration and shape change, we show that there are
two pathways that lead to platelet shape change: one independent of and
without an increase in cytosolic Ca2+, the other dependent
of and with an increase in cytosolic Ca2+. The importance
of each pathway is agonist- and dose-dependent. Thrombin receptor
activation by the heptapeptide ligand YFLLRNP as well as activation of
the PG-endoperoxide/thromboxane receptor by low concentrations of
U46619 induced shape change in the absence of an increase of cytosolic
Ca2+ (Fig 1a and d). The
threshold for shape change by thrombin varied depending on the donor
but, in general, concentrations of 0.01 to 0.04 U/mL lead to shape
change that occurred in the absence of an increase of cytosolic
Ca2+ (Fig 1b). In contrast, thrombin at higher
concentrations (0.05 to 0.2 U/mL, dependent on the platelet
preparation), ADP, CRP, and ionomycin induced shape change that was
associated with an increase of cytosolic Ca2+ (Fig 1c, e
through g).
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| Fig 1.
Effect of Rho-kinase inhibition by Y-27632 and chelation
of cytosolic Ca2+ by BAPTA plus EGTA on shape change
(left) and cytosolic calcium levels (right). Platelets loaded with
Fura2-AM were stimulated by various agonists. Shown are untreated
control samples (C), samples preincubated with 20 µmol/L Y-27632 (Y),
20 µmol/L BAPTA-AM + 2 mmol/L EGTA (B+E), or both (B+E+Y).
The arrow indicates addition of the agonist. Decrease of light
transmission together with the disappearance of oscillations are
indicative of shape change. (a) YFLLRNP (300 µmol/L). (b) Lower
concentration of thrombin (0.01 U/mL). (c) Higher concentration of
thrombin (0.05 U/mL). (d) U46619 (50 nmol/L). (e) ADP (2 µmol/L). (f)
CRP (0.05 µg/mL). (g) Ionomycin (100 nmol/L). Results are
representative of 4 to 6 independent experiments.
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Next we used EGTA and BAPTA-AM treatment of platelets to inhibit any
cytosolic Ca2+-increase due to Ca2+-influx
through the plasma membrane and Ca2+-mobilization from
intracellular stores. We found that shape change through the pathway
that was independent of an increase in cytosolic Ca2+ was
delayed, but that the maximal response was not reduced (Fig 1a, b, d
and Fig 2). The slower kinetic of shape
change after BAPTA-AM/EGTA treatment might be due to the lower
Ca2+-concentration in resting platelets that was 10 nmol/L
in the presence as compared to 50 to 70 nmol/L in the absence of
BAPTA-AM/EGTA (Fig 1, right panel). In contrast, shape change through
the Ca2+-dependent pathway stimulated by the ADP, CRP, and
ionomycin was completely inhibited by BAPTA-AM/EGTA treatment (Fig 1c,
e through g; Fig 2). Interestingly, shape change induced by the higher
concentration of thrombin that was associated with a cytosolic
Ca2+-increase was barely inhibited by BAPTA-AM/EGTA (Fig
1c).
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| Fig 2.
Quantitative comparison of the effect of Rho-kinase
inhibition and chelation of cytosolic Ca2+ on platelet
shape change induced by various agonists. Shown are untreated controls
( ), samples incubated with Y-27632 (20 µmol/L;  ), and samples
pretreated with BAPTA-AM (20 µmol/L) and EGTA (2 mmol/L;  ). (a)
Stimulation conditions that were not associated with an increase in
cytosolic Ca2+ (see Fig 1): lower concentrations of
thrombin (0.01 to 0.04 U/mL), YFLLRNP (300 µmol/L), U46619 (50 nmol/L). (b) Stimulation conditions that induced a significant increase
in cytosolic Ca2+: higher concentrations of thrombin
(0.05 to 0.2 U/mL), ADP (2 µmol/L), CRP (0.05 µg/mL), ionomycin
(100 nmol/L). Values are mean ± SD of 4 to 6 independent experiments.
Asterisk (*) indicates absence of shape change in all experiments.
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The role of Rho-kinase in mediating shape change was investigated using
Y-27632, which has been shown to specifically inhibit the kinase
activity of p160ROCK purified from platelets.15
Pretreatment of platelets with the Rho-kinase inhibitor Y-27632 (20 µmol/L) inhibited selectively the shape change that proceeded through the pathway that was independent of an increase in cytosolic
Ca2+: the responses to YFLLRNP, low-dose thrombin, and
U46619 were drastically reduced or abolished. Shape change upon
stimulation with YFLLRNP was inhibited by 67% ± 7%, with 0.01 to
0.04 U/mL thrombin by 76% ± 6%, and with U46619 by 100% ± 0% (mean ± SEM). However, shape change induced by the higher
thrombin concentration, ADP, CRP, and ionomycin was only slightly
affected (Figs 1 and 2). Y-27632 (20 µmol/L) reduced the shape change
upon stimulation with 0.05 to 0.02 U/mL thrombin by 20% ± 6%,
with ADP by 28% ± 5%, CRP by 9% ± 12%, and with ionomycin
by 21% ± 5% (mean ± SEM). Also, responses to lower ADP
concentrations (100 to 250 nmol/L), which induced 50% of the maximal
level of shape change, were associated with an increase of cytosolic
Ca2+ and were only marginally inhibited by Y-27632 (data
not shown). Y-27632 did not affect the Ca2+-increase
observed after addition of these agonists (Fig 1, right panel).
The shape change induced by the higher concentrations of thrombin was
delayed, but only weakly inhibited, by either Y-27632 or BAPTA-AM/EGTA
treatment, showing that thrombin at this concentration can induce shape
change through the separate activation of two pathways (Figs 1c and
2b). Treatment with both Y-27632 and BAPTA-AM/EGTA completely inhibited
shape change, indicating that thrombin at this concentration stimulated
the two pathways separately (Fig 1c). Interestingly, the increase of
cytosolic Ca2+ was smaller after the higher concentrations
of thrombin than after ADP, although the extent of the shape change
after the two stimuli was similar (Fig 1c and e). This was also
observed after inhibition of Rho-kinase, indicating that thrombin
required a smaller increase of cytosolic Ca2+ to produce
shape change than did ADP.
Concentration-response curves of Y-27632
(Fig 3) showed that lower drug
concentrations were sufficient for the inhibition of shape change by
U46619 as compared with YFLLRNP. The IC50 value (concentration that inhibits 50% of the response) of Y-27632 was 1 ± 0.2 µmol/L for the inhibition of shape change by U46619 as compared with an IC50 value of 9.6 ± 1.1 µmol/L for
the inhibition of shape change by YFLLRNP (mean ± SEM). The
IC50 values for the 28% and 21% reduction of shape change
by ADP and ionomycin were 3.9 ± 0.2 µmol/L and 4.6 ± 0.3 µmol/L, respectively (mean ± SEM). Y-27632 did not
induce a significant inhibition of shape change by CRP.
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| Fig 3.
Concentration-response curves of Y-27632 effect on
platelet shape change induced by different agonists. Samples were
preincubated with the given concentrations of inhibitor for 30 minutes
at 37°C. Values are the percent maximum induced by the agonist in
the absence of Y-27632. (a) ADP (2 µmol/L;  ) and ionomycin (100 nmol/L;  ). (b) YFLLRNP (300 µmol/L; ) and U46619 (100 nmol/L;
). Data are mean ± SE of 4 to 6 independent experiments.
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We measured MLC-phosphorylation during the two pathways of shape
change, and determined the action of Y-27632. Rho-kinase inhibition by
Y-27632 dramatically reduced the early peak of MLC phosphorylation
after YFLLRNP and U46619. After YFLLRNP, but not after U46619
stimulation, Y-27632 also accelerated the subsequent dephosphorylation
of phosphorylated MLC, possibly due to higher myosin phosphatase
activity (Fig 4a and b). In contrast, the
effects of Y-27632 on MLC-phosphorylation by ADP, ionomycin, and CRP
were much less pronounced. ADP induced a rapid, transient
MLC-phosphorylation with a peak at 5 seconds after addition. Y-27632
reduced the maximum level by 28% ± 4%, and subsequently the
amplitude of MLC-phosphorylation (Fig 4c). Stimulation of
MLC-phosphorylation after addition of CRP and ionomycin was more
sustained, and Y-27632 also slightly reduced the maximum level and the
amplitude of MLC-phosphorylation (Fig 4d and e). These results indicate
that Rho-kinase plays a major role in the phosphorylation of MLC during
shape change induced by stimuli that do not produce a detectable
increase in cytosolic Ca2+, and only a minor role in
phosphorylating MLC upon induction of shape change by the
Ca2+-dependent stimuli.
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| Fig 4.
Effect of Rho-kinase inhibition and chelation of
cytosolic Ca2+ on MLC-phosphorylation induced by
different agonists. (a through d) MLC-phosphorylation levels in percent
of the maximal increase upon stimulation of untreated controls. The
maximal increase of MLC-phosphorylation stimulated by the different
agonists was similar. Shown are controls ( ), samples pretreated with
20 µmol/L Y-27632 (), or 20 µmol/L BAPTA-AM and 2 mmol/L EGTA
( ). Values are mean ± SD of 3 to 4 independent experiments. (e)
Autoradiographs of phosphorylated MLC after stimulation with 0.05 µg/mL CRP in untreated control samples (top), samples pretreated with
30 µmol/L Y-27632 (middle), or 50 µmol/L BAPTA-AM and 2 mmol/L EGTA
(bottom). The results shown are representative of 2 experiments.
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Reduction of basal Ca2+ and inhibition of the increase in
cytosolic Ca2+ by BAPTA/EGTA prevented MLC-phosphorylation
by ADP, CRP, and ionomycin. BAPTA/EGTA treatment also delayed and
reduced the maximum level of MLC phosphorylation by YFLLRNP and U46619.
The delayed stimulation of MLC phosphorylation corresponded to the slow
shape change response of the [32P]-prelabeled platelets;
importantly, the stimulation of MLC-phosphorylation preceded shape
change (data not shown), further strengthening the cause/effect
relationship of MLC-phosphorylation and shape change.8
The role of Rho kinase in the response to agonists that induced shape
change without an increase in cytosolic Ca2+ was also
characterized by fluorescence microscopy of platelets stained for
F-actin. Treatment with Y-27632 did not have an effect on the discoid
shape and the diffuse weak fluorescence of resting platelets, but
inhibited the enhanced fluorescence of F-actin and change in platelet
morphology (pseudopod formation, irregular surface) after stimulation
with YFLLRNP. Shape change after stimulation with ADP, in contrast, was
barely affected (data not shown).
To evaluate the role of Rho, the activator of Rho-kinase, in the 2 pathways of shape change, platelets were preincubated with C3 exoenzyme
(400 µg/mL), which has been shown to ADP-ribosylate and inactivate
90% of RhoA in intact human platelets.19 This incubation,
which did not result in preactivation of platelets (data not shown),
reduced the shape change upon stimulation with YFLLRNP by 54% ± 4%, and with ADP by 21% ± 7% (mean ± SEM). Shape change
induced by ionomycin was not inhibited (Fig
5).
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| Fig 5.
Quantitative comparison of C3-exoenzyme treatment on
platelet shape change induced by various agonists. Platelets were
incubated with vehicle () or C3-exoenzyme (400 µg/mL; ) before
stimulation with YFLLRNP (300 µmol/L), ADP (2 µmol/L), or ionomycin
(200 nmol/L). The shape change responses (measured by the maximal
decrease in light-transmission) induced by the different agonists were
similar. Values are mean ± SD of three experiments with different
platelet donors.
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DISCUSSION |
Shape change in human, rabbit, and mice platelets by
certain agonists can be induced through a pathway that is independent of an increase of cytosolic Ca2+. Agonists that have been
shown to induce this "Ca2+-independent pathway" of
shape change in human platelets include the heptapeptide ligand of the
thrombin receptor, YFLLRNP, and low concentrations of
thrombin3,5,6 (and present study); in human and rabbit
platelets, the endoperoxide analogs U46619 and U440694,7
(and present study); and in G q-deficient mice platelets,
thrombin and U46619.23 We report in the present study that
Rho-kinase mediates the pathway of shape change that is independent of
an increase in cytosolic Ca2+, but plays only a minor role
in the Ca2+-dependent pathway of shape change. Upon
platelet stimulation by YFLLRNP and U-46619, inhibition of Rho-kinase
by Y-27632 dramatically reduced both the onset and the duration of
MLC-phosphorylation, the key event in the initiation of platelet shape
change. Two novel kinases have been isolated that phosphorylate MLC and
contain catalytic domains resembling that of Rho-kinase. These kinases bind to and are activated by the Rho-related protein CDC42, promoting cytoskeletal reorganization.24 CDC42 promotes filopod
formation in fibroblasts, and translocates to the cytoskeleton in
activated platelets.25 It might be possible that Y-27632
also inhibits these novel kinases, thereby contributing to the
inhibition of shape change that is independent of an increase in
cytosolic Ca2+.
During the Ca2+-dependent pathway of platelet shape change,
induced by ADP or ionomycin, we observed a weak inhibition of shape change that was associated with a small reduction of phosphorylation of
MLC in the presence of Y-27632. These results show that Rho-kinase plays only a minor role in phosphorylating MLC and shape change under
these conditions. The weak activation of Rho-kinase might be caused by
the increase of Ca2+ after ADP and ionomycin stimulation,
or mediated by ADP receptor activation; it cannot be excluded that
small amounts of ADP are released by ionomycin during shape change.
BAPTA/EGTA treatment prevented MLC-phosphorylation and shape change
induced by stimuli of the Ca2+-dependent pathway. It also
delayed shape change, and reduced and delayed MLC phosphorylation
induced by stimuli that did not increase cytosolic Ca2+.
Because BAPTA/EGTA treatment also reduced the basal level of cytosolic
Ca2+, this may indicate that the Rho/Rho-kinase pathway,
albeit not regulated by cytosolic Ca2+, still requires the
concentration of cytosolic Ca2+ in resting platelets for
optimal activity.
Y-27632 did not completely inhibit MLC-phosphorylation induced by
YFLLRNP or U46619 (Fig 4c and d), but almost completely abolished shape
change. This may indicate that a certain level of MLC phosphorylation
is required for shape change, and/or that Rho-kinase phosphorylates
other proteins involved in platelet shape change. It is noteworthy that
Rho-kinase phosphorylates vimentin, an intermediate filament protein,
and ezrin/radixin/moesin proteins that are involved in the interaction
of actin filaments with the plasma membrane.26,27
Phosphorylation of these cytoskeletal proteins interferes with their
function and is expected to affect the cytoskeletal rearrangements
during shape change.
We also found that inactivation of Rho by C3-exoenzyme preferentially
inhibited shape change by YFLLRNP as compared to ADP and ionomycin,
corroborating our findings with the Rho-kinase inhibitor Y-27632. Shape
change was not completely inhibited, possibly due to residual amounts
of active Rho. After submission of our manuscript, a study using mouse
platelets has been published reporting an inhibition of U46619-induced
shape change in Gq-deficient platelets by C3 exoenzyme
and the Rho-kinase inhibitor Y-27632.28 These results
support our conclusions in human platelets that the Rho/Rho-kinase
pathway is pivotal in mediating the MLC phosphorylation and platelet
shape change, when they are not regulated by an increase in cytosolic
Ca2+. Recently, inhibition of RhoA has been found to
inhibit the formation of focal adhesions in platelets that spread on
fibrinogen.19 Spreading occurs subsequently to shape
change, and depends on integrin IIb 3
signaling. It is tempting to speculate that Rho-kinase activated during
shape change is involved in the formation of focal adhesions through
modulation of integrin IIb3 activity or signaling.
Our study has delineated two pathways of myosin phosphorylation and
platelet shape change, one mediated by Rho-kinase and the other by the
Ca2+-dependent activation of MLC kinase. Each pathway can
be activated independently of each other, dependent on the type and
concentration of the agonist. The tyrosine kinase-linked collagen
receptor glycoprotein (GP) VI uses the Ca2+-dependent
pathway to stimulate shape change. In contrast, stimuli that activate
7-transmembrane G protein-coupled receptors induce shape change via
Rho-kinase, which is not regulated by an increase in cytosolic
Ca2+ or via the Ca2+-dependent activation of
MLC kinase. The ability of a particular 7-transmembrane receptor via
either pathway is presumably related to couple to G12/13
and Gq. As depicted in the model in
Fig 6, thromboxane receptor activation by
U46619 or thrombin receptor activation by YFLLRNP or low concentration
of thrombin will activate the heterotrimeric G-proteins
G12/1329 that are linked to the activation of
Rho,30 and Rho-kinase leading to MLC phosphorylation and
platelet shape change. This pathway is also present in mouse platelets.28 Activation of the purinergic P2Y1
receptor by ADP will activate mainly the Gq/phospholipase
C/Ca2+ pathway31 that leads to cytosolic
increase of Ca2+ and the Ca2+-dependent
stimulation of MLC phosphorylation and shape change. This is in
accordance with the result in Gq knock-out mice that showed a loss of the Ca2+ and shape change response to
ADP.23 In human platelets, ADP seems also to activate
weakly Rho/Rho-kinase as indicated by the small inhibition of
ADP-induced shape change and MLC phosphorylation by C3 exoenzyme and
Y-27632. Activation of GPVI by CRP will sequentially activate the Src
and Syk-tyrosine kinases with the subsequent tyrosine phosphorylation
and activation of phospholipase C 2,32 thereby increasing
cytosolic Ca2+ and stimulating Ca2+-dependent
MLC-phosphorylation and shape change. Direct
Ca2+-mobilization by ionomycin will also activate MLC
kinase. Higher concentrations of thrombin will activate more strongly
the Rho-kinase pathway and stimulate the Gq/phospholipase
C/Ca2+ pathway. Under these conditions, inhibition of
either Rho-kinase or the Ca2+ pathway alone is insufficient
to inhibit shape change, indicating that each pathway compensates for
the loss of the other. Inhibition of both pathways is needed to
completely inhibit shape change after the higher dose of thrombin (Fig
1c), showing that a single agonist can induce shape change through the
separate activation of two pathways (Fig 6).
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| Fig 6.
Model depicting the dichotomous regulation of platelet
shape change by Rho-kinase and Ca2+-dependent MLC-kinase.
See text for details. Abbreviations: MLC- , phosphorylated myosin
light-chain; MLC-kinase, Ca2+/calmodulin-dependent myosin
light-chain kinase; MLC-Pase-, phosphorylated myosin light-chain
phosphatase; PLC, phospholipase C; PLC2-Y,
tyrosine-phosphorylated phospholipase C2; Src/Syk, Src-family
tyrosine kinases and tyrosine kinase Syk.
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FOOTNOTES |
Submitted January 8, 1999; accepted May 3, 1999.
Supported by Ernst und Berta Grimmke-Stiftung, Deutsche
Forschungsgemeinschaft (Si 274/6-1, GRK 438, Ae11/5-1, and SFB
413), August-Lenz-Stiftung, Anglo-German Foundation (ARC-X-96), and the
Wellcome Trust. S.P.W. is a British Heart Foundation Senior Research fellow.
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 Wolfgang Siess, MD, Institut für
Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Klinikum
Innenstadt, Universität München, Pettenkoferstr. 9, D-80336
München, Germany; e-mail:
wolfgang.siess{at}klp.med.uni-muenchen.de.
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ABSTRACT
INTRODUCTION