Convulxin induces platelet shape change through myosin light chain
kinase and Rho kinase
Silvia Riondino, Pier P. Gazzaniga and Fabio M. Pulcinelli
Department of Experimental Medicine and Pathology, Universita
´
La Sapienza, Rome, Italy
Once platelets are activated, the first event to occur is a rapid
change in shape, associated with Ca
2+
/calmodulin-
dependent myosin light chain (MLC) phosphorylation and
with Rho kinase activation. The purpose of this study was to
investigate which is the biochemical pathway that leads to
platelet shape change in response to convulxin, a selective
GpVI activator, and to verify whether MLC phosphoryla-
tion is essential for this process. The inhibition of the Ca
2+
-
dependent pathway by means of the Ca
2+
chelator BAPTA,
the Ca
2+
/calmodulin inhibitor W-7 or the cAMP enhancing
drug iloprost reduced about 50% of platelet shape change in
response to convulxin. The treatment with either the Rho
kinase inhibitors Y27632 or HA 1077 had no effect on
platelet shape change induced by convulxin. When both
Ca
2+
/calmodulin-dependent and Rho kinase-dependent

2+
/calmodulin-dependent pathway, subsequent to Ca
2+
mobilization and mediated by MLC kinase [3], and the
other is Ca
2+
-independent, subsequent to the small GTP-
binding protein RhoA activation, and mediated by Rho
kinase [4–6]. The Ca
2+
/calmodulin-dependent pathway
which leads to MLC phosphorylation depends upon the
stimulation of a G
q
-coupled receptor and phospholipase C
(PLC) activation. This datum has also been supported by
experiments on G
aq
-deficient mice platelets [7]. On the other
hand, the Ca
2+
-independent pathway follows the stimula-
tion of a G
12
/G
13
coupled receptor [4].
The second messenger cAMP also exerts an efficacious
action on cytoskeletal rearrangements, by inhibiting actin
assembly and focal adhesion in many cell types [8,9] through

the presence of the aIIb/b3 antagonist, peptide RGDS, or
actin polymerization inhibitors, such as cytochalasin D [16].
The purpose of the present study was to investigate the
biochemical pathways followed by convulxin in inducing
Correspondence to P. Gazzaniga, Department of Experimental
Medicine and Pathology, Universita
´
La Sapienza, Viale Regina
Elena 324, 00161 Rome, Italy.
Fax: + 39 064452955, Tel.: + 39 064454820,
E-mail: [email protected]
Abbreviations: A3P5P, adenosine 3¢-phosphate 5¢-phosphate;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid;
CP/CPK, creatine phosphate/creatine kinase; HA, 1077
(1-(5-isoquinolinesulfonyl)homopiperazine); MLC, myosin light
chain; MLCK, myosin light chain kinase; PLC, phospholipase C;
W-7, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide.
(Received 3 January 2002, revised 8 October 2002,
accepted 11 October 2002)
Eur. J. Biochem. 269, 5878–5884 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03305.x
platelet shape change. In particular, the roles of Rho kinase
and of MLC kinase have been studied by means of specific
inhibitors in order to asses whether the Rho/Rho kinase
pathway is an additional route in mediating platelet shape
change in response to convulxin, independently of the
Ca
2+
-dependent pathway.
MATERIALS AND METHODS
Platelet preparation

containing 10 m
M
Hepes (pH 6.5) and resuspended in
Tyrode’s buffer containing 0.2% (w/v) albumin (bovine
serum fraction V-BSA), 0.1% (w/v) glucose and 10 m
M
Hepes (pH 7.35) (all from Sigma Chemicals).
To enhance cAMP levels, platelets were treated with
iloprost (3 l
M
for 2 min at 37 °C) (generous gift of
Italfarmaco S.p.a., Italy). This platelet preparation was
further incubated with the ADP scavenger system creatine
phosphate/creatine kinase (CP/CPK) (4 m
M
and
10 UÆmL
)1
, respectively, Sigma Chemicals) before the addi-
tion of the agonist. Samples were treated with N-(6-amino-
hexyl)-5-chloro-1-naphtalenesulfonamide (W-7) (90 l
M
)to
inhibit the Ca
2+
/calmodulin pathway [18,19] (Sigma Chem-
icals). HA 1077(1-(5-isoquinolinesulfonyl)homopiperazine)
(10 l
M
)[20,21](BioMolRes.Laboratory,Inc.,PA,USA)

millimetres per minute.
Changes in intracellular calcium mobilization
The fluorescent changes in intracellular calcium mobiliza-
tion were studied in Fura-2 (Molecular Probes, Eugene,
OR, USA) loaded platelets (3 l
M
at 37 °C for 30 min) and
monitored in a Kontron SFM 25 spectrofluorimeter
thermostatically regulated at 37 °C. Excitation and emission
wavelengths were 340 n
M
and 510 n
M
, respectively. Intra-
cellular free calcium concentration was calibrated according
to Grynkiewicz [22].
Preparation of BAPTA-loaded platelets
The concentrated platelets were incubated for 30 min at
37 °Cwith1m
M
aspirin, 100 l
M
1,2-bis(2-aminophen-
oxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester
(BAPTA-AM) (Molecular Probes), then washed by centri-
fugation. The platelet preparation was further treated with
CP/CPK (4 m
M
and 10 UÆmL
)1

M
urea, 20 m
M
Tris,
(pH 8.6), 122 m
M
glycine, 5 m
M
dithiothreitol to which
0.1% (w/v) bromophenol blue dye was added. The suspen-
ded pellet was sonicated in a PBI Briansonic 220 sonication
bath for 30 min. Gel electrophoresis was performed in a
10% (w/v) polyacrylamide-urea minigel apparatus (Hoefer
Scientific Instruments, San Francisco, CA, UA). The
running buffer used in the top chamber was composed of
20 m
M
Tris and 122 m
M
glycine at pH 8.6 containing 4 m
M
urea. The electrophoresis was stopped 1 h after the blue
marker had migrated off the end of the gel. Gels were
stained in 0.25% (w/v) Coomassie Brilliant Blue (Sigma),
destained and dried in an SE 1200 Easy Breeze Air Gel
Dryer (Hoefer) apparatus. The gels were then scanned using
a Gel Doc 2000 (Bio-Rad Laboratories, Hercules, CA,
USA) scanner and the optical densities corresponding to the
phosphorylated form of MLC were analysed using the
program

M
), was strongly inhibited in platelets
treated with the cAMP-enhancing drug iloprost (3 l
M
)
(Fig. 1).
Table 1 summarizes the variation in shape change
induced by 5 ngÆmL
)1
convulxin, indicating the magnitude
and slope of aspirinated platelets in the presence of Ca
2+
/
calmodulin or Rho-kinase inhibitors, that are also repre-
sentatively shown in Fig. 2. The results shown in Fig. 2A
demonstrate that when the two pathways were separately
inhibited by treating the platelets with either the calmodulin
inhibitor W-7 (90 l
M
) or with the Rho kinase inhibitors
HA 1077 (10 l
M
) or Y27632 (30 l
M
), we observed only a
slight reduction in the rate of shape change (more
pronounced when the Ca
2+
-dependent pathway was inhib-
ited) as compared to control, untreated, convulxin-stimula-

To avoid any possible interference with platelet shape
change caused by released ADP on its receptor P2Y
1
,we
incubated our CP/CPK-treated platelet preparation with
Fig. 1. Changes in intracellular calcium concentration in 5 ngÆmL
)1
convulxin-stimulated aspirinated platelets. Stimulation in the (a)
absence and (b) presence of 3 l
M
iloprost. The figure is representative
of three experiments performed.
Table 1. Effects of different inhibitory treatments on platelet shape change in response to 5 ngÆmL
)1
convulxin stimulation.
Treatment Magnitude Slope
None 14.07 ± 3.34 6.74 ± 2.11
+ HA 1077 (10 l
M
) 13.36 ± 2.87 5.59 ± 2.81
+ Y-27632 (30 l
M
) 13.96 ± 2.31 6.09 ± 2.77
+ W-7 (90 l
M
) 5.87 ± 3.34* 3.38 ± 1.22**
+ W-7 (30 l
M
) + HA 1077 (5 l
M

+ W-7 (90 l
M
) 4.24 ± 1.18** 2.83 ± 1.75**
*P < 0.05; **P < 0.01.
5880 S. Riondino et al.(Eur. J. Biochem. 269) Ó FEBS 2002
the selective P2Y
1
inhibitor adenosine 3¢-phosphate
5¢-phosphate (A3P5P) (100 l
M
for 1 min at 37 °C) and
obtained the same results as those observed after CP/CPK
treatment alone.
This result suggests that the ADP secreted is promptly
scavenged by CP/CPK and that the residual ADP is at too
low a concentration to activate the P2Y
1
-receptor coupled
pathway.
Because MLC phosphorylation has been suggested to be
involved in early processes during platelet activation [24] we
measured the appearance of a 20-kDa band, indicating
MLC phosphorylation consequent to the activation of the
two pathways, Ca
2+
-dependent and Ca
2+
-independent,
and determined the action exerted by the different inhibi-
tors. Rho kinase inhibition by HA 1077 or Y27632 did not

2+
-independent
pathways were inhibited by the combined treatment with
HA 1077 or Y27632 and W-7 or BAPTA, we did not
observe any phosphorylation of MLC.
DISCUSSION
The initial functional response of platelets to stimuli is
represented by shape change, during which phase platelets
are subject to cytoskeletal reorganization consequent to the
phosphorylation of several contractile proteins. It has
already been demonstrated that such a phenomenon can
occur both in the presence and in the absence of an increase
of cytosolic Ca
2+
concentration in response to thrombin,
ionomycin, the stable prostaglandin endoperoxide analogue
U44069 and the thrombin receptor activating peptide
[25–27]. Other authors [4] observed that the thromboxane
A2 synthetic analogue, U46619, was not able to induce a
detectable elevation of intracellular Ca
2+
concentration in
the absence of G
aq
and that Rho kinase inhibitor Y-27632
and C3 exoenzyme inhibited the U46619-induced MLC
phosphorylation in G
aq
-deficient mouse platelets. These
observations led them to conclude that a Rho/Rho kinase

37 °C(B)ortreatedwith3l
M
iloprost for
2minat37°C (C). The figure is
representative of four experiments
performed.
Ó FEBS 2002 Convulxin-induced platelet shape change (Eur. J. Biochem. 269) 5881
substratum in many cell types [9,29]. Protein kinase A, once
activated, phosphorylates myosin light chain kinase
(MLCK), thus inhibiting its activity and causing a decrease
in MLC phosphorylation [8].
As regards the inhibition exerted by cAMP on the
biochemical pathway leading to platelet shape change
following convulxin activation, the results shown in this
paper demonstrate that it is possible to evoke platelet shape
change even in the presence of elevated intracellular
concentrations of cAMP. This observation leads us to
hypothesize that the inhibitory action of cAMP is not
exerted at the level of Rho kinase pathway.
The conclusion that shape change responses were not due
to ADP release arises from two observations. First, the
degranulation process was dramatically reduced in convul-
xin-stimulated platelets treated with iloprost. This showed a
reduction of about 86% of the intracellular ATP as
compared to convulxin-stimulated platelets untreated with
iloprost, as already demonstrated in a previous study from
our group [12]. Second, in all preparations platelets were
treated with the ADP scavenger system CP/CPK to ensure
that even traces of ADP released could not interfere with the
experiments.

2+
-dependent pathway at different
stages by means of the Ca
2+
/calmodulin inhibitor, W-7, or
with the cytosolic Ca
2+
chelator BAPTA, we observed a
good degree of platelet shape change in response to
convulxin. This indicates that the inhibition exerted by
these inhibitors on the Ca
2+
-dependent pathway did not
affect the Rho kinase-dependent one.
In fact, while the inhibition of Rho kinase by means of the
inhibitors HA 1077 or Y-27632 was without any effect on
platelet shape change and MLC phosphorylation induced
by convulxin, a complete inhibition of platelet shape change
was achieved only when both pathways were concomitantly
inhibited, combining HA 1077 or Y-27632 treatment with
iloprost, W-7 or BAPTA treatment. These results suggest
that both pathways are involved in platelet shape change
induced by convulxin and that, since Rho kinase inhibitors
are less powerful in interfering with such a process, the
Ca
2+
-dependent one is more effective.
The fact that BAPTA treatment reduced convulxin-
induced MLC phosphorylation, while the rate of inhibition
of platelet shape change was similar to that observed after

was subtracted and the results expressed as percent of the maximal
MLC phosphorylation induced by convulxin alone (78.6 ± 11.4 mean
grey value) (A).
5882 S. Riondino et al.(Eur. J. Biochem. 269) Ó FEBS 2002
and platelet shape change, one mediated by Rho kinase
and the other by the Ca
2+
-dependent activation of MLC
kinase. These two pathways could be activated independ-
ently of each other, depending on the type and concen-
tration of the agonist used. The Rho kinase-dependent
pathway was stimulated by the activation of thrombin
and thromboxane receptors as opposed to the Ca
2+
-/
MLC kinase-dependent pathway that was stimulated by
ADP and collagen receptor activation. Like collagen,
convulxin has also been described as a GpVI agonist [13].
The different behaviour of this agonist that emerged in
our study might be explained by the fact that it is more
efficacious than collagen-related peptide [15].
Taken together our findings suggest that convulxin is able
to cause shape change by activating both pathways, the
Ca
2+
-dependent and Rho/Rho kinase-dependent. The
former appears to be more important, while the latter
seems to represent an additional route in mediating platelet
shape change and becomes preferentially involved in
response to stimuli that do not produce any increase in

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