Convulxin induces platelet shape change through myosin light chain kinase and Rho kinase Silvia Riondino, Pier P.. Pulcinelli Department of Experimental Medicine and Pathology, Universit
Trang 1Convulxin 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 Ca2+
/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 inhibiphosphoryla-tion of the Ca2+
-dependent pathway by means of the Ca2+chelator BAPTA,
the Ca2+/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
Ca2+/calmodulin-dependent and Rho kinase-dependent
pathways were concomitantly inhibited by the combined use
of Y27632 plus BAPTA, W-7 or iloprost, platelet shape change was completely abolished Our findings suggest that convulxin-induced platelet shape change occurs via both pathways, the Ca2+/calmodulin-dependent, which appears
to be more important, and the Rho kinase-dependent one The pattern of MLC phosphorylation was not modified by Rho kinase inhibitors Conversely, the inhibition of the
Ca2+-dependent pathway caused a strong reduction of MLC phosphorylation in BAPTA-treated platelets, and a total inhibition in W-7 or iloprost-treated platelets Our results demonstrate that following Rho kinase-dependent pathway platelet shape change can occur without the involvement of MLC phosphorylation
Keywords: convulxin; cyclic AMP; myosin light chain kin-ase; platelet shape change; Rho kinase
One of the first events to follow platelet functional response
to various stimuli is the rearrangement of cytoskeletal
proteins, such as actin and myosin, with the consequent
change from the discoid to the spheroid shape and the
production of pseudopodia [1] It has been demonstrated
that a crucial event in triggering shape change is myosin
light chain (MLC) phosphorylation [2] The signal
origin-ating from agonist-induced activation can lead to MLC
phosphorylation through two distinct pathways One is the
Ca2+/calmodulin-dependent pathway, subsequent to Ca2+
mobilization and mediated by MLC kinase [3], and the
other is Ca2+-independent, subsequent to the small
GTP-binding protein RhoA activation, and mediated by Rho
kinase [4–6] The Ca2+/calmodulin-dependent pathway
which leads to MLC phosphorylation depends upon the
stimulation of a Gq-coupled receptor and phospholipase C
(PLC) activation This datum has also been supported by
experiments on Gaq-deficient mice platelets [7] On the other
hand, the Ca2+-independent pathway follows the stimula-tion of a G12/G13coupled 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 decreased phosphorylation of various proteins [10,11] including MLC [12]
In a previous study of our group, it was found that it was possible to by-pass the inhibitory effect of cAMP on the multistep cascade of biochemical and morphological events that result in platelet aggregation, provided a Gq-coupled receptor and a Gi-coupled receptor were activated [12] In the same study it was demonstrated that the activation of PLC was sufficient to elicit full aggregation, in spite of elevated intracellular concentrations of cAMP, in the presence of an activated Giprotein
The activation of a PLC isoform, PLC c2, might be achieved using the snake venom toxin convulxin Convulxin activates platelets through the collagen receptor complex glycoprotein VI (GpVI)/Fc receptor c-chain, leading to tyrosine phosphorylation and activation of syk and PLCc2 [13,14] Following activation of GpVI by convulxin, several adapter molecules are tyrosine phosphorylated and/or recruited into intracellular signalling complexes; many of the adapter proteins associate with the transmembrane adapter LAT [15]
Francischetti and his coworkers demonstrated that cAMP did not inhibit the early onset of convulxin-induced tyrosine-phosphorylation of PLCc2, which also occurs in 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: pierpaolo.gazzaniga@uniroma1.it
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)
Trang 2platelet 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
Ca2+-dependent pathway
M A T E R I A L S A N D M E T H O D S
Platelet preparation
Blood samples were obtained from informed healthy
volunteers who denied having taken any drugs in the two
weeks before blood sampling and collected in citric acid/
citrate/dextrose-containing tubes [17]
Platelet rich plasma was obtained after centrifugation
(180 g for 15 min) and further centrifuged (800 g for
20 min) to concentrate the platelets (6· 108plateletsÆmL)1)
The concentrated platelets were incubated for 15 min at
37C with 1 mMaspirin (Sigma Chemicals Co., St Louis,
MO, USA) then washed twice in Tyrode’s buffer (137 mM
NaCl, 2.68 mMKCl, 0.42 mMNaH2PO4, 1.7 mMMgCl2)
containing 10 mM 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 mM
Hepes (pH 7.35) (all from Sigma Chemicals)
To enhance cAMP levels, platelets were treated with
iloprost (3 lM for 2 min at 37C) (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 mM 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 lM) to
inhibit the Ca2+/calmodulin pathway [18,19] (Sigma
Chem-icals) HA 1077(1-(5-isoquinolinesulfonyl)homopiperazine)
(10 lM) [20,21] (BioMol R es Laboratory, Inc., PA, USA)
and Y27632 (10–30 lM) (Calbiochem-Novabiochem San
Diego, CA, USA) were used as Rho-kinase inhibitors
Platelet stimulation was achieved by means of convulxin
(5 ngÆmL)1) (Latoxan, Rosans, France), purified according
to Polgar et al [13]
Platelet shape change
In vitro platelet shape change was evaluated in a four
sample PACKS-4 (Helena Laboratories, Beaumont, TX,
USA) aggregometer using siliconized glass cuvettes at 37C
under continuous stirring Once the full function of the
platelet preparation was tested in response to convulxin
(5 ngÆmL)1), in order to prevent platelet aggregation and
allow shape change to occur, platelets were pretreated with
RGDS (120 lgÆmL)1) which inhibits fibrinogen binding to
the integrin aIIbb3 The magnitude of the shape change was
detected by measuring the maximum decrease in light
transmission and the slope value The last parameter was
obtained from the tangent to the curve and was measured in
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 lMat 37C for 30 min) and monitored in a Kontron SFM 25 spectrofluorimeter thermostatically regulated at 37C Excitation and emission wavelengths were 340 nM and 510 nM, 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
37C with 1 mM aspirin, 100 lM 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 mM and 10 UÆmL)1, respectively) before the addition of the agonist If the DnM of Ca2+obtained in response to agonist stimulation was higher than 10, the platelet suspension was not used
Myosin light chain phosphorylation MLC phosphorylation was analysed according to Daniel
et al [23] Briefly, aspirinated platelets were resuspended in Tyrode’s buffer at a concentration of 2· 109 cellsÆmL)1 Aliquots of 500 lL were stirred at 37C in an aggrego-meter After 30 s from convulxin stimulation (5 ngÆmL)1) the reaction was stopped by adding 6.6MHClO4; the resulting precipitate was kept for 45 min in ice The pellets were centrifuged at 10 000 g for 2 min and washed twice with ice-cold deionized water The proteins were further centrifuged
at 10 000 g for 2 min and the resulting pellets were dissolved
in 50 lL of sample buffer containing 8Murea, 20 mMTris, (pH 8.6), 122 mM glycine, 5 mM 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 mMTris and 122 mMglycine at pH 8.6 containing 4 mM 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 programNIH IMAGE1.62 and reported as mean grey values The results were expressed as the percentage of maximal MLC phosphorylation induced by convulxin
To confirm the identity of the detected band, further sample proteins were transferred to Immobilion-P (Milli-pore) membranes and identified with anti-MLC mono-clonal Ig (Sigma Chemicals), followed by horseradish peroxidase-conjugated secondary antibody and visualized with ECL chemiluminescence reaction reagent (Amersham) and Kodak X-ray film (X-OMAT AR)
R E S U L T S
To investigate the correlation between the increase in intracellular Ca2+ concentration and shape change in
Trang 3convulxin-induced platelet activation, we studied platelet
shape change and MLC phosphorylation in experimental
conditions in which either Ca2+/calmodulin or Rho-kinase
pathways were inhibited
The increase in the cytosolic Ca2+concentration, which
occurs normally after convulxin (5 ngÆmL)1) stimulation
(743.6 ± 192.2 DnM), was strongly inhibited in platelets
treated with the cAMP-enhancing drug iloprost (3 lM)
(Fig 1)
Table 1 summarizes the variation in shape change
induced by 5 ngÆmL)1convulxin, indicating the magnitude
and slope of aspirinated platelets in the presence of Ca2+/
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 lM) or with the Rho kinase inhibitors
HA 1077 (10 lM) or Y27632 (30 lM), we observed only a
slight reduction in the rate of shape change (more
pronounced when the Ca2+-dependent pathway was
inhib-ited) as compared to control, untreated,
convulxin-stimula-ted platelets Then, in order to minimize the cytosolic
concentration of calcium, we treated our platelet
prepar-ation with 5,5¢-dimethyl BAPTA (Fig 2B), and studied
both the magnitude and the slope of the shape change
Under these conditions we found that both were slightly
decreased, with a delay in the initiation of the process A
more pronounced reduction was observed in the presence of
high concentrations of iloprost (3 lM) (Fig 2C) A similar
degree of reduction was observed after the simultaneous
treatment with calmodulin inhibitors and iloprost,
suggest-ing that these agents were actsuggest-ing on a common pathway
However, when iloprost was added to 5,5¢-dimethyl
BAPTA-loaded platelets, both the extent and the rate of
shape change were significantly inhibited (Fig 2C)
A complete inhibition was obtained when both pathways,
Ca2+-dependent and Rho-dependent, were simultaneously
blocked by adding the calmodulin inhibitor W-7 or iloprost
or when the increase in cytosolic Ca2+concentration was
minimized by BAPTA, to HA 1077- or Y27632-treated
platelets
Interestingly, a complete inhibition of platelet shape change was achieved by notably lowering the concentrations
of HA 1077 or Y-27632 when used in combination with W-7 (Table 1)
To avoid any possible interference with platelet shape change caused by released ADP on its receptor P2Y1, 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)1convulxin stimulation.
*P < 0.05; **P < 0.01.
Trang 4the selective P2Y1 inhibitor adenosine 3¢-phosphate
5¢-phosphate (A3P5P) (100 lM for 1 min at 37C) 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 P2Y1-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, Ca2+-dependent and Ca2+-independent,
and determined the action exerted by the different
inhibi-tors Rho kinase inhibition by HA 1077 or Y27632 did not
significantly modify the pattern of MLC phosphorylation
after convulxin stimulation (63.0 ± 10.9 for HA 1077 and
66.33 ± 12.6 for Y-27632 vs 78.6 ± 11.4 mean grey
value), equal to 19.8% and 15% reduction, respectively
(Fig 3A)
The identity of the 20 kDa band detected was confirmed
by immunoblot analysis by means of an anti-MLC
mono-clonal Ig (data not shown)
Inhibition of the increase in cytosolic Ca2+by BAPTA
(Fig 3B) reduced by about 40% the maximum level of
MLC phosphorylation (45.7 ± 9.6 mean grey value vs
78.6 ± 11.4) induced by convulxin It is worth noting that,
since BAPTA treatment might delay the time course of
MLC phosphorylation [5], we performed experiments at 60,
120, 150 and 180 s, but we did not observe any further MLC
phosphorylation (data not shown) Inhibition of calmodulin
by W-7 (90 lM) completely abolished MLC
phosphoryla-tion (Fig 3A), while a certain degree of platelet shape
change was still preserved although significantly reduced
(Table 1)
The increase in intracellular cAMP levels by iloprost completely inhibited MLC phosphorylation (4.2 ± 0.7 mean grey value) (Fig 3C)
When both the Ca2+-dependent and Ca2+-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
D I S C U S S I O N 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 Ca2+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 Ca2+concentration in the absence of Gaqand that Rho kinase inhibitor Y-27632 and C3 exoenzyme inhibited the U46619-induced MLC phosphorylation in Gaq-deficient mouse platelets These observations led them to conclude that a Rho/Rho kinase pathway regulating MLC phosphorylation operated in platelets and that Rho kinase mediates the Ca2+ -independ-ent pathway The dichotomous regulation of MLC phos-phorylation and shape change by Rho kinase and calcium in human platelets was demonstrated by other studies carried out with several agonists, different from convulxin [5,6,28] Elevated cAMP and the consequent activation of protein kinase A affects cell morphology, inducing the loss of actin stress fibres and their detachment from the underlying
Fig 2 Platelet shape change in response to
5 ngÆmL)1convulxin (A) Shape change in the
presence of the calmodulin inhibitor W-7
(90 l M ) or Rho kinase inhibitors HA 1077
(10 l M ) and Y-27632 (30 l M ) in aspirinated,
RGDS-treated, control platelets The
response to convulxin stimulation in the
presence of the cited inhibitors of platelets
loaded with 100 l M BAPTA for 30 min at
37 C (B) or treated with 3 l M iloprost for
2 min at 37 C (C) The figure is
representative of four experiments
performed.
Trang 5substratum 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
Furthermore, experiments employing the inhibitor of the ADP receptor P2Y1(the only receptor involved in platelet shape change induced by ADP [28,30]), A3P5P, demon-strated no additional inhibition as compared to CP/CPK alone (data not shown)
Iloprost treatment strongly affected convulxin-induced MLC phosphorylation, only partially acting on the Ca2+ -dependent pathway which, in fact, was inhibited as dem-onstrated by fluorimetric studies on intracellular Ca2+ mobilization This result also demonstrates that in response
to convulxin Ca2+ mobilization can be inhibited by elevating the levels of intracellular cAMP, as shown in studies using different agonists [31] However, the inhibition
of Ca2+ mobilization alone is not sufficient to cause a complete inhibition of MLC phosphorylation, as demon-strated by the experiments with BAPTA, which only diminished MLC phosphorylation by about 50% The enhanced capability of cAMP of inhibiting MLC phos-phorylation can be explained by the direct action of cAMP
on MLCK [8]
Antagonizing the Ca2+-dependent pathway at different stages by means of the Ca2+/calmodulin inhibitor, W-7, or with the cytosolic Ca2+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 Ca2+-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
Ca2+-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 W-7 or iloprost treatment, might be explained by the demonstration that a weak activation of MLC phosphory-lation is not sufficient to elicit shape change [6]
Bauer and coworkers [5] suggested that the GpVI-specific agonist, collagen-related peptide, causes shape change by a mechanism that is entirely dependent on calcium, since it is not affected by the Rho kinase inhibitor Y-27632 Their conclusion delineated the exist-ence of two distinct pathways of myosin phosphorylation
Fig 3 Myosin light chain phosphorylation in response to
aspirin-trea-ted, convulxin stimulated (5 ng mL)1, for 30 s at 37 °C) platelets (A)
Phosphorylation in the presence of calmodulin inhibitor W-7 (90 l M )
or Rho kinase inhibitors HA 1077 (10 l M ) and Y-27632 (30 l M ) The
effects of the same inhibitors on (B) BAPTA-treated (100 l M for
30 min at 37 C) or (C) Iloprost-treated (3 l M for 2 min at 37 C)
platelets The quantitative comparison of the effects of the cited
inhibitors is reported Results are shown as mean ± SEM of four
experiments The level of MLC phosphorylation of resting platelets
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).
Trang 6and platelet shape change, one mediated by Rho kinase
and the other by the Ca2+-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 Ca2+-/
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
Ca2+-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
cytosolic Ca2+ concentration, or when the Ca2+
/calmo-dulin-dependent pathway is not operating (Fig 4)
More-over, our results demonstrate that following Rho A/Rho
kinase activation, convulxin-induced platelet shape change can occur without the involvement of MLC phosphoryla-tion
A C K N O W L E D G E M E N T S
This work was partially supported by Grant 60% Ateneo 2000.
We thank Prof Luisa Lenti for her precious help in convulxin purification.
R E F E R E N C E S
1 Siess, W (1989) Molecular mechanisms of platelet activation Physiol Rev 69, 58–178.
2 Daniel, J.L., Molish, I.R., Rigmaiden, M & Stewart, G (1984) Evidence for a role of myosin phosphorylation in the initiation of the platelet shape change response J Biol Chem 259, 9826–9831.
3 Hathaway, D.R., Eaton, C.R & Adelstein, R.S (1981) Regula-tion of human platelet myosin light chain kinase by the catalytic subunit of cyclic AMP-dependent protein kinase Nature 291, 252– 256.
4 Klages, B., Brandt, U., Simon, M.I., Schultz, G & Offermanns, S (1999) Activation of G12/G13 results in shape change and Rho/ Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets J Cell Biol 144, 745–754.
5 Bauer, M., R etzer, M., Wilde, J.L., Maschberger, P., Essler, M., Aepfelbacher, M., Watson, S.P & Siess, W (1999) Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets Blood 94, 1665–1672.
6 Paul, B.Z., Daniel, J.L & Kunapuli, S.P (1999) Platelet shape change is mediated by both calcium-dependent and -independent signaling pathways Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change J Biol Chem.
274, 28293–28300.
7 Offermanns, S., Toombs, C.F., Hu, Y.H & Simon, M.I (1997) Defective platelet activation in Ga(q)-deficient mice Nature 389, 183–186.
8 Lamb, N.J., Fernandez, A., Conti, M.A., Adelstein, R., Glass, D.B., Welch, W.J & Feramisco, J.R (1988) Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase.
J Cell Biol 106, 1955–1971.
9 Glass, W.F & Kreisberg, J.I (1993) Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP J Cell Physiol 157, 296–306.
10 Han, J.D & Rubin, C.S (1996) Regulation of cytoskeleton organization and paxillin dephosphorylation by cAMP Studies
on murine Y1 adrenal cells J Biol Chem 271, 29211–29215.
11 Troyer, D.A., Bouton, A., Bedolla, R & Padilla, R (1996) Tyr-osine phosphorylation of focal adhesion kinase (p125FAK): reg-ulation by cAMP and thrombin in mesangial cells J Am Soc Nephrol 7, 415–423.
12 Riondino, S., Gazzaniga, P.P & Pulcinelli, F.M (2001) Evidence for platelet aIIbb3 activation despite elevated cytosolic cAMP Thromb Haemost 85, 320–325.
13 Polgar, J., Clemetson, J.M., Keherel, B.E., Wiedemann, M., Magnenat, E.M., Wells, T.N.C & Clemetson, K.J (1997) Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor J Biol Chem 272, 13576–13583.
14 Faili, A., Randon, J., Francischetti, I.M., Vargaftig, B.B & Hatmi, M (1994) Convulxin-induced platelet aggregation is accompanied by a powerful activation of the phospholipase
C pathway Biochem J 298, 87–91.
15 Asazuma, N., Wilde, J.W., Berlanga, O., Leduc, M., Leo, A., Schweighoffer, E., Tybulewicz, V., Bon, C., Liu, S.K., McGlade, J., Schraven, B & Watson, S.P (2000) Interaction of linker for
Fig 4 Hypothetical model depicting the biochemical pathways leading
to platelet shape change following convulxin activation The double bars
indicate the suggested sites of action of the inhibitors employed in our
study.
Trang 7activation of T cells with multiple adapter proteins in platelets
activated by the glycoprotein VI-selective ligand, convulxin J Bio.
Chem 275, 33427–33434.
16 Francischetti, I.M.B., Carlini, R & Guimaraes, J.A (1998) cAMP
does not inhibit convulxin-induced tyrosyl-phosphorylation of
human platelet proteins, including PLCc2, but completely blocks
the integrin aIIbb3-dependent dephosphorylation step:
compari-sons with RGDS peptide, cytochalasin D, and phenylarsine oxide.
Arch Biochem Biophys 354, 255–269.
17 Aster, R.H & Jandel, J.H (1964) Platelet sequestration in man: I:
methods J Clin Invest 43, 834–855.
18 Levin, R.M & Weiss, B (1979) Selective binding of antipsychotics
and other psychoactive agents to the calcium-dependent activator
of cyclic nucleotide phosphodiesterase J Pharmacol Exp Ther.
208, 454–459.
19 Hidaka, H., Asano, M & Tanaka, T (1981) Activity-structure
relationship of calmodulin antagonists, Naphthalenesulfonamide
derivatives Mol Pharmacol 20, 571–578.
20 Takayasu, M., Suzuki, Y., Shibuya, M., Asano, T., Kanamori,
M., Okada, T., Kageyama, N & Hidaka, H (1986) The effects of
HA compound calcium antagonists on delayed cerebral
vaso-spasm in dogs J Neurosurg 65, 80–85.
21 Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T.,
Morishita, T., Tamakawa, H., Yamagami, K., Inui, J., Maekawa,
M & Narumiya, S (1997) Calcium sensitization of smooth muscle
mediated by a Rho-associated protein kinase in hypertension.
Nature 389, 990–994.
22 Grynkiewicz, G., Poenie, M & Tsien, R.Y (1985) A new
gen-eration of Ca 2+ indicators with greatly improved fluorescence
properties J Biol Chem 260, 3440–3450.
23 Daniel, J.L & Sellers, J.R (1992) Purification and characteriza-tion of platelet myosin Methods Enzymol 215, 78–88.
24 Daniel, J.L., Molish, I.R & Holmsen, H (1981) Myosin phos-phorylation in intact platelets J Biol Chem 256, 7510–7514.
25 Rink, T.J., Smith, S.W & Tsien, R.Y (1982) Cytoplasmic free Ca2+ in human platelets Ca2+thresholds and Ca-independent activation for shape-change and secretion FEBS Lett 148, 21–26.
26 Simpson, A.W., Hallam, T.J & Rink, T.J (1986) Low con-centrations of the stable prostaglandin endoperoxide U44069 sti-mulate shape change in quin2-loaded platelets without a measurable increase in [Ca 2+ ]i FEBS Lett 201, 301–305.
27 Negrescu, E.V., de Quintana, K.L & Siess, W (1995) Platelet shape change induced by thrombin receptor activation Rapid stimulation of tyrosine phosphorylation of novel protein sub-strates through an integrin- and Ca (2+) -independent mecha-nism J Biol Chem 270, 1057–1061.
28 Wilde, J.I., Retzer, M., Siess, W & Watson, S.P (2000) ADP-induced platelet shape change An investigation of the signalling pathways involved and their dependence on the method of platelet preparation Platelets 11, 286–295.
29 Lampugnani, M.G., Giorgi, M., Gaboli, M., Dejana, E & Marchisio, P.C (1990) Endothelial cell motility, integrin receptor clustering, and microfilament organization are inhibited by agents that increase intracellular cAMP Laboratory Invest 63, 521–531.
30 Gachet, C (2001) ADP receptors of platelets and their inhibition Thromb Haemost 86, 222–232.
31 Quinton, T.M & Dean, W.L (1992) Cyclic AMP-dependent phosphorylation of the inositol-1,4,5-trisphosphate receptor inhibits Ca 2+ release from platelet membranes Biochem Biophys Res Commun 184, 893–899.