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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the 2nd and 1st Departments of Internal Medicine,
Mie University School of Medicine, Mie, Japan.
Protein kinase C (PKC)-potentiated inhibitory phosphoprotein of
myosin phosphatase (CPI) was detected in human platelets. Like smooth
muscle CPI-17, in vitro phosphorylation of platelet CPI by PKC
inhibited the activity of myosin phosphatase containing the PP1 Phosphorylation of the 20-kd myosin light chain
(MLC20) at Ser-19 is thought to be one of the primary steps
in the activation of actomyosin contractile events, which involve
platelet shape change and secretion.1-7 The extent of
MLC20 phosphorylation is regulated not only by protein
kinases, such as Ca++-dependent MLC kinase8
and Ca++-independent Rho-kinase,9 but also by
myosin phosphatase.10,11 Platelet shape change, the
earliest response induced by agonists, is a prerequisite for full
platelet activation, including secretion and
aggregation.1,2 Platelet shape change is regulated by MLC20 phosphorylation either through RhoA/Rho-kinase
activation or through Ca++-dependent MLC kinase
activation.4 Activation of protein kinase C (PKC) is not
likely to be involved in platelet shape change.1 Inhibition of myosin phosphatase activity appears to be an important mechanism for increasing the Ca++ sensitivity of
MLC20 phosphorylation and platelet secretion, as is the
case for smooth muscle.10,11 Myosin phosphatase is composed of 3 subunits Another possible candidate for the inhibition of myosin phosphatase in
platelets is PKC, an event that may be independent of the
RhoA/Rho-kinase pathway. In permeabilized platelets, GTP Materials
Measurement of PKC-potentiated inhibitory phosphoprotein of
myosin phosphatase in human platelets
Immunoprecipitation of platelet CPI Platelet suspension (109/mL) was dissolved in ice-cold lysis buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 2 mM EDTA, 2 mM sodium vanadate, and an appropriate amount of protease inhibitor cocktail tablets [Boehringer Mannheim GmbH]). Immunoprecipitation of platelet lysates was performed after clarification of the samples by centrifugation at 15 000g for 10 minutes, and the soluble fraction, precleared with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech AB), was incubated with 30 µL anti-CPI-17 antibody at 4°C for 1 hour.27,28 The immune complex was precipitated by adding protein A-Sepharose CL-4B (Amersham Pharmacia Biotech AB), incubating it for an additional 1 hour at 4°C, and washing the beads 3 times with lysis buffer. These preparations were used to detect the in vitro phosphorylation of CPI and for assay of the phosphatase activity.In vitro CPI phosphorylation by PKC and its effect on myosin phosphatase activity Phosphorylation of the anti-CPI-17 immunoprecipitate was carried out using purified platelet PKC in the presence of 50 µM ATP with 10 mM MgCl2, 0.5 mM CaCl2, 50 µg/mL phosphatidylserine, 12.5 µg/mL diolein, 0.5 µM okadaic acid (Wako Pure Chemicals, Osaka, Japan), and 20 mM Tris-HCl, pH 7.5, at 30°C for 2 minutes.32 The kinase reaction was initiated by adding ATP and terminated by adding sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Samples were subjected to SDS-PAGE, and CPI phosphorylation was analyzed by immunoblotting, using an anti-pThr38-CPI-17 antibody.To analyze phosphatase activities, thiophosphorylation of the
anti-CPI-17 immunoprecipitate was carried out using platelet PKC in
the presence of 1 mM ATP Detection of agonist-induced phosphorylation of MLC20 at Ser-19, MBS at Thr-696, and CPI in human platelets Venous blood was freshly drawn from a healthy donor who had not taken any drugs for at least 2 weeks. Human platelets (109/mL) were resuspended in Ca++-free Tyrode-HEPES buffer that contained a final concentration of 0.14 M NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1% D-glucose, 3.75 mM NaH2PO4, and 15 mM HEPES, pH 7.4. Washed platelets (300 µL) were stimulated with various agonists, without stirring, at 37°C in an PA-200 aggregometer (Kowa, Tokyo, Japan), as described.5,29 Precipitates treated with 10% trichloroacetic acid were subjected to SDS-PAGE for determination of the extent of MLC20, MBS, and CPI phosphorylation. Quantitative estimation of the level of phosphorylation was made densitometrically by scanning the immunoreactive bands after immediately photographing the visualized band. The area of an individual peak was measured above the background of the densitometric tracing and was estimated as an arbitrary unit.Measurement of ATP secretion and cytosolic Ca++ concentration in platelets. ATP secretion was measured, using a Lumi-aggregometer (Chrono-Log, Havertown, PA), as described.5 The platelet reaction mixture consisted of 400µL washed platelet suspension (5 × 109/mL) in Ca++-free HEPES-Tyrode buffer, 50 µL Chrono-Lumi luciferase luciferin reagent, 50µL inhibitor (or vehicle), and STA2 or PMA. Washed platelet suspensions pretreated with or without the inhibitor at 37°C for 3 minutes were activated by 0.5 µM STA2 or 100 nM PMA for 3 minutes, under conditions of nonstirring. For measurement of cytosolic Ca++ concentrations, platelets loaded with fura 2-AM (1 µM) were resuspended in Ca++-free HEPES-saline buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 5 mM glucose).36 Fluorescence and ATP secretion were monitored simultaneously, in parallel samples. Fluorescence of fura 2 was measured at 340- and 380-nm excitation and at 550-nm emission using a Hitachi F-4000 fluorescence spectrometer, according to the method of Tsien et al.37 Two-milliliter aliquots of fura 2-loaded platelet suspensions (1 × 108/mL) were transferred to quartz cuvettes, maintained at 37°C in a circulating water bath, continually stirred with a small magnetic stirrer for 2 minutes, and stimulated with STA2 or PMA under conditions of non-stirring. In all experiments, the fluorescence of untreated and unloaded cells was used for subtraction of the autofluorescence. To minimize the time-dependent effects on platelet responsiveness and leakage of fura 2, experiments were designed to be completed within 1 hour.Statistical analysis Data were analyzed using the StatView statistical software package (Version 5; SAS Institute, Cary, NC). Data are shown as mean ± SE and were compared statistically by Student t test (unpaired). P < .05 was considered significant.
Detection and level of CPI, PKC-potentiated inhibitory phosphoprotein of myosin phosphatase, in human platelets Recent reports have suggested that CPI-17 is almost exclusively expressed in smooth muscle tissues.19-21 We determined whether CPI is present in human platelets by immunoblot analysis using an anti-CPI-17 antibody.30 As shown in Figure 1A, the anti-CPI-17 antibody detected a major immunoreactive band with an apparent molecular mass of 22 kd, higher than that in smooth muscle CPI-17 with an apparent molecular mass of 20 kd in SDS-PAGE.19 To determine whether the immunoreactive protein at 22 kd was the CPI present in human platelets, we analyzed immunoprecipitates with anti-CPI antibody using platelet extracts (Figure 1B). The procedure was to subject the protein A-Sepharose precipitates to SDS-PAGE and follow that by immunoblotting with polyclonal antibodies against the PP1 catalytic subunit and MBS
of myosin phosphatase. PP1 isoform and MBS of myosin phosphatase
were detected at 38 kd and 130 kd, respectively, in the anti-CPI-17
immunoprecipitate of human platelets (Figure 1B). We further examined
phosphatase activity of the immunoprecipitates using
[ -32P]-phosphorylated MLC as a substrate.
Approximately 85% of the total phosphatase activity in the precipitate
was attributed to PP1 activity (Figure 1C). We then asked whether the
22-kd immunoreactive band was phosphorylated by PKC, using the
anti-CPI-17 immunoprecipitate. CPI phosphorylation was analyzed by
immunoblot using an antibody that specifically recognizes CPI-17
phosphorylated at Thr-38 (anti-pThr38-CPI-17). As shown in
Figure 1D, CPI phosphorylation at a Thr residue corresponding to Thr-38
in CPI-17 was increased approximately 4-fold with the addition of PKC,
and this phosphorylation was inhibited by a non-isoform-selective PKC
inhibitor, GF109203X.38 We then investigated the effect of
CPI phosphorylation on myosin phosphatase activity in the
immunoprecipitates. Because thiophosphorylation of proteins, unlike
regular phosphorylation, is highly resistant to protein
phosphatases,39 we used the thiophosphorylation form of
CPI to determine the effect on myosin phosphatase activity. Myosin
phosphatase activity was decreased by CPI thiophosphorylation and was
recovered by the inhibition of CPI thiophosphorylation with GF109203X
(Figure 1E). These results indicate that the immunoreactive protein at
22 kd is human platelet CPI.
We determined the amount of CPI in whole platelets by immunoblot analysis using purified CPI-17 from aorta smooth muscle as standard. The amount of CPI in platelets was 0.33 ± 0.04 µM (mean ± SE), which is almost the same as that in smooth muscle.21 Agonist-induced phosphorylation of CPI and MBS in intact platelets To determine whether the agonist-induced phosphorylation of CPI occurs in intact platelets, human platelets were stimulated with various agonists under conditions of nonstirring, and CPI phosphorylation was analyzed by immunoblot analysis using an anti-pThr38-CPI-17 antibody. In parallel, we also examined the phosphorylation of MLC20 at Ser-19 and MBS at Thr-696 by immunoblot analysis using the respective phosphospecific antibodies. When platelets were activated with agonists in the absence of stirring, they changed shape and secreted ATP from dense bodies, without aggregation.5,29 Figure 2 shows ATP secretion and cytosolic Ca++ elevation response to STA2 and PMA in a nominally Ca++-free medium and under conditions of nonstirring. STA2 (0.5 µM) treatment led to ATP secretion and a rapid increase in the cytosolic Ca++ level. This Ca++ increase reached a peak level within 15 to 20 seconds and was followed by a rapid decrease. As expected from the cytosolic Ca++ response to STA2, the maximum extent of MLC20 phosphorylation at Ser-19 was obtained within 20 seconds of STA2 (0.5 µM) stimulation, followed by dephosphorylation, as shown in Figure 3. CPI was rapidly phosphorylated and then dephosphorylated after STA2 stimulation, similar to the phosphorylation of MLC20 at Ser-19, whereas STA2-induced MBS phosphorylation was slightly delayed. Thus, treatment of intact platelets with STA2 led to a rapid and transient increase in phosphorylation of CPI at a Thr residue (corresponding to Thr-38 on CPI-17), in addition to MBS phosphorylation at Thr-696. To determine whether phosphorylation of CPI can be induced by agonists other than STA2 in platelets, CPI phosphorylation was examined after stimulation with thrombin, PMA, and ionomycin. As shown in Figure 4, treatment with 0.1 U/mL thrombin led to a rapid increase in phosphorylation of CPI at a Thr residue and of MBS at Thr-696. On the other hand, though the Ca++ ionophore ionomycin or PMA, which activates PKC, could induce the phosphorylation of CPI, these artificial agonists failed to increase MBS phosphorylation. Ionomycin-induced CPI phosphorylation was almost completely inhibited by 0.1 mg/mL aspirin, suggesting that most of the CPI phosphorylation resulted from the potential of ionomycin to promote cyclooxygenase products.40 It is now apparent that PMA and the Ca++ ionophore cannot activate the RhoA signaling pathway. Incubation of fura 2-loaded platelets with PMA led to ATP secretion, with no change in cytosolic Ca++ concentrations (Figure 2), as reported,41 and PMA (100 nM)-induced ATP release was slower and less than that induced by STA2 (0.5 µM). We then examined the time course of phosphorylation of CPI and MLC20 at Ser-19 during PMA-induced platelet activation. As shown in Figure 5, after stimulation with 100 nM PMA, CPI was rapidly phosphorylated, and this phosphorylation preceded MLC20 phosphorylation at Ser-19 and ATP secretion.
Inhibitory effects of GF109203X, Y-27632 alone and in combination on PMA- and STA2-induced phosphorylation of CPI, MBS, and MLC20 PKC cannot directly phosphorylate MLC20 at Ser-19, the MLC kinase site8,42,43; therefore, the phosphorylation of this site induced by PMA in intact platelets appeared to result from the inhibition of myosin phosphatase through phosphorylation of CPI at a Thr residue. In the next set of experiments, we determined the participation of PKC in PMA- or STA2-induced MLC20 phosphorylation of intact platelets, using a specific PKC inhibitor, GF109203X (Figure 6). As shown in Figure 6A, preincubation of platelets with GF109203X attenuated the PMA-induced phosphorylation of CPI and MLC20 at Ser-19, with similar IC50 values (1.52 ± 0.33 µM, n = 3, for CPI phosphorylation; 1.17 ± 0.21 µM, n = 3, for MLC20 phosphorylation, respectively). Inhibition of PMA-induced MLC20 phosphorylation by GF109203X correlated well with CPI phosphorylation. GF109203X at 2 µM completely abolished PMA-induced ATP secretion, as reported.38 The Rho-kinase inhibitor Y-2763244 diminished neither PMA-induced phosphorylation of MLC20 nor CPI (data not shown). GF109203X also dose dependently inhibited the STA2-induced phosphorylation of CPI and MLC20. However, GF109203X caused a nearly complete inhibition of STA2-induced CPI phosphorylation, whereas the drug did not inhibit STA2-induced MLC20 phosphorylation to the same extent seen with the inhibition of CPI phosphorylation (Figure 6B). GF109203X decreased MLC20 phosphorylation by approximately 60%, even at 2 µM, a concentration at which the drug abolished STA2-induced CPI phosphorylation. GF109203X pretreatment resulted in a marginal decrease in STA2-induced phosphorylation of MBS at Thr-696.
We reported that the Rho-kinase inhibitor Y-27632 could inhibit the
STA2-induced MBS phosphorylation dose dependently (1-20 µM) in intact platelets.27 Smooth muscle CPI-17 was
shown to be phosphorylated in a cell-free system not only by PKC but
also by the downstream kinases RhoA, Rho-kinase,30 and
protein kinase N.45 We also examined the effect of the
Y-27632 on the STA2-induced phosphorylation of CPI, MBS,
and MLC20 in intact platelets. As shown in Figure
7, Y-27632 almost completely inhibited
the STA2-induced phosphorylation of MBS at Thr-696, whereas
the drug had only a minor inhibitory effect on the
STA2-induced CPI phosphorylation, which suggests that
Rho-kinase preferentially phosphorylates MBS, compared to CPI, in
intact platelets. Y-27632 partly inhibited the STA2-induced
MLC20 phosphorylation at Ser-19 in intact platelets. As
shown in Figure 8, Y-27632 inhibited
STA2-induced MLC20 phosphorylation at Ser-19 to
approximately 40% of the level seen without Y-27632, an inhibition
likely because of the inhibition of MBS phosphorylation, though slight
inhibition of CPI phosphorylation was evident at 20 µM Y-27632. The
addition of GF109203X (5 µM) to Y-27632 (20 µM) led to a
significant inhibition of CPI phosphorylation and further suppression
of STA2-induced MLC20 phosphorylation. This additional inhibition of MLC20 phosphorylation by GF109203X
appeared to be mainly due to the inhibition of CPI phosphorylation
because the additional inhibition of MBS phosphorylation by GF109203X was slight. STA2-induced ATP secretion was partially
inhibited by either Y-27632 (20 µM) or GF109203X (5 µM) treatment,
and treatment with Y-27632 or GF109203X led to a further decrease in
ATP secretion (Figure 8). Inhibition of STA2-induced ATP
release by these compounds appeared to correlate with the extent of
MLC20 phosphorylation at Ser-19.
Our experiments revealed the existence of a CPI-17-like protein in human platelets and its functional role in regulating MLC20 phosphorylation at Ser-19 in case of agonist-induced platelet secretion. CPI-17 was originally reported to be exclusively expressed in smooth muscle tissues,19-21 but we detected a single major immunoreactive band with an apparent molecular mass of 22 kd, a slightly higher molecular mass than noted in smooth muscle CPI-17 (molecular mass, 20 kd),19 in human platelets. Myosin phosphatase co-immunoprecipitated with CPI from human platelets, suggesting that platelet CPI interacts with myosin phosphatase. Phosphorylation by PKC of smooth muscle CPI-17 at a Thr residue (Thr-38) is sufficient to induce the inhibition of myosin phosphatase activity.20,28 We showed that in vitro phosphorylation of platelet CPI by PKC at a Thr residue, corresponding to Thr-38 on CPI-17, resulted in a decrease in myosin phosphatase activity in the anti-CPI-17 immunoprecipitate of platelets. Inhibition by the PKC inhibitor GF109203X of PKC-induced CPI-17 phosphorylation reduced inactivation of the phosphatase activity. Thus, similar to smooth muscle CPI-17, platelet CPI appears to be a phosphorylation-dependent inhibitor of myosin phosphatase. Concentration of CPI in human platelets was estimated to be approximately 0.3 µM, similar to the concentration in porcine aorta smooth muscle.21 Stimulus-induced phosphorylation of CPI at a Thr residue appears
to be one mechanism by which PKC enhances the intracellular pathway of
regulated platelet secretion. PMA can induce ATP secretion in intact
platelets without producing any elevation in cytosolic Ca++
concentration, as determined using the fluorescent Ca++
probe fura-2,41 albeit the secretion is slower and of
lesser amount than the extent of secretion stimulated by agonists such as thrombin and STA2. Our study shows that PMA increases
both CPI phosphorylation at a Thr residue and MLC20
phosphorylation at the same site (Ser-19) phosphorylated by MLC
kinase8 and Rho-kinase.9 This activating MLC
kinase site is not directly phosphorylated by PKC, but phosphorylation
of MLC20 on PKC sites in vitro inhibits smooth muscle
ATPase activity.42,43 The mechanism of Ca++
sensitization of platelet secretion by PMA and by GTP Platelet agonists such as TXA2 and thrombin function through heterotrimeric G protein-coupled receptors, which activate Gq-type,46 G12/G13-type,47,48 and Gi-type49 G proteins. Gq-mediated phospholipase C activation, stimulating the synthesis of inositol triphosphate and diacylglycerol, is thought to represent the main signal pathway leading to full platelet activation such as secretion, whereas G12/G13 couple receptors to the RhoA/Rho-kinase signaling pathways.2,47,48 TXA2 mimetic STA2 and thrombin induce an elevation of intracellular Ca++ concentrations36,41 and most likely lead to Ca++/calmodulin-dependent MLC kinase-mediated MLC20 phosphorylation at Ser-19 of platelets.1,2,5,8 In addition to MLC kinase-dependent MLC20 phosphorylation, Rho-kinase-mediated MLC20 phosphorylation (both directly and indirectly through MBS phosphorylation) contributes to the platelet secretion induced by these agonists.27 Our findings suggest that PKC-mediated CPI phosphorylation and the resultant inhibition of myosin phosphatase is also functionally involved in platelet secretion. STA2 and thrombin, at doses sufficient to elicit platelet secretion, led to the phosphorylation not only of CPI but also of MBS at Thr-696 in intact platelets; these findings differ from results with PMA or ionomycin bypassing the receptor-operated signaling pathway. STA2-induced MLC20 phosphorylation at Ser-19 was diminished, but not abolished, by pretreatment of GF109203X, even at the high concentrations with which STA2-induced CPI phosphorylation was completely inhibited, in parallel samples. These data suggest that PKC-induced CPI phosphorylation is an important, but not the only, mechanism of the STA2-induced increase in MLC20 phosphorylation at Ser-19. We reported that G protein-coupled receptor agonists such as epinephrine, STA2, and thrombin induce MBS phosphorylation and inhibit myosin phosphatase activity and that both reactions are inhibited by a prior treatment with Y-27632.27 We also reported that myosin phosphatase activity is inhibited when chicken gizzard MBS is phosphorylated by Rho-kinase at Thr-695, which corresponds to Thr-696 of human MBS,25 although this site of MBS can be phosphorylated by other, as yet unidentified, kinases.17 Phosphorylation of Thr-696 occurred under in vivo conditions in which RhoA/Rho-kinase was thought to be activated after G protein-coupled receptor activation of human platelets. Our data support the hypothesis that Thr-696 is a major functional site of phosphorylation by Rho-kinase in human platelets. The Rho-kinase inhibitor Y-27632 almost completely attenuated the STA2-induced MBS phosphorylation at Thr-696, whereas this inhibitor slightly inhibited STA2-induced CPI phosphorylation. Smooth muscle CPI-17 was reported to be an in vitro substrate not only for PKC but also for downstream kinases of RhoA Rho-kinase30 and protein kinase N,45 both of which are sensitive to Y-27632. However, our present findings suggest that platelet CPI may be a substrate, but not a preferential one, for Rho-kinase, compared with MBS in intact human platelets. Y-27632 alone partially inhibited STA2-induced MLC20 phosphorylation at Ser-19, as was the case with GF109203X. The Y-27632-insensitive component of STA2-induced MLC20 phosphorylation was further suppressed by the addition of GF109203X, an event mainly attributed to a significant inhibition of CPI phosphorylation and myosin phosphatase suppression. Inhibition of STA2-induced ATP secretion by Y-27632 or GF109203X apparently correlated with the extent of MLC20 phosphorylation at Ser-19. Combined treatment with Y-27632 and GF109203X substantially inhibited STA2 (0.5 µM)-induced MLC20 phosphorylation and ATP secretion, suggesting that Ca++-dependent activation of MLC kinase contributes to a lesser extent to MLC20 phosphorylation at Ser-19 and to platelet secretion under these conditions. The extent of MLC kinase activation appears to depend on the type and concentration of agonist,27 whereas activation of Rho-kinase and PKC can lead to an increase in Ca++ sensitivity of platelet secretion through myosin phosphatase inhibition. In summary, these pharmacological studies suggest that Rho and
PKC participate in parallel pathways to increase MLC20
phosphorylation mainly through the inhibition of myosin phosphatase
following heterotrimeric G protein-coupled receptor activation, as
illustrated in Figure 9. RhoA activates
Rho-kinase, which phosphorylates MBS and inhibits myosin phosphatase
activity. Activation of PKC can also inhibit myosin phosphatase by
phosphorylating CPI. These mechanisms operate independently of the
activation of Ca2+-dependent MLC kinase. The dual
activation of MLC20 phosphorylation by kinase activation
and phosphatase inhibition may be important for a rapid and potent
platelet response to agonist.
We thank Dr R. S. Adelstein (National Instititutes of Health) for helpful discussions, and we thank M. Ohara for language assistance.
Submitted October 31, 2000; accepted February 12, 2001.
Supported in part by grants for research from the Ministry of Education, Science, Technology, Sports and Culture, Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Masakatsu Nishikawa, 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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Top
Abstract
Introduction
a catalytic subunit of the type 1 protein phosphatase 
Bq/mmol) from
NEN Life Science Products (Boston, MA). Polyclonal antibodies against
the PP1
- and 
-isoforms) was
prepared to apparent homogeneity from fresh platelets, as described.
), MBS at Thr-696 (
), or MLC20 at Ser-19
(
). Results are expressed as fold increase in phosphorylation of
each protein relative to the level at 0 second. Densities of the
immunoreactive bands determined with each antiphosphoprotein antibody
were normalized according to that of each total protein. Similar
results were obtained in 3 other experiments using different donor
platelets.
,
phosphorylated CPI; DG, diacylglycerol; Ins(1,4,5)P3,
inositol 1,4,5-triphosphate; GF109203X, inhibitor of PKC;
MBS- P