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INTRODUCTION |
Elevation of cytosolic free Ca2+ is essential for
efficient activation of platelets. Platelet agonists such as thrombin
promote Ca2+ release from internal stores and entry through
plasma membrane channels (1). The predominant mechanism for maintenance
of low intracellular Ca2+ in unstimulated platelets and the
removal of Ca2+ after intracellular store depletion is
efflux catalyzed by the plasma membrane Ca2+-ATPase
(PMCA)1 (2, 3). PMCA is a
highly regulated enzyme, in terms both of diversity of gene expression
and direct modification of enzyme activity (4). In the platelet, two
isoforms are expressed, PMCA1b and PMCA4b (5, 6), and enzyme activity
has been shown to be modulated by cAMP-dependent
phosphorylation and tyrosine phosphorylation (6). Recently, Rosado and
Sage (3) confirmed that PMCA is phosphorylated on tyrosine residues
during platelet activation and showed that small GTPases of the Ras
family participate in the signaling pathway in thapsigargin- and
ionomycin-stimulated platelets.
In addition to PMCA, many other platelet proteins are phosphorylated on
tyrosine residues during platelet activation. This phosphorylation
promotes association of membrane proteins such as the integrin
IIb
3 and tyrosine kinases such as p60src,
p125FAK with the cytoskeleton (7, 8) leading to
cytoskeletal rearrangement and a dramatic change in platelet shape.
Thus PMCA tyrosine phosphorylation could potentially promote
association of PMCA with the cytoskeleton.
Another potential mechanism for interaction of PMCA with the
cytoskeleton has been described recently. The last four residues of
PMCA4b (ETSV) contain the E(T/S)XV motif that has been
identified as a target for binding to PDZ domains (9, 10). PMCA1b as well as the -2b and -3b splice variants exhibit a similar C-terminal sequence of ETSL. Kim et al. (10) demonstrated that
C-terminal peptides (10 amino acids in length) and glutathione
S-transferase fusion proteins containing C-terminal
sequences of both PMCA2b and PMCA4b interact with PDZ domains, whereas
a PMCA2a peptide does not. PDZ domains are central organizers of
protein complexes at the plasma membrane (10, 11) and mediate
interactions with the cytoskeleton (12, 13). Thus both of the PMCA
isoforms found in the platelet, PMCA1b and PMCA4b, could interact with PDZ domains in the cytoskeleton or other organized structures.
In this study, we investigated the association of platelet PMCA with
the cytoskeleton of resting and thrombin-activated platelets. We
compared the cellular distribution of PMCA during platelet activation
with that of p125FAK and investigated whether platelet
aggregation and tyrosine phosphorylation of PMCA are required for this
association. Finally, we asked if the C-terminal region containing the
PDZ domain-binding residues is involved in cytoskeletal association.
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EXPERIMENTAL PROCEDURES |
Materials--
Antipain, apyrase (grade V), aspirin, leupeptin,
sodium orthovanadate, thapsigargin, human thrombin,
phenylmethylsulfonyl fluoride (PMSF), the synthetic peptide
Arg-Gly-Asp-Ser (RGDS), and saponin were purchased from Sigma, and
calpeptin was from Calbiochem. Chemiluminescence reagents were from
PerkinElmer Life Sciences. Anti-focal adhesion kinase
p125FAK and p60src were from Upstate Biotechnologies (Lake
Placid, NY), anti-phosphotyrosine PY20 (horseradish peroxidase,
conjugated and nonconjugated) was from BD Transduction Laboratories
(Lexington, KY), and anti-PMCA monoclonal antibody (5F10) was from
Affinity Bioreagents (Neshanic Station, NJ). A monoclonal antibody
specific for PMCA4, JA9, was provided kindly by John T. Penniston (Mayo Clinic, Rochester, MN). Horseradish peroxidase-conjugated sheep anti-mouse IgG antibody was from Amersham Pharmacia Biotech.
Isolation and Analysis of Platelet Subcellular
Fractions--
Platelet concentrates were obtained from the American
Red Cross (Louisville, KY) and prepared as described previously (14). Briefly, platelets were isolated by centrifugation and resuspended in
Tyrode's-HEPES buffer containing 138 mM sodium chloride,
2.9 mM potassium chloride, 12 mM sodium
bicarbonate, 0.36 mM sodium phosphate, 5.5 mM
glucose, 1.8 mM calcium chloride, 0.4 mM
magnesium chloride, 10 mM HEPES, pH 7.4, 0.1 mM
aspirin, and 0.2 unit/ml apyrase. Platelet suspensions were activated
with 1.0 NIH unit of human thrombin/ml at room temperature. In some
experiments platelets were preincubated for 5 min with the synthetic
peptide RGDS (0.5 mM) in Hanks'-HEPES buffer (138 mM sodium chloride, 5.4 mM potassium chloride,
12 mM sodium bicarbonate, 0.36 mM sodium phosphate, 5.5 mM glucose, and 10 mM HEPES, pH
7.4). Platelets were lysed according to Fox et al. (15) by
addition of an equal volume of ice-cold lysis buffer containing 2%
Triton X-100 (TX100), 10 mM EGTA, 100 mM
Tris-HCl, pH 7.4, 2 mg/ml leupeptin, 10 µg/ml antipain, 10 µg/ml
pepstatin A, 1 mM dithiothreitol, 1 mM sodium orthovanadate, and 1 mM PMSF. Lysates were centrifuged at
16,000 × g at 4 °C for 5 min to sediment the
cytoplasmic cytoskeletal components (low speed pellet). The supernatant
then was centrifuged at 100,000 × g for 2.5 h at
4 °C in a TL100 rotor (Beckman Instruments) to sediment membrane
cytoskeleton components (high speed pellet). Sedimented low speed
material was washed once with 1 volume of lysis buffer diluted 1:1 with
H2O and then resuspended in sample dilution buffer
containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5%
-mercaptoethanol, 11% glycerol, 0.002% bromphenol blue, and 1 mM PMSF. Aliquots of the supernatant from the 100,000 × g centrifugation were solubilized with 3 volumes of
sample dilution buffer, and the high speed pellet was resuspended in
sample dilution buffer. All samples were heated to 42 °C for 20 min.
Boiling was omitted to prevent aggregation of PMCA. The samples were
analyzed by SDS-polyacrylamide gel electrophoresis immediately
or stored at
20 °C.
Synthetic Peptides--
A peptide corresponding to the
C-terminal 10 residues of isoform 4b of human PMCA (hPMCA4b) with the
sequence SSLQSLETSV (10) and a peptide with a scrambled sequence of the
same amino acid composition with the sequence VTSLQSLSES were
synthesized chemically in the Macromolecular Structure Analysis
Facility of the University of Kentucky (Lexington, KY). The amino acid
sequences were confirmed by liquid phase sequencing.
Permeabilization of Platelets with Saponin--
To introduce the
peptides into platelets, we used saponin to permeabilize the cells
according to Authi et al. (16). Platelets were isolated as
described above, centrifuged for 2 min at 6,000 × g,
resuspended in Hanks'-HEPES buffer, and centrifuged again. The cells
then were resuspended in prewarmed (37 °C) buffer containing 140 mM KCl, 1 mM glucose, 1 mM
MgCl2, 0.42 mM NaH2PO4,
6 mM NaHCO3, and 10 mM HEPES, pH
7.4. Saponin (20 µg/ml) and the peptides were added and the platelet
suspension was incubated for 2 min. The platelets then were activated
with thrombin (1 unit/ml) under constant stirring, and the reaction was
stopped by adding an equal volume of lysis buffer. The low speed
cytoskeletal material was isolated as described above.
Electrophoresis, Immunoblotting, and Quantification--
Samples
were analyzed on 7.5% SDS-polyacrylamide gels according to Laemmli
(17). After electrophoresis, proteins were transferred electrophoretically to 0.45-µm nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 h by incubation in 5% (w/v) nonfat dry milk (Carnation) in Tween 20-containing Tris-buffered saline (7.5 mM Tris, pH 7.5, 37.5 mM NaCl, and 0.1% (v/v)
Tween 20) at room temperature. Membranes were rinsed twice and washed
in Tween 20-containing Tris-buffered saline twice for 10 min each. The membrane was divided into two parts and PMCA was probed in the upper
part of the membrane (above 90 kDa) with a dilution of 1:1,000 anti-PMCA antibody (5F10 or JA9) in antibody dilution buffer (2% bovine serum albumin in Tween 20-containing Tris-buffered saline). To
probe for tyrosine-phosphorylated proteins or focal adhesion kinase
p125FAK, the same portion of the membrane was stripped with
buffer containing 62.5 mM Tris, pH 6.8, 2% SDS, and 0.7%
-mercaptoethanol and probed either with a 1:2,500 dilution of
PY20-horseradish peroxidase or with 0.2 µg/ml
anti-p125FAK monoclonal antibody in antibody dilution
buffer for 1 h. The lower portion of the same membrane (below 90 kDa) was probed with 0.5 µg/ml anti-p60src monoclonal antibody. In
the case of monoclonal nonconjugated antibodies (5F10 and JA9), the
membranes were rinsed and incubated with a dilution of 1:10,000 of
sheep anti-mouse-horseradish peroxidase antibody. Membranes were
rinsed as above and then analyzed with chemiluminescence reagents. The
signal was captured on x-ray films (Eastman Kodak Co.) and quantified
after scanning on an Hewlett-Packard flat-bed scanner using Un-Scan-It
software (Silk Scientific). Total signal was determined by adding the
pixels from supernatant and low and high speed pellets after correcting for the volume of these three platelet fractions. Units of the total
signal were in pixels/ml of platelet sample. Analysis of statistical
significance was performed using the Student's unpaired t test.
Ca2+-ATPase Activity Assay for PMCA in Low Speed
Pellets--
Ca2+-ATPase activity was assayed using a
system in which ATP hydrolysis is coupled to NADH oxidation by pyruvate
kinase using phosphoenolpyruvate and lactate dehydrogenase (18). The
low speed pellets obtained as described above were washed once in buffer containing 20 mM HEPES, pH 7.5, 130 mM
NaCl, 1 mM MgCl2, 5% glycerol, 1 mM dithiothreitol, and 1 mM PMSF. The pellets
were resuspended with sonication for 15 s on ice in 500 µl of
the same buffer with 10 µg/ml leupeptin instead of PMSF. The material
was assayed for Ca2+-ATPase activity in buffer containing
20 mM HEPES, pH 7.5, 130 mM NaCl, 10 mM MgCl2, 1 mM ATP, 0.01 mM CaCl2, 50 nM calmodulin, and the
coupled assay components. The difference in the rate of ATP hydrolysis
in the presence and absence of 5 mM EGTA was used to
calculate the Ca2+-ATPase activity. Protein content was
determined with the BCA protein assay kit (Pierce) using bovine serum
albumin as a standard.
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RESULTS |
Association of PMCA with the Platelet Membrane
Cytoskeleton--
Most of the cytoplasmic actin filaments in
unstimulated platelets are aggregated into large complexes that
sediment from TX100-lysed platelets at 16,000 × g (low
speed pellet) (19). However, the membrane-associated skeletal fragments
and associated proteins such as integrin
IIb
3, spectrin, vinculin, and p60src
require 100,000 × g for sedimentation from TX100-lysed
unstimulated platelets (high speed pellet) (20). To investigate the
redistribution of PMCA during platelet activation, platelets were lysed
in a buffer containing 1% TX100, and the cytoplasmic cytoskeletal
components were isolated by centrifugation at 16,000 × g (low speed pellet) followed by centrifugation at
100,000 × g to isolate the membrane cytoskeleton (high
speed pellet). Analysis of PMCA distribution after activation with
thrombin was compared for unstirred platelets, stirred platelets, and
stirred platelets in the presence of RGDS, an inhibitor of platelet
aggregation, to determine whether PMCA localization was dependent on
cell-cell interaction as has been demonstrated for integrin
IIb
3 and p60src (21). As shown in Fig.
1a, the majority of PMCA in
unstimulated platelets (0 min) was associated with the TX100-soluble
supernatant (70%) and the high speed pellet (20%) with only 10% in
the low speed pellet. Thrombin-mediated activation resulted in
redistribution of PMCA to the low speed pellet; 30 min after addition
of thrombin, 80% of the PMCA was redistributed to the low speed
pellet. Stirring enhanced the rate and extent of PMCA redistribution
(Fig. 1b) resulting in ~75% of total PMCA being
associated with the low speed pellet 5 min after thrombin addition.
Inhibition of aggregation with RGDS (Fig. 1c) inhibited this
redistribution with only 20% of total PMCA associated with the low
speed pellet 30 min after thrombin addition. The amount of PMCA
detected in the immunoblots of the supernatant fractions in Fig. 1,
a and b (see insets) is small because
of the large volume of supernatant and resultant dilution compared with
the much smaller volumes and higher concentrations of low speed and
high speed pellets. The second band in the inset labeled
PMCAfrag is apparently a proteolytic fragment of
PMCA because it was eliminated by the addition of calpeptin (30 µM) to thrombin-activated platelets (data not shown).
These results demonstrate extensive redistribution of PMCA to the
cytoskeleton that requires cell-cell contacts as shown by stimulation
with stirring and inhibition with RGDS. These results are nearly
identical to those reported for
IIb
3 and
p60src by Fox et al. (20).

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Fig. 1.
Effect of activation and aggregation on
distribution of PMCA in the TX100-soluble supernatant and
TX100-insoluble low speed and high speed pellets. Suspensions of
platelets were incubated with thrombin (1 unit/ml) for the indicated
times and either agitated occasionally (a), constantly
stirred (b), or stirred after preincubation with 0.5 mM RGDS for 5 min prior to the addition of thrombin
(c). The activation was stopped at the indicated times by
lysis with an equal volume of lysis buffer containing 2% TX100. Low
speed pellet, high speed pellet, and supernatant fractions were
obtained as described under "Experimental Procedures." Proteins
were separated by SDS-polyacrylamide gel electrophoresis, transferred
onto nitrocellulose membranes, and probed with antibodies against PMCA
(5F10). PMCA was detected by enhanced chemiluminescence. An example of
each blot is given in the insets at the top of each graph.
PMCAfrag indicates a lower molecular weight band
that is not present in calpeptin-treated platelets. Graphs
show the relative amount as the percentage of total PMCA in each
fraction of lysed platelets and are presented as mean ± S.E.
(n = 3-4). *, p 0.05; **,
p 0.01; and ***, p 0.001 (t test) compared with unstimulated platelets (0 time).
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Similar results were obtained when redistribution of
p125FAK was analyzed as shown in Fig.
2. Comparison of Figs. 1 and 2 indicates that both PMCA and p125FAK are redistributed to the
cytoskeleton in an aggregation-dependent reaction initiated
by thrombin-dependent activation under the conditions used
in our experiments.

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Fig. 2.
Effect of platelet activation and aggregation
on distribution of p125FAK in the TX100-soluble and
TX100-insoluble low speed and high speed pellets. The
nitrocellulose membranes probed for PMCA in Fig. 1 were stripped and
reprobed for p125FAK. p125FAK was detected by
enhanced chemiluminescence. Examples of blots are given in the
insets at the top of each graph. Graphs show the
content of p125FAK in each fraction of lysed platelets
expressed as a percentage of the total and are presented as mean ± S.E. (n = 3-4). The 90-kDa fragment of
p125FAK was included in the calculation. *,
p 0.05 and **, p 0.01, compared
with unstimulated platelets (t test).
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Because we reported earlier that platelets contain both PMCA1b and
PMCA4b (6), it is important to determine which isoforms are associating
with the cytoskeleton. Kim et al. (10) demonstrated that
both PMCA1b and PMCA4b bind to PDZ domains, although PMCA4b was
observed to have significantly higher affinity. To determine the
relative distribution of the two isoforms in the cytoskeleton, monoclonal antibodies 5F10 and JA9 were employed (22, 23); 5F10
recognizes both isoforms, whereas JA9 only binds PMCA4. As shown in
Fig. 3, we used the ratio of the signals
obtained with JA9 and 5F10 to estimate the relative amounts of these
two isoforms in whole platelets and the cytoplasmic cytoskeletal
fraction of activated platelets. The JA9/5F10 ratio is equivalent to
x(PMCA4b)/(y(PMCA1b) + x(PMCA4b)),
where x and y depend on antibody affinity,
antibody dilution, and chemiluminescence development conditions.
Because x and y are unknowns caused by lack of
pure standards, the ratios can be used only to compare relative amounts
of the two isoforms on a single immunoblot where x and
y are the same for all samples on the blot. The results show
that compared with the ratio in whole platelets (Whole Plt),
there is not a significant change in the ratio of PMCA4b to total PMCA
in the pellet (Pellet) fractions in thrombin-activated
platelets. This suggests that both PMCA1b and PMCA4b become associated
with the cytoskeleton during platelet activation. Another possibility
is that there is little PMCA1b in the platelet as reported by Paszty
et al. (5) so that even if PMCA1b were not associated with
the low speed pellet, the JA9/5F10 ratio would not change
significantly.

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Fig. 3.
Relative amounts of PMCA1b and PMCA4b in the
low speed pellet of activated platelets. Suspensions of platelets
were incubated with thrombin (1 unit/ml) with stirring for 10 min, and
activation was stopped by lysis with an equal volume of lysis buffer
containing 2% TX100. Whole solubilized platelets (Whole
Plt) and low speed pellets (Pellet) were separated by
SDS-polyacrylamide gel electrophoresis, transferred onto a
nitrocellulose membrane and probed with a monoclonal antibody against
PMCA4 (JA9), stripped, and reprobed with a monoclonal antibody that
recognizes both PMCA1 and PMCA4 (5F10). PMCA was detected by enhanced
chemiluminescence and quantified as described under "Experimental
Procedures." The ratios of signals obtained with JA9 and 5F10 were
averaged for three bags of platelets. There was not a significant
difference in the JA9/5F10 ratio between whole platelets and the low
speed pellet.
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Role of Tyrosine Phosphorylation in PMCA
Redistribution--
Because p60src and p125FAK associate
with the cytoskeleton as a result of tyrosine phosphorylation, we
investigated the relative levels of PMCA tyrosine phosphorylation in
the TX100 fractions after lysis of thrombin-activated platelets.
Tyrosine phosphorylation was assessed using anti-phosphotyrosine
immunoblotting with the PY20 antibody, whereas the extent of
PMCA tyrosine phosphorylation was estimated by comparing the
phosphotyrosine and PMCA signals at the migration position of PMCA.
Thus the extent of PMCA phosphorylation is presented as the PY20/PMCA
ratio. As shown in Fig. 4, thrombin addition under stirred conditions promoted PMCA tyrosine
phosphorylation in the supernatant (PY20/PMCA ratio of ~3.0 at 5-30
min) and in the high speed pellet (PY20/PMCA ratio of ~6.0 at 5-15
min) from an initial ratio (0 min) of ~1.0. No increase in the
PY20/PMCA ratio was observed in the low speed pellet. Although in the
insets (one example of the signal with PY20 for each
fraction) it appears that the majority of tyrosine phosphorylation
occurs in the low speed pellet, this is also the location of the
majority (85%) of the PMCA in activated platelets thus resulting in a
relatively low PY20/PMCA ratio (~1.0). These results suggest that
unlike p60src and p125FAK, PMCA redistribution to the
cytoskeleton is not dependent on tyrosine phosphorylation of PMCA,
because the PY20/PMCA ratio is not increased in the low speed
pellets.

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Fig. 4.
Extent of PMCA tyrosine phosphorylation in
the low speed pellet, high speed pellet, and the soluble supernatant of
activated platelets. Suspensions of platelets were incubated with
thrombin (1 unit/ml) and stirred for the indicated times, and
activation was stopped by lysis with an equal volume of lysis buffer.
Low speed pellet, high speed pellet, and supernatant fractions were
obtained as described under "Experimental Procedures." Proteins
were separated by SDS-polyacrylamide gel electrophoresis, transferred
onto nitrocellulose membranes, and probed with antibodies against PMCA
(5F10). PMCA immunoblots then were stripped and reprobed for
tyrosine-phosphorylated proteins with the PY20 antibody.
Phosphotyrosine was detected by enhanced chemiluminescence.
Graphs show the ratio of the PY20 signal (phosphotyrosine)
at 135 kDa divided by the PMCA signal in each fraction of lysed
platelets (ratio PY20/PMCA). An example of each blot probed
with PY20 is given in the insets at the top of each graph.
Data are presented as mean ± S.E. (n = 4). *,
p 0.05 (t test) compared with
unstimulated platelets (0 time).
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Role of the C-terminal PDZ-binding Domain in PMCA
Redistribution--
Another possible mechanism for PMCA association
with the cytoskeleton is interaction of the C-terminal PDZ-binding
domain in PMCA1b and PMCA4b with cytoskeletal PDZ domain-containing
proteins. Kim et al. (10) demonstrated that a peptide
representing the C terminus of PMCA4b, SSLQSLETSV, bound with high
affinity to the PDZ1+2 domain of the human homolog of
Drosophila discs-large protein. We therefore tested the
ability of this peptide to disrupt PMCA-cytoskeletal interactions in
saponin-permeabilized thrombin-activated platelets. A scrambled peptide
composed of the same amino acids, VTSLQSLSES, was used as a negative
control. The results presented in Fig.
5a show that the C-terminal
peptide (PDZ) prevented association of PMCA with the low
speed pellet in permeabilized thrombin-stimulated platelets, whereas
the scrambled peptide (scr.) had a much smaller effect on
PMCA association with the cytoskeleton. Addition of saponin alone had
no effect on the redistribution of PMCA to the cytoskeleton as shown by
the lack of effect of saponin addition in the absence of added
peptides. Although the C-terminal peptide prevented PMCA
association with the cytoskeleton, it had very little effect on the
translocation of p60src and p125FAK to the cytoskeleton
compared with the effect of the scrambled peptide (Fig. 5b).
This was expected because p60src and p125FAK associate with
the cytoskeleton by means of tyrosine phosphates rather than through
PDZ domain interactions. Fig. 5c shows that the PDZ peptide
significantly inhibited PMCA association with the cytoskeleton in four
different platelet preparations, whereas the effect of the scrambled
peptide was not significantly different from the control.

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Fig. 5.
Inhibition of the
association of PMCA with the low speed pellet by the C-terminal PMCA
peptide SSLQSLETSV. Suspensions of platelets were preincubated for
2 min with 20 µg/ml saponin and 0.1 mM of the peptide
with the sequence of the last 10 C-terminal residues of PMCA4b isoform
(PDZ) or a peptide with a scrambled sequence
(scr.). The platelets then were incubated with thrombin (1 unit/ml) for 10 min under stirred conditions. After solubilization with
1% TX100, the low speed pellet was isolated, and proteins were
separated by SDS-polyacrylamide gel electrophoresis, transferred onto
nitrocellulose membranes, and probed with antibodies. a, in
the first lane platelets were not resuspended in
permeabilization buffer prior to activation. In the remaining lanes the
platelets were resuspended in permeabilization buffer, and the effects
of the addition of saponin, the C-terminal peptide
(PDZ), and the scrambled peptide (scr.) on
association of PMCA with the cytoskeleton after thrombin activation are
shown. b, samples were permeabilized and activated as in
a, in the presence of either the C-terminal peptide
(PDZ) or the scrambled peptide (scr.).
Nitrocellulose membranes were divided into two portions; the upper
portion was probed with a monoclonal antibody against
p125FAK (pp125FAK) and the lower portion
with a monoclonal antibody against p60src (pp60src).
c, platelets were permeabilized, treated with peptides, and
activated with thrombin and low speed pellets were isolated as
described above. The amounts of PMCA chemiluminescence in anti-PMCA
5F10 immunoblots of the low speed pellets obtained in the absence of
peptides (Control), with C-terminal peptide
(PDZ), and scrambled peptide (SCR) were
quantified as described under "Experimental Procedures." The
amounts of PMCA in the low speed pellet after treatment with the
C-terminal peptide or the scrambled peptide were compared with control
and results are stated as a percentage of control. Experiments were
carried out with four separate bags of platelets. Data are presented as
mean ± S.E. (n = 4). *, p 0.05 compared with unstimulated platelets (t test).
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PMCA Activity in the Cytoskeleton--
Having established that
PMCA becomes associated with the actin cytoskeleton during platelet
activation by means of binding to cytoskeletal PDZ domains, it became
important to establish a physiological role for this phenomenon. If
association of PMCA with the cytoskeleton causes localization of the
pump in specific areas of the activated platelet, then it is essential
that the pump retains its biological activity. We therefore measured
the Ca2+-ATPase activity in the isolated cytoskeleton. As
shown in Fig. 6, cytoskeletal
Ca2+-ATPase activity was measured as a function of time
after thrombin addition (shaded bars) in the presence
of 200 nM thapsigargin to inhibit sarco(endo)plasmic
reticulum Ca2+-ATPase-type ATPases. Maximal activity
was observed 15 min after the addition of thrombin, and the specific
activity is in the range observed for platelet plasma membranes, 10-40
nmol/min/mg (24). This activity correlates well with the amount of PMCA associated with cytoskeleton (open bars). The enzymatic
characteristics of the cytoskeletal Ca2+-ATPase activity
were examined and the results are presented in Table
I. The Ca2+-ATPase activity
in the low speed pellet was inhibited 43% by 10 µM
orthovanadate, an indication that a portion of the ATPase activity is
due to PMCA because this ATPase is inhibited strongly at 10 µM vanadate, whereas sarco(endo)plasmic reticulum
Ca2+-ATPase-type ATPases require 100 µM
vanadate for complete inhibition (25). Furthermore, the ATPase activity
is partially stimulated by calmodulin (18%), an activator of PMCA.
This relatively modest level of stimulation may indicate the presence
of endogenous calmodulin in the isolated cytoskeleton because no
attempt was made to strip the pellet of calmodulin. The low level of
inhibition by thapsigargin (11%) indicates low abundance of
sarco(endo)plasmic reticulum Ca2+-ATPase in the pellet.
Taken together these data indicate that a portion of the
Ca2+-ATPase activity in the isolated cytoskeleton is due to
the presence of PMCA.

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Fig. 6.
Ca2+-ATPase activity in the low
speed pellet of thrombin-activated platelets. Suspensions of
platelets were activated with thrombin (1 unit/ml) under stirring
conditions. Activation was stopped by lysis with an equal volume of
lysis buffer containing 2% TX100. The low speed pellets were prepared
as in Fig. 1, washed, and resuspended in assay buffer containing 200 nM thapsigargin. The difference in the rate of ATP
hydrolysis in the absence and presence of 5 mM EGTA was
used to calculate the Ca2+-ATPase activity (shaded
bars). The amount of PMCA associated with the low speed pellets
was determined by immunoblotting with anti-PMCA 5F10 and quantification
of chemiluminescent signals as described in Fig. 1 for six bags of
platelets (open bars). The data shown are presented as
mean ± S.E. (n = 6). *, p 0.05 compared with unstimulated platelets at 0 min (t
test).
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Table I
Properties of Ca2+-ATPase activity in the low speed pellet
The low speed pellet was prepared from thrombin-activated platelets and
the Ca2+-ATPase activity measured as described under
"Experimental Procedures." A preparation of low speed pellet from a
single bag of platelets was assayed in quadruplicate and activities are
averages of four assays ± S.E. The complete assay contained
ATPase assay components plus 50 nM calmodulin.
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DISCUSSION |
PMCA is the major Ca2+ efflux mediator in human
platelets, and it therefore plays a fundamental role in platelet
intracellular Ca2+ homeostasis (2, 3). Changes in the
activity and/or distribution of PMCA might have a crucial impact on
platelet functions. Our earlier finding that platelet PMCA is
phosphorylated on tyrosine residues and inhibited in hypertensive
individuals resulting in increased cytosolic Ca2+
demonstrates the importance of this protein for proper platelet function (14).
After the initial increase of intracellular Ca2+ after
activation by strong agonists such as thrombin, platelet shape changes dramatically because of rearrangements of the cytoskeleton. The association of many proteins with the actin cytoskeleton such as the
integral membrane protein integrin
IIb
3
and cytosolic nonreceptor tyrosine kinases such as p60src and
p125FAK requires tyrosine phosphorylation (20). However, in
this report we show that tyrosine phosphorylation of PMCA does not
promote association of this protein with the cytoskeleton because the greatest extent ((tyrosine phosphates)/(PMCA)) of PMCA
tyrosine phosphorylation in thrombin-activated platelets is either in
the supernatant or high speed pellet (Fig. 4). Thus it is the more active, less phosphorylated form that associates with the cytoplasmic cytoskeleton. This implies that the purpose of redistribution of PMCA
is to direct the active Ca2+ pump to specific locations in
the activated platelet, possibly to reduce intracellular
Ca2+ in focal contact regions thus preventing proteolytic
degradation by the Ca2+-activated proteinase calpain (19).
The concomitant inhibition of the pump not associated with the
cytoskeleton by tyrosine phosphorylation presumably allows for higher
accumulation of cytosolic Ca2+ in the bulk of the activated platelet.
We showed that the C-terminal PDZ-binding domain of PMCA is involved in
the redistribution of PMCA1b and PMCA4b to the actin cytoskeleton (Fig.
5). The C-terminal peptide of PMCA4b specifically inhibited PMCA
association but had no effect on the tyrosine phosphorylation-mediated association of p60src and p125FAK with the cytoskeleton.
Furthermore, a scrambled peptide composed of the same amino acid
residues had no effect on PMCA association with the low speed pellet.
This provides strong evidence for the role of the C terminus of
b-spliced PMCA isoforms in association with the platelet cytoskeleton
during activation. Kim et al. (10) speculated that the role
of the PMCA PDZ-binding domain in neuronal cells may be to direct the
pump into dendritic spines and restrict signaling by the
N-methyl-D-aspartate receptor. We propose a
similar redistribution of platelet PMCA into focal contacts, possibly in filopodia, to regulate cytosolic Ca2+ in these microdomains.
PDZ domains have been shown to be involved in multiprotein complexes
often linking plasma membrane proteins to stable subcellular structures
(11, 12). In contrast, PMCA in the platelet only becomes associated
with the cytoskeleton after activation and cytoskeletal rearrangement.
The mechanism for this behavior could be similar to that described for
neuronal F-actin binding proteins such as neurabin-I and -II (26).
These PDZ domain-containing F-actin binding proteins become associated
with polymerized actin, but dissociate upon depolymerization of the
cytoskeleton. Thus, PMCA could associate with the cytoskeleton in
activated platelets by interacting with a PDZ domain containing F-actin
binding protein that binds to the reorganized cytoskeleton after
activation. Although neurabins have not been reported in platelets, a
protein with similar properties, CLP-36, has been described recently
(27). This PDZ domain-containing protein binds to
-actinin in the
cytoskeleton of activated platelets by means of a distinct intervening
sequence thus leaving the PDZ domain available for interaction with PDZ domain-binding proteins such as PMCA.
In conclusion, we have demonstrated that PMCA is not associated with
the cytoskeleton in resting platelets and that association increases
dramatically upon activation with thrombin. Tyrosine phosphorylation
does not promote the translocation, but the C-terminal PDZ-binding
domain is required for PMCA redistribution. This is the first direct
demonstration of a physiologically relevant role for the C-terminal PDZ
binding sequence in PMCA4b.