(Received for publication, August 10, 1994; and in revised form, October 24, 1994)
From the
Two membrane-associated phosphoinositide-specific phospholipase
Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were
activated by brain G-protein subunits have been isolated
from human platelets. The truncation of mPI-PLC-1 that was mediated by
µ-calpain induced much higher activation by
subunits
(Banno, Y., Asano, T., and Nozawa, Y.(1994) FEBS Lett. 340,
185-188). On the basis of size and immunological
cross-reactivity, mPI-PLC-1 (155 kDa) was PLC-
3, and mPI-PLC-2
(100 kDa) was its truncated form. The cPI-PLC (140 kDa) was recognized
by the antibody selective for internal sequences of PLC-
3 but not
by the antibody raised against its carboxyl terminus, indicating that
it may be related to PLC-
3.
Treatment of human platelets with
A23187 and dibucaine, activators of calpain, caused cleavage of
actin-binding protein and talin in a time-dependent manner. At the same
time, decrease of PLC-3 (155 and 140 kDa) and concomitant increase
of the 100-kDa product of cleavage were observed on immunoblots with
the antibody to internal sequences of PLC-
3. Furthermore,
stimulation of platelets by natural agonists, thrombin and collagen,
caused the cleavage of PLC-
3 (155 and 140 kDa) and an increase of
100 kDa PLC-
3 in a time- and dose-dependent manner. The cleavage
of these PLC-
3 enzymes was completely blocked by calpain
inhibitor, calpeptin, indicating that the PLC-
3 modification may
be a consequence of platelet activation leading to activation of
calpain. This is the first demonstration that PLC-
3 is indeed
cleaved by calpain upon platelet activation by physiological agonists.
The cleavage of PLC-
3 evoked by thrombin and collagen but not ADP
was correlated with irreversible aggregation, suggesting that the
PLC-
3 modification may play a role in secondary irreversible
aggregation in agonist-stimulated human platelets.
Phosphoinositide-specific phospholipase C (PI-PLC) ()is an important signal-transducing enzyme to generate two
second messengers, inositol trisphosphate and
diacylglycerol(1, 2, 3) . There are nine
distinct isozymes in total (
1,
2,
3,
4,
1,
2,
1,
2, and
3)(4, 5, 6, 7) . The PI-PLC
isozymes are activated by differential mechanisms upon receptor
stimulation. Tyrosine phosphorylation is thought to be implicated in
the activation of PLC-
isozymes(8, 9, 10) . Receptors containing
seven membrane-spanning domains have been known to couple to effector
enzymes via guanine nucleotide-binding protein
(G-protein)(11) . Recent investigations provide evidence that
receptor-mediated activation of PLC-
isozymes is caused by two
distinct mechanisms, one through
subunits of the G
family insensitive to pertussis toxin, and the other through the
subunits of pertusis toxin-sensitive
G-proteins(12, 13, 14, 15, 16, 17) .
Both G
and
subunits activate PLC-
isozymes to different extents. The
subunits of the G
family activate PLC-
isozymes in the order of PLC-
1
> PLC-
3 > PLC-
2, whereas
subunits stimulate
PLC-
isozymes in the order of PLC-
3 > PLC-
2 >
PLC-
1(18, 19) .
It has been known that in
human platelets multiple forms of PI-PLC (,
1,
2 and
) were present in cytosol and membrane
fractions(20, 21) . We previously demonstrated that
membrane-associated platelet PI-PLC activity was enhanced by either
GTP
S-activated G
or G
to nearly the same
extent(22) . This (22) and other recent findings (23, 24) suggest that
subunits of
heterotrimeric G-proteins may be involved in the PI-PLC activation in
human platelets. Furthermore, it was recently shown that truncated
PLC-
isozyme was found to be activated remarkably by G-protein
subunits(25) . We have also demonstrated enhancement
of the cleaved PLC-
activity by
subunits and suggested
that truncation of the PLC-
by µ-calpain greatly enhanced its
activation by G-protein
subunits in
vitro(26) . In human platelets, agonist-induced activation
of calpain (27, 28, 29) and limited
proteolysis of some specific substrates have been known to occur in the
course of a platelet
activation(30, 31, 32, 33) .
In
this study, we have demonstrated by using specific antibody that
stimulation by physiological agonists of human platelets evoked the
cleavage of PLC-3 to a 100-kDa truncated form via calpain
activation. We then propose that the PLC-
3 is an endogenous
substrate for calpain in human platelets and that the limited
proteolysis would be a prerequisite for activation by
subunits. In addition, it has also been suggested that the PLC-
3
modification may be implicated in secondary irreversible aggregation.
The G-protein subunits from
bovine brain were purified as described previously(35) .
Protein concentrations were routinely determined with the Bio-Rad protein assay kit using bovine serum albumin as standard.
Figure 1:
Immunoblot analysis of isolated
mPI-PLC-1, mPI-PLC-2, and cPI-PLC. The isolated mPI-PLC-1 (A),
mPI-PLC-2 (B), and cPI-PLC (C) were subjected to
SDS-6% polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and probed with the various antibodies. Lane
1, anti-PLC-3 COOH-terminal domain (Ab-
3C); lane
2, anti-PLC-
3 peptides(550-561) (Ab-
3M); lane
3, anti-PLC-Y domain (Ab-Y); lane 4, anti-PLC-X domain
(Ab-X); lane 5, anti-PLC-
1 (Ab-
1). Arrows indicate molecular mass standards (from top): 205 kDa
(myosin), 112 kDa (
-galactosidase), 79.5 kDa (bovine serum
albumin)
Figure 2:
A23187-induced cleavage of ABP, talin, and
PLC-3. A, platelet suspension (5
10
cells/ml) was incubated with A23187 (1 µM) for the
indicated periods with stirring. Calcium (1 mM), EGTA (3
mM), or calpeptin (30 µM) was added 5 min before
the addition of A23187. Platelets were lysed by the addition of 3
concentrated SDS sample buffer, containing 10 mM EGTA
and 10 mM EDTA. Whole platelet lysates (10 µg of protein)
were analyzed by SDS-PAGE (6%). The results of Coomassie Blue staining
are shown. B, the platelet lysates from A were separated by
SDS-PAGE, and proteins were transferred to nitrocellulose. Ab-
3C
and Ab-
3M were used to detect PLC-
3. C, platelets
were stimulated with various concentrations of A23187 for 10 min and
then lysed and analyzed for the cleavage of PLC-
3 by Western
blotting as in B.
Another calpain activator,
dibucaine, also induced timedependent cleavages of actin-binding
protein and talin (Fig. 3A) and also of PLC-3 (155
and 140 kDa) (Fig. 3, B and C). The PLC-
3
cleavage induced by dibucaine (1 mM) was inhibited by 30
µM calpeptin, suggesting that the cleavage of PLC-
3
induced by dibucaine was mediated through calpain activation. On the
other hand, the presence of 3 mM EGTA could not completely
block the cleavage of actin-binding protein, talin, and PLC-
3
induced by dibucaine. This result was consistent with those of other
studies, which showed that dibucaine is a direct activator of
calpain(32) . The cleaved enzymes produced by dibucaine had a
similar molecular mass (100 kDa) to that produced by A23187 and that
the cleaved enzyme was recognized by Ab-
3M but not by Ab-
3C (Fig. 3, B and C).
Figure 3:
Dibucaine-induced cleavages of ABP,
taline, and PLC-3. A, platelets (5
10
cells/ml) were treated with dibucaine (1 mM) for the
indicated periods in the presence of calcium (1 mM) or EGTA (3
mM) or after pretreatment of calpeptin (30 µM).
Platelets were lysed and analyzed for cleavage of ABP and talin by
Coomassie Blue staining. B, the platelet lysates of the same
sample employed in A were analyzed by Western blotting as Fig. 2B. Ab-
3C and Ab-
3M were used to detect
PLC-
3. C, platelets were stimulated with various
concentrations of dibucaine for 10 min and then lysed and analyzed for
the cleavage of PLC-
3 by using Ab-
3C and
Ab-BM.
Figure 4:
Thrombin-induced cleavage of PC-3.
Platelets (5
10
cells/ml) in modified Hepes-Tyrode.
buffer were treated with 1 unit/ml of thrombin for the indicated times
with stirring. Calcium (1 mm), EGTA (3 mM), and calpeptin (30
µM) were added 5 min before stimulation. Total platelet
lysates (10 µg of protein) were resolved by SDS-PAGE and
immunoblotted with Ab-
3C, Ab-
3M, Ab-Y, and
Ab-X.
Collagen, another agonist that causes activation of
calpain(28) , also caused the proteolytic modification of
PLC-3 in a time- and dose-dependent manner (Fig. 5, A and B); the 155- and 140-kDa PLC-
3 were gradually
decreased, and the 100-kDa form was concomitantly increased. The
cleavage of the PLC-
3 enzymes caused by collagen stimulation was
also completely blocked by the addition of 3 mM EGTA and
pretreatment with 30 µM calpeptin. These results again
suggest that the truncation of PLC-
3 was a consequence of platelet
activation by which calpain was activated.
Figure 5:
Collagen-induced cleavage of PLC-3. A, platelets (5
10
cells/mlin) in modified
Hepes-Tyrode buffer were treated with 10 µg/ml of collagen for the
indicated times with stirring. Calcium (1 mM), EGTA (3
mM), and calpeptin (30 µM) were added 5 min
before stimulation. Total platelet lysates (10 µg of protein) were
resolved by SDS-PAGE and immunoblotted with Ab-
3C and Ab-
3M. B, platelets (5
10
cells/ml) in modified
Hepes-Tyrode buffer supplemented with 1 mM calcium were
stimulated with various concentrations of collagen for 10 min and then
lysed and analyzed for the cleavage of PLC-
3 as in A.
As shown in Fig. 6, A and B, the cleavage of PLC-3 in
response to various concentrations of thrombin was correlated with the
extent of platelet aggregation. Platelets treated with thrombin (1
unit/ml), collagen (20 µg/ml), and A23187 (1 µM)
caused irreversible aggregation (Fig. 7). On the other hand,
platelet-activating factor caused reversible aggregation. When
platelets were stimulated with weak agonists such as ADP in the absence
of fibrinogen, they exhibited no aggregation. The platelet-activating
factor and ADP did not evoke PLC-
3 cleavage (data not shown).
These results suggest that the PLC-
3 cleavage may correlate to
irreversible aggregation.
Figure 6:
Cleavage of thrombin-induced PLC-3
and platelet aggregation. A, platelets (5
10
cells/ml) in modified Hepes-Tyrode buffer supplemented with 1
mM CaCl
were treated with various concentrations
of thrombin for 10 min with stirring. Total platelet lysates (10 µg
of protein) were resolved by SDS-PAGE and immunoblotted with Ab-
3C
and Ab-
3M. B, platelet aggregations in A were
measured in aggregometer and expressed as the percentage of maximum
aggregation. Maximum aggregation is defined as aggregation seen 5 min
after thrombin stimulation.
Figure 7:
Platelet aggregation by various agonists.
Platelets (5 10
cells/ml) in modified Hepes-Tyrode
buffer supplemented with 1 mM CaCl
without
fibrinogen were treated with various agonists (1 unit/ml thrombin, 20
µg/ml collagen, 1 µM A23187, 1 µM platelet-activating factor, 10 mM ADP) for 2 min.
Aggregometer tracings are shown in ordinate displaying light
transmission.
We have demonstrated here that agonist stimulation of human
platelets induced cleavage of PLC-3 (155 and 140 kDa) to generate
100-kDa product as examined by immunoblot analysis. The cleavage of
PLC-
3 (155 and 140 kDa) was a consequence of platelet activation
and resulted from an agonist-induced intracellular event that was
concurrent with the activation of calpain. The truncated PLC-
3
form (100 kDa) was isolated from platelet membrane fraction and was
immunoreactive with Ab-
3M, Ab-Y, and Ab-X but not with Ab-
3C.
Thus it was conceivable that a single cleavage of the PLC-
3
occurred at the linkage between the carboxyl-terminal region and Y
domain of the intact PLC-
3. The truncated PLC-
3 (100 kDa) was
activated to a much higher extent by
subunits of G-protein
as compared with the intact PLC-
3. The 155- and 140-kDa PLC-
3
was detected in the resting human platelets by Ab-
3M. The 155-kDa
PLC-
3 was isolated from the membrane fraction, and the 140-kDa
enzyme was from the cytosolic fraction of human platelets. Agonist
stimulation of human platelets caused cleavage of both PLC-
3
enzymes at almost the same time, thereby producing 100-kDa enzyme. The
cleavage was completely blocked by calpain inhibitor, calpeptin,
suggesting that both enzymes share a sequence attacked by calpain.
Although 140-kDa PLC-
3 could be considered to be defective in the
carboxyl-terminal region of 155-kDa PLC-
3, there is no direct
evidence that 140-kDa PLC-
3 was indeed derived from 155-kDa enzyme
by limited proteolysis. A recent study (39) has demonstrated
that brain PLC-
1 exists in two immunologically indistinguishable
forms of 150 and 140 kDa, and analysis of PLC-
1 genomic DNA
indicated that PLC-
1a (150 kDa) and PLC-
1b (140 kDa) were
derived from a single gene by alternative RNA splicing. These results
could imply that the platelet 140-kDa PLC-
3 may be generated by
alternative splicing. However, the reason why the 140-kDa enzyme has
lost the immunoreactivity to Ab-Y has remained unknown.
On the other
hand, it has been reported that the PLC-1 was activated by the
G
class containing
q,
11,
16, and
14 (12, 13, 14) but that both
PLC-
2 and PLC-
3 were efficiently activated by G-protein
subunits(15, 16, 17, 18, 19) .
Experiments using a series of specific deletions and truncations of
PLC-
1 have demonstrated that the region required for activation by
G
is localized to the sequence corresponding to
residues 903-1142 of PLC-
1(14) . The truncated 100-kDa
PLC-
1 produced by calpain could no longer be activated by
G
(40) . Furthermore, a 110-kDa PI-PLC purified
from brain cytosol, a truncated form of PLC-
3, was found to be
markedly activated by
subunits but not affected by
G
(25) . Lee et al.(41) showed that the PLC-
2 mutants that lack the
carboxyl-terminal were activated by
subunits but not by
G
. It was also reported that PLC-
3 but not
PLC-
1 was activated by
q/11 even in the presence of a
saturating concentration of
subunits(19) . These
observations indicated that
subunits could activate
PLC-
subtypes by interacting at a site different from that of
G
. In human platelets, G
2 is a major
pertussis toxin substrate, whereas both G
1 and
G
3 are much less so(42) . It is also shown that
platelets express pertussis toxin-insensitive G-protein, G
,
which is coupled to the thromboxane A2 receptor(43) . Thus one
would assume that G
2 may be responsible for the activation
of PI-PLC in thrombin-stimulated platelets(44) . In Triton
X-100 extraction of human platelets, PLC-
2 and PLC-
3 were
immunodetectable by polyclonal antibodies to PLC-
2 and PLC-
3,
respectively, but PLC-
1 and PLC
4 were not undetectable. (
)PLC-
3 is reported to be activated by
subunits of pertussis toxin-sensitive G-proteins as well as
subunit of the G
family(18, 19) . These
results lead us to speculate that in thrombin-stimulated human
platelets the
subunits of G
2 may be involved in
activation of PLC-
2 and PLC-
3.
Despite substantial
evidence that human platelets contain high amounts of calpain (31, 46) and various substrates of calpain, such as
platelet factor XIII(30) , actin binding protein,
talin(47) , PI-PLC(48) , and protein kinase
C(49) , were cleaved in vivo and in vitro,
the physiological relevance of these cleavage events has remained
obscure. Fox et al.(27, 28) demonstrated
that agonist-induced calpain activation was involved in the shedding of
procoagulant-rich microvesicles from aggregating platelets and also
indicated that calpain activation was caused by signaling through the
binding of integrin GpIIb-IIIa to adhesive ligand(50) . Recent
studies have indicated that calpain activation and cleavage of specific
endogenous protein substrates, such as pp60(32) and protein phosphotyrosine phosphatase 1B (33) were correlated with irreversible aggregation in human
platelets. However, close association of calpain with platelet
functions is still controversial, since it was shown from results using
two inhibitors that calpain-mediated proteolysis in platelets is not an
obligatory event leading to shape change, adhesion, aggregation, and
5-hydroxytryptamine release(51) .
As for PI-PLC, an earlier
work has indicated that the higher molecular forms (400 and 270 kDa) of
platelet cytosolic PI-PLC were converted into the 100-kDa form without
substantial loss of activity by incubation with a calpain in
vitro, suggesting that platelet PI-PLC enzymes were a substrate of
calpain(48) . However, it was not shown whether PI-PLC(s) was
endogenous substrate for activated calpain in platelets stimulated by
natural agonists in vivo. There were some evidences suggesting
activation of PI-PLC through agonist-induced calpain activation by
using calpain inhibitors. Ishii et al.(52) demonstrated that thrombin-induced PI hydrolysis
leading to inositol phosphate production as well as aggregation and
secretion were inhibited by a membrane permeable thioprotease
inhibitor, E-64d, suggesting that calpain, a major thiol protease of
platelets, may participate in platelet functions through the activation
of PI-PLC. Although these results provided a strong indication for a
role of calpain in PI-PLC activation, the inhibitor was not specific
for calpain in intact cells. On the other hand, Ariyoshi et al.(45) have observed in experiments using a specific potent
calpain inhibitor, calpeptin, that its high concentration (300
µM) inhibited inositol 1,4,5-trisphosphate formation,
aggregation, and [Ca]
elevation
in thrombin- or collagen-stimulated platelets, whereas its lower
concentration (30 µM) did not affect these events. It was
also shown that this amount of calpeptin (30 µM) was
enough to inhibit the agonist-induced calpain activation.
Our
studies demonstrated here that PLC-3 is an endogenous substrate
for calpain in agonist-induced platelet activation and that cleavage of
PLC-
3 mediated by calpain activation occurs in the irreversible
aggregation induced by potent agonists such as thrombin and collagen
but not in reversible aggregation by weak agonists such as ADP in the
absence of fibrinogen. The generation of the cleaved form of PLC-
3
(100 kDa) induced by thrombin stimulation seems to correlate in time
with calpain activation and irreversible aggregation, which occur 30 or
60 s after the addition of thrombin(50) . Thus, these
observations lead us to consider that the modification of PLC-
3
may play a role in the secondary irreversible aggregation of platelets.