(Received for publication, November 27, 1995, and in revised form, September 18, 1996)
From the The intracellular thiol protease calpain
catalyzes the limited proteolysis of various focal adhesion structural
proteins and signaling enzymes in adherent cells. In human platelets,
calpain activation is dependent on fibrinogen binding to integrin
Calpains are a family of calcium-dependent cysteine
proteinases widely expressed in mammalian cells (1-3). Activation of these enzymes occurs in response to a wide range of physiological stimuli and is associated with limited proteolysis of several key
cellular proteins, including the c-Fos and c-Jun transcription factors
(4), the cytoskeletal proteins talin and actin-binding protein
(filamin) (5), and multiple signaling enzymes, including protein kinase
C, pp60c-src, and the tyrosine phosphatase
PTP-1B1 (6-8). Although calpain-mediated
proteolysis has been implicated in a broad range of pathophysiological
processes, including postischemic tissue damage and degenerative
diseases (3), the precise role of these enzymes in cell function has
not been established.
Calpains have been localized to points of attachment between cells and
the extracellular matrix (focal adhesions) and in the cytoskeletal
fraction of thrombin-stimulated platelets (9, 10). The recruitment of
calpain to these sites is thought to promote its activation by membrane
phospholipids and calcium and to co-localize it with target substrates
(11). A growing number of these substrates have been identified in
focal adhesions, in which they may participate in the assembly of
cytoskeletal signaling complexes and in anchoring integrin adhesion
receptors to the contractile cytoskeleton. Once anchored, integrins
form a stable transmembrane linkage between extracellular matrix
proteins and cytoskeletal elements, thereby allowing the extracellular
transmission of cytoskeletal contractile forces necessary for fibrin
clot retraction, wound healing, and tissue morphogenesis.
Studies in thrombin-stimulated platelets have demonstrated
calpain-catalyzed proteolysis of multiple focal adhesion structural proteins and signaling enzymes (7, 12). Many of these proteins translocate to the cytoskeleton in an aggregation-dependent
manner and participate in the formation of integrin-rich cytoskeletal signaling complexes. The formation of these complexes is thought to be
critical for the tyrosine phosphorylation of multiple cytoskeletal proteins and for the stable incorporation of integrin
Calpeptin was obtained from Biomol Research
Laboratories (Plymouth Meeting, PA). Ionophore A23187 and calpain
inhibitor I were from Calbiochem. All other materials were from sources
we have described previously (13, 14).
Anti-phosphotyrosine MAbs PY20 and 4G10 were
supplied by ICN Biomedicals Inc. (Costa Mesa, CA). Anti-talin MAb 8d4
was purchased from Sigma.
Anti-pp60c-src MAb 327 was a kind donation from Dr.
Joan Brugge (University of Pennsylvania). Anti-GP IIb MAb SZ22 was
kindly donated by Dr. Michael Berndt (Baker Medical Research Institute,
Melbourne, Victoria, Australia). Both anti-calpain preautolytic and
postautolytic polyclonal antibodies were kind donations from Dr.
Takaomi C. Saido (Tokyo Metropolitan Institute of Medical Science).
Both anti-mouse and anti-rabbit peroxidase-conjugated IgG were from
Silenus Laboratories (Hawthorn, Victoria, Australia).
Human platelets were obtained from healthy volunteers who
had not taken antiplatelet medication in the preceding 2 weeks and washed as described previously (14). Washed platelets were finally resuspended in modified Tyrode's buffer (10 mM Hepes, 12 mM NaHCO3, pH 7.5, 137 mM NaCl, 2.7 mM KCl, and 5 mM glucose), and preincubated with either calpeptin (2-100 µg/ml), calpain inhibitor I (2-10 µM), or vehicle (0.3% Me2SO) for 30 min at
room temperature. Washed platelets (3 × 108/ml) were
stimulated with 0.01-1 µM ionophore A23187 and/or
thrombin (1 unit/ml) in the presence of the indicated concentration of CaCl2. Fibrinogen (0.3 mg/ml) was included in the platelet
suspensions when ionophore A23187 was used as a single agonist. All
aggregations were initiated by stirring the platelet suspensions at 950 rpm for 10 min at 37 °C in a four-channel automated platelet
analyzer (Kyoto Daiichi, Japan). The extent of platelet aggregation was defined arbitrarily as the percentage of change in optical density as
measured by the automated platelet analyzer.
Washed platelets were
lysed with 1 volume of 10 × Triton X-100 lysis buffer (200 mM Tris-HCl, pH 7.4, 10% Triton X-100, 10 mM
EGTA, 20 mM EDTA, 2 mM sodium vanadate, 250 µg/ml phenylmethylsulfonyl fluoride, and 50 µM calpain
inhibitor I) to 9 volume of platelets, then gently agitated for 60 min
at 4 °C. Triton X-100-soluble and -insoluble (cytoskeletal)
fractions were prepared by centrifugation for 4 min at 15,000 × g, as described previously (15). Whole cell lysates were
prepared by lysing activated platelets in Laemmli reducing buffer
(0.125 M Tris-HCl, pH 6.8, 5 mM EDTA, 20%
glycerol, 4% SDS, 0.002% bromphenol blue, and 10%
Equal quantities of platelet extracts were
separated by 7.5% SDS-PAGE, under reducing conditions, then
transferred to PVDF membranes. Western blots were performed as
described by Towbin et al. (16), using specific primary
antibodies, followed by horseradish peroxidase-conjugated secondary
antibodies. Blots were developed using ECL according to the
manufacturer's instructions (Amersham International).
Clot retraction studies using platelet-rich
plasma (PRP) or washed platelets were performed as described (13), with
some minor modifications. Platelet-rich plasma (3 × 108 platelets/ml) was anticoagulated with 2 mM
EGTA and 2 mM MgCl2 and activated with thrombin
(5 units/ml) at room temperature in the presence or absence of 10 mM calcium. Reaction mixtures were either left unstirred or
were stirred for a maximum period of 45-60 s to induce platelet
aggregation. Washed platelets were pretreated with calpeptin (2-100
µg/ml), calpain inhibitor I (2-10 µM), or vehicle
(0.3% Me2SO) for 30 min prior to activation with thrombin
(1 unit/ml) and/or ionophore A23187 (0.01-1 µM). For studies with ionophore A23187 alone, atroxin (0.1 µg/ml) was added to
induce clot formation. In all studies with ionophore A23187, the
platelets were left unstirred during the assay. When thrombin was used
as the agonist, platelets were stirred for a maximum period of 45-60
s, unless otherwise indicated. Reproducible clot retraction results in
thrombin-aggregated washed platelets were dependent on not stirring the
platelets excessively, as the formation of large aggregates resulted in
their sedimentation within the tube and ineffective clot retraction.
For calcium dose-response studies, the free ionized calcium
concentration was maintained using Ca2+ and EGTA buffers
(17). In both the PRP and washed platelet assays, clot retraction was
assessed after 60 min of platelet activation. The extent of clot
retraction (percentage of clot retraction) was quantitated by measuring
the residual volume of serum (after removal of the fibrin clot) in each
reaction mixture and expressing this as a percentage of the total
reaction volume.
SDS-PAGE was performed according to
the method of Laemmli (18). Fibrinogen was purified from fresh frozen
plasma as described previously by Jakobsen and Koerulf (19). Protein
concentrations were measured using the Bio-Rad protein assay with
bovine serum albumin as a standard.
Studies in human platelets have demonstrated that several
cytoskeletal-associated signaling enzymes are substrates for activated calpain. These enzymes include the Src family of nonreceptor tyrosine kinases and the nonreceptor tyrosine phosphatase PTP-1B (7, 20, 21). As
these enzymes are highly expressed in platelets and translocate to the
cytoskeletal fraction of aggregated platelets, they are likely to play
a major role in regulating the tyrosine phosphorylation status of
cytoskeletal proteins. Although the precise role of these
phosphorylation events has yet to be clearly established, studies with
tyrosine kinase inhibitors have suggested a potentially important role
for these enzymes in regulating the cytoskeletal attachment of integrin
pp60c-src is the major protein
tyrosine kinase identified in platelets, constituting 0.2-0.4% of
total platelet protein (22). In our initial studies, we examined the
time course for pp60c-src cleavage in ionophore
A23187- and thrombin-stimulated platelets and correlated this with
platelet aggregation. As demonstrated in Fig. 1,
pp60c-src cleavage occurred well after the
initiation of platelet aggregation by ionophore A23187 (1 µM), with no cleavage detected at 1 min, 20% proteolysis
by 2 min, and complete proteolysis after 5 min of platelet stimulation.
Consistent with a role for platelet aggregation in calpain activation
and pp60c-src cleavage (5, 6, 12, 23), we found
that not stirring ionophore A23187-stimulated platelets prevented
platelet aggregation and significantly delayed
pp60c-src cleavage. In contrast, in
thrombin-stimulated platelets, pp60c-src cleavage
was completely dependent on integrin
We examined the possibility
that calpain-mediated proteolysis of pp60c-src may
regulate the cytoskeletal association of this kinase. Studies were
performed in ionophore A23187-stimulated platelets, as this agonist has
the unique ability to stimulate platelet activation in the absence of
calpain activation when used at low concentrations (0.01-0.1
µM), whereas at higher concentrations (1 µM) it induces both platelet and calpain activation. The
results from these experiments are presented in Fig. 2, A
and B, and demonstrate that low
concentrations of ionophore A23187 (0.01-0.1 µM)
stimulate the redistribution of pp60c-src from the
Triton X-100-soluble (Fig. 2A, I) to the cytoskeletal (Fig.
2A, II) fraction of aggregated platelets. With higher
concentrations of ionophore A23187 (1 µM),
pp60c-src was completely proteolyzed to its 52- and
47-kDa forms (Fig. 2A, I) and was no longer associated with
the cytoskeleton (Fig. 2A, II). Time course studies in
ionophore A23187-stimulated platelets (1 µM) revealed an
initial increase in the cytoskeletal content of
pp60c-src, which was followed by its proteolysis
and dissociation from the cytoskeleton (Fig. 2B, I). This
time-dependent cytoskeletal dissociation of
pp60c-src correlated with the appearance of the 52- and 47-kDa proteolytic fragments in the Triton X-100-soluble fraction
(data not shown). An important role for calpain in regulating the
cytoskeletal association of pp60c-src was confirmed
by the ability of calpeptin to prevent pp60c-src
cleavage and its subsequent dissociation from the cytoskeleton (Fig.
2B, II).
Recent studies in thrombin-stimulated platelets have suggested that the
calpain-mediated cleavage of nonreceptor tyrosine kinases and
phosphatases in human platelets leads to a substantial reduction in
protein tyrosine phosphorylation (24). Many of these proteins are
phosphorylated in an aggregation-dependent manner and are
specifically localized within the cytoskeletal fraction of aggregated
platelets (25, 26). We therefore performed antiphosphotyrosine
immunoblot analysis on the cytoskeletal extracts of ionophore
A23187-stimulated platelets to examine for time-dependent changes in cytoskeletal protein phosphorylation. As demonstrated in
Fig. 2B, tyrosine-phosphorylated proteins of 138, 110-90,
80, 71, 60-50, and 38 kDa were consistently identified within the cytoskeletal fraction of aggregated platelets. Consistent with previous
reports (24), we found that the tyrosine phosphorylation of
cytoskeletal proteins was a transient phenomenon, with
dephosphorylation occurring within 2 min of platelet stimulation (Fig.
2B, I). The pretreatment of platelets with either calpeptin
(Fig. 2B, II) or calpain inhibitor I (data not shown)
completely abolished calpain-mediated cleavage of
pp60c-src and the tyrosine phosphatase PTP-1B (data
not shown) and dramatically reduced these dephosphorylation events.
These observations are consistent with the hypothesis that the cleavage
of cytoskeletal-associated nonreceptor tyrosine kinases and
phosphatases regulates the tyrosine phosphorylation status of multiple
cytoskeletal proteins.
Our previous studies have suggested that the
phosphorylation of cytoskeletal proteins on tyrosine residues by
nonreceptor tyrosine kinases may be important for regulating the
cytoskeletal attachment of integrin
To confirm that these inhibitory effects on platelet-mediated clot
retraction were due to calpain activation, we examined clot retraction
in a washed platelet assay system. With this assay it was much easier
to examine the effects of pharmacological inhibitors of calpain, as
these platelet suspensions contained no plasma components, such as
albumin or plasma lipoproteins, which may bind these lipophilic
compounds and sequester them from platelets. Furthermore, to exclude
the possibility that the reduction in clot retraction observed in
aggregated platelets was a technical artifact due to uneven platelet
dispersion throughout the fibrin clot, we activated calpain in the
absence of platelet stirring and aggregation by stimulating platelets
with ionophore A23187 (1 µM). This agonist was
particularly useful in these studies, as it has the unique ability to
activate calpain in the absence of platelet aggregation (27). As
demonstrated in Fig. 3B, platelet stimulation by thrombin (1 unit/ml) alone (Fig. 3b, lane 1) or thrombin with
calpeptin (100 µg/ml) (Fig. 3B, lane 2)
resulted in 90 ± 7% and 90 ± 9% (n = 5)
retraction of fibrin clots, respectively. However, in the presence of
ionophore A23187, thrombin-stimulated clot retraction was reduced to
49 ± 9% (n = 5) (Fig. 3B, lane 3). This reduction in clot retraction was prevented by pretreating platelets with 100 µg/ml calpeptin (83 ± 5%) (Fig.
3B, lane 4).
To further strengthen our hypothesis that calpain is the responsible
protease mediating relaxed fibrin clot retraction, we performed
ionophore A23187 dose-response studies. We monitored calpain activation
in these studies using antibodies against the inactive large subunit of
calpain (80 kDa) and the autoproteolytic activated form (76 kDa).
Previous studies in human platelets have demonstrated
calcium-dependent autoproteolytic conversion of the 80-kDa
subunit of calpain to its 76-kDa active form (3). The antibodies used
in these studies have been raised against synthetic peptides
corresponding to the N-terminal sequence of the large subunit of both
forms of calpain (28). Immunoblot analysis of whole cell lysates with
these antibodies represents a sensitive, specific, and direct means of
monitoring calpain activation within the cell (29). As shown in
Fig. 4, concentrations of ionophore A23187 that activate
platelets without activating calpain (0.01 and 0.05 µM),
as monitored by calpain autolysis and pp60c-src
cleavage, resulted in 72.7 ± 3.5 and 70 ± 5.4% retraction
of fibrin clots, respectively. Higher concentrations of ionophore A23187 (0.25 and 1 µM) resulted in calpain activation and
pp60c-src cleavage and were associated with a
substantial reduction in clot retraction (46 ± 2.8 and 37.4 ± 5.2%, respectively). The chelation of extracellular calcium by the
addition of 1 mM EGTA and 2 mM
MgCl2 to the platelet reaction mixtures abolished calpain activation by 1 µM ionophore A23187 and restored
effective clot retraction (72.3 ± 2.9%).
Further evidence supporting a role for calpain in the regulation of
clot retraction stemmed from calcium dose-response studies. Previous
reports have demonstrated an absolute requirement for extracellular
calcium for calpain activation induced by both pharmacological and
physiological agonists (2). As demonstrated in Fig. 5, A and
B, calpain activation in both ionophore
A23187 and thrombin-stimulated platelets required the presence of low
micromolar concentrations of extracellular calcium. Higher
concentrations of extracellular calcium were associated with a
progressive increase in calpain activation, as monitored by either
calpain autolysis or calpain substrate proteolysis, with maximal
activation observed with 1.0 mM calcium (Fig. 5,
A and B). In each of these experiments, there was
a strong correlation between the extent of calpain activation and the
reduction in clot retraction. Furthermore, in all of the studies
reported here the inhibitory effects of calpain were limited to the
clot retraction process, with normal platelet aggregation and
[14C]serotonin release in response to thrombin or
ionophore A23187 (data not shown).
Calcium dose response for calpain activation
and relaxed clot retraction. A, washed platelets (3 × 108/ml) were activated with ionophore A23187 (1 µM) for 30 min, in the presence of EGTA (1 mM) and MgCl2 (2 mM) or the
indicated concentrations of CaCl2, without stirring.
Platelets were lysed in Laemmli reducing buffer, and the whole cell
lysates were examined for pp60c-src proteolysis
(upper panel) or calpain activation (
The ability of cells to transmit cytoskeletal
contractile forces to extracellular matrices is dependent on the
anchorage of integrins to the cytoskeleton. The stable association of
integrins with actin filaments requires a number of intermediary
proteins, including talin,
We correlated the effect of talin cleavage on the cytoskeletal
attachment of integrin To examine in more detail the relationship between
calpain activation and the cytoskeletal attachment of integrin
In further studies, we examined the ability of calpeptin to restore
clot retraction by thrombin-aggregated platelets. Reproducible clot
retraction results in these experiments were dependent on limiting the
duration of platelet stirring for a maximum of 45-60 s after the
addition of thrombin. Stirring the platelet reaction mixtures for
longer periods resulted in the formation of large platelet aggregates,
which tended to sediment in the tube and retract fibrin clots poorly.
Consistent with our studies in platelet-rich plasma, the stirring of
thrombin-stimulated washed platelets for 45-60 s dramatically reduced
the extent of clot retraction (Fig. 7B). The pretreatment of
these cells with calpeptin, prior to the addition of thrombin, restored
the ability of these aggregated platelets to retract fibrin clots. As
with ionophore A23187-stimulated platelets, the effect of calpeptin on
the clot retraction process was dose-dependent (Fig.
7B) and correlated with its ability to inhibit calpain
activation (data not shown).
The studies presented in this article define an important role for
calpain in the regulation of multiple postaggregation events in human
platelets. We have demonstrated under a variety of different experimental conditions that the calpain-catalyzed cleavage of several
focal adhesion structural proteins and signaling enzymes in human
platelets leads to a selective defect in the ability of platelets to
retract fibrin clots. This reduction in clot retraction was associated
with reduced incorporation of integrin
Although there are a number of cysteine and serine proteases in human
cells, we have provided several lines of evidence suggesting that
calpain is likely to be the responsible platelet protease regulating
fibrin clot retraction. First, relaxed fibrin clot retraction was only
observed under experimental conditions that promoted calpain
activation. These conditions include the requirement for extracellular
calcium in both thrombin- and ionophore A23187-stimulated platelets and
the necessity for platelet aggregation when thrombin was used as a
single agonist. Second, dose-response studies in ionophore
A23187-stimulated platelets revealed a close correlation between
calpain activation and relaxed clot retraction. Third, pretreatment of
platelets with two different inhibitors of calpain restored clot
retraction in a dose-dependent manner. Fourth, an excellent
correlation was observed between the extent of clot retraction and the
degree of calpain activation under all experimental conditions
examined. Finally, the ability of calpain to proteolyze cytoskeletal
proteins involved in regulating cellular contractile processes is
consistent with a role for this protease in the regulation of clot
retraction.
Although previous studies have indicated an important role for calpain
in the regulation of postaggregation responses, such as the release of
procoagulant-rich microparticles from the platelet membrane, a role for
calpain in the regulation of fibrin clot retraction has not been
established (23, 31). One of the major technical obstacles in examining
the role of calpain in clot retraction is the need to induce platelet
aggregation to activate the protease. The formation of platelet
aggregates may lead to uneven platelet dispersion throughout the fibrin
clot, resulting in an artifactual decrease in clot retraction. To
overcome this technical problem we have used washed platelets treated
with the pharmacological agonist ionophore A23187. This agonist has
well characterized effects on platelet function and has the advantage
of activating calpain in the absence of platelet aggregation (27).
Several lines of evidence suggest that the results we have obtained in ionophore A23187-stimulated platelets are not unique to this agonist and are likely to be physiologically relevant. First, the inhibitory effects of calpain activation on clot retraction were not limited to
ionophore A23187-stimulated platelets, as we observed a similar functional defect in thrombin-stimulated platelets under assay conditions that favored calpain activation. Second, calpain-mediated proteolysis of Src family kinases, PTP-1B and talin is also observed in
platelets activated by physiological agonists, such as thrombin and
collagen, and is associated with the dissociation of integrin Previous studies in thrombin- and collagen-stimulated platelets have
suggested that calpain-mediated cleavage of cytoskeletal proteins is
responsible for the dissociation of as much as 16% of total platelet
integrin In previous studies we have demonstrated that tyrosine phosphorylation
events in human platelets play a key role in regulating the
cytoskeletal attachment of integrin The studies reported here on human platelets clearly have important
implications for adhesion processes in other cells. Calpain is a
ubiquitous protease, which has been localized to focal adhesions in a
variety of adherent cells (9). The ability of calpain to cleave a
growing number of focal adhesion proteins suggests a potentially
important role for this enzyme in the regulation of these cellular
structures. Our studies indicate that one of the functions of this
protease is to regulate the transmission of cytoskeletal contractile
forces to extracellular matrices. Furthermore, our studies suggest that
calpain may also have an important signal-terminating role within the
cell. The ability of integrins to promote calpain activation, leading
to the proteolysis and down-regulation of cytoskeletal-associated
signaling enzymes, suggests a potentially novel means by which these
adhesion receptors can limit their own signaling function. Whether
these proteolytic events have flow-on effects to other signaling
pathways linked to cell adhesion will be an important area for future
investigation.
Department of Medicine,
IIb
3 and subsequent platelet aggregation,
suggesting a potential role for this protease in the regulation of
postaggregation responses. In this study, we have examined the effects
of calpain activation on several postaggregation events in human
platelets, including the cytoskeletal attachment of integrin
IIb
3, the tyrosine phosphorylation of
cytoskeletal proteins, and the cellular retraction of fibrin clots. We
demonstrate that calpain activation in either washed platelets or
platelet-rich plasma is associated with a marked reduction in
platelet-mediated fibrin clot retraction. This relaxation of clot
retraction was observed in both thrombin and ionophore A23187-stimulated platelets. Calcium dose-response studies
(extracellular calcium concentrations between 0.1 µM and
1 M) revealed a strong correlation between calpain
activation and relaxed clot retraction. Furthermore, pretreating
platelets with the calpain inhibitors calpeptin and calpain inhibitor I
prevented the calpain-mediated reduction in clot retraction. Relaxed
fibrin clot retraction was associated with the cleavage of several
platelet focal adhesion structural proteins and signaling enzymes,
resulting in the dissociation of talin, pp60c-src,
and integrin
IIb
3 from the contractile
cytoskeleton and the tyrosine dephosphorylation of multiple
cytoskeletal proteins. These studies suggest an important role for
calpain in the regulation of multiple postaggregation events in human
platelets. The ability of calpain to inhibit clot retraction is likely
to be due to the cleavage of both structural and signaling proteins
involved in modulating integrin-cytoskeletal interactions.
IIb
3 into the contractile cytoskeleton.
In this report, we have investigated the effects of calpain activation
on a number of postaggregation events in human platelets. Our studies
demonstrate that the calpain-catalyzed proteolysis of focal adhesion
structural proteins and signaling enzymes leads to a selective defect
in the ability of platelets to retract fibrin clots. This
calpain-mediated relaxation of clot retraction was associated with the
detachment of integrin
IIb
3 from the
contractile cytoskeleton and the dephosphorylation of multiple
cytoskeletal proteins on tyrosine residues.
Materials
-mercaptoethanol) and then boiled for 10 min.
IIb
3 (13). We therefore aimed to
investigate the effects of calpain activation on the level of tyrosine
phosphorylation of cytoskeletal proteins and to correlate these effects
with specific platelet postaggregation events, including the
cytoskeletal attachment of integrin
IIb
3 and fibrin clot retraction.
IIb
3-mediated platelet aggregation (data
not shown) (6). Inhibition of calpain-catalyzed cleavage of
pp60c-src by calpeptin had no effect on platelet
aggregation induced by ionophore A23187 (Fig. 1) or thrombin (data not
shown). These observations are consistent with the hypothesis that
calpain-mediated proteolysis of pp60c-src and other
target substrates is likely to regulate platelet responses that
occur after platelet aggregation.
Fig. 1.
Time course of platelet aggregation and
pp60c-src cleavage in ionophore A23187-stimulated
platelets. Washed platelets (3 × 108/ml) were
pretreated with 0.3% Me2SO (DMSO) or calpeptin
(100 µg/ml) for 30 min at room temperature and then activated with ionophore A23187 (1 µM) for the indicated time points
while stirring. Platelet aggregation was examined in a four-channel
platelet aggregometer, and the extent of aggregation was determined, as
described under "Experimental Procedures." Results represent the
mean ± S.E. (bars) of three separate experiments.
Platelet aliquots were removed at the indicated time points and lysed
in Laemmli reducing buffer, and total lysates were examined for
pp60c-src proteolysis by immunoblot analysis, as
described under "Experimental Procedures." The extent of
proteolysis was assessed by performing densitometry on the
pp60c-src immunoblots. These results are from one
experiment, representative of three.
[View Larger Version of this Image (33K GIF file)]
Fig. 2.
Effect of calpain activation on the
cytoskeletal association of pp60c-src and the
tyrosine phosphorylation of cytoskeletal proteins. Washed
platelets (3 × 108/ml) were pretreated with 0.3%
Me2SO or calpeptin (100 µg/ml) for 30 min at room
temperature in the presence of CaCl2 (1 mM). Platelets were stimulated with the indicated concentrations of ionophore A23187 while stirring. Platelets were lysed at the indicated time points and fractionated into Triton X-100-soluble and cytoskeletal extracts. The platelet extracts were separated by 7.5% SDS-PAGE, transferred to PVDF membranes, and examined for
pp60c-src content and protein tyrosine
phosphorylation by immunoblot analysis. A, effect of calpain
cleavage on the subcellular localization of
pp60c-src. I, Triton X-100-soluble
fraction; II, cytoskeletal fraction. B,
correlation between cytoskeletal localization of
pp60c-src and tyrosine phosphorylation of
cytoskeletal proteins in the absence (I) and presence
(II) of calpeptin.
[View Larger Version of this Image (23K GIF file)]
IIb
3
and the cellular retraction of fibrin clots (13). The ability of
calpain to regulate the phosphorylation status of cytoskeletal proteins
and to cleave focal adhesion structural proteins involved in anchoring
integrins to the cytoskeleton has suggested a potential role for this
protease in the regulation of clot retraction. We therefore performed a
series of experiments in PRP and washed platelets to correlate calpain
activation with changes in clot retraction. Previous studies examining
the role of activated calpain in the regulation of clot retraction have reported no differences in the rate and extent of clot retraction in
the presence or absence of calpeptin (23). However, it is unlikely that
calpain was substantially activated in these studies, as the platelets
were not aggregated during the clot retraction assay. In preliminary
studies, we examined clot retraction in a PRP assay system, as PRP is
the normal physiological medium used for platelet aggregation and clot
retraction studies. The stimulation of platelets with thrombin (5 units/ml) alone (Fig. 3A,I, tube
1) or thrombin (5 units/ml) in the presence of calcium (10 mM), without stirring (i.e. no aggregation; Fig.
3A, I, tube 3), resulted in 88 ± 2% (Fig.
3A, II, lane 1) and 84 ± 6% (Fig. 3A, II, lane 3) (n = 5)
retraction of fibrin clots, respectively. However, when platelets were
stimulated with thrombin (5 units/ml) and calcium (10 mM)
while stirring for 45 s (i.e. conditions that promote
platelet aggregation and calpain activation; Fig. 3A, I, tube 4), the extent of clot retraction was markedly
reduced to 32 ± 11% (n = 5) (Fig. 3A,
II, lane 4). It was unlikely that this defect in clot
retraction was purely a technical artifact related to the formation of
large platelet aggregates, as an 83 ± 5% (n = 5)
(Fig. 3A, II, lane 2) retraction of fibrin clots was observed in thrombin-aggregated platelets when calcium was omitted
from the reaction mixture (Fig. 3A, I, tube
2).
Fig. 3.
Effect of calpain activation on
platelet-mediated fibrin clot retraction. A, I. Thrombin (5 units/ml) was added to platelet-rich plasma (3 × 108/ml) in the presence or absence of CaCl2 (10 mM) as indicated. The platelet suspensions were either left
unstirred or stirred for 45 s to induce the formation of platelet
aggregates. Tube 1, thrombin (5 units/ml) without stirring;
tube 2, thrombin (5 units/ml) with stirring; tube
3, thrombin (5 units/ml) and calcium (10 mM) without
stirring; tube 4, thrombin (5 units/ml) and calcium (10 mM) with stirring. II, clot retraction was
quantitated by measuring the residual volume of serum after removal of
the clot, as described under "Experimental Procedures." These
results represent the mean ± S.E. (bars) of five
separate experiments. B, washed platelets (3 × 108/ml) were pretreated with 0.3% Me2SO or
calpeptin (100 µg/ml) in the presence of CaCl2 (1 mM) for 30 min at room temperature. Platelets were
activated with thrombin (5 units/ml) in the presence or absence of
ionophore A23187 (1 µM) without stirring. Clot retraction was quantitated as described above. These results represent the mean ± S.E. (bars) of five separate experiments.
[View Larger Version of this Image (32K GIF file)]
Fig. 4.
Ionophore dose response for calpain
activation and relaxed clot retraction. Washed platelets (3 × 108/ml) containing 1 mg/ml fibrinogen were activated
with the indicated doses of ionophore A23187 for 60 min in the presence
of CaCl2 (1 mM) or EGTA (1 mM) and
MgCl2 (2 mM) without stirring. Platelets were
lysed in Laemmli reducing buffer, and the whole cell lysates were
examined for calpain autolysis and pp60c-src
proteolysis by immunoblot analysis (upper panels). These
results are from one experiment, representative of three. In parallel experiments, washed platelets were stimulated with the indicated concentration of ionophore A23187 in the presence of atroxin (0.1 µg/ml) and fibrinogen (1 mg/ml). The extent of clot retraction was
determined as described in Fig. 2. These results represent the
mean ± S.D. (bars) of four separate experiments
(histogram).
[View Larger Version of this Image (39K GIF file)]
Fig. 5.
) by immunoblot analysis. % Inactive Calpain, amount of the intact form of
calpain (80 kDa) in whole cell lysates, as determined by densitometric measurements of calpain immunoblots. These results are from one experiment, representative of three. In parallel experiments, platelets
were activated with ionophore A23187 (1 µM) in the
presence of atroxin (0.1 µg/ml) and exogenous fibrinogen (1 mg/ml).
The extent of clot retraction was quantitated after 60 min
(histogram), as described in Fig. 2. These results represent
the mean ± S.D. (bars) of four separate experiments.
B, washed platelets (3 × 108/ml) were
activated with thrombin (1.0 units/ml) in the presence of the indicated
doses of CaCl2 and stirred for 45-60 s to induce platelet
aggregation. After 30 min of stimulation, platelets were lysed with
reducing buffer, and the whole cell lysates were analyzed for calpain
activation (
) as described above. In parallel experiments, platelets
were activated with thrombin in the presence of exogenous fibrinogen
(1 mg/ml) and the indicated concentrations of CaCl2. The platelets were stirred for 45-60 s, then left unstirred for 60 min. The extent of clot retraction (Histogram) was
quantitated as described in Fig. 2. These results represent the
mean ± S.D. (bars) of four separate experiments.
[View Larger Version of this Image (27K GIF file)]
IIb
3 from the Contractile
Cytoskeleton
-actinin, and vinculin (30). We
investigated whether the calpain-catalyzed cleavage of talin was
associated with the dissociation of either talin or integrin
IIb
3 from the contractile cytoskeleton.
Immunoblot analysis of total cell lysates, with an antibody that
recognizes the native 230-kDa form of talin and the largest 190-kDa
talin fragment, revealed rapid proteolysis of talin in ionophore
A23187-stimulated platelets (Fig. 6A).
Cleavage of talin from its 230-kDa native form to the 190-kDa fragment
was observed within 15 s of platelet stimulation and was complete
by 3 min. In agreement with previous studies (23), we observed that
calpeptin (100 µg/ml) pretreatment of platelets dramatically slowed
the rate of talin cleavage but did not consistently prevent cleavage
altogether. The effect of this cleavage on the cytoskeletal association
of talin was investigated by fractionating platelets into Triton
X-100-soluble and -insoluble (cytoskeletal) extracts. Ionophore A23187
stimulation of platelets was associated with a progressive increase in
the cytoskeletal content of talin throughout the first 60 s of
platelet activation (Fig. 6B). The association of both the
230- and 190-kDa forms of talin with the cytoskeleton was transient,
however, with complete dissociation of the 190-kDa fragment observed
after 4 min of platelet stimulation. This cytoskeletal dissociation was
not due to further proteolysis of the fragment, as the total cell
levels of the 190-kDa fragment remained constant throughout the period
of examination (Fig. 6A). An important role for calpain in
regulating the cytoskeletal attachment of talin was confirmed in
calpeptin-treated platelets, in which reduced proteolysis of talin
prevented its dissociation from the cytoskeleton (Fig. 6B).
Fig. 6.
Time course for talin cleavage and the
dissociation of talin and integrin IIb
3
(GP IIb) from the contractile cytoskeleton. Washed platelets
(3 × 108/ml) were pretreated with 0.3%
Me2SO or calpeptin (100 µg/ml) for 10 min at room
temperature and then stimulated with ionophore A23187 (1 µM) for the indicated time points while stirring.
Platelets were lysed and fractionated into Triton X-100-soluble or
cytoskeletal extracts, as described under "Experimental
Procedures." Whole cell lysates or cytoskeletal extracts were
subjected to immunoblot analysis using monoclonal antibodies against
talin or GP IIb. Cytoskeletal actin was quantitated by densitometry
after staining the PVDF membranes with Coomassie Brilliant Blue.
Results are from one experiment, representative of three. A,
talin proteolysis in whole cell lysates; B, association of
talin with the cytoskeleton; C, association of GP IIb with
the cytoskeleton; D, comparative time course for
cytoskeletal dissociation of GP IIb and talin.
[View Larger Version of this Image (26K GIF file)]
IIb
3. Time course
experiments revealed a close correlation between the cytoskeletal
dissociation of talin with that of integrin
IIb
3 (GP IIb) (Fig. 6, B-D).
As with talin, pretreating platelets with calpeptin resulted in both a
higher and more sustained level of integrin
IIb
3 incorporation into the cytoskeleton.
Previous reports have suggested that the dissociation of integrin
IIb
3 from the membrane cytoskeleton does
not correlate with talin cleavage (23). Our studies are consistent with
these findings, as we observed extensive proteolysis of talin within the first 30 s of platelet stimulation, yet the bulk of integrin
IIb
3 (GP IIb) did not dissociate from the
cytoskeleton until 2-3 min after platelet activation (Fig. 6, compare
A and B with C). To exclude the
possibility that the dramatic reduction in the cytoskeletal content of
talin and integrin
IIb
3 was due to a
global reduction in total cytoskeletal protein, the talin and integrin
IIb
3 immunoblots were stained with
Coomassie Brilliant Blue. The total amount of filamentous actin (Fig.
6D) and a range of other cytoskeletal proteins (not shown)
increased approximately 2-3-fold following ionophore A23187
stimulation of platelets and was largely maintained throughout the
period of examination. Hence it is unlikely that the calpain-mediated
reduction in the cytoskeletal content of integrin
IIb
3 and talin is attributable to gross changes in the total amount of cytoskeletal protein.
IIb
3, and Clot
Retraction
IIb
3, we performed experiments on
ionophore A23187-stimulated platelets that had been resuspended in
either calcium-free or calcium-containing buffers. Platelet stimulation
with 1 µM ionophore A23187 for 5 min in the presence of 1 mM CaCl2 resulted in complete conversion of
inactive calpain to its activated form. Under these assay conditions, integrin
IIb
3 was no longer detectable
within the cytoskeleton by immunoblot analysis (Fig.
7A). In contrast, the resuspension of
platelets in buffers containing 1 mM EGTA and 2 mM MgCl2, prior to ionophore A23187
stimulation, prevented calpain activation and restored the association
of integrin
IIb
3 with the contractile cytoskeleton. Under each of these experimental conditions, the ability
of integrin
IIb
3 to associate with the
cytoskeleton correlated well with the ability of platelets to retract
fibrin clots. Further evidence supporting a role for calpain in
regulating the cytoskeletal attachment of integrin
IIb
3 stemmed from calpeptin dose-response
studies. As demonstrated in Fig. 7A, pretreating platelets
with doses of calpeptin greater than 2 µg/ml inhibited calpain
activation in a dose-dependent manner. In these
dose-response experiments there was an excellent correlation between
the extent of calpain activation, the amount of integrin
IIb
3 incorporated into the cytoskeleton,
and the extent of clot retraction. These results were not unique to
calpeptin-treated platelets, as calpain inhibitor I also restored clot
retraction by ionophore A23187-stimulated platelets in a
dose-dependent manner (data not shown).
Fig. 7.
Correlation between calpain activation, the
cytoskeletal association of integrin
IIb
3, and clot retraction. Washed platelets (3 × 108/ml) were pretreated with either 1 mM EGTA and 2 mM MgCl2, 0.3% Me2SO, or the indicated concentrations of calpeptin for 30 min at room temperature in the presence of fibrinogen (1.0 mg/ml). All
assays, except those containing EGTA (1 mM) and
MgCl2 (2 mM), were performed in the presence of
CaCl2 (1 mM). A, platelets were activated with ionophore A23187 (1 µM) in the presence of
atroxin (0.1 µg/ml) without stirring. Clot retraction was quantitated after 60 min of platelet stimulation. These results represent the
mean ± S.D. (bars) of four separate experiments. In
parallel experiments, ionophore A23187-stimulated platelets (30 min)
were lysed and fractionated into Triton X-100-soluble or cytoskeletal extracts. Whole cell lysates were subjected to immunoblot analysis using an antibody against the 80-kDa preautolytic form of calpain. Immunoblot analysis was performed on cytoskeletal extracts using a
monoclonal antibody against GP IIb. B, washed platelets were activated with thrombin (1 unit/ml) while stirring for 45-60 s to
induce platelet aggregation. Clot retraction was quantitated after 60 min of platelet stimulation. These results represent the mean ± S.D. (bars) of four separate experiments.
[View Larger Version of this Image (28K GIF file)]
IIb
3 into the contractile cytoskeleton
and the dephosphorylation of multiple cytoskeletal proteins on tyrosine
residues.
IIb
3 from the membrane cytoskeleton (31).
Third, the ability of calpain to regulate the cytoskeletal attachment
and signaling function of pp60c-src and PTP-1B is a
feature of both ionophore A23187- and thrombin-stimulated platelets (6,
7, 21). Finally, the calpain-induced dephosphorylation of multiple
platelet proteins that we have observed in ionophore A23187-stimulated
platelets has recently been reported in thrombin-aggregated platelets
(24).
IIb
3 from the membrane
cytoskeleton of aggregated platelets (31). Although talin is considered
to play a critical role in anchoring integrins to the contractile cytoskeleton, these studies have highlighted that the calpain-mediated cleavage of talin does not appear to correlate with the release of
integrin
IIb
3 from the cell surface. Our
time course experiments in ionophore A23187-stimulated platelets are
consistent with this possibility, as they clearly demonstrate that the
cytoskeletal dissociation of integrin
IIb
3 lags well behind the initial
cleavage of talin. These observations suggest a role for additional
calpain-mediated proteolytic events in dissociating
integrin-cytoskeletal contacts. A recent report has demonstrated that
the cytoplasmic domain of
3-integrins is cleaved at
multiple sites by calpain in vivo, raising the distinct
possibility that direct proteolysis of integrins can regulate their
association with the cytoskeleton (32).
IIb
3
(13). The studies reported in this article are consistent with these
findings, as they demonstrate a close correlation between cytoskeletal
protein dephosphorylation and the cytoskeletal dissociation of integrin
IIb
3. Furthermore, studies in human
fibroblasts have demonstrated that the tyrosine phosphorylation of
cytoskeletal proteins, following the ligation and aggregation of
integrins on the cell surface, is essential for stable interaction
between filamentous actin and integrins (33). Although these
observations highlight the importance of tyrosine phosphorylation
events in regulating cytoskeletal-integrin contacts, they provide
limited insight into the molecular events modulating this interaction.
For example, the level of tyrosine phosphorylation of talin,
-integrin, and vinculin is low in normal adherent cells, suggesting
that direct phosphorylation of these proteins is an unlikely mechanism
by which integrins become anchored to the cytoskeleton. In contrast,
the vinculin-binding proteins paxillin and tensin are prominent
tyrosine-phosphorylated proteins in focal adhesions (34). The
phosphorylation of paxillin may also be important for regulating its
association with other cytoskeletal proteins, such as vinculin and
talin. An attractive hypothesis is that the phosphorylated forms of
paxillin and/or tensin bind to vinculin and unmask its talin and actin
binding sites. This "active conformation" of vinculin may in turn
stabilize the interaction between integrins and the underlying
cytoskeleton (35). It is likely that the calpain-mediated cleavage of
tyrosine kinases and phosphatases leads to a reduction in the level of
phosphorylation of cytoskeletal proteins, such as paxillin and tensin.
The dephosphorylation of these proteins may undermine the stable
association of vinculin with actin filaments and talin, ultimately
leading to the disassembly of integrin-cytoskeletal contacts.
*
This work was funded by a grant from the National Health and
Medical Research Council of Australia. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a National Heart Foundation Postgraduate Science
Research Scholarship. To whom correspondence should be addressed. Tel.:
61-3-9895-0328; Fax: 61-3-9895-0332.
1
The abbreviations used are: PTP,
protein-tyrosine phosphatase;MAb, monoclonal antibody; PAGE,
polyacrylamide gel electrophoresis;GP, glycoprotein; PVDF,
polyvinylidene difluoride; PRP, platelet-rich plasma.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.