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The Role of Calpain in Stimulus-Response Coupling: Evidence That Calpain
Mediates Agonist-Induced Expression of Procoagulant Activity in Platelets
By Joan E.B. Fox, Clifford C. Reynolds, and Cary D. Austin
Although calpain (the Ca*+-dependent protease) is widely
distributed, its function is poorly understood. One cell in
which it becomes activated as a consequence of activation
of the cell is the blood platelet. The aim of the present
study was t o determine whether activation of calpain was
responsible for any of the responses of platelets t o stimulation. Platelets were incubated with calpeptin, a membranepenetrating inhibitor of calpain, before being exposed t o an
agonist. Concentrations of calpeptin that totally inhibited
the agonist-induced hydrolysis of actin-binding protein
(ABP) by calpain had no effect on many other responses
associated with platelet activation: phosphorylation of
myosin light chain, phosphorylation of P47, platelet shape
change, aggregation of platelets, secretion of granule
contents, or retraction of fibrin clots. However, these
concentrations of inhibitor decreased the agonist-induced
generation of procoagulant activity (assayed as the ability
of platelets t o catalyze the conversion of prothrombin t o
thrombin in the presence of factor V, and factor XJ. When
thrombin was the agonist, the amount of ABP that was
hydrolyzed was small; only a small component of the total
agonist-induced procoagulant activity was inhibited by
calpeptin. When collagen was the agonist, more ABP was
hydrolyzed and the amount of procoagulant activity generated was greater: calpeptin decreased the collageninduced procoagulant activity t o levels comparable with
those induced by thrombin in the presence of the inhibitor.
We suggest that there are at least t w o mechanisms by
which procoagulant activity is generated on activated
platelets and that the agonist-induced activation of calpain
mediates one of these mechanisms. These results show
that activation of calpain is a component of the stimulusresponse pathway in platelets.
0 1990 by The American Society of Hematology.
C
detected until 30 to 60 seconds after platelets are stimulated
with t h r ~ m b i nafter
, ~ aggregation has o ~ c u r r e d ,it~ has
. ~ been
suggested that activation of calpain is most likely to be
involved in the later events of platelet activation, eg, in the
cytoskeletal changes that are required for clot retraction to
occur.* Others have noted a correlation between the extent of
hydrolysis of ABP within platelets and the appearance of
procoagulant activity on the surface of platelet^.^ Despite the
fact that the calpain substrates that have been detected so far
are not hydrolyzed until after platelets have a g g ~ g a t e d , ~ . ~
other investigators have suggested that calpain plays an
essential role in mediating the thrombin-induced aggregation
of platelets’0-12or in regulating the activity of protein kinase
C (PKC)I3 or the activity of MLC kinase.I4 Yet others have
pointed out that the amount of substrate hydrolyzed by
calpain in aggregating platelets is so small that it is unlikely
that activation of calpain has any physiologic conseq~ence.’~
Evaluation of the role of calpain in signal-response coupling in platelets has been hampered by the absence of
membrane-penetrating inhibitors of the protease. Although
some workers have used leupeptin to evaluate the role of
calpain in mediating the responses of thrombin, their studies
have been difficult to interpret because leupeptin does not
readily cross cell membranes. Further, interpretation of these
studies has been complicated by the fact that leupeptin
inhibits the amidolytic activity of t h r ~ m b i n , ’ ~an
. ’ ~activity
that is required for thrombin to initiate stimulus-response
coupling.20 Recently, several groups have synthesized new
peptide i n h i b i t ~ r s ~that
l - ~ ~ are similar to leupeptin but
contain hydrophobic groups that enable them to cross the cell
membrane; they do not contain the argininal residue that is
responsible for the inhibitory action of leupeptin on
thrombin.” In the present study, we have used one of these
inhibitors (calpeptin22)to determine whether calpain mediates any of the platelet responses to activation.
The results of this study show that concentrations of
calpeptin that totally inhibit detectable hydrolysis of ABP
have no effect on agonist-induced phosphorylation of MLC,
phosphorylation of P47, shape change, aggregation, secretion
ALPAIN IS A cysteine protease that has a widespread
distribution, is optimally active at neutral pH, and is
dependent on the presence of Ca2+.’V2Because it is present in
the cytosol of cells, it could be responsible for mediating
cellular events that are initiated by an increase in the
concentrations of cytoplasmic Ca2+.The only cell in which
calpain has been shown directly to become active as a
consequence of stimulation of the cell is the latel let.^"
Activation of calpain results in the selective hydrolysis of
several proteins. These proteins include the cytoskeletal
proteins actin-binding protein (ABP)? talin? and spectrin,’
an unidentified protein of molecular weight (mol wt) 87,000,3
and several calmodulin-binding proteins that may represent
myosin light-chain (MLC) kinase, caldesmon, and the calmodulin-dependent phosphatase.6
The various responses that platelets undergo after stimulation by an agonist include a change in shape, extension of
filopodia, secretion of granule contents, expression of receptors for adhesive ligands, aggregation with other platelets,
and expression of procoagulant activity on their surface. The
platelets subsequently retract their filopodia, thus retracting
the clot of fibrin that binds externally to the developing
platelet aggregate. Because activation of calpain is not
From the Gladstone Foundation Laboratories for Cardiovascular
Disease, Cardiovascular Research Institute, Department of Pathology, University of California, San Francisco.
Submitted April 23, 1990: accepted August 24, 1990.
Supported by Research Grant No. HL306.57 (J.E.B.F.)from the
National Institutes of Health and by an Established Investigator
Award from the American Heart Association (J.E.B.F.).
Address reprint requests to Joan E.B. Fox, PhD, Gladstone
Foundation Laboratories for Cardiovascular Disease, PO BOX
40608, San Francisco, CA 941 40-0608.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 I990 by The American Society of Hematology.
0006-4971/90/7612-0018$3.00/0
2510
Blood, Vol76, No 12 (December 15). 1990: pp 2510-2519
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CALPAIN IN PLATELET FUNCTION
251 1
sequence Arg-Gly-Asp-Ser (RGDS). This RGDS peptide was a
generous gift of Dr David Phillips (COR Therapeutics, Inc, South
San Francisco, CA); it was added in saline to a final concentration of
300 pmol/L. Aliquots of the suspension (400 pL) were then
transferred to siliconized aggregometer tubes that contained 120 pg
of fibrinogen (Kabi Vitrum, Stockholm, Sweden). Thrombin was
added to a final concentration of 1.0 NIH U/mL, and the subsequent
retraction of clots was recorded photographically at intervals.
Analytical procedures. Platelet suspensions (3 x IO’ platelets/
MATERIALS AND METHODS
mL) were solubilized in the presence of a reducing agent and
analyzed on one-dimensional sodium dodecyl sulfate (SDS)Isolation of platelets. Venous blood was drawn from healthy
polyacrylamide gels by the method of Laemmli” using 3% acrylaadult donors, and platelets were isolated from it by centrifugation, as
mide in the stacking gel and a 5% to 20% exponential gradient of
de~cribed.’~
Platelets were resuspended in a Tyrode’s buffer containacrylamide in the resolving gel. Proteins were stained with Coomassie
ing 138 mmol/L sodium chloride, 2.9 mmol/L sodium bicarbonate,
Brilliant Blue. The distribution of ”P in polypeptides was deter0.36 mmol/L sodium phosphate, 5.5 mmol/L glucose, 1.8 mmol/L
mined by autoradiography of dried gels. Western blotting was
calcium chloride, and 0.49 mmol/L magnesium chloride, pH 7.4.
Platelets were labeled with [3ZP]phosphateas described e l s e ~ h e r e . ~ ~ performed by the method of Towbin et a128as described previ~usly.~
Affinity-purified ABP and talin antibodies were raised and characterIncubation of platelets with agonists. Platelets were incubated
ized as reported previ~usly.~
at 37OC in the absence or presence of the calpain inhibitors leupeptin
The concentration of platelets in suspensions of washed platelets
or calpeptin. Leupeptin (Vega Biochemicals, Tucson, AZ) was
was determined with a Coulter counter (Coulter Corp, Hialeah, FL).
added in saline; calpeptin was added in dimethyl sulfoxide (DMSO)
(J.T. Baker Chemical Co, Phillipsburg, NJ). The final concentration
RESULTS
of the DMSO was 0.2%. Calpeptin (2-Leu-Nle-H) was a generous
gift from Dr Toshimasa Tsujinaka (Osaka University Medical
To determine the concentration of calpeptin needed to
School, Osaka, Japan). Platelets were subsequently incubated with
inhibit agonist-induced activation of calpain, platelets were
20 pg of collagen/mL (Horme, Munich, West Germany), 1.0
incubated with various concentrations of the inhibitor for 5
National Institutes of Health (NIH) unit of thrombin/mL (kindly
minutes before the addition of an agonist. Preincubation of
provided by Dr J. W. Fenton I1 of the New York Department of
platelets with 10 pg of calpeptin/mL almost totally abolished
Health, Albany), or a combination of the two agonists. In some
the subsequent collagen-induced hydrolysis of ABP (Fig 1A)
experiments, platelets were incubated with the nonphysiologic agoby calpain. In contrast, higher concentrations of calpeptin
nist, ionophore A23187 (0.4 pmol/L) (Sigma Chemical Co, St
were required to inhibit the collagen-induced hydrolysis of
Louis, MO), which was added in a final concentration of 0.2%
(vol/vol) DMSO. Unless indicated otherwise, all incubations were
talin. Figure 1B shows the result of a typical experiment, in
performed in the presence of stirring.
which it can be seen that even when the calpeptin concentraAssessment of platelet aggregation and secretion. Luciferintion was 50 pg/mL, almost 50% of the talin was still
Luciferase reagent (Chronolog Corporation, Havertown, PA) was
hydrolyzed; and even at a calpeptin concentration of 200
added to the platelet suspension (3 x 10’ platelets/mL), which was
pg/mL, hydrolysis of talin was still not totally inhibited. The
then stirred with an agonist in a dual-channel lumiaggregometer
concentrations of calpeptin required to inhibit the thrombin(Chronolog). The agonist-induced aggregation of platelets was
induced hydrolysis of ABP or talin were similar to those
assessed by a change in transmittance of light through the suspenrequired to inhibit the collagen-induced hydrolytic events
sion, as detected in one channel of the lumiaggregometer. The
(data not shown). Because calpeptin is a hydrophobic molesecretion of adenosine triphosphate (ATP) from platelet granules
was assessed by the change in luminescence of the Luciferincule, it appears likely that it would preferentially partition
Luciferase reagent as measured in the second channel of the
into the plasma membrane. Thus, the preferential inhibition
lumiaggregometer. The amount of secreted ATP was calibrated by
of ABP hydrolysis may occur because this membrane-bound
the subsequent addition of known amounts of ATP.
pr~tein’~-~’
is hydrolyzed by a membrane-associated form of
Assessment of procoagulant activity. Procoagulant activity was
calpain while talin, which appears to be a soluble protein:3 is
assayed by a modification of the assay described by Bevers et a1?6
hydrolyzed by a soluble form of the protease.
Platelets were stirred with various agents at a concentration of 0.1 x
Leupeptin, a calpain inhibitor that does not readily cross
IO’ to 1.0 x lo9 platelets/mL. Platelets were then diluted to a final
cell membranes, totally inhibited the hydrolysis of ABP and
concentration of 0.02 to 0.25 x 10’ platelets/mL with a Tyrode’s
talin induced by thrombin (data not shown), presumably
solution that contained 0.6 nmol/L factor X, (Sigma), 1.1 nmol/L
because it inhibited the proteolytic action of thrombin.I6-l9
factor V, (Enzyme Research Laboratories, South Bend, IN), and 1.3
pmol/L prothrombin (Sigma). The amount of thrombin formed was
However, leupeptin had little inhibitory effect on the collagendetermined by removing 20 to 50 pL of the incubation, transferring
induced hydrolysis of ABP or talin (data not shown) unless it
the sample into 1 mL of the chromogenic substrate S2238 (150
was preincubated with platelets for 30 minutes at 37OC in the
pmol/L) (Helena Laboratories, Beaumont, TX), and determining
presence of 0.2% DMSO and at a concentration of 1 mg of
the rate of change of absorbance at 405 nm. A standard curve was
leupeptin/mL. Under these extreme conditions, a partial
obtained using purified thrombin. When activation of platelets was
inhibition of hydrolysis was observed (Fig 1A and B),
induced with thrombin, control assays that contained the same
presumably
because some leupeptin penetrated the memconcentration of thrombin (in the absence of platelets) were inbrane.
cluded.
Experiments were performed to determine whether any of
Assessmenf of clot refraction. Platelet suspensions (1 x IO9
the agonist-induced intracellular changes or functional replatelets/mL) were incubated for 5 minutes in the presence or
absence of calpain inhibitors or a synthetic peptide consisting of the
sponses were inhibited if the agonist-induced activation of
of granule contents, or retraction of clots. However, these
concentrations of inhibitor partially inhibit the generation of
procoagulant activity by the platelets. We suggest that there
are at least two mechanisms by which an agonist induces
procoagulant activity on platelets, and conclude that the
agonist-induced activation of calpain mediates one of these
mechanisms.
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FOX, REYNOLDS, AND AUSTIN
2512
A
Actin-binding Protein
B
-
Talin
Talin-
ABP-
-200K
?
200K
I
Control
LA &
1b
20
I
I
1h2A0,
Calpeptin (pg/ml)
Control
Leupeptin
,O k
20 60
1b
160 2b0,
Calpeptin (pg/ml)
I
Leupeptin
Fig 1. lmmunoblots of SDS-polyacrylamide gels showing the effect of calpain inhibitor on collagen-induced hydrolysis of ABP and talin.
Platelet suspensions (1 x lo9platelets/mL) were praincubated for 5 minutes in the presence or absence of the indicated concentrations of
calpeptin or for 30 minutes with 1 mg of leupeptin/mL (A and B). Samples were then stirred for 15 minutes alone (control) or with 20 pg of
collagen/mL. Incubations ware terminated with an SDS-containing buffer that contained reducing agent, and proteins were separated on
SDS-polyacrylamide gels. Hydrolysis of ABP (A) or of talin (B) was detected on immunoblots after transfer of proteins from
SDS-polyacrylamide gels t o nitrocellulose paper. 200K. 200,000 mol wt products of degradation of ABP (AI or talin (B) that are generated
when calpain hydrolyzesABP or talin within platelets.'
calpain was inhibited. One of the intracellular events that
occurs after platelet stimulation is activation of PKC. This
enzyme induces the phosphorylation of an unidentified cytoplasmic protein of mol wt 47,000, termed P47.34Concentrations of calpeptin (Fig 1 ) that totally inhibited detectable
A
-Collagen
hydrolysis of ABP had no obvious effect on the collageninduced (Fig 2A) or thrombin-induced (Fig 2B) phosphorylation of P47.
Another intracellularevent that occurs when platelets are
activated is phosphorylation of the 20,000 mol wt light chain
B
Thrombin
minus calpeptin plus calpeptin
minus calpeptin plus calpeptin
*-,
MLC0 I 0.5 I 5 I 15,O 10.5 I 5 I 15
0.2 1.0 10
0.2 1.0 10
.,. 4
M
.
ur
MLC-
z..
0 10.5 I 5 I 15,O 10.5 I 5 I 15
0.2 1.0 10
0.2 1.0 10
Time (min)
Fig 2. Effect of calpeptin. a calpain inhibitor, on the collagen- or thrombin-induced phosphorylation of MLC and P47. Suspensions of
platelets (3 x 1
0' platelets/mL) were prelabaled with ['*P]phosphate. The 32P-labaladplatelets were incubated in the presence or absence
of 50 pg of calpeptinlmL for 6 minutes. Suspensions were then stirred with collagen or thrombin for the indicated times, solubilized in an
SDS-containing buffer in the presence of reducing agent, and electrophoresed on SDS-polyacrylamide gels. Phosphopolypeptides were
detected by autoradiography and quantitated by densitometry of the bands of interest. MLC, myosin light chain; P47, a cytoplasmic protein
of mol w t 47,000 that becomes phosphorylated in activated platelets."
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2513
CALPAIN IN PLATELET FUNCTION
of myosin. This phosphorylation reaction is mediated in large
part by the Ca2+-dependent activation of MLC kinase.8
Neither the collagen-induced phosphorylation (Fig 2A) nor
the thrombin-induced phosphorylation (Fig 2B) was detectably inhibited by calpeptin at a concentration (50 pg/mL)
that totally inhibited detectable hydrolysis of ABP. Quantitation of the data presented in Fig 2 confirmed that calpeptin
did not inhibit the agonist-induced phosphorylation of P47 or
MLC (Table 1).
Agonist-induced intracellular changes mediate several
functional responses, including a change in platelet shape
and the aggregation of platelets. The shape-change response
can be detected as a small decrease in transmittance of light
through a platelet suspension and is followed immediately by
an increased transmittance as the platelets aggregate. Concentrations of calpeptin that totally inhibited detectable
hydrolysis of ABP (50 p g of calpeptin/mL) had virtually no
effect on platelet shape change or aggregation as assessed by
the changes of light transmittance through a suspension of
platelets aggregating in response to thrombin (Fig 3A),
collagen (Fig 3B), or a combination of the two agonists (Fig
3C). The lack of an inhibitory effect on the shape-change
response was confirmed by fixing the platelet suspensions and
examining them under the electron microscope (data not
shown). Similarly, these concentrations of inhibitor had no
effect on the thrombin-induced (Fig 4A) or collagen-induced
(Fig 4B) secretion of the contents of dense granules. In some
experiments, higher concentrations of calpeptin (100 to 200
pg/mL) inhibited both aggregation (Fig 3) and ATP secretion (Fig 4). However, the extent of this inhibition varied
from experiment to experiment and was overcome by higher
concentrations of agonists (Fig 3C). Leupeptin, even under
conditions in which it induced partial inhibition of the
collagen-induced hydrolysis of ABP and talin (Fig 1A and B)
had no effect on collagen-induced aggregation (data not
shown) or ATP secretion (Fig 4B). However, as reported by
others, leupeptin completely inhibited thrombin-induced aggregation (data not shown) and ATP secretion (Fig 4B),
presumably because it inhibited the amidolytic action of
thr~mbin.'~.'~
Another functional response of platelets to stimulation is
the retraction of fibrin clots. Because several of the proteins
hydrolyzed by calpain (ABP, talin, and spectrin) can associate with the cytoskeleton,8 it has been suggested that
activation of calpain might regulate the contractile activity
that is responsible far the retraction of clots by platelet
aggregates. To assess this possibility, platelet suspensions
were incubated with various concentrations of calpeptin (or
other agents) for 5 minutes before the addition of fibrinogen
and thrombin. The subsequent retraction of the fibrin clot by
the platelets was recorded photographically. Consistent with
the known inhibitory action of leupeptin on the amidolytic
action of thrombin, leupeptin inhibited clot retraction (Fig
5A, lane 2). Similarly, as shown by others:' the synthetic
peptide RGDS, which inhibits binding of fibrinogen to the
glycoprotein IIb-IIIa complex on the platelet surface, inhibited the retraction of clots in the assay system used (Fig SA,
lane 3). In contrast, calpeptin, even at concentrations of 200
pg/mL, had no detectable effect on the process (Fig 5B).
Yet another functional response of platelets is the generation of procoagulant activity on their surface. One way that
activated platelets accelerate coagulation is by providing a
binding site for the prothrombinase complex. This complex,
which consists of factor V, and factor X,, catalyzes the
conversion of prothrombin to thrombin. Verhallen et a19have
noted that the platelet-induced acceleration of prothrombinase activity correlates with the extent of activation of
calpain within the platelets. They have suggested that there
might be a causal relationship between the two events. We
also have observed a relationship between these two events.
As demonstrated by the generation of hydrolytic fragments
of ABP (Fig 6 ) , thrombin does not induce activation of
calpain unless the platelet suspensions are stirred (Fig 6,
compare lanes 1 and 2 with lanes 3 and 5). When suspensions
are stirred, thrombin induces a small amount of activation of
the protease (as detected by a small amount of the 200-Kd
Table 1. Effect of Calpeptin on Agonist-Induced PhosphorylationReactions
MLC
P47
Agonist
Minus Calpeptin
Plus Calpeptin
Minus Calpeptin
Plus Calpeptin
Collagen, 0 time
Collagen, 10 s
Collagen, 30 s
Collagen, 60 s
Collagen, 5 min
Collagen, 10 min
Collagen, 15 min
22,562
26,103
54.543
60.43 1
54,005
49,636
45.9 16
26,805
40,060
6 1,822
63,520
60.8 18
56,221
56.837
37,302
39,306
84,365
75,421
73,566
70,375
58,413
48.444
65,996
60.5 17
74.070
83,346
64,075
53,593
Thrombin, 0 time
Thrombin, 10 s
Thrombin, 30 s
Thrombin, 60 s
Thrombin, 5 min
Thrombin, 10 min
Thrombin, 15 min
16,072
5 1,200
53.81 1
55,937
57,695
53.079
50,298
15,280
52,292
54,161
56,633
57,029
54,837
48,895
25,587
29,682
30,487
33,518
36,373
35,756
32,638
3 1,637
29,927
3 1.064
30.849
37,693
33.31 1
34.287
The phosphorylationof P47 or MLC was quantitated by densitometry of the autoradiograms shown in Fig. 2. The numbers given represent arbitrary
densitometry units.
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2514
FOX, REYNOLDS, AND AUSTIN
Calpeptin (pg/ml)
A Thrombin
0
1
2
B Collagen
t
3
4
-
Calpeptin (pg/ml)
nonphysiologic Ca2' ionophore, A23187 [Fig 6, lane 61).
Like Verhallen et a1: we observed that the generation of
procoagulant activity in a platelet suspension (Table 2) was
greatest in response to the agonists that caused the greatest
extent of hydrolysis of ABP. However, there was not a direct
correlation between hydrolysis of ABP and generation of
procoagulant activity; as reported by
some procoagulant activity (Table 2 and Fig 7) was always generated in
suspensions of platelets that were incubated with thrombin
under conditions in which no detectable hydrolysis of ABP
occurred (ie, in the absence of stirring [Fig 61).
To determine whether activation of calpain was responsible for generating any of the procoagulant activity induced
by agonists, platelets were incubated with 50 pg of calpeptinl
mL. This concentration of inhibitor had little or no effect on
the procoagulant activity in control platelet suspensions (Fig
7). Similarly, it did not inhibit the generation of procoagulant activity in a suspension of platelets that was incubated
with thrombin but was not stirred (Fig 7). As shown in Table
A Thrombin
Calpeptin (pg/ml)
E
m
1
C
,t
0
1
2
C Collagen and Thrombin
3
4
Calpeptin (pg/ml)
200
50
0
100
t
0!
a
C
m
CI
c)
.$
C
E
;
0
5
0I
I
I
I
a
1
1.2
Calpeptin (pg/ml)
Bcol'agen
Fig 3. Effect of calpeptin on agonist-induced aggregation of
platelets. Suspensions of platelets (3x 10' platelets/mL) were
incubated in the presence or absence of calpeptin for 5 minutes
and then stirred in the presence of an agonist in a lumiaggregometer. The aggregation of platelets was recorded as a change of
transmittance of light through the suspension.
hydrolytic fragment) (Fig 6, lane 3). As noted previously, the
amount of ABP that is hydrolyzed is very small and can be
detected only by the appearance of hydrolytic products on
two-dimensional gels3 or on immunoblots (Fig 6). We find
that collagen routinely induces a greater degree of activation
of calpain than does thrombin (eg, Fig 6, lane 4), whereas a
combination of collagen and thrombin induces sufficient
activation to result in hydrolysis of amounts of ABP that can
be readily detected as a decreased amount of intact protein
(Fig 6, lane 5) (as can the hydrolysis induced by the
'-Leupeptin
0
I
I
I
1
2
3
Time (min)
Fig 4. Effect of calpain inhibitors on agonist-induced secretion
of ATP by platelets. Suspensions of platelets (3x 10' platelets/
mLI were incubated for 5 minutes in the presence or absence of
the indicated concentrations of calpeptin or for 30 minutes with 1
mg of leupeptin/mL. They were then stirred in the presence of an
agonist in a lumiaggregometer. The secretion of ATP from the
platelet granules was detected using Luciferin-Luciferase reagent
as described under Materials and Methods.
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2515
CALPAIN IN PLATELET FUNCTION
A
B
with that required for total inhibition of detectable hydrolysis of ABP (maximal inhibition of the generation of procoagulant activity was observed at 10 to 20 pg of calpeptin/mL)
(Fig 8 ) .
Experiments were performed to eliminate the possibility
that the inhibitory effect of calpeptin resulted from an
inhibition of the assembly or activity of the prothrombinase
complex. Platelets were incubated with collagen or dibucaine,
two agents that have been shown to induce the shedding of
microvesicles from the platelet plasma
These
microvesicles contain binding sites for the prothrombinase
comple~.’~~’~
As shown in Table 3, calpeptin had no effect on
the amount of thrombin generated when these microvesicles
provided the binding site for the prothrombinase complex.
0 min
DISCUSSION
Despite the fact that calpain is present in a wide variety of
cells, a physiologic role for this protease has not previously
been elucidated. The only cell in which activation of calpain
has been shown to occur as a consequence of cellular
activation is the platelet. However, the potential role that
5 min
ABP--
< . I
10 min
-200K
-1OOK
-91K
1
2
3
1
2
3
4
Fig 5. Effect of calpain inhibitors on clot retraction. (A)
Suspensions of platelets (1 x lo9 platelets/mL) were incubated
for 5 minutes in the presence of 0.2% DMSO (lane 1). 1 mg of
leupeptin/mL (lane 2). or 300 pmol/L RGDS (lane 3). Fibrinogen
and thrombin were added t o the platelet suspensions, and the
retraction of the fibrin clots that formed was recorded photographically at the intervals shown. (B) Platelet suspensions were
preincubated for 5 minutes in the presence of 0.2% DMSO (lane 11,
50 pg of calpeptin/mL (lane 21,100 pg of calpeptin/mL (lane 3).or
200 pg of calpeptin/mL (lane 4). Fibrinogen and thrombin were
then added t o the suspensions, and the retraction of clots was
recorded.
2 and Fig 7, thrombin induced the generation of more
procoagulant activity if the platelets were stirred during the
incubation, but calpeptin decreased the generation of procoagulant activity to the level generated in the unstirred
suspension (Fig 7). Collagen, or a combination of collagen
and thrombin (Table 2 and Fig 7), caused the generation of
yet more procoagulant activity, and, again, this increased
generation of procoagulant activity was prevented if the
platelets were preincubated with calpeptin (Fig 7). The
concentration of calpeptin required to inhibit the agonistinduced generation of procoagulant activity was comparable
1 2 3 4 5 6
Fig 6. lmmunoblot showing the hydrolysis of ABP in platelet
suspensions. Platelet suspensions were incubated in the presence
or absence of thrombin, collagen, or ionophore A23187. Incubations were terminated by the addition of an SDS-containing buffer
and electrophoresed on SDS-gels containing 7.5% acrylamide.
ABP and its 200,000 (200K). 100,OOO (100K). and 91,000 (91K) mol
wt hydrolytic fragments were detected on immunoblots using ABP
antibodies. Lane 1, suspensions were incubated alone in the
absence of stirring for 15 minutes; lane 2, suspensions were
incubated with 1.0 NIH U of thrombin/mL for 15 minutes in the
absence of stirring; lane 3. suspensions were stirred in the
presence of 1.0 NIH U of thrombin/mL for 15 minutes: lane 4,
suspensions were stirred for 15 minutes in the presence of 20 pg
of collagen/mL; lane 5, suspensions were stirred for 15 minutes in
the presence of a combination of 20 pg of collagen/mL and 1.O NIH
U of thrombin/mL; lane 6, platelets were stirred in the presence of
0.4 pmol/L ionophore A23187 for 10 minutes.
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FOX, REYNOLDS, AND AUSTIN
2516
Table 2. Generation of Procoagulant A c t i v i i by Agents That
Activate Calpain
R d a i v e Extent of
Hychiysis
of ABP
Aaonist
None
Thrombin, stirred
Collagen
Collagen thrombin
A23 187
Thrombin, unstirred
+
NOM
ProcoagulantActivity
(md thrombin/min/lO' plateletsl
( x 10'0)
5.7
12.5
28.9
39.3
40.3
7.7
+
++
+++
++++
NOM
f 0.8
f 2.3
f 0.6
i 0.8
i 0.6
f 0.3
Incubations from the experiment shown in Fig 4 were terminated by
solubilization of the suspensions in an SDScontaining buffer, and the
extent of hydrolysis of ABP by the Ca2+-dependentprotease was
assessed by the appearance of the hydrolytic fragments (mol
wt = 200,000, 100,OOO. and 91,000) on immunoblots (seeFig 6) and
to
Parallel incubations
is indicatedon an increasing scale of
were diluted to 1 x lo7platelets/mL with buffers containing factor V,,
factor X, and prothrombin. llw amount of thrombin generated in a
subsequent 5-minute incubation was determined with a chromogenic
substrate as described in Materials and Methods. Values shown are the
mean i SE obtained from three different incubations.
+ + + + +.
calpain plays in mediating the responses of platelets has been
a matter of controversy. Because only small amounts of
calpain substrates are hydrolyzed in thrombin-activated
platelets: some investigators have suggested that activation
of calpain has no physiologic consequence." Others have
suggested that hydrolysis of small pools of cytoskeletal
proteins may be sufficient to induce changes in the function
of the cytoskeleton in aggregated platelets.3**Until recently,
investigation of the consequence of activation of calpain in
platelets was hindered by the absence of inhibitors of calpain
L
14.0
El mlnus calpeptin
Iplus calpeptin
v
-_
u)
.-*z
rB
12.0
I
,m
Calpeptin Concentration(pglml)
Fig 8. Effect of calpeptin on the agonist-inducedgeneration of
procoagulant activity in platelet suspensions. Platelet suspensions
(0.26 x 10' plateletslml) were incubated for 5 minutes in the
presence of calpeptin at the concentrations shown. Suspensions
were subsequentlystirred in the presenceof 20 pg of collagen/mL
1-(
or 1.O NIH U of thrombin/mL (M
for
)10 minutes. The
procoagulant activity generated in the platelet suspension was
determined as described under Materiels end Methods. The open
triangle. indicated by an arrow, indicates the procoagulant activity
present in a suspension of unstimulated control platelets.
that could enter the cell. Although leupeptin is a fairly
specific inhibitor of calpain, it crosses membranes poorly and
it inhibits the amidolytic activity of t h r ~ m b i n , ' an
~ ' ~activity
that is required for thrombin to initiate its effects on
platelets.20Thus, studies using this inhibitor to evaluate the
involvement of calpain in mediating the responses of thrombin are difficult to interpret.
In the present study, we have used calpeptin, an inhibitor
of calpain that has a specificity similar to that of leupeptin
but readily penetrates the membrane and does not inhibit the
Table 3. Effect of Calpeptin on Microvesiclelnduced
ProcoagulantA c t i v i i
ProcosgulsntActivity
(md thrombin/l5 min/mL of
microwsicks [ x 10'7)
Control Thrombin Thrombin Collagen
(unstirred)
Collagenplus
Thrombin
Fig 7. Effect of calpeptin on the agonist-induced generation of
procoagulant a c t i v i i in platelet suspensions. Platelet suspensions
(0.25 x ' l d platelets/mL) were incubated in the absence or
presenceof 60pg of calpeptin/mL for 5 minutes. Agonist was then
added and suspensions incubeted for a further 15 minutes. The
procoagulant activity generated in the platelet suspensions was
subsequently determined as described under Materials and Methods.
MiMOVeSCks
Minus Calpeptin
Plus Calpeptin
Microvesicles 1
Microvesicles 2
Microvesicles 3
0.22 f 0.01
1.50 f 0.05
1.97 f 0.05
0.26 f 0.02
1.50 f 0.02
1.84 f 0.03
Platelet suspensions (1 x lo9 platelets/mL) were stirred for 15
minutes alone (Microvesicles 11, with 20 pg of collagen/mL (MiawesC
cles 2). or with 0.5 mmol/L dibucaine (Microvesicles 3). Intact platelets
were removed by centrifugationat 15,600 g for 1 minute. The resultant
miaovesicle-containingsupernatant was preincubatedfor 10 minutes in
the presenceof 0.2% DMSO or 20 pg of calpeptin/mL and 0.2% DMSO.
Factor V,, factor X,, and prothrombinwere then added, and the thrombin
generated was measured. Values given are the mean i SE from three
different incubations.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
CALPAIN IN PLATELET FUNCTION
amidolytic activity of thrombin.22The use of this inhibitor
has enabled us to demonstrate that when the agonist-induced
hydrolysis of ABP was totally inhibited, most of the platelet
responses to collagen or thrombin proceeded normally. However, the generation of procoagulant activity was inhibited.
This finding demonstrates that activation of calpain mediates
one of the responses of platelets to activation.
The expression of procoagulant activity is an important
functional response of platelets. It is critical for efficient
hemostasis because it accelerates the formation of fibrin
polymers throughout a platelet aggregate, converting the
loose aggregate into a tight clot that is not easily disrupted by
the shear forces that characterize circulating blood. The
importance of platelet procoagulant activity is underscored
by the bleeding disorder in a patient in whom the expression
of this activity is de~reased.~’.~‘
One of the ways that platelets
activate the coagulation “cascade” is by accelerating the
final reaction, the conversion of prothrombin to thrombin by
the prothrombinase complex.
The prothrombinase complex consists of factor V, and X,
and is only active when the factors are brought together on a
phospholipid surface such as is provided by the membrane of
activated platelets. There has been some controversy as to the
mechanism by which activation of platelets can increase the
activity of this complex. One group of investigators has
reported that thrombin-activated platelets have the same
number of binding sites for factor V, as control
They suggest, therefore, that factor V, is bound to the
surface of unstimulated platelets and that the increased
prothrombinase activity on suspensions of activated platelets
results from an activation-induced change in the conformation of the prebound factor V,, which allows an increased
binding of factor X,. Others have detected an activationinduced increase in the number of factor V, binding site^.^^.^'
The reason for the conflicting results is not clear. The work
described in the present study suggests that there are two
distinct mechanisms by which procoagulant activity is generated and that activation of calpain is responsible for inducing
one of them. Because calpain is activated only when platelets
aggregate, the calpain-induced component only occurs in
suspensions of platelets that are stirred. Because the previous
experiments that detected increased binding sites for factor
V, were performed under conditions in which calpain was
a~tivated:~ while the experiments that failed to detect
increased binding were performed in unstirred platelet
suspensions,)6 it appears likely that activation of calpain is
responsible for activating the prothrombinase complex by the
mechanism that involves an increased number of binding
sites for factor V, on the platelet surface.
The present study suggests a mechanism by which activation of calpain could exert its effects. Calpain induces the
hydrolysis of several proteins in platelets. The two major
substrates are ABP3*4and talin.4 (Hydrolysis of other minor
components, including spectrin,’ calmodulin-dependentphosphatase,6 and an unidentified protein of mol wt 87,0003 that
may represent caldesmon; has also been detected on twodimensional gels,’ immunoblot~,~
or calmodulin overlays.)6
In the present study we assessed the effect of calpain
inhibitors by their ability to inhibit the hydrolysis of ABP
2517
and talin. Interestingly, the calpain-induced hydrolysis of
ABP was totally inhibited by 10 to 20 wg of calpeptin/mL,
while the hydrolysis of talin was not completely inhibited
even at 200 pg of calpeptin/mL. The differential effect of
calpeptin on the hydrolysis of different substrates may result
from the fact that the calpeptin is a hydrophobic molecule
and therefore partitions into the plasma membrane. Thus,
the inhibitory effects that we observed at 10 to 20 pg of
calpeptin/mL may result from an inhibition of membraneassociated calpain, whereas the inhibitory effects that were
observed only at higher concentrations of calpeptin could
result from an inhibition of soluble calpain. If this interpretation is correct, it indicates that the response that is inhibited
by the lower concentration of calpeptin (ie, the generation of
procoagulant activity) results from the calpain-induced hydrolysis of a membrane-associated protein.
The two known calpain substrates that are associated with
the membrane in platelets are ABP29-32
and spectrin.’ Both
proteins are components of the membrane skeleton that lines
the lipid bilayer. Comfurius et a144 noted a correlation
between the hydrolysis of ABP within the platelet and the
externalization of negatively charged phospholipids. This
correlation led them to suggest that the platelet cytoskeleton
was responsible for maintaining the asymmetry of the
phospholipids, and that hydrolysis of the cytoskeleton during
platelet activation allowed the phospholipids to “flip” to the
outer bilayer, where they could provide new binding sites for
the prothrombinase complex. Although we have not determined whether expression of procoagulant activity results
from externalization of phospholipids, our data provide
evidence that procoagulant activity is generated as a consequence of an action of calpain and indicate that hydrolysis of
a membrane-bound cytoskeletal protein may indeed be the
critical event. Work is under way to test the hypothesis that
procoagulant activity is generated as a consequence of the
calpain-induced disruption of the platelet membrane skeleton.
Other investigators have suggested that activation of
calpain mediates thrombin-induced aggregation or secretion
of granule contents.’’-” Their conclusionshave been based on
the inhibitory action of leupeptin on the responses of platelets
to thrombin. Because leupeptin had no effect on collageninduced responses,16 it appears likely that the inhibitory
action of leupeptin resulted solely from an action of the
inhibitor on the amidolytic action of thrombin. The present
study supports this conclusion. Leupeptin had little effect on
the collagen-induced hydrolysis of ABP or talin; we conclude
that leupeptin does not readily enter platelets and that any
inhibitory action it has on platelet function must result from
inhibition of an extracellular event. Further support for the
conclusion that leupeptin does not inhibit thrombin-induced
aggregation and secretion as a consequence of inhibiting
intracellular calpain came from the observation that we
could effect partial entry of leupeptin into platelets by
incubating them with 1 mg of leupeptin/mL in the presence
of DMSO. Because these conditions were much more extreme than those used by the workers who concluded that
calpain mediates thrombin-induced aggregation and secretion,I0-l2we assume that we were inhibiting intracellular
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
FOX, REYNOLDS, AND AUSTIN
2518
calpain to a greater extent than in those studies that reported
a functional consequence. Under these extreme conditions,
the thrombin-induced hydrolysis of ABP and talin was
partially inhibited, but concentrations of calpeptin that
caused comparable levels of inhibition had no effect on
thrombin-induced aggregation or secretion. Therefore, we
suggest that the previously reported actions of leupeptin did
not result from inhibition of intracellular calpain.
Although our study provides evidence that the previously
reported actions of leupeptin on thrombin-induced aggregation and secretion resulted from inhibiting the ability of
thrombin to initiate stimulus-response coupling, it does not
exclude the possibility that activation of calpain plays some
role in regulating aggregation or secretion. For example, in
our experiments inhibitory actions of calpeptin on aggregation and secretion were observed at concentrations at which
the calpain-induced hydrolysis of talin was inhibited. Because talin is thought to be a soluble protein23 in unstimulated platelets, this finding suggests that the calpain-induced
hydrolysis of soluble proteins may play some role in regulating aggregation and secretion. Because these responses are
well under way before hydrolysis of any of the known calpain
substrates can be detected, it appears unlikely that calpain is
responsible for initiating the responses. However, it is possible that the calpain-induced hydrolysis of soluble platelet
proteins may modulate them. Because calpain can hydrolyze
MLC kinase:'
it is conceivable that activation of calpain
could modulate secretion; the report by Tsujinaka et alZ2that
calpeptin inhibited the thrombin- and collagen-induced phosphorylation of myosin is consistent with this possibility.
Further, aggregation may be modulated as a consequence of
the calpain-induced regulation of thromboxane synthetase
activity:6 a possibility that is consistent with the observation
in the present study that higher concentrations of calpeptin
had variable effects on platelet aggregation and secretion but
appeared to be more often inhibitory when collagen was used
as the agonist.
One response that was not inhibited, even a t high concentrations of calpeptin, was clot retraction. Because talin
appears to be involved in linking integrins to stress fibers:' it
is possible that in activated platelets it is involved in linking
the cytoskeleton to the glycoprotein IIb-IIIa complex and
thus to the externally bound fibrin clot. Therefore, calpainmediated hydrolysis of talin has been considered a possible
mechanism by which retraction of fibrin clots might be
regulated. The present experiments suggest that this is not
the case. It is conceivable that the assay used to assess clot
retraction was not sensitive enough to detect an inhibitory
action of calpeptin. However, this seems unlikely because
inhibition of clot retraction was detected when platelets were
incubated with leupeptin, which inhibits the amidolytic
action of t h r ~ m b i n , ' ~or
. ' ~RGDS,which inhibits the association of the glycoprotein IIb-IIIa complex with fibrinogen:'
Future studies will be required to investigate the functional
consequences of the calpain-induced hydrolysis of talin in
platelets.
In summary, the present study shows for the first time that
activation of calpain is involved in stimulus-response coupling in platelets. The selective inhibition of the calpaininduced hydrolysis of ABP and the generation of procoagulant activity by a membrane-penetrating calpain inhibitor
are consistent with the hypothesis that calpair generates
procoagulant activity as a consequence of hydrolysis of the
membrane skeleton. Because concentrations of calpeptin
that inhibit the generation of platelet procoagulant activity
do not inhibit the amidolytic activity of thrombin and do not
inhibit any of the other functional responses of platelets,
membrane-penetrating calpain inhibitors may provide a
novel therapeutic method of decreasing the incidence of
thrombotic events without causing the hemorrhagic complications that are associated with current anticoagulant therapy.
ACKNOWLEDGMENT
We express our sincere gratitude to Dr Toshimasa Tsujinaka for
his generous gift of the calpeptin that made these studies possible.
We are also grateful to Dr John W. Fenton I1 for generously
providing the a-thrombin. We thank Charles Benedict and Tom
Rolain for graphics, AI Averbach and Sally Gullatt Seehafer for
editorial assistance, and Victoria Davis and Tony Gridley for
manuscript preparation.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1990 76: 2510-2519
The role of calpain in stimulus-response coupling: evidence that
calpain mediates agonist-induced expression of procoagulant activity
in platelets
JE Fox, CC Reynolds and CD Austin
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