|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4222-4231
By
From The Center for Blood Research and the Department of Pediatrics,
Harvard Medical School, Boston, MA.
Platelets function to protect the integrity of the vascular wall. A
subset of platelet activation responses that are especially important
for thrombus formation include exposure of phosphatidylserine and
release of microparticles, which generate procoagulant surfaces. The
resemblance of these platelet activation processes to events occurring
in nucleated cells undergoing apoptosis suggests a possible role for
caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human
platelets and its activation by physiological platelet agonists. Using
cell-permeable specific inhibitors, we demonstrate a role for a
caspase-3-like protease in the agonist-induced (collagen plus thrombin
or Ca2+ ionophore) platelet activation events of
phosphatidylserine exposure, microparticle release, and cleavage of
moesin, a cytoskeletal-membrane linker protein. The role of caspase-3
in platelet activation is restricted rather than global, because other
activation responses,
PLATELETS ARE THE major players of
hemostasis and thrombosis. Among other responses to physiological
agonists, activated platelets expose negatively charged
phosphotidylserine (PS) on the platelet exterior and release
PS-positive microparticles, both of which contribute to fibrin
deposition by providing competent surfaces for assembly of coagulation
factors and thrombus formation.1 Increased levels of
circulating microparticles are associated with various thrombotic
disorders, including activated coagulation, transient ischemia,
myocardial infarction, and postsurgery in cardiopulmonary bypass
patients.2-6 For these reasons, studies to delineate the
mechanisms of PS exposure and microparticle release are extremely
important for understanding platelet function.
Exposure of PS and release of microparticles occur only within the
larger process of platelet activation. Conversely, the activation
response of platelets is distinguished by a multiplicity of subevents
and regulatory mechanisms. Particularly noteworthy is the sequential
nature of the response and the temporal arrangement of individual
steps. Several parallels can be discerned between the programmed
activation response of platelets and programmed death of
nucleated cells Caspases, which are cysteinyl proteases that cleave after aspartic
acid, are key effectors of apoptosis. The family consists of at least
11 enzymes in humans.7-9 In response to apoptotic signals,
the proform of an initiator caspase containing an adapter domain, such
as caspase-8, -9, or -10, is recruited to a surface membrane complex or
a mitochondrial-derived complex, where it is autoproteolytically
processed to the two subunit active form. The initially activated
caspase processes/activates other procaspases that lack adapter
domains, including caspase-3 and -7, and the latter, the effector
caspases, incapacitate essential homeostatic pathways by limited
cleavage of specific targets.
Another hallmark of nucleated cell apoptosis is cleavage of select
cytoskeletal proteins by effector caspases.10,11 We focus
in this study on one cytoskeletal protein, moesin, the only member in
human platelets12,13 of the ERM
(ezrin-radixin-moesin) family proteins, which stabilize surface
projections by linking the underlying actin cytoskeleton with the
plasma membrane.14,15 In response to agonist, platelet
moesin undergoes rapid phosphorylation, localizes transiently to newly
formed filopodial/lamellipodial projections,12,16 and
subsequently is proteolytically cleaved,16a a reaction
expected to terminate moesin's linker function and facilitate late
platelet cytoskeletal changes required for clot retraction and
microparticle release. Moesin cleavage requires calpain
(Ca2+-activated protease), which is required also for
microparticle release17,18; however, pure calpain alone
does not cleave moesin,13 suggesting a requirement for a
second protease.
The present report breaks new ground by demonstrating the presence and
novel function of caspase in human platelets. We identify the zymogen
form of the effector caspase, caspase-3, as a component of human
platelets and demonstrate that procaspase-3 becomes activated when
isolated platelets are stimulated by physiological agonists. A specific
cell-permeant inhibitor of caspase-3 is shown to abrogate agonist-induced PS exposure and microparticle release. The caspase inhibitor also prevents cleavage of the structural protein moesin in
activated platelets. Comparative experiments with protease inhibitors
showed that both caspase and calpain function in agonist-induced late
events of platelet activation (PS exposure, microparticle release, and
moesin cleavage) and demonstrate that the two proteases have distinct roles.
Platelet isolation.
Freshly drawn blood from normal healthy donors, who gave written
consent, was collected in acid-citrate-dextrose (ACD; NIH formula A) in
plastic and fractionated immediately at ambient temperature. Cells were
counted using a MAX-M Blood Cell Analyzer (Coulter Corp, Hialeah, FL).
The blood was centrifuged at 200g for 12 minutes to separate
platelet-rich plasma (PRP). Additional ACD was added (1 part ACD per 3 parts PRP) and platelets were pelleted at 800g for 15 minutes.
The platelets were resuspended in platelet buffer (10 mmol/L
Tris-hydroxymethyl-methyl-2-aminoethane sulphonic acid [TES], pH 7.2, 136 mmol/L NaCl, 2.6 mmol/L KCl, 0.5 mmol/L
NaH2PO4, 2 mmol/L MgCl2, 0.1%
glucose, and 0.1% bovine albumin) and, after the addition of ACD (20%
of final volume) and prostacyclin (1 µg/mL; Calbiochem, San Diego,
CA), were centrifuged at 800g for 10 minutes. The isolated
platelets contained no detectable erythrocytes and less than 1 leukocyte per 4,000 platelets. For activation experiments, platelets
were suspended in platelet buffer and allowed to recover for 90 minutes
at 37°C to ensure their resting state.
Peptidase assay.
Platelets (5 × 108) in 1 mL of platelet buffer with 2 mmol/L CaCl2 and 3 µmol/L A23187 were incubated with
stirring in flat-bottom polyethylene vials (14-mm diameter) at 37°C
for the indicated time and were lysed by adding 1/3 vol of 4% Triton
X-100, 8 mmol/L EGTA, 20 mmol/L dithiothreitol, 200 µg/mL aprotinin,
200 µg/mL benzamidine, and 200 µg/mL leupeptin. The Triton lysates
(200 µL) were combined with 0.1 mmol/L of
N-acetyl-Asp-Glu-Val-Asp-p-nitro anilide (DEVD-pNA; Enzyme Systems
Products, Livermore, CA) or N-acetyl-Tyr-Val-Ala-Asp-p-nitroanilide
(YVAD-pNA; Sigma, St Louis, MO). The reactions were
incubated for 2 hours in flat-bottom 96-well plates at 37°C with
monitoring of OD405 nm. The mean Immunoblotting.
Platelets (5 × 108/mL) were lysed by adding an equal
volume of 2% sodium dodecyl sulfate (SDS), 120 mmol/L Tris-HCl, pH
6.8, 4% mercaptoethanol, 100 µg/mL leupeptin, 4 mmol/L EGTA, and 2 mmol/L diisopropyl fluorophosphate (DFP) and heating for more than 3 minutes at 100°C. The lysates were fractionated by
SDS-electrophoresis on 8 or 12% polyacrylamide gels (Novex, San Diego,
CA).19 Polypeptides were transferred to nitrocellulose at
constant 80 mA at approximately 22°C for 16 hours. The membranes
were blocked with 2% normal rabbit serum in phosphate-buffered saline
with 0.05% Tween-20 for 20 minutes, washed, and incubated for 2 hours
with clone 38 moesin monoclonal antibody (MoAb; 100 ng/mL) or C31720
caspase-3 MoAb (250 ng/mL; both from Transduction Laboratories,
Lexington, KY), or with caspase-3 MoAb CPP32/p20-E8 (1 µg/mL; Santa
Cruz, Santa Cruz, CA) or B27D8 µ-calpain MoAb20 (1 to
10,000 dilution of ascites), or, after blocking with 2% normal goat
serum, with rabbit antibodies to caspase-3 (1 to 1,500 dilution;
Stratagene, La Jolla, CA). The membranes were washed, incubated with
125I-labeled secondary antibody, and exposed to
Phosphor-screen. Bands were quantified using Phosphor-Imager Storm 860 and Image Quant v1.1 program (Molecular Dynamics, Sunnyvale, CA). As a
control for the detection of active caspase-3, isolated mononuclear
cells were lysed at 1.5 × 107/mL in 0.5% NP-40, 10 mmol/L Tris HCl, pH 7.4, 150 mmol/L NaCl, 2 mmol/L DFP, 2 mmol/L EGTA,
and 50 µg/mL leupeptin and, after clarification by centrifugation,
the lysate was incubated for 1 to 3 hours with 25 µg/mL of granzyme
B21 (kindly provided by Dr Zhinan Xia, Center for Blood
Research, Boston, MA).
Activation of platelet caspase.
Platelets (5 × 108) in 1 mL of platelet buffer with 2 mmol/L CaCl2 in flat-bottom polyethylene vials were
incubated while stirring with 3 µmol/L A23187, 1 U/mL of human
thrombin, 10 µg/mL of collagen (native collagen fibrils from equine
tendons; Collagen Reagent Horn; Nycomed Arzneimittel GmbH, Munich,
Germany), or the combination of thrombin and collagen at 37°C for
20 minutes with stirring. The reaction was terminated by solubilizing
with SDS in preparation for immunoblotting.
Treatment with caspase and calpain inhibitors.
For inhibition experiments,
carbobenzoxy-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone
(DEVD-fmk), carbobenzoxy-Tyr-Val-Ala-Asp(OMe)-fluoromethyl ketone
(YVAD-fmk), carbobenzoxy-Phe-Ala-fluoromethylketone (FA-fmk), or
diluent was added to platelets during the final 60 minutes of
preincubation, and calpeptin and E64d were added during the final 10 minutes. For moesin cleavage experiments, fluoromethylketone stocks
were prepared in dimethylsulfoxide, the recommended solvent, or
dimethylformamide. For PS exposure experiments, we found that preincubation of platelets with dimethylsulfoxide (0.01% to 1.0%), but not with dimethylformamide, significantly increased A23187-induced exposure of PS (data not shown). Therefore, fluoromethylketone stocks
for PS exposure and all other experiments were prepared in
dimethylformamide (0.2% final concentration). Using this diluent, incubation of platelets with fluoromethylketones for 1 hour did not
alter resting platelet values of any of the parameters studied. Calpeptin and E64d stocks were prepared in ethanol or dimethylformamide.
Platelet activation and flow cytometry.
For PS exposure and microparticle release experiments, 107
platelets in 200 µL platelet buffer with 2 mmol/L CaCl2
were placed in siliconized 7 × 45 mm glass cuvettes at 37°C
in an aggregation meter (DP-247E; Sienco, Morrison, CO). A23187 (1 µmol/L), thrombin (human; 1 U/mL; Sigma), or thrombin (1 U/mL) plus
collagen (20 µg/mL) was added from a diluted stock and, after an
initial mixing, incubation was continued without stirring at 37°C
for 1 to 20 minutes. The platelet suspensions were transferred to an
approximately 22°C water bath and, after 1 minute, 50 µL was
combined with fluorescein isothiocyanate-labeled annexin V (annexin
V-FITC; 1 µg/mL; PharMingen, San Diego, CA) and phycoerythrin
(PE)-labeled anti-CD41 (GPIIb) MoAb (150 ng/mL; Coulter/Immunotech,
Miami, FL) and incubated for 10 minutes at approximately 22°C.
Samples were diluted fivefold with platelet buffer with 2 mmol/L
CaCl2 for immediate analysis by flow cytometry.
Moesin degradation assay.
After preincubation of 5 × 108 platelets in 1 mL
platelet buffer in flat-bottom polyethylene vials, CaCl2 (2 mmol/L) and A23187 (3 µmol/L) were added and incubation was continued
for 10 or 20 minutes at 37°C with stirring. The reaction was
terminated by solubilizing with SDS.
Statistical analysis.
The Student's paired t-test was used to calculate P values.
Detection of caspase-3 in platelets.
To test for the presence of caspase, isolated platelets were lysed with
Triton X-100 and peptidase activity was measured by chromogenic assay
with the p-nitroanilid derivative of Asp-Glu-Val-Asp as
substrate (DEVD-pNA). Substrates based on DEVD are specific for
effector caspases, including caspase-3.7,8 DEVD-pNA
cleaving activity was detected at low levels in lysates of resting
platelets and was significantly increased on stimulation of platelets
with the Ca2+ ionophore A23187 (P < .01),
reaching maximal levels at 5 minutes (Fig
1A). In parallel assays with YVAD-pNA, a caspase-1 substrate, no activity was detected (data not shown).
Physiological agonists activate platelet caspase-3.
Because A23187 is a potent but nonphysiological stimulus, we asked
whether processing of platelet caspase-3 zymogen is induced also by
physiological agonists. Thrombin, collagen, and the combination thrombin plus collagen were each found to induce processing of procaspase-3 (Table 1). The extent of
procaspase-3 processing varied; the order of agonist efficiency
was A23187 > thrombin + collagen > either collagen or
thrombin.
Caspase inhibitor abrogates agonist-induced phosphatidylserine
exposure.
We next examined whether caspase is involved in the movement of
negatively charged PS from the inner to the outer platelet membrane
leaflet, an activation reaction synonymous with generation of the
procoagulant surface. Platelets were treated with DEVD-fmk, a
cell-permeant inhibitor of caspase-3-like proteases, and then stimulated with agonist. Exposed PS was measured at fixed time points
by binding of annexin V-FITC.18 In response to the potent stimulant A23187, exposure of PS was rapid and extensive; 80% ± 4% (n = 4) of platelets became PS positive in 5 minutes (eg, Fig 2A). Incubation with DEVD-fmk
substantially inhibited/delayed A23187-induced PS exposure (Fig 2B). At
100 µmol/L, DEVD-fmk caused 74% ± 2% inhibition of PS exposure
at 3 minutes and 40% ± 3% at 5 minutes, and FA-fmk, a chemically
similar compound lacking caspase inhibitory activity, failed to inhibit
PS exposure (Fig 2B). Lower concentrations of DEVD-fmk were also
inhibitory, eg, 25 µmol/L inhibited A23187-induced PS exposure by
35% ± 5% at 3 minutes and 52% ± 2% at 5 minutes
(n = 3).
Caspase inhibitor abrogates agonist-induced microparticle release.
PS exposure in activated platelets is closely linked with the release
of microparticles (Zwaal and Schroit1 and Discussion). The
effect of DEVD-fmk on microparticle release was examined using a flow
cytometric assay to quantify microparticles (Materials and Methods).
Platelet pretreatment with DEVD-fmk, but not with FA-fmk, was found to
substantially inhibit/delay microparticle release in response to A23187
(Fig 3A). The extent of inhibition by 100 µmol/L DEVD-fmk was 42% ± 1% at 3 minutes and 32% ± 2% at
5 minutes (Fig 3A). Lower DEVD-fmk concentrations were also inhibitory;
25 µmol/L caused 25% ± 2% inhibition at 3 minutes and 8% ± 3% inhibition at 5 minutes (n = 3). For thrombin plus collagen-treated
platelets, microparticle release was also inhibited by DEVD-fmk.
DEVD-fmk at 25 µmol/L caused 57% ± 3% inhibition at 5 minutes,
47% ± 4% at 10 minutes, and 58% ± 7% at 20 minutes (Fig
3B); FA-fmk had no inhibitory effect (data not shown).
Caspase inhibitor fails to prevent agonist-induced platelet
aggregation and secretion of
Caspase inhibitor abrogates agonist-induced cleavage of platelet
moesin.
The effect of DEVD-fmk was also examined on another late platelet
activation response, cleavage of the cytoskeletal linker protein
moesin. In the absence of inhibitor, A23187 induced cleavage of 60% ± 5% (n = 4) of moesin molecules in 20 minutes
(Fig 5A, lanes 1 and 2). Cleavage of moesin
was substantially inhibited in platelets pretreated with DEVD-fmk (lane
3). The extent of inhibition of A23187-induced moesin cleavage
increased over the DEVD-fmk range of 5, 25, and 50 µmol/L and was
complete at 50 and 100 µmol/L (Fig 5B). A23187-induced moesin
cleavage was not inhibited in platelets pretreated with FA-fmk and
minimally affected in platelets pretreated with YVAD-fmk, a related
inhibitor with primary specificity for caspase-1-like protease. These
findings strongly suggest that a caspase-3-like enzyme is required for the platelet activation event of moesin cleavage.
Caspase activation and calpain activation are independent events.
Previous studies demonstrated an important role for another protease,
Ca2+-activated neutral protease (calpain), in a subset of
platelet activation events. Specific cell permeant reagents, including E64d and calpeptin, which prevent conversion of procalpain to calpain,
inhibit agonist-induced cleavage of select platelet cytoskeletal proteins, release of microparticles, and generation of prothrombinase activity.17,18 To determine whether DEVD-fmk alters
platelet functions by acting on calpain, the latter protein was
measured in DEVD-fmk-pretreated platelets. Reciprocal experiments were performed on calpeptin-pretreated platelets. Neither calpeptin nor
DEVD-fmk detectably altered the resting platelet content of µ-procalpain (Fig 6, top panel, first 3 lanes) or procaspase-3 (lower panel, first 3 lanes). Moreover, A23187
induced the conversion of procalpain to calpain in platelets
preincubated with DEVD-fmk, but not in calpeptin-pretreated platelets
(Fig 6, top panel, last 4 lanes). Similarly, A23187 induced the
processing of procaspase-3 in platelets preincubated with calpeptin,
but not in DEVD-fmk-pretreated platelets (lower panel, last 4 lanes).
These findings indicate that the alteration of platelet function by
DEVD-fmk does not rely on acting through calpain; similarly, the
effects of calpeptin do not entail acting through caspase-3.
Effects of calpain inhibitor and caspase inhibitor on PS exposure and
microparticle release.
To compare the roles of calpain and caspase, platelets were pretreated
with calpeptin and examined for agonist-induced PS exposure and
microparticle release. Similar to previous reports,17,18 calpeptin pretreatment significantly inhibited microparticle release in
response to thrombin plus collagen (Fig
7B). The extent of inhibition of microparticle release was similar for
calpeptin (69% ± 6% inhibition at 20 minutes; Fig 7B) and
DEVD-fmk (58% ± 7% inhibition at 20 minutes; Fig 3B), and the
inclusion of DEVD-fmk together with calpeptin did not further inhibit
residual microparticle release (Fig 7D).
Our studies with specific reagents demonstrate the presence of at least
one caspase family protease in platelets. Peptidase activity
corresponding to the effector protease caspase-3 was detected in
lysates of resting platelets and was significantly increased in
A23187-activated platelets. Specific antibodies identified the zymogen
procaspase-3 in resting platelets, and stimulation with agonist induced
processing of procaspase-3. Active platelet caspase-3 appears to be
short-lived, because it did not accumulate at levels adequate for
detection by immunoblot.
The authors thank Drs Zhinan Xia and Judy Lieberman for providing
granzyme B, Dr Anthony Bretscher for advice, Dr Dianne Kenney for
advice and the use of equipment, and Drs John Hartwig and Andrey
Prodeus for critical reading of the manuscript. We are grateful to the
blood donors for their cooperation.
Submitted September 9, 1998; accepted January 25, 1999.
Supported by National Institutes of Health Grant No. AI39574.
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.
Address reprint requests to Eileen Remold-O'Donnell, PhD, The Center
for Blood Research, 800 Huntington Ave, Boston, MA, 02115; e-mail:
remold{at}cbr.med.harvard.edu.
1.
Zwaal RFA, Schroit AJ:
Pathophysiologic implications of membrane phospholipid asymmetry in blood cells.
Blood
89:1121, 1997
2.
Abrams CS, Ellison N, Budzynski AZ, Shattil SJ:
Direct detection of activated platelets and platelet-derived microparticles in humans.
Blood
75:128, 1990
3.
Holme PA, Solum NO, Brosstad F, Roger M, Abdelnoor M:
Demonstration of platelet-derived microvesicles in blood from patients with activated coagulation and fibrinolysis using a filtration technique and Western blotting.
Thromb Haemost
72:666, 1994[Medline]
[Order article via Infotrieve]
4.
Lee Y, JY W, Horstman LL, Janania J, Kelley R, Ahn YS:
Elevated platelet microparticles in multiinfarct dementias and transient ischemic attacks.
Thromb Res
72:295, 1993[Medline]
[Order article via Infotrieve]
5.
George JN, Pickett EB, Saucerman S, McEver RP, Kunicki TJ, Kieffer N, Newman PJ:
Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery.
J Clin Invest
78:340, 1986
6.
Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC, Maquelin KN, Roozendaal KJ, Jansen PGM, ten Have K, Eijsman L, Hack CE, Sturk A:
Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant.
Circulation
96:3534, 1997
7.
Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, Ghayur T, Brady KD, Wong WW:
Substrate specificities of caspase family proteases.
J Biol Chem
272:9677, 1997
8.
Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP, Chapman KT, Nicholson DW:
A combinatorial approach defines specificities of members of the caspase family and granzyme B.
J Biol Chem
272:17907, 1997
9.
Salvesen GS, Dixit VM:
Caspases: Intracellular signaling by proteolysis.
Cell
91:443, 1997[Medline]
[Order article via Infotrieve]
10.
Nath R, Raser KJ, Stafford D, Hajimohammadreza I, Posner A, Allen H, Talanian RV, Yuen P, Gilbertsen RB, Wang KKW:
Non-erythroid
11.
Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ, Williams LT:
Caspase-3-generated fragment of gelsolin: Effector of morphological change in apoptosis.
Science
278:294, 1997
12.
Nakamura F, Amieva MR, Furthmayr H:
Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets.
J Biol Chem
270:31377, 1995
13.
Shcherbina A, Bretscher A, Kenney DM, Remold-O'Donnell E:
Moesin, the major ERM protein of lymphocytes and platelets, differs from ezrin in its insensitivity to calpain.
FEBS Lett
443:31, 1999[Medline]
[Order article via Infotrieve]
14.
Bretscher A, Reczek D, Berryman M:
Ezrin: A protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures.
J Cell Sci
110:3011, 1997[Abstract]
15.
Tsukita S, Yonemura S, Tsukita S:
ERM (ezrin/radixin/moesin) family: From cytoskeleton to signal transduction.
Curr Opin Cell Biol
9:70, 1997[Medline]
[Order article via Infotrieve]
16.
Shcherbina A, Kenney DM, Bretscher A, Remold-O'Donnell E:
Dynamic association of moesin with the membrane skeleton of thrombin-activated platelets.
Blood
93:2128, 1999
16a. Shcherbina A, Bretscher A, Rosen FS, Kenney DK,
Remold-O'Donnell E: The cytoskeletal linker protein moesin: Decreased
levels in Wiskott-Aldrich syndrome platelets and identification of a
cleavage pathway in normal platelets. Br J Haematol (in press)
17.
Fox JEB, Austin CD, Reynolds CC, Steffen PK:
Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets.
J Biol Chem
266:13289, 1991
18.
Dachary-Prigent J, Freyssinet J-M, Pasquet J-M, Carron J-C, Nurden AT:
Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: A flow cytometry study showing a role for free sulfhydryl groups.
Blood
81:2554, 1993
19.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680, 1970[Medline]
[Order article via Infotrieve]
20.
Samis JA, Zboril G, Elce JS:
Calpain I remains intact and intracellular during platelet activation
21.
Xia Z, Kam C-M, Huang C, Powers JC, Mandle RJ, Stevens RL, Lieberman J:
Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.
Biochem Biophys Res Commun
243:384, 1998[Medline]
[Order article via Infotrieve]
22.
Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC:
Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.
Nature
371:346, 1994[Medline]
[Order article via Infotrieve]
23.
Fernandes-Alnemri T, Litwack G, Alnemri ES:
CPP32, a novel human apoptotic protein with homology to Caenorhabditis |
TOP
ABSTRACT
INTRODUCTION
granule secretion, shape change, and
aggregation were unaffected by caspase-3 inhibitors. Experiments with
two classes of protease inhibitors show that caspase-3 function is
distinct from that of calpain, which is also involved in late platelet
activation events. These findings show novel functions of caspase and
provide new insights for understanding of platelet activation.
the apoptotic process. Common characteristics include
the prominence of coordinated morphological changes and, to a large
extent, the quality of irreversibility. In platelet activation, as in
apoptosis, transbilayer migration of phosphatidylserine to the outer
membrane leaflet is an important event, facilitating interaction with
phagocytic cells and, in the case of platelets, generating, in
addition, the procoagulant surface. Also, the process of platelet
microparticle formation is morphologically similar to the membrane
blebbing phase of nucleated cell apoptosis. These shared features
prompted us to examine a possible role in platelet activation for
caspases, a protease family intimately associated with the programmed
death of nucleated cells.
OD per minute was
calculated from the linear range of the reaction.
granule secretion, 107 platelets were
activated as described above, and the reaction was stopped after 0, 30, or 60 seconds by adding an equal volume of 2% paraformaldehyde. Aliquots of the fixed platelets were incubated for 10 minutes with 1 µg/mL PE-labeled anti-CD62P (clone AC1.2 MoAb; Becton Dickinson, San
Jose, CA) and examined by flow cytometry.
-converting-enzyme-like protease(s) in apoptotic cells: Contributory roles of both protease families in neuronal apoptosis.
Biochem J
319:683, 1996