From the
The processing of amyloid precursor protein (APP) and production
of
The major constituent protein (
The mechanism of proteolytic processing of APP is an important
problem in the study of Alzheimer's disease, since mutations in
the APP gene are etiologically linked to some familial forms of the
disease (Ashall and Goate, 1994). The pathogenic mutations occur at or
near the cleavage sites involved in the release of the
APP is modified post-translationally by different
proteolytic pathways. Most studies of APP metabolism have been carried
out in cell lines transfected with APP
We
have demonstrated that human platelets contain APP
In the present study, we investigated the processing of
APP
In order to determine whether the
disappearance of the APP
Proteolysis and release of platelet membrane
glycoproteins GPIb to the medium as a result of A23187 plus calcium
treatment have been reported (McGowan et al., 1983). To
confirm this result, we separated the platelets from the incubation
medium and analyzed the appearance of glycocalicin (GC, an
extracellular region of GPIb) in the medium by Western blotting.
Increased amounts of GC were detected when platelets were treated with
thrombin plus collagen, PMA, or A23187, and maximum release of GC was
found with A23187 plus calcium treatment (Fig. 1C). The
release of GC to the medium was partially inhibited by pretreatment of
platelets with calpeptin, TLCK, E64d, and EGTA, but not by other
protease inhibitors including PMSF, DFP, pepstatin
(Fig. 2B).
The
cleavage of the 22-CTF in whole platelets was also examined after
stimulation with other agonists. When platelets were treated with
thrombin (1 unit/ml, or even 10 units/ml) and collagen (20 µg/ml)
or PMA (1 µM) in the presence or absence of calcium,
degradation of the 22-CTF was not observed (Fig. 4A, lanes 2 and 3; 6 and 7,
+Ca
The involvement of a
lysosomal or acidic compartment in the cleavage of 22-CTF was also
examined. When whole platelets were preincubated with NH
We also examined cleavage of other
platelet proteins as a result of A23187 treatment. Talin is an
endogenous substrate of calpain and its degradation is used as an index
of calpain activity upon platelet activation (Fox et al.,
1985, 1990). Complete cleavage of talin was only observed in the
calcium-containing medium, but not in the calcium-free medium
(Fig. 4B, lanes 4 and 8), where under the same
conditions the disappearance of the 22-CTF of APP and the appearance of
the 17-CTF occurred (Fig. 4A). A combination of thrombin
and collagen treatment or PMA treatment in the presence or absence of
calcium resulted in partial degradation of talin (Fig. 4B,
lanes 2, 3, 6, and 7), in agreement with previous reports
(Fox et al., 1990; Yano et al., 1993), but the
cleavage of 22-CTF was not apparent (Fig. 4). In contrast,
platelet plasma membrane glycoprotein IIIa and the cytoskeleton protein
actin were not cleaved under any of these conditions (data not shown).
The cleavage of talin was also inhibited by calpeptin, TLCK, E64d, or
EGTA (Fig. 5C).
This study provides evidence that
A 22-kDa polypeptide was
found in platelets which reacts with a variety of antibodies to the
The protease
responsible for the degradation of APP
Since APP
Other studies support
our view that calcium is involved in the regulation of APP processing.
Buxbaum et al.(1994) showed that APP processing is regulated
by intracellular calcium levels, independent of regulation by protein
kinase C activation. Calcium might also be involved in the
amyloidogenic processing of APP (Mattson et al., 1992;
Peterson et al., 1986; 1991; Saito et al., 1993).
Increasing intracellular calcium levels with A23187 mediates a 3-fold
increase in production of
The platelets were
preincubated with the reagents for 15 min at room temperature and
activated with A23187 (1 µM). The samples were analyzed by
Western blotting as described under ``Materials and
Methods.'' The final concentrations of inhibitors are shown.
``+'' indicates that the cleavage of substrate was
inhibited or that product was formed, ``+/-''
indicates partial inhibition, and ``-'' indicates lack
of inhibition of substrate cleavage or no product formation. Numerical
values are not assigned as these were not quantitative assays.
Platelet lysates were preincubated with the
indicated reagents followed by addition of calpain I and analyzed by
Western blotting as described in the ``Materials and
Methods.'' ``+'' indicates the 17-CTF product was
detected, and ``-'' indicates lack of product
formation, ``+/-'' indicates that a small amount
of the 17-CTF product was detected compared to the control with
protease alone. Numerical values are not assigned as this was not a
quantitative assay.
We are grateful to Dr. Sam Gandy (Cornell University
Medical College, New York) for the generous supply of the rabbit
polyclonal antibody 369A. We thank Dr. Michael Berndt for critical
review of this manuscript. We are grateful to Dr. Jon Currie and Valcy
Malone of the Mental Health Research Institute of Victoria, Melbourne,
Australia, for coordination of volunteers.
Note Added in Proof-A calpain inhibitor (MDL 28170) has been
reported to block
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
A4 amyloid are events likely to influence the development and
progression of Alzheimer's disease, since
A4 is the major
constituent of amyloid deposited in this disorder. Our previous studies
showed that human platelets contain full-length APP (APP
)
and are a suitable substrate to study normal APP processing. In the
present study, we show that a 22-kDa
A4-containing
carboxyl-terminal fragment (22-CTF) of APP is present in unstimulated
platelets. Both APP
and 22-CTF are proteolytically
degraded when platelets are activated with thrombin, collagen, or
calcium ionophore A23187. Complete cleavage of APP
and
22-CTF require the presence of extracellular calcium. Following
stimulation in the presence of calcium, a new CTF of 17 kDa is
generated, and the NH
-terminal epitope of
A4 amyloid
is lost. Preincubation of platelets with the cell-permeable cysteine
protease inhibitors calpeptin,
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane
ethyl ester (E64d),
Na
-p-tosyl-L-lysine chloromethyl
ketone, or calcium chelator EGTA before platelet stimulation inhibits
the degradation of both APP
and 22-CTF. Divalent metal
ions including zinc, copper, and cobalt inhibit the degradation of
APP
and 22-CTF. This study suggests that a
calcium-dependent neutral cysteine protease is involved in the
proteolytic processing of an amyloidogenic species of APP in human
platelets.
A4) of amyloid plaques,
which are a pathologic hallmark of Alzheimer's disease, is
proteolytically derived from a larger glycoprotein precursor, the
amyloid precursor protein (APP).
(
)
The
A4
amyloidogenic region spans 39-44 residues, the carboxyl-terminal
third of which is part of the transmembrane domain of APP (Kang et
al., 1987). A closely related family of membrane proteins (APLP1
and APLP2) (Sprecher et al., 1993; Wasco et al.,
1992) do not contain this amyloidogenic
A4 sequence. The family of
APP glycoproteins now extends to at least 10 isoforms generated by
alternate splicing of the APP gene, principally of exons 7, 8, and 15.
APP
, the prototypic isoform (Kang et al., 1987)
is predominantly expressed in the brain, and lacks a Kunitz-type
protease inhibitor domain encoded by exon 7. The Kunitz-type protease
inhibitor domain (Kitaguchi et al., 1988; Ponte et
al., 1988; Tanzi et al., 1988) is of particular interest
since it might function in an autoregulatory manner by controlling
proteolytic events near the cell membrane. The exclusion of exon 15
through alternate mRNA splicing generates a series of APP molecules
(L-APP), which were originally discovered in cells of the
lymphocyte/monocyte lineage (König et al., 1992).
Subsequent studies have shown that the L-APP isoforms are
predominantly expressed in non-neuronal cells, where the absence of the
exon 15 domain might also be expected to influence events relating to
juxta-membranous domains of APP (Sandbrink et al., 1994).
A4 protein.
Mutations near the NH
terminus (
-secretase site) and
near the
-secretase site of
A4 cause increased secretion of
A4 in transfected cells (Cai et al., 1993; Citron et
al., 1992; Haass et al., 1994). Mutations just outside
the COOH-terminal
-secretase site of
A4 (codon 717) cause an
increase in the ratio of
A4
to
A4
(Suzuki et al., 1994). Longer
A4 (
A4
) shows a greater tendency to
aggregate. However, the molecular mechanisms of
A4 deposition and
accumulation in the more common sporadic forms of the disease are not
well understood.
or APP
cDNA and show that about 15-30% of the newly synthesized
full-length APP (APP
) passes through a constitutive
secretory pathway. This generates the release of a large
NH
-terminal extracellular fragment of 100-130 kDa
into the extracellular compartment and an approximately 10-kDa
membrane-associated COOH-terminal fragment (CTF) (Knops et
al., 1992; Kuentzel et al., 1993; Weidemann et
al., 1989). This process involves cleavage in the middle of the
A4 sequence by a putative
-secretase activity and therefore
precludes formation of intact
A4 peptide (Esch et al.,
1990; Sisodia et al., 1990). Some of the remaining APP
may be reinternalized from the cell surface and degraded in a
lysosome/endosome compartment to generate a family of 8-22-kDa
CTFs (Golde et al., 1992; Haass et al., 1992; Knops
et al., 1992). In addition, low levels of intact
A4
or
A4
are
normally secreted by cells in culture (Cai et al., 1993; Haass
et al., 1993; Seubert et al., 1993; Suzuki et
al., 1994). This processing may occur either at an early step of
the endosomal-lysosomal pathway following endocytosis (Koo and Squazzo,
1994; Shoji et al., 1992), or in a late acidic compartment of
the secretory pathway (Busciglio et al., 1993). Proteases
termed
- and
-secretases are required to produce the
NH
- and COOH termini of
A4, respectively.
-Secretase activity is either integrated into the cell membrane or
operates after release of the transmembrane domain of APP from the
membrane. Numerous proteases have been implicated in APP processing
(reviewed by Evin et al.(1994)). The proteases which cleave
APP in vivo have not been identified. On the basis of
specificity and amino acid sequence alone, it is difficult to predict
which proteases are important for APP cleavage in vivo because
some proteases may require a specific secondary or tertiary
conformation of the substrate (Sisodia, 1992). Alternatively, the
subcellular compartmentation of many proteases may exclude them from
action on APP. For this reason, it is important to study APP processing
in a cellular system where normal compartmentation is preserved.
(Li
et al., 1994). Therefore platelets may be a useful model to
study normal APP processing. Unlike transfected cell lines in which the
processing of APP may not totally reflect processing in vivo (Zhong et al., 1994), purified platelets provide a model
system which may be closer to that which occurs in vivo.
Platelets are the main source of hematogenous APP and platelet APP is
readily released upon physiological stimulation (Bush et al.,
1990; Cole et al., 1990; Smith and Broze, 1992; Van Nostrand
et al., 1991). The released APP potently inhibits coagulation
factor XIa (Smith et al., 1990), suggesting a role for APP in
hemostasis.
in stimulated platelets. We describe the
identification and degradation of a
A4-containing 22-kDa CTF of
APP following platelet activation. Our data suggest that a
calcium-dependent neutral cysteine protease is involved in the
degradation of the amyloidogenic species of APP in platelets.
Materials
Calpeptin was obtained from
Calbiochem-Novabiochem Pty. (Alexandria, Australia) and dissolved in
dimethyl sulfoxide. Rainbow protein molecular weight
markers were purchased from Amersham Corp. (Aylesbury, United Kingdom).
Electrophoresis-grade acrylamide, N,N`-methylene
bisacrylamide, N,N,N`,N`-tetramethylethylenediamine, and
ammonium persulfate were obtained from Bio-Rad. Purified calpain I and
II were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Leupeptin
was from Auspep (West Melbourne, Australia). PGE
, collagen,
calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA),
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane
(E64),
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane
ethyl ester (E64d),
Na
-p-tosyl-L-lysine chloromethyl
ketone (TLCK), N-tosyl-L-phenylalanine chloromethyl
ketone (TPCK), diisopropyl fluorophosphate (DFP), 1,10-phenanthroline,
phosphoramidon, phenylmethylsulfonyl fluoride (PMSF), iodoacetamide,
and EGTA were from Sigma. Thrombin was from Boehringer Mannheim
(Mannheim, Germany). Protein A-Sepharose CL-4B was purchased from
Pharmacia LKB (Uppsala, Sweden). Protein concentrations were determined
by the BCA method (Pierce, Rockford, IL). Immobilon P (PVDF) was from
Millipore Corp. (Bedford, MA). All other chemicals were of analytical
reagent grade.
Antibodies
Monoclonal antibody (mAb) 22C11, which
recognizes an epitope near the amino terminus of APP (Weidemann et
al., 1989; Hilbich et al., 1993), was from Boehringer
Mannheim GmbH (Germany). Polyclonal rabbit antibody 92/9 was raised
against purified human APP (Moir et al., 1992).
Affinity-purified rabbit antibody to the cytoplasmic domain (residues
645-694) of APP (Ab 369A) (Buxbaum et al.,
1990; Nordstedt et al., 1991) was kindly provided by Dr. Sam
Gandy (Cornell University Medical College, New York). An anti-CT
antibody (raised to 704-730 of APP
) was a gift from
Dr. B. Cordell (Scios Nova Inc., Mountain View, CA). Monoclonal 6E10
which recognizes residues 1-17 of
A4 (Kim et al.,
1990) was purchased from the New York State Institute for Basic
Research in Developmental Disabilities, Staten Island, NY. Polyclonal
antibody ``730'' was raised against residues 1-40 of
A4. Monoclonal antibodies to talin and thrombospondin were
purchased from Sigma. Antibody to GPIb (WM23) has been previously
characterized (Berndt et al., 1985; Li et al., 1994).
Affinity purified anti-mouse and anti-rabbit alkaline
phosphatase-conjugated secondary antibodies were obtained from Promega
Corp. (Madison, WI).
Platelet Preparation and Activation
Human
platelets were prepared from fresh blood as described previously (Li
et al., 1994). In brief, platelets were isolated by
centrifugation and washed twice in buffer (0.38% sodium citrate, 0.6%
glucose, 0.72% NaCl, pH 7.0) in the presence of 3 µM
PGE. Platelets were then resuspended (1
10
/ml) in Tyrode's buffer (136.9 mM NaCl,
2.68 mM KCl, 11.9 mM NaHCO
, 0.42
mM NaH
PO
with 2 mM
CaCl
, 1 mM MgCl
, 5.5 mM
glucose, pH 7.4) (calcium-containing medium). In some cases, platelets
were resuspended in EHS buffer (10 mM HEPES, 150 mM
NaCl, and 1 mM EDTA, pH 7.4) (calcium-free medium). The
platelets were then incubated with 1 µM A23187, or 1
µM PMA, or a combination of 1 unit/ml thrombin and 20
µg/ml collagen for 15 min at room temperature with stirring in the
presence of calcium (Tyrode's buffer), unless indicated. After
the incubation, EGTA (200 mM, pH 9.0) was added to a final
concentration of 5 mM just before centrifugation to separate
the platelets from the incubation medium. The platelet pellets were
lysed in a modified EHS buffer containing 5 mM EDTA, 1% (v/v)
Triton X-100, 0.2 mg/ml leupeptin, and 1 mM PMSF followed by
brief centrifugation at 9000
g in a microcentrifuge to
remove cytoskeletal elements.
Preparation of Platelet Lysate for Calpain
Digestion
To obtain platelet lysate for calpain digestion,
platelets were lysed in Tyrode's buffer (with 1% Triton X-100,
lacking calcium and any protease inhibitors). Digestion of the CTF of
APP with calpain was performed as follows. Calcium chloride (final
concentration, 2 mM) and calpain I or II (final concentration,
20 µg/ml) were added to 20 µl of lysate (equivalent to 2
10
platelets) and incubated for 30 min at 30
°C. The reaction was terminated by adding sample buffer (42
mM Tris-HCl, 1% (w/v) glycine, 2% (w/v) SDS, and 5% (v/v)
-mercaptoethanol). In some cases, platelet lysates were
preincubated with the indicated reagents for 5 min before addition of
calpain.
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western
Blotting
Lysate equivalent to 2 10
platelets
were dissolved in sample buffer, reduced, boiled, and separated on 15
or 8.5% discontinuous polyacrylamide gels in the presence of SDS. The
proteins were then transferred electrophoretically to Immobilon P
membranes (PVDF, Millipore) as described previously (Li et
al., 1994). Nonspecific binding of primary antibody was blocked
with 3% (w/v) bovine serum albumin and the PVDF membranes were
incubated with primary antibody overnight at 4 °C.
Affinity-purified secondary anti-mouse or anti-rabbit IgG conjugated to
alkaline phosphatase (1:10,000) were used for immunodetection of the
bound primary antibody. The alkaline phosphatase-conjugated secondary
antibody was visualized by chromogenic reaction using Fast-Red/Naphthol
AS-MX phosphate as substrates for the alkaline phosphatase. Where
indicated, chemiluminescence was used to detect the primary antibody
(369A) on PVDF membrane: a horseradish peroxidase-linked secondary
antibody was used and visualized by enhanced chemiluminescence
(Boehringer Mannheim) according to the instructions provided by the
manufacturer.
Reproducibility of Experiments
The figures and
tables present representative results of experiments which were
performed at least three times.
Processing of Full-length APP in Whole
Platelets
Previous studies have identified 140-150-kDa
APP in platelets (Li et al., 1994; Gardella
et al., 1992; Schlossmacher et al., 1992). Processing
of this species may generate amyloid-containing fragments. Proteolytic
cleavage of APP
was therefore examined in whole activated
platelets. Platelets were incubated with different agonists in the
presence or absence of calcium, lysed, and APP immunoreactivities were
analyzed by Western blotting with an affinity-purified antibody to the
cytoplasmic domain of APP (antibody 369A). This antibody has been shown
to label the 140-150-kDa APP
and smaller fragments
of APP, and does not detect COOH-terminal truncated soluble APP
(Buxbaum et al., 1990; Li et al., 1994; Nordstedt
et al., 1991). The present study showed that resting platelets
(PGE
-treated) contained APP
(Fig. 1A, lanes 1 and 5), in agreement
with our previous report (Li et al., 1994). The APP
was also detected by mAb 6E10 which recognizes an epitope of
A4
(data not shown). Activation of platelets
by physiological stimulants (a combination of thrombin and collagen) or
by nonphysiological agonists (PMA or calcium ionophore A23187) in
either calcium-free (EHS buffer containing 1 mM EDTA) or
calcium-containing medium (Tyrode buffer containing 2 mM
CaCl
, 1 mM MgCl
) resulted in decreased
immunostaining of a APP
band (Fig. 1A, lanes
2-4 and 6-8). A23187 together with calcium
was the most potent treatment as it induced complete cleavage of
APP
(Fig. 1A, lane 8, and
Fig. 2A, lane 4). Platelet activation and degranulation
progressed under these conditions with or without calcium, as indicated
by release of APP into the medium (Fig. 1B).
Figure 1:
Western blotting
analysis of cleavage of APP in intact platelets and
appearance of APP
and GPIb in the medium, indicating that
the presence of A23187 plus calcium induces maximum APP
degradation and GPIb release. Washed platelets were activated
with the agonists indicated in the presence of 2 mM CaCl
(+Ca
) or 1 mM EDTA
(-Ca
) and platelet lysates and medium
were prepared. Samples were separated by a 8.5% SDS-PAGE gel,
transferred to PVDF membranes, and then probed with indicated
antibodies. The primary antibody was detected with alkaline
phosphatase-conjugated secondary antibody by chromogenic reaction.
Panel A, lysates derived from 2
10
platelets and analyzed with an antibody to the COOH terminus of
APP (369A). Panel B, platelet incubation medium derived from 2
10
platelets were analyzed with an antibody to the
N terminus of APP (mAb 22C11). Panel C, platelet incubation
medium derived from 1
10
platelets were analyzed
with an antibody to GPIb (WM23). The relative mass of standard protein
markers is shown on the left. The localization of APP and GC
immunoreactivities are indicated on the right. T/C, thrombin
plus collagen; A, calcium ionophore
A23187.
Figure 2:
Western blot analysis of APP degradation within platelets and GPIb appearance in the medium in
the presence of protease inhibitors and metal ions, showing that
APP
degradation and GPIb release are partially inhibited
by calpeptin and EGTA. A, fluorogram to show the inhibition of
APP
degradation. Washed platelets were treated with the
agonists in the presence of the indicated agents. Platelet lysates were
prepared and analyzed by Western blotting as described in the legend to
Fig. 1 except that the primary antibody 369A was detected by
chemiluminescence. B, inhibition profile of GPIb release.
Platelet incubation media were analyzed with an antibody to GPIb
(WM23). T/C, thrombin plus collagen; A, A23187;
Cal, calpeptin.
Platelet
APP could also be detected by Western blotting using the
chemiluminescence visualization technique, which is more sensitive than
chromogenic visualization (Fig. 2A). Following
activation, APP
disappeared and two faint bands of
90-95 kDa labeled by 369A appeared, these bands were not present
in the PGE
-treated platelets (Fig. 2A, lanes 1 and 4). By semi-quantitation using densitometric
analysis, only about 60-70% of APP
was cleaved when
platelets were stimulated by thrombin plus collagen or by PMA, in
contrast to nearly 100% of APP
being degraded in
A23187-treated platelets.
was due to a proteolytic process,
washed platelets were preincubated with various protease inhibitors for
15 min before addition of A23187. Cleavage of APP
in
A23187-treated platelets was inhibited by calpeptin, TLCK, E64d, and
EGTA as well as metal ions, Zn
, Cu
,
and Co
(Fig. 2A and ).
Pretreatment of platelets with calpeptin, EGTA, or Zn
protected 40-60% of APP
from degradation.
Cu
and Co
blocked almost all of the
APP
degradation (90%). A lower concentration of calpeptin
(2 µg/ml) also partially inhibited APP
degradation
(data not shown). APP
degradation induced by thrombin or
PMA was also inhibited by calpeptin and EGTA, similar to that found by
A23187 induction (data not shown).
Cleavage of APP
There is considerable evidence suggesting a
role for lysosomal and acidic compartments in the processing of APP
(Busciglio et al., 1993; Caporaso et al., 1992; Haass
et al., 1992; Knops et al., 1992). Involvement of a
lysosomal or acidic compartment in the degradation of APPIs Not
Inhibited by Agents That Affect the Acidic
Compartments
was examined by preincubating whole platelets with
NH
Cl, chloroquine, or monensin before addition of A23187.
The APP immunoreactivity in the platelet lysate was analyzed by Western
blotting. None of these agents inhibited the cleavage of APP
(), suggesting that the enzymes of lysosomal or
acidic compartments were not involved in the degradation of APP
of platelets.
Identification of APP CTF Containing
Initially, we examined the A4
A4-containing CTF of APP
by Western blotting of total platelet lysates. A lysate from
PGE
-treated platelets was found to contain a 22-kDa
COOH-terminal fragment of APP which was recognized by antibody 369A
(Fig. 3A, lane 1). Two different approaches then
confirmed that this 22-kDa band was a CTF of APP. First, when the
antibody was preincubated with the peptide homologue used to raise
antibody 369A, the staining was abolished (Fig. 3B).
Second, other antibodies including the polyclonal Ab 730
(anti-
A4
) (Fig. 3A, lane 2),
anti-CT (raised to 704-730 of APP
)
(Fig. 3A, lane 3), 92/9 (anti-APP) (Fig. 3A,
lane 4), and mAb 6E10 (anti-
A4
)
(Fig. 4A) recognized this 22-kDa fragment. Since the
epitope for 6E10 is within the sequence 1-17 of
A4 (Kim
et al., 1990), this result indicated that the 22-kDa fragment
also contains most of the
A4 sequence, in addition to the
COOH-terminal sequence of APP. The 22-kDa fragment was reproducibly
detected in platelets from several normal subjects. The 369A antibody
also recognized other CTFs with molecular mass of 15, 16, and 18 kDa
(Fig. 3). Since these bands were present in both lysates from
PGE
- and A23187-treated platelets in similar amounts and
therefore were unrelated to the activation process, they were not
examined further.
Figure 3:
Characterization of a 22-kDa A4
containing fragment in lysates of PGE
-treated platelets by
Western blot. Platelets isolated in the presence of PGE
were lysed and lyastes derived from 2
10
platelets were loaded onto a 15% SDS-PAGE gel and analyzed as
described in the legend to Fig. 1. A, APP CTFs were analyzed
with different antibodies. Lane 1, antibody to the COOH
terminus of APP (369A); lane 2, antibody to the 1-40
sequence of
A4 (730); lane 3, antibody to the
704-730 sequence of APP
(anti-CT); and lane
4, antibody to purified human brain APP (92/9). B, the
immunoreactivity to the 22-kDa band was abolished in the presence of
the peptide used to raise the 369A antibody (lane 1). The
relative mass of standard protein markers is shown on the
left. The localization of APP COOH-terminal immunoreactivities
are indicated on the right. Only the 22-kDa band was
recognized by all four antibodies.
Figure 4:
Western blot showing that cleavage of
22-CTF of APP in activated platelets required external calcium. Washed
platelets were stimulated with the indicated agonists in the presence
of 2 mM CaCl (+Ca
)
or 1 mM EDTA (-Ca
) and
analyzed as described in the legend to Fig. 1. Panel A,
platelet lysates were analyzed with antibody 369A or 6E10; panel
B, platelet lysates were analyzed with anti-talin. ``*''
indicates the new 17-kDa fragment generated upon platelet activation.
T/C, thrombin plus collagen; A,
A23187.
Cleavage of the 22-kDa CTF of APP in Whole Platelets is a
Calcium-dependent Process
We next examined the degradation of
the APP 22-CTF associated with platelet activation. Platelets incubated
with various agonists were lysed and the APP immunoreactive
polypeptides in the lysate were analyzed by Western blotting using
antibody 369A. When platelets were incubated with the calcium ionophore
A23187 (1 µM) in a calcium-containing medium, the 22-kDa
band (Fig. 4A) disappeared and a 17-CTF was observed
(Fig. 4A, lane 8, +Ca).
The 17-CTF was not present in the PGE
-treated platelets
(Fig. 4A, lane 5, +Ca
),
and was not recognized by antibody 6E10 (Fig. 4A, lane
4, +Ca
). This process was
dependent on the presence of external calcium, since platelets treated
with A23187 in a calcium-free medium did not lead to the degradation of
the 22-kDa CTF, nor to the generation of the 17-kDa CTF
(Fig. 4A, lanes 4 and 8,
-Ca
), even though platelets were
stirred in both situations to maximize platelet activation.
and
-Ca
). Under these conditions, however,
the platelets were activated, as indicated by increased APP secretion
following degranulation (Fig. 1B, lanes 2, 3, 6, and
7).
Effect of Protease Inhibitors and Metal Ions on the
Proteolytic Cleavage of the 22-CTF of APP
The effect of
different protease inhibitors was examined to identify the classes of
proteases which may be involved in the cleavage of the
A4-containing 22-CTF. Platelets were preincubated with protease
inhibitors or metal ions for 15 min, then the platelets were treated
with A23187. Platelet lysates were prepared and the APP
immunoreactivities were observed by Western blotting using antibody
369A. When platelets were preincubated with calpeptin, TLCK, E64d, or
EGTA, degradation of the 22-CTF in A23187-treated platelets was
inhibited (Fig. 5A). Inhibition of the 22-CTF cleavage
was also detected with lower concentration of calpeptin at 2 µg/ml
(data not shown). Cleavage of the 22-kDa CTF was not inhibited by the
serine protease inhibitors PMSF or DFP, or the metalloprotease
inhibitors 1,10-phenanthroline or phosphoramidon, or the aspartic
protease inhibitor pepstatin A (). Cleavage of the 22-CTF
still occurred in the presence of the cysteine protease inhibitors
iodoacetamide, or the chymotrypsin-like serine protease inhibitor TPCK,
but the intensity of the 17-kDa band was diminished compared to the
A23187-treated platelets (). Preincubation of platelets
with Zn
, Cu
, or Co
inhibited the cleavage of 22-CTF, while Fe
did
not have any inhibitory effect ().
Figure 5:
Western blot of the 22-CTF degradation in
the presence of protease inhibitors, showing that calpeptin and EGTA
prevent the disappearance of the 22-CTF and the appearance of the
17-CTF, and inhibit talin degradation. Washed platelets were
preincubated with the indicated reagents, then treated with A23187.
Platelets were separated from incubation medium and the platelet
lysates were analyzed by Western blot using antibody 369A (panel
A) or anti-talin (panel C); platelet incubation media
were analyzed with 22C11 (panel B). ``*'' indicates
the new 17-kDa fragment generated upon platelet activation.
Cal, calpeptin.
The inhibitors
calpeptin and EGTA which inhibited the degradation of 22-CTF of APP did
not significantly affect the secretion of -granule proteins
including APP (Fig. 5B), indicating that platelets under
these conditions were physiologically competent and that the processing
of the 22-CTF was specifically associated with the calcium influx.
Previous studies have shown that the concentration of calpeptin used
does not interfere with platelet aggregation and secretion (Fox et
al., 1990). However, the inhibitory effect of metal ions
(Zn
, Cu
, or Co
)
on the 22-CTF degradation may be due in part to their effects on
inhibiting platelet activation, since they inhibited the release of
both APP and thrombospondin (data not shown).
Cl,
chloroquine, or monensin before the addition of A23187, cleavage of the
22-CTF was not inhibited (). This suggests that the enzymes
of lysosomal or acidic compartments were not involved in the
degradation of the 22-CTF.
Digestion of CTF of APP by Purified Calpain
The
previous results suggested that the cleavage of the 22-CTF was mediated
by a calcium-dependent neutral cysteine protease, most of which is
composed of calpain in platelets (Phillips and Jakabova, 1977). To
avoid the problem that some inhibitors may not have been able to
penetrate the platelet membrane and interfere with intracellular
processing, a platelet lysate was prepared in Tyrode buffer containing
1% Triton X-100 without any added calcium or protease inhibitors. Under
these conditions, a 20-CTF of APP was identified in the initial lysate
by antibody 369A. From the previous experiments (Fig. 3-5),
it was apparent that platelets lysed in the presence of protease
inhibitors (leupeptin, PMSF, and EDTA) contained a 22-CTF which
disappeared after repeated freeze-thawing of the lysate. This finding
suggests that the 22-CTF is a labile intermediate in the APP processing
pathway. Interestingly, intact talin was not recovered in the lysate
prepared without protease inhibitors, suggesting that activation of
endogenous calpain due to the lysis procedure was sufficient to cleave
the 22-CTF and talin, but not sufficient to produce a 17-CTF of APP. To
investigate the possibility that calpain may be involved in the
degradation of APP fragments, platelet lysates prepared without
protease inhibitors were incubated with purified calpain I or II in the
presence of 2 mM calcium. On analysis of the APP fragments by
Western blotting with antibody 369A, the addition of calpain I resulted
in the generation of a 17-CTF, while calpain II did not generate a
17-CTF (Fig. 6, lanes 3 and 4). The generation
of the 17-CTF by calpain I was inhibited by preincubation of the lysate
with calpeptin, E64, TLCK, EGTA, or Cu, partially
inhibited by Zn
, but not by iodoacetamide, DFP,
Co
, Fe
, or pepstatin A
(). The study of APP
processing in cell-free
lysate was not possible because there was no detectable APP
in the platelet lysates prepared without protease inhibitors,
indicating that APP
was rapidly degraded upon platelet
lysis as previously reported (Li et al., 1994; Schlossmacher
et al., 1992).
Figure 6:
Western blot of calpain digestion of
platelet lysates, showing that purified calpain I can produce a 17-CTF.
Platelet lysates were preincubated with the indicated reagents followed
by the addition of calpain. Samples were separated on a 15% SDS-PAGE
gel followed by Western blotting using antibody 369A. ``*''
indicates the 17-kDa fragment generated by exogenous calpain I.
C, platelet lysate without incubation; C`: 0.2%
MeSO was added to the lysate during incubation.
Cal: calpeptin.
A4 containing CTF of
APP and APP
are present in platelets and are
proteolytically processed following platelet activation. APP
is cleaved when platelets are stimulated by thrombin, collagen,
or a combination of both, or by PMA or A23187. Stimulation of platelets
by PMA or by a combination of thrombin and collagen leads to the loss
of 60-70% of APP
. In contrast, treatment with A23187
in the presence of calcium induces complete degradation of
APP
, and induces the generation of two new CTFs of
90-95 kDa identified by 369A, which are not present in
PGE
, thrombin/collagen, or PMA-treated platelets. This
indicates that the APP
can be cleaved in the
NH
-terminal extracellular domain. It is not clear whether
the degradation of APP
produces any large
NH
-terminal fragments (by cleavage at the COOH-terminal
side), since it is difficult to differentiate any newly generated
NH
-terminal fragments from the existing sAPP species
(e.g. APP
, APP
) in
PGE
-treated platelets. The cleavage of APP
is
partially inhibited by cysteine protease inhibitors, calpeptin, TLCK,
E64d, or the calcium chelator EGTA, but not inhibited by serine,
metallo, or aspartic protease inhibitors (DFP, PMSF, phosphoramidon,
1,10-phenanthroline, or pepstatin) and acidic compartment inhibitors
(monensin, chloroquine, or NH
Cl). Although metal ions
(Zn
, Cu
, and Co
)
inhibit APP degradation, this may be due to complex effects on the
physiological function of platelets, since these ions affect the
secretion of platelet
-granule contents, including APP and
thrombospondin.
(
)
A4 and COOH-terminal region of APP, including 6E10 and 369A. This
indicates that the 22-CTF contains the
A4 sequence, and is
therefore derived from APP, rather than from the related molecules
APLP1 and APLP2 (Sprecher et al., 1993; Wasco et al.,
1992), and also is potentially amyloidogenic. Because the 22-CTF is
present in resting platelets, it is likely to have been produced by a
normal processing event. Previous studies have also identified APP CTFs
in platelets and lymphoblastoid cells in familial Alzheimer's
disease (Ghiso et al., 1994; Matsumoto and Fujiwara, 1993). In
both studies, the NH
terminus of the CTF was found to begin
30 residues NH
-terminal to the
A4 sequence. By
immunoreactivity with specific antibodies, a 22-kDa APP fragment
containing the
A4 epitope was also found in transfected 293 cells,
brain microvessels, and human umbilical vein endothelial cells (Haass
et al., 1992; Knops et al., 1992; Tamaoka et
al., 1992). When platelets were treated with A23187 plus calcium,
the 22-CTF was not detected. Concomitant with the loss of the 22-CTF,
there was the appearance of a 17-CTF. This 17-CTF did not react with
mAb 6E10 (which recognizes
A4
), indicating
that the NH
terminus of
A4 may have been removed with
the loss of this epitope. Since the 17-CTF band reacts more strongly
with antibody 369A than the 22-CTF band, this suggests that larger
fragments (including APP
) could also be the precursors of
the 17-CTF. Low levels of 22-CTF cleavage and 17-CTF production in
thrombin/collagen-treated platelets may occur, but this may be beyond
the sensitivity of the Western blotting detection used in this study.
The cleavage of 22-CTF is also inhibited by calpeptin, TLCK, E64d, the
calcium chelator EGTA, or metal ions (Zn
,
Cu
, Co
), but not by serine,
metallo, or aspartic protease inhibitors, and not by reagents that
affect the acidic compartment and lysosomes.
and 22-CTF in the
stimulated platelets remains to be identified. The characteristics of
inhibition of degradation of APP
and 22-CTF are consistent
with the involvement of a calcium-dependent neutral cysteine protease,
since degradation of APP
and 22-CTF occurred at neutral pH
and was inhibited by cysteine protease inhibitors (calpeptin, TLCK,
E64d), and the calcium chelator EGTA. Calpeptin inhibits a number of
cysteine proteases (Tsujinaka et al., 1988; Sasaki et
al., 1990) and is widely used for studying the function of
platelet calpain which constitutes most of the calcium-dependent
protease activity in platelets (Ariyoshi et al., 1991; Fox
et al., 1990; Oda et al., 1993). Calpeptin is known
to inhibit lysosomal cathepsin B as well as calpain (Sasaki et
al., 1990). However, cathepsin B is most active under acidic
conditions and does not require calcium. Involvement of cathepsin B in
the platelet APP degradation is unlikely, since the platelet APP
degradation was not affected by NH
Cl and chloroquine.
Addition of purified calpain to the platelet lysate generated a 17-CTF,
similar to that found in the A23187-treated platelets. The generation
of the 17-CTF was also inhibited by calpeptin, E64, TLCK, and EGTA.
Taken together our data suggest that a calcium-dependent mechanism
involving a neutral cysteine protease with characteristics similar to
calpain participates in the degradation of APP
and 22-CTF.
However, the involvement of other proteases in APP degradation seems
likely.
is membrane-associated (Li et
al., 1994), it could be expected that the proteolytic activity (a
calcium-dependent neutral cysteine protease) involved in APP processing
also co-localizes to membranes. The main calcium-dependent neutral
cysteine protease in platelets is calpain and it is usually located
intracellularly in the cytoplasm or associated with membrane (Saido
et al., 1993, 1994). Our results confirm the presence of a
calcium-dependent neutral cysteine protease activity (calpeptin and
EGTA sensitive) inside platelets which degrades talin. This activity
would correspond to the previously described calpain activity (Fox
et al., 1985, 1990; Yano et al., 1993). We could also
demonstrate an activity resembling calcium-dependent neutral cysteine
protease outside platelets by studying the release of GPIb (the
receptor for von Willebrand factor). Increased amounts of GC were
detected in the medium as a result of platelet activation, and this
process was partially inhibited by calpeptin, TLCK, E64d, or EGTA. This
is consistent with the finding that GPIb is cleaved by a cysteine
protease (calpain) within the extracellular juxta-membranous segment to
release GC (McGowan et al., 1983; Yoshida et al.,
1983). The activity which cleaves GPIb therefore shares similar
characteristics with the one that processes APP. Whether cleavage of
APP and GPIb involves the same or different calcium-activated neutral
cysteine proteases remained to be determined.
A4 in human kidney 293 cells transfected
with APP
(Querfurth and Selkoe, 1994). An increase in the
activated form of calpain is found in Alzheimer's disease brain
regions where neurons are most affected (Nilsson et al., 1990;
Saito et al., 1993). Although we have been unable to
demonstrate any qualitative differences between Alzheimer's
disease and control platelets in their Ca
-activated
processing of APP,
(
)
the possibility remains that
a chronic disturbance of calcium homeostasis in neurons (especially if
caused by the toxicity of the
A4 protein itself) (Furukawa et
al., 1994) might perturb a calpain-like enzyme mediated cleavage
of APP. This may modulate a cycle of calcium-mediated APP processing,
and thereby affect an amyloidogenic pathway contributing to the
evolution of Alzheimer's disease.
Table:
Effect of various protease inhibitors, acidic
compartment inhibitors, and metal ions on the cleavage of 22-CTF and
APP in stimulated platelets
Table:
Effects of various protease inhibitors
and metal ions on the generation of the 17-CTF by calpain I in
platelet lysates
A4, the APP amyloid cleavage product; CTF,
COOH-terminal fragment; APP
, full-length APP; sAPP,
secreted APP; APP
, secreted APP product generated by
-secretase; APP
, secreted APP product generated by
-secretase; PGE
, prostaglandin E
; PMA,
phorbol 12-myristate 13-acetate; E64,
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane;
E64d,
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane
ethylester; TLCK, Na
-p-tosyl-L-lysine
chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine
chloromethyl ketone; DFP, diisopropyl fluorophosphate; PMSF,
phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel
electrophoresis; GPIb, glycoprotein Ib; PVDF, polyvinylidine
difluoride; mAb, monoclonal antibody; GC, glycocalicin.
A4 amyloid production in an APP-transfected cell
line (Higaki, J., Quon, D., Zhong, Z., and Cordell, B.(1995) Neuron
14, 651-659).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.