From The Center for Experimental Therapeutics and The Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, July 14, 2000, and in revised form, March 2, 2001
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ABSTRACT |
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The main component of Alzheimer's disease (AD)
senile plaques is amyloid- Deposition of amyloid Platelets may also be a source of A Materials--
Collagen, arachidonic acid, thrombin,
calcium ionophore (A23187), and phorbol 12-myristate 13-acetate (PMA)
were purchased from Sigma Chemical Co. (St. Louis, MO). The peptide
aldehyde protease inhibitor MG-132 was purchased from Peptides
International (Louisville, KY). Wortmannin, LY294002, and BAPTA were
purchased from Biomol (Plymouth Meeting, PA). Calpain inhibitor I
(Ac-Leu-Leu-norleucinal) and II (Ac-Leu-Leu-methioninal) were from
Alexis Biochemicals (San Diego, CA). Lactate dehydrogenase (LDH) was
measured enzymatically using a kit from Sigma.
[3H]Serotonin (specific activity, 24.7 µCi/mmol) was
purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Platelet
factor 4 (PF4) was measured using an enzyme-linked
immunosorbent assay (Asserachrom-PF4). The A Platelet Isolation--
Human platelets were isolated as
previously described (13). Briefly, whole venous blood was collected
from healthy volunteers (all males, 22-40 years old) without stasis in
a syringe containing one part of sodium citrate (3.8%) as
anti-coagulant and nine parts of whole blood, using a 19-gauge needle
to reduce any possible mechanical activation of cells during
collection. The protocol was approved by the local Ethical Committee,
and a consent form to participate in this study was signed by each
individual. Donors were nonsmokers and had not ingested any drug known
to affect platelet function for at least 2 weeks prior to the study.
Platelet-rich plasma was prepared by centrifugation of whole blood at
900 rpm for 15 min at room temperature. The platelet-rich plasma
supernatant was carefully removed and was used to isolate platelets by
stepwise centrifugation as previously described (13). Washed platelets were resuspended in a modified Hanks' balanced salt solution (pH 7.4)
at a final concentration of 3 × 108 platelets/ml
unless otherwise specified, as previously described (13).
Ex Vivo Study--
Four healthy volunteers (mean age 30 ± 8 years, 2 male and 2 females, all non-smokers) who had not taken any
medication during the previous 2 weeks were each given 1 g of
aspirin. Blood samples were taken prior to and 3 h following
aspirin ingestion using the methods described above. For plasma
preparation, whole venous blood was anti-coagulated with sodium citrate
(3.8% v/v) prior to ultracentrifugation at 3000 rpm for 30 min at room
temperature. For preparation of serum, venous blood was collected
without anti-coagulant and incubated in glass tubes at 37 °C for
1 h prior to centrifugation at 3000 rpm for 30 min. Serum and
plasma were analyzed for thromboxane B2 (TxB2),
A Platelet Aggregation--
Platelet aggregation was studied in a
light transmission aggregometer (Chrono Log, Haverton, PA) at 37 °C
in plastic cuvettes with constant stirring at 1000 rpm, as previously
described (13). Threshold concentrations of various platelet agonists
were determined by measuring the lowest concentration of agonist that
caused irreversible platelet aggregation with a trace amplitude of 65%
to 85% of maximal light transmission. In our experiments, aggregation
was induced by using threshold concentrations of collagen (5 µg/ml),
arachidonic acid (50 µM), thrombin (1 IU/ml), the calcium
ionophore A23187 (1 µM), or PMA (100 nM).
Based on previous published studies, inhibitors were used at the
following concentrations: indomethacin (10 µM),
GF109203X (5 µM), wortmannin (100 nM),
LY294002 (20 µM), BAPTA (200 µM), EGTA (2 mM), calpain inhibitor I (25 µM), calpain inhibitor II (25 µM). Platelet aggregation was
continuously measured for 15 min following the addition each agonist.
At the end of the incubation time, the reaction was stopped by placing
samples in ice. They were then centrifuged at 4 °C for 10 min at
12,000 × g to separate platelet pellet from supernatant.
Serotonin and PF4 Release--
Supernatant samples
were assayed for release of dense body [3H]serotonin and
Platelet TxB2 Formation--
Platelet thrombane (Tx)
A2 formation was measured in the supernatant as its
hydrolysis product TxB2 by gas chromatography/mass spectrometry assay, as previously described (13).
Release of Lactate Dehydrogenase--
Lactate dehydrogenase
(LDH) release was measured using an enzymatic assay (14). Aliquots were
taken 15 min after the addition of the agonist, and the supernatant was
obtained by centrifuging for 3 min at 12,000 × g. LDH
in the supernatants was compared with total LDH released from control
platelets after lysis by sonication.
Measurement of A APP Western Blots--
Platelet-associated APP was measured
following lysis of platelet pellets in 100 µl of radioimmune
precipitation buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet
P-40, 5 mM EDTA in TBS, pH 8.0). 10 µl of lysate was
resolved on 7.5% polyacrylamide gels for immunoblotting with 6E10 mAb.
Immunoreactivity was quantitated by PhosphorImager analysis following
application of anti-mouse 125I secondary antibody. To
measure APP released by platelets, and APP levels in serum and plasma,
samples were mixed with sample buffer and loaded directly on 7.5%
polyacrylamide gels. APP was quantitated by immunoblotting with 6E10 as
described above.
Statistical Analysis--
Data were analyzed using analysis of
variance. Pair-wise comparisons were made using Student's t
test where appropriate. Data are displayed as means ± S.E.
APP and A
Platelet activation also resulted in increased release of
A
To test the correlation between platelet activation, APP, and A Role of Platelet Cyclooxygenase--
To test the role of
cyclooxygenase (COX) in the formation and release of A
Inhibition of platelet COX activity by indomethacin in collagen- and
arachidonic acid-stimulated platelets significantly reduced secretion
of APP (Fig. 3A), which was
associated with an increased recovery of full-length APP in the lysates
(data not shown), suggesting that COX inhibition prevented APP cleavage
and not simply APP release. In contrast, indomethacin did not inhibit
release of A
Although threshold concentration of collagen induces a
COX-dependent platelet aggregation, higher levels of
collagen can restore full platelet aggregation even when COX is
inhibited (19). Thus, to further confirm that human platelets can
secrete APP independently from COX activity, we asked if higher
concentrations of collagen can restore cleavage and release of APP
despite inhibition of COX. As predicted, higher concentration of
collagen (4-fold threshold concentration) in the presence of 10 µM indomethacin induced a full aggregation response even
in the absence of TxB2 generation (Fig.
4A, trace c). In
the same samples serotonin release increased by ~40% while
PF4 levels did not change significantly compared with
threshold concentration of collagen in the presence of indomethacin (not shown). Similar results were obtained using 100 µM
aspirin (not shown). Interestingly, high concentration of collagen, in the presence of indomethacin, fully restored APP release in the supernatant (Fig. 4, B and C), confirming our
hypothesis that APP cleavage and secretion can occur also via a
COX-independent pathway.
Ex Vivo Study--
At baseline, plasma TxB2 levels
were almost undetectable (1.5 ± 0.5 ng/ml), whereas serum levels
were high (180 ± 20 ng/ml), consistent with the intense platelet
activation during serum formation. Three hours after aspirin intake we
found that plasma TxB2 levels were unchanged (1.2 ± 0.4 ng/ml), while serum TxB2 levels were reduced by 95%
(10.1 ± 1.8 ng/ml, p < 0.001), suggesting that ex vivo platelet COX activity was effectively suppressed.
Aspirin intake did not significantly affect APP levels (data not shown) supporting our previous in vitro observation that APP
secretion is largely COX-independent. Similar results were observed for A Role of Protein Kinase C--
Although APP formation and release
from a variety of cell lines is enhanced by protein kinase C (PKC)
activation (20, 21), to the best of our knowledge, the involvement of
PKC in human platelets has not been examined. To investigate the role
of this enzyme we first treated platelets with the PKC-activator
phorbol 12-myristate 13-acetate (PMA). PMA activated platelets
irreversibly and caused increased formation of TxB2 (Table
II). The addition of the PKC inhibitor,
GF109203X (5 µM), fully blocked PMA-induced platelet
activation (Table II). By contrast, GF109203X did not significantly
reduce platelet aggregation and activation induced by collagen (Table
II). Platelets activated by PMA released A Role of Other Intracellular Signaling Events--
To elucidate
further the molecular mechanisms involved in PKC activation and
subsequent cleavage and release of APP and A In the present study we demonstrate that human platelets release
A Previous reports have shown that, in other cellular systems,
arachidonic acid metabolism may play a role in regulating the secretion
of APP (24, 25) and that prostaglandin E2, a product of
this pathway, increases expression of APP by astrocytes (26). Because
COX is the key enzyme of the arachidonic acid metabolism in platelets,
we investigated its role in platelet A peptide (A
), a proteolytic fragment of
the amyloid precursor protein (APP). Platelets contain both APP and
A
and may contribute to the perivascular amyloid deposition seen in AD. However, no data are available concerning the biochemical mechanism(s) involved in their formation and release by these cells. We
found that human platelets released APP and A
following activation
with collagen or arachidonic acid. Inhibition of platelet cyclooxygenase (COX) reduced APP but not A
release following those
stimuli. In contrast, activation of platelets by thrombin and calcium
ionophore caused release of both APP and A
in a COX-independent fashion. Ex vivo studies showed that, despite suppression
of COX activity, administration of aspirin did not modify A
or APP
levels in serum or plasma, suggesting that this enzyme plays only a
minor role in vivo. We examined the regulation of APP
cleavage and release from activated platelets and found that cleavage
requires protein kinase C (PKC) activity and is regulated by the
intracellular second messengers phosphatidylinositol 2-phosphate
and Ca2+. Our data provide the first evidence that in human
platelets COX is a minor component of APP secretion whereas PKC plays a major role in the secretory cleavage of APP. By contrast, A
release may represent secretion of preformed peptide and is totally independent of both COX and PKC activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-peptide
(A
)1 in senile plaques and
in the walls of cortical and leptomeningeal blood vessels is a hallmark
of Alzheimer's disease (AD) (1). A
is derived from proteolytic
cleavage of the 695-770 amino acids of amyloid-
precursor protein (APP) (2). Cellular APP metabolism is complex, and amyloidogenic cleavage may occur in at least three cellular organelles (3). Although a number of cell types produce A
from APP, the precise
cellular origins of A
deposited in the AD brain and cerebral vessels
have not been identified. Because neurons secrete substantial amounts
of A
, intracerebral deposition of A
may occur locally (4).
However, it has recently been postulated that cerebral vascular amyloid
deposits may be derived in part from circulating A
(5, 6). Indeed,
human platelets contain high levels of membrane-associated and soluble
forms of APP, which, upon stimulation with thrombin or calcium
ionophore, are cleaved by an
-secretase-like activity. The cleaved
APP is readily released by platelets and may contribute to more than
90% of circulating APP (7-9). The normal function of APP in the
circulation is not known, however, there is evidence to support a role
for APP in the acute phase of inflammation. Indeed, the secreted form
of APP inhibits coagulation factor IXa and platelet aggregation induced
by adrenaline or ADP in vitro (10).
detected in whole blood (5).
Recently, it has been reported that A
, like APP, is also released
upon platelet stimulation with agonists such as thrombin or collagen
(11, 12). To date, however, the biochemical mechanism(s) involved in
A
and APP formation and release from human platelets are unknown.
Knowledge of these mechanisms could provide a rational basis for
developing tools to modulate this phenomenon in AD and other
amyloidogenic diseases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
monoclonal
antibodies (mAbs) Ban50, BA27, and BC05 were generously provided by Dr.
N. Suzuki and Tekeda Pharmaceutical. The A
mAb 6E10 was purchased
from Senetek PLC (St. Louis, MO).
1-40, A
1-42, and APP levels.
-granule platelet factor 4 (PF4) as previously described
(14). Platelets were incubated for 30 min at room temperature with 0.2 µCi/ml tritiated serotonin, washed twice, and resuspended in Hanks'
balanced salt solution containing 5 µM imipramine to
inhibit serotonin re-uptake. Platelets were treated with agonist for 15 min, and 700 µl of the samples was then transferred to a vial
containing 70 µl of 5 mM EDTA, 5 mM
theophylline, 500 µM aspirin, and 0.2 µg/ml
PGE1. After centrifugation for 2 min at 12,000 × g, 100 µl of supernatant was transferred to a
scintillation vial and 2 ml of aqueous scintillation fluid was added.
The amount of tritium released was determined by using a scintillation
counter and expressed as a percentage of the total amount of tritiated serotonin accumulated by the platelets. Each experiment was performed in duplicate. PF4 release was determined by a standard
ELISA kit, according to the manufacturer's protocol. PF4
concentration is expressed as a percentage of the total amount released
from control platelets after their lysis by sonication. Each experiment
was performed in duplicate.
1-40,
A
1-42--
Sandwich-ELISA was performed as described
previously using mAbs specific for different species of A
(15, 16).
BAN-50 (a monoclonal antibody specific for the first 10 amino acids of A
) was used as a capturing antibody, and horseradish
peroxidase-conjugated BA-27 (a monoclonal antibody specific for
A
1-40) and horseradish peroxidase-conjugated BC-05 (a
monoclonal antibody specific for A
1-42) were used as
reporter antibodies. To calibrate the sensitivity of the ELISA for
detecting A
, synthetic A
1-40 and
A
1-42 peptides (Bachem Bioscience Inc., King of
Prussia, PA) were used to generate standard curves. The BAN-50, BA-27, and BC-05 mAbs were prepared and characterized as described previously (17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Formation and Release by Human
Platelets--
Isolated human platelets incubated for 15 min at
37 °C with constant stirring did not show any significant increase
in light transmission, indicating no detectable aggregation.
Supernatants from these samples had undetectable levels of
TxB2, serotonin, and PF4 (Table
I) and contained only low levels of APP
(Fig. 1A) and A
(Fig.
1D). As expected, lysates of unstimulated platelets contained high levels of APP (Fig. 1B). Because APP was
detected in these lysates using both an APP ectodomain antibody (Fig.
1B) and an APP cytoplasmic-domain antibody (data not shown),
the APP contained in platelets lysates must exist in a full-length,
uncleaved form. The addition of collagen (5 µg/ml) or arachidonic
acid (50 µM) to platelets caused irreversible platelet
aggregation (Table I), with significant production of TxB2,
and increased release of serotonin and PF4 (Table I), but
no change in levels of LDH (data not shown). Similar results were
obtained when thrombin (1 IU/ml) or calcium ionophore A23187 (1 µM) were used (Table I). Platelets activated by each of
these agonists released higher levels of soluble APP (sAPP) than
unstimulated platelets (Fig. 1A). Concomitantly, recovery of
intracellular full-length APP was decreased by ~50% (Fig. 1,
B and C). Because these agonists decreased levels
of full-length APP inside platelets and increased levels of soluble,
cleaved APP in supernatants, platelet activation must result in
cleavage and secretion of APP.
Effects of collagen, arachidonic acid (AA), thrombin, and A23187 or
vehicle on platelet aggregation (light transmission), TxB2
formation, serotonin, and platelet factor 4 release in the absence or
presence of indomethacin (10 µM) (n = 5 experiments
for each agonist)
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Fig. 1.
Platelets release APP and
A in response to a variety of agonists.
Human platelets were treated with threshold concentrations of collagen,
arachidonic acid, thrombin, or A23187 for 15 min and centrifuged to
separate cells from releasate. A, soluble APP
(sAPP
) in supernatant (releasate); and B,
full-length APP (APPFL) in platelet lysates were
detected by immunoblotting with 6E10. Blots shown are representative of
four separate experiments. C, cell-associated APP was
quantitated by PhosphorImager (*p < 0.01).
D, A
1-40 in the supernatant was measured by
sandwich-ELISA (**p < 0.001). Means and standard
errors for four separate experiments are shown.
1-40, as detected by sandwich-ELISA (Fig.
1D). Although both A
1-40 and
A
1-42 levels increased with platelet activation,
A
1-42 levels were typically 10-fold lower than A
1-40 levels. These low levels of A
1-42
precluded accurate quantitation by sandwich-ELISA and are therefore not reported.
release, we treated platelets with 0.1-20 µg/ml collagen and
measured platelet aggregation, TxB2 generation, and APP
and A
release. Increasing doses of collagen resulted in a
dose-dependent increase of platelet aggregation (light
transmission) and TxB2, A
, and APP formation and release
(Fig. 2, A-D).
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Fig. 2.
Release of APP and A
in response to increasing doses of collagen. Human platelets
were treated with indicated concentrations of collagen for 15 min, and
centrifuged to separate cells from releasate. A,
representative tracings showing the effect of increasing concentration
of collagen on platelet aggregation response (LT, %) and thromboxane
(TxB2, ng/ml) formation. B, soluble APP
(sAPP
) in supernatant (releasate) was detected by
immunoblotting with 6E10. Blot shown is representative of three
separate experiments. C, APP levels in the supernatant were
quantitated by PhosphorImager. D, A
1-40 in
the supernatant was measured by sandwich-ELISA. Means and standard
errors for four separate experiments are shown.
and APP by
human platelets, we utilized two different COX inhibitors, indomethacin
and aspirin (18). Inhibition of platelet COX was confirmed by blockade
of TxB2 formation and the effects on platelet aggregation
and activation. As expected, incubation of platelets with indomethacin
inhibited TxB2 formation following stimulation with
collagen, arachidonic acid, thrombin, and A23187 (Table I).
Indomethacin inhibited also platelet aggregation and activation
following platelet treatment with collagen or arachidonic acid. In
contrast, it did not reduce platelet aggregation or serotonin or
PF4 release following activation by thrombin or A23187
(Table I). Similar results were obtained with 100 µM
aspirin (not shown).
(Fig. 3B). Indomethacin also did not
influence A
or APP release in thrombin- and A23187-activated
platelets (Fig. 3, A and B). Similar results were
obtained with 100 µM aspirin (not shown). These results
suggest that human platelets release A
in a complete COX-independent
manner, whereas depending on the stimulus, they can cleave and release
APP in either a COX-dependent or COX-independent manner.
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Fig. 3.
Platelets release of APP but not
A is partially COX-dependent.
Human platelets were treated with threshold concentrations of collagen
(C), A23187 (A23), thrombin (Thr), or
arachidonic acid (AA) for 15 min, and in the presence of
indomethacin (I). A, soluble APP
(sAPP
) in supernatant (releasate) was detected by
immunoblotting with 6E10. Blot shown is representative of four separate
experiments. B, A
1-40 in the supernatant was
measured by sandwich-ELISA. Means and standard errors for four separate
experiments are shown.
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Fig. 4.
High concentrations of collagen overcome
inhibition of APP secretory cleavage by indomethacin.
A, platelet aggregation response (LT, %) and thromboxane
(TxB2, ng/ml) formation in platelets stimulated with
threshold concentration of collagen (a), threshold
concentration of collagen plus indomethacin (10 µM)
(b), 4× threshold concentration of collagen plus
indomethacin (10 µM) (c). B,
soluble APP (sAPP
) in supernatant (releasate) was
detected by immunoblotting with 6E10. Blot shown is representative of
at least three separate experiments. C, APP levels were
quantitated by PhosphorImager. Means and standard errors for three
separate experiments are shown (*p < 0.01).
secretion (not shown).
and APP in the
supernatant, addition of GF109203X did not significantly influence the
release of A
but significantly reduced APP secretion (Fig.
5, A and C).
Interestingly, even though GF109203X did not affect collagen-induced
platelet activation, it prevented APP but not A
release in
collagen-activated human platelets (Fig. 5, A and
C). Similar results were observed when A23187 and thrombin
were used as agonists (not shown). Furthermore, platelet preincubation
with PMA, which is known to down-regulate PKC activity, and subsequent
stimulation with collagen resulted in reduction of APP but not A
secretion (not shown). Taken together these results strongly suggest an
involvement of PKC activity in APP but not A
formation and release
from activated human platelets. LDH levels in all the samples incubated
with GF109203X were not different from controls (not shown).
Effect of PKC activation and inhibition on platelet aggregation (light
transmission), TxB2 formation, serotonin and platelet factor 4 release (n = 4 experiments for each agonist/antagonist)
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Fig. 5.
APP secretory cleavage by human
platelets is dependent on PKC activation. A, human
platelets were treated with collagen, collagen plus GF109203X (5 µM), PMA, and PMA plus GF109203X (5 µM).
Soluble APP (sAPP
) in platelet releasate was
determined by Western blotting. B, human platelets were
treated with A23187, A23187 plus wortmannin, LY294002 (Lyn),
or BAPTA, thrombin, thrombin plus wortmannin, LY294002
(Lyn), or BAPTA. Soluble APP
(sAPP
) in
platelet releasate was determined by Western blotting. C and
D, A
1-40 in the supernatant of corresponding
experiments was measured by sandwich-ELISA. Means and standard errors
for four separate experiments are shown.
release from activated
platelets, we investigated two intracellular signaling molecules,
Ca2+ and phosphatidylinositol 3-kinase (PI3K). We
found that the Ca2+ chelator BAPTA inhibited APP secretion
following platelet stimulation with A23187 or thrombin (Fig.
5B). Similar results were observed with EGTA (not shown).
Furthermore, treatment of platelets with the PI3K inhibitors wortmannin
or LY294002 was sufficient to inhibit APP secretion following platelet
stimulation with A23187 or thrombin (Fig. 5B). Similar
results were obtained when PMA was used to activate platelets (not
shown). Interestingly, pharmacological inhibition of these two
molecules did not inhibit A23187 and thrombin-induced platelet
aggregation, by contrast it suppressed PMA-induced platelet aggregation
(not shown). Taken together these findings suggest that both
Ca2+ and PI3K are important intracellular mediators of
PKC-dependent APP cleavage and secretion in human
platelets. BAPTA and EGTA both inhibited A
release from platelets
activated with A23187 and thrombin, suggesting that calcium is an
important intracellular mediator of A
secretion in these cells. In
contrast, wortmannin and LY294002 did not inhibit significantly A
secretion from platelets activated by A23187 or thrombin (Fig.
5D). Finally, because APP processing (22) and A
production (23) can be modulated by the activation of the neutral
cysteine proteases calpains, we investigated the effects of two
membrane-permeable calpain inhibitors. We found that calpain inhibitors
I and II were both effective in reducing APP secretion and A
release
secondary to thrombin and A23187-induced platelet activation (Fig.
6, A and B). No
increase in LDH was detected in the supernatant, suggesting that these
compounds did not have any toxic effect on platelets.
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Fig. 6.
APP secretory cleavage and
A secretion by human platelets are inhibited
by calpain inhibitor I and II. A, human platelets were
treated with A23187 or thrombin, alone or in the presence of calpain
inhibitor I (CI) or II (CII). Soluble APP
(sAPP
) in platelet releasate was determined by Western
blotting. B, A
in the supernatant of corresponding
experiments was measured by sandwich-ELISA. Means and standard errors
for four separate experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and APP upon their activation by several agonists. We found that
resting platelets contain high levels of full-length APP, which is
cleaved and released into the supernatant following platelet
activation. Moreover, we provide novel evidence suggesting that APP
release from platelets is only partially regulated by COX activation.
In contrast, we found that A
secretion by human platelets is totally
insensitive to inhibitors of this pathway.
and APP formation by a
variety of in vitro and ex vivo experiments. We found that this pathway does not play a crucial role in regulating APP
and A
release from human platelets. In vitro we observed that inhibition of platelet COX did not affect A
and partially reduced APP cleavage and release only when platelets were challenged with agonists that use mainly this metabolic pathway to activate them,
such as collagen and arachidonic acid (14). By contrast, no difference
was noted in the presence or absence of indomethacin or aspirin when
platelets were challenged with agonists that do not use primarily the
arachidonic acid metabolic pathway, such as thrombin and calcium
ionophore (Fig. 7). This was corroborated by the observation that, even in the presence of indomethacin, very
high concentrations of collagen, which are known in this setting to use
a COX-independent mechanism to activate platelets, fully restored APP
release. Ex vivo we found that serum had higher levels of
APP and A
than plasma. Our study supports the notion that platelets
are a source of both peptides in the circulation (5, 8, 11). However,
the pharmacological suppression of platelet COX activity by aspirin, as
assessed by the dramatic reduction of serum TxB2, did not
interfere with platelet capacity to release APP or A
, confirming our
in vitro observations.
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Fig. 7.
APP and A peptide in
human platelets. This diagram illustrates the metabolic pathway(s)
involved in APP and A
formation and release by human platelets. For
details, see text.
The inability of COX inhibitors to modulate the formation and release
of A and APP by human platelets could have some clinical relevance.
Epidemiological studies have suggested that non-steroidal anti-inflammatory drugs, which mainly block COX activity, may delay the
onset or even reduce the incidence of AD (27, 28). Our data suggest
that these drugs do not likely exert their effect by altering A
- or
APP-circulating levels derived from platelets. Rather, these compounds
may act downstream of A
and APP production.
We found that the major species of A released by activated human
platelets is A
1-40, as determined by immunoreactivity with mAb BA27 (specific for A
1-40). This is consistent with the fact that the major component of vascular amyloid is A
1-40 (29). Our finding is in agreement with that of others (11) who identified this peptide as the main component of A
from platelet releasates. The same authors were not able to detect any
A
1-42 in platelet supernatants, perhaps due to the
limit of detection of their methods.
Several reports have shown that APP secretion is increased by the
activation of PKC (20, 21). The majority of these studies to date have
been conducted using cultured cells, and no data are available on the
involvement of PKC in APP and A formation and release by human
platelets. We found that direct activation of PKC by PMA resulted in
release of A
and APP from human platelets. On the other hand,
addition of a PKC inhibitor and down-regulation of PKC activity, both
decreased APP but not A
release. Although some caution must be taken
when interpreting the action of this potentially nonspecific compound,
our findings strongly suggest that PKC is involved in APP formation and
secretion by human platelets. Parenthetically, we have previously shown
that the same concentration of GF109203X used in these experiments is
specific to prevent PKC activation and translocation in human platelets
(30). The effect of PKC inhibition on APP release was further confirmed when collagen was used as platelet agonist, suggesting that APP secretory cleavage induced by a more physiological agonist also requires PKC activity. Furthermore, we have provided evidence that,
along with PKC activation, two other intracellular signaling molecules,
Ca 2+ and phosphatidylinositol 2-phosphate, are
involved in regulating APP secretory cleavage in human platelets.
Interestingly, pharmacological inhibition of both signals coincided
with selective inhibition of PMA-induced platelet aggregation and APP
secretion, suggesting that these molecules are necessary for PKC
activation and APP cleavage and release. This confirms our previous
studies in which we have shown that PKC activation in human platelets
can be in part modulated by Ca2+ and PI3K activity
(31).
We also investigated the effects of two membrane-permeable calpain
inhibitors and found that calpain inhibitors I and II were both
effective in reducing APP secretion and A release. Although we did
not directly examine how calpains function to increase secretion of APP
and A
, we hypothesized that calpains act downstream of PKC and may
be required at a late step of secretion, because they were required for
both APP and A
release (Fig. 7). This hypothesis will be tested by
further experiments, which will examine in detail the role of calpains
in APP and A
release from human platelets.
Because the ADAM (a disintegrin and metalloprotease) family members
tumor necrosis factor cleaving enzyme (TACE) and ADAM 10 have been
implicated in the PKC-regulated cleavage and secretion of APP in
cultured cells (32, 33), it will be interesting to determine if they
are also involved in PKC-regulated APP formation in platelets.
In contrast with the secretory cleavage of APP by human platelets, we
hypothesize that A secretion may represent calcium-dependent release of preformed A
rather than increased
- and
-secretase processing of APP. In support of this hypothesis, we found that two
different calcium chelators significantly reduced A
release from
human platelets, whereas a known inhibitor of
-secretase (MG132) did
not.2 This finding was
further supported by the observation that stimulation of
-secretase
cleavage of APP (e.g. by PMA treatment) did not result in a
corresponding decrease in A
production. Indeed, any stimulus of
platelets, regardless of its effect on APP processing, increased A
secretion. For example, treatment of platelets with PMA in the presence
of GF109203X resulted in a reduced platelet aggregation (Table II), but
this was still sufficient to stimulate A
release.
In summary, human platelets release A and APP when activated
by a wide range of agonists. This mainly occurs independent of the COX
metabolic pathway and involves multiple signal transduction mechanisms
that lead to differential regulation of APP and A
secretion. Our
data suggest that PKC activation is crucially involved in human
platelets, along with PI3K activity and Ca2+, in APP
cleavage and secretion (Fig. 7). On the other hand, calcium plays an
important role also in the release of A
. Finally, activation of
cysteine proteases calpains I and II seems to have a relevant function in both events. Interestingly, regulatory kinases, such as
PKC, can be modulated by these proteases during platelet activation (34). However, future studies investigating this modulation and
interaction are warranted.
Because abnormal secretion of A and APP may contribute to the
accumulation of amyloid peripherally in amyloidogenic diseases and in
the amyloid angiopathy that occurs in AD, platelets could be a valuable
tool for further exploration of these mechanisms.
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ACKNOWLEDGEMENTS |
---|
We gratefully thank Dr. N. Suzuki and Tekeda
Pharmaceutical for providing the monoclonal antibodies for the A
sandwich-ELISA.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from NIA, National Institutes of Health Grant AG11542 and the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Medical Scientist Training Program Predoctoral
Fellowship from the National Institutes of Health.
§ To whom correspondence should be addressed: Center for Experimental Therapeutics, 812 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-6446; Fax: 215-573-9004; E-mail: domenico@spirit.gcrc.upenn.edu.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M006285200
2 D. M. Skovronsky, V. M.-Y. Lee, and D. Praticò, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid-
peptide;
A
1-40, A
1-42, 40- and 42-amino
acid long forms of A
, respectively;
AD, Alzheimer's disease;
APP, amyloid-
precursor protein;
ADAM, a disintegrin and metalloprotease;
TACE, tumor necrosis factor-
converting enzyme;
PKC, protein kinase
C;
PMA, phorbol 12-myristate 13-acetate;
PI3K, phosphatidylinositol
3-kinase;
ELISA, enzyme-linked immunosorbent assay;
mAb, monoclonal
antibodies;
LDH, lactate dehydrogenase;
PF4, platelet
factor 4;
sAPP, soluble APP;
COX, cyclooxygenase;
Tx, thromboxane;
BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetraacetoxymethyl ester;
LY294002, 2,4-morpholinyl-8-phenyl-4H-1-benzopyran-4-one;
GF109203X, bisindolylmaleimide.
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