At a later time point 24 h, opsonized zymosan was found to induce increased expression of CD47 adhesion molecule, platelet aggregation, mitochondrial membrane depolarization and phosphat
Trang 1by opsonized zymosan A – activation and long-term
commitment to cell death
Paola Matarrese1, Elisabetta Straface1, Giuseppe Palumbo2, Maurizio Anselmi2, Lucrezia
Gambardella1, Barbara Ascione1, Domenico Del Principe2and Walter Malorni1
1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita’, Rome, Italy
2 Department of Pediatrics, University of Rome Tor Vergata, Italy
Several mechanisms are brought into play in order
to control the balance between platelet production
and destruction Among these, recent studies have
identified a form of apoptosis Platelets have been
shown to be able to undergo apoptosis in response
to various stimuli [1,2] It has been reported that platelet differentiation recapitulates morpho-func-tional events that are typical of apoptosis, such as trans-bilayer migration of phosphatidylserine (PS) to the outer membrane leaflet [3] Platelets also express
Keywords
aggregation; apoptosis; mitochondrial
membrane potential; platelets; zymosan
Correspondence
P Matarrese, Department of Therapeutic
Research and Medicines Evaluation, Section
of Cell Aging and Degeneration, Istituto
Superiore di Sanita’, viale Regina Elena 299,
00161 Rome, Italy
Fax: +39 6 49903691
Tel: +39 6 49902010
E-mail: paola.matarrese@iss.it
(Received 1 November 2008, revised 25
November 2008, accepted 3 December
2008)
doi:10.1111/j.1742-4658.2008.06829.x
Changes in the mitochondrial membrane potential play a key role in deter-mining cell fate Mitochondria membrane hyperpolarization has been found to occur after cell activation, e.g in lymphocytes, whereas depolar-ization is associated with apoptosis The aim of this study was to investi-gate the effects of an immunological stimulus, i.e opsonized zymosan A,
on human platelet mitochondria by means of flow and static cytometry analyses as well as biochemical methods We found that opsonized zymo-san induced significant changes of platelet morphology at early time points (90 min) This was associated with increased production of reactive oxygen species, and, intriguingly, mitochondrial membrane hyperpolarization At a later time point (24 h), opsonized zymosan was found to induce increased expression of CD47 adhesion molecule, platelet aggregation, mitochondrial membrane depolarization and phosphatidylserine externalization Although these late events usually represent signs of apoptosis in nucleated cells, in opsonized zymosan-treated platelets they were not associated with mem-brane integrity loss, changes in Bcl-2 family protein expression or caspase activation In addition, pre-treatment with low doses of the ‘mitochon-driotropic’ protonophore carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone counteracted mitochondrial membrane potential alterations, production of reactive oxygen species and phosphatidylserine externaliza-tion induced by opsonized zymosan Our data suggest that mitochondrial hyperpolarization represents a key event in platelet activation and remo-deling under opsonized zymosan immunological stimulation, and opsonized zymosan immunological stimulation may represent a useful tool for under-standing of the pathogenetic role of platelet alterations associated with vascular complications occurring in metabolic and autoimmune diseases
Abbreviations
AM, acetoxymethyl ester; DHR123, dihydrorhodamine 123; DIC, differential interference contrast; FCCP, carbonyl cyanide p-(trifluoro-methoxy) phenylhydrazone; IVM, intensified video microscopy; JC-1, 5-5¢,6-6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazol-carbocyanine iodide; MMP, mitochondrial membrane potential; OPZ, opsonized zymosan; PRP, platelet-rich plasma; PS, phosphatidylserine; ROS, reactive oxygen species.
Trang 2many components of nucleated-cell apoptosis such as
caspases [1,4]
Mitochondria are generally considered to be key
players in cell life and death In addition to energy
supply, they have also been demonstrated to be
involved in execution of apoptosis via the release of
apoptogenic factors such as cytochrome c [5] Human
blood platelet mitochondria play a critical role as they
work efficiently as energy factories for both resting
and stimulated cells Mitochondria are also involved in
non-ATP-related functions such as oxygen radical
generation and apoptotic-like events In fact,
mito-chondrial damage has been reported to be a key step
in platelet apoptosis, and mitochondrial membrane
potential (MMP) is lost during storage and under
other conditions [4,6,7] Changes in the mitochondrial
permeability transition pore have been hypothesized to
play a role in production of so-called coated platelets,
a sub-population of platelets observed upon
dual-agonist activation, e.g thrombin plus collagen, that
express surface PS and can be induced by activation of
the pro-apoptotic Bcl-2 family member Bax [3] Hence,
mitochondria integrity and function appear to act as
general regulators of platelet fate in terms of both
acti-vation and apoptosis, two processes that, in platelets,
appear to share some common features, e.g PS
exter-nalization
Zymosan A, a complex polysaccharide obtained
from Saccharomyces cerevisiae, is a complement
activa-tor that can be used as a tool to investigate the role of
activated platelets in several diseases, including
immune complex-mediated inflammation and its
vascu-lar complications [8] Once opsonized, zymosan A has
been suggested to activate platelets in a
complement-and fibrinogen-dependent way In particular,
comple-ment components, such as C5, C6 and C7, are
necessary, and IgG binding is also required for
zymo-san opsonization [9,10] Hence, opsonized zymozymo-san
(OPZ) could represent a suitable model for the study
of platelets as ‘inflammatory’ cells In view of this, we
decided to investigate whether this immunological stimulus may induce mitochondria dysfunction and influence platelet fate We found that mitochondria modifications have a dual role, controlling both plate-let activation and death
Results Characterization of morphological modifications induced by OPZ
Treatment with OPZ induced two main modifications
of platelet morphology: (a) cell remodeling typical of activation at early time points (after 30 and, more markedly, 90 min), and (b) platelet aggregation after
24 h With regard to cell remodeling, we decided to analyze the actin cytoskeleton organization This appeared to be significantly modified by OPZ (Fig 1A): redistribution of actin filaments, forming long actin-positive protrusions (arrows), was detected
in platelets exposed for 90 min to zymosan (central panel), and, more especially, to OPZ (right panel) Scanning electron microscopy analysis indicated that exposure to OPZ for 30 (not shown) and 90 min (Fig 1B) appeared to activate platelets: the typical round morphology of platelets was altered to an activated morphology characterized by emission of thin protrusions A series of analyses were also per-formed using CD47, the thrombospondin receptor, a surface molecule involved in cell adhesion [11] Flow cytometry analyses revealed no changes at early time points, but platelet alterations were observed 24 h after OPZ administration A platelet sub-population overexpressing CD47 was detected in zymosan-trea-ted, and especially OPZ-treazymosan-trea-ted, platelets (a represen-tative flow cytometry analysis is shown in Fig 1C) This increased expression was also detected by immunofluorescence analysis, and platelet aggre-gates were detectable after both zymosan and OPZ treatments (Fig 1D) The number of aggregates was
Fig 1 Characterization of platelet modifications induced by OPZ (A, B) Morphological alterations (A) IVM analysis of actin microfilaments Actin-positive protrusions (arrows) are visible in treated platelets (90 min) Insets in the middle and right panels show bright-field micrographs that have been electronically inverted to highlight these thin protrusions (arrows) Magnification ·1500 (B) Scanning electron microscopy Exposure to zymosan A (opsonized and non-opsonized) for 90 min changed round-shaped resting platelets (control, left panel) into activated platelets characterized by the emission of long thin protrusions Magnification ·4000 (C–F) CD47 expression and cell-aggregation analyses (C) Histograms representing flow cytometry evaluation of surface expression of the adhesion molecule CD47 are shown in the upper panels Numbers represent the percentage of highly positive platelets In the lower panels, dot plots of the physical parameters of the platelet popu-lation (one representative experiment) are shown (D) IVM analysis showing the intracellular distribution of CD47 molecule in zymosan-trea-ted cells (central panel) and OPZ-treazymosan-trea-ted cells (right panel) (E) DIC (Nomarski) micrographs showing cell aggregates in zymosan-treazymosan-trea-ted platelets (central panel) and OPZ-treated platelets (right panel) in comparison with untreated platelets (left panel) In (D) and (E), arrows indi-cate platelet aggregates (F) Quantification of cell aggregation by morphometric analysis performed using DIC Values are means and SD of the results obtained in three independent experiments *P < 0.01 versus control platelets; P < 0.01 versus zymosan-treated platelets.
Trang 3Opsonized zymosan Zymosan
Untreated 5
10
20
1000
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
Untreated
Untreated A
B
C
D
E
F
5 µm
5 µm
Zymosan 24 h
Zymosan 90 min
Opsonized zymosan 24 h Opsonized zymosan 90 min
Fluorescence
0
FSC-Height
1000
0
FSC-Height
1000
0
FSC-Height
10 µm
5 µm
*
Trang 4evaluated The results of morphometric analysis by
fluorescence microscopy (CD47-stained samples) and
differential interference contrast (DIC, i.e Nomarski
microscopy, Fig 1E,F) clearly indicated that the
number of aggregates, which was negligible in
con-trol samples, was significantly increased in
OPZ-trea-ted samples in comparison with non-opsonized
zymosan-treated cells (Fig 1F)
OPZ-induced MMP alterations
On the basis of previous studies, which reported
increased MMP (hyperpolarization) in conjunction
with cell activation, e.g in lymphocytes [12,13], and
MMP loss (depolarization) in conjunction with
apop-tosis [5], we analyzed this parameter in platelets treated
with opsonized and non-opsonized zymosan A at
vari-ous time points Quantitative flow cytometry analysis,
performed using a JC-1 probe (a representative
experi-ment is shown in Fig 2A), clearly indicated the
pres-ence of a significantly higher percentage of cells with
hyperpolarized mitochondria (see boxed areas) after
treatment with OPZ (third row) in comparison with
either untreated platelets (first row) or platelets treated
with non-opsonized zymosan (second row)
Impor-tantly, this hyperpolarization of mitochondrial
mem-brane started early after OPZ addition (second
column) and peaked after 90 min (third column)
Interestingly, 24 h after OPZ addition (third row,
fourth column), flow cytometry analysis clearly
revealed a significant percentage of cells
(approxi-mately 35%) displaying mitochondrial membrane
depolarization These effects were also evident by
pool-ing together data obtained from four independent
experiments: Fig 2B,C shows the percentage of cells
with hyperpolarized or depolarized mitochondria,
respectively Altogether, these results indicate that
mitochondria of platelets treated with OPZ underwent
a marked increase in MMP at early time points (until
90 min), followed by a significant MMP loss at later
time points (starting from 24 h) Importantly, in our
experimental system, the decrease in MMP was not
paralleled by an alteration of Bax (Fig 2D) or Bak
(Fig 2E) expression levels
OPZ-induced ROS production Mitochondrial hyperpolarization has been related to hyperproduction of reactive oxygen species (ROS) [13,14] A quantitative time-course analysis of ROS generation during zymosan A treatment was thus per-formed using flow cytometry In accordance with the MMP data, increased ROS production was detected in OPZ-treated platelets using dihydrorhodamine 123 (DHR123) A representative experiment is shown in Fig 3A [compare control platelets (left) and non-ops-onized zymosan-treated platelets (middle panel) with OPZ-treated platelets (right)] The results obtained from four independent experiments are reported in Fig 3B In OPZ-treated cells, increased ROS produc-tion was detectable at earlier time points (30 and
90 min), but the values detected after 24 h were similar
to those found in control samples
OPZ induces PS externalization (but not caspase activation)
We analyzed PS externalization in platelets under vari-ous experimental conditions Flow cytometry evalua-tion of cell-surface expression of PS was performed using annexin V⁄ trypan blue double staining Fig-ure 4A shows the results of a representative experi-ment, and Fig 4B shows mean values obtained from four independent experiments These analyses clearly indicated that, in the absence of any stimulus, platelets displayed very low levels of PS at their surface up to
24 h after isolation (first row, bottom right quadrant), and non-opsonized zymosan treatment induced a small increase of the percentage of annexin V-positive cells (second row), whereas a time-dependent increase in PS externalization was observed in OPZ-treated cells (third row, bottom right quadrant) Interestingly, neither non-opsonized nor OPZ-treated platelets were positive for trypan blue dye (see percentages in the upper right quadrant), indicating that the plasma membrane of most cells was undamaged at least up to 24 h (Fig 4A, third row, fourth column) and 48 h (not shown) after OPZ administration These results were clearer when data obtained from four independent experiments were
Fig 2 OPZ induces MMP alterations (A) Biparametric flow cytometry analysis of MMP after staining with JC-1 in untreated platelets (first row), platelets treated with zymosan A (second row) and platelets treated with OPZ (third row) at various time points The numbers in the boxed areas represent the percentages of cells with hyperpolarized mitochondria The percentages of cells with depolarized mitochondria are shown below the dashed line The results obtained in a representative experiment are shown (B, C) Mean percentage (and SD) of plate-lets with hyperpolarized (B) or depolarized (C) mitochondria obtained from four cytofluorimetric experiments Statistical analyses indicate a significant (P < 0.01) increase in cells with hyperpolarized or depolarized mitochondria at early (up to 90 min) and late (24 h) time points, respectively, only in platelets challenged with OPZ (D, E) Bax (D) and Bak (E) expression levels as evaluated by FACS analysis The y axis shows the median values of fluorescence as the mean and SD from four independent experiments.
Trang 5pooled (Fig 4B) Data on cell viability obtained by the
trypan blue exclusion test were confirmed using
calcein-acetoxymethyl ester (AM) (Fig 4C)
On the basis of these results, analysis of the activa-tion state of the main execuactiva-tioners of apoptosis, i.e caspases, was required We found that neither zymosan
Untreated
Zymosan
Opsonized zymosan
40 75
60
45
30
15
0
30 20 10 0
5 4 3 2 1 0
5
D
B
A
C
E 4
3
2
1
0
T 90 min T 24 h
7.1
11.1
11.5
25.1
15.2
16.7
12.2 11.2
14.9 12.2
30.4 7.3
6.9
10.6
7.1 5.6
J-monomers
Trang 6(Fig 5A) nor OPZ (Fig 5B) induced activation of
caspases 3 and 9 at any time point considered In
par-ticular, 90 min after zymosan A administration, when
we observed the maximum OPZ-induced MMP
hyper-polarization (Fig 2) and ROS production (Fig 3),
activation of caspase 9 (which depends on the release
of mitochondrial apoptogenic factors) [5] and
cas-pase 3 (the main enzyme involved in cascas-pase-depen-
caspase-depen-dent apoptosis) was negligible Even if the zymosan A
exposure time was prolonged to 24 h, at which time
PS externalization and mitochondrial membrane
depo-larization were observed (see Fig 4), no significant
activation of these caspases was detected As a positive
control, platelets treated with 1 UÆmL)1 of thrombin
for 1 h (in the presence or absence of the specific cas-pase inhibitors) were studied (Fig 5C) Data obtained
in positive controls or in platelets treated for 24 h with opsonized and non-opsonized zymosan A were confirmed by western blot analysis (Fig 5D)
MMP plays a key role in OPZ-mediated effects
We examined the effects of an agent that is capable of specifically influencing MMP homeostasis and cell fate [12,14]: the protonophore uncoupler carbonyl cyanide fluorophenyl-hydrazone (FCCP), which is known to hinder the mitochondria hyperpolarization pheno-menon at low doses [15] In particular, the ability of
T0
T 30 min
T 90 min
T 24 h
Untreated
A
B
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3
M = 32.2
M = 44.2
M = 58.6
M = 22.5
M = 17.3
M = 21.4
M = 19.1
M = 22.7
M = 18.4
M = 20.3
M = 17.1
M = 31.6
10 4
Zymosan Opsonized zymosan
Untreated
*
*
70 60 50 40 30 20 10 0
Green fluorescence intensity
Fig 3 OPZ induces production of reactive oxygen species Quantitative cytofluorimetric analysis of ROS production was performed using DHR123 (A) Results obtained in a representative experiment The values represent the median fluorescence (B) Mean values (and SD) obtained from four independent experiments *P < 0.01 versus control and zymosan-treated cells.
Trang 7FCCP to counteract the OPZ-induced effects in terms
of ROS production, MMP alterations and PS
external-ization was studied (Fig 6) In our experiments, a very
low dose (20 nm) of FCCP was able to hinder both the early and late events induced by OPZ In platelets pre-treated with FCCP, the increase in ROS
T0 T 30 T 90
Minutes
T 24 h
Minutes
T 24 h
T0 T 30 T 90
Minutes 0
10
20
30
40
0 10 20 30 40
0 10 20 30 40
T 24 h
Untreated
Untreated
5.3 1.4
1.1 4.1 0.5
A
B
C
2.1
0.4 1.9
0.6 5.9
0.8 6.1
1.3 6.5
2.1 6.7
4.4 34.9
1.3 17.3
0.6 14.3
0.5 7.4
Zymosan
Zymosan
Exposure time
Opsonized zymosan
Opsonized zymosan
Untreated 24 h
Calcein-AM (green fluorescence)
Zymosan 24 h Opsonized zymosan 24 h
Annexin V
Annexin V positive Trypan blue positive
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
Fig 4 OPZ induces PS externalization (A) FACS analysis after double staining with annexin V ⁄ trypan blue Dot plots from a representative FACS experiment are shown Numbers represent the percentages of annexin V-positive cells (bottom right quadrant) or annexin V ⁄ trypan blue double positive cells (upper quadrant) Note the very low percentage of cells that are positive for trypan blue (B) Results obtained from four independent experiments, reported as means and SD (C) FACS analysis after staining with calcein-AM (which is retained in the cytoplasm of live cells) of platelets treated with zymosan (central panel) or OPZ (right panel) for 24 h Untreated platelets incubated for 24 h at 37 C (left panel) were used as the control Numbers represent the percentage of calcein-negative cells One representative experiment is shown.
Trang 8production induced by OPZ administration for 90 min
was significantly reduced (Fig 6A, compare shaded
gray histograms with black histograms) Fittingly, the
mitochondrial membrane hyperpolarization state
observed 90 min after treatment with OPZ was
signifi-cantly inhibited by low doses of FCCP (compare
boxed areas in Fig 6B, first and third panels, with
Fig 2B) The same protective effect of FCCP was
observed with respect to mitochondrial membrane
depolarization induced by 24 h treatment of OPZ
(compare areas under the dashed line in Fig 6B,
sec-ond and fourth panels, with Fig 2C) Similarly, the PS
externalization observed in platelets treated for 24 h with OPZ (see Fig 4) was negligible when platelets were pre-exposed to FCCP (Fig 6C)
Discussion Serum opsonized zymosan (from yeast cell walls) is known as a model phagocytic stimulus that interacts with both immunoglobulin and complement recep-tors, is ingested, and activates oxidative mechanisms Because OPZ engages at least two types of receptor, the signaling pathways triggered by this stimulus are
Caspase 9
Caspase 3
Caspase 3
Caspase 9 activation control
Caspase 3 activation control
Caspase 9
Caspase 3
LEHD-fmk
DEVD-fmk
Positive control
Green fluorescence
Green fluorescence
T 90 min
T 90 min
T 24 h
T 24 h
Zymosan
Opsonized zymosan
T0
T0
A
B
10 0 10 4
10 0 10 4 10 0 10 4
74.6
69.3 13.4
2 32
20
32 20
10 0 10 4
Fig 5 OPZ does not induce caspase activa-tion Analysis of the activation state of casp-ases 3 and 9 in intact living platelets treated with zymosan (A) or opsonized zymosan (B)
at various time points (C) Activation state
of caspases 3 and 9 in a positive control represented by platelets treated for 1 h with thrombin (1 UÆmL)1) in medium containing
1 m M Ca 2+ Values are the percentage of cells containing these caspases in their active form Results obtained in a represen-tative experiment are reported (D) Western blot analysis of caspase 3 in thrombin-trea-ted platelet (lane 2; compare with control in lane 1); untreated platelets (lane 3); platelets treated for 24 h with zymosan (lane 4) or with OPZ (lane 5).
Trang 9complex The stimulus may be considered as an
immunological stimulus, which is also able to affect
human platelets by inducing an oxidative burst [10]
Here we show that OPZ can trigger platelet
meta-morphosis [10], consisting of morphological and
bio-chemical changes, that is typical of activation In
fact, platelets rapidly changed from a discoid form
to an activated shape characterized by emission of
long actin-positive protrusions At the last time point
(24 h), OPZ treatment was found to lead to CD47
overexpression and platelet aggregation It has been
suggested that platelet activation and adhesion are
associated with morphological modifications, CD47
overexpression and platelet aggregation [10,11]
How-ever, these changes were accompanied by an early
and transient production of ROS, which probably
serve, as in other cell systems, as signaling molecules
of biological significance Activation of platelets by
OPZ was associated with a transient burst in ROS,
which was possibly due, at least in part, to the
energy metabolism [16] Indeed, zymosan A, an
acti-vator of the alternative complement pathway, has
been hypothesized to activate platelets in plasma in
a complement- and fibrinogen-dependent way
[9,10,17] Hence, immunological stimulation by OPZ
could trigger a complex cascade of events leading to platelet remodeling and activation
The main result reported here is that OPZ-stimu-lated platelets underwent a time-dependent hyperpo-larization of mitochondria, which started early (30 min) and lasted until 90 min Such mitochondrial hyperpolarization was previously observed in T cells, and was considered as an activation-associated event [12] Although the mechanism underlying this MMP increase remains unclear, it has been suggested that it could be associated with ROS signaling and could represent an early metabolic change ‘preparing’ the cell for the death process [18] On the basis of our results, we can hypothesize that mitochondrial hyper-polarization could be associated, as in lymphocytes, with platelet activation In accordance with results obtained in other cell types [14,15], the fact that the
‘stabilizing’ effect of the ‘mitochondriotropic’ drug FCCP prevented OPZ-induced mitochondrial mem-brane hyperpolarization as well as platelet morpho-logical remodeling seems to suggest that the hyperpolarization state of mitochondria might repre-sent an early transient key event sustaining platelets towards an activated phenotype, probably creating a pseudo-hypoxic redox state characterized by normoxic
Zymosan M = 15
A
B
T 90 min
10 0
10 1 10 2 10 3 10 4
Green fluorescence
J-monomers
Annexin V
10 0
10 1 10 2 10 3 10 4
T 90 min
T 90 min
10.1
7.4 5.4
1.6
Opsonized zymosan M = 63 FCCP + Zymosan M = 21
FCCP + Zymosan
FCCP + Opsonized zymosan M = 32
FCCP + Opsonized zymosan
Fig 6 The mitochondrial membrane
poten-tial plays a key role in zymosan-mediated
effects Quantitative flow cytometry
evalua-tion of (A) ROS producevalua-tion, (B) MMP and
(C) PS externalization in platelets pre-treated
with a low dose of FCCP (20 n M ) before
addition of opsonized or non-opsonized
zymosan A Pre-treatment with FCCP
signifi-cantly reduced OPZ-induced ROS production
(A, compare shaded gray histograms with
black empty histograms), mitochondrial
membrane hyperpolarization (compare
boxed areas in B, first and third panels, with
Fig 2B), mitochondrial membrane
depolari-zation (compare areas under the dashed line
in B, second and fourth panels, with
Fig 2C), and PS externalization (compare
numbers in C with those in Fig 4) The
results shown were obtained in one
experi-ment (representative of four) Values in (A)
represent median fluorescence; those in (B)
and (C) represent percentages of cells.
Trang 10decreases of ROS and a shift from oxidative to
glyco-lytic metabolism [19] As in other systems, low doses
of FCCP could inhibit ROS signaling events that lead
to the programmed mitochondrial destruction termed
mitoptosis [20] In nucleated cells, mitochondria
hyperpolarization occurs early after the apoptotic
commitment, and is followed by MMP loss It is
widely accepted that the latter could contribute to
apoptosis [5] Our model system provides some
fur-ther clues on this matter, and underlines the
differ-ences between nucleated and non-nucleated cells We
found that PS externalization, a typical early marker
of apoptosis in nucleated cells, also occurs early in
OPZ-stimulated platelets, together with ROS
produc-tion and mitochondria hyperpolarizaproduc-tion Platelets,
however, maintain their integrity for a long time (at
least 48 h) despite MMP loss and increased PS
exter-nalization As recently reported for other stimuli,
including engagement of immunoreceptors [21], OPZ
induced non-apoptotic externalization of PS
Further-more, OPZ-treated platelets dis not show either
cas-pase activation or an increase in Bcl-2 family
proteins, nor cell death On this basis, we can also
hypothesize that, in some immunopathological
instances, the increased number of platelets could be
due to a defective death of these cells (although
PS-positive) rather than de novo production of these
cells Conversely, other stimuli, such as collagen plus
thrombin, have recently been demonstrated to induce
PS externalization, a decrease in MMP, increased
expression of the Bcl-2 proteins Bax and Bak, caspase
activation and cell death [2,3] In addition, the
physi-ological platelet agonist thrombin also induces Bid,
Bax and Bak translocation to the mitochondria and
endogenous generation of hydrogen peroxide, which
stimulates cytochrome c release and activation of
caspases 3 and 9 [22] Thus, OPZ appears to be a
valuable activating agent triggering a long-term
com-mitment to apoptosis (as also suggested by
OPZ-in-duced PS externalization) rather than a typical
apoptotic inducer The previously hypothesized role
of PS externalization and its role as an adhesion
fac-tor in cell–cell interaction therefore requires
reap-praisal [4,23] For instance, platelet binding to
dysfunctional endothelium was found to be inhibited
by the phosphatidylserine-binding protein annexin V
and enhanced by platelet agonists [24]
Give the importance of the loss of functional
integ-rity of platelets in the pathogenesis of cardiovascular
complications often associated with diabetes and some
autoimmune diseases, e.g antiphospholipid syndrome
or Kawasaki disease [25,26], the results reported
here indicate that OPZ could represent a prototypic
immunological stimulus for study of the pathogenic mechanisms of these diseases
Experimental procedures Platelet isolation and treatments Blood samples were collected from healthy volunteer blood donors who had taken no drugs for at least 10 days The platelets were obtained by mixing fresh blood samples with
a 1⁄ 6th volume of acid ⁄ citrate dextrose (38 mm citric acid,
75 mm Na3 citrate, 135 mm glucose) as anticoagulant All the experiments were performed in platelet-rich plasma (PRP), which was prepared by centrifugation of blood sam-ples at 150 g for 10 min at room temperature Opsonized zymosan was obtained as previously reported [27] Briefly, zymosan A (Sigma Chemical Co., St Louis, MO, USA) was boiled for 20 min and then washed for 5 min three times in NaCl⁄ Pi After washing, boiled zymosan A was added to platelet-poor plasma (obtained by centrifugation of PRP at
1000 g) and incubated at 37C for 30 min After washing three times, OPZ was ready to use Control samples were prepared using non-opsonized zymosan A Stimulation of the platelets was achieved by incubating PRP with
4 mgÆmL)1OPZ at 37C for various durations After incu-bation with zymosan, PRP was centrifuged at 700 g for
5 min, and washed platelets were prepared for various analyses For experiments with FCCP (Molecular Probes, Leiden, The Netherlands), PRP was incubated for 10 min with 20 nm FCCP before addition of zymosan A (both opsonized and non-opsonized) Samples treated with FCCP alone were also studied
Platelets were analyzed at various time points (5, 10, 20,
30 and 90 min and 6, 8 and 24 h) after treatment with non-opsonized zymosan A or OPZ The main changes were detected after 30, 90 min and 24 h: only the results obtained at these time points are shown here In addition,
we also analyzed platelets treated with zymosan A (opson-ized and non-opson(opson-ized) and immediately washed three times These were considered as controls to test the responsiveness of platelets immediately after interaction with zymosan A, and this time point is indicated as T0
Scanning electron microscopy Samples were collected and plated on poly-l-lysine-coated slides, and fixed with 2.5% glutaraldehyde in 0.1 m cacody-late buffer (pH 7.4) at room temperature for 20 min After post-fixation in 1% OsO4 for 30 min, samples were dehy-drated through a graded ethanol series, critical point-dried in
CO2 and gold-coated by sputtering using a Balzers Union SCD 040 apparatus (Balzers, Weisbaden, Germany) The samples were examined using a Cambridge 360 scanning elec-tron microscope (Leica Microsystem, Wetzlar, Germany)