Báo cáo khoa học: Mitochondria regulate platelet metamorphosis induced by opsonized zymosan A – activation and long-term commitment to cell death docx

Mitochondria regulate platelet metamorphosis induced
by opsonized zymosan A – activation and long-term
commitment to cell death
Paola Matarrese
1
, Elisabetta Straface
1
, Giuseppe Palumbo
2
, Maurizio Anselmi
2
, Lucrezia
Gambardella
1
, Barbara Ascione
1
, Domenico Del Principe
2
and Walter Malorni
1
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:
(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.
FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS 845
many 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 zymosan
(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-treated, 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-treated cells (right panel). (E) DIC (Nomarski) micrographs showing cell aggregates in zymosan-treated
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.
Mitochondria in platelet activation P. Matarrese et al.
846 FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS
Opsonized
zymosan
ZymosanUntreated
5
10
Aggregates
(number/field)
15
20
1000
1000

200
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
2.3
14.2
24.7
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
200
0
2000
Count
0
0
FSC-Height
SSC-Height
1000
1000

0
0
FSC-Height
SSC-Height
1000
1000
0
0
FSC-Height
SSC-Height
10
µm
5
µm
*°
°
*
P. Matarrese et al. Mitochondria in platelet activation
FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS 847
evaluated. 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.
Mitochondria in platelet activation P. Matarrese et al.
848 FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS
pooled (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 executioners of apoptosis, i.e.
caspases, was required. We found that neither zymosan
Untreated
Zymosan

Opsonized
zymosan
Untreated
Zymosan
Opsonized zymosan
T
0
T 30 min
40
75
60
45
30
15
0
Cells with
depolarized mitochondria (%)
Protein expression
level (a.u.)
Protein expression
level (a.u.)
Cells with
hyperpolarized mitochondria (%)
30
20
10
0
Bak Bax
5
4

3
2
1
0
5
D
B
A
C
E
4
3
2
1
0
T 90 min
T 24 h
T
0
T 30 min
10
4
10
0
10
4
9.7
10.4
7.1
11.1

11.5
25.1
15.2
16.7
12.2
11.2
14.9
12.2
29.4
56.1
9.6
11.8
64.7
34.7
30.4
7.3
6.9
10.6
7.1
5.6
10
0
10
4
10
0
10
4
10
0

10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4

10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0

10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
T 90 min
T 24 h
T
0
T 30 min T 90 min
T 24 h
T
0
T 30 min T 90 min
T 24 h

T
0
T 30 min T 90 min T 24 h
J-monomers
J-aggregates
P. Matarrese et al. Mitochondria in platelet activation
FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS 849
(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 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
T
0
T 30 min
T 90 min
T 24 h
T
0
T 30 min T 90 min T 24 h
Zymosan Opsonized zymosanUntreated
A
B
2000
2000
2000
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
ROS production (a.u.)
Untreated
*
*
70
60
50
40
30
20
10
0
Green fluorescence intensity
Events
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.
Mitochondria in platelet activation P. Matarrese et al.
850 FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS
FCCP 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
T
0

T 30 min T 90 min T 24 h
T
0
T 30 T 90
Minutes
T 24 h
T
0
T 30 T 90
Minutes
T 24 h
T
0
T 30 T 90
Minutes
0
10
20
30
40
% of cells
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
2.7 3.4
5.3
200
Events
Calcein-AM (
g
reen fluorescence)
0
2000
2000
Zymosan 24 h
Opsonized zymosan 24 h
10
4
10
0
10
4
10
0
10

4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
Trypan blue
Annexin V
Annexin V positive
Trypan blue positive
10
0
10
4
10

0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10

4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10

0
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.
P. Matarrese et al. Mitochondria in platelet activation
FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS 851
production 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
T
0
T
0
10
0
A
B
CD
0

EventsEvents
Events
2000 2000 2000 200
0 2000 200
0 200
0 200
0 200
0 200
0 200
0 200
0 200
0 200
0 200
0 200
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0

10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
74.6
69.3 13.4
9.4
1

345
2
32
20
32
20
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
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
1m
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).
Mitochondria in platelet activation P. Matarrese et al.
852 FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS
complex. 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
0
10
4
10
0
10
4
10
0
10
4
10
0

10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
J-aggregatesTrypan blue
10
4
10
0
10
4
10
0
10

4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
0
10
4
10
1
10
2
10
3
10
4
Green fluorescence

J-monomers
Annexin V
10
0
0 200
Events
0 200
Events
10
1
10
2
10
3
10
4
T 90 min
T 90 min
9.3
12.3 15.2 9.7
10.1
1.5
1.3
2.1
7.4
5.4
1.6
3.1
4.0
16.1 15.5 19.3

T 24 h T 90 min T 24 h
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 production, (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.
P. Matarrese et al. Mitochondria in platelet activation

FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS 853
decreases 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 hyperpolarization. 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
a1⁄ 6th volume of acid ⁄ citrate dextrose (38 mm citric acid,
75 mm Na
3
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 ⁄ P
i
. After washing, boiled zymosan A was added to
platelet-poor plasma (obtained by centrifugation of PRP at
1000 g) and incubated at 37 °C 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
4mgÆmL
)1
OPZ at 37 °C 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-opsonized) 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 T
0
.
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% OsO
4
for 30 min, samples were dehy-
drated through a graded ethanol series, critical point-dried in
CO
2
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).
Mitochondria in platelet activation P. Matarrese et al.
854 FEBS Journal 276 (2009) 845–856 ª 2009 The Authors Journal compilation ª 2009 FEBS
Analytical cytology
For CD47 surface detection, control and treated samples
were stained with specific fluorescein isothiocyanate-conju-

gated monoclonal antibodies (Chemicon International Inc.,
Temecula, CA, USA) at 4 °C for 30 min. After washing,
platelets were analyzed on a flow cytometer for quantitative
analysis.
For total CD47, F-actin, Bax and Bak detection, the
samples were fixed with 0.3% paraformaldehyde in NaCl ⁄ P
i
for 30 min at room temperature, washed in the same buffer
and permeabilized with 0.2% Triton X-100 (Sigma) in
NaCl ⁄ P
i
for 5 min. For actin detection, samples were
stained with fluorescein–phalloidin (Sigma) at 37 °C for
30 min. For CD47, Bax and Bak detection, polyclonal anti-
bodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA)
were used, followed by AlexaFluor 488-conjugated anti-rab-
bit IgG (Molecular Probes). After washing, all samples
were analyzed for immunofluorescence by intensified video
microscopy (IVM) using a Nikon Microphot microscope
(Nikon, Tokyo, Japan) equipped with a CCD camera (Carl
Zeiss, Oberkochen, Germany). Micrographs were captured
using IAS2000 software (Delta Sistemi, Rome, Italy).
Morphometric analyses
On the basis of a previous study [28], quantitative eval-
uations of platelet aggregates were performed using DIC
(Nomarski microscopy). Parallel analyses were also
performed by fluorescence microscopy using anti-CD47
monoclonal antibodies. In both cases, at least 20 fields were
evaluated for each sample (10· objective ⁄ 0.65 numerical
aperture). The samples were analyzed at various time points

after treatments (T
0
, 30, 90 min and 24 h).
Mitochondrial membrane potential in living cells
The mitochondrial membrane potential (MMP) of control
and treated cells was studied using 5-5¢,6-6¢-tetrachloro-
1,1¢,3,3¢-tetraethylbenzimidazol-carbocyanine iodide (JC-1;
Molecular Probes). As described previously, living cells
were stained with 10 lm of JC-1 [12].
Production of reactive oxygen species
Cells (5 · 10
5
) were incubated in 495 lL of Hank’s
balanced salt solution, pH 7.4, with 5 l L of DHR123
(Molecular Probes) in polypropylene test tubes for
15 min at 37 °C (final concentration 10 lm). DHR123
dye freely diffuses into cells and is primarily oxidized by
H
2
O
2
in a myeloperoxidase-dependent reaction, producing
green fluorescence. As this oxidation does not occur in
dead cells (no green fluorescence), this staining was also
used to evaluate cell loss. Furthermore, as DHR123 accu-
mulates in mitochondria, production of ROS at the mito-
chondrial level can be detected [29].
Cell-death assays
Quantitative evaluation of apoptosis was performed by flow
cytometry after double staining using fluorescein isothiocya-

nate-conjugated annexin V and 0.05% trypan blue for
10 min at room temperature, and analyzed by fluorescence-
activated cell sorting (FACS) in the FL1 and FL3 channels
to determine the percentage of dead cells. The results
obtained were confirmed by treating control and treated
platelets with 5 lm calcein-AM (Molecular Probes) at
37 °C for 30 min.
Activation of caspases
The activation state of caspases 3 and 9 was evaluated
using a CaspGLOW fluorescein active caspase staining kit
(MBL, Woburn, MA, USA). We also performed positive
controls by using platelets treated for 1 h with thrombin
(1 UÆmL
)1
) in a medium containing 1 mm Ca
2+
, which has
been previously reported to induce cell death and caspase
activation [30]. The activation state of caspase 3 was also
verified by western blot analysis using a specific antibody
(Santa Cruz). Immunoreactive bands were visualized using
a secondary horseradish-peroxidase-conjugated anti-rabbit
IgG and a western blot detection kit, chemioluminescent
system (Upstate Biotechnology, Lake Placid, NY, USA).
Data analysis and statistics
All samples were analyzed using a FACScan cytometer
(BD Biosciences, Heidelberg, Germany) equipped with a
488 nm argon laser. At least 20 000 events were acquired.
Statistical analyses were performed using Student’s t test
or one-way variance analysis. All results were verified in

at least three experiments and are reported as
mean ± standard deviation (SD). Only P-values < 0.01
were considered as statistically significant.
Acknowledgements
We thank Ms Roberta Terlizzi and Dr Zaira Maroccia
for their valuable technical help. This study was
supported by grants from the Italy–USA Collaborative
Project on Rare Diseases (7qr1 ⁄ 7qr1) and the Ministero
della Salute to W.M.
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