Full text of DOI:10.1177/107602960100700307

Report
Original
Expression
Singlet Oxygen Inhibits Agonist-Induced P-Selectin
and Formation of
Platelet
Aggregates
M.S.,
†J.
†C. Schultz,
Ph.D., D.HC.
Stief, M.D., †W. P. Jeske, Ph.D.,
*†T.W.
Walenga, Ph.D.,
and
M.D.,
†J. Fareed,
‡V. Kretschmer,
Medicine
and
and Molecular
‡Institute
Haemostaseology,
Diagnostics,
of Transfusion
*Department of Clinical Chemistry
Hospital ofPhilipps
USA
Medical
and
Center,
Illinois,
Chicago-Maywood,
University
Germany;
†Loyola
University, Marburg,
in
the
of
formation
mediators of activated
decrease
(at
aggregates
platelet
dependent
3
Summary:
polymorphonuclear
Major
level for
of
100%saline
to
control
the
the
mM
ADP,
dose
are
source for
oxidants
CT 8, 12, and 13%
and
TRAP,
respectively;
chloramine,
oxidant
HOCl and
(PMN)
leukocytes
the 50%effective
which
a
nonradical
are
the
collagen,
photon-emitting
TRAP-
In ADP- and
increase in
was 1.0 to
1.5 mM
an
a
Wewere
As
in
chloramine).
interested
platelet-
possible
singlet
oxygaecnti)vit.y
a
sourcewe
mimics the
the
chose
stimulated
platelets,
of
stable
platelet
oxidant-mediated
modulating
1O2,
1O2
mM CT:
to
of the
fourfold
ofthe
mM
methionine
natural
oxidant chloramine T®
three-
mild
which
(at 3
addition
mannitol
(CT),
Freshly
fragments appeared
the
of
initial
radical
not
The
30
drawn
whole
native
to 3 mMCT for
chloramine
N-chloro-taurine.
blood
to
in contrast
modulations
oxidative
value).
oxy-
to excess
from
was incubated
did
donors
37°C.
at 0
blood
5)
saline,
(n
Then
scavenger
of
activa-
these
I
at
minute
adenosine
thrombin
10
6.25
added
antagonize
platelet
μM
μM
diphosphate
The
confirmed
or
ac-
tion.
were
results
concentra-
(ADP), 5
using
equimolar
collagen,
receptor
μg/mL
that
This
of and
NaOCl
and the
were al-
activated
were
shows
study
tions
tivator
mixtures
N-chloro-taurine.
the
(TRAP)
peptide
lowed to
for 3 minutes at
inhibits
of
incubate
37°C.
of CD62P and
expression
decreasing
platelets,
Aliquots
1O2
the
of
of
were
fixative,
1%
Activated PMN
might
blood
the
fixed
After removal
in
formation
platelet aggregates.
para-formaldehyde.
state.
ac-
an antithrombotic
into
with
it
were
anti-CD61-FITC
and
by
modulate hemostasis,
labeled
shifting
action
platelets
and the direct
such
chloramines
cytometry. An
flow
the
The
tion
antibodies and
anticoagulant
anti-CD62P-PE
physiologic signal
1O2new
analyzed
as
of
of
decrease in
oxidant
(released
vancomycin)
by
expression
concentration-dependent
for
in
intervention
and 20%
be a
3
mM CT to
-selectin
100%
of the
re-
23,
(at
39,
pharmacologic
principle
might
appeared
Words:
saline control level for
and
atherothrombosis.
TRAP,
ADP,
Key
oxygen—Hemostasis—
Singlet
collagen,
was
an
also
concentration-
There
Platelets—CD62P—P-selectin—Anticoagulation.
oxidant
spectively).
AND
MATERIALS
METHODS
Hemostasis-the
of
and degrada-
of
system
a
generation
tion of thrombi---is
coordinated
plasma
polymor-
interplay
whole
from
donors
blood
(n ==
Freshly drawn native
blood cells.
mediators of activated
j with
Major
to
mM chloramine TO
3
incubated
at 0
(CT;
a
5) was
~PM~T~’~rre
phonuclearleukocytes
the oxidants hypochlo-
minute at
for
1
St. Louis, MO,
37’C (as
USA)
Sigma,
the nonmdi-
Re-
! rite
: cal
which am a
oxidant
for
source
and
chlc~r~ani.r~e,
stable
source wechose the mild oxidant chloramine
10,
photon-emitting
singlet oxygen (*0~).
the
chloramine
10
that
mimics
natural
N-chloro-taurine
[2]).
(final
assay
we
is a
that
demonstrated
singlet oxygen
Then
concentra-
cently,
powerful
tion and
saline,
~M
physiologic
inhibition of
antithrombotic, both
activation
coagula-
by
(ADP;
tion)
BioData Corp., -
(Bio/Data
activator
adenosine-5&dquo;diphosphate
off~brin~~~rsi~
we be-
(1). Therefore,
Horsham, PA,
or
USA),
6.25J.LM
5 >g/mL collagen
interested in
We
a
came
of
possible platelet-regulating activity
thrombin
receptor
peptide
Corp.),
(TRAP;
‘
a
flow
whole-blood
have
cytometric
~0~.
applied
Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-
the activation
of P-selectin (CD62P) to
assay
study
at different chlor-
were
and the mixtures
added,
3 minutes at
Tyr-Glu-Pro-Phe; Sigma)
of normal
platelets pr~axi~~z~~d
for
were allowed to incubate
37°C.
Fifty-
fixed in
1,000
concentrations.
blood
ofactivated
were
¡amine
microliter aliquots
control
One
(auto)
native blood to
consisted
~L 1 % para-formaMehyde.
Address
and
to T. W.
Stief, M.D.,
Philipps
correspondence
reprint requests
of 50
1%
at
of an addition
1,000
>L
>L
of
and
Clinical
Molecular
Department
Chemistry
Marburg, Germany;Diagnostics,
Afteran incubation of30 minutes
para-formaldehyde.
e-mail: thstief@
D-35033
University,
the tubes were
4°C,
at
rotations/minute
1,600
centrifuged
post.med.uni-marburg.de.
219
Downloaded from cat.sagepub.com at RMIT UNIVERSITY on March 13, 2015

220
~
for
15
the
were
removed,
added. One hundred
and 400
i
,
RESULTS
minutes,
supernates
buffer was
of this
~L~,~~~d~’s
~L
bloodsolution
was taken out and incubated with 100
Becton
p.L
Oxidative inhibition
‘
of
agonist-induced
&
anti-CD61 -fluoresc~inisothiocyanate(fITC;
P-selectin
expression
’...’
.
......<..,
.......’......,...1
San
and
anti-CD62P-
antibodies
Dickinson,
José, CA,
Becton
USA)
An oxidant
decrease in the
concentration-dependent
&
(PE;
Dickinson)
phycoerythrin
(both
flow
III
of P-?electin
at
3 mM
CTto
39, 23,,l
expression
appeared
1:30 diluted in
and
buffer),
Tyrode’s
analyzed
the forma-
by
and 20% of the 100%
control level for
saline
cot-
ADP,
for the
of
CD62P,
the formation of
cytometry
expression
and
I
and
TRAP,
2).
lagen,
respectively (Fig.
Fig.
:
tion of
or
platelet
aggregates,
an
platelet
EPICS
XL-MCL
Fullerton,
(Coulter,
fragments, using
CA, USA).
To
Oxidative inhibition
of
formation
3
!
agonist-induced
a~f
platelet aggregates
action,
or
investigate a possible
oxy-radical scavenger
singlet oxygen
oxidant-quenching
mannitol
The
formation of
agonist-induced
platelet aggregates
30mM ofthe
(Sigma)
in
decreased
of the
oxidant concentration
dependence
ofthe
methionine
was
scavenger
to the
(Sigma)
I
used
CTto
and
8, 12,
mM
13% of the
and
pp~~,~
(at 3
saline,
added to blood
addition of the oxidant. To
1
prior
control level for
the
ADP,
TRAP,r~spectiv~ly, j
collagen,
50% effective dose
confirm the chloramine T’3 results
I
with other
oxidants,
and N-chloro-
was J.0 to
mMI
1.5
the
(EDso)
oxidants
i~la~3Ct
physiologic
(Sigma)
in 10-fold molar
3
and
I
chloramine)
4).
Fig.
taurine
Sigma)
(Fig.
excess of
(1‘~a(:?~I
taurine;
i
were used for blood
oxidation.
Blood from nine rhesus
of
Oxidative inhibition
ADP-induced P-selectin
nittlcittti)
(tVlcccczcc~
monkeys
was
drawn after intramuscular ketamine narcosis
c~r
(80
formation of
in
expression
platelet aggregates
I
The
blood
incubated with
0
or mMchlo-
3
was
m~lk~~.
ramine T for
monkeyplatelets
.
,
.......,.’ for’ma.t.io.n1
I
minute
and the mixtures
10
ADP was
Then,
allowed
(3’7°C).
were
shows that
5
both the
ADP-induced
~M
i
Figure
to incubate for 3
added,
minutes at 37°C.
of
and
P-selectin in
the
of
platelet aggregates
expression
in
of activated
In
Fifty- m icrol iter
aliquots
was
mM
of 3 CT
monkey
abrogated
increased from 27.7%
platelets
presence
blood
were fixed in
one
l °l~
1,000
f.LL
para-formaldehyde,
[basal
[without CT’l;
(platelets aggregates
value]
P-
control consisted of
native
50
addition,
blood
(auto)
u.L
pL
to 30.0%
[with CTI
and to 52.9%
to
~l~
All animals
l ,(lQt)
para- formaldehyde.
selectin
increased from
22.!%
and to 69. I %l
CT]
[basal value.]
expression
received care in
on
with th~
study
American Conven-
compliance
to
%
28.3
[with
[without CT]).
tion
Animal Care. The
was
the
by
approved
ins~itutic~nal ethics committee.
I
I
induction c~f agonist-induced
Oxidative
Statistics
platelet fragmentation
All
values are means
an increase in
values
data;
After
standard
the
1
deviation;
stimulation,
platelet fag-
platelet
I
, w~re
t-test for
ments
3
mM CTta three- ta faurfald o~ the ~
(at
compared by
was
using
appeared
unpaired
I
0.05
considered
initial
using ADPorTRAPas
value,
stimulants)
p <
~). ~
statistically significant.
(Fig.
FIG. ’1,
induced
drawn native wt~ol~ blood
of
Oxidative inhibition
P-s~l~ctin
agonist-
;:
expression.
Freshly
from
donors
~n ~ -
I-
was
0to
incubated at
3 mMchtoramine T*
10
5)
1
for minute
at 37°C. Then,
ad~~c~sine .
~M
I
diphosphate (diamonds), 5 pg/mL collagen,~!
(diamonds),
5
jjg/mL coMagen
~
or
thrombin
ac-
.1
6.25 pM
receptor
(squares),
fi~atc~~
(TRAP)
peptide
(triangles)
and the mixtures were incubated for
added,
minutes
were -
the
at37°C.
of
3
Fifty-microliter aliquots
in
1%
activated blood were fixed
1,000 uL
of
After
were
removal
para-formaldehyde.
’.
the
incubated ~vifih
fixative,
samples
anti-
ar~fi-~~~’t-~IT~ and ~.nti-~t~~2P-~’E
bodies and
flow
for
¡
analyzed by
cytometry
CD62P
control
the
value
of
(100%
expression
=
= ~
Gr~nc.
mM chloramine
added).
f
concentration.
Downloaded from cat.sagepub.com at RMIT UNIVERSITY on March 13, 2015

221
thrombin
and
activator
Atypical
IG. 2.
peptide (C)
flow
with
adenosine
diphosphate (A),
receptor
(B),
collagen
cytometric analysis
the agonists
at
and 3 mMchloramine TO.
0, 1,
2,
of
inhibition
induced formation of
FIG.
3.
Oxidative
agonist-
platelet aggregates.
from
drawn native
blood
whole
do-
Freshly
mM
3
nors
was incubated at
0
to
(n
=
5)
1
chloramine T&copy; for
minute at 3l°e.
Then,
10
5
thrombin
adenosine
diphosphate (diamonds),
pM
or 6.25
(squares),
pM
pg/mL coliagen
receptor activator peptide (tri-
the
were
and
mixtures were
added,
minutes at
angles)
for
3
37°C.
incubated
Fifty-
were
of activated blodd
microliter
fixed
aliquots
1
in
%
Af-
1,000
pL
para-formatdehyde.
were
removal of the
the
ter
fixative,
samples
incubated with anti.CD61.FITC and anti-
and
formatton
flow
CD62P-PEantibodies
analyzed
by
of
for the
platelet
aggre-
cytometry
value = OmMchlora-
concentration.
control
{~ Qt~°l~
gates
mine
Conc. =
~:ctded}.
Downloaded from cat.sagepub.com at RMIT UNIVERSITY on March 13, 2015

222
and thrombin
activator
4.
Atypical
flow cytometric analysis with
FIG.
the
receptor
(A), coliagen (B),
diphosphate
agonists adenosine
and mMchloramine T<1!).-
3
at 0,
1,2,
peptide (C)
I
CONCLUSIONS
ofthe oxidant
Characterization
addition to the
of30 mM of the
The
blood
oxy-radical
the
oxide
(~--G);
like
of the
mM
to
in contrast
30
mannitol,
prooxidant nitric
Singlet oxygen,
singlet
these
scavenger
shown to inhibit
function
The
has
been
did not
(7,8).
methionine,
platelet
antagonize
oxygen scavenger
the
the
that
nonradical
platelet
shows
activation.
at
Oxidation
current
inhibits
CD62P.
of
oxidative modulations
singlet oxygen
study
the
platelet
of
activation
3 mMNAOCIor
3 mM taurine-chloramine
antigen
expression
compa-
gave
and is
is located in the
P-selectin
rable
to
n+4 chloramine T~.
3
results
alpha granules
di-
inhibition
FIG.
5
Oxidative
of adenosine
P-selectin
(ADF~~-i~duceti’
~~pres- ~
phosphate
in mon-
of
The
sion orformation
platelet aggregates
of
formation
.’
platelet aggrs-
key platelets,.’
in
the
of P-ssl~ctin‘
and
expression
gates
¡
to
was determined
prior
(gray
platelets
monkey
the
i
addition
andafter
AC~P’ to
columns)
(white
columns)
blood
nine
drawn
of
a
of 10
freshly
pM
‘
columns show the
The black
rhesus
monkeys.
of 3 mM chlor~min~
time
influence ofthe addition
to the adTdiO-
incubation
and 1-minute
tion of
prior
r
ADP.
I
Downloaded from cat.sagepub.com at RMIT UNIVERSITY on March 13, 2015

223
induction of
FIG. 6. Oxidative
agonist-induced
drawn native
Freshly
fragmentation.
platelet
=
5) wasincubated
from
whole blood
donors
(n
TOfor minuteat37’C.
3 mMchloramine
1
at 0 to
adenosine
10
or
Then,
monds)
diphosphate (dia-
activator
thrombin
pM
6.25
~M
receptor
andthe mixtures
were
added,
37°C.
at
minutes
were incub~t~d
’
for
3
incubated
were
Fifty-
blood werefixed
After re-
microliter
of activated
aliquots
1 ~l~
in
moval
bated
1,000
para-formaidehyde.
pL
were incu-
anti-CD62P-PE
the
of the
fixative,
samples
‘
with anti-CD61-FITC and
for
con-
flow
and
formation of
antibodies
the
trol value
analyzed
cytometry
by
(100%
platelet fragments
=
=
Conc.
mM
0
chloramine
added).
concentration.
B&dquo;1111B,11&dquo;1
’.’ vmVIGlHlUv....VII.....
&dquo;,
I
Selectins
activation.
released to the
surface
upon
in
role
platelet
NEngl Med
by neutrophils.
J
destruction
1989;
Weiss SJ.
320:365.
Stief TW,
of
interaction
2.
3.
Tissue
the
an
important
mediating
or
play
with
(9-11).
platelet
(PMN
monocytes)
phagocytes
platelets
Nonradical oxidants
et al.
of
Stief MH, Ehrenthal W,
induce
the
PMN
can activate
Activated
thus,
(12);
uro-
platelets
the activation
of plasmatic single-chain
phagocyte type
kinase.
Thromb Res 1991;64:597.
of activated
An
a
main activation
by
product
regulation
N.
of nitric
Reaction
Woolf
MM.
AA, Epperlein
Noronha-Dutra
4.
is
of
PMN,
importance.
physiologic
singlet oxygen,
to
with
oxide
potentially cytotoxic
produce
peroxide
hydrogen
an en-
results in
often
enhanced cell-cell interaction
for
as
nitric
a model
oxide-mediated
FEBS
killing.
oxygen
1993;321:59.
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singlet
Lett
of
~ han~~d
(13).
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generation
et al.
Bechara
Medeiros
molecular
hydrogen
5.
6.
EJ,
in the reaction
MH,
P,
Singlet
mMHOllchloramines
generate upto 5
of
for inhibition of the formation
Activated PMN
I
with
of
peroxynitrite
oxygen production
The
(2).
platelet
ED5(,
1994;355:287.
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1 delta
the
activated PMN
of
is
I
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(
Quantitative
singlet
g) oxygen
aggregates
of
reaction
from
nitric oxide and
10:329.
acidified
by
aqueous peroxynitrite produced
inhibit
be able to
thrombocytes by
efficiently
should
anion.
Biolumin Chemilumin 1995;
superoxide
is
that
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with
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RJ,
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phils
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oxide
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I
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a
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singlet
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by platelets
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into an
~si1.ed~
1I&dquo;tiV~,.d.~MN.. ,’,
al.
physiologic sig-
t
t
~
Y
,
:n1 salf
action of
13.
DC,et
SB, Toman C, Anderson
Adherence-dependent
Shappell
mod.U~ ?e.fl1O~~j,S’
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(released
by
102
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J
Immunol 1989;144:2702.
(CD11b/18).
be
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ramines
as
such
might
principle
vancomycin)
in
X.
Mao
Y,
the
14.
15.
L, Shi
superox-
B19i l I2n27t.
Singlet9oo5xy;g3en6g:eneration
Zang
intervention in atherothrombosis.
for pharmacologic
Mol
ide
r
e
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Biochem
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p1h9y9s9;R2e6s2C:o6m4m7u.n
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