From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Labeling
of the
Releasable
of Washed
Human
By. H.-J. Reimers,
In
rabbit
tive
platelets,
pool
ATP
able
ATP
showing
pool
this
human
incubated
After
1 day.
10
hr,
leasable
up
to
about
with
of the
were
at
37#{176}C.
total
had
plate-
become
(4.2
re-
units/mI).
Ly-
did not occur, since less than
of the
platelet-bound
lets
labeled
with
the
hr in vitro
‘4C-ADP
1%
in the
treatment.
10
51Cr
this
ambient
The
released
from
plate-
radioisotope
fluid
upon
apthrombin
‘4C-ATP/14C-ADP
adenine
ratio
nucleotides
of
(7.6)
was similar
to the 14C-ATP/’4C-ADP
ratio
of the nonreleasable
adenine
nucleotides
(7.1)
2 hr after
the labeling
with
‘4Cadenosine.
However,
upon prolonged
incubation
(10 hr) in vitro,
the ‘4C-ATP/
‘4C-ADP
ratio of the releasable
adenine
A
DENINE
readily
NUCLEOTIDES
labeled
by incubation
or 32Porthophosphate.L3
exchange
conclusion
released
for up to
ATP
and
are
however,
significant
Da Prada
amount
practically
exposure
labeled
and
The
the
adenyl‘4C-ATP/
pool
of platelets
can
‘4C-adenosine,
4C-adenine,
nucleotides
the Department
in this
May
Supported
by
of a Fellowship
A ddress
for
reprint
accepted
Research
of the Ontario
requests:
to
labeled
pool
Dr.
these
Toronto,
August
Council
Heart
H. -J.
conditions.
l4C..adenosine
ATP
of rabbit
and the releasable
McMaster
of Toronto,
13. 1976:
Medical
under
l4C..adenine
granule
pool
have
been able to
‘4C-ATP
in isolated
platelets
of Pathology,
University
Submitted
with
‘4C-adenosine,
released
from
the
unlabeled
of intact
of Biochemistry,
2 hr
ADP
and Pletscher4
of newly
formed
shown
that radioactively
the nonreleasable
metabolic
award
the
to 2.7.
charge
in the metabolic
of platelets
with
However,
thrombin
From
decreased
energy
pool
do
be
not
readily
with those
in the storage
granules
of human
platelets.3
This
is based
on the finding
that radioactive
adenine
nucleotides
are not
upon
addition
of collagen
or thrombin
to human
platelets
that
have
been
incubated
phosphate.3
The
after
J. F. Mustard
14C-ADP
ratio of the metabolic
adenine
nucleotide
pool did not change
significantly
during
the time
of observation.
The 14C-ATP content
of the platelets
decreased
by less than 1%/hr
of incubation
at 37#{176}C.These
observations
are interpreted to mean that the ‘4C is transferred
from the metabolically
active,
nonreleasable adenine
nucleotide
pool of human
platelets
into
the
releasable
adenine
nucleotide
pool as ATP and
is partially
hydrolyzed
there to yield ADP. The transfer
of ATP across the storage
organelle
membrane of platelets
may be similar
to transport processes in the chromaffin
cells of the
adrenal
medulla
and may represent
a general phenomenon
in cells that
possess
storage
organdIes
containing
adenine
nucleotides.
to
labeled
14C-adenine
thrombin
sis of platelets
peared
platelets
or
12%
and
ate
studies
extended
and
nucleotides
releas-
N ucleotides
Platelets
Packham,
ac-
the
The
been
Human
for
‘4C-ATP
with
now
14C-adenosine
M. A.
metabolically
within
have
platelets.
with
let
the
equilibrates
Adenine
University,
Ont.,
In
or 32P-orthoby collagen
or
rabbit
platelets,
demonstrate
a small
but
amine
storage
granules
for
2 hr.
platelets
pool
Hamilton,
We
have
recently
equilibrates
between
within
I day.5 In con-
Ontario,
and
the
Department
Canada.
26, 1976.
of Cana4a
Foundation
Reimers,
Grants
to 11.-f.
Department
MT
1309
and
MT
2629
and
by
the
Reimers.
of Pathology,
McMaster
University,
Hamilton,
Oni. L85 4J9, Canada.
Cc) 1977 by Grune
& Stratton,
inc.
Blood,
Vol. 49, No. 1 (January), 1977
89
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
90
REIMERS
trast to rabbit
platelets
about
equal
amounts
ments
that preferentially
of ATP
and ADP.
to determine
if radioactively
of human
platelets
ifADP
is formed
tide
can
from
enter
ATP
release
ATP,
human
Therefore
we have
labeled
ATP
and
ADP
platelets
designed
in the
El
release
experi-
metabolic
the granule
pool,
and thus become
after its transfer
into the releasable
AL.
pool
releasable,
and
adenine
nucleo-
pool.
MATERIALS
Preparation
of Washed
Suspensions
human
of
donors
1 wk
prior
taming
acid)
twice-washed
blood
0.35%
platelets
buffer,
but
The
and
no
were
platelets
were
HEPES
S m M
apyrase.
prepared
et al.6 The
of Mustard
collection.
albumin
METHODS
Platelets
by the method
to
AND
Platelet
from
the
blood
not
ingested
donors
had
finally
resuspended
of
1-2
zM.
the
8-14C-adenosine
adenine
platelet
adenine
after
was added
nucleotides.
As
in two
well
as
each
addition
small
Tyrode’s
labeled
to
amounts
accumulating
to
for
at
least
solution
con-
‘-2-ethanesulfonic
were
nucleotides
healthy
drugs
(N-2-hydroxy-ethyl-piperazone-N
immediately
8)4C-adenosine
apparently
any
in
8-14C-adenosine
(>50
zCi/imol;
Amersham
Searle
Corp.,
Arlington
platelet suspension followed
by a second
10 zCi 8-’4C-adenosine
30
tion
of
by
the
suspension
its
phosphorylated
zCi
Ill.)
to
the
The concentra-
platelet
increase
10
adding
Heights,
mm later.
was
incorporation
derivatives
into
of
adenosine,
platelets convert
adenosine
into
inosine
and convert
inosine
to hypoxanthine.7
With
higher
adenosine concentrations,
a larger
proportion
of adenosine
was converted
into
inosine
and hypoxanthine.8
In some
experiments,
platelets
were
labeled
with
40 MCi
U-14C-adenine
(>255
uCi/imol;
Amersham
Searle
Corp.)
at a final
concentration
of 7 zM;
the
results
with
both
labeling methods were similar.
Platelets
were
incubated
at 37#{176}Cfor a total
of 60 mm
after
the
first
addition
in fresh
of
8-’4C-adenosine
Tyrode’s-albumin
109/liter and
kept
medium once after
cubation medium
14
C-inosine
in
or
at
37#{176}Cthroughout
5 hr
platelet
10
were
also
labeled
Na251Cr04
lets
concentrations
Release
platelet
20 N NaOH
100
l
release
before
of the
mediately
Detroit,
induce
was
mm
Iowa)
and
suring
final
at
experiments.
them
in the
Amersham
of
In
first
of
kept
in
the
medium.
In
the
platelet
Corp.)
per
x
fresh
same
in-
‘4C-hypoxanthine
and
these
experisuspensions
two
experiments,
washing
fluid
Searle
500
in
the
with
5
x
platelets
200
zCi
l0’#{176}plate-
concentrations
after
all
additions
to the
platelet
suspensions,
use
so that
the
EDTA
solution
M
release
incubated
The
of the
in
EDTA
final
the
(0.1
pH
of the
to
1 ml
release
37#{176}Cin
presence
M)
and
was
platelet
of the
reaction
absence
adjusted
of
to pH
suspension
platelet
with
ml
of
45
sec
fluid
in the
density
at
was
supernatant
of the
a
8.3
mM
8.3 with
would
upon
EDTA
(bovine
ethylene-
approximately
be 7.35
suspension.
thrombin
platelet
of thrombin
(170
was
removed
fluids
extracts
Tyrode’s
was
thrombin,
Fisher
rapidly
an
and
addition
added
Parke,
stored
at
259
Rotator;
to
in ice.
The
In
a
imDavis
some
in 3%
amounts
siliconized
of
total
acid
nucleotides
Co.
Inc.,
centrifuge
(Brinkmann,
experiments,
for
Dubuque,
Scientific
Eppendorf
perchloric
glass
sample)
Corp.,
Fisher
an
Microcentrifuge
determined
nm.
in
in a control
Thermolyne
transferred
Eppendorf
were
placed
solution
(Thermolyne;
cycles/mm,
then
g in
12,000
suspension
(or
Dribath
device
suspension
supernatant
optical
I
units/mI
a shaking
The
for
nucleosides
4.2
a constant-temperature
on
centrifuged
reaction,
with
Pa.).
the
studied
was
(EDTA).
Mich.).
mounted
The
reaction
induction
the
Pittsburgh,
Ont.).
are
acid
0.1
before
& Co.,
and
Cr;
were
pH
of
resuspended
Reaction
The
3
zCi/zg
platelets
accumulation
the
platelet-suspending
( 1 g/liter).
The
all
were
stated.
diaminetetraacetic
tube
and
hr
throughout
incubating
160
given
otherwise
To
by
5
the
resuspended
at a concentration
Platelets
experiments,
measure
centrifugation,
buffer
15 mm.
unless
of
5tCr
(approximately
for
All
with
to
by
HEPES
experiment.
In three
hr
suspension
after
glucose was replenished
remained between 7.35 and 7.7
ments,
recovered
5 mM
the
of incubation.
for up to
the
U)4C-adenine,
containing
solution
tube
Rexdale,
riucleotides
extracts
by
and
nucleosides
mea-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LABELING
in the
IN
HUMAN
supernatant
fluids
nucleosides
as
amounts
of material
Tyrode’s
solution
and
PLATELETS
ADP
91
were
a measure
of
expressed
the
absorbing
were
in samples
made.
In
‘4C-ATP
treated
and
its
metabolites
the
total
platelet
in
samples
were
Randerath
and
experiments
in
tivity
due
method
to
of
‘V emission
which
isotope
Sheppard
and
adenine
nucleotide
and
labeled
In
the
(AEC)’5
of
‘4C-ATP,
small
due
to the
that
was
scintillation
5tCr
and
14C-adenosine,
‘4C-hypoor
the
Thyrode-
methods
of
counting.’3
the
counting
was
Philips
radioac-
according
also
In
to
the
by
its
determined
platelets
and
at the
of
mM
EDTA.
evaluated
by
was
determined
with
the
from
the
radioactive
formula
the
‘4C-ATP/’4C-ADP
and
‘4C-AMP
corresponding
released
times.
‘4C-ATP
ratio
were
and
of
the
corrected
No
correction
14C-ADP
metabolically
for
the
was
that
releasable
made
would
have
for
the
occurred
Lysis
Platelet lysis’3’17 was
thrombin-induced
5tCr and 8-’4C-adenosine.
51Cr
liquid
51Cr
a
and
thrombinusing
scintillation
by Mills16
‘4C-ADP,
‘4C-AMP
of8.3
of
by
experiments,
in
4C-inosine,
fluids
as ATP
in EDTA-
ADP+2ATP
AMP+ADP+ATP
charge
breakdown
after
lets
energy
‘4C-ATP,
and
in the presence
Platelet
the
platelet
‘4C-ADP,
error
even
of
total
both
as well
measured
chromatography
liquid
the
as described
concentrations
calculation
pool,
ADP,
determined
the
with
Charge
charge
2
For
was
treated
counter.
AEC=’x
active
Luminescence
and
for
samples
and
‘4C-IMP,
with
these
ATP
were
counted
by
scintillation
Energy
energy
al.12
Marlow.14
platelet
paper
nucleotides
corrections
in control
supernatant
by
determined
crystal
Adenylate
adenylate
et
were
was
the
experiments
Cain
platelets
each
in a well-type
Platelet
The
and
and
platelet
centrifugation,
‘4C-AMP,
suspension
in all
al.9
et
(14C-ADP,
fluid
total
by
total
Appropriate
supernatant
obtained
Holmsen
the
reaction.
experiments,
of
of
release
in the
fluids
isolated
Struck1’
the
nm
other
of supernatant
ethanol extracts by the method
Liquid Scintillation
Counter.’0
xanthine)
of
at 259
percentages
as
extent
appeared
in the
Lysis
in the
supernatant
measuring
release of adenine
was
calculated
supernatant
fluid
fluid
from
the
appearance
nucleotides
as
after
the
5tCr
platelets
percentage
incubation
unstimulated
of
from
of
with
platelets
was
in
the
supernatant
fluid
that
had
been
labeled
the
total
51Cr
in
thrombin.
The
subtracted
small
before
this
the
with
plate-
amount
of
calculation
made.
A TPase
The
A ctivity
external
ATPase
the
conversion
following
Ltd.,
Montreal)
in the presence
acid
extracts
separated
of aliquots
by paper
of washed
of 0.2 zM
to ‘4C-ADP
or absence
Statistical
Results
activity
of
of the
platelets
and ‘4C-AMP
EDTA.
At
platelet
chromatography
stimulated
(10
8-’4C-ATP
in a platelet
time intervals
suspension
as described
with
izCi/0.013
mg;
thrombin
New
suspension.
of 2, 4, 6,
were
prepared
above
and
and
was
England
These
8, 10,
ATP,
determined
by
Nuclear-Canada,
experiments
and
15 mm,
ADP,
and
were
done
perchloric
AMP
were
counted.
Methods
were
evaluated
by Student’s
t test
and
simple
linear
regression
analysis.’8
RESULTS
Properties
Two
of Platelets
hours
about
85%
ADP,
and
metabolically
after
After
the
Prolonged
labeling
of the platelet-bound
about
1% in AMP.
active
pool at this
Incubation
of platelets
with
radioactivity
The
adenylate
time was 0.931
‘4C-adenosine
was
±
or
‘4C-adenine,
found
in ATP,
ll%-12%
energy
charge
(AEC)
of
0.007 (arithmetic
mean
±
in
the
SD
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
92
REIMERS
ofseven
experiments),
the ‘4C-ATP/’4C-ADP
mean
and range
of seven
experiments).
pension
at
metabolically
37#{176}C,neither
active
pool
‘4C-ATP/’4C-ADP
period,
hour;
ments
total
and
ratio
the AEC
nor
the
changed
significantly
at
10 hr:
7.3
the amount
of platelet-bound
the amount
of platelet-bound
in which
the incubation
was
radioactivity
less than
I .5%
ratio
was
incubation
During
During
(5.2-9.2)).
‘4C-ATP
‘4C-ADP
continued
dition
of ADP
Effect
ofEDTA
To
(in the
make
absence
presence
of
it possible
ofReleased
to determine
in an experiment
in which
presence
of 8.3 mM
EDTA,
less
verted
to ‘4C-ADP
and ‘4C-AMP
lease
studies
Release
The
8.3 mM
EDTA
than
per
was
(0.02
thrombin
ATP
used
0.5%
minute.
ratio
was
added
hydrolysis
concentration
to the
ofthrombin
amounts
3.0%;
of ATP
in the
the
total radioactivity.
metabolic
pool
used
Upon
adenine
and
after
of the platelets,
‘4C-ADP
could
However,
‘4C-ADP
and
(4.2
ADP
supernatant
concentration
to inosine
1% of ‘4C-IMP
Immediately
sion
and
to
esti-
to a suspension
the absence
of
minute.
In the
the radioactive
Therefore
in all
to diminish
releas-
ATP
was
subsequent
ofreleased
conre-
ATP.
Studies
concentration
(0%-0.9%)
in the
In
per
units/ml)
released
of
stimulation
nucleotides
as calculated
a very
released
was
between
with thrombin,
were converted
from
was
with
In these
the
radioactive
formed
during
‘4C-adenosine
small percentage
upon
addition
with
longer
incubation
times,
increasing
became
releasable.
There
appeared
to
the same.
light
at this
in compari-
experiments,
was
0.1%
of
2.1%
±
(8.3 mM)
did not
3.0%)
(means
and
of platelets,
the
remained
absorb
be small
‘4C-hypoxanthine
of ‘4C-inosine
‘4C-AMP
labeling
only
be
present.
fluid
55.4%
nm; EDTA
(58.6%
±
incubation
amount
of this material
that could
be released
by thrombin
Although
other
compounds
besides
ATP
and ADP
that
wavelength
may have
been present,
their
amounts
would
and
ad-
it was necessary
of ATP
was
units/ml).
hydrolyzed
the platelet
material
which
absorbed
light at 259
have a significant
effect
on the amount
released
SEM
of 20 determinations).
During
the
10-hr
son
after
upon
or thrombin.
‘4C-ADP
zM)
of
apyrase
aggregated
Nucleotides
(0.5
was
supernatant
containing
collagen,
‘4C-ATP/
‘4C-ATP
of the
0.013;
±
observation
in the
the released
compounds,
after
its release.
Hydrolysis
of unlabeled
platelets
10 sec after
EDTA,
about
3#{176}/a of
the radioactive
10-hr
only
they
Adenine
the
by measuring
of ‘4C-ATP
ratio
0.933
at the end of the experiments,
the platelet
suspension
was
of apyrase),
of fibrinogen6),
on Breakdown
able pool of platelets
prevent
breakdown
mated
the
the
geometric
platelet
sus-
decreased
by less than
1% per
remained
unchanged.
In experiup to 10 hr, less than 4% of the
was found
in ‘4C-hypoxanthine
of the total
radioactivity
at 37#{176}C
(in
(5.8-7.8;
of the
‘4C-ATP/’4C-ADP
(AEC
at 10 hr:
found
in ‘4C-inosine.
Both
metabolites
were detectable
fluid. The radioactivity
in ‘4C-IMP
was less than
1%.
When
platelets
were resuspended
in fresh
medium
10 hr incubation
7.1
ET AL.
the
between
and
1.3%
initial
0.1%
of the
8.1% (6.7%-lO.2%)
to hypoxanthine
and
of the
0.3%
metabolites.
Less
than
thrombin
stimulation.
or ‘4C-adenine
and
resuspen-
of the platelet-bound
of thrombin
(4.2
‘4C-ATP
units/ml).
be
amounts
a linear
of ‘4C-ATP
relationship
and
be-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LABELING
IN HUMAN
93
PLATELETS
14
12
0
LU
U)
Ui
-J
UI
Fig. 1. Release
Samples of washed
of ‘4C-ATP
+
14C-ADP.
human platelets labeled
with
14C-adenosine
were
stimulated
thrombin
(4.2 units/mI)
in either
the
or
(0-0)
presence
EDTA. The ‘4C-ATP and
.___u)
4C-ADP
-I
10
4
a
0
0.
U-
0
0
8
with
absence
z
6
UI
U
a
of 8.3 mM
in the super-
0.
UI
4
0.
4
U
natant
fluid was expressed
as a percentage
of
the total
in the platelets
at the time of the
thrombin
stimulation.
EDTA
did
not
significantly
affect the percentage
of ‘4C-ATP
+
‘4C-ADP
released
at any
time.
Means
and
2
0
2
4
SEM of seven experiments.
tween
incubation
time
and
the percentage
‘4C-ADP
that could
be released
with thrombin
10 hr
(Fig.
I).
6
10
8
12
HOURS
Figure
1 shows
also
that
of
the
during
EDTA
total
platelet
the observation
did
not
‘4C-ATP
period
significantly
+
of
change
the amounts
of radioactive
‘4C-ATP
and
‘4C-ADP
released.
The
increased
amount
of ‘4C-ATP
and ‘4C-ADP
that appeared
in the suspending
fluid
upon
stimulation
with thrombin
was not accompanied
by the appearance
of 51Cr in
the supernatant
fluid of platelets
prelabeled
1% of the 51Cr appeared
in the supernatant
12% of the ‘4C-ATP
and
‘4C-ADP
when
(4.2
that
units/mI)
was used
had been incubated
Figure
2 shows
that
nucleotides
decreased
with
were found
radioactivity
prolonged
incubation
the
ratios
was
Only
more
very
of the
platelets
evident
small
if the
amounts
in the supernatant
fluid after
thrombin
of the released
ATP
was greater
than
activity
of the released
soon after
labeling
with
labeling;
Less than
approximately
of thrombin
to induce
the release
reaction
in a platelet
suspension
for 10 hr after the labeling
procedure.
the ‘4C-ATP/’4C-ADP
ratio
of the releasable
adenine
decrease
in the ‘4C-ATP/’4C-ADP
ratio
was induced
in the presence
of EDTA.
‘4C-AMP
specific
with
this radioisotope.
fluid,
together
with
the high
concentration
ADP
(Fig.
‘4C-adenosine
of the
specific
3).
This
difference
or ‘4C-adenine
radioactivities
were
in vitro.
The
release
reaction
of radioactive
stimulation.
the specific
The
radio-
was more
pronounced
than
at later
times
after
10.8:1,
90 mm
after
add-
8
0.
0
4
6
EDTA
0.
I..
Fig. 2.
nucleotides.
4
4
14C-ATP/14C-ADP
ratio
Aliquots
of suspensions
platelets labeled with 14C-adenosine
up to 10 hr at 37#{176}C
were treated
2
2
4
HOURS
6
8
10
of
released
adenine
of washed human
and incubated for
with thrombin
at
various
times
in the presence
or absence
EDTA. The ‘4C-ATP/
‘4C-ADP
ratio is plotted
dinate.
Geometric
means of values
obtained
experiments.
of 8.3 mM
on the orfrom seven
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
REIMERS
94
10
El
AL.
ATP
AD P
2
0
2
0
4
6
8
10
12
HOURS
Fig.
3.
Specific
radioactivity
of
released
ATP and
ADP (one
of four
similar
experiments).
pIes of washed human platelets labeled with 14C-adenosine
or 14C-adenine
to 10 hr at 37#{176}C
were treated
with thrombin
(4.2 units/mI)
at various
times
and
Sam-
stored for up
in the
presence
of
8.3 mM EDTA. Radioactive
‘4C-ATP
and ‘4C-ADP
were separated
chromatographically
on paper.
Samples
of the supernatant
fluids were taken
for determination
of the ATP and ADP concentration
with the firefly
method.
Appropriate
corrections
were
made
for the metabolites
present
in the suspending
fluid before
thrombin
treatment.
(These
values
for ATP were
between
0.03
and 0.29
.tmol/1O11
platelets;
for ADP, between
0 and 0.37
imol/1O
platelets.)
The platelet
ATP
content
in this experiment
was 4.42 ± 0.25
imol/1O11
imol/10’1
platelets.
With
the thrombin
concentration
ATP/1011
platelets
and
1.27
± 0.08
z/mol
ADP/1011
minations
in this
experiment.)
The total
radioactivity
1,131,800
cpm. The plateletcount
was 500 x 109/liter.
ing
labeled
the
(Fig.
adenine,
and
2.3:1,
10.5
platelets,
the ADP content
2.04
± 0.36
used, platelets
released
1.62
± 0.6 jzmol
platelets.
(Means
± SEM
of ten deterin 1 ml of platelet
suspension
was
hr
after
adding
the
labeled
adenine
3).
DISCUSSION
In the
present
study,
human
‘4C-adenine
and incubated
induced
to release
their
These
platelets
maintained
10-hr
incubation
period.
Metabolic
The
man
and
their
Functional
adenylate
platelets
energy
used
platelets
in vitro
at
storage
granule
metabolic
Properties
charge
in these
of the
studies
and
distribution
of about
formed
#{149}
similar
stored
ADP
content
of platelets
of adenine
nucleotides
60:40,
one can calculate
per minute
to the value
at 37#{176}C(0.29
by 10”
reported
‘4C-adenosine
functional
after
similar
with
or
up to 10 hr before
they were
by the addition
of thrombin.
active
to
properties
Prolonged
metabolically
was
the
over
a
Incubation
pool
value
of washed
of 0.915
±
hu0.008
in citrated
platelet-rich
plasma,
even
for prolonged
periods
of time
at 37#{176}C.
4% of the metabolic
pool
nucleotides
10 hr of incubation
at 37#{176}C.Using
the
given
in the
between
that less
platelets
in the
by Holmsen’9
nmol/min/lO”
labeled
and
of Platelets
reported
by Mills’6
for human
platelets
when
the washed
platelets
were stored
In the present
experiments,
less than
were converted
to hypoxanthine
during
ATP
were
37#{176}Cfor
contents
legend
of Fig.
releasable
than
0.17
and
nmol
3 and
present
experiments.
and Holmsen
and
platelets).
In
contrast,
assuming
nonreleasable
hypoxanthine
a
pools
was
This
figure
is
Weiss2#{176}for PRP
when
platelet-rich
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LABELING
IN
plasma
HUMAN
was
thine
stored
from
the
hypoxanthine
modified
at 23#{176}C,Lages
ATP
formation
when
solutions
part of the
nucleotides.22
Packham,
hypoxanthine
and
then
activity,
less than
4%
Although
leads
95
metabolic
Tyrode’s
experiments,
or guanine
nod
PLAIELEIS
within
for
60
hypoxanthine
We have
washed
platelets
but no apyrase,
contain
some
platelets
were
ATPase
lets
extended
reinjection
had
never
into
left
The unchanged
‘4C-ATP
in the
possible
the
charge
during
of the platelets
in Tyrode’s
aggregation
activity
resuspended
10 hr of incubation,
as to thrombin.
We
that
that,
the experimental
used
in these
0.35%
tested.
solution
response
as
they
were
still
have also shown
periods
of
animals,
for
time
activity).
medium
responsive
previously
of time
to ADP
and colthat rabbit
platesolution
similar
to
survived,
that
of
ratio
poo1,
and the almost
as well as the
constant
unaltered
Pool
These
Nucleotides
storage
of these
become
‘4C-ATP
of
labeled
(Loss
of 5tCr
releasable
poo1
becomes
slowly
that have
been
is based
on our
platelets,
with
amounts
the
platelets
in vitro of platelets
This conclusion
suspension
negligible
reaction.
the Metabolic
that
human
increased
releasable
upon
treatment
and ‘4C-ADP
that appear
the platelet
since only
release
demonstrate
organelles
longed
storage
or ‘4C-adenine.
From
of
thrombin
of experiprelabeled
Pool
experiments
storage
platelets
amount
proportion
Transfer
of Adenine
How-
containing
circulation.2425
‘4C-ATP/’4C-ADP
metabolically
active
the Releasable
apyrase
found
containing
no
apyrase
prepa-
ADPase
in Tyrode’s-albumin
a length
is the
platelets
containing
0.35%
ADP
quickly.
In
to
suspending
peexperi-
and
authors
in a medium
and ADP
(the
as well
in fresh
albumin
These
be released
with
for the analysis
from
platelets
the
in our
into adenine
this (Reimers,
of the total
platelet
adenine
nucleotides
that
could
during
the 10-hr
observation
period,
were essential
ments
in which
the release
of ‘4C-ATP
and ‘4C-ADP
with ‘4C-adenosine
or ‘4C-adenine
was followed.
Into
15.4%
in different
since
the value
for ‘4Cabout
3% of the total
radioincreased
to a final value
of
were suspended
of released
ATP
apyrase
after
lagen as well
for
hypoxanand
suspended
reincorporated
evidence
for
containing
longest
time
suspended
lost their
the present
experiments,
platelets
apyrase
to minimize
breakdown
kept
was
some
2.7%
3.0%
believe
that ADP-induced
platelet
aggregation
viability.
Mustard
et al.6have
shown
that
also that
albumin,
upon
were
observations),
was initially
and gradually
adenylate
energy
a high viability
investigators23
test ofplatelet
used
of
between
10 hr of incubation.
the constant
one to assume
when
and
at 23#{176}C.It was
formed
obtained
at 37#{176}C
in Tyrode’s
solution
to ADP
for up to 6 hr, the
rations
formation
platelets
mm
suspended
aggregate
ever,
found
60 mm,
gel-filtered
Mustard:
unpublished
in the supernatant
fluid
declined
to almost
zero,
after
ments,
some
most sensitive
et al.2’
pool
thrombin
of 51Cr
indicates
incubated
finding
amounts
with
thrombin.
in the suspending
cannot
be
appear
in the
platelet
‘4C-ATP
and ‘4C-ADP
released
under
the conditions
rived from the metabolically
active
poo1 of adenine
of ADP
with
that,
lysis.’7)
and
increased
fluid
upon
attributed
suspending
Thus
ATP
upon
in
pro-
‘4C-adenosine
upon
prolonged
of ‘4C-ATP
The
and
labeled
‘4C-ADP
amounts
treatment
of
of
to platelet
lysis,
fluid during
the
it is unlikely
of our experiments
nucleotides.
that
are
de-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
96
REIMERS
The
about
adenine
nucleotides
accumulated
1% of the total
labeled
platelet
previously
tide
pool
twice
Prada
found
an accumulation
of rabbit
platelets,5
as fast as that
and Pletscher4
the
observed
with
also concluded
Similarly,
there
be a transfer
nucleotide
pool
of ‘4C-ATP
human
that
platelets
‘4C-ATP
‘4C-ATP
had
of adenine
the
per
adenine
was
about
experiments.
transferred
Da
from
into
the amine
storage
orin which
they
showed
that
a
in the amine
storage
for 2 hr with
‘4C-
et al.26 obtained
some
nucleotides
from
nonreleasable
adenine
nucleotide
releasable
nucleo-
accumulation
in these
could
be
AL.
pool
at a rate of
hour.
We have
releasable
accumulated
been
incubated
Kotelba-Witkowska
into
in the
rate
nucleotide
pool
from
experiments
small amount
of newly
synthesized
organelles
when
the intact
platelets
adenosine.
the releasable
nucleotides
of ‘4C-ATP
but
the metabolically
active
adenine
ganelle
pool of rabbit
platelets
may
within
adenine
El
the
pool
evidence
that
adenine
upon
prolonged
incubation
of human
platelets
in vitro.
From
experiments
with rabbit
platelets
in which
platelet
adenine
were labeled
with 32P in vivo and in vitro
or with
‘4C-adenosine
nucleotides
in vitro,
we
concluded
that the accumulation
of ‘4C-ATP
in the releasable
adenine
nucleotide pooi
represented
an exchange
of ATP
between
the cytoplasm
and
the
amine
storage
organelles.5
It is likely
that the accumulation
of ‘4C-ATP
and
‘4C-ADP
in the
ofadenine
ment(s)
releasable
pool
periments
do
not
allow
uptake
of 12% of the
into the amine
storage
amount
of releasable
ATP
bolically
periods
intact
oftime.
nucleotide
platelets
Based
pool
adenine
will
over extended
Partial
releasable
ratio
the
release
a final
and
reaction
represents
conclusion
would
available
incubation
of
into
Its
these
ratio
of the
was
not
that
in this
allow
platelets
when
Transfer
Into
Ca27Mg2-activated
ATPase
The
since
active
increase
pool
in the
been
detected
with
functionally
and
pool
of human
with
reactions
Releasable
of the
platelets
induced
in the
has
experiments,
(7.6:1)
presence
of platelet
‘4C-ATP/’4C-ADP
granules
ratio
been
reported
was based
of collagen
the
was
‘4C-ATP/
comparable
nucleotides
or ‘4C-adenine,
of
EDTA,
ATP
after
its
to French
is not
of the
‘4C-
studied
Pool
platelets
nonreleasable
adenine
with ‘4C-adenosine
or
are
of ATP
and AD P.3 This conclusion
nucleotides
released
upon
addition
In the present
adenine
nucleotides
meta-
prolonged
adenine
‘4C-adenosine
platelet
the
exchange
regard,
have
thrombin.
EDTA
inhibits
the breakdown
of
by inhibiting
platelet
ecto-ATPase.27
(According
ml of thrombin.)
an
active
compartthe present
ex-
suspending
media
for
labeling
of the releasable
account
nucleotide
to a platelet
suspension.
of the releasable
vided
the
ADP
equal
amounts
of the adenine
‘4C-ATP/’4C-ADP
after the labeling
prior
to
probably
and
methods.
become
of A TP After
to the
shortly
at
to be kept in artificial
on the present
results,
adenine
to contain
about
on measurements
also
metabolically
However,
nucleotides
from
the metabolically
pool(s)
would
cause
only a small
have
to be taken
periods
of time.
Hydrolysis
or thrombin
‘4C-ADP
after
platelets
nonreleasable
compartment(s).
us to arrive
adenine
granule
certainty
using the present
Recently,
methods
have
The
of human
nucleotides
between
the
and the releasable
granule
released
adenine
(7.1:1)
pro-
added
just
release,
et al.,28
by 5 units/
nucleotides
re-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LABELING
IN HUMAN
leased
upon
10 hr)
decreased
PLATELETS
addition
of thrombin
EDTA).
ratio
that
appeared
the change
leased
leasable
platelets
was
bolically
active
seems
into
highly
the
and a slower
exchange
in the releasable
pool
equilibrium
The findings
concerning
platelets
are in contrast
were observed
storage
granules
In experiments
lets was labeled,
ratio was
nucleotides
platelets
and
was
apparent
conversion
lets,
why
granules.
platelets
not
the
to the
been
ATP/ADP
findings
to be released
from
rabbit
found
was
only
of ATP
this
does
However,
one
release
appreciable
release-inducing
agents,
unless
of
the
are
of ‘4C-ADP
This pos-
ratio
in the
meta-
than
the
in the
any
pool
which
of human
ATP
and
about
5: 1 #{149}29Studies
with
isosupported
this observation.30
adenine
nucleotide
as a precursor,5
an
to
kinetics
in the releasable
platelets
from
pool of rabbit
plateeven higher
‘4C-ATP/
the
ratio
initial
ofthe
releasable
labeling
of the
‘4C-ATP/’4C-ADP
ratio
releasable
pool(s)
appreciable
released
Unof the
of human
extent
in rabbit
of this difference
as well as ATP,
nucleotide
in the
Packham,
and Mustard;
do not know
the cause
we
the consequences
amounts
of ADP,
whereas
complex
re-
of the rethe meta-
reached.
(7.6:l)(Reimers,
present
time,
occur
a linear
labeled
specific
radioactivity
of ‘4Cat the end of the experiment,
in a ratio
of
platelets
have
to ADP
not
part
from
and the transfer
of the experiment.
the
ratio
in rabbit
smaller
platelets
At the
was
total
pool
during
the observation
ratio
of ‘4C-ATP/’4C-ADP
(6.4:1).
This ‘4C-ATP/’4C-ADP
found
as late as 66 hr after
slightly
pool ofrabbit
observations).
nor
had
in
change
of the ‘4C-ATP/
‘4C-ADP
by assuming
a rapid
exchange
of
because
increasing
in which
the releasable
using
‘C-adenosine
‘4C-ADP
adenine
metabolic
published
of ADP
was still
the
to
well as the
practically
there
of the
‘4C-ATP/’4C-ADP
Neither
can
be explained
(up
1 .9: 1 measured
nucleotides
as
pool
remained
Furthermore,
percentage
the
pool,
the
incubation
at least
the major
after
its transfer
releasable
since
ATP
ATP
that
experiment.
time and
of
of EDTA;
adenine
active
of ‘4C-ATP
is high
is true at later
stages
unlikely,
pool remains
constant.
of the released
material
indicating
presence
indicate
that
from
‘4C-ATP
the transfer
the reverse
bolic
ratio
periods
in the releasable
with
time
in the
must
formed
pool
assumed
in which
low initially,
but
ADP
lated
in the
during
the course
of the
between
the incubation
from
the
‘4C-ADP
different
The
amounts
of
in the metabolically
ATP
and ADP
period.
Therefore
sibility
after
to 2.7: 1 (measured
the absence
of
‘4C-ATP/’4C-ADP
unchanged
relationship
97
plateplatelet
is that
human
in response
to
from
rabbit
platelets
is mainly
ATP.525
results
obtained
in the present
study
may also be relevant
to the accumuand/or
maintenance
of adenine
nucleotides
in other
amine
storage
or-
The
lation
ganelles
the
such
phate3”32
or
granules
of
some
species
granules
served
within
sults
as
nucleotides
the
of
granules
‘4C-adenosine,3335
and
adrenal
as fowl36
in addition
to ATP,
human
of
granules
bovine
such
in platelets.29
the granules
with
chromaffin
chromaffin
it
the
has
been
has
been
medulla
take
up
have
large
amounts
indicating
species
lend
some
support
concluded35
with
Labeling
of
32P-phos-
that
chromaffin
ATP
preferentially.
of ADP
in their
However,
chromaffin
differences
Peer et al.35 have
proposed
of some
species
as a possible
platelets
medulla.
demonstrated
adrenal
degradation
explanation.
to this
hypothesis.
similar
to those
ob-
of ATP
to ADP
Our
present
re-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
98
REIMERS
El
AL.
REFERENCES
I.
Holmsen
Collagen-induced
H:
adenosine
diphosphate
incubated
with
2.
Ireland
Effect
Biochem
3.
H,
nucleotide
Subcellular
Day
thrombin
of
human
Biochim
15.
the
pools
with
release
186:254-266,
Da
Prada
storage
of
M,
nucleotides
Sci9:127l-1282,
5.
blood
Synthesis
and
platelets.
Life
Transfer
Mustard
of
adenine
and
blood
iF,
Packham
nucleotides
nonreleasable
platelets.
i
Cell
modifiers.
Bio-
the
adenylate
platelets
induced
Nature
by
(New
Biol)
Baldini
and
M:
Subcellular
dis-
characterization
human
of
platelets.
Blood
bind-
35:727-
1970
18.
Williams
for
FB:
Olivetti
Holmsen
Biol
i (ed):
Statistical
Analysis.
Underwood
H:
and
Platelet
Man-
Programma
Platelet
adenine
platelet
101,
nucleotide
malfunction,
Aggregation.
in
Paris,
Caen
Masson
et
Cie, 1971, p 109
6.
Mustard
Packham
iF,
MA:
washed
7.
Perry
from
Ireland
related
humans.
Br
i
20.
NG,
suspensions
of
283- 296,
DM,
Mills
of
DCB:
Detection
adenosine
diphosphate
plasma.
in
Lips
H:
adenosine
Biochem
blood
443:33-48,
Holmsen
Blood
and
21.
Lages
99:
Studies
on
and
modification
of
plasma.
Anal
10.
Jenkins
the
platelets.
firefly
of
RL,
Mustard
platelets.
A
assay
MA,
iF:
Kinloughof
Randerath
K,
Proc
DUnnschichtvon
Nucleini
Celluloseschichten.
Chro-
Kushmerik
Biochim
Davies
Mi,
nucleotides
and
Biophys
RE:
muscular
Acta
con-
14:735-746,
MA,
ham
platelet
14.
taneous
RL,
Mustard
iF:
Lab
Invest
lysis.
Sheppard
measurement
G,
Chahil
A, Pack-
MUrer
fect
lease
The
‘4C
simulby
liquid
J Lab
Clin
Brunst
RF:
into
adenine
human
platelets.
1975
H, Zucker
platelets
Diath
MB:
from
Methand
blood
Haemorrh
33:648-654,
Buchanan
H-i,
H-i,
in vivo.
142:1222-1225,
Packham
Mustard
and
Mustard
platelets
survival.
1973
MA,
iF:
Kinlough-
Effect
rabbit
platelets
thrombin
on
of
of
MR.
rabbit
Biol Med
RL,
675-689,
1975
CG:
iD,
by
Holmsen
Reimers
metabolism
influencing
and
contain-
K’.
381:144-156,
Acta
of washed
treatment
trations
Conditions
of 51Cr,
hypoxanthine
Reimers
25.
32:352-358,
Marlow
McLaren
of
Thromb
Soc Exp
26.
Kinlough-Rathbone
or
GE,
HJ,
Rathbone
1963
13.
in a medium
separating
24.
1961
DF,
Hypoxanthine
traction.
H:
H:
Isola-
1975
37:395-412,
Trennung
an
6:365-367,
Cain
Struck
Holmsen
platelets:
Mg2’,
nucleotides
Day
for
with
disease.”
1975
Biophys
plasma.
Interactions
Blood
guanine
ods
MC,
human
Ca2’,
Rivard
23.
for
1972
Scrutton
I 1-825,
Biochim
nucleo-
patients
pool
characterization
85:8
evidence
adenine
some
gel-filtered
iF: Survival
chromatographische
12.
Med
and
Determina-
1971
matogr
Further
of
1972
B,
added
22.
platelets:
luciferase
Packham
with
no
Biochim
Hi:
46:489-501,
Rathbone
s#{228}urederivaten
Hi:
pool
from
Incorporation
blood
in
Biochem
CSP,
metabolism
E, Day
ADP
polylysine
11.
Weiss
39:197-209,
and
ing
AMC,
1976
H, Storm
ATP
H,
platelets
and
J
Trieschnigg
and
human
Acta
of
JPM,
Transport
in
Biophys
in
tion
ii,
Holmsen
Holmsen
a deficient.storage
tides
Haematol
1966
Sixma
for
thrombocytopathy-”Storage
substances
9.
Ardlie
of
1972
determination
8.
DW,
Preparation
platelets
22:193-204,
tion
in
sites
metabolism
1975
M,
ing
compartments
67:61-71,
of
1973
of 51Cr
1968
19.
between
charge
mechanism.
in
blood
diphosphate.
Steiner
739,
Isot
1968
Changes
in
tribution
ual
H-i,
releasable
of rabbit
in
A:
1970
Reimers
MA:
the
Pletscher
Radiat
energy
feedback
DCB:
243:220-222,
17.
The
with
charge
J Appl
as a regulatory
7:4030-4034,
adenosine
re-
1969
4.
Interactions
energy
VI.
Int
DW:
pool
chemistry
Adenine
platelets.
Acta
Atkinson
16. Mills
E:
platelet
Biophys
1971
1967
blood
the
counting.
22:125-127,
the adenylate
on
of nucleotide
in
scintillation
vitro.
washed
Storm
of
localization
functions
action.
of
Hi,
metabolism
different
in
1965
J 105:857-867,
Holmsen
of
platelets
17:230-246,
nucleotides
platelets.
blood
phosphate
Invest
DM:
radioactive
from
radioactive
Scand J Lab Clin
release
of
repeated
with
low concentheir
function,
Br
i
Haematol
25:
1973
Kotelba-Witkowska
EH:
B,
Storage
human
of
active
on metabolically
reaction.
Br
Chambers
DA,
i
ATP
Holmsen
H,
platelets:
and
Haematol
on
Efthe
re-
22:429-435,
1972
27.
Salzman
EW,
Neri
LL:
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LABELING
IN
HUMAN
PLATELETS
Characterization
of
human blood platelets.
119:173-178,
nucleotide
lets.
Subcellular
during
the
Holmsen
Mills
Da
man
Prada
and
M,
of
L,
plate-
Naunyn
Bovine
33.
Stevens
P,
RE:
Studies
Stitzel
1970
lease
of
and
platelet
isolated
perfused
species.
Nature
col Exp
Therap
Pharmacol
Robinson
RL,
on
Sub-
synthesized
chromogranins.
‘Arch
adenosine
Blood
medulla:
newly
nucleotides
Schmiedebergs
1972
behaviour
adrenal
of
43-61,
interaction.
for
1147-1158,
Winkler
P.
the
von
273:
Dyke
K,
and
re-
in
the
synthesis
triphosphate-8-3H
cat
adrenal
gland.
18 1:463-471,
A
Lagercrantz
stimulated
study
hormone
of
secretion
Biochem
adrenal
the
and
R:
of
adrenal
gland.
35.
H:
i
Pharma-
1972
me-
in adrenal
19:
1970
315,
SchOpf
iAL,
HOrtnagl
H,
the
Li,
H,
and
medulla
granules.
of
their
Biochem
1975
Snider
Synthesis
K,
bovine
13:40-55,
Winkler
H:
Dyke
drug-induced
perfused
Pharmacology
SR.
Gibb
nucleotides
uptake
Pharmacol
chro-
into
25:311-
1976
36. Hillarp
NA,
catecholamine
H,
in
von
and
storage
Baumgartner
maffin
RL,
Robinson
ATP-8-3H
Peer
iW,
neuro-
P.
Synthesis,
release
ma-
exocytosis
Pharmacol
Stevens
Stitzel
Biochem
nucleotide
34.
of
subcellular
Adv
adenine
from
spleen:
Mechanisms
in
1974
Hedquist
release.
transmitter
A:
platelets.
and
effluent
hypothesis
32.
other
10:310-320,
Stjhrne
and
DB:
Pletscher
blood
Catecholamines
in
and
storage
Psychopharmacol
dulla
H:
distribution
1969
organelles
terial
Hortnagl
cellular
H:
blood
206:438-448,
Thomas
5-hydroxytryptamine
31.
of
bin-platelet
Acta
in
222:991-992,
30.
Biophys
Stormorken
localization
throm
DCB,
nucleotides
H,
metabolism
Biophys
29.
Biochem
catecholamines,
PC,
Adenine
Biochim
of
“Ecto-ATPase”
Arch
1967
28. French
VII.
99
45:328-338,
Thieme
granules.
1959
G:
Acta
Nucleotides
Physiol
in
Scand
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1977 49: 89-99
Labeling of the releasable adenine nucleotides of washed human platelets
HJ Reimers, MA Packham and JF Mustard
Updated information and services can be found at:
http://www.bloodjournal.org/content/49/1/89.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.