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Platelet
By
Tagging
with Tritium
Diisopropylfluorophosphate
EDWARD
F
M.
RICHARD
ADELSON,
A.
ARNOLD
LEAR
AND
ing
which
process
it changes
isopropylphosphate
cule
until
Although
(DIP).3
from
The
that
molecule
breaks
its major
attachment
Cmo
KAUFMAN,
BERDEGUEZ,
J. RHEINGOLD
JACK
OR THE
PAST
10 years,
P32 labeled
used
to tag platelets.”2
This
compound
Labeled
diisopropylfluorophosphate
attaches
to various
has
proteins,
diisopropylfiuorophosphate
DIP
is irreversibly
down
and
has generally
bound
( DFP)
to
protein
mole-
to the
of
side
effects.
fects
are
with
up
overactivity
to 3 milligrams
However,
often
of
when
the parasympathetic
of DFP
in humans
the
dose
The
short
half-life
the
survival
creasing
DFP
of
results
p32
specific
activity
has
atom
one
in a short
toward
the
pound.
Furthermore,
as will
This
whether
be shown
in the in vitro
studies
low specific
activity
has resulted
the
survival
curves
of platelets
increasing
Leeksma
the
From
Medicine,
a serine
with
cholin symptoms
nervous
system.#{176} Usualwill produce
little
or no
reaches
4 milligrams,
the drawback
of phosphorus
side
ef-
shelf-life
end
for
of
the
of a relatively
low
in the molecule.
the
and
Hematology
Washington,
Research
D.
the
Cohen,’
dose
Alfos
Laboratory,
of
DFP
reported
in some
labeled
et
George
compound,
usual
studies.
The
specific
activity
of the tag cannot
the dose of DFP,
because
of the pharmacologic
exponential.
has
seen.4
P32 labeled
diisopropylfluorophosphate
specific
activity
since
there
is only
reducing
of
di-
then
the DIP
is not reutilized.5
been
considered
to be to the
cholinesterase
molecule,
DFP
will
attach
to any
protein
that
group.4
Its major
pharmacologic
action
depends
on its combining
inesterase
and inhibiting
the action
of that
compound,
resulting
associated
ly doses
been
dur-
to
2-week
be increased
by intoxicity
of the com-
gives
diminishing
here.
difference
of
with
DFP32
al.,7
further
10-day
Zucker
et
Washington
results,
opinion
as to
are linear
or
al.,8
and
Unicersity
others
School
of
C.
USPHS
Grant
HE
02274-09,
and bt, the U. S. Army
Medical
Research
&
Command,
under
contracts
DA
49-193
MD-2273
and
DA
49-193
MD-2325.
Presented
in part at the Second
Conference
on Blood
Platelets
at Oak
Ridge
National
Laboratorier,
July 10-11,
1964;
and at the Xth Congress
of the International
Society
of
Hematology
at Stockholm,
Sweden,
August
30-September
4, 1964.
First submitted
November
16, 1964; accepted
for publication
April 18, 1965.
EDWARD
ADELSON,
M.D.:
Associate
Clinical
Professor
of Medicine,
George
Wa.shington
University
Medical
School,
Washington,
D. C. RICHARD
NI. KAUFMAN,
M.D.:
Instructor
in
Medicine,
George
Washington
University
Medical
School,
Washington,
D. C. Cmno BERDEcuEz:
Research
Associate,
Blood
Research
Laboratory,
George
Washington
University
Medical
School,
Washington,
D. C. ARNOLD
A. LEAR,
M.D.:
Assistant
Clinical
Professor
of Medicine,
George
Washington
University
Medical
School,
Washington,
D. C. JACK
J.
RHEINCOLD,
M.D.:
Clinical
Professor
of Medicine,
George
Wa.shington
University
Medical
Supported
by
Development
School,
Washington,
D. C.
744
BLOOD,
VOL.
26,
No.
6
(DECEMBER).
1965
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PLATELET
have
TAGGING
WITH
reported
reported
the
linear
curves
DFP
P32
survival
which
could
LABELED
be
curves.
appear
to
increased,
745
DFP
Mizuno
be
it would
of platelet
survival.
Recently,
tritium
labeled
et
aJ.
exponential.
be
and
If
Hjort
the
possible
et
al.1#{176}
have
specific
to
activity
obtain
more
of
reliable
curves
manufactured
with
This
40 times
is nearly
DFP
a specific
has
become
activity
more
of
commercially
5 to
radioactive
8.6
than
the
available.0
millicuries
best
per
activity
It
is
milligram.
available
with
DFP:2.
The long half-life
of tritium
results
in a further
twofold
increase
in the
radioactive
levels
toward
the end of the survival
studies.
Cline
and Berlin’24
have
reported
the use of tritium-tagged
DFP
to label
red cells.
We have
applied
a similar
and
vival
technic
in vivo
curves
tagging
of dog
to
platelets.
We
shall
report
the
results
of platelets
with
tritium
labeled
DFP
platelets
tagged
in vivo with
DFP-H3.
of
and
in
vitro
present
sur-
METhODS
For
in
in
vivo
tagging
propylene
grams.
of
glycol
The
dog
platelets,
we
intravenously
injection
was
into
given
over
injected
0.1
mongrel
a
to
(logs
period
of
0.2
milligrams
weighing
of
tritiated
DFP
approximately
15
kilo-
2 minutes.
For our in vitro
studies,
the acid
citrate
anticoagulant
by differential
centrifugation.
whole
blood
was
obtained
from
of Aster
and Jandl.’5
Platelet-rich
human
volunteers,
using
plasma
was separated
To
amounts
(lihmte(1
in
propylene
The
cuhated
at
room
For
both
temperature
the
centrifugation.
platelet
for
to
C.
basket
al.17
inside
a
oxygen.
light
was
aimed
heat,
the
platelets
was
then
and
alcohol.
at
out
was
then
moved
arm
was
unclamped
The
fluid
we
0.15
Gm.
POPOP
absolute
placed
in
a
efficiency
to
had
cellophane
the
bath
of
following
had
apparatus
placed
in
onto
to
raise
averaged
23
for
an
Nuclear
and
per
quench
aliquot
counted
for
cent
by
was
Corporation,
in
the
and
5
a
cc.
there
internal
removed
Boston,
of
fluid
scintillation
left
with
sample
into
an
tritiated
the
and
the
side
flask.
133
at
the
8 per
ice
flask
ml.
withdrawn,
counter
in
dry
C. The
0
were
behind
approximately
Massachusetts.
to
flask
The
naphthalene,
the
the
of
flask.
(2,5-diphenyloxazole),
cc.
with
infrared
The
PPO
fluid
each
the
Gm.
of
absorbed
Gm.
standardization
from
of
a mixture
temperature
the
wire
flushed
poured
liquid
was
a platinum
were
15
of
by
water.
of
by
ap-
method
fluid
5.0
Exactly
dried
the
pigment
bottom
tape
of
beam
tritiated
100
dioxane.
made
black
the
scintillation
a
by
scotch
burned
on
The
minutes.
and
of
containing
the
counted
previously
to
a vat
were
then
and
The
converted
vapors
of
been
C.
30
temperature
placed
Ogg
bag.
a
modification
was
4
g for
nails
to
were
al.’6
composition:
ml.
vial,
was
in-
differential
at
and
on
bags
that
the
order
cc.
platelets
platelets
hag
was
2000
hung
the
et
Thomas
in
20
867
DFP
was
by
minutes
casing
were
dry
flask
a
and
water
and
corrected
burning,
#{176}New England
the
8
at
The
[1,4-bis-2-(5-phenylaxazolyl)-henzenej,
polyethylene
was
with
for
cellophane
warm
Oliverio
water
exactly
to
tritium
chamber
ice
tritiated
DFP-H3
separated
g
saline.
platelets
the
marking
the
the
and
correction
vial
ink
of
and
were
350
of
bags
in
and
the
an
alcohol,
mathematical
counting
of
used
ethyl
india
in
set
filter
placed
condensed
to
was
technic,
was
the
This
wet
using
burned,
taken
ink.
the
heavy-walled
flask
plasma
centrifuged
made
india
cellophane
2-liter,
The
various
at
an(l
bags
which
this
platelets
times
to
technic,
Following
pure
Prior
The
work,
separated
three
with
platelet-rich
centrifuged
was
lamp
combustion
et
vivo
was
containing
infrared
37
Schoniger
in
washed
added
of
minutes.
transferred
bags
an
proximately
Each
and
identification
cellophane
exposure
45
blood
was
and
plasn#{236}a we
mixture
plasma
button
marked
Kelly
vitro
whole
microscopy
The
this
for
platelet-rich
phase
and
in
The
supernatant
The
glycol.
0
flask.
cent
C.
The
quench.
water.
hemoglobin
con-
A
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746
ADELSON
ET
AL.
Id
0.
IdOl
I.-,-
Q.
(o
0
U
MICROCURIES
Fig.
tent
1.-A
was
sample
determined
was
counts
for
semilog
plot
by
prepared
per
per
hemoglobin
PER
of in vitro
the
and
minute
OF DFP-H3
tagging
method
of
burned.
milligram
contamination
in
of dog
Crosby
The
of
OF PLATELET-RICH
ml
and
of
and
platelet
sample.
each
platelets
with
Furth.’8
radioactivity
hemoglobin
PLASMA
DFP.
Simultaneously.
the
an
tritiated
red
cells
appropriate
a red
was
cell
expressed
correction
in
was
made
plasma.
a plateau
was
RESULTS
Figure
increasing
1 shows
amount
the results
of adding
of DFP
was attached
reached
at about
the curve
leading
on
semilog
not
300
up
plot,
a simple
counts
to this
suggesting
per minute
plateau
did
that
exponential
DFP-H3
to the
the
per
not
of DFP
There
may
An
platelets.
That
portion
to form
a straight
108
appear
absorption
relationship.
to platelet-rich
platelets
until
onto
be
the
several
of
line
platelet
was
mech-
different
anisms
whereby
DFP
absorbs
onto the platelet.
For the in vivo work,
a dose of DFP-H3
that would
yield
approximately
30
counts
per minute
per 108 platelets
was selected
to keep
the amount
of DFP
on the platelet
at a level
equivalent
to the lower
part
of the in vitro
curve.
Each
determination
was
ly described.
A correction
corrected
could
but there
was
less than
counts
per minute
per
each
dog,
and all other
one white
cell per thousand
platelets.
The
highest
platelet
was
arbitrarily
considered
100 per
cent
in
counts
were
expressed
in terms
of the 100 per cent
figure
for
From
the
the
all
ideal
curve
corresponding
the
using
for
observations
platelet
a digital
survival
computer.
of
by
Figure
Since
this
graph
assumes
for lack of fit to determine
test was 0.955.
The
95
showed
that
assumed
to be
We
least
then
squares
lack
of
fit
was
an
the
least
contamination
white
cell
per
per
of
exponential
the validity
per cent
level
not
cent
squares
2 shows
on semilog
scale
together
with
the mean
error
for each
mean
is shown.
The half-life
test
this
for
as previous
contamination,
-
dog.
individual
curve
for hemoglobin
not be made
survival,
fitting
this
regression
derived
exponential
curve
plotted
cent
survivals.
The
standard
the survival
curve
is 2.4 days.
relationship,
when
the
carried
we
of this assumption.
of F for our data
significant
we
to an
The
was
regression
out
F ratio
a
2.28.
of
This
curve
was
exponential.
attempted
using
to fit our
the
digital
data
computer.
to a linear
This
regression
time
the
by
lack
the
of
method
fit had
of
an
F
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PLATELET
TAGGING
WITh
P32
LABELED
747
DFP
±2.1
-J
>
>
.6
CE
C/)
I-
w
-J
Id
I0±
.6
-J
0
I-
z
Id
U
CE
0±
Id
0.
I .7
0±42
3
4
TIME
Fig.
2.-A
semilog
of all the observations
DFP-H3
platelet
tag.
each time
that mean.
ratio
lack
interval.
the
5
6
The
number
next
to each
95 per cent
linear
model
level
was
7
8
DAYS
plot of the regression
curve
obtained
of per cent platelet
survival
in six
The open
circles
are the mean
per
of 2.36.
Since
the
of fit exists,
and the
ly explain
IN
open
by a least squares
fitting
normal
dogs
with
in vivo
cent platelet
survivals
for
circle
of F was
discarded
is the
again
since
standard
error
for
2.28,
a significant
it cannot
adequate-
data.
DIscussIoN
With
tritium
pharmacologic
ium
we
This
radioactivity.
have
used
dose
in experiments
of
labeled
DFP,
effect
of the
0.6
DFP
with
the limiting
factor
in dosage
compound,
but rather
permissible
In preliminary
milligrams
is to
be
DFP32.48
of
studies
DFP-H3
compared
on humans
containing
with
doses
no
with
malignant
5 millicuries
of up
to
longer
levels
of
4 milligrams
is the
of tntdiseases,
tritium.
used
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
748
ADELSON
Figure
platelets
until
1 shows
in vitro.
a plateau
the manner
in which
increasing
amounts
Platelet
radioactivity
rises
at a gradually
is reached
at a level
Since
the rising
protein
log plot,
the absorption
function.
Any portion
more
molecules
are
et al.2#{176}
have
pointed
turnover
than
the
of the
of DFP
of 300
graph
onto
counts
with
a serine
group
involved
than
simply
out that some
of these
platelet
itseff,
per
does
not
the platelet
and
ET
of DFP
decreasing
minute
per
10
form
a straight
is not a simple
AL.
label
rate
platelets.
line of semiexponential
will be labeled,4’12
and
therefore
the platelet
cholinesterase.
Hjort
proteins
might
have
a more
rapid
this
could
result
in
some
elution
of the
tag.
While
vival
elution
does
occur
curves,20’2122
Although
platelets,
we
the
of the
alternate
which
chose
for
maximum
our
has
be
In
of the
advantages
smaller
doses
tag
spite
The
which
creased
radioactivity,
more
levels
reliable
These
could
studies
of
of DFP32
the
concentration
shown
mind.
a dose
of this
days
red
to occur
To lessen
of DFP-H3
the
gives
over
DFP2
is that
less
long
half-life
of the
is another
advantage.
which
the
danger
counting
of
of
one-
nature
elution.
An
by aging,
tritium
of elution
label
and
tritium
compound
A third
advantage
improves
only
exponential
figure
2 may
still be due
to
life span
is not determined
but by random
destruction.
therefore
surDFP.
in the case
the chances
that
precaution,
cell
of
per-
of phar-
results
in a
is the in-
statistics
and
gives
curves.
higher
levels
of radioactivity
not be carried
can be carried
out
transfusion
let cohorts
experiments
tagged
with
ilar
used
to that
the
and
few
lowering
not been
borne
in
of DFP-H3
of DFP,
first
by
in vivo
label.
macologic
toxicity.
longer
sheff-life,
on
the
lessened
curve
of platelet
survival
in
explanation
is that
platelet
would
result
in linear
curves,
One
mits
during
is
elution
of DFP
tag
this possibility
should
elution,
tenth
it
viability
by
will
also
with
the lower
out using
platelets
permit
specific
labeled
many
experiments
can be carried
out with
DFP-tagged
DFP
can be prepared
and studied
Cline
and.
Berlin
of DFP-tagged
for
platelets
red
can
cells;’
be
that
activity
of DFP2.
Survival
in vitro
with
DFP;
cross
and
platelets;
in a manner
the
effect
vitro
or
platesim-
of
storage
studied.
SUMMARY
A method
is described
tritium
labeled
a modification
sultant
tritiated
for
tagging
diisopropylfiuorophosphate,
of the
Schoniger
water
by
liquid
platelets
combustion
scintillation
as indicated
curve
of in
ponential
a half-life
struction
The
with
of platelets
DFP-H3
tag
doses
of DFP
this technic.
by
vivo
of 2.4
or by elution
of the
has several
advantages
or higher
levels
in
tagged
and
vivo
with
platelets
by
re-
the
counting
by the platelet
suggests
up the DFP.
However,
a plateau
tagged
days.
in
the
counting.
The curve
of in vitro
uptake
of DFP-H3
than one protein
in or on the platelet
takes
point
is reached,
The
survival
either
burning
technic
in the
platelets
This
can
uptake
curve.
in five normal
be
explained
that more
a saturation
dogs
by
is
ex-
random
de-
tag.
of radioactivity
over
the
DFP:2
or both
may
tag.
be
Either
achieved
smaller
with
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
PLATELET
TAGGING
WITH
P2
LABELED
SUMMARIO
Es
describite
con
methodo
tin
IN
pro
un
del
resultante
Le
per
modification
del
aqua
curva
del
le plachettas
participa
in
Le
es
esser
elution
del
uso
a phosphoro
alte tal
phato.
nivellos
un
de
que
Ic occurrentia
de
marcate
de
de
Istos
medie
un
certe
attingite
per
de
liquido.
a tritium
le plachetta
un
puncto
de
acceptation.
de
in
vivo
in
de
2.4
tritium
certe
‘ivo
e le examine
aleatori
saturation
cinque
normal
dies.
de
Isto
pote
plachettas
como
o de
marca
ha
de
de
diisopropylfiuorophosphato
diisopropylfluorophosphato
de
radioactivitate
nivello
un
o in
plachettas
Schoniger,
valor
destruction
a
de
esser
vitro
marcate
scintillatori
in le curva
pro le mesme
objectivo,
es que
plus micre
doses
le attingimento
pote
de
contation
plachettas
tempore
un
in
del
diisopropylfiuorophosphato
un proteina
in o super
plateau
de
con
le uso,
32.
pro
de
diisopropylfluorophosphato
super
suffice
combustion
plachettas
arsura
le attingimento
de
longevitate
de
avantages
de
vitro
plus
que
le
medio
Tamen,
exponential,
explicate
per
del marca.
Le
in
in le presentia
curva
canes
acceptation
suggere
le processo.
es reflectite
per
de
tritium,
technica
a tritium
INTERLINGUA
le marcage
a
diisopropylfluorophosphato
per
749
DFP
plure
o que
plus
de diisopropylfiuorophos-
dose
ACKNOWLEDGMENTS
The
ington
authors
wish
University
to
express
their
Computer
thanks
Center
to
who
NIr.
assisted
Thomas
us
Gerig
with
the
of
the
George
statistical
Wash-
analysis
of
the
data.
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1965 26: 744-750
Platelet Tagging with Tritium Labeled Diisopropylfluorophosphate
EDWARD ADELSON, RICHARD M. KAUFMAN, CIRO BERDEGUEZ, ARNOLD A. LEAR and JACK J.
RHEINGOLD
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