From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
Induction
Rabbits
iii
Human
By
T
HE
obtained
thromboplastin
factors.
The
related
to
the hypothesis
thromboplastin
be,
I)rel)are
is
active
Preparations.
was
capable
certain
between
the
a
that
VII
blood
showed
tissue
tissue
works
equally
co-factors.5’#{176} It was
the
purpose
of the
and
tissue
thromboplastin
results
appear
All
biologic
was
prepared!
tilis
brain
for
use
andl
to
reagents
and!
in
1)100(1
adlde(1
in
50
10
i)e
sedlinlented
Tile
in
tile
and
saline
was
eadl,
washed
thromi)oplastin
were
and
the
was
previously
at
20,000
generation
test
was
able
brain
which
active
also
dlifference
factor
prothrombin
con-
the
absence
to explore
the
immunologic
of these
relation-
methods.
statedl.
crude
and
The
as
dlescribedi
imidazol
as
sedlinlent
silown,”
leaving
by
reported
was
serum
Pool
aIld
and
Robinson5
a supernatant
in
the
+
a
final
clotting
metilod
total
reagents
volume
could
activity
WitilOut
to
The
Ca
thromboplastic
accordling
from
reagents
elsewhere.
suspended
1)100(1
performed
and
i)uffer.
cephalin,
serum,
thromboplastin
extracted
was
Plasma
Aiton.
with
plasilla,
Brain
cephalin
7.2
resulting
rpm,
in
phosphatide
from
As
well
madle
to p11
with
dierived
product
were
requires
optimal
otherwise
and
Bell
of
brought
end
important
for
study
unless
Quick,1
method
sedimented
was
saline.
isotonic
by
associates2
the
MATERIALS
ongm,
generation
isotonic
1111. voluilles
1111. of
human
described!
to
prepared
of
AND
tilromboplastin
thromboplastin
thromboplastin
METHODS
is
such
thromboplastic
An
.
chain
any
Wilkinson3
from
by
dissimilarity.
and!
he
thromboplastinase,
activity,
was
present
antigenic
of
as
according
dliiutedi
Blood
were
stored
nlateriai
were
illdicate
of
thromboplastin
co-factors
as
of tissues
nature
Biggs
that
bacterial
thromboplastic
reviewed
formation
might
intermediates
factor
vell
of complex
the
and
blood
clotting
activity
conjecture.
)
similar
that
plasma
been
a series
what
( AHF
is that
view
thromhoplastill
Nour-Eldin
ship
blood
has
from
of
factor
thromboplastin
between
and
blood
and
thromboplastin
Stuart
platelets
throml)o1)lastic
results
of
and
thromboplastins
and
to the
transition
that
activity
way
thromboplastin
formation,
blood!
of
this
matter
tissue
antihemophihc
two
given
interaction
of
CINTRON
thromboplastic
has
thrombopbastin,
Gollub
et al.4
of destroying
against
version;
tile
Preselitly
Against
H.
JOSE
contain
accepted
tissue
may
factor
from
AND
(lerived
from
blood
the
end
Prodilct
suggested
of blood
v,
cells
extracts
development
relation
to
blood
tissue
generally
that
\\Thether
chemically
SPAE’r
that
historical
It is now
is preforme(l;
reactions.
H.
Activity
Thromboplastin
from
is formed
Quick.1
by
Blood
ThEODORE
CONCEPT
to that
of
activity.
in
by
use
laboratory.”
tiliS
animals
Experimental
given
originally
weekly
were
New
intraperitoneal
4
animals
injections
were
so
injected1,
Zealand
of
but
rabbits
2 ml.
of
following
of
about
iluman
the
3
in weight.
thromboplastin
second
injection
From
the Department
of Hematology,
Laboratory
Dicision,
York, N. 1’.
This study was supported
by Grant
#H2442
(C) of the National
Institutes
of Health,
U.S.P.H.S.
772
Kg.
blood
Monteflorc
heart
These
were
suspension.
1 dljedi.
Hospital.
institute,
Of
the
New
National
From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
ACTIVITY
A(;AINsT
HUMAN
rcniaining
3, only
2 dleVeiope(l
dlerive(i
are
fronl
after
llOte(l
case
four
sera
into
an
to
Vohinle
of
factors”
were
equal
to
and
5 to
injection.
given
an
aii(l
(k’scrii)cdl
then
to
of
adsOrl)edl
flow
incubation.
aluminum
with
as
prepared
each
obtained
allowed
hour
first
In
were
was
a(lditional
was
be
was
sera
blood
similarly
to
7 injections.
Test
The
ear.
Serum
by
that
iflctii)ated!
for
was
one
tile
also
oi)taine(l
citrate
Ilour
dialysedl
equal
to
C.,
and
37
at
for
24
hoiir.
as
the
l)y
isotonic
that
an(i
starting
sulfate
isotonic
\‘\Tart’
saiiile
of tile
barium
a final
clotting
(lescribed
one-fifth
against
in
“Serum
with
tinles
plasm:i,
clissolvedi
pl1Snla.
sulfate,
tilree
adsorbed!
was
starting
bariilfll
waShed!
sOdliunl
from
precipitate
of
with
then
cent
sul)ernatant
of
PreciPitatioli
The
adsorption
was
10 per
was
by
Conley.11’
one-fiftil
sulfate
of
The
and
serum
bariuill
suspension
Tlw
C.
after
last
tile
concentrated!
Ratnoff
from
The
centrifugation.
37
factors.”
were
of
in a vOiunle
serum.
tile
of
data
tilronlboplastin
rabbits.
obtained
resuspended
after
centrifugation
‘sertini
saline
1
at
1)100(1
appeared
artery
appropriate
method
buffered
1 week
central
the’
mdi
against
activity
clotted
factors”
the
Stragneil.1
testing
tile
clotting
to
activity
nlaxinlal
tile
off after
remove
aIltithrolili)oplastili,
i)efinite
tube,
control
“Plasma
according
ilv
glass
untreated
011(1
for
from
drawn
was
hydroxidie9
froul
obtainedi
collected
uncoate(l
Serum
significant
but
773
TIIROMBOPLASTIN
aninlals.
injections,
were
blooti
from
tileSe
BLOOD
removed
saline.
RESULTS
Following
repeated
Pldlstmn,
rabbits
untreated
i-\%ro
sera
of the
sera
WileIl
tion
test.
into
each
case,
the
plasma,
It is evi(lent
that
effect
their
and
serum
reagellts,
volumes
from
the
the
intervals
considerably
it
the
two
certain
how
milCh
)layed
ill
the
with
brain
and
serum
of a role
were
As
representing
sera
were
( Cutter
)
.
and
tested
in figure
activity
than
the
naturally
tested
were
the
the control
normally
in
of
occurring
rabbits
12
against
above
and
seconds.
Equal
reagent,
and
the
plasma
experimental
sera
were
activity.
sera
presented
i)e anticipated’2;
some
antiit is not
tllromboplastin
seen
investigated
the
tile
substrate
by
human
control,
inactivator
in figure
of
sera
thromboplastin
against
scra had
the
2.
incubation
brain
for clotting
activity
3, tile experimental
that
evidently
reagents.
(lescribed
activity
of
and
antithromboplastic
occurring
volumes
inhibitory,
recaicified
that
mixture
calcium.
\Vhen
rabbit
plasma
in place
of human
thromboplastin
in their
was
the
injected
plasma
against
naturally
initial
inhibitory:
as
the
genera-
the
human
from
of thromboplastic
Equal
similarly
Mixtures
sera
the
control
activity
mixed
shown
antithromboplastic
the
this
loss
thrombopbastin.
plasma.
with
tested!
experiments
which
would
progressive
not
found
in
experimental
before
markedly
controls.
mixture
1)reI)tredl
it is evident
than
were
substrate
was
Again
potent
previous
activity,
the
in
1)ut
against
thrombo-
thromboplastin
added
were
of
incubated
thromboplastin
Anti-tissue
rabbits
blood
reagent
not
effect
of the
tile
was
specific
effect
hufliall
reagents,
rabbits
was
mixture
shown.
more
of
serum
recalcified
were
of the
mixture
tllronlboplastill
clotted
sera
tilrombol)lastic
in
the
l)lOod
tilis
the
sera of the
the
generating
injected
activity
that
of test
The
tllrombin
used
of
against
1 shows
cephalin
than
activity
1)0th
of test
and
injected
clotting
)rohahly
ml.
from
iluman
50
iluman
0.1
2 demonstrates
(hiute(l
III
generating
greater
:edimented
at
the
sera
anticoagulant
Figure
activity
Figure
serum
the
injections
was strikingly
reagents
were
sera
ill(luce(!
developed!
controls.
added
In
following
intraperitoneal
recaicified
no greater
inhibition
present
inhibitor.11
for antithromhin
evaluated
for
clotting
activity
against
activity
with
human
plasma
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774
ANI)
SPAET
( :IN’i
RON
70
34
60
32
-
50
28
26
Q)
!
24
#{176}
:
22
c,
20
I-_
K
c::1
20
d
p
0
-=------o
C0-
8
5
0
INCUBATION
TIME
(minutes)
INCUBATION
0
TIME
(minutes)
32
30
28
26
24
22
20
0
K
l6
TIME
INCUBATION
Fig. 1 (top,
left).-Inhibition
shaded
area
shows the effect
1111(1 B: Sera
fF0111 experimental
with
human
I)a
and
Fig.
TGT
of thromboplastin
of
5
sera
with
a and
from
in
incubated
control
the
1)otent
Sera
absence
of
antithrolnbin
were
tested!
factors”
concentrates.
as
follows
The
of
mixture
from
human
from
human
reagents;
C:
experimental
Both
brain
control
with
rabbit
thromboplastin
rai)bits
by
rabbits
The
A
Saline
rabbits
thromboplastin
experlllleiltal
sera.
reagents.
(A
aild
by
(A
rabbit
Th
and!
sera.
;
serum
( D).
control
activity,
and
for activity
: Equal
TGT
human
rabbit
by
on
TCT
Sera
saline
caiciumil.
animals
reagents.
of sedimented
human
blood
incubated
with
: Sera
from experimental
( C ) ; saline control
(D )
with:
(C );
rabbit
generation
control
b : Sera
cephalin
rabbit
sera. Thromboplastin
B ) ; serum
fronl
control
rabbit
Fig. 3 (at bottom).
Inactivation
Thromboplastin
from
rabbits
reagents;
serum,
but
human
2 (top,
right).-Inactivation
(minutes)
experiniental
nld
no (lifferences
against
human
volumes
of
test
was
incubated!
sera
at
could!
he
“plasma
were
37
mixed!
C.
sera
control
for
had!
detected.
factors”
with
15
or
the
minutes,
ilighh’
“serum
respective
diluted
From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
AGAINST
ACTIVITY
with
9 volumes
the
thromboplastin
were
unaffected
HUMAN
of
BLOOD
buffered
saline,
generation
by incubation
the
showed
experimental
sera
When
mixture
experimental
containing
hibition
was
against
tile
detected.
Commercial
human
blood
were
plasma
factors”
as
and
inhibitor
)
by
reagent
“serum
lack
X. Figure
compared
of activity
5 demonstrates
to the controls,
insignificant
rabbit
inhibitory
generation
serum
the
in
factors”
a thromboplastin
that
factor
( Ortho
to
the
indicating
possibly
and
likely
or
appropriate
that
sera,
added
reagent
AHF
to concentrate
of the
a slight,
Coombs’
serum
thromboplastin.
Attempts
any
It is therefore
“plasma
used
4 shows
Stuart
factor
or factor
“plasma
factors.”
As
sera
human
then
Figure
with
PTC,
on tile
effect.
and
test.
by the inhibitor
against
the effect
of the sera
775
THROMBOPLASTIN
reagent,
inhibitor
was
no
not
in-
active
V.
had
no
demonstrable
activity
ammonium
sulfate
fractionation
against
were
unsuccessful.
DISCUSSION
Rabbits
activity
of
given
against
untreated
active
only
repeated
this
injections
reagent
control
significantly
animals.
against
of human
human
This
greater
induced
blood
blood
thromboplastin
than
inhibitory
thromboplastin;
that
found
activity
it had
developed
little
in
the
appeared
or
no
sera
to be
effect
on
46
40
30
28
1J
20
15
L
c%)
2
INCUBATION
Fig.
4.-Activity
following
incubation
control
rabbit
(C)
of human
;
with
saline
“serum
: Sera
control
6
4
TIME
factors”
from
(D).
experimental
(minutes)
in the
thromboplastin
(A and
rabbits
generation
B) ; serum
test
from
From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
776
SPAEF
AND
CINTRON
25
c::120
B
15
..
0..
Io
.
4
2
-
INCUBATION
Fig.
5.-Activity
of human
following
incubation
control
rabbit
(C)
human
blood
rabbit
blood
recent
findings
in
sera
of
piastin.
cross
reaction
demonstrated,
prepared
The
different
as
by
of
by
ether
and
appears
the
present
activity’
typical
activity
appear
between
in their
blood
clotting
accordingly
suggested
or clearance
form
that
the two
phosphatide
protein
moiety.
);
test
serum
from
and
in
tissue
tissue
or
the
an!
and
of
of
tissue
between
have
thromboplastin
et
is
but
by
suggest
antibody,
injec-
the
The
latter
inselective
would
been
reagents.
thus
Differ-
previously
noted
co-factors.5’6
not
is rather
are
appears
al.
an
“immunizing”
pbasma
does
quite
on
properties.
thromboplastin
these
two
for
\Vilkin-
based
Shirakura
course
little
have
obthromboand
is
demonstration
specific
absorption.
requirement
thromboplastin,
thromboand
fixation
was
absorbed
by tile
rabbits
and
forms
of thromboplastin
composition,16
each
blood
of different
injected
the
activity
injected,
of Nour-Eldin
those
the
with
Japanese
investigators
use of a human
blood
a product
or
inhibitory
complement
specifically
thromboplastin
their
agreement
preparation
during
tissue
and
of blood
Although
similar
study
in
reagent
method
against
blood
dissimilarity
and
the
with
to yield
specificity,
fixation
activity
of
thromboplastin
development
of the inhibitors
to reHect
antigenic
ences
that
here
induced
tions,
species
and
reagent
vestigators
of complement
generation
B
thromboplastin
reported
the
inhibition
could
be
precipitation
of
are
who
against
During
activity
tissue
data
injections
mainly
method
inhibitory
shown
active
the
present
findings
the
present
associates,’4
It is of interest
to those
reported
principles
The
that
reagent.
similar
thromboplastin
( A and
rabbits
human
intramuscular
were
was
evident.
and
inhibitory
appropriate
tamed
data
son.15
The
and
given
sera
in the
experimental
precursors,
of Shirakura
(minutes)
(D).
thromboplastin.
rabbits
factors”
from
thromboplastin
The
plastin
;
“plasma
with : Sera
saline control
6
TIME
represent
a true
probably
to have
tissue
lipoproteins
its own
It is
a storage
product.
with
unique
SUMMARY
Rabbits
two
animals
were
injected
developed
with
sedimented
serum
activity
human
against
blood!
human
thromboplastin,
blood
thromboplastin
and
From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
;CTIVITY
ill
AGAINST
significant
HU MAN
excess
activity
was
against
ha(I
and!
TIIBOMBOPLASTI
naturally
over
specific
irecursors
BLOOD
no activity
against
tissue
Conilios
esseva
tractate
human.
thromboplastina
rentia
I lb
de
(id
de
pro
be reagente
activitate
dhsveloppava
de
nuile
sedimentate
iste
illo
non
activitate
thromboplastin
thromboplastina
un
activitate
significativemente
inhibitori.
human.
antithromboplastic
affect
thromboplastin.
dliSveIO)pava
human
to
IN’rERLINcuA
injectiolles
allilnaleS
sanguine
natural
specific
con
Duo
iN
This
failed
reagent,
SuwuARIo
sanguine
inhibitor.
occurring
human
777
N
in
activitate
afficeva
contra
de
seral
contra
del
excesso
antithromboplastic
precursores
Ic
occuresseva
de thrombopbastina.
tilromhol)lastma
tissutab.
REFERENCES
1.
Quick,
J.:
A.
Hemorrhagic
Philadelphia,
Biggs,
2
Lea
R.,
lane,
&
i)ouglas,
R.
plastic
1953.
:3. Nour-Eldin,
Diseases.
Febiger.
A.
S.,
and
action
G. : The
substances.
of
J.Physiol.
9.
Macfar-
F.,
and
ical
J.
F.:
in
of
and
1’o
din-
H.,
lndl
:
Properties.
101:799,
J.:
air1
Proc.Soc.Exper.Biol.&
Med.
\V.
Press,
1957.
and
Alton,
N.,
is
Pool,
j. C.,
plasma
( AHG
H.
G.
substitute
for
in the
thromboplastin
test.
Nature
ail(l
Robinson,
174:880,
Brit.J.Haenlat.
14.
placc’ntal
R.:
Johns
An
prothrombin
1952.
Garner,
E.
S.:
in
machunlan
34:1807,
The
toxin
ins-
method.
22:791,
1955.
active
principle
Thromboplastin;
:
in the
inactivator
blood:
Antithrom-
149:l23,
Anl.J.Physiol.
Slmirakura,
‘I’.,
amid1
gen-
1954.
globulin
5:17,
1959.
16.
22:232,
of
F.,
jol.
1:32:164,
and
of
its
human
and
with
properties.
F.:
bovine
ether
J.Phys-
1956.
Spaet,
pllosphatidles:
J.Clin.Invest.
Fine-
J.
\Vilkinson,
of
A. J., and
idientification,
tiSsue
Acta
thromboplastin
t study
kt
between
sero-
1959.
SeI):ratiOfl
and1
T.,
on
tilronlbop!astin.
Nour-Eldin,
\larcus,
Sakai,
Studies
(hiferentiation
plasma
platelet
T.,
T. :
I)lasnla
issatol.
15.
Hasegawa,
Nlaekawa,
logical
dud1
brain
: A
J. : Assay
antiliaenioplulic
).
1951.
L.:
of
Bull.
tilronlboplastin
C.
the
fraction
89:245,
J.Clin.Invest.
Schneider,
The
in
Stragnell,
and
of
1ll(’
it
suspensions
eration
H.,
L.:
1947.
R.
and
Alexander,
B.:
studies
on
Proconvertill
dchdiellcy
and the role of proconvertin.
In Brinkhous,
K. M., Ed. :
pililia
and
Hemophiloid
Dis’ases.
Chapel
Hill,
University
of
North
(‘xtract
stage
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From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
1960 15: 772-777
Induction in Rabbits of Activity Against Human Blood Thromboplastin
THEODORE H. SPAET and JOSE R. CINTRON
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