From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
The
Clot
Plasma.
L. Is/I.
By
Accelerating
Relation
of Normal
M.D.,
TOCANTINS,
{IOXTRARY
Effect
of Dilution
to the Mechanism
and
Hemophilic
TO
R.
T.
fluids.’
hibiting
Stable
agents,
surfaces
like
plasma
which
glass.2
H.
blood,
0.85 per
these
Among
inhibitors
may
dence
philic
bloodh
may,
from
is only
or plasma
by
be the
plasma
the
if adequately
t.he employment
processing
that
appreciation
disrupt
the
the
native
and
cent XaCl
lipid
coagucitrated
or other
diluted,
clot
of recently
it has
been
as normal
of t.he degree
of fluidity
equilibrium
that maintains
these
of blood
and how
the blood
fluid.
blood
sufficiently
diluted
normal
euglobulin.
technics
to observe
seems
from
eviagents,
hemo-
as rapidly
developed
possible
of inwith
antithnomboplastin
fractions.3
It
aid of activating
or plasma.
When
hemophilic
euglobulin
is prepared
from
plasma
it is as active
a clot accelerator
as a similarly
prepared
It
A.B.
HOLBURN,
seems
to owe its fluidity
ex vivo
to the presence
lose much
of their
activity
on dilution
or contact
blood,
plasma
and
that
with or without
and
and
IMPRESSION,
or hemophilic
diluted
with
brain,
to be presented
separated
R.
AXD
GENERAL
lation
of properly
collected
normal
is accelerated
when
moderately
‘a-.,,
plasma
B.S.
CARROLL,
A PREVAILING
on Blood
of Coagulation
Blood
of blood
facts,
and
even
slight,
collection
t.o have
a full
dilution
can
METHODS
Unless
otherw-ise
10 subjects
hsemisol)lsilics
w-it.h
from
silicotie
its
the
collected
cold
asl)irated
from
blood
of no
hour,
and
15
than
05
upper
Native
a 16 gauge
for
less
the
at.
of
ml
with
directly
3,000
was
10 nil.
per
into
19 per
a tense.
r.p.m.
second.
It
differ
in
were
cent
trisodium
The
blood
veins;
was
the
by
the
upper
accoumit:
(1)
carefully
and
pipets
allowing
sihicotie
was
it was
centrifuged
plasma
graduated
obtailsed
and
not
collected
turgid
of
chilled
from
of t.he
who
had
did
was
of i)lood).
siliconized
was
and
Centrifugations
three-fourths
plasma
nseedle
mimsutes
to
puncture
measured
collection.
through
calculation
any
volume
or all
of
the
atiticoagulant
of
of the
sample
volumes
From
the
Philadelphia,
This
study
the
National
Submitted
the
final
following
of
added
respective
of
a rat.e
response
blood
tubes.
used
solution
a quick
droppers,
coated
men
one-half
of those
blood
its
The
generally
citrate
Over
l)lOOd
coated
to
tubes;
three-fourths
the
of
the
removed.
Ins the
ture,
after
diluted,
silicone
anticoagulant
needles,
was
hemophihics.
in
normal
of severity.
\Vhsets
the
years.
other
ed from
collect
grades
past.
tested
of the
one
blood
several
the
ml.
after
centrifuged
of
The
coated
hour
vein
for
arid
at
for
on
various
immediate
blood
bubblirsg,
an
the
u-as
plasma
the
(0.2
silicone
within
done
of
not
stored
r.p.m.
w-ith
tested
of
18 gauge
without
removed
h)ut
(4 t.o 7 C.).
at 3,000
u-as
transfusiomis
in a syringe
through
usually
had
syringes,
placed
work
hemophilia
recent
that.
coated
citrate
flow-
tiot
its the
essentially
all
hereditary
had
transsfusiotss
clonic
stated,
to
of
Division
percentage
blood
the
each
plasma
variables
uere
(calculated
blood;
of its
(3)
the
into
from
the
t.otal
volume
ingredients,
of Hematology,
concentration
taken
namely,
Department.
in
hematocrit);
of
a given
the
(2)
the
plasma,
of Medicine,
clotting
the
total
mixplasma
volume
mixture
recalcifying
Jefferson
clotting
percentage
solution,
Medical
aided
by
Institutes
October
a grant
of
5,
1950;
from
the
Division
of Research
Grants
and
Health.
accel)ted
for
publication
720
December
26,
1950.
the
acti-
College,
Pa.
was
of
and
Fellowships
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
M.
TOCANTINS,
R.
T.
CARROLL
AND
R.
H.
721
HOLBURN
vating
agent and diluting
fluid. Example:
9.0 ml. of normal
blood was aspirated
into a
syringe
containing
1 ml. of 0.1 M. sodium
oxalate.
The packed cell volume
(hematocrit)
of
this mixture
was 30 per cent. This meant that the 7.0 ml. of plasma in the sample u-as diluted
by 1.0 ml. of the anticoagulant
solution
(or a dilution
of 6:7). In preparing
the initial
clotting mixture,
0.5 ml. of t.he plasma
was added to 0.05 ml. of thromboplastin
followed
at
once by 0.05 ml. of 0.2 M. CaCl2
thus
involving
a further
dilution
of the
plasma
of 5:6;
thus
X
X
100 = 71 or a final plasma
concentration
in the clotting
mixture
of 71 per
cent.
By usimig 19 or 38 per cent citrate
solutions
as anticoagulants
and
inscreasing
the
volume
of plasma
in the clotting
mixture,
it is possible
to work
with
mixtures
of higher
plasma
concentrations.
Example:
10 ml. of blood
is aspirated
into a syringe
containing
0.1 ml. of 38 per cent trisodium
citrate.
Let us say that the packed
cell volume
of the blood
is 40. This means that the 6.0 ml. of plasma
is diluted
59:60
by the anticoagulant.
In preparing the clotting
mixture,
1.0 ml. of plasma
is added to 0.1 ml. of thromboplastin,
followed at once by 0.05 ml. of 0.4 M. CaCI2. The plasma in the clotting
mixture
is therefore
diluted
100:115.
Thus J X +1! X 100 = 85, or a concentration
of the plasma
ins the initial
mixture
of 85 per cent.
The optimal
amount
of calcium
required
to recalcify
citrated
or oxalated
plasma
was
determined
for each sample
of undiluted
plasma.
On table
1 is an example
of how the inTABLE
of
1.-Adjustment
Concentrations
of
the
Plasma
Concentration,
Ingredients
while
Total
Volume
Tube Number
a Clotting
Mixture
Constant
and
Optimal
1
.
(ml.)
Plasma
of
Maintaining
to
the
Obtain
Varying
Thromboplastin
Calcium
Content
2
3
s
4
0.5
0.35
0.25
0.055
Thromboplastin
(ml.)
0.85%
NaCl (ml.)
0.1
0.1
0.1
0.1
0.1
CaCl2
Total
0.05*
0.44
0.055t
0.65
8.3
0.53
0.Olt
0.65
1.5
of plasma
concentra-
0
(ml.)
Volume
Plasma
(ml.)
Concentration
Blood
Dilutions
40.20
by
ml. blood + 0.2 ml. 38 per
ansticoagulamit
59:60.
* Molar
concentration
0.2
t Molar
concentration
0.02
gredients
tion,
may
while
tration.
in the
solution
be adj usted
using
Unless
proportions
above
cent
amounts
of
tube
CaCl2,
the
sample
mentioned,
recalcification
to obtain
of
was
required
(e.g.
decreasing
constant
mixture
between
citrate.
unusually
1, table
the
actual
amount
of plasma
amid
high
one-tenth
tube
degrees
volume
of
thromboplastin
or
t.heir
low,
concen-
plasnias
volume
1). As the
amounts
of CaCl2 u-ere adjusted
to the actual
quantity
using as standard
the amount
of CaCl2 originally
shown
time in the undiluted
plasma.
A constant
optimal
ratio
clotting
added.
trisodium
M.
hematocrit
for optimal
0.275
0.025*
0.65
37.8
M.
in each
optimal
the
0.65
75.6
(percent)....
hematocrit:
of plasma
0.165
0.035*
0.65
53.0
0.01
of
0.2
M.
CaCl2
diluted,
the
of plasma in the clotting
mixture,
to have given the optimal
clotting
was
in
it
plasmas
citrated
thereby
amid
u-crc
maintained
the
quantity
in
of
each
calcium
Definitions
Stable
silicone
plasma:
technic
Plasma
throughout.
collected
with
If centrifuged
special
for
precautions
one
hour
from
normal
subjects,
using
the
at 3000
recalcified
with 0.05 ml. CaCl2,
should
not clot in silicone
seconds;
when centrifuged
for two hours
at. 16,000
r.p.m.
hours
or longer.
Asbestos
plasma:
hiasma
that
has been placed
in contact
asbestos
fibers per 1 nil. plasma)
for a fixed period
of time.
r.p.m.,
0.5 ml. of t.his plasma
coated
tubes
in less than
1,000
it should
remain
fluid for eight
with
asbestos
fibers
(10 mg.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
722
CLOT
Actuated
Plasma:
Plasma
si miii lam’ activators
have
.Vonactir’ated
t.he
Purified
soluble
extract
w-itls
Thromboplastin:
1.
Effect
(It-icc!
Blood
was
ethiyl
ON
l)lat
elet
has
beets
BLOOD
5, cephiahims.
glass
particles,
or
fromii
obtaimsed
at. least
from
i)rain
Rate
dric(l
braims
amid
separated
five
precipitations
of
the
ether
.
itsg, thermolabile,
(humans
added.
acetomse
et.hamsol.
ethanol
the
ether
absolute
product.
on
(‘itrated
no activator
which
with
absolute
organs
of Dilution
a nil
without.
to
racted
A clot accelerat
acetotie
Natu-e
The
cold
DILUTION
-
wit hi cold
(‘ephalin:
lipid
froni
ext
OF
thromnboplastin,
ad(lc(l
Plasma
lipid
ether
I-FFECT
to which
beets
Plasma:
The
(‘ephalin:
frons
ACCELERATING
u-titer
usually)
with
of Coagulation
soluble
0.85
lipoprot.ein,
of Normal
extracted
NaCI.
per cemit
and
Hemophilic
Blood,
Plasma
into
collected
anticoagulants,
chilled
with
tubes
the
from
a normal
precautions
and
already
hemophilic
specified,
patient,
centrifuged
for
>70,0001
Contents
of mixture:
0.55 ml. Blood
(or native
plasma)
(undiluted
or dil.
with O.85%NaCI)
In
Ct)
Silicone
surfaces
38#{176}C
K
NORMAL
NATIVE
K
K
PLASt4’
HEMOPH/L/C
C,)
St
10
20
50
FINAL
OF
1.-Effect.
FIG.
hemophiil
of dilution
i c blood
and
hat
(0.85
ive
ten minutes
at. 5 C. and
priate
dilutions
of blood
rate
of coagulation
tion
of both
Indiluted
to have
native
a greater
clotting
t.imes
of
tration,
while
in
perhaps
centration
because,
actually
30
per
cent
of blood
10
20
CENT
THE
per
cent
BLOOD
NaCl)
50
00
CONCENTRATION
(OR
on
the
PLASMA)
rate
of coagulation
of normal
mixture
and
plasma
separated
with 0.85
measured.
(fig.
with silicone
coated
per cent. NaC1 were
Dilution
1), the
accelerates
hemophilic
more
pipets.
made
the
has
to its red cell cont.ent,
about.
t.he same amount
concentration.
evident
in both types
of blood
per cent.
It seems
that. even
Most
of the
clot
Approand the
coagula-
pronouncedly.
plasma
takes
longer
to coagulate
than blood,
but dilution
accelerating
effect on native
plasma
than on blood.
The
norma!
blood
are obt.ained
between
50 and 70 per cent
normal
plasma
they
are between
30 and 50 per cent.
owing
and
plasma.
the plasma
or plasma
of each
types
100
PER
seems
shortest
concenThis
is
blood
diluted
of plasma,
to 60 per cent conas plasma
diluted
t.o
accelerating
effect
of dilution
is
and plasma
at concent.rations
between
70 and
slight
dilution
of blood
or plasma
is sufficient
100
to
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
cause
by
M.
TOCANTINS,
a pronounced
some
than
change
workers5’
that
H.
of whole
ID
that
6
t.he
1 both
normal
per cent
have
and
its
rate
IL
H.
of coagulation.
is because
the
test
723
HOLBURN
It
has
l)een
of plasma.
seems
is usually
done
pointed
to
plasma
at
out
he shorter
con-
(0.1 ml. plasma,
0.1 ml. 0.85 per cent
NaCl,
ml. plasma,
0.1 ml. CaCl2).
As shown
in figure
plasmas
clotting
AXD
of coagulation
cent
(0.1
hemophilic
shorter
CARROLL
rate
This
1)100(1.
centrations
of about
30 per
0.1 ml. Cad2)
or 50 per cent
T.
times
when
tested
in concentrations
than
either
of their
respective
under
70
undiluted
bloods.
Dilution
with
of stable,
native
citrated,
plasma
(fig.
one hour at 3000
NaCh immediately
is collected
2).
normal
In this
and each clotting
before
recalcification.
with
a painstaking
FIG.
2.-Coniparisomi
produces
the
technic,
and
a minimum
CONCENTRATION
of the parabolic
results
plasmas
similar
were
curves
OF
of diluted
0.85
nonactivated
per
plasma
of dilution
PLASMA
to
those
centrifuged
mixture
was diluted
with
If well centrifuged
normal
r.p.m.
FINAL
philic
plasma
experiment,
for
cent
itself
with
the
(%)
normal
timid
hicmo-
plasmisa.
citrate
and
of about
calcium
80
solutions
per
or
cent
is
above.
involved,
Citrated
it will
often
hemophilic
not
clot
plasma
in concentrations
diluted
al)out.
five
times
with 0.85 per cent
XaCl clotted
at nearly
the same rate as undiluted
normal
plasma.
Ordinarily,
in order
to bring
the coagulation
of these
two l)lasmas
to
about
the same rate, the hemophihic
plasma
must 1)e diluted
ten or twenty
times.
When
the plasmas
are unusually
stable,
because
of good collection
techsriic,
P’#{176}longe!
centrifugation
plasma
is required
plasma.
These
coagulability
(“anti-hemophilic
and low platelet,
content,
before
its rate of coagulation
finolings
are
of hemophihic
globulin”)7-
plastinogen”).9
If this
ate
and
the
defect,
were
delay
incompatible
with
the explanation
that. the delayed
blood
is due to an insufficiency
of an accelerator
or the precursor
of an accelerator
(“Thromboso, dilution
the
a greater
dilution
of hemophilic
reaches
that of undiluted
normal
rate
of either
of coagulation.
blood
or plasma
should
accentu-
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724
CLOT
2.
Response
ACCELERATING
to Human
Brain
EFFEOT
OF
DILUTION
and
Thromboplastin
ON
BLOOD
Cephalin
of Plasma
at Different
in various
strengths
were
Concentrations
Solutions
of aqueous
to 0.5 ml.
so adjusted
extracts
of either
stable,
that
the final
of human
brain
normal
or hemophilic
concentration
of the
plasma,
the
plasma
in any
added
ingredients
test mixture
being
was
uniform,
and not below
70 per cent. A difference
in response
between
hemophilic
and normal
plasma
is evident
even with stronger
t.hromboplastin
solutions
(fig.
3). This difference
becomes
more
accentuated
as the concentration
of thromboplastin
is reduced,
and,
the coagulation
plastin
solution
plasmas
eventually,
the
hemophihic
of normal
plasma
is still being
of constant
strength
is used
is reduced
by dilution,
the
resulting
plasma
curves
___________________
remains
accelerated.
while
the
assume
5QQ
fluid
while
If a strong
concentration
thromboof the
the
parabolic
familiar
CONTENTS
OF
MIXTURE:
400-1
50.0001
-
CLOTTING
MIXTURE:
0.5 ml. Plasma
opoo-
0.1
ml.
Th,-omboplastsn
ml.
0.05
O0S0
300-i
3eC
200
M.CaCl2
0.2
SILICONE
SURFACES
38C.
000
do
0.0001
0.00$
Mgm.
0.1
I.
in Clotting
Mixture
0.01
Thrombopiastin
extract)
somi of
and
of thromboplastin.
tested in mixtures
hemophilic
plasma
tiomo of the
the
plasmas
curves
the
of
plasma
and
52
ii
a plasma
the
hemophihic
and
of
difference.
Tpt.
per
sigmsifies
cent,
the
per
100
response
of
plasma.
Dilu-
The
figures
thromboplastin.
of thromboplastin
brain
by variable
of normal
that
100.
aqueous
activated
74
than
74
PLASMA.
4 is a compari-
Figure
hemophilic)
is slower
concentration
(human
plasma.
conscentration
OF
4
thromboplastims
(normal
thromboplastin
obliterates
represent
effect
of normal
with
to a strong
0
CONCESTRATIOS
FIG.
the
3 show-s
ons the rate
of coagulatiomi
the
effect
of dilution
on
amounts
Whens
along
4.-Figure
6
CUT
PER
3
FIG.
3
FIGS.
3
FINSL
ml. of the clotting
mixture.
character
(fig. 4). The principal
effect
of an excess
of thromboplastin
on the rate
of coagulation
of normal
plasma
is in cutting
down
t.he initial
sharply
declining
slope of the parabolic
curve,
best shown
in nonactivated
diluted
plasma
(fig. 2).
The
clotting
time
is still
observed
between
20 and 30 per cent final
of the plasma.
Regardless
of the concentration
of the thromboplastin,
a significant.
difference
is evident
between
hemophilic
and normal
plasma
when
both are tested
in mixtures
of high plasma
concentrations;
t.his difference
minimum
concentration
is eventually
The
of the
effaced
response
plasma.
activated
by
concentration,
anti-thromhopla.stin,
as the
plasmas
of activated
On figure
5 is shown
a
thromboplastin.
strong
are
plasma
thromboplastin
next.
the
the
depends
response
to dilution
In
is least
on
diluted.
to dilution
effective
hemophilic,
clotting
on
and
on the
of four
mixtures
plasma
most
original
stability
types
of plasma
of
high
containing
active
on
plasma
the
the
lipid
asbestos
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
plasma.
As
M.
t.he
thromboplastin
the plasmas
TOCANTINS,
plasmas
dilution.
mitted
are
in the
is gradually
tion, the four plasmas
to t.hromboplast.in
R.
normal
content
varied
however,
clot at. about
between
these
are
AND
R.
H.
while
the same
plasmas
725
HOLBURN
the
is maintained
constant,
until
at or below
3 per
concentration
the
cent
being
of the
difference
plasma
between
concentra-
rate. The wide differences
in response
can,
therefore,
be eliminated
by
to illustrate
how potent
the inhibitors
mixtures
with a high plasma
concentration.
one-stage
“prothrombin
time”
about
26 per cent., discrepancies
content
CARROLL
diluted,
mixtures
reduced
Figure
5 serves
to act in clotting
inhibitor
T.
are
when
Since
permost
met.hods
are done
in plasma
concentrations
of
are bound
to result
when
plasmas
of differing
examined.’0
of prothrombin
The
and
plasmas
used
Ac-globulin,
yet
in these
their
tests
all had
“prot.hrombin
a
times”
widely.
01
K
‘.4
C,)
FINAL
FIG.
normal,
5.-Effect
hemophihic
Asbestos
mg.
normal
normal
per
plasma
sigmsificant
ml.
to
was
found
thromboplastin
cephalintm
plasma”’
amount
dilution
of the
Response
The
buffered
and
exposure
for
Hunnams
content34
to the
generally
found
per
response
beef
to
the
asbestos
and
of cephalin.
lipid
(10
added
imi the
hemophilic
Purified
of hemophilic
of the two plasmas
between
5 and
a standard
effect
be
fibers
to
plasma.
No
4 plasmas.
of normal
to be true
loose
of the
of
anstithroniboplastin.
antithromboplastims
cemst
betu-een
to thromisboplastims
lipid
hour
lipid
of hemophihic
plasma
with
clot accelerating
action12
an
against
thromboplastin,
can
plasma
human
than
of normal
to a constant
10 per cent are
cephalin
which,
reduced
to
brain
like
suspension
the
action
or offset
by
Per
NaC1
of
simple
plasma.2
of Plasma
curves
one
brain
Gm.
respomise
comst.ainirsg
plasma
ion of 0.05
antithrombin
CONCENTRATION
ons the
is much
less act.ive
as clot, accelerator
but dilution
equalizes
the response
of cephauin,
when
plasma
concentrations
reached.
Incubation
greatly
reduces
its
hemophilic
plasma
3.
after
in regard
was
PLASMA
cemst NaCl)
at. 20 C.).
a comicenstrat
in
CENT
plasmas
plasma
plasma
give
differenice
What
asbestos
amid
plasma:
asbestos
PER
(0.85 per
of dilutiomi
obtained
fibrinogen,
to Dilvtion
after
5 per
with
dilution
cent
Fluids
other
of normal
glucose
solution,
than
and
0.85
hemophilic
acacia,
Cent
plasma
buffered
with
0.85
per
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
726
CLOT
cent
NaCl
those
(pH
ACCELERATING
7.2)
obtained
imidazole
with
coagulability
EFFECT
buffer
plain
of normal
0.85
plasma
OF
DILUTION
solution
per
(pH
cent
cannot,
NaC1.
therefore,
ON
7.4)
were
The
effect
accelerator
inhibitors,
thereby
globulin
and
allowing
even
faster
essentially
platelets
to
on
the
to some
experiments.
plasma
from
conversion
similar
of dilution
be attributed
of the saline
solution
employed
in most
of these
dilution
therefore,
that
dilution
of stable
normal
and hemophilic
t.hrombin,
BLOOD
the
property
It seems,
releases
pro-
restraining
of prothrombin
effect.
by
the
of
existing
and consequently
a prompter
the formed
thrombin.
That. this occurs
Ac-globulin,
platelets
and fibrinogen
are
transformation
of fibrinogen
to fibrin
in spite
of the fact that prothrombin,
themselves
diluted,
is an indication
of
the strong
role
obvious
excess
played
by the inhibitors
of these
procoagulants.
in undiluted
4.
Dilution
Curves.
expressing
the
accelerators
by
Analjsis
The
of
curves
relation
plasma,
of clotting
time
to
where
there
plasma
is an
concentration
have
a parabolic
course,
whether
or not the plasma
is activated,
of the nature
of the activating
agent
(figs. 2, 4). Three
distinct
detected
in these
curves.
In the first. or descending
section
of the
and regardless
phases
may he
curve,
the rate
of coagulation
is accelerated,
owing
chiefly
coagulants
(prothromhin,
platelets,
Ac-globulin)
release
effect
coagulants
(anticephalin,
40 per cent,
in the plasma
the
reduction
antithromhin).
about
20 per
of inhibitors
by
prothrombin,
Ac-globulin,
flat. or stationary
section
thse curve,
the lengthening
perhaps
from
to partial
the retarding
of proof anti-
A point
is reached
(in the blood
about
cent.) when
the clot enhancing
effect
of
dilution
is offset
by
a progressive
diminution
platelets
and fibrinogen.
This
is represented
of the curve.
After
t.his, comes
the ascending
clotting
time resulting
chiefly
from too great
in
by the
portion
of
a diminu-
tion in prothrombin
and other
proeoagulants.
If a suitably
strong
thromboplastin
is employed
to activate
the plasma
(fig. 4) the resulting
plasma
dilution
curve
the same
t.hree sections
but, because
of the excess
of coagulant
introduced
the system,
both
the ascending
and
pressed.
These
tentative
explanations
especially
receive
t.he
some
descending
support
from
sections
the
has
into
are defollowing
experiments:
A. Heparin
dilution
with
the
curve
amid
original
is added
but.
In
6).
When
cent
spite
plasmas
a similar
fact
that
rims is amit.ithromboplastic
plastic
only)
they
interpretation
cipally
that.
by
inhibitors
of
a similar
the
content
first
or
clot
at
When
a
the
extend
the
on
mode
of action
of t.hese
as
antithrombic,
on
descending
the
first
two
of
the
(descending
plasma
same
the
the
The
latter
dilutions
higher
is
to
the
the
of
curve,
(fig.
rate
differs
(hepa-
is antithromboto
support
is influenced
concentrat.ion
clot
their
brain4
dilutiomi
is taken
curve
of
the
humans
the same
inhibitor
This
of
sectiomi
inhibitors
lipid
effect.
compared
regardless
from
of t.he
clotting
the
then
concentration,
rate,
section
curve.
plasma.
resistant
phase
at approximately
dilution
section
of
the
and
was
ext.racted
while
t.he
plasma,
plasmas
cent
anticoagulant
all clot
more
first
a 1 per
approximately
lipid
is observed
effect
niormal
the
to
plasmas
in inihibitors
its t.he plasma,
heparinized
of
accelerating
the
printhese
effect
dilutioms.
B.
in
the
as well
have
to stable
these
however,
the
the
added
of heparin
effect
concentration,
of the
of
diluted,
concentration.
to t.he plasma,
I per
at
time
amounts
fig.
heparinized
hieparin
was
clotting
Increasing
dilution
normal
concentrations
activated
a control.
the
7).
ins various
on
Beef
plasma
a sihicomiized
w-as
glass
obtained
pitcher
from
containing
freshly
slaughtered
19 per
cent.
animals,
sodium
citrate.
the
blood
Dilution
being
curves
collected
of the
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L. M.
TOCANTINS,
R. T. CARROLL
R.
AND
platelet-poor
plasma
were then worked
out, using
(1) 0.85 per cent NaCl,
(2) buffered
beef fibrinogen,
H.
each of the
(3) buffered
CONTENTS
CONTENTS
OF
0.5 ml. Plasma
300
0.1
ml
200
do
US
MIXTURE:
(aRt d1 I
0.5
IT4
ThambopIasI:t
HOLBURN
ml
727
following
diluting
agents:
beef plasmna Ac-globulin,
OF MIXTURE.
Plasma
laar.dI
I
300
0.1 ml. Ag. haman man
en,racl
0.05 ml. CaCI2 laar M. conc I
2OC;ONEsURFAcEJc2O
4
ONESURFAcESc,
00
50
30
20
CO
.,ASM4
0
do
624
FINAL
PER
CENT
5274
CONCENTRATION
FIG.
FIG.
to
of
A small
obtain
the
FIG.
lipid
thromplastin
of added
of dilutions
cent
obtaimi
the
ons normal
Toronto
Heparin
thromboplastin
A small
(homogenized
to
PER
CENT
00.
CONCENTRATION
PLASMA
7
plasnias
plasma
amid!
solution
OF
is added
containiing
to normal
Plasma
shown.
amit.ithromboplastin.
plasma
effect
cotscentration
effect
FINAL
FIG.
the
of 1 per
volume
7.-The
10.
PLASMA
6
6.-Comparisoni
heparin.
OF
by
of
exposure
to
concemstrations
ml. plasma
to
volume
diluted
a
10
plasmas
per
cent
supersonic
with
varyimig
suspemision
is-ayes)
added
comitent
of the
to
the
lipid
stable
of
anti-
miormal
shown.
(var. dii.);
.i mi beef
thrombopl.
;0.05
ml.CaCI2_(varMconcentr))
200
-‘.
100
Diluting
Diluting
fluid:
fluid:
Buffered
Fibrinogen
O.85%NQCI
(3
K
,-.
-4
Dilut. fluid:
Ac-Globulin
Prothrombin
C,)
15u
300u
‘iper
J
ml.
00
10
FINAL
CONCENTRATION
IN
FIG.
8.-Effect
componemsts.
nogen
by
Ow-ren’s
are
technic
by
salt
is used
buffered
purified
alone.
purified
of
courtesy
of Dr.
thromboplastin
clotting
technic.6
those
with
using
throughout
beef
To
each
H.
ansd
of these,
componsents
Seegers.
the
calcium.
accelerates
various
of
isolation
separated
using
buffered
of the
Ac-globulin
show-ms
coagulat
on
clot
and
is-as
8 dilutioni
ions until
a plasma
(5)
beef
fibri-
plasma
prothrombin
as shown.
tinig
the
Sili-
reagents.
buffered
added,
of
clotting
amid
purified
together
of plasma
figure
of
from
purified
toget.her
amount
mixtures
prothromaslnni
ansd Ac-globulin
proper
As
PLASMA
Purified
Ac-globulin
results
ins the
prothrombin
THE
with
plasma.
prothrombin
except
amid
plasma
beef
The
OF
MIXTURE
beef
from
W.
cent)
CLOTTING
activated
origimsated
fractionation
identical
prothrombin
beef brain
these
dilutiomi
All insgrediensts
supplied
alone
cone
(4)
of
(per
purified
followed
plasma
comicent
by
with
rat ions of
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
728
CLOT
about
30 per
cent
taming
purified
dilution
curve
Some
tion
in
cause
of the
shorten
coagulation,
though
plasmas
affecting
is usually
of clot ting
ant.ithrombin
to
dilution
(fig.
than
those
without
old
though
added
normal
of the plasma,
a lipid
7)
will,
like
100,000
\
will
(2)
will
shorten
of stable
plasmas
0.2
SILICONE
of stable
also accelerate
ml Human
or I:luE)
O(undIl.
Platelets 1810 01061 nO.1 ml. O.65NaCl
0.05 ml. CXCI2 (Variable
M.concent.I
M.CaCI2
(0.000
SI
I
38C
-
ace.
o.
NORMAL
(000-
HEMOPHIL1C...,..#{176}
/
000
-.J
NORMAL
(OC
do
0.001
P L
I
A
T
per
Milhons
0-01
E
cmm
L
ai
0.1
E
9 amiol 10.-Figure
of coagulatiomi
human
platelets
on
slow-er
response
of heniophihic
Platelets
Platelets
blood
and
to normal
and
the
were
excess
Response
isolated
CONCENTRATION
i
effect
(per
of is-ashed
plasma.
rate
of
plasma
of platelets
of Normal
normal
Figure
coagulatiomi
to
or
and
the
of
10
effect
(0.85
accelerating
action
of the
Hemophilic
centrifugation
platelets
the
diluted
by dilution
on
of the
per
cent
of platelets
the
rate
addition
NaC1)
cami
be
plasma.
Plasma
from
PLASMA
I
cent
humami
10 show-s
00
OF
FIG.
by fractional
washed
three
or hemophilic
each component
concentration
mixture
the
plasma.
a large
FINAL
the
hemophilic
by eit.her
S
hemophihic
and
corrected
5.
and
b
01
-
9
9 show-s
of miormal
is-ashed
T
clotting
FIG.
FIGS.
00
-
0.0001
The
normal
the rate
in antithrombin.
SURFACES.
10000
normal
an
plasmas
C.
do
of
of
delays
display
of such
Dilution
rate
often
is added,
(5)
If
usually
their
activity
NaCl,
plasma
plat.
susp.
ml.
0.05
Lj
antithrombin
dilution
of such
cent
a diminuprincipal
MlXTURE
ml.
ml.
0.5
0.1
HEMOPI-IILIC
normal
antithrombin
per
suspected.
to
the
of clotting.’-
ant.it.hromboplastin
0.85
been
rate
con-
parabolic
(3) Exposure
hours
dilution
the
with
tubes,
0
do
have
is unchanged.
tis-o
the
is rich
serum
CLOTTING
in glass
antithromboplastin.
serum
though
the
mixture
of
attributed
this
is not
that.
blood
Yet
in the
phase
be
delays
for about.
which
except
as might
tested
activity.
B,LOQD
third
naturally
believe
activity
antithrombin
Plasmas
24 hour
and
to asbestos
to
less3
w-it.h
plasma
must
to
dilution
syringes
(4)
rate,
rising,
hypercoagulable
activity,
response
rate of clotting.
accentuated!
the
the
ON
diminution,
collected
glass
DILUTION
prolongs
that
to prothrombin
anticephahin
in uncoated
without.
dilution
effect
of dilution
reasons,
however,
are
or hemophilic
clotting,
the
low
is collected!
normal
due
(1) in poorly
is-ith
OF
ami indication
accelerating
There
change:
EFFECT
Further
is principally
but
blood
not
is reached.
prothrombin,
of the clot
antithronsbins.
contenit,
ACCELERATING
to Dilution
normal
and
hemophilic
times
with 0.85 per cent NaCl.’4
They
were t.hen added
recalcifled
plasma
in silicone
coated
tubes,
the volume
of
of the clotting
in each tube was
of an increasing
number
plasma,
hut
even
more
mixture
being
so adjusted
that
the final plasma
75 to 80 per cent.
As shown
in figure
9 addition
of platelets
so that
of
accelerates
hemophilic
the rate
plasma.
of coagulation
The
relation
of normal
between
clotting
time
and platelet
concentration
is approximately
linear
for both
types
of plasma
between
the limits
of 0.001
and 0.8 M. platelets
per cu. mm.
The
slopes
of the two
lines are, however,
markedly
different,
the addition
of platelets
producing
a steeper
fall
in the
clotting
time
of the
shown
by
hemophihic
than
of t.he normal
plasma.
If
enough
platelets
are
added,
as
Eagle,’3
hemophilic
plasma
will
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
clot
M.
as rapidly
as normal
as normally
found
platelet
concentration
hemophilic
TOCANTINS,
will
to
sufficient
concentration,
not
antagonistic
be possible
indicate
CARROLL
clot
five
that
platelets
supply
eventually
1 per cent
undiluted
eliminates
concentration
Addition
distilled
of
platelets
that
to each,
which
plasma
does
incompatil)le
support
plasma
not
by
alter
with
the
explanations
the hypothesis’5
factor
necessary
platelets.
If it were
existing
deficiency
platelets.
Moreover,
so,
dilution
trifuged
removed
action
and
platelet
sediment
subsequent.
re-suspended
at. the
for
of one
increasing
number
of
specimens
content
platelets
(fig. 11). The numbers
per cu. mm.
of plasma.
influences
the coagulability
platelets
the platelet
was counted
would
clotted
at
concentrat.ion
but
that with 3,072,000
about.
1,000
with
only
platelets
is modified
plasma
cit rated
minutes
hour
likewise
only
(10
not
by
and
dilution
ion
of an
the
prevailing
by the
collected
24 hours.
con-
following
and cen-
In addition,
of platelet
original
that.
specimen
were
done
studies
excessive
platelet
rich plasma
was
was cent.rifuged
for 20
of the
in
any
or utilize
the
of hemophilic
rich
plasma
the
plasma
volume,
four fold.
The
in each of the
7
adjacent
to each
curve
indicate
the platelet
It may
he seen at once,
that
the number
of
of the sample,
principally
in the
lotting
seconds,
160
not
accentuate
is supplied
blood
was
cent.rifugation
one-fourth
clotted
normal
are
they
addit
mixtures
of high plasma
concentrations
(89 per cent.).
For example,
while
89 per cent normal
plasma
with
mm.
to about
and
findings
naturally
simple
by
concentration
and
in
and
of disintegl’ate(l
but.
minutes.
The
this plasma
60, 360
approximately
in
thereby
difference
even less able to mobilize
the rate of coagulation
for two
minutes;
end
These
ahove,79
of platelets
ones
diluted.
effect of
of platelets,
hemophilic
shown.
reduced
in the cold at 5,000 r.p.m.
and centrifuged
for five
it. should
rate after
to he diluted
diluted
in
of an
of diluted
dilution
not
original
number
of suspensions
or
plasma
plasma
between
be
when
excess
plasma
is lacking
or deficient
in a
of the clot accelerating
factor
in
of stabilizing
inhibitors
in the
Stable
normal
and hemophilic
minutes
was
not
the
same
to
curves
of the
of an
so, however,
at the same
plasmas
have
is reached.
mentioned
and render
the
the difference
plasma
which
action
narrows
the
that.
hemophilic
for the utilization
and normal
plasma
would
number
of platelets.
Further
proof
that
the
centration
experiment:
also
extracts
the
cent
were
the same,
results
were
in-
rate of coagulation
10, progressive
figure
waves)
significantly
80 per
accelerator,
on the
to
platelet.
platelets
if the plasmas
were suitably
consisted
in comparing
the
The
point
supersonic
as many
of about
the
on
plasmas
water
(destroyed
times
plasma.
If this were
and normal
plasma
difference.
l)efore
that
729
HOLBURN
this
experiment
were used.
The
a clot
three
times
washed
normal
human
platelets
normal
and hemophilic
plasma.
As shown
only accelerates
their
rate of clotting
but
of the
ten
mixture
of offsetting
inhibitor
in hemophilic
to clot hemophilic
plasma
response
and
H.
.
if a clotting
is capable
R.
to produce
this effect
Below
a certain
500 per cu. mm. of the mixture)
the
The
results
of
or normal
platelets
adding
an equal
number
of platelet.s
This was tried in the next experiment
the
AND
Between
blood are required
(on figure
9, below-
concentration
is maintained.
whether
washed
hemophilic
terpreted
T.
plasma.
in the
plasma
R.
a
mixture
platelets
in about.
of
took
400
normal
660,000
platelets
plasma
longer
than
20,000
seconds.
Yet when
of
per
the
cu.
same
seconds,
and
these normal
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
730
CLOT
with
plasmas
widely
concentration,
dilution
curve
the
the clot
Moreover,
accelerating
its
to lose
and
character
to display
The
content
to
role
the
offset
effectively
BLOOD
were diluted
to about
brought
close together.
content
were
manner
to hemophilic
a tendency
is evident
plasma
for the
30 per cent
The same
(fig.
right
11). As the
limb of the
with the high platelet
lost. It is also seen that
in platelet
poor plasmas.
within
or near
the normal
a dilution
of the
curve
resembling
that
normal,
by increasing
11).
of platelets
stabilizing
plasmas
in
ON
effect
of dilution
is more
striking
plasma
can be made
to clot,
(fig.
principal
agents
DILUTION
slope until,
in the mixtures
of the curve
is almost
its sharp
hemophilic
OF
of coagulation
striking
increases,
parabolic
platelet
platelet
rates
even more
of platelets
content,
EFFECT
varying
their
applies
in an
concentration
range,
ACCELERATING
of high
as clot accelerators
effect
of a first
concentration.
seems
to be
inhibitor,
therefore,
phase
the
plasma.,
consequently
When
as
acting
and
most
(r
U)
K
K
K
(‘5
FINAL
Fig.
11.-Rate
heniophilic
the
of coagulatioms
plasma
inhibitor,
unlikely
rupting
cent,
over
1,000
r.p.m.
for
the
five
faster
out5
clots
minutes.
need
of
high
plasma
markedly
cent
of
affect
plasma.
rifugation
less
for
clotting
Elsewhere,
of
plasma
(0.85
per
cent
NaC1)
of normnal
and
platelets
seems
to be lessened.
It is also
dilution
is mediated
through
a dison the platelets,
since normal
and hemophilic
cont.ent,
diluted
to concentrations
of about
of
when
normal
about
t.he
tests
than
concentration
the
dilutions
effect
than
at
CONCENTRATION
content.
that
The
concentration
hemophilic
speed
much
pointed
plasma
t.ion
clot
been
PLASMA
of serial
platelet
diluted,
are
has
r.p.m.
tures
varying
the (‘lot, accelerating
of the diluting
fluid
regardless
of their
platelet
10 per
not
of
CENT
that.
effect
plasmas,
It
PER
(fig.
plasma
centrifuged
same
rate
were
done,
however,
cent..
When
are
used,
of
evidence
lower
and
minutes
tubes
length
hypercoagulable,
and
and
at
mix-
of centrifuga-
even
that
at
at 3,000
surfaces
and,
presented
layers
five
in glass
plasma
be
for
centrifuged
silicone
speed
normal
shall
its
the
11).
as that.
30 per
time
renders
undiluted
more
so,
prolonged
of
high
while
its
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
topmost
layers
lighter
lipid
lower
6.
M.
may
R.
molecules
the
of
Contacting
T.
CARROLL
be incoagulable.
inhibitor
sections
The
TOCANTINS,
H.
H.
inequality
rfhis
upward
AND
as much
731
IIOLIIURX
is due
to displacement
as to packing
of
of platelets
in the
plasma.
and
Surface
the
Response
and
of Normal
Hemophilic
Plasma
to
J)ilution
If glass
each
particles
particle
(chemically
passed
hemophilic
clean
through
plasma,
the
and
a Xo.
rate
washed
40
B.
ground
of S.
of coagulation
glass,
sieve)
of both
plasmas
(fig.
12) resemble
those
of normal
and hemophilic
t.hromboplastic
agent
has been
added.
If, however,
the
plasmas
is reduced
by dilution
while the quant.ity
of glass
the
rate
of coagulation
of the
plasmas
two
to
so that
normal
is accelerated.
or
The
plasma
to which
a
concentration
of both
particles
is maintained
curves
constant,
screened
added
are
gradually
shortens,
until
5000
‘T
05
GLASS
0.05tT
.
4
PLASMA
POWDER
SUSPENSION
.
0.05m1
2OOOO\
02M
do
CaCil
Colloaion
surfaces
2OOO-
3R’C.
2
l00
0.01
0.1
(MOM.
PER
CENT
IC.
IN
12 amid!
normal
and
of coagulation
MIXTURE
The
be
resistance
reduced
(fig.
the
same,
and
when
effect
of glass
13 show-s
Figure
NaC1)
especially
particles
t.he
normal
Blood
a plasma
and
on
effect
of
the
glass
:00
NTROT05
13
plasm-.
plasma
concentration
of coagulation
on
the
rate
plasma.
to
collected
rate
particles
ansd hemophihic
of hemophilic
of the plasma.
dilution
50
LONJI
I
the
per cent
0
L.,MA
activations
with
of about
2 per
by
siliconsized
cent
glass
cams
vessels.
is teached
13).
Another
example
stability
of plasma
normal
and
asbestos
per
for
of normal
by
it becomes
(0.85
j
LINT
FIG.
show-s
plasma.
I
4
PIll
12
/2
of diluted
:
(000.
NOL
CLOTTING
13.-Figure
hemophilie
(00.
M
TT
POWDER
FIG.
FIGS.
025%G
ioo
I.
GLASS
of
200
.
varying
hemophilic
ml.
periods
I
platelets
samples
and thromboplastin)
testedi
at a high
depressed
the first
normal
and
concentration
influence
down
tested
of
to
plasmas
packed
to dilution
stable
plasma
the
its response
of plasma
of time
response
fibers
were
of
and
were
cont.act.
dilution
exposed
in a silicone
coated
(0 to 210 minutes).
at
the
at
plasma
(descending)
hemophilic
(89 per
i)ottom
once.
of
Contact
the
with
cert.ain
is illustrated
t.o loose
tube,
After
surfaces
in figure
asbestos
on
the
14. Stable
fibers
(10
mg
without
agitation,
at 20 C.)
the stipulated
interval,
the
tube,
the plasma
removed
and
with t.he fibers
(like the presence
modified
principally
the
concentration.
Increasing
phase
of the parabolic
plasma,
not exposed
to
cent),
clotted
in a longer
rate
its
of
of clotting
of the
exposure
to asbestos
dilution
curve.
asbestos,
tested
time
than
when
While
in high
diluted
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
732
CLOT
ten
fold,
they
after
had
trat.ions
as
ACCELERATING
the
plasmas
a longer
rate
asbestos.
and
by the
Hemophilic
plasma
concent.ration
minutes
of contact.
hemophilic
for only
plasma
120 minutes
boplastin,
the
equalized
by
did
Two
rate
OF
when
not
clot
BLOOD
asbestos
for
to that
of the
exposure
ten
samples
minutes,
The
concen-
of exposed
by
above
to asbestos,
minutes
120
extent.
were
not altered
tested
in a mixture
before
and
ON
with
diluted
in each
method,’6’8
which
when
hundred
DILUTION
in contact
Ac-globulin
two-stage
plasma,
to
been
had
of coagulation,
of prothrombin
measured
EFFECT
plasma
exposure
50 per
clotted
of contact
were
to
cent
after
required
60
for
reach
the rate of coagulability
of normal
plasma
exposed
(fig. 14). As after addition
of platelets,
cephalin
and throm-
of coagulation
appropriate
of normal
exposure
and!
to a suitable
hemophilic
plasma
cont.acting
surface.
may
also
be
.-.
U)
(3
K
‘4
(‘5
FINAL
FIG.
of contact
plasma
to dilution.
Plasma
to dilution
7.
inal
CONCENTRATION
14.-Effect
Normal
and
After
cx vivo.
Hem.ophilic
Such
plasma,
have been
hemophilic
plasma
tion
which
lation
of
has
separated
method.
globulin
although
from
to
asbestos
for
wide
and
the
210
separated
by
a similar
effective.
acceptance,
is due
,“22
the
hemophilic
the
and
response
hemophilic
of hemophilic
120 minutes.
Fractions
it has been known
effect
on hemophilic
dilution
and
effect,7IL
This seemed
that
to
PLASMA
of normal
minutes,
for
Eu globulin
OF
response
exposed
of Frank
and Hartmann’9
has a clot accelerating
blood
CENT)
on
plasma
of normal
t hromboplast
in
table
2 are shown
normal
fibers
Plasma
fractions,
gained
PER
(
asbestos
shown
to exert
are much
less
hemophilic
termed
“plasma
tinogen.”9
On
exposure
is like that
Ever since the work
plasma
euglobulin
and
with
the
delay
a deficiency
that the norblood! in vivo
acidification
21
while those
to support
in the
of a plasma
of normal
derived
from
the explana-
inception
fact.or
of coaguvariously
“anti-hemophilic
globulin,”23
“thromboplasresults
of addit.ion
of euglobulin
solutions,
plasmas
by
the
dilution
and
acidification
1.0 Gm. per cent concentration,
it.is obvious that the normal euhas a clear clot, accelerating
effect
on normal
plasma
and an analogous,
slightly
less pronounced
effect,
on hemophilic
plasma.
The hemophilic
At
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
euglobulin,
normal
M.
on the
plasma,
Putting
aside,
2, when
the previously
of the clotting
having
were
acid
TABLE
2.-The
the
moment,
Gm.
per
action
and
a clear
two
The
collected
from
finding
diluted.
From
that
to bring
Clotting
Time
Eu globulin
of Normal
Fractionated
and
on,
and
Heinophilic
of Euglobulin
1.0
Xormiial
0.005
1.0
Hemophilic
0.005
NaCl
of each
clotting
ml.
M
precipitate
0.2
was
out
up in 0.85
immediately
throughout.
solution
mixture:
separated
Plasma
per
0.5
Silicone
ml.
plasma,
technic,
38
by centrifugation,
cent
after
ml.
0.1
dried
and
XaC1,
making
the
the
and
pH
adjusted
solutions.
normal
cent
resulting
by
Solution
of
Plasma
Time
to plasma,
plasma,
Plasma
(Secs.)
testing
the
the
calcium
the
hemophilic
Hemophilic
Plasma
287
1164
1820
264
>14,400
1190
1904
1,374
>14,400
euglobulin
weighed.
solution
(or
0.8&
A fairly
uniform
and hemophilic
plasmas
452).
The residue
was
to 7.4 and
Silicone
clotting
required
euglobulin
tubes
mixtures.
the
and
for optimal
tests
were
pipets
carried
were
used
Recalcification
about
optimal
precipitated
citrate.
When
mixture is not altered
by the presence
of the euglobulin
of one per cent or below.
On figure
15 are shown
the
On
1 per
the
C.
amounts
of calcium
designed
to bring
w-ith Fraction
I (Cohn)
the euglobulin
acidificat
ion does
not seem
to contain
is added
and
Activated
or Hemophilic
point
in uncoated
water,
to 5.9
the
to the
processed
obtained
from
100 ml. of normal
normal:
450 mg.,
hemophilic:
in preparing
(lone with
In contrast
tion
and
using
up
distilled
con-
euglobulins
subjects,
mixture
Control
CaCl2.
gravimetric
yield
was
(mean
of 6 experiments,
taken
the
195
Hemophilic
0.05
is
9m.percntin
Normual
Contents
NaC1),
plasma
euglobulins
two
t.ubes
were
Normal
0.85%
the
in
on variable
Clotting
Type
hemophilic
examine
As shown
in the
which
coated
cold
of the
Normal
from
they
pH
from
the
us
euglobulins
hemophilic
1 to 10 with
t.he
that
of the
two
in silicone
point
diluted
were
added
stored
on
becomes
as active
an accelnormal
euglobulin.
tfhis led us
plasmas
normal
and
effect
plasma.
plasma,
let
concentrations.
concentrations
citrated
accelerating
of euglobulin
of the
733
HOLBURN
clot
stressed
concentration
amounts
plasmas.
H.
on hemophilic
of normal
at lower
of various
of fixed
plasmas
was
cent
R.
action
little
the clotting
when
tested
throughout,
The
acetic
the
AND
inactive
hemophilic
euglobulin
of hemophilic
plasma
as the
were
technic
t.hey
accelerator
the
of the
glassware.
hand,
no significant
a 0.005
derived
when
though
plasmas,
centrations
were
other
further
two
silicone
CARROLL
accelerates
euglobulins,
use(l,
erator
examine
T.
for
tal)le
on the
R.
has
euglobulin
itself
effect
of the two
to
TOCANTINS,
was
clotting
times.
by plasma
dilitthe euglobulin
clotting
time
of the
solution
in concentrations
results
of these
experiments.
is as effective
a clot
accelerator
as the normal euglobulin, at concentration
1 and 3). The slight
difference
between
0.05 per cent or below
(fig. 15, No.
the act.ion
of the two cuglobulins
on
normal
of 0.1
plasma
appear
at
concentrations
of
or above,
when
the
hemophilic
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
734
CLOT
euglobulin
apparently
(and
do
not
change
is especially
plasma
(fig.
cent.)
when
1). At
it still
the
hemophilic
tration
of
philic
plasma
where
the hem.ophilic
clotting
prolong
the
high
itself
has
has
of heniophilic
no
clot
(fig.
plasma
(a.)Var.
l)egin
of
effect
normal
1 and
on
hemophilic
the
As
accelerating
reached
behavior
to hemophilic
(above
0.1 pei
plasma.
a clot
as the
15, No.
anomalous
accelerating
are
to behave
in an
the euglobulin
is added
euglobulin
on normal
concentrations
is as ffective
BLOOD
This
euglobulin
of the
is reduced
until
en globulin
euglobulin)
concentrations
effect
euglobulin
ON
coagulation.
a significant
appears,
DILUTION
hemophilic
concentrations
euglobulin
though
OF
the normal
i.e., greater
or actually
evident
15, No.
hemophilic
the
plasma,
the
a lesser
extent
manner,
to
anomalous
either
EFFECT
ACCELERATING
concen-
effect
(below
on
0.01
en globulin
hemo-
per
cent)
in accelerating
3).
Glob. concentr.
(b.) Var.
Plas.concentr.
-
(tj
(0
3,OOO-
3.NORM.
GLOB.
4. NORM.
GLOB.
K
lpOOn.j
IOO
xnnn
CONCENTRATION
(gms. per cent
Fnd.
l.-Response
of normal
ams(l hemsiophilic
euglobin
tration
of
ml.
plasma
solution
finsa.l
plasmiia
(various
msiolar
so
plastin”),
iii
a
because
response
or normal
euglobulin
various
74 per
the
to the
concentrations,
of
normal
the
per
38
the
of
accompanying
hemophilic
in a fixed
Comitent
0.06
that
content
M.
of
cent
plasmas
CaCI2.
from
Actual
normal
plasma,
ml.
0.1
plasma
clotting
niixtures
for
euglobimi
solution,
0.05
euglobulins
differ
material
anticoagulants.
plasma
does
or hemophilic
fractions
mnl.
concen2 and
nil.
4: 0.5
CaCI2
C.
coagulant
concentration
two
0.2
100
I amid 3: 0.5
for
ml.
tubes,
results
their
of
in
cemit.
ml.
to globulin
mixtures
0.05
Silicone
of
accelerator
plasmnas
clotting
of
0.1
these
from
its slower
of
mixture
concentrations).
hut
clot
hemophilic
and!
emit
dilutions),
because
muds
0
FINAL(per
cent) PLASMA
CONCENTRATION
OF
CLOTTING
MIXTURE
concentrations),
clotting
It. is evident,
not
Const
-
(various
(various
.1
OF GLOBULIN
of plasma
in clotting
mixture)
.01
.001
not
per
eventually
each
other
thrombo-
Moreover,
a deficiency
seem to l)e the cause
for
euglohulin.
(0.06
from
(“plasma
If either
cent.)
hemophilic
is added
respond
alike
to plasma
to
both
euglohulins
(fig. 15, Nos.
2 and 4). Though
at high plasma
concentrations
(75
per cent in the charts)
significant
differences
in the response
of t.he two plasmas
td) the
two euglol)uhns
are observed,
the responses
become
the same
at a plasma
concentration
of 13 per
cent for normal
(fig. 15, No. 4), and of 6 per cent for
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
M.
TOCANTINS,
hemophilic
euglobulin
centration
and
R.
(fig.
below,
15,
the
T.
CARROLL
No.
AND
may
rather
to the
philic
euglOl)ulin
be
traced
hemophilic
presence
and
at
normal
with
to a deficiency
of inhibiting
fraction
the tests.
Erroneous
fractions
at a single
plasma
concentrations,
contact
not
and
agents
in the
735
IIOLBURN
6 per
cent
euglobulin
plasma
con-
the
same
have
plasma
(fig. 15, Nos.
2 and
4).
in a study
published
elsewhere,
between
hemophilic
and normal
in a clot
in greater
hemophilic
accelerating
factor,
concentration
plasma
impressions
are obtained
by
fixed
concentration,
(2) using
(3) employing
as a substrate
glass
H.
Incidentally,
2).
activity,
whether
added
to hemophilic
normal
These
and other
points
to be elaborated
further
seem to indicate
that the differences
in behavior
euglobulin
R.
but
in the
used
as
a
hemo-
substrate
for
(1) testing
the euglol)ulin
clotting
mixtures
of low
hypercoagulable
plasma
in
surfaces.
1)IsCussIox
It
is evident
plasma
can
from
the
foregoing
l)e markedly
that
shortened
by
effect
of dilution
requires
stable
technic.
Anything
that contributes
of blood,
its exposure
to glass,
coagulant
solutions
recalcifying
tion.
Stable
plasma
.
with
(citrate,
mixtures,
human
glass
hut
fish,
rapidly
of
of the
or nullify
in silicone
marine
clots
and
reduce
plasma
of certain
rate
coagulation
dilution.
The
of blood
and
demonstration
of the
blood
or plasma,
ol)tained
with
a painstaking
to disturb
this stability
such as poor
collection
inadvertent.
dilution
of the blood
with
anti-
oxalate),
may
the
simple
tubes
which
after
clot
by
to
fluid
complex
buffer
accelerating
seems
remains
addition
plasma
the
effect
behave
like
indefinitely
of a suspension
the
when
and
of dlilunormal
in contact
of porphyrized
glass
or
by
simple
dilution
with
sea water.2425
There
are points
of analogy
betw-een
the effect
of dilution
on the coagulability
of blood
and on the peptic
activity
of gastric
juice.
Generally
speaking,
the
higher
the pepsin
content
of gastric
juice,
t.he moi.e it must. l)e diluted
before
it
ceases
to
quired
present
to reach
this
in equilibrium
gain
dissociates
activity
upon
dilution.
inhibitors
and
that
dietermine
the
amount
to
use
such
on
point.
with
dilution,
that.
the
rate
not.
effect
of the
phase
clotting
might.
of the activated
have
differed
when
are no longer
thromboplastin
that,
inhibitor
plasmas
undiluted
if the
are
t.he
(figs.
impure
pepsin
found
directly
proportional
that
of prothromhin
appears
used!
plasmas
to
are
is
pepsin.
Northrop
is
is usually
in gastric
a (‘omplex
active
operative.
is
(anticephalin)
pepsin
forming
into
Likewise,
it
to the amount
dilution
100
that.
in solution,
of digestion
inhibitors
or
show-n
present
It. is significant.
first
has
solution
taken.
he proportional
to use a dilution
at which
depending
on whether
A I to
it. is dissociated
of pepsin
a dilution
transformation.
dilution.26
Bucher
et al.26 suggest
that
an inhibitor
(or inhibitors)
Northrop27
aft.er
amount
of enzyme
rate of coagulation
further
in
This
hound
In
by
order
to
it necessary
to the
order
that
the
it is important
varies,
of course,
prothromhin
accelerate
sufficiently
is overcome,
IC-
juice
is
wisich
and
(hluted,
the
the
rates
same,
regardless
of how
much
5, 10). Considerations
such as
of
they
these
probably
forced
those28 engaged
in the measurement
of prothromhin
by t.he onestage
method,
to use dilutions
of plasma
which
yielded,
after
activation
with
a
strong
thromboplastin,
a clotting
time which
varied
inversely
as the amount
of
prothrombin.
Dilution
as originally
used
in the
two-stage
prothrombin
titrat.ion
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
736
CLOT
method’7
was,
effect
before
ACCELERATING
on
the
of antithromhin,
its activity
other
hand,
have
now
reached
OF
DILUTION
intended
which,
when
could
be measured
prothrombin
conversion
by
action
of first phase
inhibitors
We
EFFECT
ON
chiefly
perhaps
time
to
in high concentration,
; dilution
must
also,
sparing
t.hromboplastin
(antithromboplastin).
the
BLOOD
when
and
a tentative
overcome
destroyed
however,
Ac-globulin
hypothesis
may
the
thrombin
accelerate
from
the
be presented
to guide
us in the understanding
of the evidence
presented
in this paper
and in
developing
future
work.
Normal
circulating
blood
and plasma
in contact
with
inert surfaces
maintain
their fluidity
and coagulability
by an equilibrium
between
clot
inhibiting
and clot
promoting
forces.29
Under
normal
conditions
circulating
blood,
clot inhibiting
forces
are dominant,
though
procoagulant
stances
ture present
in excess,
to be sure, of the amount
required
to bring
rapid
clotting.
The
may
be mediated
may
be
bound
coagulants,
to
oi
01
inactivate
The
incubation
Clotting
of these
either
holding
them
role
and
of
words,
first
exposure
disturbing
These
in
phase
considered
tected
an
and
unlikely
inactive
clot
form
accelerating
(antieffect
are
the
developments.
present.
in the
by cytozyme;
and
rapidly
transformed
proserozyme
may
He
inhibitor.
directed
or plasma
between
principally
of
inception
clotting,
in
to the
other
also
represent
showed
released
from
certain
the
combined
activating
of Bordet
therefore,
reactivity
globulin
and
globulin
releasing
that
of
that
the
may
inhibitor
proserozyme
contact
or
the
latter
be looked
(factor
V).
of serum
in t.he
be
form
upon
His active
Ac-globulin
which
may
dilution
two
slowly
plasma
to
Bordet
actually
dissociates
during
of serum
prothrombin
(factor
VI).
was
were
Expressing
the inactive
in the
pres-
(serozyme)
It seems
responsible
for
conjugated
with
plasma
the inhibitor
from
the
Ac-globulin.
pro(sub-
clotting
as prothrombin
felt
or
substances
and
prothrombin
with
an active
prothrombin
(serozyme)
to thrombin
by cytozyme.
Bordet
prothrombin
t.hat
of the
Bordet3#{176} distinguished
plasma
(proserozyme)
helped to convert prothromhin
to thrombin.
findings in the light of later developments
and terminology,
serum
of
may reduce
antithrombin
with cert.ain surfaces, dilution of the blood,
more
recent
one inactive,
ence
of plasma
Ac-globulin
prothrombin
in the presence
slow
remarks
preventing
Contact
“excitoproductrices”)
in the
Bordet’s
plasma
present
or
inhibitors.
that
by
stances
found
precursor
The
may result. from exposure
of the blood
what
is probably
a loose conjugation
The
delaying
to thrombin
in the serum
and
found
their
action).
substances
ways:
(1) the inhibitors
their
transformation
into
to t.he by-products
of blood and tissue
cell disint.egration,
are some
forces
which
may determine
or help to determine
these
changes.
findings and hypothesis
reach some
measure
of reconciliation
Bordet’s
theories
types
of prothrombin:
converted
or stabilizing
the
subabout
activity
of cephalin, thromboplastin
and thrombin
after
plasma
or serum
makes
this explanation
also plausible.
be initiated
by release
of the procoagulant
into its active
procoagulant..
inhibitors
inhibitors
this explanation.
(2) The inhibitors
coagulants
(antit.hromboplastin,
coagulant
form,
a change
which
to any force which
is-ill dissociate
inhibitor
in
globulin
plasmas
favors
effect
of released
reduced
with stable
may t.hen
clot
or both of the following
thereby
preventing
pi’ocoagulants,
antiaccelerator
on stable
the
action).
of action
virtually
prot.hrombin,
dlilut.iOn
mode
through
in
is
not.
the
Ac
Ac
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
L.
What.
M.
TOCANTINS,
R.
of hemophilic
then
a normal
prothrombin
is unusually
slowly
the plasma
is diluted
surfaces,
or
is acted
cephalin
hemophilic
01.
dissociated
and
tested
little
upon
and
normal
plasma
diluted
1 to
no differences
be
will
tubes
Clearly,
the
and
globulin,
and
philic
plasma
hasten
first.
on normal
and
1 to
10 or 20
diluted
accelerating
effect
of
plasma
hemophilic
when
as
plasma.32
and
normal
the
Ac-globulin
plasma
undiluted
(1) the
1)001
obtained
(factor
hemophilic
blood
slow
conversion
content
of
of serum
up
the
from
Ac-globulin
Summing
rupts
dilutions
a degree
acquire
stability
normal
dilution
of blood
i)y
plastin
required
the
presence
(or platelets)
t.o free the
hemophilic
activity
(like contact
or plasma
by
tubes6-
obviously
hemophilic
(huantitatively
18
offset
and
the
and
excess
(1cm-
Ac-globulin
that
tests
are
carried
of excess
of inhibitors
Ac-globulin,
alike.
contact
potency
Isemoand
with
glass
comparable
make
the
VI)
of the accelerator
In hemophilia,
obtained
and
with
conversion
in nonactivated
accounting
for
(2) the relatively
(us-
results
in
conversion
this
dilutions,
out
of
from
in hemophilic
in
either
highly
thromboplastin;
and
since,
Thus
the
the inhibitor
more
the
is
thrombo-
with
certain
surfaces
are
therefore
inhibitor.
Once
this is achieved,
the
as
addition
the
he the
actually
of
(factor
from
between
may
would
Greater
active
qualitatively
hemo-
originates
slow,
thereby
to thrombin,
of inhibitor.
plasma
action
normal
This
Release
to thrombin.
the
The
or after
That
amply
fresh hemophilic
serum.33
or excessive
thromboplastin)
dissociating
an accelerator/inhibitor
is as
why
at
Ac
in
or a longer
contact.
accelerator
from
the
accelerator
becomes
clear then,
found
to be normal.
glass
of an
and
plasma
differences
complex.
amount.
Ac-globulin
or plasma
unusually
hemophilic
prothrombin
complex
(Ac-globulin/ant.icephalin?).
rapid
conversion
of prothromhin
slowed
plasma.
V) to serum
Ac-globulin
then,
the
Ac-globulin.
has been
euglobulin
and more
prolonged
of clot, accelerating
greater
to
glass
inhibitor
euglobulin
therefore
similar
require
with
from
the
in the Ac-globulin
with a greater
of
contact
the
Therefore,
expression
of a qualitative
change
philic
Ac-globulin
may be conjugated!
surfaces
plasma
prothrombin
antid’ephalin
perhaps
plasma
method!)
globulin”
if prepared
from
a sufficiently
plasma,
will have as strong
a clot accelerating
hemophilic
normal
method)
normal
accelerat.e
Phase
the
of hemophilic
and
to
of dilution,
the
time”
hemophilic
dissociation,
‘
dliluted!
between
If
thromboplastin
and normal
prothrombin
two-stage
conversion
of the latter
into active
serum
has a normal
content
of plasma
Ac-globulin
effect
thromboplast.in,
convelsion
if hemophilic
effects
its
this
globulin”).
“prothrombin
Smith
put
that
time
by a strong
is employed
to
Yet.
“antihemophilic
(1 to 200 or 500) hemophilic
onstrated.6-
clot,
to
upon
Likewise,
in the
tend
platelets,
of prothrombin
cephalin
all
for some
of
one-stage
rate
cumulative
in excess,
a disadvantage,
Quick
737
HOLBURN
(“antihemophilic
of conversion
when
H.
as shown
by several
workers,
its
into thrombin.
Coagulation
is deinto contact
with asbestos
or similar
as acted
observed.
rate
even
thromboplastin
number
(as
R.
known
excessive
the
or mole
in the
been
but,
as well
be
has
content
transformed
or brought
(as in the
will
200
It
AND
euglobulin
between
observed,
conversion.’2
an
is diluted
in glass
ale
by
normal
or no difference
CARROLL
plasma?
plasma
has
prothrombin
layed unless
plasma
T.
edlualize
in
t.he
normal
plasma
diluted
both
t.he
dissociated!
plasma.
has
plasmas
in
procedures
performance
form,
It
been
of
they
the
are
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
738
ACCELERATING
CL(1)T
EFFECT
OF
DILUTION
ON
BLOOD
SUMMARY
1)ilution
with
physiologic
coagulation
of properly
silicone
coated
vessels,
platelets,
thromboplastin,
When
normal
rate
of clotting
plasma
of the
deficiency
in
in
excess
of
thrombin
any
a
effect
vithi
a procoagulant
of released
coagulants
accelerates
the
used,
the
and
rate
of coagulation
by
the
support
slows the
mechanisms
its
of hemophilic
appropriate
blood
or
concept
dilution.
plasma
of a
of the
presence
conversion
of prothromhin
: (1) reducing
or inactivating
(antit.hromhoplast.in
maintaining
20 per cent,
in prothrombin.
of diminution
that
of normal
plasma
the existence
in hemophilic
factor,
thereby
fluids
of about
because
inhibitor
which
of the following
l)y one or both
other
a concentration
agent
to
procoagulant
stabilizing
the
glass
principally
equal
against
and
and hemophilic
blood! and plasma
in
the aid of activating
agents
such
as
particles
or plasma
euglobulin
fractions.
without
under
activating
can he made
findings
speak
solution
normal
diluted
is
is prolonged,
Regardless
plasma
These
saline
collected
with
or
cephalin,
activity)
it in an
inactive
(2)
form
to
conjugation
(anti
Acglohulin
activity).
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1951 6: 720-739
The Clot Accelerating Effect of Dilution on Blood and Plasma. Relation to
the Mechanism of Coagulation of Normal and Hemophilic Blood
L. M. TOCANTINS, R. T. CARROLL and R. H. HOLBURN
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