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Fibrin
Suspension
Assay
By
A
GROWING
of methods
plate
is
methods,2
nics
using
The
other
purpose
fibrinolytic
technic
fibrin
of
this
study
by
the
particles
was
the
also
natural
been
to
examine
substrate.
devised,
the
in turbidity
fibrin
has given
rise to a variety
agents
in vitro.
The
fibrin
as
Other
well
as tech-
as casein.
change
of
agents
of such
have
such
Activity
Kuui
employing
clots,
substrates
activity
suspended
in fibrinolytic
the activity
useful
formed
employing
of
a
Fibrinolytic
AND D.
A. E. DR
INTEREST
for assessing
assay1
for
under
possibility
resulting
the
of
from
influence
measuring
the
of
fibrinolytic
used
as the
dissolution
enzymes.
METHOD
In
this
ever,
work,
fibrin
gardless
of
from
To
was
the
5 hours.
final
wash
mass
When
Blendor
fibrin
cheesecloth.
dried
by
to
in
a very
approximately
fine
16
approximately
5
mass
and
grinding
liter
it
mortar
below
fibrinogen
a
volume
allowed
from
of
in
with
water
was
expressed
into
gel
distilled
used,
for
N.I.H.
a period
water
fibrin
small
distilled
20
to
were
re-
described
approximately
fibrin
subdivided
was
and
by
This
as
in
repeatedly
excess
then
slowly
How-
unchanged
Bovine
solution,
the
washings
was
prepared
laboratory.
this
washed
the
was
substrate.
remains
to
remove
remaining
by
in
pressing
portions
and
powder
in
the
dried
in
pentoxide.
powder
hours.
and
brittle,
a
diameter.
.t
per
six
technic
dissolve
To
washing,
The
thoroughly
to
solution.
then
phosphorus
our
allowed
Usually
After
over
or
was
by
fibrin.
added
was
The
fibrin
bovine
cent
mass
overnight.
dessicator
subdivided
of
The
was
were
origin
desired.
Human
of
1 per
contaminants.
in
a vacuum
a
if
) produced
source
thrombmnf
other
fibrin
make
used
employed.
fibrmnogen
to
and
be
( human
the
or bovine
human
may
fibrin
as
fibrin,
bovine
4 to
salts
of
used
sufficient
of
of
type
prepare
units
either
source
Fibrinogen
I
water
of
any
the
Dried
fraction
fibrin
from
grinding
process
The
reduced
pestle.
to
The
in
a
a mortar
yields
fibrin
fibrin
grinder
a powder
powdered
coarse
powdered
may
or
with
an
be
stored
was
ball
mill
average
a
Waring
then
further
for
a period
particle
in
a
size
dry
state
of
until
required.
Suspensions
medium
may
in
were
made
such
a suspension
2
by
parts
with
turbidity
of
various
was
1 part
the
with
diluted
to
a final
tained
24.2
Gm.
25
and
pH
of
ml.
1963;
Research
accepted
(Armour
Thrombin-(
8.0.
stock
with
powdered
of
ml.
this
Tris
in
the
been
For
at
prepared
solution
distilled
Tris
water.
Laboratories,
stock
Dcc.
turbidity
follow,
M
the
initial
transmission.
Tris-HCI
solution
40
of
diluted
ml.
solution
buffer
of
pH
7.4
of 0.1
N
HC1
of
Tris
con-
liter.
Unitersity
publication
.05
suitable
suspensions
\Vhen
cent
buffer
plus
The
per
per
in
a
of
m/t.
to
20
a
all
The
620
procedure
example,
with
study,
buffer.
transmission
aminomethane
for
fibrin
In
approximately
have
and
Pharmaceutical
Parke,
per
cent
was
fibrin
7.0
the
5 minutes.
described
(hydroxymethyl)
Medical
28,
as
incubation
bovine
100
for
per
a concentrated
of
mixing
fibrin
14
between
volume
Tris
to
by
rpm
powdered
solution,
prior
ml.
of
#{176}Bovine Fibrinogen
(Topical)
test
human
From
Connaught
tario, Canada.
Submitted
June
Bovine
of
of
of
15,000
approximately
mixture
of
prepared
at
1 mg.
read
levels
made
simply
Blendor
adding
Suspensions
at
be
a \Varing
of
‘loronto,
loronto,
On-
3, 1963.
Co.).
l)avis
& Co.,
Ltd.)
729
BLOOD,
VOL.
23,
No.
6
(JuNE),
1964
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
730
DYER
Most
of
the
aggregation
sions
7
work
of
prepared
days
in
during
large
which
does
occur.
Redispersion
tion
during
tests
water
bath.
The assay
Six
to
ml.
with
a
transferred
was
placed
\Vith
tested.
permits
be
carried
each
by
allowing
contents
of
assist
of
as
control.
the
30
by
delay
is needed
the
with
same
between
at
were
Tris
ference
the
a
2.
buffer.
Response
the
called
Human
per
estimated
settling
even
to
distribu-
in
a
shaking
to
of
was
each
an
was
well
to
37
each
aliquot
of
continually
with
at
fibrinolytic
added
tube
which
rapid
pace
same
period.
and
a
5
mixed
Vortex
C.
length
of
care
must
our
work
In
additions
same
additions
level
mixer
A glass
bead
incubation.
between
the
each
mixed
of
of
fibrin
time
and
therefore
this
be
was
ac-
suspension.
This
allows
sufficient
addition
of an inhibitor
at the
When
this approach
is feasible,
fibrin,
as
the
was
average
recorded
as
readings
the
of
per
of
inhibitor
incubation.
5 and
a wave
cent
In
60
minutes.
may
be
added
this
study,
length
of
transmission
620
oryzae,
mj.,
for
the
using
as
control
the
in
a
a blank
against
calculated
for
in-
However,
transmission
in turbidity
change
cent
tubes
containing
the
agent
under
test.
The following
two
fibrinolytic
agents
were
studied:
1. A proteolytic
enzyme
extracted
from
Aspergillus
50
up
some
rapidity.
between
presentation
no
suspen-
periods
out
solutions
maintained
It is possible
with
this
procedure
to vary
the
length
cubation
has been
carried
out for periods
of between
most cases,
a 15-minute
interval
was
selected.
Turbidity
was measured
as per
cent
transmission
at
Coleman
Universal
Spectrophotometer
set
at 100 per
o
ensure
carried
to
Then
bath
the
for
successive
levels
of
for
To
above
time between
successive
tubes
to measure
turbidity.
As an alternative,
the reaction
may be stopped
by the
end of incubation.
Readings
then may be made
at leisure.
no
these
storage
always
and
the
during
seconds
tube
At
Aliquots
intervals
shaking.
volume
tube
water
precisely
of
each
of
agitation
for
7.4.
During
solution
a bulk
proceeds
tube
at
was
each
used
from
or
storage.
KADAR
follows:
of
added
a lapse
incubation
as
control
ml.
8.0
or
testing
easily
out
was
fibrinolysis
each
for
incubation
to a shaking
to
pH
unchanged.
the
2.5
buffer
tube
technic,
the
to
of
The
used
remained
agents,
was
either
incubation
accomplished
assigned
of
at
been
turbidity
A volume
incubate
complished
have
stirrer.
in
to
volumes
immediately
this
taken
during
was
were
magnetic
and
out
either
fibrinolytic
The same volume
of fibrin
suspension
tUl)e.
carried
can
with
tubes
be
been
occurs
time
procedure
test
agent
has
particles
AND
the
dif-
tubes
purpose
and
of
this
Aspergillin-O.
with
glycerin.
The
cent
glycerin
and had an
by the method
of Sguoris
plasinin
activated
activity
et al.3
equivalent
solution
of
to
115
plasniin
was
stabilized
caseinolytic
units
with
per
ml.,
RESULTS
Aspergillin-O
and
A suspension
containing
Human
of human
this
Fibrin
fibrin
suspension
was
were
prepared
incubated
Aspergillin-O.
The lowest concentration
the final mixture.
A two-fold
increment
five dose
levels
between
bation,
read as per cent
The
identical
Lion indicates
the readings
measurements
sistent
finding
turbidity
in
.005
and
transmission,
of
the
that
no spontaneous
table
1 reveals
at each
level.
Low
with
this
technic.
in Tris
with
buffer
a
at pH
purified
of enzyme
was .005 mg.
between
doses
was used
that
tubes
read
before
and
replicate
particularly
of
after
1.
after
fibrinolysis
occurred.
Examination
good
replication
was
achieved
variation
between
This
feature
is
Tubes
per ml. in
to provide
.08 mg. per ml. The
turbidity
is shown
for each tube in table
control
8.0.
preparation
incuincuba-
of all
between
tubes
is a conattractive
be-
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FIBRIN
SUSPENSION
1.-Turbidimetric
Table
Control
Control
incubation
Tube
-----
mg./ml.
Change
r/ T
Average
-------
2
3
4
5
6
‘I’
20.0
20.5
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.5
20.0
20.0
20.1
27.5
28.0
28.5
29.0
27.5
28.0
28.1
8.0
34.0
35.0
35.0
.16.0
35.5
35.0
35.1
15.0
0.02
mg./ml.
45.0
46.0
44.0
48.0
47.0
46.0
46.0
25.9
0.04
mg./mI.
56.0
57.0
56.0
57.0
57.0
56.0
56.5
36.4
60.5
62.5
62.0
62.5
63.0
63.0
62.3
42.2
0.08mg/mi.
cause
it means
sion
that
only
minimal
replication
Bovine
Fibrin
is required
to yield
good
preci-
of estimates.
Aspergillin-O
and
Bovine
fibrin
to that
analysis
can
this
of
be
seen
for
The
results
graphically
ment
was
on
incubated
described
obtained
data
that
each
reaction
1. For
fibrin
l)e seen
that
and
that
values
table
with
Plasmin
To
the
test
sion.
A
with
prior
ml.
with
either
portion
of
evident
that
This
dose-response
and
statistical
table
2.
lambda
It
was
of
experi-
figure.
a dose-response
embraces
a four-fold
parallelism
is significant
as
by
between
shown
of these
curves
some
difference
fibrin
illustrated
the
this
yields
curve
plasmin
in
Since
0.5
ml.
curves
of
50
the
buffered
human
by
the
presented
exists
in
in
the
Aspergillin-O.
each
solution
7.4.
The
and
plasmin
intervals.
interval.
suspen-
prepared
in acid
dilution
pH
time
the
glycerin
by
with
slightly
with
stable
plasmin
at 5-minute
at
modified
mixing
cent
made
at
fibrin
measured
recorded
per
to
is reasonably
were
of
was
prior
plasmin
a suspension
turbidity
procedure
activity
testing
the
are
for
in
is a lack
bovine
of
for
in
fibrin
included
portion
that
the
loss
0.01 N HCI.
to incubation,
and
in
for
results
substrate
there
linear
implies
human
human
used.
ceeded
at a physiologic
p11.
In one test,
the mixture
25 minutes
fibrin
value
bovine
each
difference
concentrations
of fibrin
is also
linear
of plasmin,
of
was
appropriate
identi-
of the
Fibrin
preventing
solution
laboratories
solution
Just
of
activity
of
the
on
above
curves.
Human
the
human
by
a plot of the
reaction
the
fibrinolysis
purpose
conditions
summary
for
of Aspergillin-O
It is also
and
that
comparison,
expressing
the slope
of the
2. This
lack
of parallelism
of
The
expressed
described
dose-response
nature
under
fibrin.
experiment.
in figure
of doses.
Aspergillin-O
human
precision
of the
human
two
for
is presented
good
It can
range
with
above
relationship
the
-------
1
mg/mI.
0.01
7.0
__________
Transmission
0
0.005
our
(;
Fibrin
after
Aspergillin
the
Readings
of Human
_____
before
incubation
the
Readings
of a Suspension
Incubated
with Aspergillin.O
--
Dose
cal
731
ASSAY
of
was
was
the
then
incubated
Figure
These
stock
with
mixed
reaction
by
medium,
profor
2 shows
results
pro-
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732
DYER
Table
of Analysis
of Aspergillin-O
Human
and Bovine
Fibrin
2.-Summary
Fibrin
vide
Slope
±
1.73
0.7360
0.02069
52.4
±
2.88
1.506
0.0287
evidence
that
and
concentration
minutes.
any
time
interval
of
the
Interpolation
plasmin
for
use
the
the
activity
the
plasmin
through
that
that
obtained
the
breakdown
were
result
or
of
that
for
simply
to
the
sensitivity
with
gains
as
cited
an
not
of
15
units
as
increased
comparable
presumably
spontaneous
are
case
of
0.15
turbidity
findings
in this
purpose
period
of
the
sufficiently
the
incubation
in
to
is
for
incubation
change
These
3 times
periods,
an
same
according
technic
periods
that
15 minutes
It appears
incubation
of
chosen
approximately
of 5 minutes.
longer
selected
The
incubation
2 reveals
minutes.
for
measurement
period
5
for
incubation
short
in
be
used.
arbitrarily
figure
resulted
incubated
that
may
enzyme
of quite
of
15 minutes
units
illustrate
with
Lambda
X = S/b
S
35.6
sensitive to permit
tion
=
KADAR
on
Bovine
of assessing labile materials.
In our
studies,
we have
of
S.D.
Reaction
Human
nature
0.50
b
=
AND
incubaachieved
the
inhibition
loss
from
of
by-
products.
Aspergillin-O
In
assay
this
was
versus
study,
Human
the
carried
Plasmin
activity
out
on
of Aspergillin-O
aliquots
of the
was
same
compared
suspension
to plasmin.
of human
fibrin.
The
A
z
2
60
-
In
In
50..
1
z
4
40..
30..
O
z
20
4
I
o
l0
I
.005
I
.01
.04
.O
.08
MG / MI/TUBE
Fig.
1.-Change
iii
turbidity
of
suspensions
bated
with various
levels of Aspergillin-O.
for 15 minutes
at pH 8.0.
of
The
human
mixtures
and
were
bovine
incubated
fibrin
incuat 37 C.
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FIBRIN
SUSPENSION
733
ASSAY
z
z
9
o10-
0.125
0.15
0.25
0.5
CASEIN
Fig.
2.-Change
various
levels
minutes
at
pH
plot
of the
that
certain
in
human
of
7.4.
results
turbidity
plasmin.
of
suspensions
were
is shown
in figure
similarities
exist
human
/ML./TUBE
fibrin
incubated
The mixtures
were
incubated
made
at 5-minute
intervals.
Readings
two enzymes.
Both
enzymes
appear
of
UNITS
in
3. Examination
both
at
of these
qualitative
and
37
with
C.
results
for
25
suggests
quantitative
properties
of the
magnitude.
incubation
could
Because
than
would
present
an
From
the
similarity
the
hydrolysis
similar
Because
of
therapy
this
a
In
technic
to human
or
fibrinolytic
as
by
relative
activity
Aspergillin-O,
of
order
of
observed.
an
longer
of
duration
incubation
duration,
likely
possibly
of Human
it
is
of
The
fibrin
of
accurately
major
Changes
in a fashion
assessing
interest
be
as
may
he
suspended
in turbidity,
the
a means
of
particularly
the
of
result
other
possiguiding
useful
in a variety
as
independently
fibrinolytic
examine
to
thereof,
technic
may
measured
note
that
Plasma
modification
agents.
proceeds
to
plasmin.
a means
time,
some
readily
than
Aspergillin-O
Presence
or serum.
be
the
same
the
comparison,
it is of interest
the two
reactions.
This
suggests
enzyme,
powdered
the
plasma
this
of
the
value.
provides
space
this,
may
anticoagulant
fibrin
of
enzymes,
particularly
of a qualitative
the slopes
for
in the
short
on
stable
different
Activity
with
activity,
influence
chosen,
physiologic
applying
respect
including
entirely
activity
different
less
one
of the
this
in
the
of human
Aspergillin-O
activity
a major
standpoint
between
to that
fibrinolytic
comparing
is inherently
plasmin
other
the
when
exert
period
bility
to possess
However,
of
factors
in
of media,
fibrinolytic
such
as
activity.
regard,
a preliminary
plasma
was
incubated
test
with
was
done
a suspension
in
which
Aspergillin-O
of l)ovine
fibrin.
added
The
final
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734
DYER
AND
KADAR
0
41
z
0
VI
VI
VI
z
4
I-
z
w
0
z
4
I
U
.01
.02
Fig. 3.-Comparison
Both reactions
were
mixture
57
summarized
plasma
the
plasma
initiating
of
form
for
during
and
/TUBE
plasmin
and
at pH
possessed
control
Aspergillin-O.
7.4.
a fibrinolytic
without
made
to
the
purpose
of
predicting
Also,
blood
levels
of
therapy
b’
plasma.
activity
These
of
results
are
in figure 4.
might
therapy.
measured
Effect
cent
in graphic
a patient’s
to
25 per cent
per
determinations
Similar
.11
MG/ML
.056
of fibrinolvsis
of human
fibrin
incubated
at 37 C. for 15 minutes
contained
approximately
.04
.028
be
by the
same
assess
the
the
inhibiting
the
desirable
potential
of
dosage
fibrinolytic
agent
prior
might
be
technic.
of pH
It was
Use
agents.
feature
For
was
that fibrin suspensions
found
of this
has
example,
been
the
to study
potency
compared
to a crude
shown
in figure 5, the
stable
remain
made
extract
the
of
a purified
at
various
a wide
over
influence
preparation
levels
range
of pH
of
of pH.
fibrinolytic
Aspergillin-O
between
pH
of
on
and
7.0
8.0.
As
the
crude
that
the
product
pH
optima
of the
as
two
A similar comparison
of
ous levels
of pH. In contrast
potency
relative
indicating
Effect
(a)
man
that
of
potency
increased
of the
the
two
two
of the
the
level
preparations
two
pure
of
pH
differed
material
was
relative
raised,
in some
to
which
that
of
suggests
respect.
purified
extracts
was also carried
out at varito the findings
of the previous
experiment,
the
purified
possessed
materials
similar
pH
remained
the
same
at all
levels,
optima.
Temperature
Heating
or bovine
before
fibrin
incubation:
suspended
The
in Tris
dispersion
buffer
is not
of
particles
adversely
of
affected
either
by
huhigh
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
FIBRIN
SUSPENSION
735
ASSAY
Buff.r
41
z
0
VI
VI
VI
z
30
4
I-
125
%
Plasma
20
w
0
z
4
I
U
1
.04
.046
.08
MG
Fig.
4.-Comparison
that of a mixture
with
or
low
temperatures.
activator
effect
of
incubation
heated
served
It was
resulting
the
This
of Aspergillin-O
human
in
a
buffer
solution
plasma.
desirable
to
destroy
tests
were
undertaken
of bovine
fibrin
for 10
Aspergillin-O.
that
the
incubation
A portion
initial
with
turbidity
Aspergillin-O
is illustrated
explanations
of
the
enzymatic
or
to examine
minutes
at
the
80 C.
same
suspension
un-
was
was
in figure
for
the
unaltered,
yet
significantly
the
change
reduced
by
6.
decreased
fibrinolysis
of
the
heated
themselves.
change
susceptibility
2. The
fibrin
tion
be
may
suspension,
a suspension
observation
present
of which
it
a
with
possible
1. Some
in
activity
per cent
25
/ TUBE
as control.
found
from
heating.
Two
Since
activity
preheating
before
fibrin
of fibrinolytic
containing
/ML
would
of the
associated
with
intramolecular
alteration
of heated
fibrin
to Aspergillin-O.
might
contain
residual
quantities
contribute
plasminogen
to the
by
fibrinolytic
heat
would
of
effect
then
might
plasminogen,
activation
of Aspergillin-O.
remove
this
reduce
Destruc-
component
of
the
system.
(b)
Heating
temperatures
enzymes
after
incubation:
presented
following
immersion
The
water
observations
by
in which
Aspergillin-O
boiling
made
bath,
with
of
means
arresting
of heat.
and
bovine
readings
also
To
fibrin
were
1 hour
are
and
exposed
fibrinolytic
test
suspensions
of bovine
and
then
immersed
bath. Turbidimetric
in the
stability of suspensions
possibility
incubation
ments
were
performed
first incubated
with
boiling
The
the
made
this
and
for
summarized
aspect,
after
in graphic
of
experi-
human
fibrin
3 minutes
immediately
20 hours
to high
activity
were
in a
prior
to
immersion.
form
in
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
DYER
736
AND
KADAR
w
a
UI
II.
5.0
-
a.
U
z
4.0
UI
>
4
3.0
-I
UI
I
I
7.0
7:4
7.2.
76
7.8
pH
Fig.
5.-The
of Aspergillin-O
figure
7.
relative
potency
at various
levels
The
findings
preparation.
This
procedure
pergillin-O,
and
at
of
these
20
However,
resulted
heating
in an
proportion
to
slope.
This
taken
before
of
after
curves
partial
rials.
assay
7 by
heating.
Perhaps
after
It permits
of
resulting
close
of
at
an
readings.
mixture
which
increased
in
overall
curves
decrease
for
the
this
is the
result
of
reprecipitation
of
which,
in
the
studies,
it does
not
incubated
negate
inactivation
for assays
involving
identical
this
modification
may
he useful
for
readings
in
readings
proportion
of fibrinolysis.
for some
to he
made
at leisure
and
As-
1 hour
approximation
between
on the
turbidity
in
bovine
action
the
the
the
the
readings
checked
extract
also
the
materoutine
makes
it pos-
if necessary.
Inhibitors
Because
cases
readings
from
comparing
the
the amount
is undesirable
work.
Effect
fibrinolysis,
products,
With
to repeat
seen
a crude
with
the
incubation
exerts
some
effect
in turbidity.
Furthermore
utilizing
heat
this
qualification,
sible
that
for
remained
of
to
arresting
shown
be
in figure
mixture,
likely
parallels
Although
this effect
of
can
seen
breakdown
possibility
It
fibrinolysis
compared
paralleled
completely
results
amount
be
and
the
heating.
that
extract
fibrin
in
by
after
increase
the
can
human
successful
evidenced
hours
two
with
was
as
of a purified
of pH.
VALUES
to halt
of
the
rapid
rate
fibrinolysis
inactivation
presents
an attractive
alternative.
at
one
of reaction,
the
end
possibility.
it would
of
incubation.
The
use
of
be
an
As
described
enzyme
advantage
inhibitors
in
above,
some
heat
presents
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
FIBBIN
SUSPENSION
737
ASSAY
,at.d
z
0
;;
30
VI
VI
z
4
20
I-
-
z
UI
0
z
4
I
U
I
I
.0056
Fig.
6.-A
sion
of
for
comparison
fibrin
divided
10 minutes
Of
a group
to be
an
of
only
out
with
cases,
human
0.2
to each
ately
type
ml.
tube
I ML / TUBE
activity
one
with
.0189
MG
portion
the
of
protease
inhibitors
agent
of Aspergillin-O.
are
shown
in
of fibrin
utilized.
fibrin
one
in
following
cent
incubation.
adding
the
bovine
solution
of DFP
Readings
inhibitor,
and
was
on
a suspen-
preheated
only
The
8.
figure
and
Aspergillin-O
studied,
Incubation
test
of
which
at
80
C.
enzyme.
blocking
of a 10 per
before
fibrinolvtic
parts,
.0126
common
this
in the
the
two
to incubation
effective
demonstrating
of
into
prior
I
.0084
The
two
with
Aspergillin-O
fibrin
in the
studies
differed
was
other
in methyl
and
found
was
of two
experiments
were
20
hours
DFP
in
carried
test.
alcohol
of turbidity
1 hour
DFP
results
In
was
made
both
added
immedi-
after
the
addi-
tion.
In
was
tive,
the
above
experiments,
the
concentration
of
2.6 mg./ml.
Further
studies
revealed
that
1.3
whereas
lower
levels
resulted
in only partial
Unlike
DFP
heating,
as
produces
only
easily
performed
a means
slight
of
change
arresting
in the
mg/mi.
inhibition.
enzyme
the
activity,
incubated
final
was
mixture
equally
the
effec-
addition
of
mixtures.
DIscUssioN
An
been
described.
estimates
dence
vantage
may
of
In
than
other
this
precision
that
be varied
method
our
to suit
experience,
methods
has
conditions
the
of rapidly
the
measuring
technic
employing
fibrin
has
as
been
presented.
The
such
as
temperature,
pH
needs
of the
experiment.
fibrinolytic
provided
a substrate.
method
and
activity
more
Statistical
also
length
has
of
has
accurate
evithe
incubation
ad-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
738
DYER
z
UNHEATED
0
1
6C
In
-
HR.
20
AFTER
HRS.
AND
KADAR
HEATING
AFTER
HEATING
In
50..
In
z
4
40
-
30
-
20
-
11
z
Ui
0
z
4
I
10
0
I
.01
.005
I
.04
MG
.02
.08
/TUBE
I ML
Fig. 7.-The
effect of using
heat
to arrest
fibrinolytic
activity.
A suspension
of
fibrin
was incubated
with
five levels
of Aspergillin-O
and turbidimetric
readings
made
following
incubation
immediately
prior
to immersion
of the mixtures
in a
boiling
water
bath,
and also 1 hour and 20 hours
after immersion.
the
In
description
guiding
for
determining
type
of results,
therapy
the
with
inhibiting
of assessment
may
prior
initiating
agents
to
be
of
has
as
larger
freeze-drying.
Thus
in quantity.
Widespread
prepared
distilled
a uniform,
stable
use
of such
a safe
dosage
desirable
as
This
of fibrinolytic
well
to
assess
developed
Tris
with
any
activity
along
with
a lyo-
buffer.
This
desired
medium
of a sample
of
We
as
a
before
amount
standard
curve
agent.
suspension
water
pH
have
for
presented.
containing
of fibrinolytic
and
was
technic
a test
turbidity
of such
a mixture
vials
containing
a standard
containers.
turbidity
of this
of
serum
shaking
fibrinolytic
the
use
example
therapy.
we
standardized
a
with
size,
be
fibrin
simply
The
been
to level
in
particle
by
thereof.
purposes,
resuspended
to
may
measuring
this purpose,
in turbidity
assay
an
predicting
subdivided
have
lyophilized
preparations
tics
by
For
substrate
general
been
finely
to the
and
patient’s
serum
during
of work
in particular,
be resuspended
or fractions
change
For
It
may
serum
lyophilized
relating
for
therapy.
of
serum
may be estimated
and
after
incubation.
made
of a patient’s
essential
preparation
preparation
including
was
agents,
potential
fibrinolytic
activity
in the
To facilitate
this
type
philized
reference
fibrinolytic
have
of
found
possess
the
the
original
preparation
for
a preparation
fibrin
that
in
same
characteris-
suspension
assay
could
can
buffer
lyophilized
prior
be
to
produced
standardize
testing
procedures.
SUMMARY
A new
method
method
is based
has
on
been
the
described
change
in
for
turbidity
measuring
resulting
fibrinolytic
from
activity.
the
dissolution
The
of
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
FIBRIN
SUSPENSION
739
ASSAY
BEFORE
60
.
.-----
1 HR.
__
20
DFP
ADDED
AFTER
HRS.
DFP
AFTER
DFP
Bovin.
50.
In
z
Fibrun
4
0.
I-
Human
.
Fibrin
30.
Ui
0
20..
z
4
I
0
10..
.01
.02
.04
MG
Fig.
8.-The
effect
and
bovine
human
turbidimetric
and
suspended
were
changes
accurate
also
following
and
of
technic
exposes
method
and
have
been
The
also
length
of
fibrin
a
hours
the
following
two
the
vast
that
sed
fibrina
describite
un
es basate
fibrinolytic
of
substrate,
agents.
Be-
measurable
yet provides
as a substrate.
such
Experiments
of
and
addi-
as
more
temperature,
illustrating
this
oryzae.
nove
provide
como
IN
methodo
de
plus
accurate
glycerina
INTERLINGUA
pro
in le alteration
particulas
studied.
glycerin.
del
fibrina
mesurar
activitate
turbiditate
sub
le
estimationes
fibrinolytic.
ab
le dissolution
de
agentes
fibrinolytic.
superficie
de
Le technica
que
Le
resultante
influentia
vaste
area
de
rapidemente.
altere
substrato,
alteranon es complexe,
methodos
utilisante
substrato.
Le methodo
ha etiam
le avantage
e duration
del incubation
pote
esser
facto
es reportate.
Le duo agentes
fibrinolytic
studiate
con
of
conditions
were
with
que le technica
expone
un
de grado
mesurabile
occurre
illo
Suspensions
of Aspergillin-O
prior
to the
added.
complex,
fibrin
varied.
agents
activated
of Aspergillus
suspendite
Viste
tiones
was
area
is not
employing
be
SUMMARIO
de
immediately
DFP
surface
can
activity.
levels
influence
advantage
fibrinolytic
plasmin
2. An extract
Es
three
after
procedure
methods
incubation
fibrinolytic
incubation
20
/TUBE
reported.
1. Human
methodo
arrest
with
under
The
other
has
to
incubated
made
occur
rapidly.
estimates
than
The
DFP
1 hour
particles
the
cause
pH
using
readings
of DFP,
tion
of
fibrin
/ML
e (2)
un
extracto
de
que
factores
como
variate.
Experimentos
esseva
Aspergillus
(1)
plasmina
oryzae.
temperatura,
que
illustra
human,
pH,
iste
activate
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
740
DYER
AND
KADAB
ACKNOWLEDGMENTS
We
in
wish
this
to
work.
excellent
express
our
Thanks
gratitude
are
technical
also
to
due
to
Miss
Mr.
A.
C.
B.
H.
Lewis
for
Holliwell
her
and
interest
Mr.
and
R.
assistance
Harding
for
their
assistance.
REFERENCES
T.,
and
Astrtip,
2.
I)late method
for estimating
fibrinolytic
activity.
Arch.
Biochem.
40:346,
1952.
Lllscher,
E.
F., and
Kaser-Glanzmann,
R.: A new method
for the quantitative
determination
of fibrinolytic
activities,
the
on
based
Mullertz,
The
1.
Allan
use
of
Ph.D.,
head,
University
Kadar,
Control
B.Sc.,
and
HEREDITARY
WITR
Two
Research
DEFIcIENcLE5
L. Pechet.
From
the
270:6,
1964.
hundred
and
Vox
OF
of
Medical
of Toronto,
Sang.
Toronto,
Assistant,
6: 116,
Canada
individuals
of Quali-
Medical
Toronto,
VII
B. Alexander,
Boston,
AND
Mass.
belonging
Research
Canada
FACTORS
TuMoRs.
A. I. Kroll,
Beth
Israel
Hospital,
forty-two
Control
Labora-
Department
of Toronto,
CLOVrING
Quality
Research
Connaught
University
CAROTID-BODY
J. Med.
Department
Pharmacology,
Laboratories,
a substrate.
1960.
Connaught
tories,
ty
as
to
four
X ASSOCIATED
F. Cochios
and
New
England
generations
of
a family
were
studied
after
the finding
of a carotid-body
tumor
and deficiencies
of factors
VII and X in one member
of the
family.
Sixty
demonstrated
mild to moderate
factor
X deficiency
and 28 of these
also had a
mild deficiency
of factor
VII. Factor
VII deficiency
was not encountered
when
factor
X levels
were
normal.
Twelve
individuals
belonging
to two
generations
of one branch
of the family
had
carotid-body
tumors
and
eight
was
of
these
autosonial
be autosomal
found
to be
-R.
G.
also
dominant
had
clotting
defects.
whereas
that
intermediate.
The
particularly
useful
for
1961.
:3. Sgouris,
J. T., Inman,
J. K., McCall,
K.
B., Hyndman,
L. A., and Anderson,
H.
I).: The preparation
of human
fibrinolysin
(plasmin).
Vox
Sang.
5:357,
fibrin
Pharmacology,
Dezso
fibrin
fluorescent
Dyer,
E.
and
S.:
The
of
the
transmission
clotting
whole-plasma
detecting
the
defects
prothrombin
coagulation
of
the
appeared
tumor
to
time
was
abnormality.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1964 23: 729-740
Fibrin Suspension Assay for Fibrinolytic Activity
A. E. DYER and D. KADAR
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