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Activated
Factor
Concentrates
Circulation
By
Un
Seligsohn,
Carol
VII:
and
K. Kasper,
Presence
in Factor
Persistence
in the
After
Infusion
Bjarne
Factor
IX concentrates
were evaluated
for
their
factor
VII content
and
for
the
presence
of activated
factor
VII through
use of a coupled
amidolytic
assay
insensitive to activated
factor
VII and a clotting
assay sensitive
to activated
factor VII. The
factor
VII content
of the concentrates
studied
(except
for one concentrate
purposely
produced
to exclude
factor
VII)
varied
between
33 and 621
U/vial.
All
concentrates
contained
activated
factor
VII. as indicated
by ratios
of factor
VII
clotting
activity
to factor
VII amidolytic
activity,
of from 1.6 to 21.5.
Higher ratios
were
found
in two
brands
of activated
concentrates
than in nonactivated
concen-
T
HE PRESENCE
concentrates
of variable
hampered
has
#{216}sterud,and
Samuel
IX
I. Rapaport
In patients
infused
with factor
IX
concentrates,
plasma
factor
VII activity
rose strikingly
in the clotting
assay but not
in the amidolytic
assay. Thus, the elevated
factor VII levels by the clotting
assay after
infusion
of factor
IX concentrates
stem
from
circulating
activated
factor
VII. A
mean
intravascular
half-disappearance
time of 144
mm was found for activated
factor VII. Its persistence
in the circulation
makes it important
to evaluate
the possible
role of activated
factor VII in the thrombogenicity
of factor
IX concentrates
and in
their
reported
effectiveness
in treating
bleeding
in hemophilia
A patients
with
inhibitors.
trates.
amounts
accurate
of activated
measurement
clotting
factors
of the content
in factor
IX
of clotting
factors
factor
in such concentrates,
can introduce
serious
because
error
clotting
forms:
factor
as the
VII may be present
in in vitro materials
in at least two
factor
VII molecule
and, after
minimal
proteolysis,
assays.
Factor
native
one-chain
even small
amounts
of an activated
clotting
into test results
obtained
with
conventional
as a two-chain
molecule
possessing
therefore
referred
to as activated
particularly
factors
levels
plasma
concentrates
known
increased
factor
to contain
(activated
factor
IX concentrates),
of factor
VII activity,
e.g., 6,000
factor
VII levels have been reported
into patients.35
Until
results
of the presence
We have recently
insensitive
From
the
Department
Administration
Angeles,
Submitted
Supported
Institute,
and
Dr. Seligsohn
Tel-Hashomer,
and
Tel Aviv
September
by
National
of factor
of Medicine.
Hospital,
Calif.
state
Orthopedic
University,
amounts
tissue
IX
of
factor”2
and
concentrates,
activated
clotting
When
factor
of California,
Hospital,
San
VII
Diego.
University
leave from
of
on such
VII
is measured
the San
Southern
Diego
test
that
is
both
by
Veterans
California.
the Chaim-Sheba
Medical
Los
Center,
Israel.
accepted
Project
Grant
Institutes
VII.6
was on sabbatical
27, 1978;
Program
large
with
factor
possible
to evaluate
the effect
VII in factor
IX concentrates.
amidolytic
assay
for factor
University
the
reactivity
Some
have been reported
to contain
very high
U/vial.3
Moreover,
striking
increases
in
after infusion
of factor
IX concentrates
now it has not been
of activated
factor
described
a coupled
to the activity
VII.
of Health.
January
5. 1979.
HL-18576
and
by a grant
from
from
the
National
the Hemophilia
Heart,
Lung,
Foundation
and
Blood
of Southern
California.
Address
reprint
requests
Hospital.
H81 1K, 225 Dickinson
© 1979 by Grune
& Stratton.
828
to
Samuel
I.
Rapaport,
M.D.,
Department
of
Medicine,
University
Street,
San Diego,
Calif
92/03.
Inc. ISSN
0006-4971/79/5305--0004$02.00/0
Blood,
Vol.
53, No. 5 (May),
1979)
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FACTOR
VIII FACTOR
IX CONCENTRATES
829
a clotting
assay and by this coupled
activity
state of factor
VII in a test
measurements
made
on different
report
the results
of measurements
amidolytic
IX
techniques
concentrates.
amounts
of activated
samples
be shown
factor
of plasma
that
VII,
factor
VII activity
after
permitted
the determination
vated factor
VII.
large
factor
was a saline
particles.
normal
It clotted
human
absorption,
plasma
DEAE
heparin-agarose
which
is responsible
NaCI
and 0.05-M
prepared
with
reference
standard,
pooling
from
Factor
normal
plasma
involving
was
Diagnostics,
containing
to contain
I unit
(U)
from
factor
significant
rise
in plasma
assays
has also
time of acti-
at 12,000
barium
sulfate
gel
bovine
serum
VII
activity
tubes
Louis,
Bio-Medical,
A
Salem,
VII
was prepared
N.H.
Bz-Ile-Glx-Gly-Arg-p-nitroanilide
was
factor
20#{176}C.
Hereditary
-
and
0. 15-M
(TBS-BSA)
Mo.).
per milliliter,
at
citrate
containing
albumin
St.
from
or barium
electrophoresis,
was a solution
(Sigma,
g to remove
X was prepared
(TBS)
in plastic
King
times
factor
polyacrylamide
of factor
George
X,),
marked
two
steps:
albumin
and was stored
X (factor
Raritan,
following
1 mg/ml
serum
males
centrifuged
in I 7 sec. Purified
saline
bovine
obtained
factor
for the
preparative
available
with
contain
METHODS
Tris-buffered
pH 7.5. TBS
infused
Use of these
half-disappearance
tissue
the
chromatography,
I 2 healthy
for activated
from Ortho
recalcified
assumed
plasma
substrate
brain
commercially
plasma
deficiency
of human
chromatography.
Tris,
AND
extract
cellulose
patients
IX concentrates
infusion
of concentrates.
of the intravascular
by a method7
column
from
factor
MATERIALS
Tissue
assay,
it is possible
to evaluate
the
We report
herein
the results
of such
types of factor
IX concentrates.
In addition,
we
of factor
VII activity
by the clotting
and coupled
in serial
It will
amidolytic
material.
The
by
factor
VII
chromogenic
(S-2222),
was purchased
N.J.
IX Concentrates
Commercial
producers
and
during
complex-human,
and
kindly
by
Dr.
X-l25,
provided
by Drs. J. Watt
Scotla:d,
X-l29,
X-l43,
lot numbers
Activated
Factor
314,
deliberately
prepared
Laboratories,
supplied
Dr.
Vienna,
Austria)
FEIBA
units
from
324,
made
Mesa,
Laboratories,
purchased
National
X-30l;
Calif.),
Blood
the Scottish
lot
Berkeley,
factor
National
as follows:
numbers
Calif.),
Paris,
concentrate
Blood
IX
581D198AA,
M-6500,
50 units of heparin,
Center,
IX
by the
factor
lot numbers
containing
Transfusion
human
or provided
used were
Coagulante-P.P.S.B.,
France,
(DE.FIX)
Transfusion
Service,
lot
kindly
Edinburgh,
and 327.
for use in treatment
to contain
Costa
by
the
and
either
preparations
IX Concentrates
were concentrates
These
from
were
The
Costa
Fraction
X-2l5,
and J. Cash
251,
1978.
(Cutter
and M-57I7;
J. P. Allain
numbers
concentrates
Laboratories,
Konyne
M-3682,
IX
to April
(Hyland
581EIO7AA;
M-6346,
provided
factor
January
Proplex
58IC206AA,
M-6418,
noncommercial
the period
Mesa,
increased
Calif.),
William
lot numbers
Thomas
was kindly
per bottle
in
supplied
(05A
and
March
F. Elsinger
05A2377,
A patients
of activated
581 Dl 34, 650D0l
February
by Dr.
1877,
of hemophilia
amounts
clotting
7, 650D03
1978.
inhibitors
was kindly
(Immuno
Three
250
were
(Hyland
I, and 650D02l,
1977.
one lot contained
that
Auto-IX
FEIBA-Immuno
in September
05A2477);
with
factors.
AG.,
lots contained
FEIBA
units
1,000
per bottle
(05A2277).
Reconstitution
and
Concentrates
instructions.
were
At
clotting
factor
At
Orthopedic
the
number
(and
ice.
were
before
of Concentrates
in
the
solutions
Hemostasis-Thrombosis
activities
within
Hospital,
pooled
for
the addition
It was shipped
the laboratory
the
Testing
reconstituted
within
about
Los
infusion
Angeles,
of heparin
3 days
for up to 3 more
3 hr after
into
days
several
vials
infusions
before
testing.
the
producers,
Diego,
of
in which
reconstituted
was
heparin
in San
according
concentrates
Concentrates
A subsample
ice to the laboratory
by
San
reconstitution.
patients.
in those
in dry
provided
Laboratory,
were
kept
concentrate
removed
and
and
to
of the
same
lot
pooling
was frozen
at
for
tested.
after
was stored
their
tested
in ice until
immediately
was given)
Diego
were
-
in dry
20#{176}C
in
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830
SELIGSOHN
Protocolfor
Infusion
Informed
consent
by
the
University
of California,
and
San
or Konyne)
factor
frequently.
and 24 hr after
samples,
the arm opposite
that
the saline
was removed
g
12,000
for
described
10 mm
Calculation
Infusion
The
factor
parts
Plasma
patients,
through
in dry
ice.
120,
a 21 -gauge
added
One
received
180,
were
stored,
in Factor
VII
300,
drawn
less
inserted
saline.
into
At the time
and a further
shipped,
of
sample
of balanced
by centrifugation
samples
240,
were
needle
to one part
immediately
The
program.
samples
and discarded,
were
prepared
concentrate
A patients
and at 5, 30, 60,
removed
of the sample
was
frozen
were
Units/mi,
IX
treatment
0.01
episodes.
before
obtained
the
of less than
Bethesda
their
were
and
citrate
two
times
at
and
tested
as
for the concentrates.
observed
Rise
rise in factor
VII
following
of bleeding
In the inhibitor
ml of blood
Nine
levels
37
forms
Angeles,
and the 2 hemophilia
drawn
were
and
received
of a prophylactic
(Proplex),
were
(2
IX
and kept open by a slow drip of isotonic
3-5
tube.
of Observed
level of factor
VII
syringe.
inhibitors
consent
Los
B (factor
B patients
infusions
the 24 hr sample,
at 4#{176}C
and
previously
VIII
for the control
samples
Hospital,
hemophilia
hemophilia
of concentrate.
was stopped,
in a plastic
factor
and the informed
Orthopedic
with
of scheduled
infusion
protocol
the
IX concentrate
used for the infusion
drip
in a plastic
anticoagulant8
with
blood
except
The
of
patients
(Auto-IX)
B patients,
patient.
Eight
factor
IX concentrate
All
sampling
patients
in the study.
received
In the hemophilia
and 360 mm
Nine
as one of a series
B patient
activated
each
committees
Diego.
A
participated
hemophilia
from
subjects
2 hemophilia
respectively)
(Proplex
human
AL.
IX Concentrates
was obtained
approved
U/mI)
ofFactor
ET
before
infusion
and
VII
Expected
level following
infusion
and the level
was calculated
from
Rise
infusion
was calculated
immediately
after
Levels
After
as the difference
infusion.
The
between
expected
the
rise in factor
the formula
Ui,j
W
where
is the
U
concentration
total
units
of factor
represents
an assumed
hematocrit.
C.F.
in 2 patients
Assay
plasma
Factor
plasma,
tube for 3 mm
were
added,
activity,
different
:1000
to 1:5000
units
per milliliter
reference
time
dilutions
of test
35%,
was carried
and
45).
This
the
40
factor
the
a correction
-
and
in kilograms,
CF.
was
for
used only
respectively.
materials
of the test
a dilution
curve
Since
were
was tested
out by incubating
25 &l of adsorbed
was noted.
plasma
Amidolytic
amidolytic
assay
X,, is described
incubated
with
stopped
activity
reference
tissue
of the
determining
test
bovine
material
materials
required.
at 1:10 to 1:100
prepared
with
25 Ml of hereditary
plasma
dilutions.
1:10
in a 12-
in TBS
differed
and
in their
concentrates
Clotting
VII
glass
were
in TBS
CaCl2
factor
were
times
to 1:80 dilutions
factor
X 75-mm
50 Ml of 35-mM
markedly
Reconstituted
standard.
Assay
for
Factor
VII
factor
VII,
in which
its measurement
elsewhere.6
factor,
by adding
from
It is carried
calcium
ions,
Na2EDTA
incubation
the initial
(U/mI)
dilutions;
for
in detail
at 3 mm
subsample
1:2000
VII
factor,
25 Ml of a dilution
the clotting
from
represents
injected
weight
VII
tested
in
converted
to
of the factor
VII
standard.
Coupled
An
for factor
50 Ml of tissue
dilutions;
CF.
hematocrit)/(lOO
33% and
milliliters
VII
at 37#{176}C.
Then
and
of the
W is the patient’s
and
patient’s
-
were
the product
concentrates,
of 40 ml/kg,
as (100
values
A one-stage clotting assay
deficiency
from
in the pooled
volume
hematocrit
for
as determined
(U/ml)
was calculated
whose
Clotting
injected
VII
CF.
X 40 X
and
in a recording
curve
for plasma,
a 1:100
made
tested
dilution.
in
is coupled
two
human
steps.
factor
In the second,
to an aliquot
a dilution
were
purified
and cooling.
mixture
Abs/min
Materials
out
of the
specific
spectrophotometer.
with
in the
1:80
following
to
1:400
dilutions
to the generation
In the
first,
X. The
the factor
generation
Results
in TBS:
test
of factor
are converted
S-2222
to factor
of the
for concentrates,
is
X is
by adding
substrate
in TBS
of factor
material
X is measured
chromogenic
dilutions
the
factor
1:500
a
and
VII
VII
to
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FACTOR
VII1
Coupled
FACTOR
IX
Amidolytic
An amidolytic
Assayfor
assay
this assay
a test material
cephalin,
and purified
adding
Na2EDTA
CONCENTRATES
for factor
human
Concentrates
were
assayed
samples
were
tested
undiluted.
prepared
with
time
that
was assumed
storage
ions
as
to have
mm
for 20
shown
The
the
concentration
IX,
native
activation
factor
of factor
Amidolytic
One
native
IX
out by incubation
(final concentration
of 50-mM
of test material
5-mm
a
(I
preparation
which
presumed
full
X,.
amounts
was calculated
plasma
curve
was prepared,
at
in a clotting
factor
sulfate
of purified
XI,7
and
before
calcium
to factor
IX,
polyacrylamide
‘25I-factor
mean
gel
IX.’#{176}
Therefore,
the final
an assumed
20#{176}C
and
-
assay9
IX was activated
dodecyl
by multiplying
by 0.75,
1:35;
standard
purified
by
a reference
had been stored
(undiluted
mg/mI),
concentrate
and the initial
value
X, reference
standard
was
tubes
concentration
of
for the extent
of
under
our
Consequently,
prepared
in TBS
activation
factor
of the reference
were
each
day
of Russell’s
that
factor
viper
added
Details
the
to
of the
test
was
X preparation
venom,
and
20 Ml
were added, and the tubes were transferred to ice. A
conditions
the
plasma)
was measured.
of 200 ILl of a purified
solution
of Na2EDTA
or undiluted
Abs/min
with 1:7.5 to 1:40 dilutions of this incubation
period
to factor
The
of the factor
trace
at 37#{176}C
in plastic
20 Ml of 0.3-M
incubation
with
sodium
20 ILlof a 66-g/ml
1.1 U/mI),
reference curve was prepared
that
standard.
from
ions,
previously.
1 :5 to about
IX,
as determined
reduced
mixture
as described
about
In
calcium
X, is stopped
of factor
activity
1X7 that
elsewhere.7
VIII,
Xa
for 5 mm
CaCl2. Then
factor
IX,.
Factor
microliters
is described
of factor
IX,
factor
60%-90%
of the standard
in the incubation
from
was incubated
containing
X
into U/mI
native
IX
after
mixtures
IX to factor
ranged
concentration
conditions,
600 ILl of TBS and 100 Ml of 5-2222
assay are given elsewhere.7
A factor
carried
IX
factor
profile
Assayfor
hundred
these
for factor
of a factor
purified
factor
X,,
bovine
at 37#{176}C
the generation
that
in TBS-BSA
out, from
radioactivity
the factor
3 mm
was converted
dilutions
of factor
purified
is assayed
in TBS
Abs/min
purified
of incubation
purified
X. After
the same
electrophoresis
to the generation
thrombin-activated
A subsample
at 37#{176}C.
Under
by
IXa
coupled
with
factor
the assay was carried
(5 U/mI).
IX,,
at a dilution
1:20 to 1:200
each
Factor
is incubated
and cooling.
831
resulted
X,
standard,
in only
mixture.
Later we discovered
33%
conversion
concentrations
as read
from
the dilution
were
by 3.
and
activated
divided
of the
factor
X
curves,
RESULTS
Factor
The
VII Activity
data
in Concentrates
on the
IX concentrates
factor
VII
activity
are summarized
except
DE.FIX
contained
measured
by the coupled
of factor
in Table
appreciable
amidolytic
brands
studied,
coupled
in F actor IX Conce ntrates
and Activated
Factor
No. Lots
Tested
Clotting Units
(VII,)
factor
studied
(33-621
U/vial),
as
only from 2.9 to 3.6
a higher
amidolytic
Range of Factor
Brand
of concentrate
amounts
of factor
VII
assay.
DE.FIX
contained
U of factor VII per vial. In every concentrate
was found in the clotting assay
than in the
Table 1. Factor VII Activity
IX concentrates
1. All
value for factor
VII
assay.
The ratio of
Factor IX Concentrates
VII Activity
per
Vial
Amidolytic
Units
(VII,,,,)
Ratio
VII,/Vll,,,,
IX concentrates
Konyne
(USA)
5
294-618
54-139
4.4-9.1
Proplex
(USA)
3
1338-3006
158-621
4.4-8.5
P.P.S.B. (France)
5
123-390
DE.FIX
4
4.8-6.6
2.9-3.6
4
1725-3330
117-266
Activated
Auto-IX
(Scotland)
factor
1000
FEIBA
250
2.9-3.8
1.6-2.1
IX concentrates
(USA)
FEIBA
40-136
(Austria)
(Austria)
3
1
960-2310
480
70-165
33
6.8-21.5
13.7-14.4
14.4
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
832
SELIGSOHN
factor
VII
amidolytic
activity
in the
assay
(VIIc/VIIam)
(p < 0.01)
for
(mean
1 3.9 ±
4.24 ± 0.6 SE).
Factor
and
IXa
Only small
concentrates:
three
in four
lots
assay
from
to factor
VII activity
1 .6 to 21.5. Significantly
this ratio
were obtained
from
1 .6 SE)
than
from
nonactivated
Factor
Xa Activities
from
of DE.FIX.
0.1 to 0.5
The
activated
factor
in
AL.
the coupled
higher
values
factor
IX concentrates
IX concentrates
(mean
in Concentrates
amounts
of factor
IX activity
from 0.4 to 3.4 U/vial
in three
of Proplex,
lots
clotting
varied
ET
were found
in nonactivated
factor
lots of Konyne,
from 0 to 1 1 U/vial
.
U/vial
activated
in five lots
factor
of P.P.S.B.,
IX concentrates
and
0.6
appeared
IX
in
U/vial
to contain
considerably
higher
factor
IX activity,
but quantitative
data
were not reliable
because
different
dilutions
of an activated
concentrate
were found
to yield significantly
different
quantitative
values
in our assay.
No factor
X, activity
could
be measured
in six lots of nonactivated
factor
IX
concentrates.
U/vial.
Two
One
lots
activity.
Very small
were found in seven
Factor
Ten
SE for
clotting
U/mI.
assay,
of Konyne
lot of an activated
factor
assay,
studies
were
Immediately
3.49 ± 0.77
after
U/ml;
X
of factor
carried
out
had
X, activity,
about
0.1
factor
X,,
no measurable
activity,
from about
factor
IX concentrates.
0.1
to 0.6
U/vial,
Infusion of Factor IX Concentrates
Following
VII activity
in these
0.73
± 0.06 U/ml;
traces
IX concentrate
amounts
of factor
other
lots of activated
VI! Activity in Plasma
infusion
contained
factor
in 9 hemophilia
patients
in the
before
coupled
B patients.
infusion
amidolytic
infusion,
the mean
values
in the coupled
amidolytic
were
Mean
as follows:
assay,
1.28
values
±
in the
± 0.09
were as follows:
in the clotting
assay,
1.44 ± 0.01 U/ml. The
elevated
factor
VII activity
found
in the clotting
assay gradually
fell over a period
of hours to the activity
found before
infusion
(Fig.
1).
In all of the infusion
studies,
the observed
rise in factor
VII activity,
as measured
in the clotting
assay,
exceeded
the expected
rise in factor
larger
difference
between
mean
observed
rise and mean
with
infusion
of Proplex
than
with
infusion
of Konyne.
VII activity
(Table
2). A
expected
rise was found
A comparison
between
observed
because
assay
by this
and expected
rises
in the
of the small changes
in factor
coupled
amidolytic
VII activity
found
was
assay
meaningless
technique.
3.
-
E
‘,,VIIc
52
o
1
-o
Fig. 1.
Plasma factor VII activlevels, as measured in the clotting assay
(VII) and coupled
ity
amidolytic
I
I
1
2
infusion
3
4
HR AFTER
5
INFUSION
612
18
24
assay
of
factor
(VII,,,),
following
IX concentrates.
EachpointisthemeanforlOinfusions.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
FACTOR
VII, FACTOR
Table 2. Expected
IX CONCENTRATES
vs. Observed
of Factor
833
Rise in Factor
IX Concentrates
VII Activity in the Clotting
and Activated
Assay
Following
Infusion
IX Concentrates
Factor
Difference Between
Observed and
Rise
Expected
Total
Dose (U)
Patients’
Hemophilia
Observed
(U/mI)
Proplex (5)
Hemophilia
t
p
988-3024
0.68
± 0.34
1.01
± 0.42
3104-9600
1.90
± 0.73
4.71
±
2.25
1.55 ± 0.23
1.498
2.49
2.658
± 0.51
>0.1
0.025
inhibitors
VIII
Auto-IX
‘Numbers
(2)
5229,
in parentheses
infusion
Two
in which
patients
activated
bleeding
with
factor
episode.
5.09,
a slight
5.23
factor
with
factor
VII activity
IX concentrates
was
hemophilia
rise
followed
A and
in factor
values
VII
for factor
A2 are
slopes
(exponential
=
18.6, A2 =
time
the
to factor
of the
pooled
measured
clotting
VIII
in the
activity
VII activity
rise in factor
of data from
were
given
an
a
in
used
intercepts
infused
(exponential
to calculate
studies
computed
0.29.
The
VII
clotting
in hemophilia
concentrate
paper
coefficients)
Their
a2
factor
amidolytic
immediately
Factor
IXa and Factor
Xa Activities
of Factor
IX Concentrates
As
expected
from
the
plasma
either
from
the
8 patients
over, only traces
of factor
plasma
of the 2 inhibitor
heparin.
Despite
small
no factor
against
the
use
amounts
with
of
or only
traces
hemophilia
and
activity,
B patients,
a1 and
calculated
heparin
infusion.
Following
factor
IXa
of factor
in
plasma
as the
a2 are
from
(300
No
test
follows:
half-
IU)
a2,
was
difference
was
in
noted
Infusion
present
in the
IX5 could
B immediately
IXa were detectable
immediately
patients
given
activated
factor
of undiluted
data
time
values
were as
mean
intravascular
before
in Plasma
IX
the
converted
The mean
+ A2e
A1eaI’
=
coupled
initial
rise in factor
VII clotting
activity
or in its rate of disappearance
between
infusions
given with heparin
and infusions
given without
heparin.
concentrates,
an
days
during
were observed
shown
in Fig. 1 were
VII clotting
activity.
plotted
on semilogarithmic
to the biexponential
equation
rate constants).
81.4,
a1 =
2.1,
2.4 hr (144 mm).
In 6 of the 10 infusion
to the
of inclusion
on two consecutive
in the clotting
assay
was
VII
Ci
added
1.22
1 hr only.
an inhibitor
activity
values
for these
percentages
are
Fig. 2. The points could be fitted
disappearance
1.37,
are given because
for
(Auto-IX)
VII levels
points
of the descending
slope of factor
into percentages
of the mean observed
A1 and
6.36
studied.
each infusion:
in one patient
up to 8.6 U/mI
and 7.6 U/ml,
and
up to 8.0 U/ml
and 7.6 U/ml.
As in the hemophilia
B patients,
assay.
The individual
in which
6.86,
of patients
numbers
IX concentrate
Very high factor
these patients
after
in the other patient
only
5278
denote
from a total of 1 1 infusions
tData
additional
was
Expected
A
to factor
the
A1
Observed!
Rise
Bt
Konyne(6)
with
Expected
Rise (U/mI)
after
IX
factor
infusion.
after
infusion
concentrate
material
IX
be measured
in these
in
More-
in the
without
studies,
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
834
SELIGSOHN
ET
AL.
100
90
80
70
=
mm
144
60
0
>
z
50
w
C,,
40
0
z
30
I-
z
U-
0
20
.
plot of the rate of
of factor
VII clotting activity after infusion of factor
IX concentrates.
Each point
represents the mean of values from 10
Fig. 2.
A
disappearance
infusions,
10
HR
the
expressed
as
percent-
age of the mean initial observed
rise (the difference
between
the
activity
AFTER
measured
factor Xa activity
was
clotting
infusion
INFUSION
approached
the
measureable
after
limits
of sensitivity
infusion
factor
VII activities
and after infusion).
of the
assay.
No
before
plasma
of concentrates.
DISCUSSION
factor IX
A
apparent
factor
than as measured
is found because
clotting
assay
concentrate
containing
VII
as measured
content
in the coupled
activated
factor
than
does
amidolytic
the activity
assay
are
measured
amidolytic
assay
studied
(factor
except
amidolytic
VII gives
native
factor
VII
the
activity
ratio)
coupled
DE.FIX
(from
contained
appreciable
amounts
varied between different brands
ent
of
lots
the
same
assay
shorter
VII,
removed)
Amounts
concentrates
appeared
factor
IX concentrates.
factor
VII
in a one-stage
for factor
clotting
whereas
will
factor
test
have
VII
VII.
times
a higher
clotting
brand
of
will
reflect
amidolytic
which
assay
factor
of factor
VII,
of concentrates
concentrate
to contain
similar
Every
concentrate
the
results
in the
amounts
studied
(Table
extent
establish
VII
had
i.e.,
1).
of factor
contained
coupled
ratio of
coupled
of activation
that
been
Activated
as did
activated
of
all concendeliberately
from 33 to 621
but also between
VII
assay
This discrepancy
in the factor
VII
not affected
by the activity
state of factor
V!!.6 The
in the clotting
assay
to the activity
measured
in the
factor
VII in the concentrate.
The data obtained
with
trates
activated
U/vial.
differ-
factor
IX
nonactivated
factor
VII,
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
FACTOR
VII,
FACTOR
as evidenced
IX CONCENTRATES
by factor
VII
835
activity
ratios
that
varied
between
factor
VII activity
ratios of activated
factor
IX concentrates
the factor
VII activity
ratios of nonactivated
concentrates
Although
the factor
VII activity
ratio allows
comparison
of activated
factor
the concentration
data
will not
VII
in different
of activated
obtainable
be
completely
with
a purified
so induced
can
be correlated
clotting
concentrates,
factor
VII
until
purified
human
factor
with
the
VII
not
provide
and
the
in reactivity
The
exceeded
amounts
a measure
in a concentrate.
factor
VII can
activator
increase
21.5.
molecular
found
of
The latter
be activated
changes
in a factor
VII
assay.
It is noteworthy,
increase
in
activity
however,
factor
preparation
that
VII
clotting
of activated
factor
ratio
of 21 .5, the
Radcliffe
plasma
factor
VII
XII
highest
amidolytic
assay
level measurable
activity
ratio
circulation
after
with
a mean
the
reported
repeated
plasma.
found
Thus,
in our
in the
that
factor
infusion
by
study,
a 35-fold
addition
of
clotting
a
even
a factor
VII
probably
reflects
the
VII molecules
in that concentrate.
a striking
difference
after infusion
as measured
activated
&‘ have
induced
to human
(Fig.
1), established
in a clotting
assay
circulating
and
activity
activation
of only a portion
of the factor
The infusion
studies,
which
revealed
from
it does
molecules
human
1.6 and
significantly
(Table
1).
of the relative
assay
and
between
in the
coupled
after
the marked
rise in plasma
factor
VII
infusion
of factor
IX concentrate
results
VII.
Activated
factor
intravascular
VII
persisted
half-disappearance
in
time
the
of 144
mm (Fig. 2). The data in Fig. 2 in the paper
of Kurczynski
and Penner3
may now
also be interpreted
as evidence
of the persistence
over several
hours
of activated
factor
VII in the circulation
of their patient
infused
with an activated
prothrombin
complex
concentrate.
lar half-disappearance
VII-deficient
represent
VII from
that
In addition,
the data of Hoag et al.,5 who reported
intravascutimes
of 85-1 20 mm in four
severe
hereditary
factor-
patients
infused
with
an
early
experimental
concentrate,
evidence
for the disappearance
over a period
of hours
the circulation.
These
observations
fit with the recent
antithrombin
III,
in contrast
to its
neutralization
probably
of activated
factor
demonstratio&2”3
of other
activated
protease
clotting
factors,
is unable
to neutralize
activated
factor
VII. The
mean that activated
factor
VII, as opposed
to factor
IXa,’4
is not cleared
ately from the circulation
Half-disappearance
factor
VII
activity
block
normal
half-disappearance
cular
factor
by a cellular
clearance
mechanism
in the liver.
times
of 290-355
mm’5”6
have
been reported
for
in normal
half-disappearance
time
activated
factor disappears
factor.
We cannot
explain
why
activity
volume
as calculated
of 40 ml/kg.
it reflects
factor
given
If
factor
of 144 mm
from
the observed
VII
for activated
of the
at about
rises
factor
twice
factor
expected
rises
activity
similar
infused
findings
concentrate.
VII
clotting
We
activity
plasma
anticoagulants
to
the intravascular
of a mean
intravasVII
suggests
the
rate
VII
that
the
of the native
clotting
in factor
and an
following
subject
and into
could
conceivably
in the
factor
of oral
in plasma
the
into a normal
discrepancy
by an activity
doses
values
represent
then our finding
the circulation
from the clotting
Hoag et al.5 reported
underestimation
huge
these
VII,
by up to threefold
an experimental
concentrate
factor
VII deficiency.
The
endogenous
subjects
VII synthesis.
times of native
after infusion exceeded
serine
data also
immedi-
VII
activity
clotting
assumed
plasma
the injection
of
patients
reflect
suspect,
of the
with moderate
activation
of
however,
concentrates
that
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
836
SELIGSOHN
to
related
their
experiment,
high
dilution
a concentrate
was
dilution
of hereditary
for factor
VII activity
the
dilution
plasma,
in
prior
factor-Vil-deficient
in the clotting
TBS-BSA,
1 25
assay
1:1000
in
buffer.
in TBS,
(In
a
in TBS-BSA,
supplemental
and
in a 1:100
plasma.
The following
values
were found
assay:
for the dilution
in TBS,
95 U/ml;
for
U/ml;
for
the
dilution
in
factor-VII-deficient
160 U/mI.)
The low values
differ
remarkably
nonactivated
for factor
1X5 activity
from
the very much
concentrates
reported
assay
for factor
IXa. The absence
X, that we found in nonactivated
Xs that
we found
in activated
activated
factor
reported
this
to
diluted
ET AL.
factor
X5 activity
that we found in nonactivated
higher
values
for factor
by
El#{246}diand
of factor
concentrates
should
who
used
are also
Varadi.’7
much
Our
be considered
of factor
of factor
less than the values
for
data on factor
IXa and
approximations
in view
the absence
of uniform
reproducible
reference
standards
for either
factor
factor
X5 activity.
Neither
factor
IX5 nor factor
Xa could be detected
by our assay techniques
plasma
of patients
vated
or activated
demonstration
The
infused
(with
or without
concentrates.
This finding
of circulating
persistence
of factor
genicity
activated
of activated
IX concentrates
of activated
factor
factor
added
heparin)
with
contrasted
strikingly
factor
VII
VII
after
situations
where
plasma
activated
blood
hemophilia
B during
VII in concentrates
thrombotic
VII
native
in
be exposed
or
in the
nonactithe ready
hours
after
infusion
thromboof exposure
not increase
blood
coagulability.
of tissue
factor,
activated
factor
a 1 :50 dilution
of our tissue
factor
factor
60 sec).
to tissue
VII in 85 sec
Consequently,
factor,
surgery
or following
trauma,
could,
at least theoretically,
e.g.,
the infusion
increase
the
and
in
clotted
clinical
in a patient
with
of activated
factor
risk of initiating
a
episode.
Test
methods
presently
concentrates
are
The nonactivated
vitro
might
containing
factor
for some
to evaluate
the potential
Clearly,
in the absence
to tissue
factor,
activated
factor
VII should
However,
in the presence
of a low concentration
VII will shorten
the clotting
time of blood (e.g.,
preparation
clotted
plasma
containing
either
with
of
IX
infusion.
in the blood
highlights
the need
VII in concentrates.
in
a clotting
1X5 or the negligible
amounts
and the very small amount
concentrates
by El#{246}diand
in concentrates
Varadi,’7
concentrates
activitiy
IX
test
for
used
to
evaluate
not sensitive
to variation
partial
thromboplastin
detecting
thrombogenic
the
thrombogenicity
in the factor
time, which
material
of
factor
IX
VII activity
of concentrates.
has been advocated
as an in
in concentrates,’8
is an
intrinsic
clotting
test independent
of factor
VII activity.
Moreover,
the model
for stasis
thrombosis developed by Wessler et al.,’9which has been used as an in vivo test for
the thrombogenicity
activated
factor
VII,
presumed
absence
of concentrates,’8
since this model
of vascular
trauma
probably
does
is based on clotting
and
resultant
not reflect
the effect
of
static
venous
blood in the
exposure
factor.
Consequently,
we believe
that measurement
of concentrates
deserves
evaluation
as an additional
of the
of the factor
indicator
blood
to tissue
VII activity
ratio
of their
potential
thrombogenicity.
The
activity
(or activities)
factor
IX concentrates
inhibitors
to factor
amount
of activated
responsible
for the
in controlling
bleeding
VII has not been
identified.
factor
VII in such concentrates
reported
effectiveness
of activated
in a hemophilia
A patient
with
Whether
or not the increased
contributes
to their hemostatic
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
FACTOR
VII, FACTOR
837
IX CONCENTRATES
effectiveness is an open question.
initiated
by tissue
factor
cannot
Patients
compensate
Nevertheless,
not exclude,
this
reasoning
does
tial amounts
of activated
factor
VII
hemostatic
effect in a hemophilic
patient
with
hemophilia
bleed because
for their defect
in intrinsic
clottting
clotting.
a priori,
substan-
the
possibility
in the circulating
with an inhibitor
that
blood
could
in the presence
factor
at a bleeding
site. It would be of interest,
in clinical
IX concentrates
in the management
of bleeding
in such
relate
serial factor
VII activity
ratios to clinical
response.
exert
a
of tissue
trials of activated
factor
patients,
to attempt
to
ACKNOWLEDGMENT
We are indebted
of California,
to Chris
San
the University
Diego,
Prodanos
for her technical
for providing
of Southern
us with
California,
purified
Los Angeles,
assistance,
bovine
to Dr.
factor
J. Brown
VIII,
for the computer
and
from
to Dr.
analysis
the University
H. Rostami
of the factor
from
VII
decay
data.
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1979 53: 828-837
Activated factor VII: presence in factor IX concentrates and persistence in
the circulation after infusion
U Seligsohn, CK Kasper, B Osterud and SI Rapaport
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