From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Activated
in Factor
Clotting
Factors
IX Concentrates
By
The
precise
factors
has
human
been
recently
factor
rate
of
be
Xa.
and
IXa,
specific
trates.
for
contained
the
trates
were
assays
of
initial
(4-34
H
UMAN
are
VII,
factor
related
small
which
respec-
amounts
IX
prepared
deficiencies
Recently,
have also
Thrombotic
concentrates
in patients
intravascular
some
IXa
throm-
in
3
of
III
the
complexed
These
IX
the
4
than
suggesting
may
results
the
degree
of
concentrates
is
concentration
of
responsible
for
be
of these
clinical
for
are
partially
concentrates
settings.
purified
the
transfusion
these
with
hemophilia
coagulation
have
B5 or liver
been
concentrates
reported
and
contain
fractions
of patients
clotting
experimental
disease,6
in two
and
with
factors
II,
activated
patients
with
following
therapy
of concentrates.7’8
immunologic
not only
plasma
therapy
vitamin-K-dependent
concentrates,
including
been used in factor-VIII-deficient
complications
have occurred
lots of these
which
or
higher
that
to
factor
antigen
factor
of the
patients
who received
activated
preparations
Previous
studies
using
clotting
assays
that
Xa
(12-
concentrates
commercially
the
activity,
thrombogenicity
in some
of
thrombin
with
several-fold
primarily
IXa.
partial
factor
hypothesis
of
unactivated
concentrates
factors.
the
activation
the
and
III
the
antithrombin
activated
support
The
with
Ill
of
with
IXa;
concen-
factor
ng/ml)
not
was
presence
S-2238,
similar
significantly
but
antithrombin
of p-nitroaniline
preparations,
inhibitors.34
nated
concen-
of
Antithrombin
rate
and
X.’2
bin.
and
nonactivated
time
concentrates
thrombin,
or combined
IX, and
initial
shown
g/ml).
activated
The
content,
and
FACTOR
that
isolated
of
in the
correlated
concentrates
(<0.2
S-2222
showed
Xa
in the
IX
of unactivated
Xa
for
the
ng/ml)
thromboplastin
of
assay
was
IXa
negative
from
factor
The
zg/ml
assays
the
release
assays
factor
76
concentrates.
use
specific
factor
factor
measure
the
measures
1.0-2.3
whereas
activated
activation,
Activated
B. Hultin
concentrates
thrombin.
which
3H-factor-X
tively,
IX
with
developed,
IXa.
for
of
factor
accomplished
factors
to
quantitation
in
Mae
VIII
factor
with
signs
these
of dissemi-
factor-VIII-inhibitor
techniques
the zymogens
suggested
but also
varying
amounts
of the activated
enzymatic
forms
of the vitamin-K-dependent
factors.9”
Since factor
IXa, in particular,
as well as factor
Xa and thrombin,
is thrombogenic
in animal
models,’2
this study was designed
to quantitate
these enzymes
in both the
unactivated
and the activated
concentrates.
A new specific
assay
for factor
IXa,
recently
reported
by this laboratory,’3
was adapted
for the assay
of the concentrates.
Specific
assays
for factor
Xa and thrombin,
using chromogenic
substrates,
were also performed
on the concentrates.
The results
of these specific
quantitative
assays
were
From
Stony
the
Brook,
Supported
the Research
Submitted
Addres.s
T-/5-040,
/979
(C)
1028
compared
Division
and
to
the
of Hematology.
the
in part
Veterans
hI-
the
Foundation.
April
9. /979;
reprint
request.s
nonactivated
Department
Administration
Veterans
State
accepted
to
Mae
Stony Brook,
N.Y. / 1794.
by Grune & Stratton,
Inc.
of Medicine,
Medical
Administration
University
July
B.
partial
of New
Center,
and
York
thromboplastin
the State
Northport.
UniversitiNew
by a biomedical
at Stony
time
of
of New
the
York
York.
research
support
grant
from
Brook.
9. 1979.
Hultin,
Division
of
Hematology,
Health
Sciences
Center,
0006-4971/79/5405--0008$02.00/0
Blood,
Vol. 54, No. 5 (November).
1979
at
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
ACTIVATED
CLOTTING
FACTORS
1029
concentrates,
since this less specific
assay
has been used
material
in the concentrates.’4
In order to clarify
discrepant
of the
antithrombin
heparin,”’6
immunologically
III content
the
of the concentrates
antithrombin
III
and functionally.
content
and
of the
MATERIALS
AND
widely
data
the
to detect
activated
on the importance
possible
concentrates
value
was
of added
measured
both
METHODS
Materials
Chromogenic
substrates
S-2222
Phe-Pip-Arg-p-nitroanilide
from
Ortho
Diagnostics
immunodiffusion
Calif.).
Mesa,
chemicals
Factor
IX
Proplex
(Lots
concentrate
for protein
Cutter
from
05A2477,
in water,
purchased
Method
IV)
(DFP)
was
and
stored
Behring
kindly
supplied
Diagnostics
from
obtained
were
(H-D-
purchased
at 4#{176}C.
M-Partigen
from
were
5-2238
HCI)
was purchased
and assays
freshly
and diluted
in buffer
reported
reconstituted
with
concentration
x 20
to assay.
=
are
total
Konyne
In order
FEIBA
was
radial
(San
by Hyland
Sigma,
Diego,
Laboratories,
St. Louis,
as previously
Mo.
Other
reported.’3
NC
as
of
water
comparison
Proplex
IX,
IXa,
within
in
and
Auto
and
tritiated
and
FEIBA
Vienna,
All
kept
on ice,
all assays on
volume
of
contain
(lots
Austria.
concentrates,
both
Hyland
IX concentrates
1 hr of assay,
a total
IX
from
factor
AG.,
between
concentration
vial.
obtained
Activated
Immuno.
ml of sterile
the
were
9007)
was a gift of Hyland,
a gift
to facilitate
of one
(lot
respectively.
(lot 650D02.3)
I 0-20
expressed
content
and
Calif.,
Auto-IX
05A2277)
here
and Konyne
Berkeley,
05A2377,
prior
concentrates
whereas
058 1Dl 53 AA)
two manufacturers:
were
20
ml.,
heparin
i.e.,
(-.1-1.5
do not.
Purification
Human
factor
VIII
reported.’3
Bovine
previously
reported.
Coagulation
Assays
and
factors
VIII,
expressed
of nonactivated
dilution
dilutions
IX,
the
Factor
(1/10-1/80)
Factor
As previously
plasma
test
X
(kindly
provided
and
antithrombin
IXa
(30-50
37#{176}C.The
to which
X
by
Dr.
were
prepared
Jolyon
at dilutions
to a buffer
control,
(200-250
sec).
rather
differed
from
Jesty)
initial
acid-soluble
the
as previously
were
purified
as
activity)
were
of
Denson,’9
using
was
according
to Kingdon
the buffer
The
results
a 1/10,
added.
The
varied
are
Results
among
reported
here
since
heparin
present
negative
if the
by less than
unactivated
et al.’4
control
is considered
control
cofactor
method
prothrombin
since
assay
(heparin
by
than
The
the buffer
the
are
batches
NAPTTs
of a
in Auto
NAPTT
20 sec. or a ratio
IX
of all
>0.9.
Assay
assay
is performed
7.5, with tritiated bovine
U/mI
performed
1/10-1/50.
Ill
measured
purified
was
concentrates,
reported,’3 this
0.05 M Tris, pH
X was
(NAPTT)
plasma
activated
tested
trichloroacetic
thrombin,
platelet-poor
of
the assay
factor VIII
Xa,
of the NAPTT
with
Specific
thrombin
reported.’3
time
as the ratio
interfered
factors
and
bovine
thromboplastin
at
Xa
as previously
barium-sulfate-adsorbed
partial
bovine
factors
Assays
for
performed
tube
were
purifications
Laboratories,
05A1877,
concentrates
1/80
reconstituted
Ill
(Hemofil,
058 1 D I 37 AA,
and
Protein
N.J.),
antithrombin
HCI),
(H-D-VaI-Leu-Lys-p-nitroanilide
Concentrates
were obtained
U/mi);
5-2251
Diisopropylfluorophosphate
reagents
Laboratories
a-d:
for
VIII
Calif.
and
and
(Raritan,
kits
Factor
Costa
(N-Benzoyl-lle-Glu-Gly-Arg-p-nitroanilide
HC1),
after
activation),
rate
of
CaCI2
factor
tritiated
by combining
(50
factor X
X
(8 mM),
activation
activation
a test sample,
g/ml),
cephalin,
and 2 mM
benzamidine-HCI
is quantitated
peptide
in timed
diluted
human
in a polystyrene
by scintillation
serial
in 0.1 M NaC1,
thrombin-activated
subsamples
counting
over
3-5
of
the
The
mm.
amount of factor IXa in a test sample is calculated
by reference to the calibration
curve of initial
rate
versus factor IXa concentration
for purified
bovine factor
IXa, performed
on the same day. Bovine
factor
IXa
have
110%
calculated
was calibrated
against
of the specific
activity
relative
to the bovine
purified
human
of human
factor
IXa
factor
factor
reference
lXa
IXa
(a gift
of Dr.
in the assay;
without
correction.
David
therefore,
Aronson)
the
reported
and
found
data
to
were
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1030
MAE
Preliminary
experiments
were
the test sample
contained
a mixture
factors
Xa,
which
IXa,
and
thrombin
has previously
IXa
could
performed
were
been shown
be accurately
to
confirm
posttreatment
the
with
over
IX. Treatment
thrombin
Since
assay
factor
of bovine
IXa
of factor
Similarly,
IX
varying
to a constant
caused
The
50%
IX,
factor
IX content
human
factor
IX
contain
5 zg/ml
FEIBA-l000
contained
was disregarded,
X content
was
concentrates,
it may
IX
level
(<20%
‘/3
o
or
a
‘/3
below
and
‘/4
in some
Since
a
cases,
‘/5
repeated
factor
Factor
as 0.1
VIII,
(2)
of factor
factor
Xa and
the sensitivity
of the
to ‘/2o dilution
assay).
at higher
assay
measures
factor
X content.
would
would
the
On
dilutions.
excess
IX
the factor
IXa
which
contained
with
was
40-50
and
X
assay.
a specific
assumed
g/ml,
to
while
which may be factor
IXa
by this method. The factor
factor
X is converted
to factor
to pooled normal
human
X content
of the four
of
factors
the basis
be necessary
to reduce
reported
of a large
the factor
assay
plasma,
sum
a
of tritiated
X.
neutralization
in every
The
factor
showed
50 g/ml
to perform
be sufficient
the concentrates
added
mixtures.
inhibition
to quantitate
in which
by comparison
X. The factor
assay
Therefore,
factor
IX antigen,
overestimated
assay.
on the
in the presence
of factor
be slightly
by a clotting
concentrates
with
Konyne
IXa
were
in the apparent
I j.g/ml,
human
and
performed
of
at a constant
factor
Majerus)
X. This
necessary
by antibody
Philip
IXa.
concentra-
activity
decrease
of cold
factor
concentration
of the
factor
be likely
with
I, increasing
IXa
assay
IXa,
normal
fraction
the
in Fig.
of Dr.
IXa
IX,
a constant
factor
it was necessary
Proplex,
may
this
factor
the dilution
assay,
with
X (a gift
would
the assay is calibrated
10 .ig/ml
of factor
IXa
of
of factor
Xa and
in several other
purified
10% inhibition
of 50 g/ml
IXa
IX,
<
of factor
X,
IXa
and tritiated
to pooled
The small
of these
while
lXa.’3
inhibitor
X, or factor
a proportional
IXa
was estimated
Auto
overestimate
of the factor
the
presence
of factor
factor
apparent
factor
and
of human
IX content
g/ml.
dilution
when
of as much
human
inhibited
As shown
gave
X showed
was estimated
slightly
50 g/ml,
inhibition
‘/4.
or
50-200
IX
is, the assay
compared
the factor
venom;’9
to contain
factor
activated
a specific
combined
mixtures.
of factor
antigen.
of the concentrates
concentrates
factor
the
in the
IXa,
to determine
I 50 g/ml.
so that
Xa by Russell’s
viper
which
was assumed
estimates,
IX
than
presence
without
of factor
were
or human
of the concentrates
and
inhibit
factor
factor
amounts
in order
of factor
more
in the
completely
mixtures
in excess
by the additional
antibody
without
decrease
of the assay
of the concentrates
and
and
conditions
50 g/ml,
I : I ; that
or lesser
with
IXa
mixtures
of 2 mM-benzamidine-HCI,
Xa much
sg/ml
(I)
on the
concentration
inhibition
factor
IX
of bovine
of approximately
a similar
content
Below
of unlabeled
X, was reduced
presence
with
might
performed
of bovine
at a constant
stoichiometry
of human
(3)
by assaying
factor
concentrations
concentrations
Since
assay:
a proportional
lXa.
for factor
Varying
IXa.
tested
the assay
concentration
activity
factor
and
of factor
Increasing
the
X in the concentrates
and
of 0.1-3.0
of the assay
IX concentrates.
in the
and factor
range
16 hr. and
on factor
concentrations
concentration
IXa
no effect
IX
factor
tions
x
was
bovine
thrombin
the
of
DFP
on the concentrates
Varying
as described
with 10 mM DFP under identical
but had
the specificity
as the factor
of factor Xa. These levels are greater
than the levels
The assays on the concentrates
were also performed
specificity
10 mM
such
assayed
to inhibit
assayed
Mg/mI
of thrombin
and 0.2 pg/mI
thrombin
found in the concentrates.
ways
to determine
of proteins,
B. HULTIN
X
of these
and
Xa
factor
factor
at a final
data
are the average
the
and
X
the factor
X below
assayed
in
IX
case to reduce
were
plasma,
types of
10 g/ml
dilution
of
of at least
2
50
Fig. 1.
Inhibition of the factor
IXa assay
by
factor
IX. A constant
concentration
of bovine factor
IXa (0.6 tg/ml)
was assayed
in the presence
of
25
varying
concentrations
100,
125. 200. and 250
o
‘
0
50
00
FACTOR
50
(pg/mI)
200
250
of bovine
factor
ig/ml).
The factor
IX (0. 50.
lXa assay
in the absence of factor IX was considered
activity,
and the other
assays
are expressed
percentage
of this 100%
activity.
100%
as a
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
ACTIVATED
CLOTTING
independent
assays.
FACTORS
The
8% by 5 independent
1031
coefficient
assays
of variation
on Auto
IX;
of the assay
thus,
on the concentrates
the reproducibility
was determined
of the assay
is comparable
to be
to that
for
purified factor IXa.’3
Assay
ofFactor
Xa
Concentrates
performed
were
by placing
transferred
was started
mM),
and
recorded
over
reproducible
5-25
ng/ml,
activity
of freshly
reconstituted
and thawing.
Assay
of
Assays
John
results
therefore,
had
had
I 2 ng/mI
of
Antithrombin
sufficiently
for
antithrombin
of 0.4-1.7
g/ml
PNP.
In
concentrates
Thus,
Xa,
usual
at 405
gave
concentration
Purified
since
which
range
bovine
factor
factor
IXa).
control
nm)
a linear
used
IXa
had
Dilutions
experiments
showed
as tenfold
after
Xa,
with
on this
97%
purified
factor
IXa
(3500
thrombin
a gift
on 5-2238.
of Dr.
The
without
Purified
while
at a final
bovine
by NPGB,
reference
of 3.5 mU/mI.
substrate,
this
thrombin
thrombin
5-2238
cs-thrombin
When
active
of human
bovine
that
bovine
ng/ml.
U/mg.
activity
exception
purified
of0.S-5
NIH
to the
the
with
assay
correction;
bovine
factor
factor
Xa at I g/ml
bovine
IXa
at 6
activity.
might
cause
false
of0.6
Finlayson),
IXa.
negative
as described
concentration
factors
measured
and
The
above
mM.
The
compared
Xa, and
traces
assay
to the
assays
for factor
assay
thrombin
to
by indicating
Xa but
was calibrated
manufacturer’s
had negligible
detect
for a 20-mI
250 g/ml).
plates.
to
the
or
heparin
pooled
factor
V Ill,
with
the substitution
with
a purified
stated
activity
small
amount
per vial
in the
normal
specific
of
plasmin
activity
for
on -2251.
of
activity
concentrates
U/mI
Ill
undiluted
(15 g/ml
in order
in
The
direct
in the
were
not
(PNP)
affect
was measured
had
the
diluted
over
a reproducible
and diluted
x 10 ml).
to allow
gave
heparin
antithrombin
measured
would
plasma
antigen
plasma
of 1-2
was
Concentrates
human
Ill
human
ml and assayed
as 150 zg/vial
cofactor
described.’3
Antithrombin
normal
the presence
1/16);
volume
Heparin
with
(2)
Pooled
to 10-15
as little
total
(1)
as previously
of thrombin
(undiluted
measure
ways.
heparin,
was calibrated
PNP,
reconstituted
could
in two
excess
M-Partigen
pg/mI
order
the assay
concentration
(2914
of the specific
for plasmin
so that
using
were
and
The
concentration
.XA,
1-2 hr at 4#{176}C
and as much
the range
is the equivalent
(‘/,so-’/,ooof
of 15-250
factor
over
ng/ml)
John
was
assay.
immunodiffusion
(final
Xa in 24 Mg/mI
was calibrated
as I ng/ml
thrombin
assay
Ill
were
Assays
Ill
of added
of
Dr.
III
presence
for
assay
relative
assayed
Purified
Antithrombin
The
range.
to assay,
after
factor
this
factor
prior
calculated
of plasmin
at a final
plasmin.
just
5-2222
bovine
Assays
spectrophotometer.
in absorbance,
range.
within
7.5.
were prewarmed
on S-2251
were
(a gift
purified
85
“thrombin”
the presence
reference
a wide
twofold
o-thrombin
(<0.2-0.3
Plasmin
5-2251
The
titration)
to have
reported
apparent
the concentrates
made
above
used.
human
were
no activity
Assa;’for
substrate
was
by NPGB
here
an assay
g/ml
a range
as described
it was found
reported
Since
increased
recording
change
purified
of apparent
were
to purified
Fenton),
(linear
to fall
ng/ml
concentrates
mM
95% active
was compared
(0.5
CIII
pH
which
on 5-2238
of 0.05
U/mg,
over
diluted
albumin
of 50 p1 of prewarmed
with
concentration
serum
Acta
release
was calibrated
were
bovine
in microcuvettes,
Beckman
cuvette
concentrates
performed
concentration
assay
versus
Thronibin
were
the
to each
on this substrate
freezing
NIH
The
Xa in diluted
in
0.2%
samples
ofp-nitroaniline
and test samples
negligible
factor
holder
rate
mm.
phosphate,
2SO-Ml diluted
by the addition
the initial
plot of A/sec
was 0.5-10
that
in 50 mM
duplicate
to a 37#{176}Ccuvette
reaction
0.16
on 5-2222
diluted
by radial
assay
no effect
results
comparison
with
vials
normal
are reported
with
over
on the assay
concentrates,
‘/2
the
a range
of
saline.
as the
the activated
factor assays.
Protein
Each
order
content.
Concentration
reconstituted
to verify
The
reported.’3
that
vial of concentrate
concentrates
concentration
was assayed
reconstituted
of purified
proteins
for protein content
to different
was estimated
volumes
were
by absorbance
by the method
comparable
at 280
of Lowry2#{176}
in
in total
nm,
protein
as previously
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1032
MAE
Statistical
Methods
The
of the
same
results
concentrates
coefficient,
factor
using
since
the
IXa,
the
former
Xa,
or thrombin
assays
rank
correlation
coefficient,
does
not
on the
depend
were
compared
rather
data
than
having
to the
the
NAPTT
B. HULTIN
results
product-moment
a normal
on the
correlation
distribution
and
a linear
relationship.25
RESULTS
Factor
IXa
Factor
Assays
IXa
was detectable
at I .0-2.3
g/ml
in the activated
concentrates
but
not
in the unactivated
concentrates
(Table
1). The specificity
of the assay
for factor
IXa in the concentrates
was supported
by several
control
experiments.
First,
it was
shown
that the factor
IXa activity
detected
in the activated
concentrates
depended
on the presence
VIII,
the assay
addition,
human
assay;
effect
mM
of thrombin-activated
was
negative,
a specific
factor
IXa,
whereas
an
on the assay
DFP
human
i.e.,
inhibitor
completely
of
inhibited
identically
(Fig.
2).
to inactivate
no
to the
assays
Xa
and
untreated
IX,
which
also
of Auto
thrombin
(and
inhibited
purified
IX in the
of normal
concentrates
human
were
any
factor
serum
treated
other
had
with
which
of benzamidine
factor
IXa assay
were
IXa
no
10
DFP-sensitive
factor
IXa. The factor
IXa assay
of BZA, gave very similar
results
concentrates,
BZA. Since
the assay
in the absence
50 ng/ml
of factor
lXa,’3 a negative
since
in the absence
of factor
of 3H-factor-X
occurred.
In
activity
prepared
fraction
Finally,
all of the
factors
on the
factor
the
enzymes
that might
be present)
but not
DFP-treated
concentrates,
in the absence
10%)
factor
VIII,
activation
performed
on the
(within
in 2 mM
(BZA)
can detect
on a DFP-treated
as little
sample
as
in
the absence
of BZA probably
represents
<50 ng/mI
multiplied
by the dilution
of
the concentrate.
On this basis,
the negative
factor
IXa assays
in Table
I are
reported
as less than
the calculated
lower
limit of sensitivity
of the assay,
which
varied
with
tested
showed
the
dilution
necessary
no or minimal
any DFP-sensitive
inactivating
the
enzyme
factor
VIII
Table
1.
to perform
activity
on S-2251.
in the concentrate
in the assay.
Activ ated
Clotting
Factor
Concentrate
the
assay.
All
of the
Thus,
it is unlikely
depressed
the
Facto rs in Factor
IXa
concentrates
that
factor
plasmin
or
assay
by
IXa
IX Concentrates
Factor
Xa
(ng/mI)
(ng/mI)
Thrombin
(ng/m)
Activated concentrates
46
1.AutoIX
2300
34
2a. FEIBA-1000
1000
22
76
b.FEIBA-1000
1200
19
26
c.FEIBA-1000
1100
8
13
d.FEIBA-25O
<27Of
4
12
Unactivated
concentrates
3. PropIex
<200t
6
14
4.Konyne
<l5Ot
18
37
Lots a-d
correspond
to the lot numbers
of FEIBA
listed
in order
tThese
negative
assays are expressed
as less than the calculated
which
varies with the dilution
necessary
to perform
the assay.
*Lot number
0581D153AA.
in Materials
lower
and Methods.
limit
of sensitivity
of the
assay,
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
ACTIVATED
CLOTTING
FACTORS
1033
S
1000
..
--
5--
Fig.
factor
2.
IX
of factor
IXa by
The factor
IXa
assay was performed
on Auto IX in
the presence (A) and absence (#{149})
of a
partially
purified
fraction
of serum
containing
a high-titer
specific
human
inhibitor
of factor
IX. The assay
is
plotted
as the counts
per minute
of
3H-activation
peptide
versus the time
of subsampling
(minutes)
from
the
assay
mixture.
An identically
prepared fraction
of normal human serum
hadnoeffectontheassayoffactor
lXa(U).
Factor
Inhibition
antibody.
Xa
by
and
unactivated
5-2222
assay,
respectively
(Table
I).
activities
on this substrate,
could
account
for only
detected
by this
these concentrates
5Q0
Thrombin
‘,.“-
----
-
3
-
-
I
--
-
-
-
.--
2
-
3
4
-
5
6
7
MINUTES
concentrates
contained
with
a range
of 4-34
small
amounts
of
and
6-18
ng/ml,
IXa and thrombin
have
very
low specific
IXa and thrombin
content
of the concentrates
fraction
(<5%)
of the apparent
factor
Xa
method.
In contrast,
by a clotting
assay
assay,
of other
similar
ng/ml
the apparent
factor
Xa activity
detected
for factor
Xa was 2-4-fold
greater
than
presumably
factors.
because
the
clotting
assay
in
the
is also
Assays
Activated
thrombin
respectively
IXa and
and
unactivated
concentrates
contained
similar
small
amounts
of
by
5-2238
assay,
ranging
from
12 to 76 ng/ml
and
14 to 37 ng/ml,
(Table
I ). On the basis of the demonstrated
minimal
activity
of factors
Xa on this substrate,
the factor
lXa and Xa content
of the concentrates
account
for
<5%
of the
of thrombin
detected
(12-76
mately.
When
the concentrates
thrombin
thrombin
apparent
also
be detectable
necessitates
dilution
chromogenic
assay
would
NAPTT
Test
The
NAPTT
is measuring
be detected
was
assay,
positive
As initially
a ‘/0 dilution
the concentrate).
of the concentrate
This reporting
to
the
on the
described,’4
assay.
The
the
presence
clotting
as well
range
NIH
U/mI,
approxiclotting
technique
for
detectable
by S-2238
of heparin
assay,
thus
results
of the clotting
assay;
however,
it is also
-y-thrombin
by the clotting
concentrates.
by this
NIH
U/mI,
only FEIBA
contained
the thrombin
level in Auto
IX
prior
of the assay.
Therefore,
with those of the chromogenic
content
represents
0.04-0.3
assayed
by a standard
by the clotting
considerable
sensitivity
consistent
thrombin
ng/ml)
were
that detects
as little as 0.2
(0.2-0.5
U/ml).
Although
should
‘-
U
Since
factor
the factor
a minimal
results
of the chromogenic
influenced
by the presence
latter
#{149}‘-
, -,
Assays
Activated
factor
Xa
could
, ‘..
as a-thrombin,22
in Auto
decreasing
assays
possible
while
IX
the
may
that
be
the
only
the
assay.
activated
the
concentrates
NAPTT
(the shorter
the
method
is known
is reported
but
not
the
unactivated
as the clotting
clotting
time, the
to be inapplicable
time
more activated
to concentrates
of
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1034
MAE
B. HULTIN
2.5
2.0
E
a’
1 ‘.5
0
Fig. 3.
The relationship
between
the factor
IXa concentration
and the NAPTT
of the factor
IX concentrates.
The factor
IXa concentration
(sg/ml)
is plotted
on the y axis,
versus
the
NAPTT
ratio (clotting
time of a 1 /80 dilution:
clotting
time of the buffer control)
on the x axis
for each concentrate:
Auto IX (U). FEIBA
lots
a-d
(a), Proplex
(.).
and
Konyne
(X).
The
NAPTTs
of FEIBA lot d, Proplex.
and Konyne
at
this dilution
were
negative
(ratio
> 0.9).
The
dashed line represents
the average
lower limit of
#{163}
#{163}
1.0
#{163}
U
0.5
lAS
Ct
O
0.25
0.5
NAPTT
that
contain
these
with
sufficient
studies,
increasing
lengthened.
1.0
RATIO
sensitivity
heparin
positive
dilution
Therefore,
determine
(44 see;
to prolong
NAPTT
from
the
the
positive
at
activated
(ratio,
‘/2
0.5);
of the factor
clotting
concentrates
IXa assay.
time
of Auto
IX, which
‘/,o to ‘/20, reached
their relative
degree
ofactivation.
ratio of 0.22 compared
to buffer),
(lot d) had a negative
NAPTT
FEIBA-250
and
the
0.75
at a
contains
a plateau
were
dilution.’4
‘/,o
heparin,
at
compared
In
shortened
‘/2o-’/4o,
then
at ‘/o in order
to
Auto IX had the shortest
clotting
time
followed
by FEIBA-l000
lot b > a > c.
at ‘/go, was borderline
at ‘/,o (ratio,
0.9),
whereas
Konyne
was
negative
at
both
dilutions
(ratio
>0.9).
Since
neither
contains
heparin,
it is reasonable
to rank FEIBA-250
as
more positive
than Konyne,
but less than FEIBA-I000
or Auto
IX. Proplex
had a
negative
NAPTT
at all dilutions,
but the presence
of heparin
prolonged
the assay
on dilutions
Konyne
factor
positive
of ‘/,o or less,
or
IXa,
Xa,
=
When
or thrombin
correlation
(Fig. 3), which
the data
were
there
(r
so that
FEIBA-250.
between
it was
the
not
results
content
possible
of the
of the
shortening
to rank
NAPTT
concentrates,
of the
Proplex
were
there
NAPTT
and
factor
concentration
(r
100-200
ng/ml
NAPTTs
of these
.40). Even
by freezing
concentrates
of the NAPTT
factor
Xa (r
=
with factor
.77, p > 0.1)
to
versus
appeared
the
was not as evident
for factor
Xa (Fig. 4) or thrombin
analyzed
by a rank correlation
method
(omitting
was a significant
correlation
.94, p <0.02)
but not with
in relation
plotted
the
to be a
IXa
content
(Fig. 5). When
Proplex,
n
6),
=
IXa concentration
or with thrombin
when factor
Xa levels were intentionally
increased
and
thawing
the
unactivated
concentrates,
remained
negative.
to
the
0. I
0
Fig. 4.
The
Xa concentration
relationship
between
the factor
and the NAPTT
of the factor IX
concentrates.
The
factor
Xa
3
.
#{163}
A
concentration
(gig/mI)
is plotted
on the y axis versus
the
NAPTT
ratio on the x axis for each concentrate
(symbols
as in Fig. 3).
.
#{163}
-o
.25
.50
NAPTT
.3’S
RATIO
x
A
1.0
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ACTIVATED
CLOTTING
FACTORS
1035
0.1
2,
A
Fig. 5.
bin
The
relationship
concentration
between
the throm-
o
the NAPTT
of the factor
thrombin
concentration
(gig/mI)
is plotted
on the y axis versus
the
NAPTT
ratio on the x axis for each concentrate
(symbols
as in Fig. 3).
and
IX concentrates.
The
Antithrornbin
The
III
and
III
unactivated
antigen
(8-41
contrast,
antithrombin
2-9 ig/ml,
or 8%-38%
whereas
levels
of antithrombin
containing
heparin
to antigen
A
os
I
O5
NAPTT
and
had
15-42
only
(8%-14%)
one
than
unactivated
A
05
1.0
RATIO
those
antigen
IX and
without
as heparin
unactivated
levels
had
(FEIBA
lower
and
approxi2).
ratios
In
was only
activated
had
(Table
anti-
2).
cofactor,
and two
activity
Proplex)
of
Table
(Konyne)
and
heparin
small
respectively,
concentrate
III
(Auto
similar
tg/ml,
III biologic
activity,
measured
of the antigen
level, in one
mately
concentrates
O
concentrates
tg/ml
concentrates;
equal
A
‘
Content
activated
thrombin
.
,
Those
of activity
Konyne),
with
ratios
of 21%-126%.
DISCUSSION
The identification
and quantitation
components
in factor
IX concentrates
nonspecific
nature
of most
clotting
of both the
had been
assays
used
therapeutic
hampered
to evaluate
Several
clotting
assays,
including
the NAPTT
and the
have been reported
to correlate
with the thrombogenicity
in the
the
of stasis
thrombosis.’423
These
same
components
in the
concentrates.2425
The
that
is detected
by the
nent
animal
model
NAPTT
was
thrombin
of factor
assays
apparent
tentatively
and thrombogenic
previously
by
the concentrates.
linked
may
generation
time,
IX concentrates
or may
2.
Anti thrombin
Concentrate
Activated
Ill (AT
Ill ) in Factor
not
thrombogenic
to factor
activity
by inhibitor
studies.9
In the stasis thrombus
model,
purified
times
more thrombogenic
than factor
Xa and 60 times
more than
Table
detect
compo-
IXa
and
IX Concentrates
ATIII
ATIII
Antigen
Activity
(g/ml)
(g/ml)
Activity/Antigen
1%)
concentrates
1.AutoIX’
24
1.8
8
41
8.8
21
b.FEIBA-1000
26
6.1
24
c.FEIBA-1000
19
7.3
38
d. FEIBA-250
8
2.8
35
40
5.5
14
42
4.6
Unactivated
concentrates
3a. Proplex
b. Proplex*
4.Konyne
heparin,
15
1-1.5
U/mI
Lots
11
19
a and b of Proplex
are the first
126
and second
lot numbers
given
Materials and Methods.
tLots
a-d
Xa
factor
IXa is 7
thrombin
on a
2a.FEIBA-l000t
Contain
the
of FEIBA
correspond
to the lot numbers
of FEIBA
listed
in order
under
Materials
and Methods.
under
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1036
MAE
molar
basis;
the
2-kg
rabbit.’2
minimal
Similar
concentrates
would
could be performed
This
study
measures
concentrates
has
These
showed
(250
FEIBA
lots of FEIBA-l000.
significantly
with the
did
not.
Although
factor
IXa
genie
U according
The
NAPTT
in the
thus
developed
probably
the
well
but
not
in
IXa,
which
Xa
IX
The
thrombin26
demon-
content
factor
lot of
content
IX
factor
IXa in factor
of sensitivity.
IXa
between
or
thrombin
activated
concentrate,
and
lesser
degree
manufacturer)
compared
concentrates
to the
2-kg
factor
as well.
factor
tg/viaI)
rabbits
Xa and
the
of
three
might
content
synergistic
IXa
IXa
times
effects
the
be thrombo-
per vial
dose
and contribute
is markedly
Since
the activated
the risk ofthrombosis
not available
NAPTTs
would
The amount
of
100-200
thus
thrombogenic
However,
VIII.’3
patients,
was
and
thrombin
minimal
of factor
of factor
were
lots with short
factor
X assay.
(20-46
below
in the presence
the action
presence
of its cofactor,
solely in factor-VIII-deficient
for factor
factor
loss
in factor
on one
protein
to the
in normal
for humans
might occur
Furthermore,
factor
clotting
assays
for factor
Xa and
the assay
of the concentrates
difference
concentrates
In contrast,
were
assay
ng in a
of
activated
used to assay
and only
minor
lots of unactivated
dose
tested
200-400
factor
IXa
content
of the concentrates
correlated
results;
whereas
the factor
Xa and thrombin
content
activated
in humans.
thrombin
potential.
be
was
relationship
for each
study,
it is probable
that those
factor
IXa by the tritiated
thrombogenic
concentrates
and
a newly
concentrates,
thrombogenic
for assay
in this
contain
measurable
minimal
assays
a marked
unactivated
IXa
dose-response
negati,e
factor
IXa
assay
is consistent
with
its lower
FEIBA-250,
activation
that
of factor
of chromogenic
substrate
and their
feasibility
for
and
The
the
activation,
can
loss of specificity
studies
activated
content.
of
demonstrated
relative
specificity
has been confirmed
the
studies
dose
be feasible
if specific
on the concentrates.
3H-factor-X
without
strated.
thrombogenic
B. HULTIN
ofall
the
for a rabbit’2
of factor
Xa and
to thrombogenic
dependent
on
concentrates
from factor
the
are
IXa
used
may
be small in this setting.
It is interesting
that no thrombotic
complications
have been
reported
for Auto-IX
and only two cases for FEI BA,78 one of which occurred
when
cryoprecipitate
was given simultaneously.7
The
persistence
of active
measurable
antithrombin
inconsistent.
However,
and
thrombin
and
the
antithrombin
of the
enzymes-factors
III
recent
Ill
apparent
equilibrium
III concentration.27’28
factors
IXa,
in
studies
by antithrombin
activated
activity
the
and
thrombin-despite
might
at
first
seem
have
shown
that
the
inhibition
of factor
in molar
excess
does
not
proceed
to completion,
concentration
Therefore,
generated
Xa,
concentrates
during
of free enzyme
it is plausible
that
preparation
depends
most, but
of the concentrates
Xa
on
not
the
all,
would
be
inactivated
by complex
formation
with antithrombin
III. The apparent
excess
of
antithrombin
III antigen
compared
to activity
in the two activated
concentrates,
and in one unactivated
concentrate
containing
heparin,
is consistent
with
the
hypothesis
that most of the antithrombin
III is complexed
with activated
factors.
Since
heparin
appears
III without
affecting
of
antithrombin
concentrates
addition
to act by increasing
the final equilibrium,29
III,
appears
of heparin
with
or
to have
to the
the
without
much
reconstituted
rate of inactivation
it is understandable
heparin,
more
effect
during
on
concentrate.3#{176}
the
by antithrombin
that the presence
purification
thrombogenicity’6’29
of
than
the
the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
ACTIVATED
CLOTTING
Another
activated
in some
factor
settings
111.27 This
neither
factor3’
1037
FACTORS
that
is factor
study
did
not
clotting
assays
was sufficiently
include
IXa is available,
it very possible
factor
IXa,
VIIa,
therapeutic
or thrombogenic
as it is not
inhibited
a factor
VIla
assay
of 2.5
hr.32 No
accurate
estimate
tive data
VIla,
or some
other
factors
are
responsible
since
of tissue
investiga-
of these
therapeutic
and
factors,
effects
survival
for the apparent
vitamin-K-dependent
these assays
should
activated
thrombotic
in vivo
of
IXa by antithrombin
III
in vivo survival.
Whether
therapeutic
VIII inhibitor
patients
assay,32
demonstrates
each of the activated
future
studies
using
on the relationship
to the
concentrates,
of the
but the slow inhibition
of factor
that factor
IXa has a measurable
feasible
for
Therefore,
the
activation
assay
in the presence
VIIa versus
factor
VII. Other
benefit
of some factor
IX concentrates
in factor
This
study,
along
with
the new factor
VIIa
assays
are
concentrates.
on
potential
by antithrombin
amidolytic
assays,
have
recently
reported
that
both
factor
IX concentrates
contain
factor
VIla.
Factor
VIIa
after
transfusion
of concentrate
and found
to have a
time
factor
makes
have
particularly
nor a 3H-factor-X
specific
for factor
tors,
using
clotting
and
activated
and unactivated
was measured
in plasma
half-disappearance
might
VIla,
is unclear.3’4
that
specific
factors
in the
provide
quantita-
in particular
factors
IXa
and
of the concentrates.
ACK NOWLEDGMENT
I wish
to
assistance,
thank
and
Penny
Hausscr,
Elizabeth
Cohen
Roberta
and
Vesta
Sugar,
Carucci
and
Raymond
for typing
Dattilo
for
excellent
technical
the manuscript.
REFERENCES
1. Breen
trates
FA,
Tullis
in treatment
disorders.
JAMA
2. SandIer
Prothrombin
208:1848,
5G.
bin complex
JL:
of Christmas
Rath
Ann
and allied
Ruder
Med
prothrombin
VIII
Penner
concentrate
inhibitors.
N
for
EngI
JA:
patients
J
Med
Activated
with
factor
inhibitors:
plex
5.
Management
concentrates.
Kasper
tions.
JA: Antihemophilic
with
JAMA
Blood
49:159-170,
CK:
Thromb
prothrombin
com-
Elodi
Diath
Haemorrh
factor
Thromb
33:640-644,
13:821-828,
Marassi
Mannucci
thrombin
A,
PM:
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1979 54: 1028-1038
Activated clotting factors in factor IX concentrates
MB Hultin
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