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Porcine
Willebrand
Factor
VIII:C
Prepared
Factor
and Heterologous
Sulfate
Polyacrylamide
By Gaylord
A
procedure
was
porcine
purify
tional
precipitation
from
heparinized
bean
ene
and
VlIl:C
overall
Affinity
hydroxide
of 24%.
an
and
Willebrand
second
affinity
antihuman
resin.
ORCINE
factor
plasma.
protein
factor
which
soy-
acidic
VIII:C
IgG
averaged
35.
concentrate
on
a
was
two
and
fold
applied
fraction
agarose.
VIII:C
of
The
von
to
human
(40
Vlll:C-agarose.
the
antibody
weights
concluded
that
reagents
represents
porcine
in
that
human
sure
sera.
These
ting
been purified
and
plasma
sources,
partially
human,’6
Willebrand
characterized
bovine,7
factor
has
from various
canine,8
and
poricne.6
However,
the purified
factor
VIII:C
protein
has not been extensively
studied.
Bovine
factor
VIII:C
was
purified
in sufficient
quantity
to study by sodium
dodecyl
sulfate-polyacrylamide
gel electrophoresis
(SDS-PAGE).
Vehar
and
Davie9
reported
that their highly
purified
preparation
of bovine
factor
VIII:C
migrated
as a triplet
on SDS/
urea-PAGE
with
apparent
molecular
weights
of
88,000,
93,000,
bin to
obtain
triplet
weights
was converted
of 73,000
and
and 85,000.
When
treated
maximum
factor
VIII:C
to a
69,000,
band of about
55,000
molecular
38,000 molecular
weight.
doublet
a faint,
weight,
with thromactivity,
the
with
molecular
diffuse
staining
and
a band
of
canin&3
by gel filtration,
porcine
by sucrose
density
gradient
centrifugation,14
and human
by immunoadsorption
of
the von Willebrand
factor/factor
VIII:C
complex
by
antibodies
Blood,
Vol. 59.
to von
Willebrand
No. 3 (March),
1982
factor.’5’16
the
from
apparent
50.000.
We
two
have
different
SDS-PAGE
gel
derived
from
been developed
to meain various
plasmas
and
based
upon
related
appear
the
immunoglobu-
spontaneously17”8
or
severe
hemophiliac.l9
to
be
to the
measured
Using
SDS-PAGE
analysis,
Weinstein
to visualize
the protein(s)
to which these
factor
during
The
VIII
by
the
clotassays.
has attempted
antibodies
are
binding.20
The immune
complexes
formed
by the addition of ‘25[I]-labeled
(antihuman
factor
VIII)-Fab
to
whole
human
plasma
were detected
by autoradiography
after
SDS-PAGE.
showed
several
The
bands,
resulting
one
of
autoradiographs
which
was
thrombin
sensitive.
In this report
which
protein(s)
VIII:C
activity.
utilized
the
Willebrand
ciated
specific
From
the
observation
factor
form
Mayo
Supported
June
Address
reprint
Presented
Societies
12-17.
© I 982
Clinic
and
by NIH
29,
1981:
requests
Mayo
in part
American
that
factor
a noncovalent,
Additionally,
of a cross
in part
Submitted
April
we have
attempted
to demonstrate
can be attributed
to porcine
factor
To carry
out this
study
we have
complex.
affinity
logy Research,
and
are
determinants
several
bovine,’2
by
antifac-
eluted
with
by
polypeptides
assays
have
VIII:C
antigen
activity
tion.
human,10’1’
and
purified
part.
arise
either
of a classical
A number
of investigations
have
shown
that
the
factor
VIII:C
activity
can be dissociated
from the von
Willebrand
factor
in the presence
of I M sodium
chloride
or 0.25 M calcium
chloride.
This phenomenon
has been
demonstrated
for several
species
and
by
techniques:
which
visualized
in
assays
lins which
treatment
antigenic
von
67.000
chro-
by the
VlIl:C.
Immunological
the factor
Willebrand’s
The
least.
factor
material
material
and
at
only
in
factor
factor-agarose
bound
and
activity
activated
polypeptides
of 76.000,
the
bands
1 30.000
Thrombin
inactive
was
from
protein
antigen
Willebrand
from
eluted
by
remnant
contained
and
column
of 1 66.000.
specifically
The
column
uniquely
represented
some
von
also
column
molecular
material
studies.
from
was
tor
hemophilia,
and von Willebrand
factor
is that protein
which
is absent
or abnormal
in human
and porcine
von
disease.
retained
fold)
matography
Willebrand
in classic
also
this
lgG-agarose
weights
neutralization
affinity
a
was
molecular
and
from
inactive
molecular
complex
is analogous
to
or abnormal
The
The
agarose
apparent
Vlll:C
factor
56.
at pH 2.8
nonimmune
by SDS-PAGE.
antibody
an
eluted
prepared
immune
with
the
to
which
76.000
with
of the
Vlll:C
to
the
benz-
With
von
Dodecyl
N. Fass
analyzed
polyethyl-
produced
a noncovalent
factor
VIII:C
is absent
and
up
product
coupled
FACTOR
form
Porcine
citrate.
of
factor
This
the
VllI:C
in
acid
treatment
plasma
of the
Blood
purified
coefficient
activity.
factor
a similarly
chromatography.
factor-agarose
in specific
material
conven-
chromatog-
8.iOO-fold
thrombin
activation
of
partially
QAE-cellulose
was
David
and
adsorption.
sulfate-agarose
coefficient
increase
collected
and
using
techniques.
aluminum
chromatography
von
P
was
concentrate
gave
by
chromatographic
#{128}-aminocaproic
dextran
activation
plasma
precipatation.
yield
VIII:C
concentrate
inhibitor.
glycol-6000
raphy.
from
and
After
factor
to
VIll:C
animal(s)
trypsin
amidine.
developed
factor
J. Knutson
by Affinity
Interaction
Antibodies:
Sodium
Gel Analysis
we have
utilized
reacting
antibody,
Mayo
Foundation,
grant
#HLI
7430
accepted
November
to David
N.
Clinic,
Rochester,
at the 65th
Annual
for
Experimental
VIII:C
and von
easily
disso-
Rochester,
and
Section
Minn.
55905.
Biology.
Minn.
Mayo
Founda-
13. 1981.
Fass,
meeting
the
the
ofthe
of HematoFederation
Atlanta,
of
Georgia,
/981.
by Grune
& Stratton,
Inc.
0006-497l/82/5903-0023$Ol.00/0
615
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
KNUTSON
616
antibody
which
developed
in a hemophiliac
to transfusions.
The protein
these
two affinity
reagents
bed
in response
removed
from solution
has been
visualized
by
by
SDS-PAGE.
(8.5
cm diameter
a coarse
sintered
resin
washed
the
was
effluent
MATERIALS
Normal
the
porcine
Institute
blood
Hills
Director.
The
was
Farm,
factor
cellulose
was
purchased
NH,
and
prepared
The
DE-52
A-15m
Richmond,
were
Rochester,
NY,
were
from
bumin).
gift of
Bovine
a-thrombin
Dr. K. G. Mann,
a specific
of
the
E. J. W. Bowie,
for
2,700
M.
E.
from
Nesheim
and
All
reagents
other
J. T.
Baker
G.
Chemical
Aldrich
Chemical
Blood
Collection
Mann,
were
Mayo
Clinic.
was
and
and
Blood
was
Drs.
Mayo
obtained
agarose
sodium
citrate,
50
zg/ml
rin,
55
0.5
M
soybean
collection,
by
the
femoral
Company,
animals
The
inhibitor.
were
given
blood
was
0.1
Five
an
M benzamidine
and
prior
to the
injection
from
blood
The
anticoagulated
of hepa-
either
one
or
two
of I 5-20
less
at 20#{176}Cto remove
volume
of Al(OH)3
Al(OH)3
was
20#{176}C.The
than
removed
solution
concentration
stirring
was
at
20#{176}C.The
x g for
iO mm
QAE-Cellulose
The
CaCl2,
plasma
at 6000
PEG
was
at
formed
was
the
adsorbed
i
/ I0
The
10 mm
at
with
a
10 mm.
x g for
plasma
at a flow
Aliquots
The
resulting
precipitate
addition
was
greater
was
collected
by
of 50%
volume
so that
made
was
the
rate
5 mM
x g for
was
benzamidine,
at 20#{176}C.
The
10
mm
dissolved
dropwise
stirred
at 20#{176}C.The
of
prior
supernatant
and
mI/hr.
0.01,
and
the
benzamidine,
rate
0.1
of 20-25
the
dextran
buffer
to
The
dialyzed
sulfate-
CaCl2,
I mM
the
CaCl2
lower
sample
was
factor-agarose
resin
was
were
washed
until
eluted
0.255
applied
at a flow
the A280
with
10
M CaC12,
mM
pH
6.0
mI/hr.
Chromatography
from
the
ml/hr.
resin
was
0.1
factor-agarose
VIII:C-agarose
washed
benzamidine,
0.5
M glycine,
of nonimmune
This
Willebrand
ofantifactor
The
I mM
with
von
column
0.5
with
M
10 mM
NaCI,
M NaCI,
pH
pH
immunoglobulin-agarose
chromatography
was
was
carried
out
6.0
2.8.
An
used
at 20#{176}C.
SDS-PAGE
and
M 1-13PO4
performed
with
some
titrated
to pH
6.8
buffer
contained
5% and
was
was
Osborn
with
were
(1 16,000),
bovine
ovalbumin
(43,000).
with
Blue
The
out
E.
albumin
(69,000),
gels
stained
were
base
gel
co/i
and
the
was
sample
of acryl-
at 5 ma/gel.
The
f3-galactosidase
catalase
with
method
buffer
concentration
carried
(210,000),
to the
The
Tris
The
was
myosin
serum
solid
6 M urea.
electrophoresis
proteins
according
modification.24
(60,000),
Coomassie
and
Brilliant
R-250.
for 20-30
centrifugation
at
histidine,
5 mM
6.0 buffer
to
by centrifugation
was
6.0
proteins
x 1.6 cm
15-20
resin.
Analytical
Factor
follows:
10 mM
buffer,
5 mM
0. 10 M NaCI,
CaCI2,
column
VIIJ:C
applied
1/5
initiated
some
to a
follows:
was
time
10 .ol sample,
cl of AP’I’T
was
Assay
VIIl:C
thromboplastin
in
of
ml of
of 60-80
a flow
from
Willebrand
the
of I 5-20
to elution
Factor
0.15
M NaCI,
pH
solution
was clarified
half
five
SDS-PAGE
at 20#{176}C.
precipitate
than
I mM
histidine,
at 20#{176}C.The
eluate
final
Chromatography
1 mM
volume
One
column
was
a
5 mM
Seventy
rate
eluate
10 mM.
and
the
histidine,
standard
was
suspension
buffer.
less
as
on
Chromatography
pH
of von
and
to a 0.9
at a flow
amide
plasma
6.0
at
method
histidine,
ml with
buffer
10 mM
than
rate
of
applied
preparation
a sufficient
to the
at a flow
histidine,
VJII:C-Agarose
of Weber
Precipitation
adsorbed
A2
ofthe
benzamidine,
Antifactor
0.1
by adding
in
10 mm
sec).
to the
5%.
x g for
adsorbed
20#{176}Cfor
at 6000
of the
(PEG)
of PEG-6000
of PEG
plasma
solids)
50-60
Glycol
at 20#{176}C,and
6000
time
(control
A precipitate
(w/v)
The
mg/mI
at 6000
pH
100
6.0
NaCI,
0.01
1 mM
as a control
centrifuged
by centrifugation
prothrombin
300 seconds
Polyethylene
mm
cells.
(70
was
pH
M
mI/hr
than
analogous
Plasma
blood
CaCI2,
units)
to less
buffer
ofAdsorbed
the
10 mM
against
concentration
were
animals.
Preparation
The
completed
from
3.8%
intravenous
collected
to
until
dialyzed
0.1
was
containing
minutes
factor
performed
at a time.
Factor-Agarose
benzamidine,
rate
or arterio-puncture
acid,
was
10 mM
M NaCI,
processed
diluted
to a 1 .6 x 2.0 cm column
Company.
of anticoagulant
-aminocaproic
trypsin
units/kg.
veni-
1 / 10 volume
into
of the
ISO
VIII:C.
was
with
with
(50-100
was
histidine,
collected
animals
6.0
next
at 20#{176}C.
Willebrand
buffer
normal
pH
the
bulk
according
sulfate-agarose
M
the
20#{176}Cand
prepared
0.1
washed
0.255
and
of
10 mM
M NaCI,
QAE-factor
chromatography
was
was
eluted
An aliquot
from
Chemical
Chemical
NaCI,
von
were
was
with
Chromatography
VIII:C
were
the
A2
of Dr.
Dansylarginine
and
Sigma
M
hemophilic
a gift
Research,
grade
Pierce
oval-
mI/hr
a gift
Hematology
resin
proteins
into
the
eluted
0.80
as
at
until
discarded
contained
equilibrated
dextran
were
were
out
The
VIII:C
of buffer
proteins
designated
benzamidine,
to the
sample
benzamidine,
was
column
I mM
applied
a generous
Clinic,
and
The
was
(DAPA)
Company,
Company,
was
Mayo
cm
QAE-factor
The
Chemical
catalase,
antibody
of reagent
weight
Sigma
units/mg.
VIII:C
Research,
K.
molecular
and
The
carried
Kisiel.23
QAE-factor
and
applied,
fraction
was
sulfate-agarose
4.5
CaCI2,
Company,
albumiun,
NIH
antifactor
Hematology
the
I 50 ml
and
by
x
the
chloroform
as described22
Research,
N-(3-ethyl-l,5-pentanediyl)amide
Clinic.
Chemical
from
serum
Labo-
N,N’-methylenebis-
(myosin)
purified
Hematology
Bio-Rad
2.4
packed
was
Sulfate-Agarose
described
The
resin
the
ml of eluate
was
Dextran
Vestling.2’
NJ.
This
activity
Dextran
Keene,
Clifton,
from
and
proteins
bovine
activity
containing
Inc.,
Eastman
J. Reynolds
from
Inc.,
and
recrystallized
The
Dr.
purchased
VIlI:C
of QAE-cellulose
volumes
I mM
70-80
chromatography
I5-20 mm.
E. Zollman,
Schuell,
purchased
from
were
at
The APTT
reagent
Plains,
NJ. QAE-
and
acrylamide
products
(fl-galactosidase,
plasma
was
The
was
KS.
Morris
Whatman,
agarose)
and
Park,
Paul
ofSophianopolous
of
CA.
Company
had
method
respectively.
standard
plasma
Schleicher
a product
(4%
acrylamide
1-propanol,
from
Dr.
maintained
0.50.
FASS
After
sample
CaCI2,
first
ml collected.
animals
Foundation,
Overland
Diagnostics,
by the
Bio-Gel
from
VIII:C-deficient
Biomedical,
of General
ratories,
METHODS
obtained
Mayo
George
King
was a product
was
AND
The
2-3
less than
5 mM
buffer.
funnel.
with
was
histidine,
x 2.5 cm)
glass
AND
of the
reagent
by
assayed
assay.25
90
were
the
fractions
90 tl factor
the
VIII:C
incubated
contained
of
one
stage
activated
partial
assay
procedure
was
deficient
plasma,
standard
zl factor
addition
VIIl:C
by
The
at 37#{176}C
for S minutes.
100
CaCl2,
deficient
.ol of 20
mM
the
assay
plasma
and
CaCl2.
was
and
The
as
100
assay
Because
modified
100 ol of APTT
as
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PORCINE
FACTOR
VIII:C,
SDS-PAGE
reagent
were
addition
of 10 ol of sample
and
reaction.
The
produced
within
incubated
modification
10%
with
100% and
fold,
10%
and
prior
the
Thus,
mixture
plasma
as
factor
the
VIII:C
VIII:C
with
diluted
by the
assayed
the
was
sample.
a-thrombin
A
factor
time
of
added
incubation
mixture
was
approximately
(For
thrombin
was
altered.)
to
same
as
40-60
of units
of factor
ratio
VIII:C
(unactivated).
the
fold.
One
porcine
assay
so
clot
The
time
activation
VIII:C
unit
contained
of factor
was
and
studies
human
plasma
Inhibitory
was
bovine
These
The
von
purified
denatured
factor-agarose
von
bovine
serum
natured
BSA-agarose
Parikh,
et al.26 Bovine
pH
9.0,
was
agarose
time
same
fraction
of IgG
coupled
to agarose
was
activated
ide
HCI
(MES)
pH
cold
added
pH
collected
of factor
final
albumin
M in 0.1
5.0
for
buffer
platelet
proceed
overnight
coupling
The
de-
described
by
activated27
ml of the denatured
M MES,
sec),
pH
6.0
Willebrand
4%
quickly
assay.28
containing
The
washed
resin
cake
100
as assayed
by the
coupling
Antifactor
VIII:C
This
plasma
Bethesda
When
porcine
plasma
plasma
Bethesda
Inhibitory
assay
IgG
was
used
contained
by
units
of
Units/mg
CaCl2
to
IgG.
by
and
removed.
bovine
a-thrombin
After
incubation
Saturated
concentration
NaOH.
collected
After
original
plasma
applied
to
equilibrated
with
until
U
the
pH
was
30
mm
at
and
Na2HPO4
x
A2
was
column
phosphate,
less
than
was
adjusted
were
toward
pH
2.8
aliquot
chilled
added
stanas
of 0.2
M
of
clot
to
a
with
KH2PO4)
The
protein
The
adsorbed
was
final
3 U
was
U
DEAE-cellulose
the
subsequently
p1-I
to
was
(DE-52)
column
I U Tris
p1-I 8.5
was
mixed
at
pH
VIll:C
from
antifactor
an
remaining
had
would
(porcine)
at
volume
for
nonimmune
lgG
the
of this
VIII:C-
equal
which
antibodies
the
for 30 mm
2 more
eluate
VIII:C
the
acidic
aliquot
37#{176}
for
assayed
2.8
to bring
to dilute
incubated
with
was
containing
used
(An
the
were
on ice and
antifactor
saline”
fraction.
incubated
in the
eluate
hr.
of
The
factor
antigenic
manifest
titer.
agarose
an
A control
to dilute
a
fraction.
RESULTS
ofthe
Factor
VJII.’C
Concentrate
The preparation
ofthe
factor
VIlI:C
concentrate
for
two step affinity.
analysis
included
measures
to
prevent
possible
proteolysis,
particularly
by thrombin.
the
blood
animal(s)
anticoagulant
was
proteins
was
collected
from
a
heparinized
by femoral
veni- or arterio-puncture
containing
the protease
inhibitors,
into an
benz-
amidine,
soybean
trypsin
inhibitor
and
acid.
After
removing
the cells,
plasma
with Al(OH)3,
an effective
adsorbent
e-aminocaproic
was adsorbed
of the vitamin
K-dependent
The first
were
factors.
two steps
isolated
fibrin
then
and
of the same
was
precipitate
0. 10 and
and
as
analogous
VIII:C-agarose
eluate
dilutions
in the antifactor
and
M phosphate,
8.0.
An
buffered
to prepare
The
were
the
6000
in 0.04
pH
lgG
IgG.
agarose
resin.
antifactor
antibody
to agarose
reduction
the
the
acid
Protein
using
concentration
0.04
plasma
VIII:C
then
Ca.)
the
activated
normal
sufficient
plasma
in a Tripartigen
to 8.0
overnight.
of
was
20#{176}C,the
with
dialyzed
15 cm
U
dissolved
titrated
and
0.04
to a final
30 mm,
sulphate
the
porcine
of proteins
IgG
added
acidic
5 of the
procedure.
and
above).
tion
I / 10 volume
was
the
or 9.6
concentration
The
at 37#{176}C
for
53
VIII:C,
with
for
volume
a 2.5
IgG
Laiolla,
recalcified
or
assay.29
units/mI
manufacturer.
by centrifugation
(0.04
of factor
was
and
stirring
Units/mI
immunodiffusion
ammonium
of 35%
buffer
washed
the
of a
hemophilia.
Inhibitory
The
plasma
Bethesda
source
Corporation,
20 ml of plasma
5 units/mI.
as the
radial
the
Inhibitory
This
and
from
using
the
allowed
classical
in the standard
plasma.
of
neutralized
dilutions
activity
used
albumin
The
coupled
Preparation
ristocetin
was
from
severe
145 Bethesda
Mancinni
provided
follows:
isolated
with
Bethesda
(Calbiochem-Behring
dards
8.0
800
units/mg
inhibitory
determined
were
patient
contained
Inhibitory
was
activity.
Thus,
antibodies
Bethesda
IgG
or 62 Bethesda
activity
CNBr
1/10
VIII:C
was
at 4#{176}C.
multitransfused
were
into
I 200
units/mI
of imidazole
acid
moist
at
I mg/mI
from
antifactor
VlIl:C
similar
BSA-agarose
was
the
buffer
factor,
aggregation
resin
and
pH
on a I .5 x
units/mg
at
same
carbodiim-
at 20#{176}C.The
60
prepared
eluate
described
apparent
0.2 M NaHCO3,
(CNBr)
to
al.,27
I / I 0 volume
to 7.4.
mixtures
Immunoglobulin-Agarose
19 yr old
2.8
IgG
normal
activated
M 2-(N-morpholino)ethanesulphonic
than
von
induced
by
method
in 10 M urea,
bromide
Five
30 mm
(less
porcine
prepared
the
activity
porcine
coupled
by the
serum
pt-I
agarose
(BSA-agarose).
by
isolated
Inhibitory
inhibitory
eluted
NaCI,
immunoglobulins
Inhibitory
using
et
was
into
1% bovine
in I ml of a
l-ethyl-3-(3-dimethylaminopropyl)
to 25 ml 0.1
purified
serum
of resin.
with
at 0.1
prepared
The
The
U
Neutralization
The
is defined
activity
Antibody
sample
to carbodiimide
albumin-agarose
was
to cyanogen
at I mg/mI
was
with
coupled
factor
were
VIll:C
of the
were
the
Willebrand
was
Willebrand
IgG
March,
37#{176}C.These
Willebrand
porcine
0.1
Bethesda
Bethesda
45%
the
fraction
von
162
antibodies
acidic
Resins:
buffer.
fraction
antifactor
and
by
7.0
M imidazole,
A-Sepharose.
or 460
contained
described
plasma.
Preparation
ofAffinity
Factor-A
garose
in this
units/mg
fraction
pH
0.05
incubation
clot
to units
as the
the
phosphate,
M glycine,
0.5 U NaCI
pH 3.0 buffer
Tris
pH 8.5. These
acidic
IgG antifactor
units/mI
as
M
against
of protein
Inhibitory
coefficient
(activated)
is defined
column
with 0.1
of 1 U
antibodies
fraction
the
2.5 cm
eluted
volume
only
The
for
0.4
dialyzed
final
I U/mI,
the
The
with
using
selected
that
eluted
in the
VlII:C
the
of
I mm.
immunoglobulins
of
purified
concentration
7.0.
ten
a sample
when
were
limits
defined
factor
then,
proteins
30 to 40 fold
was
at 37#{176}C
for
usually
as the
selected
diluted
prior
the
unactivated,
as it was
were
at least
assay
incubating
containing
treatment;
with
diluted
the
activity
after
to a final
incubated
in
fraction
thrombin
diluted
the
the
agreed
the
diluted
of CaCl2
than
VIlI:C
measured
same
was
concentration
greater
activity
was
mixture
final
which
between
were
by
to initiate
curve
were
M CaCl2
Factor
followed
CaCI2
procedure,
fractions
0.255
thrombin.
without
5 minutes
a standard
All
4.5%
617
100 jzl 20 mM
VIII:C.
containing
to assay.
37#{176}Cfor
unmodified
factor
those
reaction
at
ANALYSIS
of the
procedure
precipita-
in the adsorbed
plasma
with
subsequent
batchwise
chromatography
PEGof the
precipitated
proteins
on QAE-cellulose.
These
steps
yielded
a product
which
was 200 to 300 fold purified,
which
had the same activation
coefficient
as the factor
VIII:C
in plasma,
and which
represented
a 40 to 50%
yield. The QAE-cellulose
product
remained
stable
at
4#{176}C
for a week, but routinely
the next step was carried
out
immediately.
The last step in the preparation
was chromatography on dextran
sulfate-agarose
(Fig.
1). In addition
to
increase
in specific
activity,
factor
VIII:C
from
the bulk
an
this step separated
the
of the von Willebrand
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
KNUTSON
618
AND
FASS
500
400
280
300
0
VlIl8
(U/mI)
0.08
200
A280
100
Villa
0.06
0-0
Fraction
0.04
number
Fig. 1 .
Dextran
sulfate-agarose
chromatography
of factor
VIIl:C.
A 2.4 x 4.5 cm column
of dextran
sulfate-agarose
was
equilibrated
with
1 0 mM Histidine.
5 mM CaCl3.
1 mM Benzamidine.
O.i M NaCI. pH 6.0 buffer.
Seventy-five
ml of the eluate
from QAE-cellulose
was diluted
to 1 00 ml with column
buffer and
applied
to the resin at a flow rate of 60-80
mI/hr
(4 mI/fraction).
The resin was washed
until the A
was less than
0.01 . and the
proteins
were
eluted
with
1 0 mM Histidine.
1 mM Benzamidine.
0.1 MNaCI.
0.255 MCaCI2.
pH 6.0 at a flow rate of 20-25
mI/hr
(2
mI/fraction).
The chromatography
was carried
out at 20C. Protein
was measured
as A
(OO)
and factor
VIII:C
activity
was
expressed
as Vlll:C U/mI (-S)
in the left panel
or as Vlll:C
U/mI
(#{149}-‘#{149})in the right
panel.
This chromatography
was repeated
for
the remaining
75 ml of the QAE-factor
VlIl:C; the final result
of this
chromatographic
step
is summarized
in Table
1.
factor.
This
tin induced
porcine
von
assay
procedure
von
Willebrand
dextran
sulfate
agarose
eluted.
This step
stable
for several
been
was
0.02
factor
was
after
provided
weeks
eluted
from
Fig. 2.
von Willebrand
factor-agarose
affinity
chromatograof factor
Vlll:C.
A 1 .6 x 2.0 cm column
of von Willebrand
factor-agarose
was equilibrated
with
1 0 mM Histidine.
5 mM
CaCI3. 1 mM Benzamidine,
0.1 M NaCl. pH 6.0 buffer.
One hundred
units
of the
factor
Vlll:C
concentrate
(eluate
from
dextran
sulfate-agarose)
was applied
at a flow
rate
of 1 5-20
ml/ hr (4
mI/fraction)
and at 20’C. The column
was washed
until the A
was
less than
0.01 , and the proteins
were
eluted
with i 0 mM
Histidine.
1 mM Benzamidine,
0.10 M NaCI. 0.255 M CaCl,
pH 6.0
buffer
at a flow rate of 1 5-20
mI/hr
(2 mI/fraction)
and at 20’C.
Protein
was measured
as A
(00)
and factor
VIIl:C activity
expressed
as Vlll:C U/mI
(*).
factor
the
von
is summarized
factor
VIII:C
had
factor
VIII:C
which
when
it was
stored
at
Willebrand
The first
noncovalent
in Table
affinity
step
reversible
for factor
association
Step
Plasma
5%
supernatant
factor
(approximately
factor
was
factor
of the
VIII:C.
antigen
Total Activity
Vlll:C, Units
Both
but after
proteins
of the Porcine
Preparation
Protein
A2,,
Willebrand
factor.
Purified
factor
coupled
to denatured
as the affinity
reagent.
In the
units/mi
Willebrand
pie times,
additional
VIII:C
utilized
the
between
porcine
1 . Summary
von
lebrand
rose (AH-Sepharose)
resulting
resin was
Chromatography
Table
Purification
Al (OH)3
i.
Factor-Agarose
and
Willebrand
served
presence
of a low concentration
of CaCl2
factor
VIII:C
activity
was efficiently
removed
from
solution
(9299% of the applied)
(Fig. 2), and there
was a two fold
increase
in the specific
activity
of the factor
VIII:C
(Table
2). When
a sample
of von Willebrand
factoragarose
factor
VIII:C
was assayed
by the IRMA
for
von Willebrand
antigen,
the concentration
of von Wil-
the
its activatability
by thrombin.
Thus,
the prodconsidered
suitable
for further
study.
The
procedure
VIII:C
porcine
von
BSA-agarose
4#{176}C.
The total
time
for this procedure,
blood
collection
through
elution
from dextran
sulfate-agarose,
was 12
to i4 hr. The overall
yield was 20-25%
and the product
retained
uct was
15
number
phy
(IRMA)
according
to a
of Ruggeri,
et al.3#{176}
The
(RIPA)
10
5
Fraction
factor
VIII:C
concentrate
had no ristoceplatelet
aggregation
(RIPA)
activity
but
Willebrand
antigen
was detectable
by the
immunoradiometric
modification
of the
(U/mI)
Factor
Vlll:C
Activation
Coefficient
was
found
to
be
8
x
by
also
an
carbodiimide
effective
affinity
resins
1 5 to 20 times
as detected
von
coupling,
affinity
resin
were
reusable
the
on
Concentrate
Specific Activity
Vlll:C Units/A2
Pisification
(x-fold)
Yield
%
Total
1.000
56,000
52,000
35
0.93
1 .000
55.900
5 1 .000
30
0.9
42,000
28
18,600
32
204
36
219
11,854
56
7,550
24
8,118
100
1
1
98
0.98
PEG-6000
200
QAE-cellulose
150
8,900
91
4.7
5.05
80
Dextran
sulfate-agarose
summary
for
1.57
32
this
St ep represents
the
result
of chromatographing
the
QAE-factor
Vlll:C
in two
batches
as describe
d in Figure
1.
the
for
multi-
eluate
contained
SDS-polyacryl-
Volume
(ml)
precipitate
The
iO
4 x iO
gm/mi).
When
coupled
to aminohexyl-Sepha-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PORCINE
FACTOR
VIII:C,
SDS-PAGE
2.
Ta ble
619
ANALYSIS
Chromatog
raphy
of Factor
Total
Volume
of the
gels,
attempt
not
was
seen
made
in the
Factor-Agarose
Specific Activity
Vlll:C, Units/A2
Total Activity
VllI:C, Units
Activation
Coefficient
15
0.74
5,520
7,550
56
6
0.16
2,160
13.500
30
eluate
second
from
to regenerate
Immunoglobulin-Agarose
The
rand
factor-
agaroseeluate
amide
von Willeb
factor
VIII:Cconcentrate
Willebrand
on Porcine
Protein
A2,
(ml)
Aliquot
Vlll:C
fresh
resin.
No
step
utilized
seven
dilutions
the immune
represented
the resin.
Chromatography
affinity
The
an
antibody
to
at which
data
or nonimmune
by two or three
remaining.
The lines are
value of the factor
VIII:C
human
assays
drawn
assays.
tion
fold
of the antibody
suggesting
that
IgG
column
units of factor
was accomplished
tial antibody
neutralizing
activity
column.
(The
eluate
from
the
with
a total
exposure
activity
was washed
prior to the column
of
solution
passage
(Table
3). (This
over the column
15 mm.)
No
factor
produced
VIII:C
from the column
with 0.5 M NaC1
being
eluted
with 0.1 M glycine,
0.5 M NaC1,
pH 2.8 buffer.
Because
of its sensitivity
to
low pH, factor
VIII:C
activity
was not detected
in the
eluate
even when the sample
was eluted
into 1 M Tris,
pH 8.5 to neutralize
the pH.
When
an identical
solution
of factor
VIII:C
the control
resin,
agarose
and the
to 85%
of the
(Table
3). This
glycine
buffer.
after
I 0 cycles
Antibody
was
applied
contained
to 1 milliliter
of
nonimmune
human
IgG coupled
to
column
washed
with 0.5 M NaC1,
80
activity
was not bound
by the resin
resin was also eluted
with the pH 2.8
Both of these
columns
were
reusable
of pH changes.
IgG column,
are
for factor
VIII:C
toward
factor
VIII:C
acid eluate
from the
approximately
which
was
from
average
reducwas I .6
immune
4% of the
poten-
of the input
to the
nonimmune
column
indistinguishable
from
that obtained
by dilution
of antibody
with immunoglobulin-agarose
column
buffer.
The
50%
titration
point was 1:177 N = 2 compared
to 1:184 for nonimmune
IgG
reduce
N
factor
3.)
=
The
VIII:C
intact,
unfractionated,
immunoglobulins.
>,
inability
activity
of its activity
against
VIII:C
sources.
The
of this
to zero
porcine,
inability
but not
is also
antiserum
antibody
to
is a characteristic
human,
factor
seen
with
the
as well
as the
acidic
100
>
90
U
Ca
80
0
Neutralization
a curve
collected
through
the
The apparent
human
factor
VIII:C
isolated
from a hemophilic
plasma. One milliliter
of the antifactor
VIII:C-agarose
removed
20 to 40 units of factor
VIII:C
(900 to I 600
VIII:Ca)
from
in a single
titre
the
were
>
70
0
60
U
Thirty
VIII:Ca)
units
of factor
VIII:C
were applied
to 1 ml ofthe
agarose.
The
column
was
washed
into I 10 volume
of imidazole
ing 1% bovine
serum
albumin
pH 8.5 to bring
the final pH
(900
units
factor
antifactor
VIII:Cand
eluted
at pH
50
Ca
E
2.8
40
E
x
30
Ca
buffered
saline containand sufficient
1 M Tris,
to 7.4. A control
nonim-
E
20
C
5)
U
10
5)
mune
IgG-agarose
column,
binding
in this case less
than 1% ofinput
factor
VIII:C,
was eluted
in a similar
fashion.
These
eluates
were used to dilute
the acidic
IgG fraction
to a Bethesda
Pairs
of
separate
of the antifactor
VIII:C
antibodies
Inhibitory
assay.
dilution
curves
were
established
occasions
Table
3.
and
the
data
of Porcine
Chromatography
combined
Factor
Vlll:C
Type of Resin
Anti-factor
VllI:C
1 .080
Non-immune
‘No
factor
1 .080
VIll:C
Vlll:C, Units
Not Bound
was
measured
0
in Fig.
on Human
Units
Eluted
-
850
in the pH 2.8
-
eluate.
Fig.
from
on
two
3.
1
1
1
1
400
200
100
50
Log dilution
prior
lgG-Agarose
Vlll:C Units
Applied
0.
tor
(900
anti-factor
25
Vlll:C
3.
Blocking
of anti-factor
VlIl:C activity
by the acid eluate
antifactor
Vlll:C agarose.
The pH 2.8 eluate from an antifac-
Vlll:C-agarose
units
antibodies
column
which
of factor
VlIl:C.)
prior
a
to
Bethesda
was
removed
30
used
to dilute
Inhibitory
units
assay.
of factor
anti-factor
using
Vlll:C
Vlll:C
porcine
plasma
as the factor
VlIl:C source.
The 50% titration
point is at a
1 :1 1 7 dilution
of the antibody
solution
().
The pH 2.8 eluate
from
the
control,
non-immune
lgG-agarose
column.
which
removed
less than 1 % of the factor Vlll:C activity.
was also used to
dilute
the anti-factor
Vlll:C antibodies.
The 50% titration
point for
this
eluate
is at a 1 :1 84 dilution
of the antibody
solution
(00).
The x-axis
represents
the log of the dilution
of the anti-factor
VllI:C
antibodies
being
titrated.
The original
antibody
solution
contained
2.6 mg lgG/ml
and 162 Bethesda
Inhibitory
Units/mI
versus
porcine
plasma.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
KNUTSON
620
SDS-PAGE
Analysis
from
The various
fractions
associated
with the immunoglobulin
columns
were analyzed
by 5% SDS-PAGE
(Fig.
4). Gels A-C
represent
the fractions
associated
with the nonimmune
IgG-agarose
and gels D-F
are
the fractions
from the antifactor
material
eluted
from the antifactor
VIII:C-agarose.
VIII:C-agarose
seen on gel F. All of the material
which
the gel entered
the gel under
nonreducing
was
columns
agarose,
is represented
by
and gel D, antifactor
gel
A, nonimmune
VIII:C-agarose.
and E show the material
washed
with 0.5 M NaC1.
By comparing
is
activity
that
the
antifactor
from the two columns
all six gels, it appears
VIII:C-agarose
removed
die band of a triplet
around
76,000
and bands
with apparent
molecular
and
I 30,000.
The
three
bands
the
mid-
on
gel
show a reduction
of apparent
molecular
treatment
with 0. 1 4 M 2-mercaptoethanol
F did
benzamidine,
concentration.
(10
U/mI
reaction
final
0.1
Twenty
U/mi)
were
concentration
was
equilibrated
column
of
then
prior
to
For this
factorS mM
to lower
of
treated
5
x
applied
with
washed
was
above.
factor
with
the
VIII:C
thrombin
(1
M.
106
to
the
DAPA
containing
and eluted
at pH
A control
experiment
was
The
reaction
antibody
column
buffer.
The
2.8 as described
done
with
an analo-
gous aliquot
ofthe
same factor
VIII:C
solution
without
thrombin
treatment.
In both the experiment
and the
control
95-99%
of the factor
VIII:C
activity
was
The
SDS-PAGE
shows
5)
at 90#{176}Cfor I 5 mm. Actually,
the band
measured
at
76,000
appeared
to increase
to 83,000
daltons
upon
reduction
suggesting
intrachain
disulfides.
The effect
of thrombin
on the factor
VIII:C
eluted
determined
M NaCl
units
FASS
final
concentration)
for 5 mm at 37#{176}C.The
was stopped
by the addition
of DAPA
at a
mixture
not
weight
upon
in I % SDS
was
thrombin
column.
from the von Willebrand
against
10 mM histidine,
removed
from solution*
when the columns
were
molecular
weight
weights
of I 66,000
seen
1 mM
to
lgGGels B
factor-agarose
experiment
the eluate
agarose
was dialyzed
CaCl2
The
bands
have
apparent
molecular
weights
of
1 66,000,
1 30,000
and 76,000.
The analysis
of the same
fraction
from
the nonimmune
IgG-agarose,
gel C,
shows no visible
bands.
The material
not binding
to the
Willebrand
by treating
the factor
VIII:C
with
applying
the sample
to the antibody
CaCl2,
The
applied
conditions.
von
AND
and no activity
was measured
washed
with 0.5 M NaC1.
analysis
that
the
of this
166,000,
experiment
130,000
and
(Fig.
possibly
the
76,000
bands
(Gel A) are sensitive
to thrombin.
Gel B
represents
the thrombin
treated
factor
VIII:C
which
bound
to and
eluted
from
the antifactor
VIII:Cagarose
column.
The apparent
molecular
weights
are
76,000,
shows
67,000
and
the thrombin
an indistinct
control.
band
at 50,000.
Gel T
DISCUSSION
I
In this report,
what
protein(s)
VIII
coagulant
antibodies
I
bodies
application;
6.0
are anti-
these
antibodies
be
addition
AB
C
DEF
S
for
are:
B. protein
gel C. protein
details).
gel
The
A. protein
washed
eluted
from
fractions
from
not binding
resin
with
the
during
0.5
non-
useful
for
the
study.
In
to this
specific
assovon
of
the sample
to be analyzed
by the immobilized
antibody.
Because
factor
VIII:C
is apparently
unstable
and is
present
in plasma
in low quantity,
we have developed
a
procedure
VIII:C
from
to concentrate
and
plasma
by using
partially
conventional
purify
factor
precipita-
sample
M NaCI.
pH
at pH 2.8. The fractions
from the
anti-factor
Vlll:C-agarose
are:
gel D. protein
not binding
during
sample
application;
gel E. protein
washed
from
resin with 0.5 M
NaCI, pH 6.0 buffer; gel F, protein
eluted at pH 2.8. Gel S shows the
standard
proteins.
(See text
for details
of the electrophoretic
system.)
The protein
seen on gel F was eluted from the anti-factor
VllI:C-agarose
column
after
23 units
of factor
VIII:C
(52-fold
activatable)
were
bound
by the resin.
buffer;
activity,
though
affinity,
we utilized
the noncovalent,
reversible
ciation
between
porcine
factor
VIII:C
and porcine
Willebrand
factor
as the last step in the preparation
text
gel
inhibitory
Even
there was sufficient
cross
porcine
factor
VIII:C
to
SDS-5%
polyacrylamide
gel electrophoretic
analysis
of
eluate
from antifactor
Vlll:C-agarose.
Analogous
samples
of
factor
Vlll:C,
affinity
purified
on von Willebrand
factor-agarose.
were
applied
to non-immune-lgG-agarose
and anti-factor
Vlll:Cimmune-lgG-agarose
by their
VIII:C.
are antihuman
factor
VIII:C,
reactivity
of this reagent
with
Fig. 4.
(see
which,
to factor
I
the
agarose
we have attempted
to demonstrate
can be attributed
to porcine
factor
activity.
To do this,
we have
used
5A
recent
thrombin
suggests
106
U
to 4.2
This
study
of
(Hultin,
that activated
DAPA.
In our
x
l0
M prior
concentration
K. for thrombin,
the
MB
activation
and
factor
assays,
to the
of DAPA
and
we would
of
Jesty,
J:
human
Blood
factor
VIII:C
1981)
VIII:C
persists
in the presence
the DAPA
in the sample
was
assay
and
is 5 orders
expect
1.4
x
lOb
of 2 x
diluted
M in the
of magnitude
expression
by
57:476-482,
less
of activity.
assay.
than
the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PORCINE
FACTOR
VIIl:C,
SDS-PAGE
ANALYSIS
621
tion was
factor(s)
8000
to 10,000
fold. It is not
has stabilized
the factor
VIII:C
possibly
maintaining
the
0.25 M CaCl2.
Tuddenham
diisopropyl
isolation
peaks
of factor
sulfate-agarose
gradient
from
VIII:C
elutes
Possibly,
the
5 mM
between
precautions
factor
AB
Fig. 5.
thrombin
SDS-5%
Vlll:C.
factor
antifactor
T
S
.
single
The
Vlll:C-agarose
column
after
20
units
of
factor
Vlll:C
(735 units of factor
Vlll:C.)
were
bound by the resin (analogous
sample
is shown
in Fig. 4. gel F). The same
amount
of the affinity
purified
factor
Vlll:C
was treated
with thrombin
(1 U/mI,
37’C. 5
mm)
prior
to chromatography
on the same antibody
column.
The
protein
which
shown
on
thrombin
tion
was
gel
control
and
eluted
subsequently
B. Gel
S shows
the
thrombin.
that factor
the
resin
proteins
at pH 2.8
and
gel
is
T is the
gel.
chromatographic
used to define
factor
VIII:C
from
standard
techniques.
One
we have
the
by
which
suggest
in a precursor
form and final factor
VIII:C
activity
is expressed
when
it is altered
by thrombin
and/or
factor
Xa, calcium
and
phospholipid.
In the preparation
of the partially
purified
factor
VIII:C,
measures
were
taken
especially
by thrombin.
vessel
puncture
rather
blood.
The animals
and the blood was
to prevent
proteolysis,
The blood was drawn
by blood
than
using
slaughter-house
were heparinized
prior
collected
into a mixture
to bleeding
of protease
inhibitors.
Additionally,
the first step in the preparation, after
removal
of cells, was to adsorb
the plasma
with aluminum
hydroxide,
an effective
adsorbent
for
vitamin
K-dependent
The
from
partially
dextran
established
20-25%,
in the
proteins.
purified
factor
sulfate-agarose
VIII:C
did
which
meet
for the subsequent
analysis.
the activation
coefficient
was
starting
plasma
(30-40
fold),
and
the
eluted
criterion
The yield was
similar
to that
the
purifica-
taken
proteolysis
or
to proteolysis
Alternatively,
the
is proteolyzed
These
not
two
and
are
which
has
arisen
however,
may
sufficient
porcine
factor
than is human
protein
and
cleavages,
by thrombin
We did
obtain
may
not cause
we see
from
a
not
be
an increase
or decrease
in activity
or activation
coefficient.
The
von Willebrand
factor-agarose
served
as an
affinity
step as indicated
by an increase
in the specific
activity.
Horowitz
et al.33 described
the properties
of
“low molecular
weight
factor
VIII”
prepared
by affinity chromatography
using
“factor
VIII-Sepharose.”
Their
material
had no detectable
factor
IX clotting
activity,
sedimented
with an apparent
S value of 7.2,
had an VII1:C/VIIIR:Ag
ratio l50,
and was activatable
by thrombin
3-4
fold. Their
procedure,
while
effective
in producing
a purified
product
for analysis
had a low capacity
and required
long exposure
factor
VIII:C
to the resin at ambient
temperature.
criterion
an appropriate
preparation
was that
had to maintain
its activatability
There
have been studies9’31’32
VIII:C
may exist in plasma
chain.
catalyzed
polyacrylamide
gel electrophoretic
analysis
of
von Willebrand
factor-agarose
affinity
purified
protein
on gel A was eluted at pH 2.8 from the
treated
VIII:C.
on SDS-PAGE
I--
not
activity
eluting
from dextran
resin
was eluted
by a linear
to 250 mM CaCl2.
The factor
0.17
M and 0.25
M CaCl2.
to prevent
significant
VIII:C
is less susceptible
--
(DFP)
is necessary
for
human
factor
VIII:C
chromatography.
step and we did
VIII:C
if the
what
stage;
product
at pH 6.0 and/or
et al.16 have indicated
that
fluorophosphate
of stable,
unactivated
by immunoadsorbent
include
DFP
at any
known
at this
difference
explained
between
their
results
by the prior purification
and
steps
ours
which
of the
The
may
be
we used
to remove
much
of the fluid
phase
von Willebrand
factor.
This von Willebrand
factor
in solution
would,
of
course,
be a competitive
binder
of the factor
VIII:C.
The efficiency
of the affinity
step,
as we have
performed
it, probably
represents
the equilibrium
between
free and complexed
VIII:C
as described
by Koutts
et
al.34 and Over
SDS-PAGE
et al.35 Horowitz
analysis
of the
et al.33 did not report
factor
VIII:C;
but
an
our
experience
shows
factor
VIII:C
at
contains
many contaminating
proteins
In the present
study the factor
VIII:C,
using
von Willebrand
factor-agarose,
this
stage
still
(Fig. 4, Gel D).
affinity
purified
had no detect-
able (<1%)
VIIIR:Ag
had a VIII:C/
activatable
by
factor
ratio
IX clotting
activity,
l5,000
and
was
thrombin
30-40
fold.
VIII:C/VIIIR:Ag
ratio
witz
et al.33 and
that
sensitivity
of the
VIII:C
concentration
of dissociation
The
difference
between
the
seen in the analysis
by Horodone
here
may
reflect
the
antigen
assays
used and the factor
being measured
and not the state
achieved,
as their
number
represents
a
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
KNUTSON
622
minimum
estimate.
An unexplained
observation
is that
the activation
coefficient
always
decreased
slightly
after
the von
Willebrand
factor-agarose
step,
even
evidence
that
factor
VIII:C
these bands
represent
activity.
This experiment
by incubating
the factor
though
plasma.
The
application
to the antifactor
this was done,
no detectible
passed through
the antibody
apparent
molecular
weights
it was
30-40
step
in this
definitive
factor
VIII:C
von
Willebrand
VIII:C
agarose
The antifactor
isolated
from
acidic
activity
PAGE
showed
fold,
similar
study
was
to that
the
of
removal
of
activity,
previously
affinity
purified
on
factor-agarose,
by the
antifactor
and not by an analogous
control
resin.
VIII:C
antibody
used in this study
was
a hemophilic
plasma.
The
particular
fraction
which
was used had the highest
specific
in inhibitory
units per mg protein.
The SDSanalysis
of the antibody
column
experiment
that
the antifactor
VIII:C
antibodies
bound
proteins,
ent
always
which
dissociated
molecular
at pH
weights
of
2.8,
and
1 66,000,
had
1 30,000,
bands
weight
did not show a reduction
of apparent
upon treatment
with 2-mercaptoethanol
presence
of 1%
Lane
et al.36
lized antibodies
philiac
patient.
from a commercial
and
affinity
resin,
from solution.
ing conditions,
chains
These
molecular
in the
SDS.
described
experiments
using
to factor
VIII:C
isolated
from
When
4.5 units of factor
VIII,
concentrate,
were applied
to 8 1 % of the activity
The SDS-PAGE
analysis,
showed
that 95% of the
gel was
molecular
associated
weights
analogous
sample
with
two
bands
with
of 62,000
and
35,000.
was
applied
immobia hemopurified
to their
was removed
under
reducprotein
on the
45%
to a column
apparent
When
an
prepared
were eluted.
bovine
factor
was represented
with apparent
85,000
which
55,000,
which
appar-
76,000.
The samples
containing
these
peptide
were also able to block factor
VIII:C
inhibition.
50,000
The
Davie9
by
coupling
rabbit
antifactor
VIII antibodies
to agarose,
the eluate
showed
eight
bands,
including
the 62,000
and the 35,000
molecular
weight
bands.
Our study
differs
from the investigation
of Lane
et
al.36 in several
aspects.
The factor
VIII:C
concentrate
which
was prepared
in our laboratory
included
pro-
VIII:C
the
with
in
protein
having
was carried
out
thrombin
prior
activated
though
the
sibly
the
been
cleaved
to
weights
bands
by Vehar
and
of stained
bands
of 93,000,
of 73,000,
88,000,
69,000,
38,000
after thrombin
treatment.
The
bound
to and eluted
from our antifactor
agarose
column
sented
by bands
the two different
FASS
VIII:C-agarose.
When
factor
VIII:C
activity
column
and proteins
with
of 76,000,
67,000,
and
VIII:C
prepared
by a triplet
molecular
resulted
AND
material
VIII:C-
after
thrombin
treatment
was repreof 76,000,
67,000,
and 50,000.
Thus,
approaches
for siudying
thrombin
factor
VIII:C
“unactivated”
unactivated
by
yielded
material
material
limited
similar
was
in our
proteolysis
results
different.
study
even
even
Pos-
may
have
though
the
material
is 30-40
fold activatable.
Factor
VIII:C
may
be very sensitive
to the first cleavage
and subsequent
cleavages
will produce
full expression
of activity.
The
difference
between
our unactivated
factor
VIII:C
and
Vehar
and Davie’s
cannot
be explained
at this time.
Vehar
and Davie
8 M urea
contained
would
enter
retic
system
a 5%
used an SDS-PAGE
in the gel so that
polyacrylamide
differed
gel.
in that
the
system
that
their
material
Our
sample
electrophopreparation
buffer
contained
6 M urea rather
than
8 M urea and
our gel buffer
contained
no urea. As seen in Fig. 4, all
the material
entered
the 5% polyacrylamide
gel. If we
left the urea
results were
with
urea.
explained
by
We applied
out of the sample
preparation
buffer,
the
identical
to those
with the sample
buffer
The
necessity
for 8 M urea
may
be
the amount
of protein
applied
to the gels.
only
1 5%-20%
of the number
of factor
inhibitors
in the initial
steps;
the factor
VIII:C
was affinity
purified
on von Willebrand
factor-agarose
prior to the antifactor
VIII:C-agarose
step; the antifactor
VIII:C-agarose
removed
20-40
units
(30-40
VIII:C
that
fold activatable)
efficiency;
and
bands
When
tease
from solution
the material
antibody
column
under
nonreducing
on film the bands
gels containing
This,
however,
intrinsic
factor
the
from
von
the equivalent
may reflect
the
VIII:C
activities
Willebrand
VIII:C
no less
eluted
than
from
entered
the SDS-polyacrylamide
conditions.
We also cannot
generated
by Coomassie
Blue
two species.
The demonstration
antifactor
with
which
that
gel
record
stained
of 4 units
of activity.
difference
between
the
of the proteins
from
thrombin
factor-agarose
antibody
85%
the
agarose
sensitive
bind
provides
peptides
factor
Vehar
found
VIII)-Fab
migrated
at
240,000
at 1 80,000
he treated
The
tion
partially
Willebrand
results
isolated
Davie
and
plasma
applied.
‘25[I]-labeied
to
proteins
molecular
(antihuman
that
weight
principally
with
1 30,000
molecular
with thrombin,
the
minor
weights.
240,000
band
disappeared
and
a band
at
weight
appeared.
These
experithat
these
bands
are in total,
or in
part, the factor
VIII:C
plasma
is a complicated
simplify
the experiments
prepared
from von
and
that
bound
molecular
weight
1 10,000
molecular
ments
suggested
to specific
additional
units
Weinstein20
antigen.
Thrombin
situation.
We
by analyzing
purified
factor
factor-agarose.
reported
here used
from
the plasma
VIII:C
treatment
of
have
tried
to
the carefully
which
eluted
an acidic
IgG fracof a severe
classic
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PORCINE
FACTOR
hemophiliac
VIII:C.
SDS-PAGE
patient.
being labeled
to screen
for
VIII:C.
This
IgG
with 12511]
monoclonal
An
ANALYSIS
fraction
was
as a probe
antibodies
accompanying
of the monoclonal
of the porcine
reacting
with
polypeptides
VIII:C
activity
coincidentally
polypeptides
monoclonal
antibody.
associated
but
activity
We
the
indistinguishable
cannot
potential
antibody
sented
1 3,500
about
5%-l0%
fold purified
porcine
column
identified
over the
which
protein
by
by
the
the
from
antibody
procedure
sensitive
conditions
VIII:Ca.
The
polypeptides
There
may
antifactor
are
inhibitor
tion
those
Fig.
eluted
4, gel
F. In
polypeptides
which
time and concentra-
activated
factor
VIII:C
to factor
also retained
about
4% of the
neutralizing
activity
and
repre-
of the stained
protein
from
active
factor
VIII:C,
indicating
the
a
concentration
of these
proteins
in porcine
plasma
of
about
200 ng/ml.
These characteristics,
in conjunction
with the molecular
weight
considerations
above,
provide the comparative
data9
which
suggest
that
the
affinity
punand visualized
with
the
addition,
the
were thrombin
the
bound
bound
and
from
antibodies
and the characfactor
VIII:C
polypeptides
SDS-PAGE
gels
represents
from the porcine
factor
VIII:C.
other
after
assay
factor
describes
distinguish
between
the polypeptides
human
inhibitor
antibodies
and those
mouse
monoclonal
antibody.
We have concluded
that the material
fled by two different
affinity
reagents
on the
derived
used,
in a binding
to porcine
manuscript37
isolation
terization
the
623
be
factor
material
seen
are attributable
in Fig. 4, gel F and Fig. 5, gels
to porcine
factor
VIII:C.
A and
B
VIII:C-agarose
removed
factor
VIII:C
seen on the SDS-PAGE
activity
(compare
gel A with gel D). This suggests
that
these proteins
are necessary
for factor
ACKNOWLEDGMENT
and the
Fig. 4,
one or more
of
VIII:C
activity.
The
polypeptides
eluted
from
monoclonal
antibody
columns
are sufficient
by themselves
for factor
VIII:C
The
authors
Heiler
blood.
the
wish
for
obtaining
We
also
IRMA
tion
for the
of this
Ms.
large
quantities
thank
assay
gratitude
to thank
Ms.
for
Marylou
Donna
von
expertise
Stewart
and
of carefully
Mujwid
Willebrand
of Ms.
Sandra
Mr.
Greg
collected
for
expertly
factor.
We
Schumann
C.
porcine
performing
also
express
in the
prepara-
manuscript.
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1982 59: 615-624
Porcine factor VIII:C prepared by affinity interaction with von Willebrand
factor and heterologous antibodies: sodium dodecyl sulfate
polyacrylamide gel analysis
GJ Knutson and DN Fass
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