Download Factor VIlI-Related Protein Circulates in Normal

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CONCISE
REPORT
Factor
VIlI-Related
Protein
High
The
size
of
agarose
was
it
human
identified
forms
VIll-related
was
identified
with
plasmas
venipuncture
plasma
as
methods.
the
a population
ANY
of
very
the
rabbit
The
distribution
the
pattern
was
complete.
multimers
and
that
the
have
factor
VIII
sought
an
structure
and
issue has been the
protein
(VIIIR),
of the factor
VIII
complex
that
is
heterologous
antibodies
to factor
VIII
and is responsible
for ristocetin
cofactor
(von
Willebrand
factor)
gel filtration
indicated
that
this concept
and ultraV1IIR
is
has
been
by the results
of recent
gel filtration4
ultrafiltration5
studies
carried
out directly
While
it is possible
that
anticoagulation
at low
temperatures
artifactual
might
aggregation
of
and
on
or
be responsible
data.
reason,
VIIIR
studies
conditions,
plasma
(SDS)
were
agarose
plasma
as
in
carried
vitro,
apparent
of
has
multimers
x
of 0.85
Mr
out
same
observations
distribution
the
of
The
106.
University
and
Hematology
with
Under
fresh
Supported
16872,
in part
and
Macy.
Jr.
ilL
Address
Medicine,
Conn.
University
by Grune
31, 1 979,
requests
of
an
and
at 37’C
VlllR:Ag
from
induced
citrate
the
circulates
artifact
and
of multimeric
heparin
in
glyoxyl
agarose,
in
by
time
normal
purification
In the
previously
course
HL
Scholar
of
is
Medicine.
Farmington.
Cleveland,
16361,
Award
and by Grant
C’onn.
Ohio.
HL
of these
Inc.
studies,
for
a new
purified
method
was
bilizing
gel,
readily
glyoxyl
agarose,”’2
controllable
reactivity
which
by
used
initially
was
for separating
as a gel matrix
through
covalent
inert
support
medium
neutral
pH and then
proteins
were
fixed
virtue
of
as an
proteins
at a
to which
the
attachment.
VIIIR,
having
been separated
according
to size by
SDS
electrophoresis
and
then
immobilized,
was
washed
free of SDS,
incubated
with
labeled
antiVIIIR:Ag
and identified
by autoradiography.
MATERIALS
Standard
agarose
lots
type,
474
and
from Sepharose
Glycidol
(2,3
545)
16626,
Co.
Co.
(St.
Louis,
obtained
from
(West
Orange,
rabbit
described.#{176}
Before
from
a patient
U/mI)
ture.
The
HL
1gM
N.J.).
use
nemia,
from
proteins
and
for
1gM
of
molecular
the plasmas
crosslinked
the
of
weight
two
with
(VIIIR:Ag
at room
<
tempera-
markers
with
patients
at
of plasma
included
Sweden),
dimethyl
borate-buffered
incubated
disease
Stockholm,
grade.
previously
volume
5 mm
sodium
Organon,
reagent
it was
g for
(IMCO,
thickness)
and
as
an equal
Willebrand’s
at 8730
used
were
Co.
Chem-
from
purified
experiments,
von
(7-mil
obtained
was
and
Sigma
Me.)
chemicals
16 hr with
fibrinogen
from
composition
was
in these
East-
Chemical
film
mucosa,
centrifuged
human
polyester
other
N.J.).
from
from
(Rockland,
at 4#{176}C
for
severe
Aldrich
Colloids
All
obtained
wasobtained
bond
(HSA
was prepared
cyanoborohydride
from
anti-VIIIR:Ag
with
and
purified
Gel
Marine
intestinal
251-Iabeled
0.01
X-100
Denmark
Piscataway,
were
sodium
obtained
Triton
Mo.),
porcine
SDS
N.Y.);
were
ical
Co.,
agarose
Fine Chemicals,
and
(Rochester,
Wise.);
Litex
glyoxyl-moditied
4B (Pharmacia
was
purified
of the Josiah
I3X-2475
and
METHODS
from
epoxypropanol-1)
Kodak
Inc.
AND
obtained
was
human
macroglobuli-
suberimidate.’4
saline
(BS)
The
has
been
published.’3
accepted
(onnecticui
demonstrated
developed
for measuring
the size of this trace protein.
The method
employs
a newly developed
protein-immo-
heparin,
Council.
to Leon
#{231}
Stratton,
0006-497I/80/5506-0029$0I.00/0
1056
Clinic,
Grants
06032.
1980
Medicine,
by a Faculty
Research
January
reprint
of
to L. W. Hover,
Medical
Submitted
kept
is not
the same as that
VIIIR.6’#{176}
(Milwaukee,
in
an
pattern
Department
Cleveland
by USPHS
19767,
Foundation
the Swedish
School
Division,
same
that
(SDS)
A series
the
dimethylsuberimidate
these
been
identified
a protein
with
multimenic
Division,
of Connecticut
the Research
sulfate
modified
as in plasma
indicate
37#{176}C
for 2 hr and
From
was
freezing
dodecyl
it to the
(VlllR:Ag).
multimers
after
size
sodium
antigen
as
it was separated
by sodium
dode-
electrophoresis.
VIIIR
a series
by
by coupling
VIll-related
man
human
plasma
kept at 37#{176}Cwhile
from cellular
elements
and analyzed
cyl sulfate
determined
electrophoresis
for
suggested
by the two studies,
it is also possible
that the
differences
reflect
problems
associated
with
size
measurements
inferred
from agarose
gel filtration
and
ultrafiltration
For this
been
These
as
R. Shainoff
of VlllR:Ag
the
Plasma
Multimers
anti-factor
x 10’.
was
STUDIES
of
platelet-related
questioned
analytic
plasma.
an
purified
and
large
has
after
Human
chelation.
activities.
While
most agarose
centrifugation
studies
have
very large
(> 106 daltons),’
storage
in plasma
immobilized
using
important
unresolved
of factor-VIlI-related
VIIIR:Ag)
other
and John
of 0.85-12
or calcium
RECENT
the component
identified
by
and
By Leon W. Hoyer
electrophoresis
understanding
function.
An
size in plasma
Weight
was
in serum.
in Normal
Molecular
protein
protein
M
and
until
freezing.
M
The
by autoradiography
anticoagulated
of
factor
electrophoresis.
Circulates
February
W. Hover,
Health
M.D..
(enter,
6, 1980.
Department
Farmington.
Glyoxyl
of
agarose”
4% agarose
was
prepared
glycidol
at room
temperature
agarose
was
washed
and
suspended
pH
7. After
by incubating
100 ml of I M NaOH,
with
then
in 500
60 mm
for
with
ml 0.06
incubation
0.002
16 hr with
distilled
M sodium
at room
water
200
gentle
and
30 ml
agitation.
The
on a B#{252}chner funnel
metaperiodate
temperature,
Blood. Vol.
ml of washed
M NaBH4,
adjusted
the
to
modified
55, No. 6 (June),
1980
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
FACTOR
VIII
agarose
was
washed
transferred
equal
MULTIMERS
mately
of the
1/3
Blood
ture
in
B#{252}chner
settled
were
obtained
( I /50
blood)
in
aliquot
a protocol
tubes
centrifuged
6.88
sodium
was added
after
to glass
dissolved
SDS,
Serum,
at 37#{176}C
for
1.25%
and 0.05
Electrophoresis
cm.
electrode
chambers
acetic
acid,
They
were
agarose,
0.25%
pH
backing
using
with
dye
0.27%
containing
0.1%
a mold
methanol,
After
marker
and
Blue
water
in acetic
liter
the
(1:4:4
were
a metal
punch.
surface
blue
marker
of
0.1
to
M
the
sodium
Ultra-Wicks
(Bio
electrophoresis
protein
R-250
acid,
assembly
connected
by cellulose
Calif.).
Coomassie
with
(Eastman
by
and
were
Kodak)
volume)7
methanol,
and
lanes
for
water
30 mm.
by
volume).
The
samples
hr at room
examined
carbonate,
pH
1% Triton
X-100.
room
for
The
cpm)
total
hr
gently
with
I liter
deionized
out
for
with
-70#{176}C
for 16-24
No Screen
film
were
fixed
ml of 0.2
dish
normal
was
3 changes
Kodak
The
XR-I
at 4#{176}C
for 3-7
BS
and
film
results
for
2
human
lgG
a magnetic
(IMCO);
the
were
gels were
dried.
using
then
each),
washed
over
washed
with
Autoradiography
two
were
Lanex
obtained
was
screens
using
at
Kodak
days.
RESULTS
SDS-agarose
electrophoresis
revealed
a series of VIIIR:Ag
0.85-12
x 106 daltons
(Fig.
of fresh human
plasma
bands of M, spanning
1). The
most
rapidly
migrating
VIIIR:Ag
had slightly
faster
1gM; no smaller
forms
were detected
ment.
The
multimeric
pattern
was
mobility
than
in any experi-
identical
ture,
or
at
incubation
the glyoxyl
was
37#{176}C.The
buffer
agarose
in which
examined.
inclusion
of
8 M
urea
in the
also had no effect.
Studies
in which
or the fixation
step (sodium
cyano-
at pH
patterns:
borohydride
satisfactory
10) were omitted
gave much
less
only a faint outline
of the largest
be detected.
of the polymers
to that
of highly
in each
purified
experiment
fibrinogen
VIIIR:Ag
1gM (0.95
x 106),16
and
1gM polymers
x
106).
The
migration
of the
smallest
band was slightly
faster
than
that of the
monomeric
obtained
from
(1.9-4.8
form
of
patients
two
different
1gM
with
Waldenstrom’s
globulinemia.
The Mr, estimated
from
made
in 12 separate
experiments,
was
106 (mean
±
SEM).
As many
as
VIIIR:Ag
bands
could
be detected
in
The
0.8-1.2
In addition,
poorly
samples
macro-
measurements
± 0.03
eight
separate
the autoradio0.85
in M, of successive
differences
x 106.
bands
resolved
x
were
VIIIR:Ag
migrated
with an Mr of ca. 8 x I 06 to I 2 x 1 06. The
complete
multimeric
pattern
was identified
in plasma
diluted
as much as 1:16 (ca. 2 ng VIIIR).
DISCUSSION
Although
most
chromatographic
and
in seven
vitro
aggregation
temperatures.5
VIIIR:Ag
that
is a
have
favored
by decalcification4
We
report
demonstrate
and
low
here
studies
of plasma
a pattern
of multimers,
all
greater
than 0.85
x 106 daltons,
in material
kept at
37#{176}C
until mixed with an anionic
detergent
(SDS),
an
alkylating
agent
(iodoacetamide),
and urea-agents
that
would
tend
to prevent
VIIIR:Ag
aggregation.
Taken together
with recent
agarose
gel filtration
studies,’7 our data,
and similar
results
obtained
independently
is very
by Ruggieri
unlikely
gates
might
SDS,
until
the electrophoresis
ments
in which
citrate
polymer size.
The method
in 7 expenianticoagulated
ultracentniVIIIR:Ag
reports
been interpreted
as evidence
that VIII:Ag
circulates
in
plasma
as a molecule
that is smaller
than
106 daltons.
They suggest
that the larger
forms are the result of in
separate
experiments
in which
fresh plasma
was anticoagulated
with citrate
or heparin
and kept at 37#{176}C
was initiated,
or hepanin
of 1 hr to 3
fuge studies have suggested
that plasma
large heterogeneous
protein,
two recent
temperature
ml, and the contents
(1 liter
periods
in 2 experiments
chamber
in which
the surface
temperature
at 10#{176}C
(running
tap water),
room tempera-
and
for 2 hr at
(100,000-500,000
10-30
of
pressed,
hr. Equivalent
with
at room
anti-VIIIR:AG
I mg/mI
6 hr.
agitated
-70#{176}C for
and
M sodium
cyanoborohydride
incubated
the incubation.
with
water
M sodium
BS gently
‘25l-labeled
during
period
0.02
subsequently
in the plastic
rocked
a 48-hr
carried
were
in BS containing
volume
migration
in 400
The gels were then rinsed and washed
with
gels
16-24
VIIIR:Ag
by immersion
10, containing
temperature
stirrer.
for
temperature
held
grams.
in
(1:4:4
was
(340,000),
18 x
samples
were
I
SDS)
10 x
bromphenol
gels
migration,
destained
the
slab
agarose,
The
at
serum
polymers
could
The migration
was compared
manner.
glyoxyl
7.0.
in the agarose
so that
added
proteins
in horizontal
Twenty-microliter
Richmond,
of
with
out
phosphate,
agarose
(each
7.0,
Laboratories,
stained
mm
The
blood
in the same
out on a temperature-controlled
60-80
measurement
from
blue
I hr. and marker
was carried
for
pH
obtained
carried
M
bromphenol
cut
4-4.5
5 V/cm
mixture
0.01
SDS,
I .0 cm slots
migrated
Rad
unmodified
spacer.
at 37#{176}C.
This
analyzed
was
M sodium
U-frame
to 0. 1 x
phosphate,
were
cast on a polyester
a plastic
added
mg/mI,
an
to 4
mg/mI
of 0.5%
and held
0.2 cm gels were
with
volume
was
and
added
prepared
phoresis
U/ml
blood
15 mm),
in BS-was
held
incubation.
with
( 10
frozen
to analysis,
In other
studies
of conditions
that might
affect
the
electrophoretic
separations,
similar
patterns
were
obtained
for fresh
plasma
samples
run in an electro-
University
37#{176}C.The
12.5
2-hr
the
heparin
g for
tubes
electrophoresis
containing
0.1%
or
to
mixture
7.0. A 1/25
in BS at 2-3
SDS-agarose
citrate)
iodoacetamide,
pH
dissolved
anticoagulated
was
prior
freshly
by venipunc-
by
were
of plasma
incubation
mg/mI
phosphate,
directly
approved
37#{176}C(3000
of a dilution
of the SDS
included
mo
mixture-approxi-
personnel
prewarmed
at
of plasma-or
volumes
laboratory
of 0.5 M sodium
polystyrene
immediately
water
all of the agarose
Committee,
volume
The
plasma
water,
ofdistilled
at 4#{176}C.
from
with
distilled
with
in a volume
until
stored
Experimentation
citrate
funnel
volume.
heated
aliquots
accordance
Human
at
a
agarose
samples,
1057
PLASMA
and suspended
2% in agarose-was
20-SO-mI
gels
on
to a beaker,
to
and
IN
and
that
the
Zimmerman,’8
large
suggest
multimers
are
that
it
aggre-
formed
in vitro.
Although
aggregated
forms
be dissociated
in vitro by the incubation
with
there is no basis
for an artifactual
increase
in
used
for
protein
identification
in these
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
HOVER
1058
studies,
autoradiography
antibody
glyoxyl
with
agarose.
analytic
after
separated
has several
technique.
incubation
proteins
major
Protein
of
labeled
immobilized
advantages
resolution
gM
is excellent
in
trophoretic
0.95x10
agarose
angles
gel that
removes
SDS
to immunoprecipitate
approach,
protein
mixtures
of acids
factory,
but
dissolution,
body.
The
lems
through
from
proteins
be
relied
on
to
prevent
antigen
especially
in the presence
of excess
glyoxyl
agarose
method
avoids
these
and permits
precise
analysis
of proteins
by their migration
in SDS-agarose.
note that this method
does not rely
tation
to identify
The absence
the specific
of detectable
antiprob-
separated
It is important
on immunoprecipi-
proteins.
protein
trailing
to
in Fig.
1
demonstrates
that there is little interaction
of proteins
with glyoxyl
agarose
at neutral
pH values,
and standard electrophoretic
separations
can be carried
out in
the presence
or absence
of SDS.”
Above
pH 10, the
aldehyde
ible
groups
Schiff
proteins
and
reversible
interaction
can
be converted
NPP
s l06
Fig. i .
SDS-agarose
electrophoresis
of VlllR:Ag.
The migration of marker
proteins.
1gM and 1gM polymers.
is indicated
in the
Coomassie
Blue-stained
gel on the left
and their
molecular
weights
are noted.
The proteins
of other
samples.
each a i :3
dilution
of plasma
in
BS. were
separated
by electrophoresis.
immobilized
in the glyoxyl agarose.
washed
free of SDS. incubated
with “I-labeled
rabbit
anti-VlllR:Ag.
and examined
by autoradiography.
The samples
include citrate
anticoagulated
plasma
from
a patient
with von Willebrand’s
disease
(VWD).
frozen
normal
human
plasma
anticoagulated
with citrate
(CF) or heparin
(HF).
fresh
citrate
(C). or heparin
(H) anticoagulated
plasma.
kept at
37’C until electrophoresis.
and pooled
(n = 24) normal
citrate
anticoagulated
plasma
(NPP).
determinants
remain
readily
available
after
the immobilization.
The basic approach
has a rather
wide potential
for application
and has been of special
antigenic
agarose
form reversgroups
of adjacent
is prevented.
This
of the modified
with
amino
further
migration
bases
H
prior
formation.’9
An
alternative
fixation
after
electrophoresis
using
and alcohols,20’2’
might
be as satis-
cannot
C
.9 x 106
a two-layered
the
HF
3.8 x l06
2.8
at right
F
SHAINOFF
in
as an
this method,
since there is direct
immunologic
identification
of the relevant
bands instead
of a second
elecstep
VWD
AND
value
in the analysis
of fibrin
complexes.’2
to a covalent
ACKNOWLEDGMENT
bond by mild reductive
amination
with sodium
cyanoborohydnide.22
Thus,
proteins
are
immobilized
in
agarose
at the point
they have reached
at the end of
the
electrophoretic
separation.
They
can
then
be
washed
to remove
SDS and incubated
with identifying
reagents.
All of the steps are relatively
gentle,
and
We thank
Carl
Joanne
Randall
and
Carta
Blomb#{228}ck kindly
and Lena
provided
of Drs.
the manuscript.
facilities
for
Coagulation
Research,
studies
were initiated.
The
courtesy
Wikstr#{246}m for technical
for preparing
1gM
G#{246}ranHoIm
for
Dr.
Karolinska
samples
Hoyer
F. Hinz,
Birger
at the Laboratory
Institutet,
were obtained
and Carl
assistance
Professor
where
these
through
the
Jr.
REFERENCES
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OD,
antihemophilic
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Kass
L, Lang
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antihemophilic
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amino
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1980 55: 1056-1059
Factor VIII-related protein circulates in normal human plasma as high
molecular weight multimers
LW Hoyer and JR Shainoff
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