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Synthesis
of Coagulation
By T.
Factor
Jannette
V by Cultured
David
Cerveny,
Bovine aortic endothelium
has been examined
with respect
to the synthesis
of coagulation
factor
V. After
cultured
cells reached
confluency,
samples
of supernatant
culture
media and solubilized
cells were analyzed
for factor V in a
two-stage
bioassay
and in a double-antibody
radioimmunoassay.
In addition.
preconfluent
cells were pulsed for 4
days with S-methionine
in methionine-free
media. After
the 4-day
pulse.
supernatant
media
were
chromatographed
on a factor
V monoclonal
antibody-Sepharose
resin to isolate 35SlabeIed
factor V. The isolated
material
and 125l-factor V standards
were analyzed
by electrophoresis and autoradiography.
The
bioassay
indicated
an
increase,
with time. of unactivated
factor V in the culture
F
ACTOR
V is a high
plasma
essential
an
protein,
cofactor
prothrombin
molecular
to the blood
clotting
vitamin
increase
in the perfusate.59
cell types:
parenchymal,
has indicated
located
in
ney).’#{176}” In addition,
in the
(factor
factor
VIII:vWF
from
factors
are
VIII:vWF
vascular
identiendo-
the cellular
site of synthesis
of
of the factor
VIII
molecule
is uncertain,
its close
association
to
suggests
that the vascular
endothe-
cofactors
es, both
are
sedimentation
and consections
has been
cultured
hum
may
play
a role.
Factor
physical
and chemical
properties
are essential
X),
The liver contains
many
biliary
(epithelial),
vascular
factor
Although
portion
VIII:C)
VIII:vWF
IX,
muscle
of liver
VIII:C
with
in coagulation
activated
coefficients
by
shares
many
factor
V: both
enzyme
factor
thrombin,
and both
in the same
range.’
have
We
radioimmunoassay.
Preparation
Bovine
ing,
Blood.
Rock
ofPrimary
aortas
Dell,
Vol. 63.
used were
The aortas
were
NaC1,
7.4
pH
(Garamycin,
were
obtained
within
No. 6(June),
from
30 mm
1984:
reagent
sealed
Corp.,
collagenase
placed
mechanical
MA)
at 200
g for
aspirated
5 mm
at
room
Walkersville,
serum
(FBS,
Davis),
and
200
jg/ml
was added
was then
plated
tissue
streptomycin
onto
100-mm
washed
6-well
cluster
after
(2-9
culture
plating,
days),
Factor
Fresh
10%
and
changes
every
bovine
Indianapo-
culture
coverslips
medium
dishes
that
was
until
or
had
accordin Costar
added
to become
days
Bio-
cell suspension
glass
3-4
was
A.
(Parke-
Co.,
The
PBS
fetal
penicillin
allowed
(Cam-
(M.
polystyrene
culture
were
and
the
confluent
confluency.
V Bioassay
two-stage
A
Costar
human
fibronectin,
prepared
et al.,’5 in Medium
199
dishes.
medium
Lilly
sterilized
and the cultures
with
with
U/mI
enzyme
and centrifuged
199
suspended.
Costar
and
been precoated
with 30 g/ml
ing to the method
of Roushlahti
day
50-mI
Supernatant
(Eli
pellet
The
endothelium.
tubes
37#{176}CMedium
20
was
closed
37#{176}CPBS,
the luminal
sterile
mg/ml
in PBS
clamped
with
centrifuge
Inc.),
and the cell
culture
of 0.6
for 30 mm.
filled
supplemented
Systems,
amphotransport,
IN)
end
temperature.
and
MD),
Sterile
bath
into
conical
pellet,
cell
solution
vessel
was decanted
the
off
products,
the
polypropylene
1 j.tg/ml
and one end of the aorta
used to dislodge
was
suspension
cell
and
and the remaining
and
agitation
bridge,
onto
closed,
in a 37#{176}C
PBS
decanted
0. 15 M
gentamycin
at 4#{176}C.
After
Indianapolis,
vessel,
indicated.
phosphate,
NJ),
Ni)
bovine
otherwise
10 ig/ml
Kenilworth,
Princeton,
sutured/tied
to incubate
was
lis, IN),
glucose,
A 37#{176}C
filter-sterilized
into the secured
solution
unless
M sodium
(Boehringer-Mannheim,
and allowed
The
were
stapling).
incorporation-all
by cultured
stock
1 mg/mI
Squibb,
vessels
(by
grade
in 0.01
(PBS),
Schering
accessory
S-methionine
V is synthesized
transported
(Fungizone,
performed
From
tion,
bioassay,
on
daily
the
as
described
1 :1 dilutions
Hematology
Rochester,
Supported
I 7430
of
by
cell
et
Nesheim
al.,’6
was
in 0.02
supernatants
M
Research
Submitted
Proc
Research
Section.
Mayo
Clinic/Founda-
MN.
in part
by National
and by the Mayo
Address
METHODS
Cultures
MN,
Mann
and
indicate
that factor
aortic endothelium.
(Fed
AND
G.
supernatant,
whereas
solubilized
cells were
negative
for
factor
V. The radioimmunoassay
indicated
an increase,
with time. of factor V antigen
in the culture
supernatants,
and the solubilized
cells yielded a constant
level of antigen
per cell. Autoradiograms
of electrophoretograms
of immunoadsorbed
35S-culture
supernatant
with 125I-factor
V/Va
standards
revealed
labeled
proteins
with electrophoretic
mobilities
compatible
with ‘25I-factor
V/Va
standards.
The
data obtained
from
three
different
sources-bioassay.
Presentedin
V.
MATERIALS
Kenneth
Endothelium
complex-
have used these chemical
and physical
similarities
as
the basis
for a hypothesis
to determine
if cultured
bovine
aortic
endothelial
cells can synthesize
coagulation
and
tericin
has likewise
been associated
endothelium,
as well as in the
of other
tissues
(skin,
kid-
supernatant
thelium.”’4
the coagulant
VII,
that the vitamin
K-dependent
liver
parenchyma.5
Factor
(von Willebrand
factor)
with the liver vascular
vascular
endothelium
fled
thrombin.’
(II,
and associated
smooth
Immunofluorescence
the
as
of
that many of the
V, VII, XI, XII,
factors
Fass,
materials
loop, activates
factor
and 94K
peptides).’
indicated
factors
K-dependent
(endothelial),
nective
tissue.
enzyme,
feedback
wt 74K
Liver perfusion
studies
have
coagulation
factors,
including
the
single-chain
which,
upon activation,
serves
for the optimum
conversion
Thrombin,
in a positive
V to factor
Va (mol
and
weight
N.
Aortic
May
part
20. 1983;
accepted
at the FASEB
42:1032,
1983,
reprint
requests
Section,
Institutes
of Health
Grant
HL-
Foundation.
absir
Mayo
January
Meeting,
/8.
April
1984.
1 983,
Chicago.
JL
4339).
to Dr.
Kenneth
Clinic/Foundation,
G. Mann,
Hematology
Rochester,
MN
55905.
Rock
Dell
of animal
pp. 1467-1474
Meats
and
exsanguination.
Process-
©
All
I 984 by Grune
& Stratton,
Inc.
0006-4971/84/6306-0030$03.00/0
1467
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1468
CERVENY,
imidazole-HCI,
0.15
deficient
prepared
plasma
factor
anti-human
bin-treated
and
tor
burro
three
IIC3
Factor
factor
V-
culture
plasma
on
in the
results
are from
IVC6,
burro serum
studies,
polyclonal
cold
are inhibitory
were
antibodies,
For
the
and
was used as a control.
V Radioimmunoassay
Radioimmunoassay
cultured
cell
antibody
technique
mogeneous
et al.”
of
single-chain
specific
ng/assay.
that
was
activity
factor
prepared
of
Aliquots
been
described
bovine
V was
had
accomplished
previously
‘251-factor
with
samples
supernatants
taken
using
by
Tracy
et
V was isolated
by
600-900
the
the
doubleal.’8
and
supernatant
was
used
at
were
used
in the
5
assay as the sample with unused FBS-supplemented
Medium I 99 as
a control.
Burro anti-bovine factor V lgG and goat anti-burro
IgG
were
prepared
tated
pellets
and
were
evaluated
as described
counted
by Tracy
in a Beckman
8000
et al)8
An indirect
immunofluorescence
gamma
counter.
and
a
secondary
antibody
bovine
aortic
the
glass
then
in PBS,
(FITC)
which
were
in cold
had
at
(4#{176}C)buffered
PBS
and
stored
jars.
at
The
-70#{176}C (for
fixed
37#{176}C
for 1 hr. then
Sigma, St. Louis,
antibodies
were
the
to
were
acetone-formalin,
in PBS-l%
followed
from
stock
polyclonal
anti-bovine
from
IgG
factor
ascitic
supplied
by
Dr.
W.
B.
mouse IgG
precipitation
and
nonimmune
burro
by
in
washing
(anti-mouse,
Laboratories,
Cochranville,
applied
antibodies
to the
and
Zeiss
Standard
and barrier
positive
samples
a uniform
endothelium
for
were
the
removal
30 mm
in the
then
removed
by washing
slide
and photographed
with
were
Ektachrome
taken
in
with
with
appropriate
400 and/or
Tri-X
and
film
of 60 sec.
experiments
were
grown
a
excitation
of negative
to
each
the
in
carried
Falcon
out
35-mm
on bovine
tissue
of
and
M Na
dishes
the
burro
and
the
anti-bovine
serum
and
albumin
applied
temperature.
and
for 30
Alternatively,
washing,
coverslips
(1:1).
were
cells
The
fluorescence
were
micros-
V/Va
and
four
on
was present
V/Va
lgG
(IB6),
III
which
(ATIII)
in the
flow-through.
been
immobilized
MN)
with
buffer).
was run in duplicate
(which
placed
been
had
column
added.
The
tubes
were
30-45
mm.
The
flow-through
this
point,
the
with
each
buffer.
and the 2-mi
fractions
HP
cpm, elution
I 00
using 0.2 M glycine,
pH
scintillation
The
counts.
dialyzed
versus
with
change
one
preparation
Elect
buffer
LS-7500
liquid
2.8.
of dialysate.
All
for electrophoretic
thrombin
with
M
counter
with
less
were
accomplished
were
(2
acetic
were
column
scintilla-
counts
was
At
anti-ATIII-
via
each)
were
ml
acid
then
by
monitored
overnight,
lyophilized
in
analysis.
utoradiography
35S-samples
of Neville27
eluates
one
scintillation
fractions
of 0.2
355-superna-
washed
fractions
peak
volume
and
When
for
glass
V content.
by monitoring
counts
Elution
the
four
10 U of bovine
scintifluor.
bound
two
an 800-mI
rophoresis/A
Na2 EDTA).
followed
into
and
were
were
the
aliquots
platform
factor
with
columns
as the
of
frits,
column
treated
All
in a Beckman
Redi-Solv
wool
poured
to determine
V-Sepharose
medium,
on a rocker
were
of
centrifuge
35S-supernatant
placed
glass
bioassayed
were
column
counts
FBS-supplemented
and
with
aliquots
conical
with
anti-factor
of column
be treated
(0.5-ml
polystyrene
35S-supernatants
was
could
resins
0.15 M
immunoisolation
buffer)
and
by Dr.
M Tris-HCI,
Each
one column
four
CNBr-
anti-human
provided
precoated
columns
one
into
was
for use as a control.
0.02
equilibrated
sealed
resin
pipette
tant
I ml
so that
The
with
prechro-
on
was generously
Rochester,
equilibrated
rinsed
that
anti-bovine
Mouse
B. Foster.’9
7.2 (column
then
of
ensure
chromatography
resins were
were
sample
The
monoclonal
CaCl2,
resin)
to
mouse
had
Sepharose
to elution.
355
after
CNBr-
et al.25 A 50-id
NaCI,
prior
was
Cell
mI/dish)
(10
bioassayed
by W.
Clinic,
pH
cells.
glycine-quenched
The antibody
5 mM
of
Immunoisolation
provided
(Mayo
streptomycin,
Amersham)
was
the aforementioned
Sepharose,
Island,
was divided equally into four aliquots
10 ml each).
using
RPM!
Grand
plus
dishes
March
35S-supernatant
(approximately
penicillin
dishes
the
as per
35S-supernatant
GIBCO,
Ci/mmole,
culture
from
collected
activity
kit,
(1,300
100-mm
FBS-supplemented
to methionine-free
medium
prechromatographed
matographed
in
changed
was
10% FBS
4
flow-through
factor
preconfluence
to
Sepharose prepared
Lyophilized
also
at 4#{176}C
with
serum
glycerol:PBS
35S-methionine
were
days
than
at room
(0.01
d
by epiillumination
with
mCi
Beckman
BSA
primary
excess
chamber
on a microscope
viewed
equipped
(2#{176}
passage)
of the
moist
0.5
tion
Cappel
in PBS-l%
of
grown
supplemented
buffer,
goat
incubation
medium
then
in
removed
from
I :200
was
Photographs
time
was
After
“Select-an-Amine”
Sepharose
and
(FITC)
anti-horse
was mounted
using Kodak
exposure
and
Gonchoroff
IgG’8
antibody
diluted
FITC-antibody
Cells
N.
ambient
(PBS)
bovine
burro
cells,
successive
10 mm
from
in PBS-1%
drops
The
NY)
Pasteur
MN),
V/Va
fluoresceinated
were
following
for FITC.
lmmunofluorescence
aortic
PA)
18 Microscope
filters
primary
prepared
generously
at
were
(from
then
by ammonium
by Dr.
199.
tubes
mouse
Rochester,
cells
1640
each
nonim-
al.,24
and
serum
factor
anti-rabbit,
coverslip
( I :2).
glycerol:PBS
with
excess
and the moist
Clinic,
donated
excess
et
and IIIA5)
mouse
secondary
incubated
The
temperature.
PBS,
cells
(Mayo
from
The
Ni),
Ey
BAE
thrombin
(Behr-
for
500
the
with
Immunoisolation
column
used
VIII/vWF
et al.’9
anti-bovine
The
made
antibodies
(IB6
Foster
generously
serum.
PBS.
antibodies
and
Foster
burro
stocks
were
by
IgG
by
prepared
and
Clinic),
(Mayo
IgG
dilutions
1:250
and photographed
M. E. Nesheim
room
fixed
was removed
nonimmune
chamber
several
antithrombin
antibody
Somerville,
monoclonal
at
inspected
activated
then applied
chamber
factor
as described
described
as
I % BSA,
Primary
Cal-Biochem,
V/Va
(BSA,
Primary
and
anti-human
purified
fluid
nonimmune
sulfate
rabbit
antiserum,
rabbit
mune
of lgG,
at
and
MANN
copy.
performed
coverslip
chamber
albumin
in a moist
immunoglobulins.
the following:
serum
all primary
mg/mI
in sealed
PBS washes.
in PBS-
mm
30
of
8-10
bovine
as
intervals
Approximately
diluted
antiserum.
over
factor
cold (4#{176}C)
in
a moist
in
by two
times
for
Concentrations
these
ing
50-250
cells
10 days)
rehydrated
MO),
approximately
included
up to
were
washed
diluted
rehydrated
temperature.
cells
on
postconfluence,
saline
7.4)
ethanol
1:250
a moist
activated
tempera-
The
pH
AND
nonimmune
burro
serum
was diluted
1:250 in PBS-l%
BSA and
similarly applied. The cells were washed and normal goat serum,
diluted 1:20 in PBS-l% BSA, was applied for I 5 mm. Another PBS
wash
was followed
by application
of fluorescein-conjugated
goat
4
Confluent
room
in
days
at -min
phosphate-buffered
NaCI,
was
supernatants
conjugated
described by Yam et al.,23 for 90 sec and finally washed
coplin
involved
been cultured
washed
containing
added
in question
antibody.28’22
cells,
coverslips,
fixed
the protein
primary
(BAE)
endothelial
against
isothiocyanate
against
fibronectin-coated
ture
directed
fluorescein
directed
was used that
technique
antibody
V IgG
(BSA)
Medium
Precipi-
Immunofluorescence
the use ofa primary
factor
affixed
method,#{176}
M
Three
3 times
as above.
anti-horse
Ho-
as per Nesheim
Bolton-Hunter
cpm/ng,
of 0.2 ml of culture
from
the
of fresh
0.15
washed
mm
CA).
rinsed
70% ethanol.
cells
polyclonal
(Oxnard,
were
volumes
phosphate,
antibodies
antibody.
I-mI
throm-
(procofac-
monoclonal
monoclonal
dishes
dishes,
of total
a measure
purified
and
is a noninhibitory
studies, nonimmune
human
of whole
For antibody-inhibition
V’8 and
of these,
Two
IB6
using
All assay
and are, therefore,
activity.
cofactor)
7.4,
pH
by immunoadsorption
V-Sepharose.’7
samples
anti-factor
used.’9
NaC1,
M
FASS,
(10%
were
SDS,
251-bovine factor
dissolved
in the sample
2% fl-mercaptoethanol,
V and thrombin-activated
preparation
and
12.5 mM
‘251-bovine
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
FACTOR
factor
V BIOSYNTHESIS
were
for
Va
heat
block
well
of
used as standards.
10-IS
a 5%-I
mm,
5%
1469
BY ENDOTHELIUM
and
Samples
were
equivalent
gradient
resis,
the
gels
fixed
were
Hance”
by
(New
water
England
placed
DuPont
a Wafer
“Lightning
x-ray
XAR-5
film
for
Plus”
exposure
a 4%
out
then
fixed
on a slab
in “En-
intensifying
were
gel drier.
Each
cassette
screens,
- 70#{176}C,
with
with
lined
Kodak
of exposure
time
of sample applied to
followed
gels
x-ray
points.
No adjustments
points
unused
presented
medium
(considering
acetic
fixed
for 45 mm,
The
in a
electropho-
acid-glacial
mm.
dried
with
Following
rigid-form
at
on the radioactivity
dependent
carried
MA)
45
least
paper and
inside
gel
was
30 mm,
Boston,
at
on electrophoresis
gel was
with
for
for
to each
added
slab
trichloroacetic
(v:v)
Nuclear,
rehydration
mounted
dried
in 100%
(1:1:3:5)
at 90#{176}C
in a
were
polyacrylamide
acrylamide
stacking gel,26 and electrophoresis
BioRad “Protean”
electrophoresis
apparatus.
acid-mcthanol-water
heated
counts
gel.
tion
contributed
RIA and
Daily
cated
an
showed
BAE
culture
level
of factor
Radioimmunoassay
higher
bioassay
antigen
sensitive
of
increasing
bioassay.
to
RL.4
samples
levels
data.
of factor
The
data
of
by
the
consistently
V antigen
as compared
activity
versus
stems
from
the RIA
being
of factor
V/Va
and not just
which
give bioactivity.
inactive,
as well as
Thus,
active
factor
V products.
the results
of the
Figure
1 presents
two assay methods
cal samples
from
taken
mdi-
V activity
divergence
data
most
likely
to all fragments
the two
quantify
supernatants
(RIA)
confluent
the
or
RIA
will
activatable,
a comparison
of
from four identi-
cultures
over
a 4-day
period
between
medium
changes.
Both RIA and bioassay indicate
an increase
in factor
V antigen
over the
4-day period.
Unused
FBS-supplemented
Medium
199
background
was
subtracted
from
the
raw
of the
lability
bovine
labile
supernatant,
as
immunoisolation.
relatively
wide
RIA
data
of
of factor
serum
factor
later
V, heat
prior
confluent
range
of 27-340
ments,
ng/ml
by
factor
cultures.
ng/ml
in the
have
V-deficient
in Table
1
determined
two
were
inhibitory
conducted
using
were assayed
using human
for the
factor
is presented
V/Va
was
at a concentra-
of the
monoclonal
a
experi-
of inhibitory
and
In these experi-
to be present
Incubation
in the
For some
plasma.
A typical
experiment
In this experiment,
factor
by bioassay
For
1983,
observed
.
of 1 33 ng/ml.
a
was
experiments,
was observed.
supernatants
V bioactivity
the
that
variables.
1981 and
these
studies
culture
from
pointed
out
V biosynthesis
and a combination
monoclonal
antibodies.
ments
culture
fluid
production
of factor
with
may
results
V was
For
immunoinhibition
both polyclonal
noninhibitory
tion
inactiva-
to use
V molecule
evidenced
It should
be
variation
in factor
144
4-day
cell
supernatant
antibodies,
IIC3
and
IVC6,
resulted
in total loss of the observed
bioactivity.
In contrast,
incubation
with
a noninhibitory
antifactor
V monoclonal
antibody
(IB6)
led to no inhibition.
A similar
burro
factor
result
antisera
V sera.
activity,
I
fetal
data
V activity
in the
by the bioassay
to this finding).
The differences
between
bioassay
may be attributable
to the fragmen-
a mean
and
to the bioassay
observed
due to presently
unexplainable
seven experiments
conducted
between
RESULTS
Bioassay
made
,
the heat
of the
tation
were
in Fig. 1 as factor
was undetectable
was
The
whereas
was
obtained
when
compared
to immune
latter
totally
abolished
the
former
had
nonimmune
burro
antifactor
V
no effect.
.3
1.2
Immunofluorescence
1.1
to
C
>
1.0
0.
IRIA
.-
>0#{149}
tto5
!
The degree
of fluorescence
appeared
to be dependent
for the factor
V antigen
on the age of the culture,
with
optimum
fluorescence
visualized
2-3 days postconfluence.
Not all cells
.6
from
cultures
were positive
for
Uo
.
.4
.‘..,
.3
I
BIOASSAY
Table
1 . Immune
lnh ibition
of Endothe
hal Cell Factor
Factor
0
Time
Fig. 1 .
Factor
sortie
endotholial
Recovered
Antibody
2
3
(days)
V production
by confluent
cultures
of bovine
cells. On day 0. the culture
media were changed
over
a confluent
primary
culture
of bovine
sot-tic endothelial
cells.
The supernatant
was sampled
on days 1 -4 and analyzed
for both
factor
V-reactive
protein
(RIA) and functional
factor
V (bioassay)
using the procedures
developed
by Tracy et al.’ The error bars at
each point represent
the mean and standard
deviation
for four
separate
culture
dishes.
The polyclonal
nature
of the burro antibovine
factor
V antibody
results
in detection
of all the fragments
of
factor
V. The divergence
of the RIA and bioassay
data over the
4.-day time course
probably
is the result of degradation
of factor
V
following
synthesis.
Specificity
Added
4
V Activity
V/Va
Percent
Recovery
(ng/mI)
100
133
None
-
11C3
Inhibitory
aEt
0
0
lVC6
Inhibitory
aEt
0
0
Noninhibitory
115
aDt
1B6
BurroaV
Nonimmune
To 90
burro
0
-
1 18
88
antibody
tme factor Va chain specificity
of the antibody
is specified.
To 90 M1 culture fluid supernatant,
10 gzl of a 10 mg/mI
antibody
solution
fluid
0
mg/mI
solution
.d culture
86
-
supernatant,
10 I
of a 0.5
was added.
was
added.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
CERVENY.
1470
factor
V antigen
technique
used.
The
studies
endothelial
antibodies
sera.
were
cells
and
Although
obtained
by
of
were
with
the
factor
indirect
V
conducted
polyclonal
positive
with both
immunofluorescent
cytoplasm
of select
stage
of cell cycle
immunofluorescence
on
pared
V
tion
with
as the monoclonal
the same exposure
(Fig.
2B
with both monoclonal
burro
anti-factor
immunofluorescence
the monoclonal
and
results
polyclonal
with
cells,
(Fig.
observed
nonimmune
).
fluorescence
intensity
clonal
reagents
were
experiments.
Figure
cence ofcells
treated
but weak.
For
cence
appeared
and
reagents,
the
and restricted
fluoresto the
the
Fig. 2.
Indirect
immunofluorescence
of the factor
V/Va
associated
with
bovine
aortic
endothelium.
Confluent
cultures
of
bovine
aortic
endothelium
were fixed in acetone-formalin
prior to
indirect
immunofluorescence
processing
with primary
antibodies:
(A) murine
monoclonal
anti-bovine
factor
V/Va
(1B6); (B) nonimmune
mouse
lgG at an equivalent
protein
concentration.
The
secondary
antibody
for both A and B was an FITC-conjugated
goat
anti-mouse
lgG and produced
the resultant
fluorescence.
The
fpctor
V associated
with bovine
aortic
endothelium
is apparent
4rom the fluorescence
observed
in A (480x).
counterstained
horse antiserum.
in Fig. 3B. For
experiment
antibody
time did
MANN
concentra-
and photographed
not show fluorescence
immunofluorescence
reagents,
relatively
high
was observed
when
the polyused in the immunofluorescence
3A presents
the immunofluoreswith burro anti-factor
V antibody
with
fluorescein-labeled
The nonimmune
the experiment
was
culture
conducted
dish
goat
control
presented
directly
35-mm
tissue
observed
Distinct
by epi illumination
fluorescence
perinuclear
cytoplasmic
staining
Fig. 3.
Indirect
ciated
with bovine
AND
at a particular
controls
pre-
at the same
to the weak
monoclonal
reagents,
the polyclonal
reagents
gave
rise to much
greater
intensity
of fluorescence.
The intensity
of immunofluorescence
observed
with
monoclonal
antibody
staining
was distinctly
positive
the monoclonal
to be diffuse
perhaps
those
2A ). Negative
Ig used
In contrast
with
FASS.
and
the
anti-
is presented
in Fig. 3A,
in
a Falcon
fluorescence
microscopy.
is observed
immunofluorescence
of the factor V/Va
assoaortic
endothelium.
Preconfluent
cultures
of
first
passage
bovine
aortic
endothelium
were fixed in 70% ethanol
in 35-mm
plastic
dishes and analyzed
with (A) 1 :250 dilution
of
burro
anti-bovine
factor
V/Va
lgG (10 mg/mI)
diluted
in a 1:250
dilution
of nonimmune
burro serum
in 1 % bovine serum albumin.
and (B) the nonimmune
burro diluent.
The secondary
antibody
for
both A and B was a goat anti-horse
Ig conjugated
with FITC and
diluted
1:200 in PBS-1%
BSA (800x).
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
FACTOR
V BIOSYNTHESIS
in some,
but
for the
various
aortic
not all, cells,
monoclonal
gradations
observed,
BY ENDOTHELIUM
a result
1471
previously
antibody
reagents.
of immunofluorescent
suggesting
endothelial
that
cells
the
synthesis
The
brand
with
cell
for
the
factor
brightly
). Positive
is used
endothelium.’#{176}2729
which
Behring
exposure
time,
was
are
V by
property
for
rather
must
be
that
is perhaps
confluent
cultures
were positive
for von
factor,
using
Behring
rabbit
anti-human
Willebrand
to
tive controls
comparison
4B).
provided
a low background
to the positive
antibodies
Although
ing that
the
factor
immunofluorescence
V is in/on
is consistent
with
not
conclusive
provide
contrast
(Figs.
2B,
the
by the endothelial
the
RIA
data
endothelial
and
evidence
indicat-
cells
bioassay
for
3B,
studied
results,
it does
concerning
synthesis
cell.
cycle.
VIII:vWF
IgG.
Large,
were observed
(Fig. 4A
control,
of factor
is not a continuous
all endothelial
cells
in culture
but
subject
to some
sort of regulation
associated
alluded
Furthermore,
staining
fluorescent
fluorescence
The
antisera
and
was
negative
nonimmune
at the
same
starting
criteria
factor
rabbit
IgG
concentration
photographed
for fluorescence.
for
For
granules
for von
as one of the identity
used
Immunoisolation
Willefactor
the
as
same
All nega-
the
experiments
material
supernatant)
described
(4-5
had
V activity
1.57
x
by
in Figs.
days
cpm/1O
108
the
5 and
postconfluent
bioassay
ml
(27
6, the
culture
as well
ng/ml).
as
The
flow-through
from the glycine-Sepharose
column
was
bioassayed
to ensure
that factor
V activity
was present
prior to immunoisolation
chromatography.
No factor
V activity
was lost; in fact, bioactivity,
as determined
by the bioassay,
glyci ne-Sepharose
increased
slightly
prechromatography
(32
ng/ml).
This
step
was
in-
cluded
in the protocol
to decrease
the amount
of
nonspecific
binding
by the 35S-labeled
products
that
were binding
to the immunoabsorbent-Sepharose.
This
nonspecific
binding
had presented
a problem
in recovery,
as factor
V represents
other cellular
products.
through
contained
1.48
applied
to
the
a trace
protein
The prechromatography
x iO cpm/lO
ml,
anti-factor
relative
to
which
flowwas
and
anti-
V-Sepharose
ATI I 1-Sepharose
columns.
The anti-ATI
I 1-Sepharose
was chosen
as a control
solely on the grounds
that the
ATIII
antigen
mass is entirely
outside
the molecular
weight
range
of factor
V/Va
antigens.
Whether
or not
250.000
10.000
[us)
cpmi
501uJ
5.000
0
5
10
15
Fraction
Fig. 5.
Fig. 4.
Indirect
immunofluorescence
of the factor
Vlll:vWF
associated
with bovine aortic endothelium.
As a positive
control.
cells from
the same
endothelial
cultures
as in Fig. 2 were
processed
for indirect
immunofluorescence
with primary
antibody
(A) Behring
rabbit anti-human
factor
Vlll:vWF
and (B) nonimmune
rabbit lgG at an equivalent
protein
concentration.
The secondary
antibody
was FITC-conjugated
goat anti-rabbit
IgG and produced
the resultant
fluorescence
(800x).
Elution
of radiolabeled
proteins
20
25
30
35
number
from immunoabsorbent
columns.
Culture
fluid supernatant
obtained
following
incubation
of confluent
bovine
aortic
endothelial
cell cultures
with
Smethionine
was first treated
on glycine-Sepharose
to remove
the
major synthetic
products
derived
from these cells in culture.
The
breakthrough
fractions
obtained
from
glycine-Sepharose
were
applied
to either
murine
anti-bovine
factor
V/Va
(IB6)-Sepharose
(X) or murine
anti-human
ATIII-Sepharose
(0). The columns
were
washed
with
solutions
A, B. and C and eluted
with solution
D. (A
and C) 20 mM Tris. 0.1 5 M NaCI, 5 mM CaCI2. pH 7.4; (B) 2 M NaCl;
(D) 0.2 M glycine.
0.1 5 M NaCI. pH 2.9; fraction
volume
2 ml.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
CERVENY,
1472
330K
p
94 K
74 K
wash
to background
cpm.
tivity
was
with
the
total
anti-factor
cpm
in
C
D
produced
indicated
tivity,
retained
implied
the resin.
been
these
endothelial
antibody-Sepharose
proposed
revealed
implying
by this
resin
factor
was
and
had
added
the
columns.
anti-factor
V-Sepharose,
for identification
of the
anti-factor
an increase
that factor
that,
potentially,
occurred.
At this
The
while
is
of bioac-
4.3
5 is an
autoradiogram
x
and
i04
total
Fig.
5).
8 x
the
anti-
cpm
in its
V/Va-Sepharose
in factor
V had not
of the
activation
immobilized
some
point,
pairs
of
I0
of
factor
Va
35S-eluate
factor
of
V-related
total
products
35S-labeled
supernatant
start
for by the factor
prechromatography
wash,
necessary
that visualization
possible
bin-treated
factor
the
V
The anti-factor
non-thrombin-
protein
A very
from
the
small
fraction
cell
culture
material
(lane A) can be accounted
V. This
small
fraction
made
the
step,
as well as the 2 M NaCl
to decrease
the nonspecific
of the recovered
factor
binding
V would
so
be
on the autoradiogram.
Lane
E is the thromanti-factor
V-Sepharose
355-eluate
and has
bands
at 94,000
and
74,000
corresponding
to the thrombin-treated
factor
Va standard
in lane F. The
molecular
Guinto
amounts
and
Esmon,3#{176} show
of methionine,
equivalent
labeling
The
two factor
which
weight,
(activated)
125J
other band in lane F
corresponds
to the factor
Va activation
apparent
molecular
weight
7 1 ,000.
The
data for the 94,000
and 74,000
peptides,
peptide
of
composition
as published
relatively
predicts
equivalent
the
intensity
observed
in lane
Va peptides
may
result
relatively
E.
from
a
variety
of cleavage
that,
in addition
mechanisms
of factor
V. It is likely
to factor
V itself,
a variety
of high
molecular
weight
B, are contributing
fragments
to the
the
appear
E that
to be some
of factor
V, as seen in lane
bands
in lane E. There
also
low molecular
weight
bands
in lane
correspond
to noncovalently
associated
cleavage
fragments
of the 94,000
and 74,000
peptides,
which
were
bound
on the antibody
column
after
thrombin
treatment
and correspond
factor
V. The thrombin-treated
Fractions
content.
355-eluant
V thrombin
by following
the 355-isotope
of the unbound
radioactivi-
from
‘25I-bovine
355-eluate
in lane D
in the region
where
occur.
products
factor
V, and thrombin
treatment
of the anti-ATIIISepharose
served
as a control
for this identification.
were monitored
Following
elution
of eluates
standards.
from
the
the control
anti-ATIII-Sepharose
has a marked
absence
of bands
on the
provided
significant
evidence
eluted
355-labeled
substance
as
with
treated
column
in lane
B has a 35S-labeled
peptide
band
at 330,000
molecular
weight,
corresponding
to
the adjacent
‘25I-factor
V standard
in lane C, whereas
by
V activity
was
the anti-ATIII-
to one of each
V molecule,
cells
level
that
factor
Bioassay
of
of inhibitor(s)
U of thrombin
from
an undetectable
flow-through
(43 ng/ml),
retained
radioacV presence
yielding
fraction
chromatogram,
and ‘25I-bovine
V-Sepharose
F
E
by
V-Sepharose
depletion
factor
V-Sepharose
peak
(see
anti-factor
unknown
at this time.
Bioassay
of the flow-through
Sepharose
V activity
of bound
pH,
Autoradiography
Fig. 6.
SDS gel radioautograph
of synthetic
products
labeled
with S-methionine
obtained
from antibody
columns.
(Lane A) The
fraction
obtained
after treatment
of the culture
fluid supernatant
with glycine-Sepharose
to remove
major
high molecular
weight
synthetic
products.
This material
was applied
to the specific
antibody
columns.
(Lane B) Material
eluted in peak D (Fig. 4) from
the murine
anti-factor
V/Va
antibody
column.
(Lane C) A radioiodinated
factor
V standard.
(Lane
D) Equivalent
eluate
(peak D)
from the murine
anti-ATIII
antibody
column.
Factor V. as well as
degradation
products
obtained
from
single-chain
factor
V. are
apparent
in lane B and are absent
in lane D. (Lane E) Protein
eluted from murine
anti-factor
V/Va-Sepharose
after prior treatment of bound protein
with thrombin.
(Lane F) A radioiodinated
factor
Va standard.
(Lane
G) An equivalent
fraction
(peak
D)
obtained
from the murine
anti-ATIII
column
after
treatment
of
bound protein
with thrombin.
The bands coincident
with the two
factor
Va noncovalently
associated
chains
(94K and 74K)
are
apparent
in lane E and are absent in lane G. These results indicate
that
intact
and thrombin-activatable
factor
V is produced
by
bovine aortic
endothelium.
as it is specifically
bound and subsequently
eluted from murine
anti-bovine
factor V/Va-Sepharose.
which
by
the
yielding
fraction
Figure
is
at low
could
not be monitored
by bioactivity;
therefore,
elution was monitored
by
content.
The glycine-eluted
355 products
came
off discretely
in the first 2-3 frac-
peak
‘up,’
ATIII
As elution
accomplished
ATIII-Sepharose
B
MANN
washed
with 2 M NaCl
to further
binding
of 355-labeled
protein.
was followed
by column
buffer
i04
,
AND
ty, the columns
were
decrease
nonspecific
This
high salt wash
tions,
ii
FASS,
to inactivation
products
anti-ATIII-Sepharose
in lane G is blank
in the region
activation
peptides.
of the
of
factor
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
FACTOR
V BIOSYNTHESIS
BY ENDOTHELIUM
1473
DISCUSSION
The
bioassay,
cence,
strate
radioimmunoassay,
demoncells
polyclonal
nature
of the burro
anti-bovine
factor
V
IgG, which
detects
all identifiable
antigen,
whether
bioactive
or not.
Immunofluorescence
was employed
to further
delineate whether
the substance
produced
by the cultured
the
in vitro
endothelium
cells
provide
cultures,
which
were
preconfluent,
confluent,
and
varying
days
postconfluent,
were
used
initially
until
determination
was made
that 2-3 days postconfluent
was the optimum
time for immunofluorescence
detec-
immunofluores-
and immunoisolation
data reported
here
that
cultured
bovine
aortic
endothelial
synthesize
factor
culture
V, and,
conditions
the
factor
V as a secreted
lines the luminal
surface
may
provide
a vehicle
factor
V.
Several
liver
at least
used,
under
endothelial
protein.
Because
endothelium
of the entire
vascular
tree,
for supplying
perfusion
ation of coagulation
perfusate.9
These
the plasma
studies
factors,
perfusates
have
it
pool
tion.
The
gener-
fine,
granular,
V, in the
exposed
cell.
Nuclei
indicated
including
factor
were probably
of
was
factor
fluorescence
exhibited
Primary
of factor
and
did
V.
diffuse
not
fluorescence;
and
passaged
V was
noted
the
cytoplasm
over
fluoresce.
Indeed,
it appeared
to be very
of the
not
as ifa
all
cell had
cells
to be
to the parenchymal
cells of the liver,
as well as to
endothelial
cells that line the vessels
and sinusoids
of
the liver. The relative
contribution
of endothelium
to
the production
of factor
V by the liver is unclear
at this
in a particular
stage
of the cell cycle,
or in a specific
metabolic
state,
to exhibit
the antigen.
The anti-von
Willebrand
factor
lgG
resulted
in fluorescence
of
bright
granules
over the area
described
by the cell,
time.
It has
synthesize
often
tin,333
been
and
demonstrated
secrete
a variety
collagen,34
aortic
endothelial
three
major
and
factor
cells,
proteins
VIII:vWF.’2’4
in vitro,
into
fibronectin,
a noncollagenous
III collagen.34
Fibronectin
1 5% of the protein
released
also
fore,
less
by
in
V were
provided
supernatants
inactivated
V-like
culture
supernatant:
vitro
plasma
BAE
cell
expected
initial
activity
could
in excess
of that
FBS-supplemented
proteins;
Va for detection.
Daily
in culture
present
in unused
medium.
bioassay
and
low. The
that
at
be found
investigate
this
heat-
only
the
treatment
factor
V to
of the confluent
munoassay,
was applied.
which
Factor
increase
with time
RIA than they had
factor
V detected
a double-antibody
had been
previously
V antigen
levels
and to much
in the bioassay.
by the
RIA
can
matrix
supernatant
of supernatant.
yielded
autoradiograms
was
in the
Preliminary
with
By best
in the
of 100-150
isolations
had
355-labeled
materi-
masked
the minute
quantities
of 355-factor
from the isolation.
Procedures
were develenhanced
the concentration
of intrinsically
factor
V. Consequently,
was inserted
in the protocol
step
lation.
phy
Although
the
flow-through
the
bioassay
indicated
high
of the
that
molecular
tography.
The
that
contained
with unactivated
immunoisolation
35S-labeled
‘25I-factor
a
prechromato remove,
or
material
that
iso-
prechromatogra-
factor
V activity
weight,
was
nonspecifically
decreased
in the
the
prechroma-
then
yielded
eluates
protein
that
comigrated
V and a thrombin-treated
comigrated
with
treated
‘251-factor
Va in the autoradiograms
polyacrylamide
gels
(Fig.
6). None
of
thrombinthe
of the
control
anti-ATIII
comigrated
standards.
antibody-Sepharose
355-eluate
peptides
with either
‘25I-factor
V or ‘251-factor
Va
Thus,
the immunoisolation
provided
cor-
roboration
of the
and
product
identity
as factor
Results
from
immunoisolation
bioassay,
(via
cate that cultured
bovine
thesize
and
secrete
into
be explained
substance
identifiable
of the
cultured
endothelial
V.
higher
levels
in the
The higher
levels of
by the
difficult.
V content
range
many
of endo-
at least decrease,
the nonspecific
Sepharose-adhering
355-labeled
materials
prior to antibody-Sepharose
cell
to
surrounding
is typical
proved
somewhat
RIA results,
factor
the
culture
ng/ml
als, which
V expected
oped that
granular
immunofluorescence
thelial
cells.2224
Immunoisolation
estimates
from
35S-labeled
radioimdeveloped,’8
were
noted
a fluorescent
This
adherent
355-labeled
materials
were
immunoisolation
eluates
following
V) that increased.
This increase
in
V could be interpreted
as endothelial
of factor
V activity.
To further
possibility,
with
cells.
unaffected,
culture
supernatants
revealed
an increase,
with time,
in the quantity
of factor
V. Not only did the amount
of
total
factor
V increase
with time,
it was the unactivated
form (factor
the level of factor
cellular
synthesis
the
radiolabeled
tography
there-
synthesis
to be
evidence
The
first stage
of the bioassay
detects
activated
form, factor
Va, and the thrombin
of the sample
in the second
stage activates
factor
secrete
VIII:vWF
antigen
reprereleased
into the culture
are comparable
to plasma
Factor
V levels in plasma
1 % of total
of factor
bioassay
factor
Bovine
and
glycoprotein,
and type
appears
to constitute
about
into the culture
superna-
analogy,
secretion
two-stage
least
than
synthesize
the
tant.34 By comparison,
factor
sents less than
1% of protein
supernatant.34
These figures
levels of these two proteins.
are
that
endothelial
cells
of proteins:
fibronec-
RIA,
immunofluorescence,
autoradiography)
data
mdi-
aortic
endothelial
cells
the culture
supernatant
as factor
V of the
coagulation
syna
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1474
CERVENY.
cascade.
Factor
significant
V synthesis
in further
cofactor
in the
by
defining
endothelium
may
the role of this
hemostatic
be
like
would
preparing
some
thank
to
of the
Kim
Winter
and
immunofluorescence
figures
and Donna
tion
body
and
ieanne
paper.
We
for their
The
this
assistance
would
in
to thank
like
with
assistance
studies.
for typing
her
assistance
for his contributions
immunoinhibition
Nemitz
for this
staining.
is appreciated
Foster
for
W.
Barry
to the monoclonal
We
would
also
like
Biol
to thank
19.
manuscript.
tion,
in
Wiley
Fairbanks
and
2.
Fass
KG,
VF
Sons,
The
(ed):
1982,
KG,
Mann
DN:
V
3.
Nesheim
KH,
ME,
Mann
KG:
KG,
Mann
20.
Bolton
York,
Elsevier
4.
Coleman
ME,
5.
isolated
LS,
Tracy
New
Tracy
York,
The
Holland,
1980,
Orlando
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iL,
Bloom
Proc Soc Exp
1976,
Mink
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in
CA
Owen
8.
CA
coagulant
rat
10.
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ir:
LW,
by
EA,
factor
71:1906,
on factor
14.
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Invest
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XI,
28.
coagulation
214:919,
RP,
Hoyer
endothelial
52:2737,
iH
by
factors
by
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Proc
NatI
NW,
McKee
VIII
RL,
by human
PA:
The
procoagulant
iaffe
Haemostas
Wagner
Rouslahti
Invest
52:2757,
29.
DD,
EA:
35:120,
Marder
effect
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E, Hayman
E,
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Blood
Sci
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platelet-endothelial
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258:2065,
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P, Shively
von
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Willebrand
1983
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aortic
IgG1,
using
of
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Protein
A-
P: Simplified
method
for
chromatograph.
weight
determination
cells.
1971
5cr
M,
Haematol
Nachman
cultured
of protein-
in a discontinuous
RL:
human
Booyse
6:469,
FM:
Proper-
1973
Synthesis
of antihemo-
endothelial
cells.
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NatI
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long
Formation
257:10038,
ME
term
ir:
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culture.
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role
Acad
of fibronectin
Sci USA
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globulin
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cells
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binding
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of the isolated
1982
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cells.
endothelial
in
CT:
Mosher
SciUSA
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EA,
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for affinity
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LW,
Chem
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1984 63: 1467-1474
Synthesis of coagulation factor V by cultured aortic endothelium
TJ Cerveny, DN Fass and KG Mann
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