From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
Radioimmunoassay
of Factor
By
Homogeneous.
develop
to
single-chain
a double
measure
lets.
factor
Standard
detection
of
as
factor
plasma
64).
No
and
age
with
human
factor
competition
were
little
as
20
with
an
correlation
was
or sex.
The
V
ng
factor
value
from
of 7.0
observed
weight
and
2.0
±
are
providing
=
in
to
described
factor
and
some
VIII
individuals,
deficiency,
acquired
Griffin’2
that
these
V activity
but
factor
has
with a combined
lack an inhibitor
C and suggested
of factor
with
recently,
not
resulted
platelets
V found
with
and
function-
found
to
be
activity.
was achieved
H2O
.
V with
The
specific
V
for
ment
and
stored
of
lO9’-SO%
converted
as
plasma
Conse-
plasma
contains
human
plasma
When
to
prepared
be
to
factor
V.
have also
activated
-
against
This
20#{176}C.
of the
procedure
radioactivity
into
cpm/ng.
factor
Va
according
based
by assay
30 and
between
from
plasma
is used
50 g
assay
In
Va)
this
has a
plasma
Va activity/mI.
thrombin
V/mI.
treatV. Bovine
In contrast,
(described
the
al.”
of factor
of factor
then
factor
on a bovine
after
content
4 to I 4 g/ml
for
et
V (factor
I U of factor
procofactor
and
human
Bloom
factor
of protein
determined
ranges
thrombin
using
to
human
contains
of total
by
previously’3
in this
plasma
report).
standard,
factor V has a specific activity 2-5 times higher
with a bovine plasma standard, i.e., 3400-8500
observed
fully
than that
U/mg of
protein.
Antisera
Specific antiserum to human factor V was produced in a burro by
I 0 weekly subcutaneous
injections of 0. I mg of human factor V in a
I : I emulsion with complete Freund’s adjuvant. Goat anti-burro
IgG
antiserum
was
prepared
in an
analogous
procedure
as described
Radioimmunoassay
Plasma
using
METHODS
activity
human
at
buffer
dialyzed
of 250-1000
described
by definition,
the
pooled,
V
was
is a measure
the imidazole-Ca4
were
radioactivities
activity
which,
using
fractions
(v/v),
ofFactor
Therefore,
than a deficiency
in the antigen
itself.
Recently,
our laboratory5
has described
the purification of homogeneous,
single-chain
human
factor
V
using a munine
hybnidoma
antibody.
This antigen
has
been used to develop
a double
antibody
radioimmunoassay
that
has been
applied
to measurement
of
AND
individuals
examined
and
in an incorporation
standard
rather
MATERIALS
that
factor
by chromatography
on QAE-cellulose
equili. HCL.
5 mM CaCl2, pH 6.5. Elution
of
protein
V-deficient
be devoid
of human
V activity
indicated
of the
two
assay, fully activated
purified
specific activity of I 700 U/mg
that
V antigen.
plasma
and platelet
concentrations
Two individuals
deficient
in factor
been studied.
data
V were
I .2 M NaCI.
Factor
quently,
many
reported
factor
V deficiencies
may
the result
of the inability
of the factor
V protein
function
of
M imidazole
the iodinated
containing
assayed
factor
V and factor
of activated
protein
factor
the
antigen
products
in 0.02
Bioassay
V inhibi-
may
brated
factor
are
with
postulated
individuals
Radioimmunoassay
1 8%-25%
factor
in both
50% gylcerol
V
to the
during
of fac-
has not been reported.
Factor
V deficiencies,
based
on bioassay
data,
well documented6
I I and
have been shown to occur
dysfunctiont9
count.
In addition,
deficient
iodination
tor V in plasma
tons.’#{176}” More
bioassay.
drawn
eluded
investigators
until
very recently35
due
protein’s
susceptibility
to proteolytic
cleavage
manipulation.
Consequently,
the concentration
liver
the
platelet
blood.
deficient
high
molecular
as a cofactor
was first
of human
in
approximately
in whole
ally
levels
consistent
of prothrombin
the factor
Xa-catalyzed
conversion
thrombin.
Even though
this activity
in I 943 by Owren,2
the isolation
and
contribute
(n
freshly
used
Platelets
G. Mann
and Kenneth
is
tocnits
14 g/ml
antigen
data
the
plasma.
.Lg/ml
plasma
and
washed
platelets
indicate
that 0.63-1
.93 sg of factor V is
present
per 2.5 x 1 0’ platelets
(461 2-1 41 28 molecules
of
factor V per platelet).
When normalized
to individual
hema-
platefor
of
4 to
between
assays.
to
Plasma
Bowie,
E. J. W.
used
allow
V/mI
V is a single-chain,
plasma
protein
that serves
ACTOR
was
that
radioimmunoassay
clotting
V
L. Eide,
in plasma
range
average
Lisa
radioimmunoassay
constructed
V concentrations
factor
F
B. Tracy,
V concentrations
curves
of
Normal
antibody
Paula
V in Human
and platelet
a double
concentrations
antibody
that described
of factor
competition
previously
V were determined
radioimmunoassay
for bovine
factor
similar
Burro
V.’5
to
anti-human
Proteins
Homogeneous,
hybridoma
single-chain
antibody
prepared
by
thrombin
as described
trations
were
with an
E,,,
280
only
Blood,
human
in our
prepared
No.
The
1
with
was purified
using
et al.5 Factor
Va was
catalytic
V (Va)
on a molecular
Kane
factor
protein
using
‘25I-factor
(July).
V
Factor
by Lundblad
V was
described.’5
Vol. 60.
factor
based
nm of9.6.’3
as described
‘251-Factor
of
factor-V
by Katzmann
prevsou5ly.’3
determined
nm for purified
an 8% difference
prepared
viously
incubation
280
human
as described
1982
and
weight
Majerus4
V of 8.9.
readings.
amounts
protein
a
of
concenof 330,000
reported
an
Em,
which
would
result
in
Bovine
thrombin
was
et al.’4
Bolton-Hunter
V was separated
reagent
from
as prethe other
From
Mayo
the
Address
tology
of
Supported
Presented
and
Hematology
Rochester.
reprint
Research,
HL-07069
bosis
Section
Foundation.
requests
Mayo
in part
to Kenneth
Clinic,
by
Research,
Mayo
Clinic
and
Minn.
G. Mann,
Rochester.
Mayo
Foundation
VIlIth
International
Section
Minn.
of Hema-
55901.
and
by
NIH
Grants
and HL-16150.
in part
Haemostasis,
at the
July
1 98!
,
Toronto,
Submitted
December 3, 1981; accepted
(C) 1982
by Grune & Stratton.
Inc.
Congress
Ontario,
February
on ThromCanada.’
16, 1982.
0006-4971/82/6001-0008$01.00/0
59
From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
TRACY
60
factor
V was
the primary
was the precipitating
plastic
tubes
contained
0.2 ml of either
Mg/mI)
I :2500
the following:
a working
goat
anti-burro
mixtures
antiserum
V standard
in 2.5%
normal
burro
incubated
centrifuged,
tivity.
for
Tris,
0.075
The
anti-burro
a minimum
Figure
in which
(0.02-10.24
Following
M
NaCI,
and
1% Triton
working
titer
determined
using
munoassay.
An
initial
binding
known
amounts
Source
pH
0.2 ml of a
that had
a 1-hr
containing
1%
and
tubes
the
were
bovine
an antibody
dilution
antiserum
(B/T).
anti-human
study
dilution
of 40%.
of purified
factor
Standard
human
in the above
I :2500
of
yielded
curves
factor
V antiserum
were
0.08-5.
1 2 zg/ml
The accuracy
was
radioim-
an
specificity
optimal
prepared
using
med.
curve
V.
V. Line
was drawn
Na,
2.85%
obtained
citrate
normal
PGE,
were
was
platelet
to the
solutions
performed
with
in
the
medium
Platelet-free
whole
following
were
method
5 MM
PGE,.
Counter.
The
radioimmunoassay
2.0-5.0
in
and
10
washed
Michaeli”
All
platelet
diluted
prevent
to remove
counts
platelet
platelets/mI.
The
platelets
X-lOO
as determined
were
wash
V-Immunodeficient
Seven
lant
parts
(0.16
0.016
light
by
0.09
was rendered
hybridoma
into
antibody
of both
a column
coupled
less than
0. 1% detectable
noassay,
remaining
of
M
to Sepharose.5
factor
V activity
procedure
bioassay
acid,
plasma
and antigen
anti-human
This
V, by both
The
factor
“b”
is plotted
versus
represents
a standard
V is added
(slope
=
back
-3.32).
V were
exam-
a typical
standard
(slope
= -3.10)
in
log concentration
factor
curve
when
obtained
to factor
V-immunodeficient
The plasma
dilution
curve,
range
of 0.3
3.5 tg factor
yielded
a slope of -3.16.
-
“a”)
Our antiserum
recognized
both factor
V and thrombin-activated
factor
V (factor
Va).
When
unlabeled
factor
Va was used to generate
a standard
curve
with
this antiserum,
the curve obtained
was supenimposable
with
that
obtained
with
factor
V. Plasma
factor
V
antigen
levels were not affected
by lyophilization
of the
anticoagu-
adenine).
factor
a murine
CPD-A
0.01 5 M citric
citrate,
0.002
deficient
through
one part
M trisodium
biphosphate.
sodium
passage
Plasma
was collected
M dextrose,
M
obtained
blood
for
reactivity
of this antiserum
with other plasma
proteins
since we can recover
quantitatively,
and measure
accurately,
the factor
V present
in plasma.
were
scattering.
Factor
of the assay, as well as the
antiserum
-
to be
by
B/BO
from
to
Both of the slope values
generated
in plasma
are within
I standard
deviation
of the mean
obtained
by averaging 6 assay
standard
curves
( 3. 1 9 ± 0. 14). These
results
indicate
that
there
appears
to be no cross-
in appro-
suspensions
plasma.
and validity
of the
curve
as a
of unlabeled,
highly
punthis is a competition
assay,
found
in the precipitate
is
the amount
of unlabeled
Typically,
the assay
is linear
factor
V per assay tube, equivalent
covering
a concentration
V/ml
of plasma
(line
5 MM
to
with
Triton
factor
plasma
was
to which
and
platelet
were
x
0.2%
plasma
blood
collected
of Orloff
into
at 1500 g for 20 mm.
venipuncture
containing
lysed
by venipuncture
blood
a Coulter
to yield
completely
citrated,
Platelets
according
priate
from
immediately
activation.
plasma
v/v).
of the citrated
obtained
added
volunteers
2 H2O (9:1,
.
by centrifugation
Platelets
used
from
Curve
dose-response
factor
V is plotted
Line “c” in Fig. 2 illustrates
using factor
V in assay buffer
which
of Samples
Blood
1 shows a representative
the bound
‘251-human
factor
V added.
0.01 6 to I .024 g
serum
X-l00.
of the burro
Standard
log function
of the addition
fled human
factor
V. Since
the amount
of radioactivity
inversely
proportional
to
incuba-
was added
16 hr at 4#{176}C.
The
7.0,
V Radioimmunoassay
V (20 ng),
solution
antiserum
of
Factor
washed with assay buffer, and then assayed for radioacreagents for this assay were diluted in assay buffer: 0.075
All
albumin
serum.
RESULTS
in 1.0 x 6.0 cm
0.2 ml of ‘251-factor
factor
at 37#{176}C,
0.2 ml of goat
tubes
M
and
Incubation
or appropriate
plasma
or platelet
dilutions,
and
dilution
of burro
anti-human
factor
V antiserum
been diluted
tion
antibody
antibody.
ET AL.
factor
V
results
in
plasma,
intentional
and radioimmu-
in the plasma.
storage
of the plasma
at
conversion
of the plasma
-
70#{176}C(6
to serum.
mo),
or
5000
3000
125I-factor
(Cpm per
assay)
V
4000
2000
1000
Fig.
1.
Standard
competicurve
obtained
with factor
V radioimmunoassay. The
ordinate
represents
‘2l-factor
V precipitated
by the
________________________________________________________
tive
inhibition
0.004
0.016
0.008
0.064
0.032
Factor
0.256
0.128
V added
1.024
0.512
( ug per assay)
4.096
2.048
antibody.
From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
FACTOR
V RADIOIMMUNOASSAY
61
I .0
0.9
C
0.8
b
0.7
a
0.6
B/B0
0.5
0.4
0.3
0.2
Assay specificity
and
recovery
study.
The
B/Bo-log
factor
V dilution
curve obtained
with (A) plasma
and a standard
Fig.
2.
curve
of
(B)
purified
factor
0.1
I
I
0.008
0.016
0
V
added
plasma
back to immunodeficient
yielded
slope values
of
- 3.1
6
and - 3.32.
respectively.
A typical
standard
curve
obtamed with (C) factor
V in assay
buffer
had a slope value of -3.10
(-3.19
± 0.14.n
- 6).
The
plasma
8.0% (mean
10.2 jsg/ml).
was assayed
=
sample
a value of 6.3
± 0.58
obtained.
plasma
was
Factor
V Plasma
5.6
=
In addition,
4 times over
tg (mean
1
1
40
100
factor
V/mI
mean
of
different
Based
ently
factor
mean
on
radioimmunoassay
of
for
64
appar-
(7.4
2.2)
±
and
31 males
levels
and
performed
(6.5
the
age or sex.
±
1.7)
between
V clotting
on 22 of these
plasma
samples
total
factor
V concentrations
determine
bioassay.
Bioassay
data
were
quantitated
These
data
are not significantly
by a paired
Student’s
t test.
Factor
V
V in whole
the amount
of
blood
was
factor
V
PGE,
in order
and possible
secretion
V. Assay
of the platelets
to prevent
any
platelet
of platelet-associated
lysed
in 0.2% Triton
X-lOO
(Table
I ) indicated
that 0.63=1 .93 zg of factor
V was present
per 2.5 x 108 platelets
(4612-14,128
molecules
of factor
V per platelet).
For each individu-
22 and
assays
were
in order
based
based
2.048
the plasma
and
platelets
of 8 different
Platelets
were collected
and washed
in the
of 5 tM
factor
normal
population
used in
was observed
between
anti-
Factor
1.024
0.512
of Platelet-Associated
present
in
individuals.
activation
healthy
individuals,
the normal
concentration
of
V in plasma
ranges
from 4 to 14 zg/ml
with a
value of 7.0 ± 2.0 (Fig. 3). Thirty-three
females
6 1 yr of age constituted
this study.
No correlation
gen
Range
results
7.5 ± 1.7.
as determined
The distribution
of factor
established
by determining
presence
Concentration-Normal
0.258
I
(l/assay)
Measurement
and
when a single
a I -mo period,
SD)
±
sg/ml),
I
V or Va (pg/assay)
I
Plasma
I
0.128
20
=
8.0% (mean
0.064
I
(mean
zg/ml),
0.032
I
10
samples
0.19
I
Factor
within-run
precision
of our assay (coefficient
of
assessed
by assaying
3 different
plasma
10 times each within
the same assay,
was 4. 1%
variation),
I
18
16
(I)
14
Ce
to
on
on the
V
>
V
10
C
‘#{248}-
activity
of highly
purified
human
factor
V, i.e., 1 .7 U
of factor
V was equivalent
to each
microgram
of
protein.
Factor
V clotting
assays
were consistent
with
the radioimmunoassay
data (Fig. 4), providing
freshly
drawn
plasma
was used
in the bioassay.
(Apparent
concentrations
time,
even
bioassay,
of factor
±
decreased
-
radioimmunoassay.)
indicate
that the
of 6.8
V in plasma
if stored
at
20#{176}C,when
but remained
constant
when
2.0
and
Statistical
radioimmunoassay
the
clotting
analyses
data
assay
data
measured
measured
I-
a,
.0
E
z
2
with
0
by
by
of these data
gave a mean
produced
08
I
3
4
5
6
FACTOR
Fig.
normal
a
2
pooled.
3.
7
8
9
1011121314
V, g/ml
Distribution
of factor V concentrations
in plasma
individuals.
Results
for 33 females
and 31 males
The mean was 7.0 ± 2.0 g factor V/mI
of plasma.
of 64
were
From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
62
TRACY
Table
14
2.
Normal
12
of Factor
V-Deficient
Range
Patient
Patients
1
Patient
2
Bioassay:#{149}
C
C
Factor
.
E
Assay
ET AL.
C
10
Factor
z
_______
C..
C
Factor
C..
plasma)
C
0.63
done according
V/mI
based
0
truly deficient
in
Patient
2 has
both by bioassay
is truly devoid of
I
Bioassay
Method
amount
platelet.
platelets
were
normalized
count in order
to his or
to determine
Our
We
studied
tional
deficiency
factor
is only
V-factor
deficient
ity of our
of plasma
sensitivity
V-Deficient
the
plasma
of factor
antibody
VIII:C
in factor
of two
patients
deficient
V. We
2).
individual;
increased
with
func-
Patient
1 is a
patient
the sensitiv-
assay to detect as little as 20 ng factor
(-0.3%
of normal
levels).
We achieved
by
decreasing
both
the
amount
of
lets
2
Table
No.
Factor
Nonpiatelet
V/mi
Whole
radioimmunoassay
range
is 4-14
quantitates
The assay
indicated
present
V/mI
this
radiola-
and the antibody
( 1 :8000) used in
standard
curve obtained
in the assay
0.004 ng to 0.5 1 2 ng factor
V per assay
to 0.02-2.56
zg/ml
plasma.
Mg
that
he
indicates
factor
V
levels
factor
factor
identically
per
ofwell
that
2.5
washed,
Triton
there
are 0.63-I
V
V
in
x
108
platelets
X-lOO-lysed
plate.93 g of factor
V
(4612-14,128
sess approximately
in whole
blood.
1 . Distribution
of Factor
V in Whole
2%-5%
of the total
In the human
system,
Percent
Platelet
Blood
2.5
Mg Factor V/
x 10’ Platelets
Molecules
of
Factor V/Platelet
1
6.36
1.36
17.6
1.13
8.272
2
2.86
075
20.8
0.63
4.6 12
3
3.14
1.12
26.3
0.76
4
7.54
2.47
24.7
1.93
moleare in
bovine
400bovine
to 50
pos-
factor
V present
however,
on an
BlOOd
Platelet
she is
of factor
V concentration
in
g/ml.
The age or sex of an
cules of factor
V per platelet).
These
results
marked
contrast
to our observations
for the
system.
Bovine
platelets
possess
approximately
800 molecules
of factor
V per platelet,
and
plasma
factor
V concentrations
range
from
30
sg/ml.
Therefore,
in the bovine
system,
platelets
beled factor V (5 ng)
the assay. The
was linear
from
tube, equivalent
indicating
and
values
plasma
and with intentionally
activated
factor
V (factor Va), collection
of plasma
to preclude
any factor
V
activation
is unnecessary.
Plasma
lyophilization,
prolonged
storage,
or intentional
conversion
of plasma
to
serum
has no effect on factor
V antigen
levels.
Patients
V (Table
mean),
of normal
individual
does not appear
to influence
the
concentration.
Since our burro
anti-human
her
the
24.9%.
ofFactor
V by bioassay
(9%
antigen
as well as activity.
no detectable
factor
V, established
and radioimmunoassay,
indicating
antigen.
double-antibody
that the normal
human
plasma
of factor
V in whole
blood contributed
by the
The amount
of factor
V contributed
by the
of each individual
ranged
between
I 7.6% and
Assay
factor
of plasma
normal
et
DISCUSSION
Fig. 4. Factor V plasma
concentrations
determined
by radioimmunoassay
and bioassay.
Twenty-three
samples
gave mean factor
V
concentrations
of 6.8 ± 2.0 g/ml
by radioimmunoassay
and
7.5 ± 1 .7 Mg/mI by clotting
assay. These data are not significantly
different
as determined
by a paired Student’s
t test.
data
platelet
on 7 sg/ml,
134%
to Bowie
1 has 9% functional
tg factor
2
al, the platelet
hematocrit
and
Mg/mI
(<0.3%)
17%
were
Patient
C
I
<0.02
(9%)
Vlll:C
Bioassays
C.. C
C
Radloimmunoassay
pg/mI
0.63
2.0 g/ml
±
(55%-145%)
C
C
<1%
Bioassay:
C.....
I:
V
(7.0
C.
-Y-
9%
Radioimmunoassay:
C
C
V
(50%-150%)
5.563
14.128
5
3.10
0.83
21.1
0.90
6.588
6
6.16
1.35
18.0
1.11
8,125
7
4.56
1.24
21.4
1.03
7,540
8
3.71
1.23
24.9
1.23
9,004
From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
FACTOR
63
V RADIOIMMUNOASSAY
individual
basis,
the hematocrit,
plasma
and platelet
concentrations
such
that
the platelets
platelet
count,
and
of factor
V exist,
contribute
approximately
18%-
25% of the factor
V present
in whole blood.
We increased
the sensitivity
of our assay
detect
factor
V concentrations
as low
0.3% of normal
concentrations.
known
factor
V deficiencies
ual,
totally
competent
Two
were
in order
as 20 ng/ml
individuals
studied.
devoid
of factor
V
with regard
to the other
factor
VIII
deficient
patient,
was found
to have 9%
factor
V by both bioassay
and radioimmunoassay.
Factor
V deficiencies
have
been
described
that
result from acquired inhibitors to factor V’#{176}”
or from
the
inhibitor
individbut
fully
coagula-
found
to be completely
deficient
in
Another
individual,
a factor
V and
tion factors,
was
factor
V antigen.
of an inhibitor
lack
of factor
to activated
V (Va)
protein
activity.’2
C, a potent
Since
these
types
of deficiencies
may result
from factor
V antigen
which
cannot
express
function,
we intend
to assay
several
factor
V deficiences
to determine
if they
are truly
devoid of both antigen
and activity,
or possibly
possess
with
One
activity,
assayable
to
or
a normal
cule.
or abnormal
nonfunctional,
factor
V mole-
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Katzmann
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From www.bloodjournal.org by guest on September 30, 2016. For personal use only.
1982 60: 59-63
Radioimmunoassay of factor V in human plasma and platelets
PB Tracy, LL Eide, EJ Bowie and KG Mann
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