From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Structural
Studies
von
Functional
Willebrand
Martin,
By S. Eric
on the
Victor
Protein
J. Marder,
Charles
Studies
of subunit
chain size, disulfide
bond arrangement.
carbohydrate
content.
and pattern
of tryptic
degradation
of von Willebrand
protein
polymers
were undertaken
in an
attempt
to
explain
their
functional
heterogeneity.
Human
von Willebrand
protein
purified
from cryoprecipitate
was
separated
by gel elution and sucrose
gradient
ultracentrifugation into groups of polymers
of different
size. ranging
from
a molecular
weight
greater
than
1 0 x 1 0’ to a
minimum
of
2.4
x
lOc.
After
disulfide
bond
reduction,
all
polymers
showed
a major
band of 208.000
molecular
weight
with about 1 % of the protein having lower molecular weights
of 1 97.000.
1 74.000.
and 1 54.000.
Major and
minor
moieties
were
recovered
from
immunoprecipitates
obtained
with
antibody.
polymers
The ristocetin
cofactor
activity
of the different
showed
increasing
specific activity
with increas-
ing
monospecific
molecular
weight.
protein
concentration.
content
of
V
that
ON
the
Willebrand
whether
von
208.000
measured
Willebrand
molecular
WILLEBRAND
protein
relative
antigen
weight
protein
value
subunit
or
chain.
is a glycoprotein
for
weight
forms
platelet
ristocetin
binding
greater
generally
than
have
interaction
as
cofactor
activity’0
to subendothelium.#{176}
106.19
greater
reflected
Larger
molecuin vitro potenby
increased
and greater
affinity
for
In vivo functional
reflec-
tions of molecules
of different
size are indicated
by the
failure
of factor
VIII
concentrates,
which
are relatively deficient
in larger
forms,
to correct
the bleeding
time
patients
IIA
von
of
variant
smaller
molecular
with von Willebrand’s
Willebrand’s
disease
weight
polymers
disease,’2
in which
by
the
predominate,9
and
by the preferential
removal
of large molecular
weight
forms
after
treatment
of a patient
with acquired
von
Willebrand’s
disease,’3
presumably
as the result
of
their
increased
binding
efficiency.
Although
some
studies
have
noted
the
importance
of disulfide
bonds6’4
and of terminal
sialic
acid
residues’5
and
penultimate
galactose
residues’6”7
as important
factors
in the normal
function
of von Willebrand
protein,
the molecular
basis for the distinction
between
high and
not been
individually
low activity
determined,
separated
broad
report,
functional
individual
mers
purified
compared
or size
polymers
from
with
regard
utory
factors
ences
in activity.
Blood,
Vol. 57.
in large and small
polymers
nor have there
been studies
polymers
within
these
that
categories.
or similar
human
cryoprecipitate
to a number
could
Studies
No. 2 (February),
explain
performed
1981
In the
groups
of possible
the
observed
include
has
of
two
present
of polyare
contnibdifferevalua-
of
Polymers
W. Francis,
and Grant
H. Barlow
This difference
in specific activity
was particularly
evident
when comparing
groups of molecular
weight
greater
than
1 0 x 1 O with those of molecular
weight
less than 5 x 10’.
There
was no difference
in the content
of the minor
reduced
bands in each polymer,
no difference
in carbohydrate concentration
or susceptibility
to neuraminidase
or
galactose
oxidase. and no difference
in the pattern
of
tryptic
degradation
or function
of the 1 1 6.000
molecular
weight
tryptic
remnant
that retains
ristocetin
cofactor
activity.
The disulfide
bond organization
of the larger
polymers
appeared
to
differ
from
that
of
the
smaller
polymers
inasmuch
as partially
reduced
polymers
obtained
from
the high specific
activity
group
expressed
more
ristocetin
cofactor
activity
than unreduced
polymers
of
similar
size present
in the low specific
activity
group.
Apparently.
to
composed
of subunits linked by disulfide bonds
may circulate
in vivo as a series
of polymers
of
molecular
Ian weight
tial
anti-von
Heterogeneity
optimal
interaction
of
the
von
Willebrand
polymers
with platelets
is dictated
not only by size but also
by tertiary
structure
as shaped by disulfide
bond organization.
tions
of
chains,
dation,
disulfide
minor
disulfide
bound
subunit
polypeptide
active
molecular
fragments
after tryptic
degracontent
and susceptibility
ofcarbohydrate,
and
bond
contributions
to overall
activity.
The
data
suggest
that each
polymer
has its own level of
ristocetin
cofactor
activity
and that,
in addition
to
overall
size, tertiary
structure
as dictated
by disulfide
bond
arrangement
is an important
determinant
of
differences
in activity.
MATERIALS
Purification
oflluman
Cryoprecipitate
Rochester
and
absorption
Penn.)
(0.3
after
and
0.01
M sodium
von
were
performed
according
Lowry.’9
VIII
procoagulant
activity
From
the Hematology
School
Supported
in
National
Heart.
Health,
Bethesda.
Submitted
Address
Medicine,
©
198!
part
by
Lung,
and
July
reprint
601
Unit,
by Grune
Grant
(Pharmacia
0.15
azide,
protein.
the
as
Protein
technique
ofMedicine.
Dentistry.
Institute,
NaCI,
6.8)
was determined
of
by the
University
Rochester.
#5-R01-HL21379-02
Blood
M
pH
N. Y.
from
the
Institutes
of
Department
of
National
Md.
1 1, 1980;
requests
Elmwood
0006-4971/81/5702-00/
and
by
King
in fl-alanine
sodium
Department
of Medicine
processed
CL-2B
to
Cross,
ethanol
Scientific,
Willebrand
determinations
of Rochester
was
phosphate,
0.05%
to obtain
with
chromatography
10 U aprotinin/mI,
Factor
it
Fisher
Sepharose
N.J.)
Red
washing
hydroxide,
precipitation
previously’6
American
6000 (Carbowax,
M (3-alanine,
described
Protein
the
and
aluminum
Piscataway,
M EACA,
by
Program
(PEG)
Chemicals,
buffer
0.02
with
METHODS
Willebrand
prepared
Blood
glycol
of Prussia,
von
was
Regional
polyethylene
Fine
AND
accepted
October
3, 1980.
to S. Eric
Martin.
M.D..
Avenue,
& Stratton.
Rochester,
N. Y. /4642.
Inc.
7$02.00/0
313
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MARTIN
314
two-stage
thromboplastin
tor assay
utilized
Antisera
protein
generation
formalinized
prepared
and
in
fibrinogen
severe
type
beads
I von
human
serum,
against
cryoprecipitate
disease
(absent
and
0%
Von
using
immunodiffusion
The
studied
I U of
immunodiffusion
plates,
tubes
as described
buffer
pH
7.2,
buffer
pH
8.6. The
of 6%
ethanol)
then
dodecyl
(Sigma
and
Chemical
Co.),
mm
before
retic
systems.
M
immunoprecipitates
Co.,
Areas
M
St.
x
Louis,
Mo.),
of agarose
that
in parallel
processed
5 mm
(type
then
were
Sucrose
Willebrand
gradients
protein
prepared
(Beckman
M tris
were
collected
0.05
(Model
from
ristocetin
Model
x
l0.
from
cofactor
at 60#{176}C
for
25
gel electropho-
free of visible
the
0.3 M fl-alanine,
sodium
azide
pancreas
0.01
pH
type
I,
(Sigma
Chemical
mg/mg
von
with
constant
sin inhibitor
at a final
was
added
protein
stirring.
Degradation
(type
ofSialic
Acid
0.02
bovine
and
Biochemical
U/mg
Co.,
protein
cofactor
-alanine
buffer
appropriate
in
lytic
absence
in parallel
oxidase
and
activity,
the
0.02
of
with
enzymes
as demonstrated
Purified
Bond
von Willebrand
at 37#{176}C
and 0.025-mi
by lack
system
was
(final
concentrations)
tested for
polyacryl-
tube
gels
were
dissolved
SDS,
0.008
Co.)
of
and
retested
were
mixed
160
for
with
incubated
conducted
of 0. 1% SDS,
of degradation
was
incubated
aliquots
drawn
slabs
with
constant
voltage
(bromophenol
blue)
reached
linear
as previously
used,
samples
Na2EDTA
system
were
in 0.008
at pH
dissolved
M boric
8.6.
was employed,
concentration
described.’8
When
in 0.6%
acid,
0.13
a complete
dithiothreitol
gradients
When
(DTT)
a nonreSDS,
M tris
E
disulilde-bond(final
concen-
2.0
Uj
C)
,
ca
0
Co
:
cc
Pooled Frachons
0.5
I
II
III
V
V
vi
U
vii
viii
ix
0
U-#{149}
-.--.-s-.-.-.-.
of I 0 U
ELUTION
of ‘4C-labeled
with
2 M
buffer
0
of proteo-
0.1 M $-ME
at timed
0.004
150-V
for
neuramini-
no evidence
in
M borate,
electrophoresis
system
were
6.8,
Reduction
and 0.05-mi
and
marker
or
0.6%
buffer
the tracking
gradient
Co.)
at 37#{176}C
used in the presence
showed
w2t
fractionator
Neb.),
1’O
‘
EACA
protein
sucrose
in the SW
SDS
samples
pH 8.6, using
tris,
prepared
0.025%
the sample
Worthington
The
when
M
slab
Test
(2 M urea,
Fig. 1 .
Sepharose
CL-2B
6.000
precipitate
prepared
Disulfide
gradient
Lincoln,
(2%:0.5%)
0.064
urea,
hemoglobin.24
Limited
acid,
tryp-
with
Chemical
and
test materials.
preparations
M
hr
samples
run
I .0 ml fractions
in nonreduced
described.’8
concentrations)
stopped
duced
53
concentration
additional
controls,
and
at a constant
a density
run
on 5%-20%
buffer,
2.5
pH
and incubated
at a final
concen-
Electrophoresis
in a continuous
Polyacrylamide
was treated
activity,
04522,
final
gel electrophoresis.
final
tris
of 5%- 1 5% were
chromatography,
cofactor
(LSOO
(5 x
were
the end of the gel.
at 37#{176}C
buffer
(Sigma
protein
two
As
and
Residues
affinity
activity)
N.J.)
37#{176}Cfor
intervals
of aprotinin/ml
by
layered
Specialties,
buffer
U/mI)
Chemical
i-aIanine
using
and
Gel
of 0.25
soybean
(Sigma
10 U aprotinin/mI
ristocetin
oxidase
activity.
dase and galactose
in
mucin
Freehold,
at
ristocetin
with
were
centrifugation
the anode
reducing
of I I .5 U/mg
for I 8 hr. After
testing
for
was exposed
to galactose
ester
incubated
Galactose
purified
submaxillary
concentration
(bovine
mg/mI.
and
X,
mixture
(103M
ultracentrifuge)
top
(2%:0.5%)
as previously
M boric
0.05%
trypsin
concentration
was stopped
protein
M EACA
(type
the
against
chloride,
which
ethyl
lot 77C-8000)
Willebrand
neuraminidase
after
to a final
and
I-S
of0.25
containing
0. 1 5 M sodium
buffer),
Willebrand
von
overnight
N-benzoyl-L-arginine
Co.)
(511)
Purified
U/mg
10,000
concentration
Removal
at a final
M phosphate,
6.8 (fl-alanine
dialyzed
other
DTT
and run in nonreduced
LS-65
Polyacrylamide-agarose
prepared
towards
was
2-IAA
Following
activity
Polyacrylamide
immunopre-
as controls.
protein
In
with
I, Sigma
0. 13 M
von Willebrand
nonreduced
at 25#{176}C.Aliquots
in fl-alanine
Instrumentation
185,
amide-agarose
(Fisher
samples
manually
rad2/sec
Hydrolysis
Purified
to
electrophoresis.
activity
3
M borate,
Ultracentrifugazion
urea-SDS-tris-borate
Trypsin
at 25#{176}C
for
gel electrophoresis.
Gradient
27. 1 rotor
with
8.0 M urea
gradient
used
aliquot
0.04
was treated
with
cofactor
serum
and
0.05-mi
application
incubated
mixed
The
6% SDS,
gel
protein
polyacrylamide-agarose
of 44,500
I recrystallized
and
polyacrylamide
for ristocetin
M isis
0.064
mixed
(f-ME)
intervals,
tested
Co.),
incubated
before
and
at timed
plastic
0.05
borate,
then
(SDS,
at 100#{176}C
for
to
chloride,
were
8.6
(2%:0.5%)
concentration)
Von
from
x 2.5 cm perforated
fl-mercaptoethanol
heated
polymers
von
0.004
sulfate
Chemical
7.3
application
were
with
and
Willebrand
of
pH
104M
SDS
activity.
M
N.Y.),
ml of bovine
Chemical
ml of 10 M urea,
at
of 0.05
Rochester,
0.025
ml of I .0 M 2-IAA,
to 0.005
buffer
final
with
volume
Co.,
Double
described
I .0 M sodium
2 days
and
mixed
cofactor
the von Willebrand
tration),
previously
of
immunoprecipitates
sodium
Scientific)
cipitate
for
standards.
tris
an equal
Kodak
V, Sigma
experiments
drawn
immuno-
by Ouchterlony22
to
in 8.5
with
fi-
and
(Cochranville,
as
0.005
polyacrylamide-agarose
as described
per ml of plasma
with
with
(fraction
of ristocetin
mm, then added
M
mixed
(Eastman
3 mm,
mg/mI)
for determination
0.32
activity,
by rocket
plasma
washing
for 2 days
and
was
(2-IAA)
(20
was mixed
normal
ct2-macroglobulin,
antigen
removal
to
to
von Willebrand’s
Laboratories
composition
the
and
prepared
according
subunit
aliquot
at 25#{176}C
for
albumin
coupled
procoagulant
measured
normal
performed
following
centrifuge
ml
was
immunoelectrophoresis
methods.23
was
Cappel
pooled
was
1gM,
columns
fibronectin
severe
0%
were
from
0.025-mi
incubated
Willebrand
Chemicals)
with
antigen
assuming
von
plasma’8
antigen,
activity)
obtained
of
The
cofac-
through
against
a patient
human
Willebrand
dilutions
crossed
disease
from
against
electrophoresis2’
Fine
Antiserum
cofactor
were
Penn.).
human
by passage
(Pharmacia
Willebrand
Antisera
lipoprotein
against
Willebrand’s
von
the ristocetin
2-iodoacetamide
adsorbed
respectively.’8
ristocetin
before.’8
rabbits
were
of Sepharose-CL4B
test2#{176}
and
platelets.’8
ET AL.
intervals.
column
VOLUME
elution
(L)
of reconstituted
PEG-
from human cryoprecipitate
(see
Materials
and Methods). Column size was 1 30 x 8.5 cm, elution
rate was 300 mI/hr using $-alanine buffer. Arrows indicate the
regions
that
were pooled
and concentrated
by dialysis
against
PEG-20.000.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
FUNCTIONAL
HETEROGENEITY
tration
M
0.02
concentration)
(Sigma
vW
Chemical
was also added,
Co.)
0. 1% SDS. When
the electrophoresis
acid
(Fisher
All
were
Fairbanks”
contained
0.00035%
protein
2%
and
Densitometric
Gel
system
at pH 8.6
were
periodic
acid
with
Zebrowski.26
0.95,
1.9,
2.85,
prepared
as previously
included
fragment
Y (I
(68,000),
and
polypeptide
3.8,
weights
agarose
55#{216})27
and
For each
D (stage
(17,000).
squares
gel, the best straight
molecular
weight
of
5.7
x
For
line
the
acrylamide
gel
X
ity,
reduced
gel
bovine
pools
inhibitor
systems,
(200,000),
distance
and
the
a linear
area
39231,
result
bands
in a Beckman
scanning
under
Coy
each
device
peak
Laboratory
used
was
Prod-
as a reflection
of
(Fig.
I)
factor
VIII procoagulant
activactivity,
and von Willebrand
from
after
and
Sepharose
CL-2B
columns
which
it was collected
into
concentrated
2%
dialysis
pools
1-VIl contained
a2-macroglobulin,
and /3-lipoprotein,
and did
rum against
cryoprecipitate
at
analysis.’8
by
at
nine
against
Double
diffusion
or immunoelectrogels against
appropriately
adsorbed
antisera
showed
that
1% 1gM,
fibrinogen,
versus
determined
emerged
volumes,
20% PEG-20,000.
phoresis
in agar
the
A (94,000)
were used
inhibitor
and myoglowas
with
stained
in the gel strip.
Material
containing
ristocetin
cofactor
antigen
I .4 void
(260,000),27
2) (lOO,000),28
for migration
Mich.),
PAS
RESULTS
fibrin
x 106,
trypsin
standards
Arbor,
Bands
and
respectively,
the
(Model
by
I .02
myosin
scanning,
a planimeter
concentration
nm,
equipped
Following
with
542
blue
deterdone
106 and
soybean
(220,000),
and
at molecu-
and
fragment
i-galactosidase
(I 32,000),
and phosphorylase
in addition
to bovine albumin,
soybean
trypsin
logarithm
polymers
Nonreduced
(43,000),
fibronectin
were
680,000
(340,000),
fragment
myoglobin
of
4.75,
described.’8
ovalbumin
chains
1gM
(PAS)
weight
nm
24 spectrophotometer
Ann
protein
ofElectrophoretic
the Coomassie
at 575
recorder.
ucts,
of
containing
scanned
calculated
method
Schiff’s
Molecular
of 340,000,
fibrinogen
the
Analysis
strips
Model
and
mercaptoacetic
to
polyacrylamide-0.5%
at molecular
albumin
least
final
system was used,
according
to glutaraldehyde-crosslinked
standards
bin.
M
in a nonreducing
tris-borate
buffer
reducing
for carbohydrate
the
in
weights
(22,000),
(1.4
at 100#{176}C
for 5
a complete
also
for
by Kapitany
comparison
all
stained
and
minations
polymers
fl-ME
heated
Scientific).
gels
as described
lar
buffer
or
and the sample
mm or at 60#{176}C
for 30 mm. Electrophoresis
was performed
towards
the anode using
containing
315
PROTEIN
less than
fibronectin,
not react with rabbit
antiseobtained
from
a patient
NON-REDUCED
>10x10[
:::
-6.4x
1O
-
4.6x10
__
3.4x10
2.4x10
-
950.000
-
680
-
440,000
-340,000
POOL
Fig. 2.
SDS-polyacrylamide
gel electrophoresis
of the concentrated
pools
obtamed
from
the Sepharose
CL-2B
elution
shown
in Fig. 1 . The top panel
shows
nonreduced
samples
in a 2% polyacrylamide: 0.5% agarose
slab gel. using sample
sizes of 2.2 g of pool
1. 9-13
xg of pool
Il-VIl.
and 16 gg of pools VIII and IX. The
bottom
panel
shows
the disulfide
bondreduced
samples
after electrophoresis
in a
5%-i
5%
discontinuous
gradient
slab gel
containing
SDS. using 1 .1 ig of pool 1 . 5-8
;&g of pools Il-VIl. and 9-10
xg of pools VIII
and
IX. Details
of electrophoresis
and
calculation
of molecular
weights
as in
Materials
and Methods;
‘lgM.”
refers
to
the heavy chain of 1gM and Aa. B. and ‘y
refer to the reduced
chains of human fibrinogen.
I
\
II
\\
Ill
IV
\
V
\
VI
I
VII
I
IX
VIII
I
I
208,000
197,000
-
197,000
174.000
-
174,000
154.000-
154.000
-
130,000
-
90,000
74,000
5-15%
REDUCED
-
65.000(M)
58.000(B)
-
47,000(y)
(1gM14)
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
MARTIN
316
with severe
type I von
bleeding
time greater
procoagulant
activity,
and
VIII
0% von
reacted
Willebrand’s
disease
who has a
than
15 mm, 0% factor
VIII
0% ristocetin
cofactor
activity,
Willebrand
antigen
by Laurell
assay.
with anti-IgM
and anti-fibrinogen
Pool
anti-
-208,000
t...
-197,000
.
serum,
and pool IX with
these
and
antiserum
as well.
Analysis
of the concentrated
pools
phoresis
2, top)
2.4
x
anti-fibronectin
106
relative
polymers
a series
of bands
to greater
than
by SDS-electro-
migration
distances
as standards.
Pools
of minimum
gels
of molecular
size
x
based
106,
of 1gM
and
I-Ill contained
est
molecular
weight
forms,
beyond
the measurable
limit
which
was estimated
as greater
IV-Vl
were composed
mostly
polymers
agarose
10
size 4.6
(440,000),
fibrinogen
-154,000
(Fig.
from
on
the
the fibrin
the high-
much
of which
was
of the standards
and
than
10 x 106. Pools
of intermediate
sized
x 106, and
pools
contained
the smallest
species
of molecular
4.6 x 106. Small
amounts
of 1gM (950,000),
tin
-174,000
.
in 2% polyacrylamide:O.5%
showed
ET AL.
(340,000),
VIl-IX
size below
fibronec-
and
fibrin
,:
dimer
-
IgG
HEAVY
-
IgG
LIGHT
(680,000)
were present
in pools VIII and IX. Densitometric
analysis
showed
2% and 14.8%
1gM,
1 and
5% fibrinogen
species,
and 0 and 0.2% fibronectin
in
pools VIII
and IX, respectively.
The disulfide
bond-reduced
subunits
of the different
groups
of von Willebrand
polymers
was studied
using
a discontinuous
system
(Fig.
subunit
197,000,
2,
SDS-polyacrylamide
bottom).
In addition
of molecular
174,000,
and
weight
154,000
gradient
to the
208,000,
bands
of
were present in all
pools, and a faint band of I 30,000
was observed
but pool
I . The minor
reduced
bands
were
strated
least well in pool I because
the protein
tration
was only 20% that of the other pools. A
90,000
and bands
corresponding
to the 1gM
chain
and to reduced
fibrinogen
chains
were
present in pool IX, faint in pool V III.
The
bands
of
recovered
along
immunoprecipitates
monospecific
197,000,
with
anti-von
174,000,
that of
obtained
and
208,000
after
Willebrand
gel
major
in all
demonconcenband of
heavy
clearly
Fig.
154,000
were
from
reduced
reaction
with
antiserum
(Fig.
3),
suggesting
that they represent
von Willebrand
protein.
Bands
that were faintly
demonstrated
in Fig. 2 were
not visible
in these immunoprecipitates.
The
electrophoretic
mobility
of von
Willebrand
protein
present
in the separated
pools corresponded
to
that
of von Willebrand
protein
present
in normal
REDUCED
5-15%
3.
SDS-polyacrylamide
gel
electrophoresis
of the
discontinuous
5%-i
a predominance
primarily
the
The
protein,
fast
absolute
Laurell
5% polyacrylamide
of the
peak.
slow
ristocetin
von Willebrand
gradient
peak
and
cofactor
antigen
gel system.
pool
densitometric
quantity
of 208,000
subunit
unit volume
of each pool after
concentration
are shown
in Table
I . Specific
ristocetin
activities
relative
to each parameter
of von
protein
concentration
are shown
in Figs.
fast
Ristocetin
of the
normal
plasma
pattern.
Pool
I had
cofactor
activity
was
greatest
IX
showed
activity,
reaction,
plasma
(Fig.
4). Pool
I showed
more
heterogeneity
than
was suggested
by SDS-gel
electrophoresis
(Fig.
2, top), and the material
in pools I and IX appeared
to
migrate
as two populations,
corresponding
to slow and
portions
disul-
fide bond-reduced
immunoprecipitate
obtained
after agar gel
double immunodiffusion
reaction of monospecific
anti-von Willsbrand protein antiserum
with pool
V. The area of the agar gel
surrounding
but
not containing
immunoprecipitate
showed no
protein bands after similar processing and electrophoresis
in this
total
and
chain
per
dialysis
cofactor
Willebrand
5A and B.
in pool
1 by
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
FUNCTIONAL
HETEROGENEITY
vW
PROTEIN
317
Table 1 . Ristocetin
Estimates
Cofactor
Activity
of von Willebrand
Fractions
Obtained
Protein
by Sepharose
Ristocetin
and Three
Independent
Concentration
CL-2B
Von W,llebrand
in Pooled
Elution
Protein
(Fig.
i)
Concentration
Cofactor
Lowry
(mg/mI)
Activity
(U/mI)
Pool
vW
antigen/mI
208,000
Reduced
(U/mI)
(Area
I
10
0.024
1.6
0.0554
II
47
0.360
17.8
0.7472
III
56
0.390
33
1.1308
lv
59
0.490
57
2.1890
1.8888
V
57
0.530
50
VI
59
0.540
42
1.5099
VII
57
0.430
63
2.3058
VIII
48
0.610
52
1.4933
lx
23
0.650
39
1.2147
and
VIII
inhibition
1gM
IX
lower.
of
activity
in pools
using
severe
diluent;
the
diluent
(Fig.
A
not
different
be proven
pools,
so the
VIII
To
rule
by
or IX,
out
the
possibility
fibrinogen,
the
Chain
U/mi)
of
fibronectin,
samples
were
or
also
tested
von Willebrand’s
disease
plasma
as the
results
were identical
as with a buffered
5A, dotted
open circles).
specific
activity
or ruled
out
relative
activity
for each
by analysis
polymer
could
of the total
of individual
polymers
of
von Willebrand
protein
was
further
evaluated
by
sucrose
density
gradient
centrifugation
of pools
III
and IX. Fractions
that contained
ristocetin
cofactor
activity
were electrophoresed
in nonreduced
SDS
2%
acrylamide:
Willebrand
0.5% agarose
protein
was
gels and
quantitated
the amount
of von
by densitometric
analysis
ofthe
stained
gel patterns
(Fig. 6). Within
the
group
of polymers
present
in pool IX (Fig.
2), the
bands
of 2.4, 3.4, and 4.6 x 106 were
associated
with
progressively
greater
specific
were less than that associated
in pool
The
minor
I 74,000,
Fig. 4.
Crossed
immunoelectrophoretic
patterns
of normal
plasma
(20 ‘xI) pool I (20 tl) and i :5 diluted
pool IX (iO xl). using
monospecific
anti-von
Willebrand
protein
antibody
in the second
dimension
gel.
activities,
with the
all
6.4
of
x
106
which
band
III.
absolute
and
relative
concentrations
of the
reduced
polypeptide
chains
of
197,000,
and I 54,000
were analyzed
by densitometric
quantitation
of each
pool
had the same
contribution
(Fig.
2, bottom).
of minor
bands,
mately
1% of the total protein,
and
tion of each
of the three
moieties
the
same
relative
All pools
approxiproporto the
Specific
activity
using
or the Laurell
reaction
208,000
band.
Therefore,
the differences
in ristocetin
cofactor
activity
for pools
of different
polymer
size
could
not be explained
by functional
enhancement
or
suggested
two populations
of molecules,
those in pools
I-Ill
with high but sequentially
lesser
activities,
and
those
in pools IV-lX
with a lower
level of relatively
similar
specific
activities.
According
to the concentration of total
protein
determined
by the Lowry
technique (Fig. SB), three levels ofactivity
were seen, with
pool
1 clearly
the highest,
Il-VII
intermediate,
and
deficiency
attributable
to these
minor
polypeptide
components.
The nonreduced
SDS polyacrylamide
gel pattern
of
45-mm
tryptic
hydrolysates
of pools II, III, VIII,
and
IX showed
the same degradation
fragments
of molecular weight
235,000,
219,000,
170,000
(faint),
154,000
(faint),
I 16,000,
43,000,
and 22,000-26,000
(Fig.
7).
all measures
the amount
of concentration.
of 208,000
subunit
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MARTIN
318
.
180
7.0
ET AL.
500
80
#{163}00
3.0
60
300
RCA units
2 .0
200,000
C
40
RCA units
VWAntigen
________
RCA
units
mg
200
A
1.0
20
100
0
A
I
II
Ill
IV
V
VI
VII
VIII
IX
B
POOL
III
IV POOL
V VI
VII
VIII
IX
Fig. 5.
Specific
ristocetin
cofactor
activity
of the pools of von Willebrand
protein
eluted from Sepharose
CL-2B gels, as shown in Figs.
i and 2. The left panel shows activity
relative
to the densitometric
assay of reduced
208.000
subunit
in each pool (solid circles)
or to the
amount
of von Willebrand
antigen
detected
by Laurell
immunoelectrophoresis
(open triangles).
The dotted
circles
indicate
results
obtained
for pools II, VIII. and IX, diluted
in severe
von Willebrand’s
disease
plasma
rather
than in buffer.
estimated
on the basis of
208,000
subunit
quantity.
The right panel shows the ristocetin
cofactor
activity
per mg of total protein
in each pool. as determined
by the
Lowry
technique.
POOL
-‘6.4x106
I
POOL
III
IX
4.6x106
I
p
3.4x
-
106
2.4x106
4
SUCROSE
GRADIENT
FRACTION
RELATIVE
RCA
10
8
7
6
5.4
4
3
1
Fig. 6.
Nonreduced
SOS polyacrylamide-agarose
gels (2%:0.5%)
of fractions
obtained
following
simultaneous
sucrose
gradient
ultracentrifugation
of pools
Ill and IX. Samples
of 0.1 ml were
diluted
in urea-SDS-tris-borate
buffer
pH 8.6. applied
to a disc gel,
electrophoresed
and measured
densitometrically
in comparison
with the ristocetin
cofactor
activity
of each fraction.
The values at the
bottom
represent
the activity
of each fraction
in comparison
with the value obtained
for sucrose gradient
fraction
6.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
FUNCTIONAL
HETEROGENEITY
vW
PROTEIN
319
235,000
-
219,000
-
170,000
-
154,000
I
116,000-
-116,000
-43,000
Fig. 7.
SDS-polyacrylamide
gel electrophoresis
of nonreduced
von Willebrand
protein
pools II. Ill, VIII. and IX after degradation wit trypsin
(0.25 g/mg
substrate)
at
37*C
for 45
mm.
Electrophoresis
was
performed
on a discontinuous
5%-i 5%
polyacrylamide
gradient
gel system
using
i 2. i 3, 20. and 22 g of the respective
digests.
The residual
ristocetin
cofactor
activity
of each pool, expressed
in absolute
units per unit volume of sample and relative
to the amount
of 1 1 6,000
fragment
in the
digest
as analyzed
densitometrically
are
indicated
at the bottom.
Residual
ristocetin
calculated
component
in proportion
of 1 16,000
significant
difference
cofactor
Ei
POOL
RCA
(u/mI)
RCA (u/i 16,000)
activity
of
each
pool
to volume
or to the active
molecular
weight’8
showed
no
between
samples
and
no trend
of
increasing
or decreasing
activity
in relation
to size of
the parent,
untreated
polymers.
The carbohydrate
content
of the different
polymers
was determined
densitometrically
by the ratio of PAS
to Coomassie
blue staining
ences
in specific
ristocetin
(Fig.
cofactor
8). Despite
differactivity,
the ratio
for each group
or individual
was similar,
most apparent
von Willebrand
polymer
in the analysis
of pools VII
and
VIII.
Fibronectin
(CIG)
had a ratio
of 0.06,
indicating
a lower
relatively
content
of carbohydrate
than
in von Willebrand
protein.
The ratio
for 1gM
(0.12)
and
fibrinogen
(0.13)
were
higher
than
for
II
III
VIII
IX
I .7
2.9
1.8
1.4
1.1
1.3
0.7
1.1
fibronectin,
but
von Willebrand
cofactor
activity
and penultimate
ent
in pools
still lower
polymers.
than
The
-
26,000
-
22,000
the value
response
obtained
for
of ristocetin
to the removal
of terminal
sialic
galactose
residues
of polymers
II and
treatment
with
(Table
2). The
VIII
was
neuraminidase
loss of activity
studied
and
with
(STI)
by
acid
pres-
sequential
galactose
galactose
oxidase
oxidase
only after
prior liberation
of sialic acid residues, and the effect of removing
both of these residues
on the ristocetin
cofactor
activity
was similar
for these
groups
of polymers,
widely
disparate
in their
molecular size. There
was no alteration
in electrophoretic
occurred
mobility
of von
polyacrylamide:agarose
bation
The
with either
relationship
Willebrand
protein
using
an
(2%:0.5%)
system
after
neuraminidase
of disulfide
or galactose
bond structure
SDSincu-
oxidase.
to activ-
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MARTIN
320
POOL
I+II
III+IV
I
>10xi06
-6.4x
V+VI
-‘0.25
:0.33
106_
POOL
f
3.4x106
2.4x
IX
VII+VIII
4.6x106
MAJOR
PROTEIN
PEAK
-
-
evaluated
pI020
by progressive
exposure
800,000-I
of polymers
in pool II to either
dithiothreitol
or fl-mercaptoethanol, followed
by alkylation
with 2-iodoacetamide.
The
ristocetin
cofactor
activity
of the partially
reduced
pool II polymers
was expressed
relative
to the activity
of untreated
pool IX polymers
(Fig. 9). Before
reduction,
pool II had
activity
than
pool
mers had decreased
I 0 x 1 6 to a group
x
sixfold
greater
ristocetin
cofactor
IX. After
2 mm, the pool II polyfrom an initial
size of greater
than
with apparent
molecular
weight
of
Although
106.
this
partially-reduced
pool
resembled
in size
the polymers
of pool
retained
more
than
four-fold
higher
specific
After
5 mm exposure
to the reducing
agent,
the
polymers
J0.13
0.06
-0.12
‘I
2.4-4
Fig. 8.
Staining
of SDS nonreduced
polyacrylamide-agarose
(2%:0.5%)
gel
electrophoresis
patterns
for proteins
and carbohydrate
in polymers
of various
pools
and in the bands
demonstrated
for the ascending
limb of the major
protein
peak of Fig. 1 (elution
at 6.0-6.6
liters).
The
protein
stain
(Coomassie
blue) is on the left and the carbohydrate
stain (PAS) on the right of each pair of
gel patterns.
Molecular
weight
values
are noted to the left and the ratios
of
PAS:Coomassie
blue values
are noted
to the
right
of appropriate
polymer
bands.
1“0.200.20
0.20
106”
ity was
in
ET AL.
pool
II
were
of
II
IX,
they
activity.
most of
molecular
.
1 x
106,
with
400,000
and 2.4 x 106.
forms
than was present
consisting
predominantly
with contributions
of I .
smaller
size of most
pool II, its ristocetin
than
lesser
amounts
of material
of
Pool IX had generally
larger
in the 5-mm
pool II sample,
of bands of 2.4 and 3.4 x 106
I and 4.6 x 1 06. Despite
the
forms present
in partially
reduced
cofactor
activity
was still slightly
higher,
(1.4:1)
decrease
increase
reduced
in the 2.4 x 106 moiety
and the relative
in that
of 400,000,
the relative
activity
of
pool II continued
to decrease.
However,
at IS
in
pool
IX.
With
the
further
mm the activity
was still only slightly
lower (0.7) than
in pool IX, although
the overall
molecular
size in pool
II was lower
than that in pool IX.
size
DISCUSSION
Table
Ristocetin
2.
E ifect
Co factor
of Neuraminidase
Activity
%
and Galactose
of Selected
Loss
Oxidase
von Willebrand
on
Polymers
Cofactor Activity
Ristocetin
Neuraminidase
Galactose
Netzaminidase
Oxidase
PoollI
36
0
PoolVlII
33
0
FOllOWed
Galactose
by
Oxidase
95
93
The factor
VlII-von
Willebrand
protein
that forms
the basis of this report
consisted
of a series
of multimeric structures
that ranged
in molecular
weight
from
2.4 x 106 to much greater
than
by comparative
electrophoretic
hyde crosslinked
1gM polymers
geneous
population
of molecules
cryoprecipitate
was analogous
10 x
mobility
106,
as measured
of glutaralde-
(Fig.
2). This
obtained
from
in size to those
heterohuman
seen by
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FUNCTIONAL
HETEROGENEITY
vW
PROTEIN
321
POOL
POOL
II
IX
>iOxlO6
-3.4x
3.4x106
106
-2.4x
2.4x106
106
1.1x106
.
.
I
#{149}1
REDUCTION
TIME
RCA
RELATIVE
1
--208,000
L
0
2’
5’
10’
15’
0
6
4.3
1 .4
0.9
0.7
1
Fig. 9.
Relationship
of ristocetin
cofactor
activity
wit molecular
size during partial
disulfide
bond reduction
of von Willebrand
protein
polymers.
The SDS-polyacrylamide-agarose
(2%:0.5%)
gel electrophoresis
patterns
show pool IX and pool II before
and after
limited
disulfide
bond reduction.
For the 2-mm
incubation,
pool II was exposed
to 5 x i0
dithiothreitol.
then mixed
with 2-iodoacetamide
(i03Mfinal
concentration).
For the 5-, iO-, and 1 5-mm samples,
the pool II protein
was exposed
to 0.1 Mfi-mercaptoethanol
then mixed
with 2-iodoacetamide
(0.09 Mfinal
concentration).
Ristocetin
cofactor
activity
of pool II before and after reduction
is expressed
relative
to
the activity
of unreduced
pool IX. all calculated
on the basis of total quantity
of 208,000
reduced
chain after complete
disulfide
bond
reduction
(Table
1).
other
investigators39
position
with
the
plasma
We did
and overlapped
in electrophoretic
protein
demonstrated
in normal
by crossed-immunoelectrophoresis
not distinguish
a moiety
weight
in our
tration
presence
in cryoprecipitate
in the latest
protein
peak
(Fig. 2).
preparation,
Electrophoretic
of the
agarose
molecular
components,
molecular
latter
that
since
purified
were
material
that
been
of the
208,000,
evident
197,000,
probably
do not
bind
nonspecifically
they
have
polymers
using
(Fig. 2) showed
weight
most
weight
suggesting
analysis
different
gel system
(Fig.
4).
molecular
106
its concen-
is lower
than in plasma.
Its
eluting
pool prior
to the major
I ) may
(Fig.
of
antiserum
components
present
using
of
masked
subunit
by 1gM
composition
a polyacrylamidea major
subunit
of
but
also
other
minor
which
were
those
of
174,000,
represent
to von
and
154,000.
unrelated
Willebrand
The
molecules
protein,
in immunoprecipitates
of the
monospecific
Wille-
anti-von
brand
antibody
(Fig. 3), and since they did not precipitate with antibody
against
plasma
of severe von Willebrand’s
disease
patients.
In fact,
they
seemed
to
precipitate
more efficiently
with anti-von
Willebrand
than
did the major
subunit
band.
Minor
have been
previously
observed
by other
investigators,’29
ies of Gorman
and the tryptic
and
Ekert29
peptide
suggested
mapping
studa structural
relationship
between
the 208,000
subunit
and
the
minor
chains
in the
molecular
weight
range
of
I 20,000-1
80,000.
In our preparations,
minor
bands
comprised
only about
I % of the total subunit
composition as measured
by densitometric
analysis
in comparison with
the 208,000
band,
and
probably
do not
represent
in vitro
were
consistently
proteolytic
procedure.
degradation
chain
that
proteolytic
demonstrated
inhibitors
Furthermore,
in vitro30’3’
degradation,
despite
throughout
detailed
show that
since
they
the use of
the
purification
studies
of plasmic
the initial
subunit
is degraded
through
a series
of smaller
do not correspond
in size to the minor
identified
represent
in vitro.
Willebrand
in Fig.
2, making
it unlikely
plasmic
cleavage
products,
either
Whether
protein
not been established.
Analysis
of the
these
bands
are
freshly
prepared
ristocetin
cofactor
chains
bands
that
they
in vivo or
also present
in von
from plasma
has
activity
of
the
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
322
MARTIN
polymers
showed
three
possible
5), with those larger
than
activity
and the smallest
the least activity.
These
activity
Willebrand
could
levels
of activity
various
(Fig.
be ascertained
antigen
content
on
or
the
by
basis
of
densitometric
pools
were
tion,
analyzed
cofactor
activity
10 x 106 having
the greatest
(less than 4.6 x 106) having
three
differences
in specific
von
also
by
activity
is known
trypsin,’8’30’34
the
with the presence
assessed
by
ET AL.
tryptic
degrada-
gel electrophoresis
and
determinations.
Ristocetin
ristocetin
cofactor
to decrease
rapidly
upon exposure
to
activity
in earily
digests
correlating
of fragments
of molecular
weight
measure
of the 208,000
subunit,
but they were not as
apparent
using
a Lowry
determination
of protein
concentration.
Further
separation
of the polymers
by
sucrose
gradient
centrifugation
(Fig. 6) was needed
to
greater
than
tion, residual
approximately
establish
specific
I 16,000.8
Fig. 7 showed
that the degradation
ucts obtained
after 45 mm of exposure
to trypsin
prodwere
the
protein
poly-
activity
weight
was
frag-
that
individual
levels of ristocetin
lapping
effect
ences
or similar
activities
pools
have
over-
reflecting
(Fig.
5). These
are not explained
the
differby the
of contaminant
proteins
such as fibrinogen32
of the lower molecular
weight
polymers,
since
there
was no difference
using von Willebrand’s
diluent
(Fig. 5A). The
ity for smaller
polymers
von
of the
of groups
of polymers
in the specific
activity
presence
in pools
tions
polymers
probably
cofactor
activity,
with
that show
Willebrand
removal
precipitate
in ristocetin
cofactor
activity
disease
plasma
or buffer
as the
findings
of lower specific
activare in concert
with observa-
preferential
binding
of larger
forms of
protein
with platelets’#{176}’ and in vivo
of larger
polymers
into a patient
brand’s
disease.’3
The
experiments
investigate
whether
were due to a strict
after
with
transfusion
acquired
of cryovon Wille-
with
the
same
and function
and/or
whether
other
factors
contributed
to the activity,
such
as might
be demonstrated
by
proteolytic
degradation,
partial
disulfide
bond reduction, or analysis
of carbohydrate
content.
Heterogene-
presence
for
In the late stages
of degradacofactor
activity
accounts
for
initial
activity
and correlates
of a fragment
all
of the
von
ment in each digest,
specific
activity
of
and
this
initial
This
polymer
size.
of molecular
Willebrand
mers.
Residual
ristocetin
accounted
for by the I 1 6,000
cofactor
molecular
weight
no difference
was seen in the
fragment
regardless
of the
in activity
of the original
result
from
differences
indicates
that
the
difference
undegraded
polymers
in the structure
or
did not
specific
activity
of the I 16,000
region
of the molecules,
that enzyme-sensitive
regions
or exposed
cleavage
are
similar
for all forms,
regardless
of size.
There
was a progressive
loss in ristocetin
activity
reported
here
were
designed
to
the differences
in specific
activity
correlation
between
polymer
size
3 14,000.’
ristocetin
5% of
with
partial
disulfide
bond
and
sites
cofactor
reduction
and
alkylation
of the polymers,
such as occurred
with those
of greater
than
10 x 106 in pool II (Fig. 9). This is in
agreement
with Counts
et al.6 who noted
the correlation of decreased
activity
with decreasing
molecular
weight
during
disulfide
bond reduction.
We have additionally
found
that the molecular
size of such partially-reduced
moieties
is not the sole determinant
of
ristocetin
cofactor
ity in the subunit
composition
does
not explain
the
observed
differences,
since
the proportion
of minor
reduced
bands of I 97,000,
1 74,000,
and I 54,000
rela-
obtained
in size
by reduction
to molecules
tive to the 208,000
band was constant
for all samples.
Thus,
smaller
polymers
do not owe their decreased
size
or function
to a greater
degree
of subunit
variation.
All polymer
groups
of molecules
contained
the same
proportion
of carbohydrate,
as reflected
in PAS-
the pool
less than
polymers
retained
activity,
higher
specific
II polymers
1.1 x
in pool
106,
IX,
for
instance,
of pool II polymers
in unreduced
pool
were
clearly
both
activity.
molecules
were similar
IX, but they
Furtherfore,
when
reduced
to a general
size
less than the mean
size
samples
had approximately
of
of
staining
of the gels after electrophoresis
(Fig. 8), and
they had similar
sensitivity
to the loss of sialic acid and
the galactose
residues
(Table
2). These
results
are in
agreement
with
observations
by Zimmerman
and
the same ristocetin
cofactor
activity
(Fig. 9). Thus,
the
disulfide
bond
organization
of the polymers
plays
a
major
role in platelet-related
activity.
This would
be
consistent
with the report
of Cooper
et al.,35 who noted
an initial
increase
in activity
of bovine
von Willebrand
protein
after
short
exposure
to mercaptoethanol,
colleagues
carbohydrate
continued
protein
patients
Taken
who
obtained
showed
content
from
with variants
together,
the
carbohydrate
difference
no
of
significant
difference
reduced
von
Willebrand
normal
individuals
and
in
from
of von Willebrand’s
disease.33
data
suggest
that differences
in
content
do
between
normal
not explain
and variant
patients,
or between
von Willebrand
different
molecular
size from normal
Possible
differences
in primary
the functional
molecules
molecules
individuals.
structure
of
of
of
the
followed
by
the
disulfide
expected
bond
decrease
reduction.
in
This
activity
with
suggests
that
a more
optimal
conformation
for platelet
interaction
may even exist with partial
disulfide
bond
reduction
than with the initial
form of the molecule.
Our results
indicate
that differences
in the specific
activity
of the
polymeric
forms
of von Willebrand
protein
found
in
cryoprecipitate
are a function
not only of their size but
also of quaternary
conformation
that may be dictated
by disulfide
bond arrangements.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
FUNCTIONAL
HETEROGENEITY
vW
PROTEIN
323
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1981 57: 313-323
Structural studies of the functional heterogeneity of von Willebrand protein
polymers
SE Martin, VJ Marder, CW Francis and GH Barlow
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