From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
Aurin
Tricarboxylic
Acid:
A Novel
Inhibitor
Factor
By Martin
Shear
stress
presence
activated
of large
(vWF)
multimers
other
exogenous
ogous
in
without
platelet
narrowed
atherosclerosis
pound.
rich
plasma
(PRP)
of 0.1
ATA
also
smoI/L.
clumping
ristocetin-induced,
in both
suspensions.
This
the
concentration
V
ON
involved
in arterial
reported
Evans
determination
association
vWF alone,
vWF-mediated
clumping
forms
lb molecules.
Subsequent
made
20 custom
synthetic
to EB.2 They
on the phenol
are
formation.
and
of
first
S
In
likely
to
be
1975,
Kirby
used for blood
the in vitro
and ristocetin,
or bovine
human
platelets.’
This
platelets
has
is the result of the binding
to platelet
surface
glycoprotein
to the report
compounds
found
that
rings altered
of Kirby,
Geratz
with structural
changes
in
the inhibitory
of
et al
simi-
substituent
effects
from
other
with
inhibits
by Grune
& Stratton,
cells);
surface
llb/Illa
tion of shear
platelets
by
stress-induced,
polyanionic
sulfonic
acid
EB and suramin.
Aurin
phenyl-methyl
compound
aggregated
in the shear
field by large
human
plasma
vWF
multimers,
or by unusually
large
vWF
multimers
derived
from endothelial
cells, without
the addition
of ristocetin
or
other
exogenous
are:
agents.
large
or
diphosphate
adenosine
The
components
unusually
(ADP)
large
released
needed
vWF
for
multimers;
from
platelets
Rice
University,
(or
The
Methodist
Hospital,
and
Houston.
Submitted
February
Supported
in part
HL
of Medicine,
4, /988;
accepted
by the National
22, /988.
July
Institutes
ofHealth
Grant
No.
35387.
or other
platelet-rich
meeting
reprint
and
©
to
ofHematology
D.
Phillips,
Hospital,
This
article
in accordance
article
must
with
this fact.
1 988 by Grune
were defrayed
therefore
18 U.S.C.
be hereby
section
Inc.
inhibi-
dyes with structural
tricarboxylic
acid
with
a structure
similarities
to
(ATA),
a tndistinct
from
vWF-mediated
of
are
ten times
tested
in
platelet
aggluplatelet
AND
METHODS
for
(PRP),
in ten
blood
3.8%
donors who had not ingested
three
weeks
before
was drawn
sodium
donation.
into plastic
citrate.
PRP
For
syringes
was made
by
For washed platelets,
the blood was drawn into acid-citrate-dextrose
(ACD) (pH 4.5, one
part in six), and PRP was obtained by centrifugation.
Platelets were
sedimented at 1,000 g for 15 minutes, washed in HEPES buffer (pH
of blood at 400 g for 15 minutes.
containing
HEPES
experiments
heparin
(Elkins-Sinn,
(Grade
V, Sigma,
buffer,
pH
7.4.
contained
(Sigma),
and
Outdated
platelets
were
procedure
of Zaleski
(55
umol/L
Cherry
St Louis,
Hill,
0.35
CaCI2
Olson.4
acetylsalicylic
used for shear
g/dL
(final
formaldehyde-fixed
10 U/mL)
and suspended
suspensions
glucose,
1 mmol/L
and
NJ;
2.5 U/mL),
Platelet
100 mg/dL
albumin
bovine
serum
concentrations).
by a modification
They
were
acid
[ASA],
treated
of the
in vitro
with
rotating
for
gently
L ethylene
MS
by page
buffer
marked
EDTA,
1 734 solely
to
diamine
tetra-acetate
1% paraformaldehyde
(50
ATA
(CR),
saline buffer,
mmol/L
pH
6.4)
I .2 x i06/zL.
fully
and
imidazole,
with
They
and Chicago
two
pH 7.1),
hours,
mmol/L
7% dimethylsulfoxide
(Fig
Sky
Direct
Blue
(CSB)
were
more
imidazole
70#{176}Cin
NaCI,
1 mmol/L
to ensure
cofactor
Yellow
-
with
three
at a concentration
periodically
in ristocetin
1),
incubated
washed
and stored at
I 10
were tested
functional
EB
[EDTA],
at 25#{176}C
for
with citrated
remained
& Stratton,
the
Baylor
Fannin,
in part
studied
30 minutes
at 25#{176}C),washed
three times in citrated
saline
buffer
(150 mmol NaCI, 6 mmol/L
trisodium citrate dihydrate, 0.9 mmol/
MD.
6565
platelets
Ib, and the
annual
1987.
Martin
have
We
from volunteer
one part
centrifugation
times
0006-4971/88/7206-0002$3.00/0
1898
December
The Methodist
costs ofthis
payment.
“advertisement”
indicate
Society
TX 77030.
The publication
charge
DC,
requests
ofMedicine
Houston,
902,
at the American
in Washington,
Address
College
in part
of
aggregation
of which
medications
plasma
containing
aspirin
Presented
be a
development
as well as shear-induced,
vWF-mediated
in the absence
of ristocetin.
Blood was obtained
aspirin
in
College
it may
sulfonic
acid dyes, is approximately
per mole than
any other
compound
and apyrase
Baylor
platelet
of ristocetin,
vWF-mediated
compounds,
some
MATERIALS
6.9)
From
Because
vWF-mediated
of
of these compounds
on platelet
agglutination.
Shear
stress-induced
platelet
aggregation
may be a model
of in vivo arterial
thrombosis,
because
human
platelets
are
aggregation
ristocetinlb.
the
be
binds
Inc.
ristocetin-induced,
tination,
aggregation
their
not
can
ATA
glycoprotein
complex.3
naphthalene
inhibiting
inhibits
the
and
are
ATA
calcium
ions; and metabolizing
vWF receptors,
glycoprotein
glycoprotein
by
acid-
with
to impede
in vivo.
naphthalene
more active
and
absence
compound
several
any
in the
and
for
However.
shear-induced,
thrombosis
blood
intact
of aggregation
aggregation
platelet
in
multimers
Arachidonic
compound.
forms
found
vWF
accounted
incubated
the
with
prototype
1988
rate
(ADP)-induced
Platelets
in vitro
arterial
(vWF)-mediated
of nonmetabolizing
termed
agglutination
vWF multimeric
larities
groups
kinetics
(EB), a dye formerly
in humans,
interrupted
of human
plasma
vWF
with formaldehyde-fixed
useful
platelet
by increasing
the
also
The
is not
attachment
forms
large
calcium.
multimeric
aggregation
plate-
or
interaction
ATA
unusually
ATA
the
multimeric
cells.
by
ATA.
vWF
prevents
vWF
of the
ristocetin
separated
to large
ATA
largest
diphosphate
induced
manner,
aggregation
thrombus
blue
that
and
inhibition.
and
by
easily
a concentra-
Willebrand
Turner
endothelial
of
inhibited
platelet-
Nancy
inhibition
adenosine
shear-
MmoI/L
at
FACTOR
and
adhesion
with
corn-
200
be overcome
following
WI LLEBRAND
volume
binding
formaldehyde-fixed
can
vWF.
plasma
vWF-mediated
and
inhibition
of
platelet
been
large
fresh
normal
patients
In a concentration-dependent
inhibits
or any
and
of the
of
in
above
competitive
platelets
from
inhibits
platelets
order
degree
aggregation
of washed
Nolasco,
to
triphenyl-methyl
in concentrations
Leticia
factor
be anal-
of von
Platelets
derived
of
(ATA),
platelet
suspensions
tion
A
acid
Association
to thrombosis
may
leads
arterioles
vasospasm.
in the
of ristocetin
that
tricarboxylic
L. Moake,
Willebrand
phenomenon
and
vWF-mediated
in buffer
let
This
Joel
aggregation
von
addition
aggregation
or
aurin
induced.
large
the
arteries
Phillips,
undergo
or unusually
chemical.
to the
the
platelets
D.
and
of the
50
they
Congo
Red
assays.
(DY5O),
purchased
Blood, Vol 72, No 6 (December),
of
that
from
Aldrich
1988: pp 1898-1903
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ATA
INHIBITS
PLATELET/VWF
H2N
INTERACTION
OH
_
1899
OH
-
NH2
and
50 zL of ristocetin.
optical
density
were
The
To analyze compounds
because of their optical
SO3Na
vWF
SO3Na
Blue
Evaru
platelets
test
in
OH
(final
COCH
HO
0
Trcarboxylic
Acid
Structures
of EB and ATA.
generously
provided
Institute.
by
Vancomycin
and ristocetin
minimally
dissolved
ate
Suramin
Dr
from
of
from
the
Eli
Bio-Data
New
York)
National
Lilly
was
Cancer
(Indianapolis),
(Hatboro,
PA).
ATA
is
soluble in water; thus, for some experiments
it was
in 95% ethanol at 200 mmol/L
and added at the appropriIn
other
experiments,
ethanol
was placed
in a plastic
stream
of N2,
and
the
that
the
indicate
soluble
platelet
tube,
ATA
and
aliquot
of
ATA
efficacious
of
AlA
evaporated
in plasma.
salt
is equally
an
the ethanol
dissolved
ammonium
in water
in
under
Preliminary
a
data
Ferranti
Commack,
NY)
mL)
plasma;
fresh
a source
vWF
counting.
forms
dynes/cm2
for
seconds.
at
Electric,
of large
multimers
the cryopre-
human umbilical
unusually
large
vWF
Model
was 50 .tm.
lets. The
Particles
aggregates.
Platelet
All counts
aggregation
PAP-2
were
100
and
platelets
could
Thus,
or
I 20
for
30
FL)
tube,
and the supernatant
and
multimeric
to the percent
In experiments
with
using
to a siliconized
in
EDTA,
SDS,
electrophoresed
0.04
a Bio-Data
glass cuvette,
with formaldehyde-fixed
In experiments
lets in HEPES
a source
tate,
buffer,
of vWF
or endothelial
multimers)
or arachidonic
were
pH
(large
7.4, were
vWF
multimers
cell supernatant
added,
along
placed
with
acid)
platelets,
purified
from
unusually
50 .tL of buffer
added.
were
350 L
in the cuvette.
containing
vWF
multimers
added
A
added.
uL of
cryoprecipilarge
for vWF
was
quantification
was
(SDS)-l%
quantified
by
patterns
were
agarose
gel electro-
in a continuous
mol/L
sodium
in order
were
buffer
acetate,
to prevent
purified
as described
I 99 with
(Gulf
to the tube,
insulin
(0.1%
2 mmol/L
adsorption
from
pooled
previously.8
of
normal
Endothelial
cell
and transferrin
(both
5 j.tg/mL;
temperature
Ni)
vWF
or test compound
conical
plastic
tube, and the ethanol
of N2. Two hundred
microliters
of
Coast Regional
and the ATA
a final concentration
Blood Center,
was dissolved
of 2 mmol/L.
The
for 30 minutes,
4B column
equilibrated
and
with
mixture
I 00 L
(Pharmacia
in HEPES
buffer,
Houston)
gentle
was
to
incubated
at
layered
on a 9 x
Fine Chemicals,
Piscata-
pH
were
were
rotation
7.4. Absorbance
at 280
nm was monitored, and 0.5-mL fractions were collected. The ristocetin cofactor activity of each fraction was assessed by adding I 95 zL
ofeluate
(or eluate dilution made with HEPES
buffer)
to siliconized
glass cuvettes with 35 oL of concentrated
formaldehyde-fixed
plateconcentration,
from
severe
with an equal
proteins
other
activity
To determine
vol of 20 mmol/L
and the ethanol
added,
incubated
than
incubation
with
stirring,
25
added.
The
a standard
curve
eluted
fractions
were
of plasma
from
a patient
with
(vWD)
vWF
were
The
containing
(which
at room
normal
was
amounts
I % of normal).
<
temperature,
the ristocetin
was again measured.
whether
ATA
adheres
ATA
in 95%
ethanol
was evaporated.
and
plasma.
volume
disease
a ten-minute
continuous
1 mg/mL)
was compared
of normal
von Willebrand’s
After
With
concentration,
dilutions
also incubated
were
I 75,000/zL).
(final
rate of aggregation
prepared
of plateFifty
sample
(ECS) containing
unusually
large vWF multimers36
from cultured human umbilical
vein endothelial cells in
cryoprecipitate
initial
platelets,
and 50 oL
were
on a
( I 0 zL)
by centrifuga-
and multimeric
plastic
ethanol
were placed in a 1.5 mL
was evaporated
under a stream
cofactor
collagen,
compound
were
Medium
ADP,
(ristocetin,
test
for a final
was placed
The samples were denatured in I .5
urea-0.02 mol/L, tris-HCI-2
mmol/L
0.02
pipettes
cryoprecipitate,
agent
or
and
largest
human
of plasma
7.4)
added
pelleted
antigen
in I % agarose
tris-HC1,
vWF
EDTA),7
overlaid
with ‘251-labeled
polyclonal
rabbit
anti-human
vWF IgG, and placed with X-ray film at - 70#{176}Cfor 24 to 72 hours.
bar was placed in the cuvette, and a stable baseline
was achieved. Fifty microliters
of an aggregating
(pH
mol/L
stored
sulfate
for
remaining
were
vWF
dodecyl
phoresis and autoradiography.6
volumes of 2% SDS-8 mol/L,
lets (final
in platelet
fresh
The
analyzed
by sodium
The
was again
saved
To the remaining
at 25#{176}C.
Samples
counting.
assay (IRMA),5
zL of ristocetin
in duplicate.
performed
minutes
immunoradiometric
way,
for by
decrease
increase
was
analysis.
the platelets
analysis.
100 mm Sepharose
aperture
be accounted
three
particle
tion,
room
(Coulter).
the percent
supernatant
To determine
whether
ATA attaches
to vWF, plasma cryoprecipitate was incubated
with AlA,
and the vWF was separated
by
column chromatography.
Twenty microliters
of 20 mmol/L
ATA in
10 oL
the
the
mul-
in the Coulter
zL,
for
for
of either HEPES
magnetic stirring
light transmission
buffer
from
of 20 U/dL.
Sigma).
±
were performed
studies
were
added
concentration
and multimeric
in a microfuge
serum-free
20% of the mean platelet
were considered single plate-
sizes
samples
aggregometer.
400 oL of PRP
was
related
purified
vein
Hialeah,
and Channelyzer
aggregates.
was directly
applied
and by counting
with
PRP
by diluting
Diagnostics,
counter
of single
of platelet
was
performed
(Coulter
counting
in the unsheared
platelets
Model
for
disappearance
the formation
single
II
Particle
volume
distribution
again
glutaraldehyde
Electronic
sample
setting
of Isoton
0.5%
ZBI
suspensions
were
for
g, and
table
removed
supernatant
was obtained
Samples
vWF
from
Inc,
10,000
shaker
vWF.
of 180 dynes/cm2
platelet
counts
in 20 mL
containing
The
washed
Particle
samples
force
a final
were
All tubes
purified
containing
A shear
for
were placed
multimers
mixture
is freely
timers, ie, vWF multimeric
forms larger than those found in normal
human plasma),
were applied
to the plate in the presence
or absence
of compounds
to be tested. Other samples
(10 .tL) were taken for
particle
added
vWF
of
formaldehyde-fixed
and the reaction
of vWF-
previously.3
plasma; or cultured
supernatant
of
the disappearance
of
75,000 to I .2 million/zL)
50 oL of large
were
iL
50 uL of ristocetin
inhibition
(Ferranti
in detail
platelets,
of normal
cells
viscometer
described
the largest
fraction
endothelial
plate
has been
containing
(normal
cipitate
and
400
suspension,
The
cone
of decrease
not be used in the aggregometer
or to follow
of 1 mg/mL,
(aluminon)
in
interaction.
The
extent
platelet-vWF
were
Chemical,
Trissel
was obtained
was obtained
concentration.
(0.6
(Mobay
Larry
and
concentration
placed
Milwaukee.
could
medium,
quantification
rotating
Chemical,
reaction
To this,
sedimented
antigen
Fig 1 .
that
properties,
concentration
tubes.
rate
Fifty microliters
of buffer or test compound were added, and 10 L
samples were taken for particle counting. One hundred microliters
of
the mixture
were placed in a microfuge
tube, the platelets were
y
Awl,
the
cyroprecipitate
HOOCL
HOOC
from
initial
recorded.
the
ATA
was
at room temperature
Ten
to the
platelet
was placed
volumes
dissolved
surface,
in a plastic
of normal
with
gentle
for ten minutes
(final
one
tube,
ACD-PRP
rotation
ATA
and
concen-
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1900
PHILLIPS
tration
- 2
made
with
acid-free
2.5
mmol/L).
1.5
each
apyrase
with or without
and
then
was
recovered,
at
HEPES
and
ATA
20%,
suspended
at a platelet
ten
10%,
and
pH 7.4, with
of
milliliter
density
I 5 minutes.
count
pellet
and
was
of 300,000/L.
The
PRP
gradient
The platelet
in HEPES-apyrase-heparin,
The
was
fatty
One
into the albumin
minutes.
8%
in HEPES,
heparin.
at I 200 g for
g for
density gradient
13%,
(Sigma)
10 U/mL
was layered
centrifuged
1000
albumin
of 40%,
bovine serum albumin
U/mL
fuged
A discontinuous
mL
pellet
centri-
resuspended
response
in
of the
platelets to 2 U/dL
of added vWF and I mg/mL
of ristocetin was
then assayed in a ristocetin cofactor assay.
Two-dimensional
immunoelectrophoresis
was performed
in 0.5%
agarose.6 The sample was electrophoresed
in the absence of any
denaturing
agent in the first dimension.
The second dimension
included polyclonal
rabbit IgG anti-human
vWF in the agarose.
Samples
resed
of normal
in the same
identical
blue,
wells,
with
and
electrophoresis
conditions.
massie
origin
plasma
After
the gels were
taped
together,
without
chamber
ATA
were
at the same
electrophoresis
and
staining
electrophotime,
with
under
Coo-
aggregation
tion
induced,
centration
of the
in
induced,
was
(Fig
ATA
At
were
screened
for
platelet
aggregation
2A). At low concentrations
inhibited
shear-induced,
higher
concentrations
(200 to 400
vWF-mediated
(5 mmol/L),
ally similar
dyes CSB,
DY5O,
activity.
There
was a direct
relationship
of ATA
platelet
their
ability
to inhibit
in PRP in the shear field
and
EB and
CR
had
between
and inhibition
of shear-induced,
aggregation
in normal
PRP
(Fig
concentration
of ATA,
the
extent
smol/L),
only
aggregation.
the
some
structurinhibitory
the concentration
vWF-mediated
2B). At a given
of inhibition
of platelet
PRP,
At
the
therapeutic
had
no
effect
clumping
(Fig
with
of ATA
between
aggregation
during
ristocetin-induced,
the concentration
of ATA
inhibition
of agglutination.
fled from cyroprecipitate.
agglutination
could not
platelet
and
the apparent
platelet/
in fresh,
purified
vWF,
to achieve
the
necessary
clumping.
vWF
seen
was also
be completely
of large vWF
concentration
double-reciprocal
Vmao
clumping
fashion
by
were
in shearobserved
overcome
multimers
At 2.0 tmol/L
ATA,
be reversed
completely,
in the
A
and
Other
of
was
As
increased,
so did the extent
At 0.5 omol/L
and 1 .0 mol/L
ATA,
this
inhibition
could
increasing
the concentration
unchanged,
2A).
vWF-mediated
agglutination
The rate of agglutination
of both vWF and ATA.
platelets.
on the concentration
increase
buffer
were
ATA
and
experiments
formaldehyde-fixed
the
shear-
that increasing
the con(a tenfold
molar excess)
of platelet
of inhibition
competition
platelet
vWF
multimers.
showed
that
con-
on
by ATA of shear-induced
platelet
the mechanism
of ATA
inhibi-
as compared
The
induced
dependent
thera-
chelation.
concentrations
degree
ristocetin-
times
in both fresh PRP and formaldehyde-fixed
suspensions
(Fig 4). When
platelets
eight-fold
A#{176}#{176}
platelet
that
with
aggregometer,
ristocetin-induced
inhibited
in a concentration-dependent
also
same
at ten
on inhibition
derived
agglutination.9
shown)
indicated
ofcalcium
to 20 mmol/L
indicating
vWF
was
aggrega-
multimers
to interfere
vancomycin
not calcium
normal
Compounds
vWF-mediated
and
PRP,
had no effect
aggregation,
In the
vWF
(not
centration
was
large
platelet
vWF-mediated
experiments
greater
matching
unusually
concentrations
RESULTS
exact
by
endothelial
cells.
has been shown
vWF-mediated
peutic
ATA
vWF
with
is shown
mediated
and photographed.
superimposed
This
from human
Vancomycin
tion
as the concentration
of
in Fig 3 for shear-induced
decreased
increased.
ET AL
of
platelet
affinity
of
plot
by
pun-
inhibition
of
even by an
purified
large
of
data
was
these
agglutination
of the
of
platelet
receptor
for vWF decreased
proportionally
with increasing
concentrations
of ATA.
Ristocetin
was not rate-limiting
above
threshold
concentration
of I mg/mL
(data not shown).
a
[-a
0
Fig 2.
Inhibitory
effects
of different
compounds
on shearinduced.
vWF-mediated
aggregation
of platelets
in normal
PRP. (A) The concentration
of test cornpounds
was 4.00 MmoI/L
unless otherwise
specified.
They were
dissolved
in normal
PAP
(platelet
count = 350.000/uL).
and a shear stress
of 180 dyne/
cm2 was applied
for 30 seconds.
Electronic
particle
counts
were
performed
before and after shearing.
The final count is plotted
on
the ordinate
as percentage
of the initial platelet
count.
(B) The
inhibition
of shear-induced.
vWF-rnediated
platelet
aggregation
by
ATA is concentration-dependent.
ATA was dissolved
in ethanol
and added
to normal
PRP (platelet
count = 350.000/L).
and a
shear stress of 1 80 dynes/crn2
was applied for 30 seconds.
Ethanol
alone has a mild inhibitory
effect on platelet
aggregation.
25OiiM
500U
rrrM
ATAO
EIOH
1%
ATA Coroarlraloe
Fig 3.
Increasing
concentrations
of ATA progressively
inhibit
shear-induced.
vWF-mediated
aggregation
of fresh, washed
platelets. The platelets
were suspended
in HEPES buffer (pH 7.4. bovine
serum albumin
[BSA] 3.5 g/dL. glucose
100 mg/dL)
at 130.000/
gtL. Endothelial
cell supernatant
was the source of unusually
large
vWF multimers.
A shear stress of 120 dynes/crn2
was applied for
3Q seconds.
Inhibition
of aggregation
was proportional
to the
concentration
of ATA and could be overcome
by increased
concentrations
of ECS containing
unusually
large vWF
rnultirneric
forms.
ECS = 3.5 U/dL
(white
bars); ECS = 7.0 U/dL
(hatched
bars).
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ATA
INHIBITS
PLATELET/vWF
INTERACTION
1901
ADP 10pM
AraeFcmild
. IOOpMATA
a
o
10
iM AlA
Coctroi
Time
Fig 4.
ATA inhibits
ristocetin-induced
clumping
in the aggregorneter.
Agglutination
PRP (350.000/.tL;
left) or formaldehyde-fixed
vWF-rnediated
of normal
platelets
platelet
platelets
in
(200.000/
ML) in HEPES buffer,
pH 7.4 (right).
in the presence
of added large
vWF multimers
purified
from cryoprecipitate
(100 U/dL).
ristocetin (1 .5 mg/mI).
and ATA.
Increasing
the concentration
of ATA
progressively
inhibits
ristocetin-induced.
vWF-mediated
platelet
clumping
in both systems.
von
Willebrand
factor
disappeared
as formaldehyde-fixed
by nistocetin
in the presence
from cryoprecipitate.
When
nation
(Fig
of platelets
Gel
5A).
demonstrated
timers
when
agglutination
were blocked.
and
was
and
the
The nistocetin-induced
is attenuated
at any
is no dissolution
not
of vWF
and
acid-induced
platelet
of PRP (350.000
platewith ATA (2 mrnol/
aggregation,
that
this
adversely
ATA
autoradiography
absent.
were
oflarge
of previously
formed
platelet
in fresh
6). Since
and
PRP
arachidonic
were
fibrinogen
acid-mediated
unaffected
is a cofactor
platelet
by the
for
addition
ADP-induced
does
surface.
when
platelets
blocked
platelets
pended
presence
5B)
mulboth
in PRP
but there
clumps
(data
aggregation
of ATA
(Fig
platelet
not
PRP
with
ATA,
the platelets
were
proteins
by albumin
density
gradient
There
shown).
ADP
of
plasma
ATA
complex.
the platelet
purified
aggluti-
multimers
clumping
of fresh platelets
by the addition
of ATA,
evidence
washed
in HEPES
buffer,
and resuspended
buffer
to a concentration
of 300,000
platelets/sL.
was no difference
in responsiveness
to nistocetin/vWF
(Fig
vWF
is indirect
affect the glycoprotein
lIb/IlIa
does not associate
closely
with
in
centrifugation,
HEPES
of purified
large vWF
In the presence
of ATA,
disappearance
point
reaction
agglutinated
of large vWF multimers
ATA
was added,
both
disappearance
ATA
the
were
consumption
electrophoresis
the
from
platelets
TOme (mm)
Fig 6.
Neither
ADPnor arachidonic
aggregation
was inhibited
by preincubation
lets/gEL) for 30 rninutes
at room temperature
1).
After
incubation
separated
from
medium
(mm)
preincubated
in
ATA
were
with
compared
preincubated
in buffer.
Separated
platelets
susonly in HEPES
buffer
did not agglutinate
in the
of nistocetin
alone,
indicating
that there was little or
no platelet
surface-bound
dient separation
ATA
attaches
cryoprecipitate
vWF
after
(data
not shown).
to vWF
multimers.
that had been incubated
albumin
density
vWF
isolated
with ATA
grafrom
before
column
chromatography
had a reduced
capacity
to support
nistocetin-induced
platelet
agglutination.
The
nistocetin
cofactor
activity
of comparable
fractions
eluted
from
a
Sepharose
4B column
chromatography
was
cipitate
with
was reduced
by two thirds
when
the
preceded
by preincubation
of cryopre-
2 mmol/L
ATA,
compared
with
a control
Fig 5.
ATA
blocks
ristocetin-induced.
vWF-mediated
agglutination
of formaldehyde-fixed
platelets
and simultaneously
prevents
disappearance
of vWF from the reaction
rnedium.
Platelets
(600.000
to 1 .2 million/giL).
large vWF multimers
purified
from cryoprecipitate
(20 U/dL).
ATA (2 MmoI/L)
or HEPES buffer. pH 7.4. and ristocetin
(1 mg/mL)
were mixed in plastic tubes and shaken on a rotating
table for
three minutes.
Aliquots
for platelet
counting
and vWF antigen
level (A) and for vWF multimeric
pattern
analysis
(B) were taken before and
after agglutination.
(B) Autoradiograph
of SDS-1 % agarose
gel electrophoretic
separation
of the vWF multirners
in the supernatant
of the
reaction
in (A). The gel was overlaid
with ‘2l-rabbit
IgG anti-human
vWF.
Pre - supernatant
prior to the addition
of ristocetin
and
agglutination.
Post - supernatant
after agglutination.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
PHILLIPS
1902
fraction
preincubated
metric
in buffer
quantitation
ofvWF
(Fig
7). The
antigen
to
withstand
reversible
chromatography
in the presence
follows
from
activity
values
of these
when
patient
with
proteins
the
in
of plasma
demonstration
fractions
the fractions
plasma
proteins
compete
with
Incubation
ofcontrol
fractions
no significant
effect
on the
proteins.
This
conclusion
the
nistocetin
nearly
with
vWF
with
normal
cryoprecipitate,
containing
nistocetin
cofactor
or in supernatant
unusually
(Fig 8). The denaturing
detection
of any effect
system,
the
dimensional
when
activity
and
large
after
autora-
with ATA
in normal
purified
from
of endothelial
vWF
cell
multimenic
forms
effects
of SDS may have prevented
of ATA
on vWF
structure
in this
however,
because
ATA changed
the mobility
of vWF
analysis
was conducted
in a non-denaturing,
twoimmunoelectrophoretic
system.
Preincubation
of normal
toward
cofactor
for the binding
of
vWD plasma
had
it was determined
that preincubation
alter
the
pattern
of vWF
multimers
in normal
cryoprecipitate,
in vWF
cultures
was
to the control
plasma
from a
correcting
for the dilution
effect.
By using SDS-l%
agarose
gel electrophoresis
diography,
did not
plasma,
strong
but
contains
all normal
plasma
These
results
indicate
that
except
other
ATA.
sufficiently
buffer
restored
incubated
severe
vWD,
which
vWF
(Fig 7).
of the
HEPES
that
was
were
for compara-
preincubation
was
plasma
the
with
positive
ATA
pole
in the
AL
immunoradio-
was similar
bly eluted
fractions
with and without
cryoprecipitate
with ATA.
The association
of ATA and vWF
ET
slowed
first
the
migration
dimension
of vWF
(Fig
9).
This
20
100
15
.75
Fig 8.
ATA does not change the migration
of vWF rnultirners
in one-dimensional
SDS-1 % agarose
gel electrophoresis.
vWF in
normal
plasma
(NP). vWF in normal
cryoprecipitate
(cryoprecipitate). vWF purified from cryoprecipitate
(purified
vWF). or unusually large vWF multimers
in ECS (ECS) were
incubated
with 2
rnmol/L
AlA
for 30 minutes
at room temperature.
analyzed
by
SDS-1 % agarose
gel electrophoresis.
overlaid
with l-rabbit
lgG
anti-human
vWF. and autoradiographed.
The ECS in the lanes at
the sides of the gel was produced
by endothelial
cells cultured
from a different
umbilical
vein source and contains
relatively
more
intermediate-sized
vWF multimers.
All of the ECS samples
contain
unusually
large vWF multimeric
forms.
Lanes of normal
pooled
plasma (NPP) are included
as standards.
must
indicate
a change
in the tertiary
structure
multimens
induced
by ATA
binding,
because
ATA
negatively
charged.
of vWF
itself is
10
5
DISCUSSION
25
2
The
0
I
4
5
6
7
Elufrd
8
Volume
9
10
11
12
13
ImIl
Fig 7.
ATA attaches
to vWF multimers.
Cryoprecipitate
was
incubated
with or without
2 mrnol/L
ATA and then chromatographed
on a Sepharose
48 column.
Lines indicate
the relative
absorbance
of each column fraction
at 280 nm ( indicates
control
eluate;
#{149}
indicates
eluate from cryoprecipitate
preincubated
with
ATA).
Bars indicate
the ristocetin
cofactor
activity
of each fraction. The black bars indicate
eluate from control
cryoprecipitate;
the hatched
bars indicate
eluate
from cryoprecipitate
preincubated with ATA. Inset: vWF antigen
levels of control
and ATApreincubated
fractions.
The same fractions
were incubated
with
plasma frorn a patient
with severe von Willebrand’s
disease.
which
contains
all normal
plasma
proteins
except
vWF
(<1 % vWF
antigen
and ristocetin
cofactor
activity).
The ristocetin
cofactor
activity
of the ATA-preincubated
fractions
(white
bars)
was
almost
completely
restored
to the ristocetin
cofactor
activity
of
the fractions
isolated
from cryoprecipitate
that had been incubated with buffer before chromatography.
ATA
experiments
inhibits
ments
neither
tribute
aggregation
indicate
report
in the shear
in defined,
reconstituted
systems
have
fibninogen3”#{176} nor arachidonate
significantly
to shear-induced
platelet
The
degree
ATA
of
ATA
of aggregation
inhibition
is dependent
ens and
calcium
tion
in this
described
platelet
only
on the concentration
the concentration
ofATA,
chelation
by the polyanionic
apparently
by interfering
inhibits
with
in the
and
lb and large
sion follows
or unusually
large
from the observation
tin-induced,
von
Willebrand
Expeni-
shown
‘
field
platelet
of any
aggrega-
glycoprotein
vWF multimers.
This
that ATA
inhibits
factor-mediated
by
multim-
is not the result
ATA.
between
that
con-
aggregation.
shear
ofvWF
shear-induced
the interaction
that
field.
conclunistoce-
agglutination
of formaldehyde-fixed
platelets.
Fixed
platelets
retain
only
the glycoprotein
lb receptor
in functional
form.’2’4
These
platelets
have no metabolic
capability
and cannot
release
granule
contents.
As
in
shear-induced
aggregation,
the
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ATA
INHIBITS
PLATELET/VWF
INTERACTION
1903
presence
of ATA.
pletely
agglutination
by AlA
by ATA
of agglutination
of fixed
structure
undenatuned,
system
Elevated
tion
shear
stresses
of platelets,
of large
vWF
multim-
The
metabolic
function
of
platelets
is preserved
in the
in
an
immuno-
ATA
spastic
arterial
elevated
shear
itself
is nega-
mediated
vessels.3”5
Aggregation
in vitro
is mediated
large
inhibits
vWF
shear
stresses
platelet
reactive
and
tional
in the
blood
or
endothelial
be important
formation
unusually
cells. ATA
multimers
may
in thrombus
aggregation,
to ADP
at pharmacologic
with aspirin
and
type
as
aggrega-
in stenotic
or
induced
by
by large
vWF
forms
derived
stress-induced,
yet leaves
arachidonic
and
inhibition
dipynidamole
is a different
hold
promise
platelets
pole
two-dimensional
the
but not
overall
in a way
positive
and
induced
to fixed
multimers
the
though
unequivocal
evidence
that these
laxis or therapy
for most arterial
attachment
vWF
the
charged).
by nistocetin
and vWF is dependent
only on the concentration
of vWF
multimens
and AlA.
ATA
inhibits
the nistocetinens.
with
of vWF
(even
multimens
of competi-
changes
toward
gel,
vWF
kinetics
associates
agarose
0.5%
of large
apparently
mobility
electrophonetic
tively
and
tertiary
their
and
which complatelet
vWF-mediated
first-order
charge
attempts
clumping
platelet
of the binding
the classic
reversibly
and
to ADP
of ATA,
vWF-mediated
on nistocetin-induced,
follows
slows
normally
platelets.
tive inhibition.
ATA
with
fresh
platelets,
that
of inhibition
PRP
of fixed
Inhibition
degree
concentrations
shear-induced,
in
to platelets
aggregate
despite
prevent
aggregation
Fig 9.
Preincubation
of vWF
rnultimers
with
negatively
charged
ATA slows the migration
of those multimers
toward
the
positive
pole in undenaturing.
two-dimensional
imrnunoelectrophoresis.
Normal
platelet-poor
plasma
was incubated
at room
temperature
for 30 minutes
with or without
200 Lmol/L
ATA and
electrophoresed
in the horizontal
dimension
into 0.5% agarose
(in
the absence
of SDS or any other
denaturing
agent).
then in the
vertical
dimension
into agarose
containing
rabbit lgG anti-human
vWF. The test and control gels were run in the same electrophoresic chamber
at the same time under identical
conditions,
and then
stained with Coomassie
blue. The origin wells of the two
gels were
precisely
matched.
and the gels were taped together
for photography.
Platelets
acid
arachidonic
of
agents
are
thrombotic
from
vWF-
the
platelet
funcacid.
Previous
in
have
vivo platelet
not provided
effective
prophydisorders.
ATA
of platelet
aggregation
inhibitor
and may
an alternative
prototype
of an arterial
antithrombotic
agent.
9. Moake
JL, Cimo
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1988 72: 1898-1903
Aurin tricarboxylic acid: a novel inhibitor of the association of von
Willebrand factor and platelets
MD Phillips, JL Moake, L Nolasco and N Turner
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