From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
REPORT
CONCISE
Participation
of ADP
in the
to Thrombin-Stimulated
Edward
By
Thrombin
similar
and
adenosine
kinetics
identical
and
dependence
phosphokinase
of
participates
CP/CPK
in the
binding
sites
a
many
and
saturable
discrete
From the analysis
to ADP-stimulated
6
the
by
char-
receptor
of the specific
binding
of
platelets,
linear
Scat-
chard
plots are derived
indicative
of a single class of
binding
sites with an affinity
of 1.5-2.3
x 106 M ‘,
and approximately
40,000
molecules
are bound
per
platelet
in the presence
of I mM calcium.
Additional
features
of fibrinogen
binding
to ADP-stimulated
platelets
include
a requirement
for divalent
ions (calcium
or magnesium)
and the complete
inhibition
of
binding
by the enzymatic
removal
of ADP.45
Recently,
1-lawiger
et
al.7
shown
have
that
supports
the binding
of fibrinogen
platelets.
The affinity
and number
were
similar
to values
that
they
stimulated
platelets.
platelet
storage
release
if platelet
nonlytic
assessed
fibrinogen
of
ADP.8
In this
ADP participates
MATERIALS
were
was
(AC
filtration.
The
equilibrated
albumin
at
study
been
‘25I-fibrinogen
follows:
to
platelets
concentration
thrombin
were
or ADP,
was
clotting
activity
completely
Blood.
of
were
Incubation
Vol.
thrombin.
56,
No.
gently
times
2%
(The
and
The
was
10 mm
mixed
and
and
measured
3 (September),
1980
are
results
which
was
molar
excess
‘25I-fibrinogen
total
binding
tical
analyses
Tl-55
Platelet
recovery
labeling
5tCr
400
2.5
mm
at
of
in
using
tip.
a 50-fold
from
the
represented
of ‘25I-fibrinogen
performed
was
tube
observed
binding
in a
platelet
‘251-fibrinogen,
(nonspecific)
nonspecific
rpm
centrifuge
binding
binding
sucrose
I 1,750
per
in the
fibrinogen
or triplicate
tl of 20%
bound
specific
subtracting
and
isolation,
the
total
5%
utilized.
a Texas
Instruments
reaction
were
labeled
serotonin
ogen
(as
cause
‘Cr
of
StatisModel
(St.
Louis,
As utilized,
apyrase
polyacrylamide
by
or ‘4C-serotonin
platelet
aggregation
kind
gift
and
and
Percent
to the
from
purchased
ofthe
did
gel
recov-
radioactiv-
manner.
were
from
highest
grade
not degrade
fibrin-
electrophoresis),
platelets,
by thrombin.
John
After
assays,
were
CP/CPK
release
induced
of Dr.
CPK
(New
in binding
in an identical
Mo.)
judged
as
relative
and
either
described.4
25l-fibrinogen.
calculated
CP,
by
with
or 2-’4C-serotonin
utilized
treated
measured
plasma
previously
for
were
apyrase,
Co.
was
the
were
platelets
Chemical
as
fibrinogen
release
hirudin,
Ill.)
Mass.)
platelets
unstimulated
of
ADP,
in platelet-rich
Heights,
Boston,
unlabeled
and
release
platelets
Arlington
Nuclear,
substituting
the
the
(Amersham,
England
and
The
did
did
not
cs-thrombin
not
block
utilized
Fenton.
RESULTS
and
binding
was
of fibrinogen
to thrombin-stimulated
initially
assessed as a function
of time
compared
with
the activity
to the
Institut
de
to a final
stimulus,
the
concentrations
also
aggregation.)
at
the
The
were
as
to inhibit
added.
addition
From
ADP-stimulated
of the
platelets.
thrombin
fibrinogen,
the
to the
platelets
To
platelet
were
as
stimu-
the
Department
Pathologie
Submitted
April
Supported
in part
publication
Molecular
Immunology.
La Jolla.
Cellulaire,
Address
28, 1 980;
(alif..
and
Research
INSERM
Hopital
de
May
5. 1980.
reprint
ofScripps
accepted
by HL-l64l
21 1 1 ofthe
(‘alif
92037.
© I 980 by Grune
of
of
Clinic,
Bicetre,
U 143,
Bicetre,
France.
Institute
22#{176}Cwithout
to the
platelet,
calculator.
isotopically
ity
for
at all concentrations
ofScripps
required
relative
the
duplicate
onto
radioactivity
as
by
times,
layered
spun
the
binding.
Institute
were
an
pretreatment
from
‘25l-fibrinogen
of unlabeled
of
was
incubated
derived
binding
measured
inhibitors
but
released
selected
and
reported
The
in
the
phosphate/creatine
binding,
exhibited
serum
at 22#{176}C,hirudin
hirudin
potential
All
100.
utilized
buffer
calcium,
platelet
were
bovine
with
Chelex
fibrinogen
with
or creatine
and
The
by counting
cry
0. At
tube
determined
opposed
ml bed
platelets
binding
ADP,
removed
microfuge.
volume)
platelets
thrombin-induced
into
time
conical
Beckman
followed
of the concentration
‘25I-fibrinogen
samples
After
a 500-al
in
(40
in Tyrode’s-albumin
added.
were
centrifugation
in detail.
1 mM
at
SOMI aliquots
restrict
containing
human
drawn
Thus,
human
fibrinogen
1-fibrinogen
125
‘25l-fibrinogen
The
platelets
blood
column
reported
with
excess
inhibited
Subsequently,
buffer
diluted
108/ml
in a tenfold
stirring.
2B-CL
washed
of
added
The
D) by differential
previously
the
study,
we have
in the binding
of
human
7.2, freed
of divalent
ions
of the
labeled
and unlabeled
have
the
METHODS
fresh
sepharose
in Tyrode’s
pH
characteristics
this
from
washed
and
apyrase
effect.
available.
within
induces
with
inhibited
without
Sigma
platelets.
AND
isolated
acid-citrate-dextrose
resides
thrombin
to thrombin-stimulated
Platelets
by gel
ADP
and
also
to washed
human
of molecules
bound
derived
with ADP-
However,
granules,
thrombin
to
release,
platelets.
stimuand the
of the
125l-fibrinogen
of ADP
markedly
was
to thrombin-stimulated
exhibit
of
removal
stimulation
binds directly
to platelets
not to unstimulated
cells,
for fibrinogen
of
system.3
fibrinogen
of fibrinogen
A. Marguerie
binding
platelets
thrombin
of Fibrinogen
Platelets
by 14C-serotonin
Enzymatic
is an essential
cofactor
for
of washed
human
platelets
ADP.’2
The molecule
lated with ADP but
acteristics
to
the
as measured
thrombin-stimulated
prior
and Gerard
supported
secretion,
concentration.
from
with
IBRINOGEN
aggregation
binding
(ADP)
Platelet
on thrombin
(CP/CPK)
platelets
F
diphosphate
affinity.
F. Plow
Binding
1 and
Immunology
requests
Clinic,
to
Grant
1852.
This
is
Departments.
Dr.
10666
& Stratton,
NA TO
N.
Edward
Torrey
F.
Pines
Plow,
Research
Road,
La Jolla,
Inc.
0006-4971/80/5603-0033$0I.0O/0
553
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
554
PLOW
lated
with
added
for
thrombin
to the
platelets
prior
stimulated
10 mm and
addition
of
were treated
excess
hirudin
‘251-fibrinogen.
in a similar
including
the addition
of hirudin.
As shown
both
thrombin
and ADP
supported
the
‘25I-fibrinogen
to platelets,
whereas
less
molecules
bound
to the unstimulated
cells.
stimuli,
one-half
attained
within
maximal
fibrinogen
The
fibrinogen
to
assessed
binding
to achieve
capacity
after
a 30-mm
of 251-fibrinogen
j.tM
rangeofO.1-0.7
in Fig. 1A,
binding
of
than
1000
With
both
saturable
plot
derived
linear
sites.
(r
The
lation
was
binding
platelets
incubation
to the cell
added,
‘25I-fibrinogen
from
=
0.998),
affinity
molecules
of the
same
this
data
(see insert
a single
was 2.0
suggesting
of binding
were
bound
platelet
of
was
6
II
C
0
0.
(Fig.
1B). The
was linear
in the
and satura-
tion was attained
at higher
concentrations
of a discrete
number
of binding
sites. The
37,000
was
binding
thrombin-stimulated
MARGUERIE
ADPmanner,
at 4.2 mm, and maximal
binding,
achieved
15 mm, did not change
during
the subsequent
30 mm.
AND
indicative
Scatchard
to Fig.
ThrOmbin (mUIml)
1B) was
class of binding
x 106M
‘, and
per platelet.
preparation
ADP
supported
stimu-
Fig.
2.
Relationship
‘2I-fibrinogen
binding
and
‘4Cwere measured
30 mm after addiM
final concentration.
Serotonin
to the radioactivity
within
nonstimwas greater
than 95% at all throm-
between
serotonin
release.
Parameters
tion of fibrinogen
at a 0.3
release
was measured
relative
ulated
platelets.
51Cr
recovery
bin concentrations.
the
of 28,000
lO6M’.
relationship
binding
of2.4x
The
and
molecules/platelet
between
‘4C-serotonin
of thrombin
two events
concentration.
were very closely
of
concentrations
‘
was
release
<0.25
with
an
251-fibrinogen
measured
binding
as a function
in Fig. 2, these
Thrombin,
at
As shown
correlated.
mU/mb,
affinity
failed
to
induce
fibrinogen
binding
or serotonin
release,
whereas
above
0.5 mU/mI,
fibrinogen
binding
and serotonin
release
occurred
in a parallel
fashion.
When
fibrinogen
binding was expressed
as a percent
of the maximum
binding
at 25 mU thrombin/mI
and correlated
with
the percent
ficient
of r
Since
exhibited
=
serotonin
release, a linear
0.993 was derived.
fibrinogen
an
binding
identical
and
correlation
the
release
reaction
on
thrombin
dependence
concentration,
the role of released
ADP
binding
of fibrinogen
to thrombin-stimulated
was considered.
CP/CPK,
were
and
both
Two enzymatic
utilized
to effect
markedly
inhibited
to thrombin-stimulated
ADP was added
to the
‘21.Fibrinogen
in supporting
platelets
systems,
apyrase
and
the removal
of ADP,
binding
of fibrinogen
(Table
1).
these enzymes
When
inhib-
ited fibrinogen
binding
by 98%;
and, with thrombinstimulated
platelets,
inhibition
exceeded
86%. Huang
Added IjaMI
Fig. 1 .
Binding of ‘‘1-fibrinogen
to thrombin-stimulated
lets. (A) Final concentration
of ‘25I-fibrinogen,
platelets.
and ADP were 0.3 gzM. 10/ml.
2.5 mU/mI.
and
tively.
(B) The
specific
binding
curve
shown
subtracting
the binding
observed
in a 100-fold
unlabeled
fibrinogen
from the total ‘251-fibrinogen
30-mm
incubation
with 10 platelets
stimulated
thrombin.
The insert is the Scatchard
plot of the
the
platelets
platelets,
coef-
platethrombin
12.5 MM, respecwas derived
by
M excess
of
binding
after a
with 2.5 mU/mI
data in Fig. 1 B.
and Detwiler9
have recently
ADP
in plasma
influences
reported
platelet
remove
ADP
such
nonsecreted
that nonsecreted
aggregation.
To
from
our
washed
human
platelets,
centrifugation
the cells were isolated
by differential
and incubated
with
CP/CPK
for
mm
gel
prior
pretreatment
to
filtration.
As
of the platelets
with
shown
CP/CPK
in
10
Table
I,
resulted
in
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
FIBRINOGEN
Table
BINDING
TO
555
PLATELETS
1 . Effect
of the Enzymatic
of Fibrinogen
to Thrombin
Removal
Removal
on the Binding
byt
Percent
of
the
‘251-Fibrinogen
0.0
None
Apyrase
676
97.9
ADP
CP/CPK
509
98.4
31,774
None
Thrombin
Apyrase
5.353
86.2
Thrombin
CP/CPK
4.776
87.7
Thrombin
Pretreated
37,877
2.1
0.0
at 2.5
(108/ml)
were
stimulated
with
12.5
pM
ADP
concentrations
mg/mI,
and
0.5
platelets
were
isolated
10 mm with
CP,
and CPK
respectively.
prior
were:
80
In pretreatment
by differential
CP/CPK
pM
0.3
of apyrase,
mg/mI.
centrifugation
ag/mI.
with
and
25.5
but
CP/CPK,
preincubated
for
to gel filtration.
‘25I-fibrinogen
was
added
and
binding
was
measured
after
30 mm.
§Calculated
enzymatic
to ADP
or thrombin-stimulated
platelets
without
treatment.
a
only
relative
2%
inhibition
of
fibrinogen
binding.
in the
platelets.
platelets
fibrinogen.
The
or
thrombin
with the inducreceptors
by
is apparently
present
granules
within
released
the capacity
of serotonin
by
of thrombin
to
and to support
also,
the
fibrinogen
binding
to thrombin-stimulated
by greater
than 86%. Furthermore,
the partiADP was not initially
accessible
to CP/CPK,
as a result
arose
of thrombin
bin releases
approximately
whereas
I M
exogenously
binding
of fibrin-
stimulation,
washed
exhibit
the
capacity
to
bind
kinetics
and
affinity
of
fibrinogen
with
thrombin
stimulation.
6 nM
added
support
fibrinogen
binding.
ness of released
ADP
could
concentration,
close proximity,
gism
Thus,
Throm-
ADP/l0t
platelets,8
ADP
is required
to
This
increased
effectivereflect
( 1 ) its high local
and/or
presentation
to
(2) its increased
platelets;
or (3)
released
or other
latter
possibility
is suggested
and epinephrine,
which
act
platelet
aggregation,
also
DISCUSSION
ADP
Following
consistent
fibrinogen
dense
the platelet
membrane;
with thrombin-stimulated
secreted
ADP
is implicated
ogen to thrombin-stimulated
human
ADP
serotonin-containing
inhibited
platelets
cipating
or thrombin
mU/mI.
tFinal
and ADP.
of
binding
of ‘251-fibrinogen
to thrombin-stimulated
platelets.
Thus,
apyrase
(which
converts
ADP
to
AMP)
and CP/CPK
(which
converts
ADP
to ATP)
CP/CPK
Platelets
similar,
class
fibrinogen
binding
very closely
correlated,
but
the enzymatic
removal
of ADP markedly
inhibited
Thrombin
38.690
very
same
thrombin.8
Not only was
induce the nonlytic
release
Bindings
ADP
were
the
thrombin
Inhibition
ADP
with
binding
tion of
Platelets
“5l-Fibrinogen
Bounds
(Molecules/
Platelet)
ADP
Stimulus’
of ADP
or ADP-Stimulated
reactivity
its syner-
constituents.
The
since low doses of ADP
synergistically
to induce
synergistically
support
fibrinogen
binding.’0
Any of these mechanisms
would
increase
the effective
concentration
of released
ADP
and explain
its capacity
to participate
in the binding
of
fibrinogen
to thrombin-stimulated
platelets.
125l
REFERENCES
I . Cross
MJ:
by adenosine
Effect
diphsophate.
2. Solum
NO,
of washed
diphosphate,
thrombin,
Invest 17(Suppl
DW,
tion.
4.
possess
Mustard
Regoeczi
Blood
5.
ogen
84):170,
with
ADP-induced
and
Haemorrh
of
platelets
I 2:324,
tors
1964
fibrinogen
induced
adrenaline.
6.
of platelets
on
the
7.
J Clin
bin-treated
Scand
Lab
Fibrinogen
and
Kinlough-Rathbone
RL,
ADP-induced
platelet
lism
Perry
aggrega-
Plow
and
EF,
Edgington
saturable
receptor
TS:
Human
specific
for
9.
platelets
fibrinogen.
Edgington
platelet
receptor
platelet
aggregation.
Huang
as
Plow
part
J Biol
EF:
of
Interaction
a multistep
Chem
255: 1 54,
10.
of fibrinreaction
1980
in
fibrinogen
54(Suppl
G: Exposure
5,
HJ,
EM,
Storm
ADP
recep-
1979
Prostacyclin
inhibits
ADP-
and
throm-
1980
E: Adenine
in
186:254,
Detwiler
S:
on human
Subcellular
functions
Acta
fibrinogen
63: 1 393,
Timmons
283:195,
VI
different
Invest
sites
Nature
Day
of platelet
J Clin
Parkinson
platelets.
ofsecreted
Med95:59,
TS,
H,
blood
Biophys
the role
1979
GA,
J,
platelets.
with
Biochim
Villaire
epinephrine.
of fibrinogen-binding
Holmsen
of
pools
1975
JS,
and
Hawiger
mobilization
8.
E:
GA,
Bennett
by ADP
by adenosine
1965
MA,
254:5357,
its
aggregation
Influence
blood
Packham
inducible
Marguerie
H:
human
collagen
52:453,
J Biol Chem
Diath
JF,
Marguerie
an
on the
Thromb
Stormorken
aggregation
3.
of fibrinogen
nucleotide
localization
the
platelet
metabo-
of
release
nucleotide
reaction.
1969
TC:
in biphasic
Reassessment
platelet
of the
evidence
aggregation.
for
J Lab
Clin
1980
Plow
EF,
Marguerie
receptor
l):257,
on
1979
G,
human
Edgington
platelets
TS:
by
Induction
epinephrine.
of
the
Blood
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1980 56: 553-555
Participation of ADP in the binding of fibrinogen to thrombin- stimulated
platelets
EF Plow and GA Marguerie
Updated information and services can be found at:
http://www.bloodjournal.org/content/56/3/553.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.