- Email: [email protected]
The single prostacyclin receptor of gel-filtered platelets provides a correlation with antiaggregatory potency of PGI2 mimics
THROMBOSIS RESEARCH 45; 645-659, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
THE SINGLE
PROSTACYCLIN
CORRELATION
Thomas
L.
Eggerman,
RECEPTOR OF GEL-FILTERED
WITH
ANTIAGGREGATORY
Cynthia
J.
Departments University of
POTENCY OF PGI2
Sara
HartzelI,
of Chemistry Washington,
PLATELETS
Selfe,
and
PROVIDES
MIMICS
Niels
H.
Andersen
and Pharmacology Seattle, WA 98195
(Received 15.4.1986; Accepted in revised form 12.9.1986 by Editor R.W. Colman)
ABSTRACT Gel-filtered
human
for
: KD=61nM,
[3H]-PG12
Platelet-rich
plasma
MD value
increases
lowers has
its
been
inhibition
for
PGD2.
El,
based
[ HI-PGI2/GFP basis
six
display
of
mimics, ICSos
PG12
greater
on PG12-binding
receptor
The
and
and
same
binding
of
density
but
PG12
which
binding
the
assay
aggregation three
and
PGE type
radioligand
analogs.
binding
affinity
and
PGD2,
antiaggregatory
site
site
sites/platelet).
binding
molecular
analogs
PGE2,
a single (1410
protei;
Antiaggregatory
6-oxo-PGEl,
expected
the to
concentration. the
only
platelets
displays nM due
evaluate
for
extent
123
iisplay
fmol/lO
prostacyclin
and
correlate
lesser than
to
(GFP)
234
(PRP) to
effective used
structures, ICsos
platelets
to
a
potency
data.
INTRODUCTION
Prostacyc
I i n (PG12),
prostaglandin of
platelet
hydrolytic are
usually
complicates The
a further
endoperoxide aggregation
(1,Z).
instability
at
not
determined
the
analysis
binding
of
product
intermediate, The
enol
physiological directly. of
[3H]-PG12
pH
binding
platelets,
Keywords: platelets, prostacyclin, receptor, relationships, aggregation inhibition. 645
arachidonic
the
ether
This
receptor to
of is
most unit
(3,,4,5),
acid
potent of
this
via
natural
the inhibitor
molecule
imparts
consequently
PG12
instability
also
hydrolytic
ievels
experiments. intact
(6)
and
structure-activity-
membrane
prepar-
A SINGLE PROSTACYCLIN
646
ations, For
neurona
human
from
is
of
adenylate presented
binding
of
antagon
i sts
of
[3H]-PGE, (16))
a single
binding
high
and
a probe
site
We now report
its
affinities
of
present
and
mimics 2 agents in
which
with
utilizing
via
studies
to
have
antiaggregatory employing
purportedly
selective
presented
(GFP)
demonstrates
that
PGE
site
that
which
a separate of
with
act
coupled
We recently
GFP binding
correlate
PRP and
each
literature,
characterization The
PGI
antiaggregatory
or
questions.
further
KD=
can
-
binding
earlier
platelets
cross-reactivities.
All
site:
(2,13,14)
correlate
(13,15),
is
MD varying
(9-12).
prostanoids
PG12/El
The
filtered
reported.
with
affinity
radioligand
agents.
many open gel
and
affinities
as
been
(9)
published
receptor
to
low
antiaggregatory
PGD
the
active
affinity
(6).
site
that
of
leaves
reported
been
has
49-2400/platelet
an additional
-
of
that
a series
[ HI-PG12
exists
None
have
sites/platelet
receptors
evidence for
report
accepted
distinct
(8)
from
45, No. 5
Vol.
membranes
sites
densities
2700-4095
cyclase.
potency
pulmonary
affinity
studies
generally
two
and
receptor
nM (9,12), It
one
(7)
high
nM and platelet
400-992
Is
platelets,
4.1-63
previous
I ccl
RECEPTOR
the
assays
their
a study
binding
[3Hf-PG12
provide
binding
relative
potencies
as
GFP aggregometry.
METHODS AND MATERIAL
Prostaglandins. Upjohn
PGEl, Cold
Co.
We I I come)
.
(NEN)
the
by
in
form
acid
the
-
DH-MP-12
saponified
were
PGE2 were
PG12
as
prepared
in
to
as
the
method PG12
laboratories
2B-CL
was obtained
from
the
the
(Burroughs
New England
Nuclear
was
described
PGD2,
these
gifts
Whittaker
6-0x0-PGFla
previously
PGBl,
as
N.
from
salt.
was obtained
according -
of
was obtained
HOE-892
analogs
obtained
was a gift
Ci/mM)
of
analog
PGs and
and
Na salt)
tetraethylammonium
hydrolysis
AG and
following
the (7-15
of
Thiaimino
Analogs.’ Hoechst
(as
9-[3H]-PG12
aqueous
The
6-oxo-PGE1
PGI2
prepared
(4).
methyl
provided
ester
with
Me ester,
the
sample.
PFB-PG12
by methods
from
and
previously
detailed
(4,17,18,19). Other
Materials.
Sepharose
by a mod i f ication(6) with
0.2%
Tyrodes-
of
(wt/vol)
the
sodium
5mM HEPES
(pH
7.35)
’
The
syntheses
PFB-PG12
previously; analogs
of
of
Tangen
Bovine
azide.
A adenosine-5’-diphosphate
and
method
was stored (ADP)
DH-MP-I2
from
(15-O-(5
from (20),
thrombin frozen
they
were
carried
as
described
out for
at
-2O’C
have
analogs
with in
at
prior
the ref.18.
been
in
prepared to
use.
in Grade
hydro-PG12)
detailed
corresponding
sa I i ne
throughout.
l)-13,14-di
not
“purified”
4’C
Davis),
was employed
i-methoxypent--l”--y
starting
and
stored
(Parke
Caibiochem
(17-C4FQ-18,19,20-trisnor-PGI2)
precisely
Pharmacia
and
PGF~u
vol.
Bovine were
albumin
and
reagent
Stock
case
All
solutions
of
in
(Na
into
vials
from
the
salt)
and
with
200
material
was
purity.
However, in
upon
previously
of
point
IC50([
of
IC,,
the
values
multiplied utilized
lower
set
after
purity -
were
addition
of3the
binding
of
ADP to
compared
[ HI-PG12
was
concentrations
from
scintillation
counting
manufacturer. dpmi3s to
experiments.
Using
the
26:l
Our
+9.85
previously +0.7
synthetic maximum *2.9.
3
-
reported(4)
were
based
PGI for For
values
-Na of uncertain 2 PG12(Na)/40mM aq.
the
purposes
-
on a smaller
of
purity NaOH
quantitation
from is
of
lots.
by
bioassay
the
the
and
inhibition
IC50
as for
the
nominal
was then
purity
actual
storage
(PG12 Na
determination
assumed
fractional the
net
final 50%
used
dpm’s
conditions
200-208
value
is
for
and
employed
nm,
AC = 13.8
reactions. at
of [3H] -
Ae205(max)
= +4.2-6.3,
small-scale
Aeo20 L
for as
using
determinations
now observed
-7O’C.
radiochemical
established
de 215_210(sh)
number
ampoules at
3
-
(vol/vol)
commercial
later
determined
estimate
CH3CN with
glass
85%
10 PM,
the
<3(*6)%
by aggregometry
at
The
to
stored
the
some
PG12
[ HI-PG12
experimental
in
and
and for
the
by the
placed
were
= glass
in
of
was determined
curves
curves3were
ratio
activity purity
the
equilibrated
argon,
unlabeled
Dose-response 90
the
to
(8~~~~
obtained
experiments
PG12 “purity”
studies,
by comparison
the
in
early
specific
listed
times
In
were
under
aliquoted
determined
revealed
as
ca
the
Reconstitution
a final
solution sealed
and as
solution
-2O’C.
as
in
Unlabeled
lyophilization
CD analysis
give
nominal
HI-PG12)
activity
this
9-[3H]-PG12.
Dose-response
salt)/
to
K2C03,
basic
(previously
revealed
receipt
;nd
ethanol
kept
K2C03
basis)
9-[3H]-PG12
bioassay
AOD at
(IC50)
specific
of
at
NaOH and
were
with
1 mg salt/ml)
acid
the
stored
assuming
later
concentration.
of
overnight)
solid
(ca
(free
thorough
storage.
absolute
described(6).
aggregation,
of
gas,
of
used
Consequently lot
chemicals
described(l7,21).
NaOH
PG12
and
4mM aq.
PL aliquots
a grain
(“Purity”)
each
other
esters
saturated
previously
40mM aq. pg of
argon
9 months
argon
as
After
with
after
EtOH:CH3CN. containing
Activity
All
Co
prostacyclin
ethanol
(CD)2spectrum
under
was diluted K2C03
100
= +11.2*1.0).
concentration
solid
Chemical
and
or
in
contain
sealed
PG12
solution
-2O’C
dichroic
Ac21o were
of
PG12
at
to
circular
+5.45*0.35,
original
Sigma
(absolute
was dissolved
each
ampoules
from
prostaglandins ethanol
prostacyclins)
PG12
loss
HEPES were
grade.
Solutions.
2-10mM
647
A SINGLE PROSTACYCLIN RECEPTOR
5
NG.
45,
The
CD
used.
This is equivalent to assuming that the purity deficit corresponds to hydrolytic and/or oxidative degradation. The assumption is viewed as a safe one since NEN determined nominal specific activity at the stage of3the Me ester and those values agreed with the specific activity of the [ HI-PGF2a employed as a starting material in the chemical synthesis of PG12.
=
A SINGLE PROSTACYCLIN RECEPTOR
648
3 [ l-l]-PGI
described
Samples
f’or
binding
Confirmation PGI2
70
rug of
and
PG12
Me,
900
2.11
pmoles
20
yL
with
dpm/pg
iodomethane
with
counts
15-O-Me-PG12 thus
described of
(4),
theory)
tracer
with
drawn
The
a measured
venipuncture
from
drug
3.22
(wt/vol)
aq.
3509
(15
centrifugation
min)
aggregometry was
employed
as
then
0.5
optical
min
60
prior
inhibitor
a dose
resulting
in
IC50
using
change rate
batch thrombin
from
of
the PRP.
gives
described
The
(GFP).
proteins (6),
test
by
a slight
of
as
tight
(10
or
for
[for of
controls
as
the
PRP data
and in
relative
Platelets filtration
modification
For
PG12 is
For
each ion
was also inverse
from
based
rat
DRCs obta
ios i ned
on ADP
potencies.
twenty
the
The
than
the
mL lots
(sepharose of
and
PG concentrat
anti-aggregatory
from
22’C)
challenge
I
6OOthe
ADP stimulated
are
cold
Table
after
challenges].
that
after
to (22)
were
(at
(rather
potencies
substance
min
and
from
prior
per
sec.
aggregation.
IC50. set
ware
aggregometry
thrombin
no
with
supernatant
90
buffer
100%
with 9:l
inhibitors
4.5
obtained
Blood,
milli-units
Aggregation
initial
Ci/mM.
(PRP).
the
(96%
the
*0.8
was mixed
was measured
OD 10-25
gel
normal
days,
was
ester
Thus,
Plasma
Opt i ca I Born
inhibitory
similar
Platelets plasma
taken in
Rich
76% of with
previously
11.9
plastic
addition
was
of
yL
by
traces as
dpm/pg.
500
indicated
prostacyclin
thrombin
was taken
was
966
and to
assayed
initial
-
remaining
10% ran
as
stirring) the
(DRC)
of
Gel-Filtered
previously
with
curve
for
or
TLC
air
density.
with
Aggregation values
a single
separated
rate
aggregation
stimulus
response
the (21).
(rate)
challenges;
the
final)
Net
37’C
50% aggregation
aggregations,
of
to
PM,
907
dpm/pg
brought
were
collected
ADP challenges]
change
the
an
(THE)
The
was
The
of
10
.
Andersen(21)
optical
[for
to
(OD)
inhibitor)
in
set
(warming
density
measured
(8-10
stimulus.
change
for
of
purity.
product
fossa
in
C3H] to
to
1275
additional of
preceding
stored
subjected
nominal
activity
PRP was
and
-
TLC. an
Platelet
the
of
esterification.
was added
product)
of
antecubital
citrate.
ADP
the
the
preincubated
a specific
the
method
using as
stimulus
sodium
by the
PL sample)
activity
with
lot
tett-ahydrofuran
by CD)
specific
during
Ci/mM)
remaining
(determined
Aggregometry,
ingestion
and and
the
was
product
radiopurity
of
pg
was 80*7X
and
of
minimum
611
Preparations by
history
crude
chromatography
radiopurity
Platelet
the CD,
(n=3).
mg dicyclohexano-18-crown-6
18% of
Me ester
a
after
determined 45
The
recovery),
instance,
above,
homogeneity
hr.
PGI2
one
12.6
19
(88%
afforded
ngl
l8*6X/year
preparation.
activity
bioassay
for
was
of
PL anhydrous
FL THF,
with
Me ester. Column
)76X.
for 200
(totaling
ran
60
activity
detailed
specific
confirmed
stirring
counting
applied
of
9 mos.
In
25.4
with
with
aliquots
scintillation the
as
of
stirred
corrected
treated
loss within
Activity.
(nominally
and
CD on an aliquot
and
used
by aggregometry
aliquot
was
THF
average
determination
PGI2(Na)
counting
were
+10X
spectroscopic A [3H]-PG12
*‘20
the
assays
[3H]-PGI, Specific
of
assaying
exact
above,
Vol. 45, No. 5
method
of
PRP were
2B-CL)
as
of
Tangen
(20).
The
GFP was
brought
(pH
7.35
12.0)
to
platelet
count
Ca-Tyrodes
to
at
binding
platelets/pL)
order 37’C).
in to
9.1
0.1-625
or
9.1
in
mM NaOH were
added
GFP giving
concentration
prepared
in
The
counted
the
amount
was
subtracted
the
method
assuming
other
prostaglandins
Buffer
A and
added
from
of
both
of
in
final
9.1
a one
at
PGI2
18009
samples
were
22’C).
The
aggregometry
nM.
at
4’C.
and
spun
17309
each
freeze
NaOH for
the
Lowry
VS.
10 min
thawed
2 hrs
method subtracted
at (24)
ambient using
using
and
ampoules, prostanoid
addition
of
treated.
esters,
In
these
reported
were
of
PG12
14
The
each
the
next
supernatant
data
DRC.
The
A 500-FL
sample
times
and
incubated
the
Protein standard. to
to
give
unlabeled to
yield
other at
by PRP of
PGI2
was
experiments.
by centrifu-
GFP and
content
and
a 100%
(standing
half-life
with
ice
The
min
duplicate
of
a
in
stimulus. 20
was
model.
establish
was prepared
4OC.
GFP value
of
was determined
frsom two
then
model
site
cooled
used
the
site
so as
the
squares
one
immediately
PM ADP as
control of
22’C
Using
affinity;
two
the
Aliquots
was
over
and
as
and
binding.
sum of
from
GFP at was
intervals
BSA as
PM unlabeled
least
were
supernatant
using
100%
was defined
density a
terminated,
binding
nspecificn
Conditions.
samples
in
5 minutes,
no cooperativity.
temperature.
the
of as
radioligand
similarly
8.3
GFP supernatant at
three
from
receptor
plot
Determination. for
Na salt
by the
for
of
regression
The
the
22’C
deternnine
One portion
at
a semi-log
at
determine
to
PG12 remaining
from
Protein
was
166
prepared
to
under
concentration
a final
Non-specific
to
Binding
added
added
down
another
prostacyclin
presence
values
GFP under
3 min
run (6).
the
sites
PRP aggregometry
X of
at
to
warming
10 PL samples.
count
and
calibrated
aq.
value
two
of
cooled
estimated
Platelet
were
or
in
for
DRC in
gation
(23)
mM NaOH were
control
then
sample
better,
concentration
spun
in
min
I.
dried
PG12
were
was employed 0.5
was
PG12,
and
analogs
were
by nonlinear
significantly
PG12
as
bound
Scatchard
PG12
followed
7.5
and
described
each
to
Prostacyclin
incubations
[ HI-PGI2
tubes
7.45
ca 2 nM.
analyzed
Stability
polystyrene
GFP
1 M NaOH was
above,
lyophilized
pH of
Table
a stock
Unlabeled
PL of
22’,
in
giving
mM-
1
for
thrombin
at
described
unlabeled
10
4 hr
with
PL of
and to
previously
min
mM NaOH from
of
of
as
PM EDTA,
preincubation
3.2
water,
adding
as
were
9.1
a final
competitive
and
prepared
10
HEPES
required
I y prepared
reported
Platelets.
0.3781
the
within
used
(4.5 not
nM [3H]-PG12.
solution
experiments
not
150
fresh were
conditions
pL with
used
A S-min
GFP are
to
5 mM HEPES,
GFP was
stimulus.
was 350
[3H]-PG12
PL of
data
which
with
A).
PL portions
Filtered
up to
about
the
using
Gel
made
1 mM Ca using: dilution
aggregometry
600
binding
PM solutions
10 PL of
750
to
brought
mM NaOH and
For
as
the
C3H]-PGI,
and
were
and
Further
(Buffer
in A)
simulate
Binding
PL of
vacuum
Buffer
pH 7.35,
CaCl2.
studies.
ADP challenges
[3H]-PGI, 200
aq.
pH 7.35
(200*50x103
in
O.lM
(50-250x103/pL)
radio!igand
to
5 mM HEPES,
and
buffer
(dissolved
649
A SINGLE PROSTACYCLIN RECEPTOR
Vol. 45, No. 5
20.8
supernatant FL
of
was assayed
The
GFP supernatant
platelet
protein
2.5 by
protein
content.
M
650
A SINGLE
PROSTACYCLIN
RECEPTOR
Vol.
45,
No.
RESULTS
As
previously
affords
Hemocytometry less
than
platelets min
reported
a GFP fraction reveals
0.1% in
of
at
at
5 min, occurs
competitor,
at of
instability in
degradation
product,
to
up to
PG12
in
this
In
the
(n=7)
A separate this
PM)
and
binding
nor
the
and
about
white
attributed
to
a half-life
antagonizes
PG12 the
18*7 PG12
inhibition
after
a 5
8.3@!,
final
specifically
bound
unlabeled occurs
with
a
hydrolytic
bioassay)
of
inhibits
the
(to
of
6 min
(by
constitute to
platelets
after
PRP
cells
[3H]-PG12
addition
determination
neither
blood of
90% of of
of
studies.
unlabeled
%-binding is
filtration
filtered
of
absence
of
pH revealed
6-oxo-PGFla, 8.3
red
of
the
decay
gel
binding
The
addition
dissociation
min
PG12.
GFP at
concentrations due
pH 7.45,
count.
by counting Upon
5 min.
21.8i4.1 of
stability
cell
pH 7.45.
in
2B-CL
radioligand
contaminating
total
a rapid
radioligand
half-life
that
the
sepharose for
GFP was assessed
incubation
cont.)
(6),
suitable
of
min.
PG12 The
binding of
(in
PRP aggregation
assay.
FIG. 1 Competitive [3H]-PG12 prostanoids.
Displacement to
GFP
in
the
Curves, presence
relative of
varying
percent
specific
concentrations
binding of
other
of
2 nY
5
A SINGLE PROSTACYCLIN
Vol. 45, No. 5
RECEPTOR
i-551
TABLE I A comparison
of
specified)
stimulus)
YS.
change)
-
for
donors
is
[n]
I&
binding
otherwise
values and
obtained
PRP (ADP stimulus, assessing
aggregation
various
using
in i tial
anti-
IC50
protocols aggregation
d saggregatory
and
data
-
(nM,
GFP
rate
Vs.
net
potencies.
PGI2
DH-MP-I2 Hoe-982
38
PGEl
920*90
6-oxo-PGEl
6pM Q1rM >8.3j~M 12,uM
6-oxo-PGFla
>16jbM
>lOOpMAA ~200j~M >>160/.d
CDLUMN
A
PG12
at
1 Ci/mM)
the In
sites/platelet. nM at PG12
zll and
Ci/mMole, other binding.
of
i nd i ng)
IC50(b
studies
in
affinity
values
present
was
employed
values
our
data
of
for
these
saturable,
reversible,
consistent
with
the
concentrations
displacement shown
present
as
column
work
(GFP
has
physiologic
and
appear
binding
(defined
a concentration effect
in
(6).
in
A of
li;30*180
of
[ HI-PGI2,
of
were
1.
The
Table
I. and
columns in
;and time
course
Non-specific
based
set
previous
and of
Table
Methods)
binding
on !&
complete
potency
detailed
2
unlabeled
aggregations)
other
by
(11-570nMr3H]-
curves Figure
relative
tihe as
obtained
nM with
varying
prostanoids
and
KD=45
with
(17,19,21,25-27)
[3H]-PG12
were
concentration
appears
the
(n=4)
D
curves
a fixed
are
62@16] -
C
study
these
obtained from
laboratories
specific
were
>>2pM 54*5[9]
>7O/rM
saturation
Competitive
A number
IC50
data The
resulting
16pM
1lOpM[4]
[3]
characteristics
binding
1.4pM[2]
78+20[5]
-
receptor
the
prostanoids.
specific
Aggregometry
(6)
w 621 150[3]
5/JM 116pM -
-
radioligand
22
1.7pM
B
study of
42PI 1.55jbM
180@
PGBl
64 [2]
194+28[8] 115*13[11]
>8. ~/LM [3]
PGE2
initial
110
-
(~20j~M)
PGD2
analysis
3.2[2]
-
17-C4Fg-PG12
our
4.1
-
-
Hoe-982 (Me ester)
In
4.9+0.4[11]
-
PGI2 (Me ester)
Scatchard
7.0+1.0[7]
-
2.25jLM
15-O+e-PG12
A-9O
-
310 [3]
[3]
s 5O--
rate
5 [31
700
(Na)
of
PRP
AOJ
PI
number
IC
GFP
66+6 [6]
(Na)
OD
The
Aggregometric IC50 3 [JH]-I2 Binding
unless
(thrombin
I.
is
dependence constitutes
652
A SINGLE PROSTACYCLIN RECEPTOR
3.0
KD= R,
z
nM
= 234
r=
: * ‘, 7; ;i;
61.0
Vol. 45, No. 5
fmof/108platelets
-0.997
2.0
?i “0 7 g
50
100
BOUND
Ifmol
’ it,=
150
PGl,/lOS
190
PlateletSi
fmof/l08
PlateletS
. .**. . .
I R:=185
fmovio8
~ r=-.945
IdO
5b BOUND
(fmol
PGI,/IOB
FIG. A -
Scatchard
830
nM unlabeled)
B -
Comparative
The
stippled
early the in
lots specific
B were
purity.
analysis
of
to
GFP,
Analysis area
of
shows
[3H]-PG12 GFP controls
performed
prior
competitive 5 min of
the -
at
GFP,
PGI2 22’C.
PRP,
binding All
and
87*9
for
this
to
routine
nM,
limits 212*18
experiment bioassay
(2.19
points
GFP+PPP
90% confidence
KD=
Platelets)
2
are (GFPP)
for
+O.ll mean
(n=2).
*SEM
Binding
The Most of
of
(n=Z).
(n=4)
solid the
+ 5-
(n=20).
Data
GFP’binding
fmol/lO*.
estimation
nM tracer
dots
using show
experiments
radioligand
Vol.
45,
at
No.
most
For
15% of
samples
binding PGI2
A SINGLE
5
the
of
is
li;early
unlabeled
PGI with
This
in
high
type
previous
controls
with
(n>20)
the
competition
PG12
with
similar
to
45.2nM)
(6),
those
Since site
our
by all
PRP
(9)
effects
and
GFP resuspended
of
gel
significantly
(123+24
show
nM,
filtration
or
3,
act has
analog
Scatchard
via not
of
all
one-site)
a KD values
(1130
the
binding
analysis
(r-=-0.997, and
[ H]-
by 8.3@
affinities that
each
linear
to
display
previous
(61.0nM)
sites/platelet,
sites,
The
is
[3H]-PG12
results
appear
Figure
as
fmole/108
212+18
(112+12
indicating
205*57
lower
nM).
The
than
GFP+PPP
no significant
PRP,
the
The
are
(n=4)
and
GFP,
using
2B.
those
and
determine
compared
platelets,
; PRP,
(n=4)
to
b i nd i ng to was
in
binding
(10-12)
controls
(GFP+PPP)
significantly
GFP+PPP
matched
PG12
fractions
we performed
expressed
nM)
a single
membrane
procedure.
GFP,
(6719
<.OOl)
characteristics
all
two
densities,
GFP
relative
Figure
In
curves
platelet-poor-plasma
different:
MD va I ue for
continued
technique.
receptor
and
run. a
saturation
filtration
in
binding
I
to
displaced
substances
sites/platelet)
the
while
to
the
also gave
not
;f
2A.
criteria,
the
observed
(1410
from
Figure
reported
competitive
binding.
GFP preparation
our
are
Table
were
is
and
those
employed. component
content,
that
selectivity for
[3H]-PG12 PGI
conditions
1. HI-PG
correlation,
densities obtained
see
total
component
experizents
653
the3non-specific
of
unlabeled
receptor
to
potency of
incubation
binding
structural
studies
study,
graph
the
anti-aggregatory
receptor.
PGI2
the
RECEPTOR
purity,
(rz0.995)
contrast,
displays
2
under
radiochemical
related In
correlate
binding
reduced
content.
appeared
total
PROSTACYCLIN
not
However, for
the
PRP
PRP binding
alteration
due
to
over
platelet
the
gel
be
used
procedure.
DISCUSSION Gel
filt&ed
preparations PRP, for
platelets
which
GFP retain
have
aggregometry
with
that
fi
Plasma
Itration.
by gel
prostaglandins 534 to
degree
4
of
is
binding
Commercially
protein
in
as
78
15 min could
available
the
removal thus
eliminated. that
stimulus.
room
is
essentially
a/.
(28)
Et4N+
purity. and PG12
have
At
This
salt
site
factor,
lower
Like
(Figure
in
PG12 that
our
and
with
radioiigand
other 67
is
concentrations undoubtedly,
preparations
2
by gel
()99.9X for
shown
the
can
study
altered
complete
of
trauma. and
present not
binding
temperature.
Q-[3H]-PG12
vary significantly in radiochemical activity was assumed (see note 3) aggregometric assay.
(20)
are
respectively,
greater.
other physical
The
density
et
severe
integrity
a competing
52X,
be even
more
and
Pifer and at
advantages to
functional
function
filtration,
nM [3H]-PG12, PPP proteins
and
thrombin
receptor
some
subjected
morphological
demonstrates
studies)
offer been
were
and
bound the
provides
found
Therefore, nominal specific content (*lo%) was assessed
to by an
654
A SINGLE PROSTACYCLIN RECEPTOR
the
explanation
for
determinations)
the
lower
observed
with
7 determinations), competing the
KB.
The
hydrolytic
determination
and
unlabeled
been
PGI,
in
t
quite
may not
reflect
association
total
100~
that
as
this
curves
to
suggest
study
that
of
(30) Siegl
we report
for
GFP.
quantitating
of
actual
additional
IC5G
analogs
and labeled have
bioassay to workers very
be approached values,
display
however,
different
the
rather
populations, apparent have
KB.
Scatchard
to
an
plot
alkaline
of
in
latter
measured
PRP provides
in
on
free
such
total
The
platelet sites
sites. resulting the
Wynalda
presence
of
(equated
and
of with
bound
increase
the
Fitzpatrick
stabilization albumin
the
discrepancy.
receptor will
-
those
concentration
Iigand
In
site
that
the
free
added
and
at
of
same.
effective
PRP.
technique
matching
we conclude
the
1.6pM
a single
cause
the
figured
and
counting found
of
criteria.
nM in
is
not
study excess
the
nM (PG12)
KB =123+24
also
to
Siegl
on our
a direct
binding
microenvironment
the
characteristics
the
the
140
and
(more
equivalent
component,
of
our
ways:
course
indicate based
two
or
comparison
based
additional
sequestration;
of
group
presumably
was
to
paper
platelets
Thomae
time
is
GFP at
using
in
period,
level
with
is
different
binding
PRP decreases
separately
addition
Albumin
the
plots
result
reference that
va I ues
radioligand
PRP
in
Scatchard
observation
washed
on this
GFP and sites
demonstrated
IC50
al.,
bound
than the
et
PRP and
by others
With in
our
using
binding
to
a
non-specific
with
site
incubation
radioligand
binding
a two extended
could
reported it.
4-5nM
noted
with
minute
sites/platelet)
Based
KB in binding
Since
attributed
Analog
linear
illustrated
presumably
method
(29)
by
are
should
of
storage
previous
values
study
occurs
we defined
at
nM (837*68
B + F),
for
of
platelet
of
storage
approach
with
the
difference
binding
consistent
unpublished
PG12.
the
strictly
a five
curves binding
, entirely
The
at
has the
This
affinity
KB = 56.7+3.5
5
of
34
nM,
estimate
confirmed
agreement
if
(12)
occupancy with
competition
similar
been
n#,
absence
sample
a year
question
period.
(with
binding.
low
subtracts
study find
concentrations)
site”
the
competition
an
has
equilibrium
affinities
to
specific
non-specific
(PGE2)
up to
displacement
incubation
another
binding
the
one
in
in
the
better
conditions
for
the
we find,
relative
non-detectable
non-specific
If
Storage
to
in
the
problems
loss/yr)
that
GFP,
data.
(
and
in
presumably
poses
suitable
suggesting
affinity
thus
20X
PG12
Turning
a 5 min
value is
are
studies,
yet
PG12 of
likely)
that
association
we continue
definition
(9),
binding
true
though
phenomenon,
that
of
(tota I range 32-105 PRP or GFP+PPP (93-190
kinetics.
Even
higher
instability
stability
<2 min, using
l/2 closely
than
observed
equilibrium
binding
PG12
KD value
sites’,
spectroscopy.
that
rapid,
The
binding
samples
and
chiroptical
equilibrium
was
of
developed,
(9,10),
28.
protein
actual
in
and
Figure
plasma
apparent GFP rather
Vol. 45, No. 5
binding
of site.
PG12
is
We also means
conclude for
that
evaluating
aggregation
the the
inhibiting
obtained
and
tested.
various
the
initial
present site
GFP
of
of
of
binding
synthetic
Table
of
assays rate
[3H]-PG12
action
properties. measures
Aggregometric
DD and
555
A SINGLE PROSTACYCLIN FlECEPTOR
Vol. 45, No. 5
I
collects
antiaggregatory
employed
change
in
assay
the
potency
endypoints turbidity
can
prostanoids
based due
to
as
binding
IC56
for
substances
the
on both
the
serve
a
with
net
data
change
in
aggregatory
PGE2a
100
,/
10
1%0-Me-PG13
/I ,/
t
t-HOE-982
Me ester
1.0
PGE, --1 \
II a 1
PPGI,
Me ester
A
/
z? & .lO 0 Q
PGDZ
?? ?? 6-0x0-El
5: 0 .Ol
IC60
I binding1
3
FIG. Graphical log-log columns co1.D ones
Correlations plot
of
A and data.
for
regression
I&G
C of Round
other
of
Receptor
Table symbols
I
(sol
id
are
employed
prostanoids
analysis
since
Affinity
w>and I&,
(bind
it
and
(w
symbols), for
17-C4Fg-PGI2 displays
py
Antiaggregatory
re,p a t ion) r =0.996. PGI2
(A)
and
was
some antagonist
Potency.
employing Open
symbols
bicyclic
excluded
data
employ
analogs; from
properties
A
from
the (26).
square
656
A SINGLE
challenge
(21).
correlation
with
Log-log used
to
assay through
is
aff
data
event
and
symbols,
a “spare
10% occupancy
to
reduced and
reduced
under the
3)
clearly
NIH
is
were GFP
known
to
act
6-oxo-PGEl based
linear
on
correlation
analog
DH-MP-12,
antiaggregatory
(25),
value
only
if
occupancy may
reflect
work
ROl-HL-23103, the
technical
Pharmaceutics
Manufacturers
occupancy
at
was supported
Some of
(NOl-HV-8-2933)
receptor
contain for
a
the
effect, under
by U.S.
the
6.
De and
PG12
support. Association.
can
the
the
esters,
time and
PGI2
calculate
rapid
mimics that
on-rate,
PGE2,
correspond In
a would
and
particularly
the
PGE2-receptors
to:
PGEl
alternative (KD=0.5-20
nM,
proaggregation study.
National
PDl-HL-22163). of
for
response).
presently
-GM-26935;
(ROl-HL-32827). contract
and
response
as
binding
one
PG12,
pharmacological
assistance
in aggregatory
with
observed
PGEl,
a full
are
indicate
initial
better
This,
expressed
interactions
This
For
a rapid
for
the
particularly
concentrations.
an opposing
of
I)
reduction
(0.927).
response.
requiring
(Table
magnitude
AOD 90-set values
on
phenomenon.
agonist
of
ratios
ful6
which
These
grants
US PHS
and
from
correlates
based
r2
a system low
(c80%
have
Acknowledgments.
acknowledge
is
be predicted
which
inhibition
disaggregation
the
aefficacy”
(5,25,27,31).
the
of
efficacy, PGE2
which
Health
Figure
receptor”
in
responsiveness
ref.6)
>
potencies
order
cases,
potencies
produces
be advantageous
the
these
two
IC50(binding)/IC50(aggregation)
indicate
PGE2
values by our
which
outlier would
range:
inhibition
a ca.
a significantly’lower
The
%30%),
In
component
gave
PC50
essentiaily
magnitude to
No.
best
determined PGD2
than The
PG12,
the
45,
3).
a distinct
site.
aggregation
produces
Aggregation
significant
show
natural
gave
affinities
is
of
Vol.
antiaggregatory
potency
PG12
than
potency.
(filled
affinity.
and
the
data
response.
higher
order
and
esterification in i ty
Figure
between
a five
potent
rate
versus
(2,13,14,27)
for
RECEPTOR
PM range.
Binding that
(see IC50
pharmacological
affinity
more
100
initial
correlation
receptor
nearly
the
affinity
significantly
over
the
that
binding
presumed
measured
which
of the
another
extends
in
the
displays
its
binding
plots
determine
and
also
We found
PROSTACYCLIN
Institutes
of
We gratefully the
continuing
analogs
employed
TLE
supported
was
support were
of
prepared
by grants
from
5
Vol.
45,
No.
5
A SINGLE
PROSTACYCLIN
657
RECEPTOR
REFERENCES
1.
GRYGLEWSKI, R., BUNTING, S., and VANE, J.R. An Enzyme MONCADA, S., Isolated from Arteries Transforms Prostaglandin Endoperoxides to an Unstable Substance that Inhibits Platelet Aggregation. Nature (fond.) 262, 663-665, 1976; GRYGLEWSKI, R.J., BUNTING, S., MONCADA, S., and VANE, J.R. Arterial Walls are Protected Against FLOWER, R.J., Deposition of Platelet Thrombi by a Substance (Prostaglandin X) which They Make from Prostaglandin Endoperoxides. Prostaglandins 12, 685-713, 1976; JOHNSON, R.A., MORTON, D.R., KINNER, J.H., GORMAN, R.R., MCGUIRE, WHITTAKER, N., BUNTING, S., SALMON, J.A., MONCADA, S., J.R., SUN, F.F., and VANE, J.R. The Chemical Characterization of Prostaglandin X (Prostacyc I in) . Prostaglandins 12, 915-928, 1976.
2.
WHITTLE, Prostacyc
B J . R ., MONCADA, I in
(PGI2)
S., and Prostaglandins
VANE, 16,
3.
CHO, M.J. and ALLEN, M.A. Chemical Aqueous Solutions. Prostaglandins
4.
DE, B., ANDERSEN, N.H., IPPOLITO, R.M., Synthesis and Chiroptical Characterization Prostaglandins 19, 221-247, 1980.
5.
J.R. Comparison 37:3-388, 1978.
Stability 15, 943-954,
of
of
the
Prostacyclin 1978.
Effects
(PGI2)
WILSON, C.H., and of Prostacyclin
Platelet
6.
EGGERMAN, T.L., ANDERSEN, N.H., and ROBERTSON, R.P. Separate and Distinguishable Binding Sites for PGI2 and PGE on Human Gel-Filtered Platelets. J. Pharmacol. Exper. Therapeutics, 1986. 5 36, 568-573,
7.
MACDERMOT, J. and BLAIR, I.A. Prostacyclin Receptors of Cell Line: Divalent Cations and Ligand-Receptor Coupling. 30, 2041-2044, 1981.
8.
MACDERMOT, J., J.A. Prostacyclin Eur. J. Pharm.
9.
SIEGL, Selective J. C/in.
10.
SCHILLINGER, Fraction of 1980.
a Neuronal Biochem.
BARNES, P.J., WADDELL ,K.A., DOLLERY, C.T., and Binding to Guinea Pig Pulmonary Membranes. 75, 127-130, 1981.
SMITH,
Binding Invest.
J.B.,
SILBER,
M.J.,
Site for [3H]Prostacyclin 63, 215-220, 1981.
E. and Platelets
PRIOR, G. Prostaglandin of Various Species.
J.L., MACDERMOT, on Human Platelets.
J.,
NICOLAOU,
K.C.,
in
JOHNSON, W.D. Diastereomers.
ANDERSEN, N.H., WILSON, C.H., DE, B., TYNAN, S.S., WATKINS, J., CALLIS, J.B., GIANELLI, M.L., HARKER, L.A., HANSON, S.R., and EGGERMAN, T.L. Analytical Methodology in the Cardiovascular and Areas. Atheroscler. Rev. 8, l-28, 1981.
A.M.,
of
and
Hybrid Pharm.
BLAIR,
AHERN,
D.
on Platelets.
12
Receptors
Biochem.
in
Pharm.
BLAIR, I.A., and LEWIS, C/in. Sci. 61, 29P, 1981.
P.J.
a Particulate
29,
2297-2299,
11.
SHEPHERD, Receptors
Prostacyclin
12.
LOMBROSO, M., NICOSIA, S., PAOLETTI, R., WHITTLE, B.J.R., MONCADA, S., and VANE, J.R. The Use of Stabler Prostaglandin to Investigate Prostacyclin (PGI2)-Binding Sites and PGI2-Sensitive Adenylate Cyclase Prostaglandins, 27, 321-333, 1984. Human PI ate I et Membranes.
in
A SINGLE
658
13.
SHAFER, Platelet
PROSTACYCL
A.I., COOPER, B., O’HARA, D Receptors for Prostaglandin’12
RECEPTOU
and HARDIN, R.I. Identification and D2. J. Biol. Chem. 254,
of
2914-2917, 1979. 14.
MILLER, O.W. D Receptors
and GORMAN, R.R. in Human Platelets.
Evidence for Distinct J. Pharmac. fxp.
Prostaglandin I Ther. 210, 134-148,
and
1379. 15.
MACDONALD, J.W.D. and STUART, R.K. in Regulation of Cyclic-AMP and E2 Evidence for a Common Prostaglandin
Interaction Aggregation Receptor.
of Prostaglandins in Human Platelets: J. Lab. C/in. Med.
El
and
84,
111-121, 1974. 16.
MACINTYRE, D.E. and GORDON, J.L. Discrimination between Platelet Prostaglandin Receptors with a Specific Antagonist of Bis-enoic Prostaglandins. Thrombosis Res. 21, 705-713, 1977.
17.
ANDERSEN, N.H., and RAO, CH.V. Prostaglandins
16.
ANDERSEN, N.H., Bi s-unsaturated
19.
SUBRAMANIAN, N., DE, B., CCRAE, D.A., Methyl Ethers of Prostaglandin F2a and and Medicine 6, 345-357, 1981. and IMAMOTO S., I and i ns. Prostag
PICKER, D.H. Prostaglandins,
Synthesis
TYNAN, 12.
of
S.S.,
Diastereomeric
14, 61-101, 1977.
IMAMOTO, S., SUBRAMANIAN, Iv., PICKER, D.H., LADNER, D.W., ANDERSEN, N.H.) EGGERMAN, T.L., HARKER, L.A., TYNAN, S.S., ROBERTSON, R.P., DE, B., OIEN, H.G., RAO, CH.V., LINDBERG, M., and NAQVI, R. Molecular Basis for Prostaglandin Potency. III. Tests of the Significance of the ‘Hairpin Conformation’ in Biorecognition Phenomena. Prostaglandins 22, 841-856,
1981. 20.
BERMAN, H.J. TANGEN, O., for Separation of Blood
I and MARFEY, Platelets
from
P.
Gel
Filtration:
Plasma.
a New Technique
Thrombosis,
30,
268-278,
1971. 21.
ANDERSEN, N.H., EGGERMAN, T.L., HARKER, L.A., WILSON, C.H., On the Multiplicity of Platelet Prostaglandin Receptors. I. Competitive Antagonism by Aggregometry. Prostaglandins, 19, 1980.
22.
BORN, G.V. its Reversa
23.
SCATCHARD, G. The Attractions of Proteins Ann. N.Y. Acad. SC;. 51, 660-672, 1949.
24.
LOWRY, O.H., Measurement
25.
Aggregation I Nature,
WILSON, C.H., Aggregometric Prostaglandins
of Blood Platelets by Adenosine 1962. 194, 927-929,
ROSEBROUGH, N.J., with Foiin Reagent.
for
FARR, A.L., and J. Biol. Chem.
Small
and DE, 8. Evaluation 711-735,
Diphosphate
Molecules
RANDALL, R.J. 193, 265-275,
and ANDERSEN, N.H. A Single Platelet TYNAN, S.S., and Antiaggregatory Prostanoids. Assay for ProLeukotrienes and Med. 9, 415-427, 1982.
and
and
Ions.
Protein
1951.
of
Vol.
26
45, No. 5
HARKER,
A
L.A.,
659
SINGLE PROSTACYCLIN IRECEPTOR
HANSON,
S .R.,
ANDERSEN,
N .H.,
WILLS,
M .T.,
DE,
B.,
LIN
MCCRAE, D.A., and WILSON, C.H. Vascular and Platelet Activities B-S., Prostacyclin and PGD Analogs in the Non-sedated Baboon Vth Internatl. Prostaglandin Conf. z bstr . Book (Florence, Italy) p.309, 1982.
27
28
TYNAN, On the N-0164
S.S., ANDERSEN, Multiplicity of for Distinguishing
Hydanto
i n Ana
N.H., WILLS M.T., HARKER L.A., and HANSON S.R. Platelet Prostaglandin Receptors. II. The Use the Loci of Action for PGI2, and PGE2, and
Iogs . Prostaglandins,
PIFER, D.D., CAGEN, L.M., and in Human Blood. Prostaglandins,
CHESNEY, C.M. 21, 165-175,
Stability
of
Prostaglandin
30
WEISENBERGER, the Xth Winter
31
KLDEZE, J. Influence of Prostaglandins on Platelet Adhesiveness latelet Aggregation., in Nobel Symposium 2, Prostaglandins, S. and B. Samuelsson (Eds.) New York, Interscience, 1967, p.241-252.
32
MILLER, D.V., prostaglandin Antiaggregatory
F. Albumin 1980.
Stabilizes
H’. (Thomae GmbH, . Biochemie) . Prostaglandin Conf. (l/83).
12
1981.
WYNALDA, M. Prostaglandins,
FITZPATRICK, 20, 853-861,
of
1984.
Z??, 683-693,
29
and
of
Persona
Prostaglandin
I2.
I communication
at
and Bergstrom
AIKEN, J.W., SHEBUSKI, R.J., and GORMAN, R.R. 6-KetoEl is not Equipotent to Prostacyclin (PGI ) as an Agent. Prostaglandins, 2U, 391-396, l98?.
THE SINGLE
PROSTACYCLIN
CORRELATION
Thomas
L.
Eggerman,
RECEPTOR OF GEL-FILTERED
WITH
ANTIAGGREGATORY
Cynthia
J.
Departments University of
POTENCY OF PGI2
Sara
HartzelI,
of Chemistry Washington,
PLATELETS
Selfe,
and
PROVIDES
MIMICS
Niels
H.
Andersen
and Pharmacology Seattle, WA 98195
(Received 15.4.1986; Accepted in revised form 12.9.1986 by Editor R.W. Colman)
ABSTRACT Gel-filtered
human
for
: KD=61nM,
[3H]-PG12
Platelet-rich
plasma
MD value
increases
lowers has
its
been
inhibition
for
PGD2.
El,
based
[ HI-PGI2/GFP basis
six
display
of
mimics, ICSos
PG12
greater
on PG12-binding
receptor
The
and
and
same
binding
of
density
but
PG12
which
binding
the
assay
aggregation three
and
PGE type
radioligand
analogs.
binding
affinity
and
PGD2,
antiaggregatory
site
site
sites/platelet).
binding
molecular
analogs
PGE2,
a single (1410
protei;
Antiaggregatory
6-oxo-PGEl,
expected
the to
concentration. the
only
platelets
displays nM due
evaluate
for
extent
123
iisplay
fmol/lO
prostacyclin
and
correlate
lesser than
to
(GFP)
234
(PRP) to
effective used
structures, ICsos
platelets
to
a
potency
data.
INTRODUCTION
Prostacyc
I i n (PG12),
prostaglandin of
platelet
hydrolytic are
usually
complicates The
a further
endoperoxide aggregation
(1,Z).
instability
at
not
determined
the
analysis
binding
of
product
intermediate, The
enol
physiological directly. of
[3H]-PG12
pH
binding
platelets,
Keywords: platelets, prostacyclin, receptor, relationships, aggregation inhibition. 645
arachidonic
the
ether
This
receptor to
of is
most unit
(3,,4,5),
acid
potent of
this
via
natural
the inhibitor
molecule
imparts
consequently
PG12
instability
also
hydrolytic
ievels
experiments. intact
(6)
and
structure-activity-
membrane
prepar-
A SINGLE PROSTACYCLIN
646
ations, For
neurona
human
from
is
of
adenylate presented
binding
of
antagon
i sts
of
[3H]-PGE, (16))
a single
binding
high
and
a probe
site
We now report
its
affinities
of
present
and
mimics 2 agents in
which
with
utilizing
via
studies
to
have
antiaggregatory employing
purportedly
selective
presented
(GFP)
demonstrates
that
PGE
site
that
which
a separate of
with
act
coupled
We recently
GFP binding
correlate
PRP and
each
literature,
characterization The
PGI
antiaggregatory
or
questions.
further
KD=
can
-
binding
earlier
platelets
cross-reactivities.
All
site:
(2,13,14)
correlate
(13,15),
is
MD varying
(9-12).
prostanoids
PG12/El
The
filtered
reported.
with
affinity
radioligand
agents.
many open gel
and
affinities
as
been
(9)
published
receptor
to
low
antiaggregatory
PGD
the
active
affinity
(6).
site
that
of
leaves
reported
been
has
49-2400/platelet
an additional
-
of
that
a series
[ HI-PG12
exists
None
have
sites/platelet
receptors
evidence for
report
accepted
distinct
(8)
from
45, No. 5
Vol.
membranes
sites
densities
2700-4095
cyclase.
potency
pulmonary
affinity
studies
generally
two
and
receptor
nM (9,12), It
one
(7)
high
nM and platelet
400-992
Is
platelets,
4.1-63
previous
I ccl
RECEPTOR
the
assays
their
a study
binding
[3Hf-PG12
provide
binding
relative
potencies
as
GFP aggregometry.
METHODS AND MATERIAL
Prostaglandins. Upjohn
PGEl, Cold
Co.
We I I come)
.
(NEN)
the
by
in
form
acid
the
-
DH-MP-12
saponified
were
PGE2 were
PG12
as
prepared
in
to
as
the
method PG12
laboratories
2B-CL
was obtained
from
the
the
(Burroughs
New England
Nuclear
was
described
PGD2,
these
gifts
Whittaker
6-0x0-PGFla
previously
PGBl,
as
N.
from
salt.
was obtained
according -
of
was obtained
HOE-892
analogs
obtained
was a gift
Ci/mM)
of
analog
PGs and
and
Na salt)
tetraethylammonium
hydrolysis
AG and
following
the (7-15
of
Thiaimino
Analogs.’ Hoechst
(as
9-[3H]-PG12
aqueous
The
6-oxo-PGE1
PGI2
prepared
(4).
methyl
provided
ester
with
Me ester,
the
sample.
PFB-PG12
by methods
from
and
previously
detailed
(4,17,18,19). Other
Materials.
Sepharose
by a mod i f ication(6) with
0.2%
Tyrodes-
of
(wt/vol)
the
sodium
5mM HEPES
(pH
7.35)
’
The
syntheses
PFB-PG12
previously; analogs
of
of
Tangen
Bovine
azide.
A adenosine-5’-diphosphate
and
method
was stored (ADP)
DH-MP-I2
from
(15-O-(5
from (20),
thrombin frozen
they
were
carried
as
described
out for
at
-2O’C
have
analogs
with in
at
prior
the ref.18.
been
in
prepared to
use.
in Grade
hydro-PG12)
detailed
corresponding
sa I i ne
throughout.
l)-13,14-di
not
“purified”
4’C
Davis),
was employed
i-methoxypent--l”--y
starting
and
stored
(Parke
Caibiochem
(17-C4FQ-18,19,20-trisnor-PGI2)
precisely
Pharmacia
and
PGF~u
vol.
Bovine were
albumin
and
reagent
Stock
case
All
solutions
of
in
(Na
into
vials
from
the
salt)
and
with
200
material
was
purity.
However, in
upon
previously
of
point
IC50([
of
IC,,
the
values
multiplied utilized
lower
set
after
purity -
were
addition
of3the
binding
of
ADP to
compared
[ HI-PG12
was
concentrations
from
scintillation
counting
manufacturer. dpmi3s to
experiments.
Using
the
26:l
Our
+9.85
previously +0.7
synthetic maximum *2.9.
3
-
reported(4)
were
based
PGI for For
values
-Na of uncertain 2 PG12(Na)/40mM aq.
the
purposes
-
on a smaller
of
purity NaOH
quantitation
from is
of
lots.
by
bioassay
the
the
and
inhibition
IC50
as for
the
nominal
was then
purity
actual
storage
(PG12 Na
determination
assumed
fractional the
net
final 50%
used
dpm’s
conditions
200-208
value
is
for
and
employed
nm,
AC = 13.8
reactions. at
of [3H] -
Ae205(max)
= +4.2-6.3,
small-scale
Aeo20 L
for as
using
determinations
now observed
-7O’C.
radiochemical
established
de 215_210(sh)
number
ampoules at
3
-
(vol/vol)
commercial
later
determined
estimate
CH3CN with
glass
85%
10 PM,
the
<3(*6)%
by aggregometry
at
The
to
stored
the
some
PG12
[ HI-PG12
experimental
in
and
and for
the
by the
placed
were
= glass
in
of
was determined
curves
curves3were
ratio
activity purity
the
equilibrated
argon,
unlabeled
Dose-response 90
the
to
(8~~~~
obtained
experiments
PG12 “purity”
studies,
by comparison
the
in
early
specific
listed
times
In
were
under
aliquoted
determined
revealed
as
ca
the
Reconstitution
a final
solution sealed
and as
solution
-2O’C.
as
in
Unlabeled
lyophilization
CD analysis
give
nominal
HI-PG12)
activity
this
9-[3H]-PG12.
Dose-response
salt)/
to
K2C03,
basic
(previously
revealed
receipt
;nd
ethanol
kept
K2C03
basis)
9-[3H]-PG12
bioassay
AOD at
(IC50)
specific
of
at
NaOH and
were
with
1 mg salt/ml)
acid
the
stored
assuming
later
concentration.
of
overnight)
solid
(ca
(free
thorough
storage.
absolute
described(6).
aggregation,
of
gas,
of
used
Consequently lot
chemicals
described(l7,21).
NaOH
PG12
and
4mM aq.
PL aliquots
a grain
(“Purity”)
each
other
esters
saturated
previously
40mM aq. pg of
argon
9 months
argon
as
After
with
after
EtOH:CH3CN. containing
Activity
All
Co
prostacyclin
ethanol
(CD)2spectrum
under
was diluted K2C03
100
= +11.2*1.0).
concentration
solid
Chemical
and
or
in
contain
sealed
PG12
solution
-2O’C
dichroic
Ac21o were
of
PG12
at
to
circular
+5.45*0.35,
original
Sigma
(absolute
was dissolved
each
ampoules
from
prostaglandins ethanol
prostacyclins)
PG12
loss
HEPES were
grade.
Solutions.
2-10mM
647
A SINGLE PROSTACYCLIN RECEPTOR
5
NG.
45,
The
CD
used.
This is equivalent to assuming that the purity deficit corresponds to hydrolytic and/or oxidative degradation. The assumption is viewed as a safe one since NEN determined nominal specific activity at the stage of3the Me ester and those values agreed with the specific activity of the [ HI-PGF2a employed as a starting material in the chemical synthesis of PG12.
=
A SINGLE PROSTACYCLIN RECEPTOR
648
3 [ l-l]-PGI
described
Samples
f’or
binding
Confirmation PGI2
70
rug of
and
PG12
Me,
900
2.11
pmoles
20
yL
with
dpm/pg
iodomethane
with
counts
15-O-Me-PG12 thus
described of
(4),
theory)
tracer
with
drawn
The
a measured
venipuncture
from
drug
3.22
(wt/vol)
aq.
3509
(15
centrifugation
min)
aggregometry was
employed
as
then
0.5
optical
min
60
prior
inhibitor
a dose
resulting
in
IC50
using
change rate
batch thrombin
from
of
the PRP.
gives
described
The
(GFP).
proteins (6),
test
by
a slight
of
as
tight
(10
or
for
[for of
controls
as
the
PRP data
and in
relative
Platelets filtration
modification
For
PG12 is
For
each ion
was also inverse
from
based
rat
DRCs obta
ios i ned
on ADP
potencies.
twenty
the
The
than
the
mL lots
(sepharose of
and
PG concentrat
anti-aggregatory
from
22’C)
challenge
I
6OOthe
ADP stimulated
are
cold
Table
after
challenges].
that
after
to (22)
were
(at
(rather
potencies
substance
min
and
from
prior
per
sec.
aggregation.
IC50. set
ware
aggregometry
thrombin
no
with
supernatant
90
buffer
100%
with 9:l
inhibitors
4.5
obtained
Blood,
milli-units
Aggregation
initial
Ci/mM.
(PRP).
the
(96%
the
*0.8
was mixed
was measured
OD 10-25
gel
normal
days,
was
ester
Thus,
Plasma
Opt i ca I Born
inhibitory
similar
Platelets plasma
taken in
Rich
76% of with
previously
11.9
plastic
addition
was
of
yL
by
traces as
dpm/pg.
500
indicated
prostacyclin
thrombin
was taken
was
966
and to
assayed
initial
-
remaining
10% ran
as
stirring) the
(DRC)
of
Gel-Filtered
previously
with
curve
for
or
TLC
air
density.
with
Aggregation values
a single
separated
rate
aggregation
stimulus
response
the (21).
(rate)
challenges;
the
final)
Net
37’C
50% aggregation
aggregations,
of
to
PM,
907
dpm/pg
brought
were
collected
ADP challenges]
change
the
an
(THE)
The
was
The
of
10
.
Andersen(21)
optical
[for
to
(OD)
inhibitor)
in
set
(warming
density
measured
(8-10
stimulus.
change
for
of
purity.
product
fossa
in
C3H] to
to
1275
additional of
preceding
stored
subjected
nominal
activity
PRP was
and
-
TLC. an
Platelet
the
of
esterification.
was added
product)
of
antecubital
citrate.
ADP
the
the
preincubated
a specific
the
method
using as
stimulus
sodium
by the
PL sample)
activity
with
lot
tett-ahydrofuran
by CD)
specific
during
Ci/mM)
remaining
(determined
Aggregometry,
ingestion
and and
the
was
product
radiopurity
of
pg
was 80*7X
and
of
minimum
611
Preparations by
history
crude
chromatography
radiopurity
Platelet
the CD,
(n=3).
mg dicyclohexano-18-crown-6
18% of
Me ester
a
after
determined 45
The
recovery),
instance,
above,
homogeneity
hr.
PGI2
one
12.6
19
(88%
afforded
ngl
l8*6X/year
preparation.
activity
bioassay
for
was
of
PL anhydrous
FL THF,
with
Me ester. Column
)76X.
for 200
(totaling
ran
60
activity
detailed
specific
confirmed
stirring
counting
applied
of
9 mos.
In
25.4
with
with
aliquots
scintillation the
as
of
stirred
corrected
treated
loss within
Activity.
(nominally
and
CD on an aliquot
and
used
by aggregometry
aliquot
was
THF
average
determination
PGI2(Na)
counting
were
+10X
spectroscopic A [3H]-PG12
*‘20
the
assays
[3H]-PGI, Specific
of
assaying
exact
above,
Vol. 45, No. 5
method
of
PRP were
2B-CL)
as
of
Tangen
(20).
The
GFP was
brought
(pH
7.35
12.0)
to
platelet
count
Ca-Tyrodes
to
at
binding
platelets/pL)
order 37’C).
in to
9.1
0.1-625
or
9.1
in
mM NaOH were
added
GFP giving
concentration
prepared
in
The
counted
the
amount
was
subtracted
the
method
assuming
other
prostaglandins
Buffer
A and
added
from
of
both
of
in
final
9.1
a one
at
PGI2
18009
samples
were
22’C).
The
aggregometry
nM.
at
4’C.
and
spun
17309
each
freeze
NaOH for
the
Lowry
VS.
10 min
thawed
2 hrs
method subtracted
at (24)
ambient using
using
and
ampoules, prostanoid
addition
of
treated.
esters,
In
these
reported
were
of
PG12
14
The
each
the
next
supernatant
data
DRC.
The
A 500-FL
sample
times
and
incubated
the
Protein standard. to
to
give
unlabeled to
yield
other at
by PRP of
PGI2
was
experiments.
by centrifu-
GFP and
content
and
a 100%
(standing
half-life
with
ice
The
min
duplicate
of
a
in
stimulus. 20
was
model.
establish
was prepared
4OC.
GFP value
of
was determined
frsom two
then
model
site
cooled
used
the
site
so as
the
squares
one
immediately
PM ADP as
control of
22’C
Using
affinity;
two
the
Aliquots
was
over
and
as
and
binding.
sum of
from
GFP at was
intervals
BSA as
PM unlabeled
least
were
supernatant
using
100%
was defined
density a
terminated,
binding
nspecificn
Conditions.
samples
in
5 minutes,
no cooperativity.
temperature.
the
of as
radioligand
similarly
8.3
GFP supernatant at
three
from
receptor
plot
Determination. for
Na salt
by the
for
of
regression
The
the
22’C
deternnine
One portion
at
a semi-log
at
determine
to
PG12 remaining
from
Protein
was
166
prepared
to
under
concentration
a final
Non-specific
to
Binding
added
added
down
another
prostacyclin
presence
values
GFP under
3 min
run (6).
the
sites
PRP aggregometry
X of
at
to
warming
10 PL samples.
count
and
calibrated
aq.
value
two
of
cooled
estimated
Platelet
were
or
in
for
DRC in
gation
(23)
mM NaOH were
control
then
sample
better,
concentration
spun
in
min
I.
dried
PG12
were
was employed 0.5
was
PG12,
and
analogs
were
by nonlinear
significantly
PG12
as
bound
Scatchard
PG12
followed
7.5
and
described
each
to
Prostacyclin
incubations
[ HI-PGI2
tubes
7.45
ca 2 nM.
analyzed
Stability
polystyrene
GFP
1 M NaOH was
above,
lyophilized
pH of
Table
a stock
Unlabeled
PL of
22’,
in
giving
mM-
1
for
thrombin
at
described
unlabeled
10
4 hr
with
PL of
and to
previously
min
mM NaOH from
of
of
as
PM EDTA,
preincubation
3.2
water,
adding
as
were
9.1
a final
competitive
and
prepared
10
HEPES
required
I y prepared
reported
Platelets.
0.3781
the
within
used
(4.5 not
nM [3H]-PG12.
solution
experiments
not
150
fresh were
conditions
pL with
used
A S-min
GFP are
to
5 mM HEPES,
GFP was
stimulus.
was 350
[3H]-PG12
PL of
data
which
with
A).
PL portions
Filtered
up to
about
the
using
Gel
made
1 mM Ca using: dilution
aggregometry
600
binding
PM solutions
10 PL of
750
to
brought
mM NaOH and
For
as
the
C3H]-PGI,
and
were
and
Further
(Buffer
in A)
simulate
Binding
PL of
vacuum
Buffer
pH 7.35,
CaCl2.
studies.
ADP challenges
[3H]-PGI, 200
aq.
pH 7.35
(200*50x103
in
O.lM
(50-250x103/pL)
radio!igand
to
5 mM HEPES,
and
buffer
(dissolved
649
A SINGLE PROSTACYCLIN RECEPTOR
Vol. 45, No. 5
20.8
supernatant FL
of
was assayed
The
GFP supernatant
platelet
protein
2.5 by
protein
content.
M
650
A SINGLE
PROSTACYCLIN
RECEPTOR
Vol.
45,
No.
RESULTS
As
previously
affords
Hemocytometry less
than
platelets min
reported
a GFP fraction reveals
0.1% in
of
at
at
5 min, occurs
competitor,
at of
instability in
degradation
product,
to
up to
PG12
in
this
In
the
(n=7)
A separate this
PM)
and
binding
nor
the
and
about
white
attributed
to
a half-life
antagonizes
PG12 the
18*7 PG12
inhibition
after
a 5
8.3@!,
final
specifically
bound
unlabeled occurs
with
a
hydrolytic
bioassay)
of
inhibits
the
(to
of
6 min
(by
constitute to
platelets
after
PRP
cells
[3H]-PG12
addition
determination
neither
blood of
90% of of
of
studies.
unlabeled
%-binding is
filtration
filtered
of
absence
of
pH revealed
6-oxo-PGFla, 8.3
red
of
the
decay
gel
binding
The
addition
dissociation
min
PG12.
GFP at
concentrations due
pH 7.45,
count.
by counting Upon
5 min.
21.8i4.1 of
stability
cell
pH 7.45.
in
2B-CL
radioligand
contaminating
total
a rapid
radioligand
half-life
that
the
sepharose for
GFP was assessed
incubation
cont.)
(6),
suitable
of
min.
PG12 The
binding of
(in
PRP aggregation
assay.
FIG. 1 Competitive [3H]-PG12 prostanoids.
Displacement to
GFP
in
the
Curves, presence
relative of
varying
percent
specific
concentrations
binding of
other
of
2 nY
5
A SINGLE PROSTACYCLIN
Vol. 45, No. 5
RECEPTOR
i-551
TABLE I A comparison
of
specified)
stimulus)
YS.
change)
-
for
donors
is
[n]
I&
binding
otherwise
values and
obtained
PRP (ADP stimulus, assessing
aggregation
various
using
in i tial
anti-
IC50
protocols aggregation
d saggregatory
and
data
-
(nM,
GFP
rate
Vs.
net
potencies.
PGI2
DH-MP-I2 Hoe-982
38
PGEl
920*90
6-oxo-PGEl
6pM Q1rM >8.3j~M 12,uM
6-oxo-PGFla
>16jbM
>lOOpMAA ~200j~M >>160/.d
CDLUMN
A
PG12
at
1 Ci/mM)
the In
sites/platelet. nM at PG12
zll and
Ci/mMole, other binding.
of
i nd i ng)
IC50(b
studies
in
affinity
values
present
was
employed
values
our
data
of
for
these
saturable,
reversible,
consistent
with
the
concentrations
displacement shown
present
as
column
work
(GFP
has
physiologic
and
appear
binding
(defined
a concentration effect
in
(6).
in
A of
li;30*180
of
[ HI-PGI2,
of
were
1.
The
Table
I. and
columns in
;and time
course
Non-specific
based
set
previous
and of
Table
Methods)
binding
on !&
complete
potency
detailed
2
unlabeled
aggregations)
other
by
(11-570nMr3H]-
curves Figure
relative
tihe as
obtained
nM with
varying
prostanoids
and
KD=45
with
(17,19,21,25-27)
[3H]-PG12
were
concentration
appears
the
(n=4)
D
curves
a fixed
are
62@16] -
C
study
these
obtained from
laboratories
specific
were
>>2pM 54*5[9]
>7O/rM
saturation
Competitive
A number
IC50
data The
resulting
16pM
1lOpM[4]
[3]
characteristics
binding
1.4pM[2]
78+20[5]
-
receptor
the
prostanoids.
specific
Aggregometry
(6)
w 621 150[3]
5/JM 116pM -
-
radioligand
22
1.7pM
B
study of
42PI 1.55jbM
180@
PGBl
64 [2]
194+28[8] 115*13[11]
>8. ~/LM [3]
PGE2
initial
110
-
(~20j~M)
PGD2
analysis
3.2[2]
-
17-C4Fg-PG12
our
4.1
-
-
Hoe-982 (Me ester)
In
4.9+0.4[11]
-
PGI2 (Me ester)
Scatchard
7.0+1.0[7]
-
2.25jLM
15-O+e-PG12
A-9O
-
310 [3]
[3]
s 5O--
rate
5 [31
700
(Na)
of
PRP
AOJ
PI
number
IC
GFP
66+6 [6]
(Na)
OD
The
Aggregometric IC50 3 [JH]-I2 Binding
unless
(thrombin
I.
is
dependence constitutes
652
A SINGLE PROSTACYCLIN RECEPTOR
3.0
KD= R,
z
nM
= 234
r=
: * ‘, 7; ;i;
61.0
Vol. 45, No. 5
fmof/108platelets
-0.997
2.0
?i “0 7 g
50
100
BOUND
Ifmol
’ it,=
150
PGl,/lOS
190
PlateletSi
fmof/l08
PlateletS
. .**. . .
I R:=185
fmovio8
~ r=-.945
IdO
5b BOUND
(fmol
PGI,/IOB
FIG. A -
Scatchard
830
nM unlabeled)
B -
Comparative
The
stippled
early the in
lots specific
B were
purity.
analysis
of
to
GFP,
Analysis area
of
shows
[3H]-PG12 GFP controls
performed
prior
competitive 5 min of
the -
at
GFP,
PGI2 22’C.
PRP,
binding All
and
87*9
for
this
to
routine
nM,
limits 212*18
experiment bioassay
(2.19
points
GFP+PPP
90% confidence
KD=
Platelets)
2
are (GFPP)
for
+O.ll mean
(n=2).
*SEM
Binding
The Most of
of
(n=Z).
(n=4)
solid the
+ 5-
(n=20).
Data
GFP’binding
fmol/lO*.
estimation
nM tracer
dots
using show
experiments
radioligand
Vol.
45,
at
No.
most
For
15% of
samples
binding PGI2
A SINGLE
5
the
of
is
li;early
unlabeled
PGI with
This
in
high
type
previous
controls
with
(n>20)
the
competition
PG12
with
similar
to
45.2nM)
(6),
those
Since site
our
by all
PRP
(9)
effects
and
GFP resuspended
of
gel
significantly
(123+24
show
nM,
filtration
or
3,
act has
analog
Scatchard
via not
of
all
one-site)
a KD values
(1130
the
binding
analysis
(r-=-0.997, and
[ H]-
by 8.3@
affinities that
each
linear
to
display
previous
(61.0nM)
sites/platelet,
sites,
The
is
[3H]-PG12
results
appear
Figure
as
fmole/108
212+18
(112+12
indicating
205*57
lower
nM).
The
than
GFP+PPP
no significant
PRP,
the
The
are
(n=4)
and
GFP,
using
2B.
those
and
determine
compared
platelets,
; PRP,
(n=4)
to
b i nd i ng to was
in
binding
(10-12)
controls
(GFP+PPP)
significantly
GFP+PPP
matched
PG12
fractions
we performed
expressed
nM)
a single
membrane
procedure.
GFP,
(6719
<.OOl)
characteristics
all
two
densities,
GFP
relative
Figure
In
curves
platelet-poor-plasma
different:
MD va I ue for
continued
technique.
receptor
and
run. a
saturation
filtration
in
binding
I
to
displaced
substances
sites/platelet)
the
while
to
the
also gave
not
;f
2A.
criteria,
the
observed
(1410
from
Figure
reported
competitive
binding.
GFP preparation
our
are
Table
were
is
and
those
employed. component
content,
that
selectivity for
[3H]-PG12 PGI
conditions
1. HI-PG
correlation,
densities obtained
see
total
component
experizents
653
the3non-specific
of
unlabeled
receptor
to
potency of
incubation
binding
structural
studies
study,
graph
the
anti-aggregatory
receptor.
PGI2
the
RECEPTOR
purity,
(rz0.995)
contrast,
displays
2
under
radiochemical
related In
correlate
binding
reduced
content.
appeared
total
PROSTACYCLIN
not
However, for
the
PRP
PRP binding
alteration
due
to
over
platelet
the
gel
be
used
procedure.
DISCUSSION Gel
filt&ed
preparations PRP, for
platelets
which
GFP retain
have
aggregometry
with
that
fi
Plasma
Itration.
by gel
prostaglandins 534 to
degree
4
of
is
binding
Commercially
protein
in
as
78
15 min could
available
the
removal thus
eliminated. that
stimulus.
room
is
essentially
a/.
(28)
Et4N+
purity. and PG12
have
At
This
salt
site
factor,
lower
Like
(Figure
in
PG12 that
our
and
with
radioiigand
other 67
is
concentrations undoubtedly,
preparations
2
by gel
()99.9X for
shown
the
can
study
altered
complete
of
trauma. and
present not
binding
temperature.
Q-[3H]-PG12
vary significantly in radiochemical activity was assumed (see note 3) aggregometric assay.
(20)
are
respectively,
greater.
other physical
The
density
et
severe
integrity
a competing
52X,
be even
more
and
Pifer and at
advantages to
functional
function
filtration,
nM [3H]-PG12, PPP proteins
and
thrombin
receptor
some
subjected
morphological
demonstrates
studies)
offer been
were
and
bound the
provides
found
Therefore, nominal specific content (*lo%) was assessed
to by an
654
A SINGLE PROSTACYCLIN RECEPTOR
the
explanation
for
determinations)
the
lower
observed
with
7 determinations), competing the
KB.
The
hydrolytic
determination
and
unlabeled
been
PGI,
in
t
quite
may not
reflect
association
total
100~
that
as
this
curves
to
suggest
study
that
of
(30) Siegl
we report
for
GFP.
quantitating
of
actual
additional
IC5G
analogs
and labeled have
bioassay to workers very
be approached values,
display
however,
different
the
rather
populations, apparent have
KB.
Scatchard
to
an
plot
alkaline
of
in
latter
measured
PRP provides
in
on
free
such
total
The
platelet sites
sites. resulting the
Wynalda
presence
of
(equated
and
of with
bound
increase
the
Fitzpatrick
stabilization albumin
the
discrepancy.
receptor will
-
those
concentration
Iigand
In
site
that
the
free
added
and
at
of
same.
effective
PRP.
technique
matching
we conclude
the
1.6pM
a single
cause
the
figured
and
counting found
of
criteria.
nM in
is
not
study excess
the
nM (PG12)
KB =123+24
also
to
Siegl
on our
a direct
binding
microenvironment
the
characteristics
the
the
140
and
(more
equivalent
component,
of
our
ways:
course
indicate based
two
or
comparison
based
additional
sequestration;
of
group
presumably
was
to
paper
platelets
Thomae
time
is
GFP at
using
in
period,
level
with
is
different
binding
PRP decreases
separately
addition
Albumin
the
plots
result
reference that
va I ues
radioligand
PRP
in
Scatchard
observation
washed
on this
GFP and sites
demonstrated
IC50
al.,
bound
than the
et
PRP and
by others
With in
our
using
binding
to
a
non-specific
with
site
incubation
radioligand
binding
a two extended
could
reported it.
4-5nM
noted
with
minute
sites/platelet)
Based
KB in binding
Since
attributed
Analog
linear
illustrated
presumably
method
(29)
by
are
should
of
storage
previous
values
study
occurs
we defined
at
nM (837*68
B + F),
for
of
platelet
of
storage
approach
with
the
difference
binding
consistent
unpublished
PG12.
the
strictly
a five
curves binding
, entirely
The
at
has the
This
affinity
KB = 56.7+3.5
5
of
34
nM,
estimate
confirmed
agreement
if
(12)
occupancy with
competition
similar
been
n#,
absence
sample
a year
question
period.
(with
binding.
low
subtracts
study find
concentrations)
site”
the
competition
an
has
equilibrium
affinities
to
specific
non-specific
(PGE2)
up to
displacement
incubation
another
binding
the
one
in
in
the
better
conditions
for
the
we find,
relative
non-detectable
non-specific
If
Storage
to
in
the
problems
loss/yr)
that
GFP,
data.
(
and
in
presumably
poses
suitable
suggesting
affinity
thus
20X
PG12
Turning
a 5 min
value is
are
studies,
yet
PG12 of
likely)
that
association
we continue
definition
(9),
binding
true
though
phenomenon,
that
of
(tota I range 32-105 PRP or GFP+PPP (93-190
kinetics.
Even
higher
instability
stability
<2 min, using
l/2 closely
than
observed
equilibrium
binding
PG12
KD value
sites’,
spectroscopy.
that
rapid,
The
binding
samples
and
chiroptical
equilibrium
was
of
developed,
(9,10),
28.
protein
actual
in
and
Figure
plasma
apparent GFP rather
Vol. 45, No. 5
binding
of site.
PG12
is
We also means
conclude for
that
evaluating
aggregation
the the
inhibiting
obtained
and
tested.
various
the
initial
present site
GFP
of
of
of
binding
synthetic
Table
of
assays rate
[3H]-PG12
action
properties. measures
Aggregometric
DD and
555
A SINGLE PROSTACYCLIN FlECEPTOR
Vol. 45, No. 5
I
collects
antiaggregatory
employed
change
in
assay
the
potency
endypoints turbidity
can
prostanoids
based due
to
as
binding
IC56
for
substances
the
on both
the
serve
a
with
net
data
change
in
aggregatory
PGE2a
100
,/
10
1%0-Me-PG13
/I ,/
t
t-HOE-982
Me ester
1.0
PGE, --1 \
II a 1
PPGI,
Me ester
A
/
z? & .lO 0 Q
PGDZ
?? ?? 6-0x0-El
5: 0 .Ol
IC60
I binding1
3
FIG. Graphical log-log columns co1.D ones
Correlations plot
of
A and data.
for
regression
I&G
C of Round
other
of
Receptor
Table symbols
I
(sol
id
are
employed
prostanoids
analysis
since
Affinity
w>and I&,
(bind
it
and
(w
symbols), for
17-C4Fg-PGI2 displays
py
Antiaggregatory
re,p a t ion) r =0.996. PGI2
(A)
and
was
some antagonist
Potency.
employing Open
symbols
bicyclic
excluded
data
employ
analogs; from
properties
A
from
the (26).
square
656
A SINGLE
challenge
(21).
correlation
with
Log-log used
to
assay through
is
aff
data
event
and
symbols,
a “spare
10% occupancy
to
reduced and
reduced
under the
3)
clearly
NIH
is
were GFP
known
to
act
6-oxo-PGEl based
linear
on
correlation
analog
DH-MP-12,
antiaggregatory
(25),
value
only
if
occupancy may
reflect
work
ROl-HL-23103, the
technical
Pharmaceutics
Manufacturers
occupancy
at
was supported
Some of
(NOl-HV-8-2933)
receptor
contain for
a
the
effect, under
by U.S.
the
6.
De and
PG12
support. Association.
can
the
the
esters,
time and
PGI2
calculate
rapid
mimics that
on-rate,
PGE2,
correspond In
a would
and
particularly
the
PGE2-receptors
to:
PGEl
alternative (KD=0.5-20
nM,
proaggregation study.
National
PDl-HL-22163). of
for
response).
presently
-GM-26935;
(ROl-HL-32827). contract
and
response
as
binding
one
PG12,
pharmacological
assistance
in aggregatory
with
observed
PGEl,
a full
are
indicate
initial
better
This,
expressed
interactions
This
For
a rapid
for
the
particularly
concentrations.
an opposing
of
I)
reduction
(0.927).
response.
requiring
(Table
magnitude
AOD 90-set values
on
phenomenon.
agonist
of
ratios
ful6
which
These
grants
US PHS
and
from
correlates
based
r2
a system low
(c80%
have
Acknowledgments.
acknowledge
is
be predicted
which
inhibition
disaggregation
the
aefficacy”
(5,25,27,31).
the
of
efficacy, PGE2
which
Health
Figure
receptor”
in
responsiveness
ref.6)
>
potencies
order
cases,
potencies
produces
be advantageous
the
these
two
IC50(binding)/IC50(aggregation)
indicate
PGE2
values by our
which
outlier would
range:
inhibition
a ca.
a significantly’lower
The
%30%),
In
component
gave
PC50
essentiaily
magnitude to
No.
best
determined PGD2
than The
PG12,
the
45,
3).
a distinct
site.
aggregation
produces
Aggregation
significant
show
natural
gave
affinities
is
of
Vol.
antiaggregatory
potency
PG12
than
potency.
(filled
affinity.
and
the
data
response.
higher
order
and
esterification in i ty
Figure
between
a five
potent
rate
versus
(2,13,14,27)
for
RECEPTOR
PM range.
Binding that
(see IC50
pharmacological
affinity
more
100
initial
correlation
receptor
nearly
the
affinity
significantly
over
the
that
binding
presumed
measured
which
of the
another
extends
in
the
displays
its
binding
plots
determine
and
also
We found
PROSTACYCLIN
Institutes
of
We gratefully the
continuing
analogs
employed
TLE
supported
was
support were
of
prepared
by grants
from
5
Vol.
45,
No.
5
A SINGLE
PROSTACYCLIN
657
RECEPTOR
REFERENCES
1.
GRYGLEWSKI, R., BUNTING, S., and VANE, J.R. An Enzyme MONCADA, S., Isolated from Arteries Transforms Prostaglandin Endoperoxides to an Unstable Substance that Inhibits Platelet Aggregation. Nature (fond.) 262, 663-665, 1976; GRYGLEWSKI, R.J., BUNTING, S., MONCADA, S., and VANE, J.R. Arterial Walls are Protected Against FLOWER, R.J., Deposition of Platelet Thrombi by a Substance (Prostaglandin X) which They Make from Prostaglandin Endoperoxides. Prostaglandins 12, 685-713, 1976; JOHNSON, R.A., MORTON, D.R., KINNER, J.H., GORMAN, R.R., MCGUIRE, WHITTAKER, N., BUNTING, S., SALMON, J.A., MONCADA, S., J.R., SUN, F.F., and VANE, J.R. The Chemical Characterization of Prostaglandin X (Prostacyc I in) . Prostaglandins 12, 915-928, 1976.
2.
WHITTLE, Prostacyc
B J . R ., MONCADA, I in
(PGI2)
S., and Prostaglandins
VANE, 16,
3.
CHO, M.J. and ALLEN, M.A. Chemical Aqueous Solutions. Prostaglandins
4.
DE, B., ANDERSEN, N.H., IPPOLITO, R.M., Synthesis and Chiroptical Characterization Prostaglandins 19, 221-247, 1980.
5.
J.R. Comparison 37:3-388, 1978.
Stability 15, 943-954,
of
of
the
Prostacyclin 1978.
Effects
(PGI2)
WILSON, C.H., and of Prostacyclin
Platelet
6.
EGGERMAN, T.L., ANDERSEN, N.H., and ROBERTSON, R.P. Separate and Distinguishable Binding Sites for PGI2 and PGE on Human Gel-Filtered Platelets. J. Pharmacol. Exper. Therapeutics, 1986. 5 36, 568-573,
7.
MACDERMOT, J. and BLAIR, I.A. Prostacyclin Receptors of Cell Line: Divalent Cations and Ligand-Receptor Coupling. 30, 2041-2044, 1981.
8.
MACDERMOT, J., J.A. Prostacyclin Eur. J. Pharm.
9.
SIEGL, Selective J. C/in.
10.
SCHILLINGER, Fraction of 1980.
a Neuronal Biochem.
BARNES, P.J., WADDELL ,K.A., DOLLERY, C.T., and Binding to Guinea Pig Pulmonary Membranes. 75, 127-130, 1981.
SMITH,
Binding Invest.
J.B.,
SILBER,
M.J.,
Site for [3H]Prostacyclin 63, 215-220, 1981.
E. and Platelets
PRIOR, G. Prostaglandin of Various Species.
J.L., MACDERMOT, on Human Platelets.
J.,
NICOLAOU,
K.C.,
in
JOHNSON, W.D. Diastereomers.
ANDERSEN, N.H., WILSON, C.H., DE, B., TYNAN, S.S., WATKINS, J., CALLIS, J.B., GIANELLI, M.L., HARKER, L.A., HANSON, S.R., and EGGERMAN, T.L. Analytical Methodology in the Cardiovascular and Areas. Atheroscler. Rev. 8, l-28, 1981.
A.M.,
of
and
Hybrid Pharm.
BLAIR,
AHERN,
D.
on Platelets.
12
Receptors
Biochem.
in
Pharm.
BLAIR, I.A., and LEWIS, C/in. Sci. 61, 29P, 1981.
P.J.
a Particulate
29,
2297-2299,
11.
SHEPHERD, Receptors
Prostacyclin
12.
LOMBROSO, M., NICOSIA, S., PAOLETTI, R., WHITTLE, B.J.R., MONCADA, S., and VANE, J.R. The Use of Stabler Prostaglandin to Investigate Prostacyclin (PGI2)-Binding Sites and PGI2-Sensitive Adenylate Cyclase Prostaglandins, 27, 321-333, 1984. Human PI ate I et Membranes.
in
A SINGLE
658
13.
SHAFER, Platelet
PROSTACYCL
A.I., COOPER, B., O’HARA, D Receptors for Prostaglandin’12
RECEPTOU
and HARDIN, R.I. Identification and D2. J. Biol. Chem. 254,
of
2914-2917, 1979. 14.
MILLER, O.W. D Receptors
and GORMAN, R.R. in Human Platelets.
Evidence for Distinct J. Pharmac. fxp.
Prostaglandin I Ther. 210, 134-148,
and
1379. 15.
MACDONALD, J.W.D. and STUART, R.K. in Regulation of Cyclic-AMP and E2 Evidence for a Common Prostaglandin
Interaction Aggregation Receptor.
of Prostaglandins in Human Platelets: J. Lab. C/in. Med.
El
and
84,
111-121, 1974. 16.
MACINTYRE, D.E. and GORDON, J.L. Discrimination between Platelet Prostaglandin Receptors with a Specific Antagonist of Bis-enoic Prostaglandins. Thrombosis Res. 21, 705-713, 1977.
17.
ANDERSEN, N.H., and RAO, CH.V. Prostaglandins
16.
ANDERSEN, N.H., Bi s-unsaturated
19.
SUBRAMANIAN, N., DE, B., CCRAE, D.A., Methyl Ethers of Prostaglandin F2a and and Medicine 6, 345-357, 1981. and IMAMOTO S., I and i ns. Prostag
PICKER, D.H. Prostaglandins,
Synthesis
TYNAN, 12.
of
S.S.,
Diastereomeric
14, 61-101, 1977.
IMAMOTO, S., SUBRAMANIAN, Iv., PICKER, D.H., LADNER, D.W., ANDERSEN, N.H.) EGGERMAN, T.L., HARKER, L.A., TYNAN, S.S., ROBERTSON, R.P., DE, B., OIEN, H.G., RAO, CH.V., LINDBERG, M., and NAQVI, R. Molecular Basis for Prostaglandin Potency. III. Tests of the Significance of the ‘Hairpin Conformation’ in Biorecognition Phenomena. Prostaglandins 22, 841-856,
1981. 20.
BERMAN, H.J. TANGEN, O., for Separation of Blood
I and MARFEY, Platelets
from
P.
Gel
Filtration:
Plasma.
a New Technique
Thrombosis,
30,
268-278,
1971. 21.
ANDERSEN, N.H., EGGERMAN, T.L., HARKER, L.A., WILSON, C.H., On the Multiplicity of Platelet Prostaglandin Receptors. I. Competitive Antagonism by Aggregometry. Prostaglandins, 19, 1980.
22.
BORN, G.V. its Reversa
23.
SCATCHARD, G. The Attractions of Proteins Ann. N.Y. Acad. SC;. 51, 660-672, 1949.
24.
LOWRY, O.H., Measurement
25.
Aggregation I Nature,
WILSON, C.H., Aggregometric Prostaglandins
of Blood Platelets by Adenosine 1962. 194, 927-929,
ROSEBROUGH, N.J., with Foiin Reagent.
for
FARR, A.L., and J. Biol. Chem.
Small
and DE, 8. Evaluation 711-735,
Diphosphate
Molecules
RANDALL, R.J. 193, 265-275,
and ANDERSEN, N.H. A Single Platelet TYNAN, S.S., and Antiaggregatory Prostanoids. Assay for ProLeukotrienes and Med. 9, 415-427, 1982.
and
and
Ions.
Protein
1951.
of
Vol.
26
45, No. 5
HARKER,
A
L.A.,
659
SINGLE PROSTACYCLIN IRECEPTOR
HANSON,
S .R.,
ANDERSEN,
N .H.,
WILLS,
M .T.,
DE,
B.,
LIN
MCCRAE, D.A., and WILSON, C.H. Vascular and Platelet Activities B-S., Prostacyclin and PGD Analogs in the Non-sedated Baboon Vth Internatl. Prostaglandin Conf. z bstr . Book (Florence, Italy) p.309, 1982.
27
28
TYNAN, On the N-0164
S.S., ANDERSEN, Multiplicity of for Distinguishing
Hydanto
i n Ana
N.H., WILLS M.T., HARKER L.A., and HANSON S.R. Platelet Prostaglandin Receptors. II. The Use the Loci of Action for PGI2, and PGE2, and
Iogs . Prostaglandins,
PIFER, D.D., CAGEN, L.M., and in Human Blood. Prostaglandins,
CHESNEY, C.M. 21, 165-175,
Stability
of
Prostaglandin
30
WEISENBERGER, the Xth Winter
31
KLDEZE, J. Influence of Prostaglandins on Platelet Adhesiveness latelet Aggregation., in Nobel Symposium 2, Prostaglandins, S. and B. Samuelsson (Eds.) New York, Interscience, 1967, p.241-252.
32
MILLER, D.V., prostaglandin Antiaggregatory
F. Albumin 1980.
Stabilizes
H’. (Thomae GmbH, . Biochemie) . Prostaglandin Conf. (l/83).
12
1981.
WYNALDA, M. Prostaglandins,
FITZPATRICK, 20, 853-861,
of
1984.
Z??, 683-693,
29
and
of
Persona
Prostaglandin
I2.
I communication
at
and Bergstrom
AIKEN, J.W., SHEBUSKI, R.J., and GORMAN, R.R. 6-KetoEl is not Equipotent to Prostacyclin (PGI ) as an Agent. Prostaglandins, 2U, 391-396, l98?.