Vol. 69, No. 1, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SYNTHESIS OF PLATELET-AGGREGATING FACTOR BY HUMAN PLATELET MICROSOMES P. P. K. Ho, C. P. Walters,
and R. G. Hermann
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206 Received January 15,1976 SUMMARY: The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable factor that induced platelet aggregation. Formation of a polar product, similar in properties to thromboxane B ~ was also demonstrated. Indomethacin and fenoprofen (2-ph~noxyphenyl-propionic acid) inhibited this enzymatic activity, while preincubation with enzyme protein was necessary for aspirin to exert an inhibitory effect.
Hamberg and Samuelsson
(1,2) reported the hemiacetal
derivative of 8-(l-hydroxy-3-oxopropyl)-9, 10-heptadecadienoic
acid
(thromboxane B2)
8-10-heptadecatrienoic acid of prostaglandin G 2 (PGG2)
1
12L-dihydroxy-5, and 12L-hydroxy-5,
(HHT) as the major metabolites and arachidonic acid in suspen-
sions of human platelets. It was later demonstrated that an unstable intermediate, designated as thromboxane A 2 was found in the conversion of PGG 2 into thromboxane B 2 (3) and was responsible for irreversible platelet aggregation. Since incubation of arachidonic acid with washed platelets also led to formation of thromboxane A2, a fatty acid cyclooxygenase was suggested to be present in the platelet in addition to another enzyme protein that converts PGG 2 to thromboxane B 2. To understand the mechanism of these conversions,
it is evident
that a cell-free system for the synthesis of thromboxanes has
iAbbreviations: PGG~, prostaglandin G2; HHT, 12L-hydroxy5,8,10-heptadecatrie~oic acid, thromboxane B2[8-(l-hydroxy-3oxopropyl)9, 12L-dihydroxy-5,10-hep£adecadienoic acid]. Copyright © 1976 by Academic Press, Inc. All rights o/reproduction in any form reserved.
218
Vol. 69, No. 1, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
to be isolated.
The present work demonstrates that arachidonic
acid is oxygenated to thromboxane-like substances with a human platelet microsomal preparation. MATERIALS AND METHODS [5,6,8,9,11,12,14,15,3H] Nuclear, acid
Arachidonic acid
(New England
Boston, Mass.) was diluted with unlabeled arachidonic
(Nu Chek Prep.
Inc., Elysian, Minn.).
activity of the sample
The specific
(20 ~M) prepared in this way was 1 Ci/
mr~ole. Bovine hemoglobin was purchased from Sigma Chemical Co., St. Louis, Missouri.
Platelet rich plasma and washed human
platelets were isolated from blood collected from donors according to the procedures described by Hamberg
et al.
(3).
Platelet aggregation was monitored with a Chronolog aggregometer. The aggregometer
tube contained 0.5 ml of suspension of platelets
preincubated at 37~C for two min with 14 ~M indomethacin.
Thin-
layer chromatography was performed with silical gel G. plates (LQ6D,
Quantum
Industries, Fairfield,
New Jersey)
and the
organic layer of ethyl acetate-2,2,4-trimethylpentane-water i00:i00 v/v/v)
(50:
as solvent.
Proteins were measured using the method by Lowry et al. (4).
Bovine serum albumin was used as the standard. RESULTS
Preparation of Human Platelet Micromes All the preparations were performed at 4°C. rich plasma
(500 g) was
Platelet
centrifuged for i0,000 x g for 20 mln,
and the platelet pellet was suspended in 100 ml of 0.i M potassium phosphate buffer, pH 8.0, by homogenization with a Teflonglass homogenizer.
The suspension was sonicated at maximum
energy in a Bronson Sonifier for three min.
(Heat System, Melville,
New York)
The sonicate was centrifuged at i0,000 x g for
219
Vol. 69, No. 1, 1976
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
15 m i n and the c l o u d y at i00,000 was
x g for
suspended
pH 8.0, dried
supernatant
90 min.
The pellet
powder
(400 mg)
was
of this powder
Demonstration Microsomal
microsomal
mixture
initiated
of 1 M citric
Platelet
acid.
tion of the e t h y l a c e t a t e treated
chromatography.
Four
substance
thromboxane
inhibited
peaks
found.
and
III)
mixture
of P l a t e l e t
A reaction
a larger
amount
the a d d i t i o n
the a g g r e g o m e t e r
mixture
5 sec,
of i0 ~i twice
after
evapora-
appeared
(Fig.
Rf v a l u e
of this polar
i).
of
product
or f e n o p r o f e n
aspirin
had no effect.
Factor the above
of m i c r o s o m a l
2 min before
for
with
incubation
to t h i n - l a y e r
indomethacin
Aggregation
reaction
stirring
of
[5,
radioactivity)
w i t h a similar
(Table i), w h i l e
The c o m p l e t e
obtained
subjected
The f o r m a t i o n
was p e r f o r m e d
5 min
then e x t r a c t e d
of r a d i o a c t i v i t y
by the a d d i t i o n
to the r e a c t i o n
After
(70-80% of the added
(Compound
B 2 was
was
The r e a c t i o n
by the a d d i t i o n
The r e s i d u e
with diazomethane
pH 8.0,
of 10 pl of 20 ~M
acid.
The m i x t u r e
buffer,
of 0.2 ml.
terminated
4 ml of ethylacetate.
After
concen-
and 0.i mg of l y o p h i l i z e d
by the a d d i t i o n
was
except
in L y o p h i l i z e d
in a total v o l u m e
the r e a c t i o n
Formation
The
The p r o t e i n
50 m M Tris-HCl
2 pM h e m o g l o b i n
powder
was
contained
at 37°C,
was
buffer,
to dryness.
at -20°C.
Activity
arachidonic
A polar
fraction)
is 50% by weight.
6,8,9,11,12,14,15-3H]
was
centrifuged
Powder
5 mM L-tryptophan,
with
further
phosphate
lyophilized
stored
of E n z y m a t i c
A reaction
mixture
was
was
(microsomal
in i0 ml of i0 m M p o t a s s i u m
and the s u s p e n s i o n
tration
solution
reaction
powder
(i mg)
was p r e i n c u b a t e d
components, was used.
at 37°C
for
of i0 pl of 0.3 mM a r a c h i d o n i c
acid.
0.1 ml was r a p i d l y
to
tube w h i c h
contained
220
transferred
0.5 ml of p l a t e l e t
VoI. 69, No. 1, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
'0
x12
E D.
-
methyl arachiclonate
.Ta
T
~,.~
~o 4
,'sa '
" ','o ' ' ' 'sil' Distance
( cm )
Fig. 1. T h i n - l a y e r r a d i o c h r o m a t o g r a m of p r o d u c t (methyl esters) o b t a i n e d after i n c u b a t i o n of [3H]-arachidonic acid as d e s c r i b e d in the text. Solvent system = organic layer of e t h y l a c e t a t e - 2 , 2 , 4 - t r i m e t h y l p e n t a n e - w a t e r (50: i00:i00, v/v/v).
Table Effect
Inhibitor
1
of Inhibitors
Amount
(~g/ml)
Percentage C o m p o s i t i o n of C o m p o u n d III
Not p r e s e n t
Percent Inhibition
22.2
-
Indomethacin
l0
2.6
88
Indomethacin
1
6.9
68
Fenoprofen
i0
2.7
88
Fenoprofen
1
11.7
47
Aspirin
i00
20.6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SYNTHESIS OF PLATELET-AGGREGATING FACTOR BY HUMAN PLATELET MICROSOMES P. P. K. Ho, C. P. Walters,
and R. G. Hermann
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206 Received January 15,1976 SUMMARY: The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable factor that induced platelet aggregation. Formation of a polar product, similar in properties to thromboxane B ~ was also demonstrated. Indomethacin and fenoprofen (2-ph~noxyphenyl-propionic acid) inhibited this enzymatic activity, while preincubation with enzyme protein was necessary for aspirin to exert an inhibitory effect.
Hamberg and Samuelsson
(1,2) reported the hemiacetal
derivative of 8-(l-hydroxy-3-oxopropyl)-9, 10-heptadecadienoic
acid
(thromboxane B2)
8-10-heptadecatrienoic acid of prostaglandin G 2 (PGG2)
1
12L-dihydroxy-5, and 12L-hydroxy-5,
(HHT) as the major metabolites and arachidonic acid in suspen-
sions of human platelets. It was later demonstrated that an unstable intermediate, designated as thromboxane A 2 was found in the conversion of PGG 2 into thromboxane B 2 (3) and was responsible for irreversible platelet aggregation. Since incubation of arachidonic acid with washed platelets also led to formation of thromboxane A2, a fatty acid cyclooxygenase was suggested to be present in the platelet in addition to another enzyme protein that converts PGG 2 to thromboxane B 2. To understand the mechanism of these conversions,
it is evident
that a cell-free system for the synthesis of thromboxanes has
iAbbreviations: PGG~, prostaglandin G2; HHT, 12L-hydroxy5,8,10-heptadecatrie~oic acid, thromboxane B2[8-(l-hydroxy-3oxopropyl)9, 12L-dihydroxy-5,10-hep£adecadienoic acid]. Copyright © 1976 by Academic Press, Inc. All rights o/reproduction in any form reserved.
218
Vol. 69, No. 1, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
to be isolated.
The present work demonstrates that arachidonic
acid is oxygenated to thromboxane-like substances with a human platelet microsomal preparation. MATERIALS AND METHODS [5,6,8,9,11,12,14,15,3H] Nuclear, acid
Arachidonic acid
(New England
Boston, Mass.) was diluted with unlabeled arachidonic
(Nu Chek Prep.
Inc., Elysian, Minn.).
activity of the sample
The specific
(20 ~M) prepared in this way was 1 Ci/
mr~ole. Bovine hemoglobin was purchased from Sigma Chemical Co., St. Louis, Missouri.
Platelet rich plasma and washed human
platelets were isolated from blood collected from donors according to the procedures described by Hamberg
et al.
(3).
Platelet aggregation was monitored with a Chronolog aggregometer. The aggregometer
tube contained 0.5 ml of suspension of platelets
preincubated at 37~C for two min with 14 ~M indomethacin.
Thin-
layer chromatography was performed with silical gel G. plates (LQ6D,
Quantum
Industries, Fairfield,
New Jersey)
and the
organic layer of ethyl acetate-2,2,4-trimethylpentane-water i00:i00 v/v/v)
(50:
as solvent.
Proteins were measured using the method by Lowry et al. (4).
Bovine serum albumin was used as the standard. RESULTS
Preparation of Human Platelet Micromes All the preparations were performed at 4°C. rich plasma
(500 g) was
Platelet
centrifuged for i0,000 x g for 20 mln,
and the platelet pellet was suspended in 100 ml of 0.i M potassium phosphate buffer, pH 8.0, by homogenization with a Teflonglass homogenizer.
The suspension was sonicated at maximum
energy in a Bronson Sonifier for three min.
(Heat System, Melville,
New York)
The sonicate was centrifuged at i0,000 x g for
219
Vol. 69, No. 1, 1976
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
15 m i n and the c l o u d y at i00,000 was
x g for
suspended
pH 8.0, dried
supernatant
90 min.
The pellet
powder
(400 mg)
was
of this powder
Demonstration Microsomal
microsomal
mixture
initiated
of 1 M citric
Platelet
acid.
tion of the e t h y l a c e t a t e treated
chromatography.
Four
substance
thromboxane
inhibited
peaks
found.
and
III)
mixture
of P l a t e l e t
A reaction
a larger
amount
the a d d i t i o n
the a g g r e g o m e t e r
mixture
5 sec,
of i0 ~i twice
after
evapora-
appeared
(Fig.
Rf v a l u e
of this polar
i).
of
product
or f e n o p r o f e n
aspirin
had no effect.
Factor the above
of m i c r o s o m a l
2 min before
for
with
incubation
to t h i n - l a y e r
indomethacin
Aggregation
reaction
stirring
of
[5,
radioactivity)
w i t h a similar
(Table i), w h i l e
The c o m p l e t e
obtained
subjected
The f o r m a t i o n
was p e r f o r m e d
5 min
then e x t r a c t e d
of r a d i o a c t i v i t y
by the a d d i t i o n
to the r e a c t i o n
After
(70-80% of the added
(Compound
B 2 was
was
The r e a c t i o n
by the a d d i t i o n
The r e s i d u e
with diazomethane
pH 8.0,
of 10 pl of 20 ~M
acid.
The m i x t u r e
buffer,
of 0.2 ml.
terminated
4 ml of ethylacetate.
After
concen-
and 0.i mg of l y o p h i l i z e d
by the a d d i t i o n
was
except
in L y o p h i l i z e d
in a total v o l u m e
the r e a c t i o n
Formation
The
The p r o t e i n
50 m M Tris-HCl
2 pM h e m o g l o b i n
powder
was
contained
at 37°C,
was
buffer,
to dryness.
at -20°C.
Activity
arachidonic
A polar
fraction)
is 50% by weight.
6,8,9,11,12,14,15-3H]
was
centrifuged
Powder
5 mM L-tryptophan,
with
further
phosphate
lyophilized
stored
of E n z y m a t i c
A reaction
mixture
was
was
(microsomal
in i0 ml of i0 m M p o t a s s i u m
and the s u s p e n s i o n
tration
solution
reaction
powder
(i mg)
was p r e i n c u b a t e d
components, was used.
at 37°C
for
of i0 pl of 0.3 mM a r a c h i d o n i c
acid.
0.1 ml was r a p i d l y
to
tube w h i c h
contained
220
transferred
0.5 ml of p l a t e l e t
VoI. 69, No. 1, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
'0
x12
E D.
-
methyl arachiclonate
.Ta
T
~,.~
~o 4
,'sa '
" ','o ' ' ' 'sil' Distance
( cm )
Fig. 1. T h i n - l a y e r r a d i o c h r o m a t o g r a m of p r o d u c t (methyl esters) o b t a i n e d after i n c u b a t i o n of [3H]-arachidonic acid as d e s c r i b e d in the text. Solvent system = organic layer of e t h y l a c e t a t e - 2 , 2 , 4 - t r i m e t h y l p e n t a n e - w a t e r (50: i00:i00, v/v/v).
Table Effect
Inhibitor
1
of Inhibitors
Amount
(~g/ml)
Percentage C o m p o s i t i o n of C o m p o u n d III
Not p r e s e n t
Percent Inhibition
22.2
-
Indomethacin
l0
2.6
88
Indomethacin
1
6.9
68
Fenoprofen
i0
2.7
88
Fenoprofen
1
11.7
47
Aspirin
i00
20.6
