1. health and fitness
2. therapy

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YC-1, a Novel Activator of Platelet Guanylate Cyclase
By Feng-Nien KO, Chin-Chung Wu, Sheng-Chu Kuo, Fang-Yu Lee, and Che-Ming Teng
YC-1 [3-~5’-hydroxymethyl-2’-furyl)-l-benzylindazolel
inhibited theaggregation of and ATP release from washed rabbit
platelets induced by arachidonic acid(AA), collagen, U46619,
platelet-activating factor (PAF), and thrombin in a concentration-dependent manner. YC-1
also
disaggregated the
clumped platelets caused by these inducers. The thromboxane
formation caused by collagen,PAF, and thrombin
was inhibited by concentrations of YC-1 that did not affect
formation of thromboxane B2 and prostaglandin D2 caused
by AA. YC-1suppressed the increaseof intracellular Ca2+
concentration and generation of inositol 1,4,5-trisphosphate
caused by these five aggregation inducers. Both the cAMP
and cGMP contents of platelets were increased by YC-1 in
a concentration- and time-dependent manner. Like sodium
nitroprusside, YC-1 potentiated formation of cAMP caused
by prostaglandin E, but not thatby 3-isobutyl-l-methylxanthine. Adenylate cyclase and cAMP phosphodiesteraseactivities were not altered by YC-1. Activity of cGMP phosphodiesterase was unaffected by YC-1. Activities of guanylate
cyclase in platelet homogenate and cytosolic fraction were
activated by YC-1, whereas particulate guanylate cyclase activity wasunaffected. The antiplatelet effect of sodium nitroprusside but not that of YC-1 was blocked by hemoglobin
and potentiated by superoxide dismutase. After intraperitoneal administration for 30 minutes, YC-1 prolonged the tail
bleeding time of conscious mice. These data indicate that
YC-1 is a direct soluble guanylate cyclase activator in rabbit
platelets. It may alsopossess antithrombotic potential in
vivo.
0 1994 by The American Society of Hematology.
P
proteins mediated by either cAMP/cAMP-dependent protein
kinase (PKA) or cGMP/cGMP-dependent protein kinase
(PKG), respectively. The platelet pathways affected by both
cAMP/PKA and cCMPPKG result in the inhibitionof platelet activation, most likelycaused by the inhibition of agonistinduced calcium mobilization. There is also evidence that
cAMP and cGMP can suppress platelet reactions in vivo,
indicating that an antiplatelet action may supplement their
effects on vascular smooth muscle in the treatment of cardiovascular disease^.^^"'
We found that YC- 1, a chemically synthetic benzylindazole compound, possessed antiplatelet activity. In the present
report, we endeavored to elucidate the mechanism of its
inhibitory activity on platelet aggregation and proved it to
be a novel soluble guanylate cyclase activator in platelets.
LATELETS AND platelet-derived vasoactive agents are
important physiologic regulators of vascular tone and
hemostasis. Much evidence indicates that interactions between platelet and vessel wall contribute significantly to the
pathogenesis of atherosclerosis, myocardial infarction, unstable angina pectoris, and thrombosis.’-4Platelets are activated by diverse stimuli in vivo, including diseased arteries.’
Mural thrombus formation can restrict the flow of blood to
vital tissues or organs leadingto peripheral, cerebral, or
coronary ischemia. The developing thrombus may embolize
with potentially lethal consequences. Activation of platelets
may lead to not only acute vascular complications such as
thrombosis but also, perhaps due to the release of growth
factors, to enduring effects such as smooth muscle proliferation, one of the hallmarks of ather~sclerosis.’,~
Therefore,
inhibition of platelet function may be a promising approach
to prevent and to treat diseases in which a pathophysiologic
participation of activated platelets appears likely.
Agents that elevate concentrations of either platelet cAMP
or cGMP are powerful inhibitors of platelet activation.‘.’
Two physiologically important cyclic nucleotide-elevating
agents, prostaglandin Iz and endothelium-derived relaxing
factor (EDRF), are produced by vascular endothelial cells
that are important antithrombotic agents through their antiplatelet mechanism. cAMP and cGMP stimulate in platelets
the stoichiometric and reversible phosphorylation of several
From the Pharmacological Institute, College of Medicine, National Taiwan University, Taipei;the Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung; and YungShin Pharmaceutical Industry CO, Ltd, Taichung, Taiwan.
Submitted April 20, 1994; accepted August 26, 1994.
Supported by a research grant of the National Science Council
of the Republic of China (NSCS3-0412-BOO2-172).
Address reprint requests to Che-Ming Teng, PhD, Pharmacological Institute, College of Medicine, National Taiwan University, No.
I , Jen-Ai Rd, 1st Section, Taipei 10018, Taiwan.
The publication costsof this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society o j Hematology.
0006-4971/94/8412-0019$3.00/0
4226
MATERIALS AND METHODS
Platelet aggregation und ATP release reaction. Blood was collected from the rabbit marginal ear vein and mixed with EDTA to
a final concentration 6 mmol/L. It was centrifuged for 10 minutes
at 90s and room temperature, and the supernatant was obtained as
platelet-rich plasma (PRP). Platelet suspension was prepared from
EDTA-anticoagulated PRP according towashing procedures described previously.” Platelets were counted by Hemalaser 2 (Sebia,
Molineaux, France) and adjusted to a concentration 3 X IO8 platelets/
mL. Platelet pellets were finally suspended in Tyrode’s solution of
the following composition in millimoles per liter: NaCl(136.8), KC1
(2.8), NaHCO, (1 1.g), MgCI2 (2.1), NaH2P0, (0.33), CaCI, (1.0).
and glucose (1 I .2) containing bovine serum albumin (0.35%). Aggregation was measured at37°C by turbidimetry as described by
0’Brien.l’ ATP released from platelets was detected by the bioluminescence method of DeLuca and McElory.” Both aggregation and
ATP release were simultaneously measured in a Lumi-aggregometer
(Chrono-Log CO, Havertown, PA) connected to two dual-channel
recorders. Platelet preparations were stirred at 1,200 rpm. To eliminate the effect of solvent on aggregation, the final concentration of
dimethyl sulfoxide (DMSO) was fixed at 0.5% (vol/vol).
Tromboxane BZandprostaglandin D2 assay. After 6 minutes of
platelet incubation with the inducer, EDTA (2 mmoliL) and indomethacin (SO pnol/L) were added to halt thromboxane and prostaglandin Dz formation. After centrifugation in an Eppendorf microcentrifuge (Model 541SC; Eppendorf, Hamburg, Germany) for 2
minutes, thromboxane B, and prostaglandin D,in the supernatant
were assayed by enzyme immunoassay.
Assay of inositol 1,4,5-trisphosphate (If.,)mass content. PlateBlood, Vol 84, No 12 (December 15), 1994: pp 4226-4233
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4227
VC-l, AN ACTIVATOR OF GUANVLATE CYCLASE
lets (10’ platelets/mL, 500 pL) prepared as described above were
incubated with DMSO (0.5%), YC-I, or prostaglandin E, at 37°C
for 3 minutes and then stimulated with the aggregation inducers.
The reactions were terminated by the addition of 100 pL of ice-cold
perchloric acid (20%) followed by 20 minutes of incubation in an
ice bath. After centrifugation at 2,OOOg for 15 minutes at 4°C the
supernatant was recovered and its pH was adjusted to 7.5 with a 10
N KOH solution. KCIO4was precipitated for 30 minutes at 4°C and
sedimented at 2,OOOg for 15 minutes at 4°C. The amount ofIP,
in the resulting supernatant was determined by radioimmunoassay
(RIA).
Measurement of intracellular calcium in platelets. The method
of Pollock and RinkI4 was followed. Platelets (3 X 10’ platelets/
mL) were incubated with fura-2IAM (5 pmoVL) at 37°C for 45
minutes and centrifuged at 500g; the resultant pellet was washed with
Tyrode solution containing 1 mmol/L EDTA. After centrifugation,
platelets were resuspended in the Tyrode solution containing Ca’+
(1 mmol/L). Fluorescence (Ex 339 nm, Em 500 nm) was measured
with a Hitachi Fluorescence Spectrophotometer (Model F4000; Hitachi, Tokyo, Japan) at 37°C. At the end of the experiment, the cells
were treated with Triton X-l00 (0.1%) followed by the addition of
EGTA (1 0 mmoI/L) toobtain the maximal and minimal fluorescence,
respectively. [Ca”], was calculated as described for fura-2 using
Ca2+-dyedissociation constant 224 nmol/L.”
Estimation of platelet cyclic nucleotides. The method of Kamiguian et all6 was used. The platelet suspension was warmed at 37°C
for 1 minute in an aggregometer (Chrono-Log CO) stirred at 1,200
rpm. YC-1, prostaglandin E , , sodium nitroprusside, or 3-isobutyll-methylxanthine (IBMX) was then added with incubation for various time intervals. The reaction was stopped by adding EDTA ( I O
mmol/L) followed immediately by boiling for 5 minutes. Upon cooling to 4”C, precipitated protein was sedimented by centrifugation in
an Eppendorf microcentrifuge (Model 5415 C). The supernatant was
used toassay for cyclic AMP and cyclic GMP by enzyme immunoassay kit.
Phosphodiesterase assay. The method of Moore et all’ was used.
Washed rabbit platelets prepared as described above were resuspended in 50 mmoVL Tris-HCI (pH 7.4, containing 5 mmoVL
MgCI’). Platelets were disrupted by sonication at 4°C (4 to 6 X I O
seconds; setting 5; Vibra cell; Sonics and Materials Inc, Danbury,
CT). The lysate was centrifuged at 39,OOOg for 20 minutes (4°C).
The supernatant contained greater than 95% cyclic nucleotide phosphodiesterase activity.
The crude cytosolic enzyme (1 mg/mL; 0.1 mL) was incubated
with Tris-HCI (0.2 mL) at 37°C for 5 minutes; 0.1 mL cAMP (final
concentration, 0.25 pmoVL containing 0.1 pCi [’HICAMP) or cGMP
(final concentration, 0.25 pmol/L containing 0.1 pCi [,H]cGMP)
was then added. After 30 minutes at 37°C the samples were heated
to 100°C for 1 minute before cooling. Ophiophagus hannah snake
venom ( I mg/mL; 0.1 mL) was then added and incubated at 25°C
for 30 minutes to convert the 5’-AMP or 5’-GMP to the uncharged
nucleosides, adenosine, or guanosine. An ion-exchange resin slurry
(1.0 mL; Dowex-l; Sigma Chemical CO, St Louis, MO) was added
tobind all unconverted cAMP and cGMP. After centrifuging, an
aliquot (0.5 mL) of the supernatant was removed for determination
in a liquid scintillation counter.
Guanylate cyclase assay. Washed rabbit platelets were prepared
as above but resuspended in Tris-HCI buffer (50 mmoVL; pH 7.4).
Platelets were disrupted by sonication as described above and the
lysate was centrifuged at 39,OOOg at 4°C for 20 minutes. The supernatant fluid was used as a source of soluble guanylate cyclase. The
pellet was washed twice in the original amount of Tris-HC1 buffer
and used as membrane guanylate cyclase. Protein content was determined witha protein assay kit (BioRad, Richmond, CA) and adjusted
to a concentration 1 mg/mL.
Fig 1. Chemical structure of YC-1.
Guanylate cyclase activity was determined as previously described.” Enzyme preparation (50 pL) was incubated in a final volume (200 pL) with other reactants as follows: GTP (0.2 mmoVL
containing 1 X IO6 cpm [cY-~’P]GTF’),MgCI2(5 mmoVL), cGMP
(2.5 mmol/L), creatine phosphate (15 mmol/L), creatine phosphokinase (30 pg) without or with sodium nitroprusside, or YC-1 in TrisHCI buffer (50 mmoVL), pH 7.4. The reaction was initiated by
adding the enzyme preparation and, after incubation at 30°C for 10
minutes, was terminated by adding HCI (0.5 N, 200 pL). The reaction mixture was then heated to 100°C for 6 minutes and cooled in
an ice bath. To each tube was added imidazole (1 mmol/L, 200 pL).
GTP and cGMP were separated on neutral alumina as described by
White and Zenser” andthe radioactivity ([”PIcGMP) was determined in a liquid scintillation counter.
Adenylate cyclase assay. Rabbit platelet membrane prepared as
described above wasfinally resuspended in Tris-HCI buffer (50
mmoVL, pH 7.4) containing EDTA (5 mmoVL). Adenylate cyclase
activity was measured as described by Insel et al.’’ Membranes (20
pL) were incubated in a final volume (60 pL) with other reactants
as follows (final concentrations): Tris-HCI, 50 mmoVL (pH 7.4);
EDTA,2.6 mmoVL; MgClz, 13 mmoVL; creatine phosphate, 25
mmoVL; creatine phosphokinase, 1 mg/mL; CAMP, 1 mmol/L; ATP,
0.5 mmoVL containing 1 X lo6 cpm [cY-”P]ATP;and other additions
as indicated in the text. The reaction was initiated by adding platelet
protein and was performed at 30°C for 20 minutes. The reaction was
terminated by adding HCI (0.5 N.0.2 mL) and boiling the sample
for 6 minutes. After cooling in an ice bath, imidazole (1 mmoVL,
200 pL) was added. ATP and cAMP were separated on neutral
alumina as previously described” and [32P]cAMPwas determined
with a liquid scintillation counter.
Tail bleeding time in conscious mice. Thirty minutes after the
intraperitoneal administration of DMSO (2.5 pWg), YC-I, or indomethacin, male mice (18 to 22 g) were placed in a tube holder with
the tail allowed to protrude. The tail was transected at 2 mm from
the tip and 1.5 cm of the distal portion was vertically immersed into
saline at 37°C. The bleeding time was then measured as described
by Hornstra et al.”
Materials. YC-I [3-(5’-hydroxymethyl-2’-furyl)-l-benzylindazole; Fig l ] was chemically synthesized as described previously.’’
Bovine thrombin (Parke Davis CO, Detroit, MI) was dissolved in
glycerol (50% vol/vol) for a stock solution of 100 NIH U/mL. Collagen (type I, bovine Achilles tendon; Sigma) was homogenized in
acetic acid (25 mmol/L) and stored (1 mg/mL) at -70°C. Plateletactivating factor (PAR I-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; Sigma) was dissolved in chloroform and diluted into 0.1%
bovine serum albumin (BSA)-saline solution immediately before
use. Arachidonic acid (AA), BSA, indomethacin, EDTA, luciferase-
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4228
KO ET AL
R
YC-l
(PM)
Fig 2. Inhibitoryeffect of YC-l on platelet aggregation inducedby
thrombin-induced platelet aggregation from 0.10 f 0.02 and
0.28 t 0.04 minutes to 0.25 ? 0.02 and 0.55 ? 0.04 minutes
(n = 6), respectively. It also induced a more rapid recovery
of PAF- and thrombin-induced aggregation (data not shown).
YC- 1, PGE, , and sodium nitroprusside, at concentrations
that maximally inhibited platelet aggregation, also markedly
disaggregated platelets that had been previously aggregated
with these five inducers. Again, sodium nitroprusside was
less potent than YC-1 and PGE, in disaggregating platelets
(see Fig 3 for PAF-induced platelet aggregation).
YC-I (1 .S to 1.50pmoVL), PGE, (1 pmol/L), and sodium
nitroprusside (10 pmol/L) also inhibited, in a concentrationdependent manner, the ATP release caused by these five
inducers. Inhibition of ATP release was parallel to inhibition
of platelet aggregation (data not shown). However, YC-I
(1.50 pmol/L), PGE, (1 and 10 pmoVL), and sodium nitroprusside (1 mmoVL) did not affect the platelet shape change
U46619 (1 pmol/L, D), collagen (10 pg/mL, A), AA l100 pmol/L, A),
PAF (3.6 nmol/L, e), and thrombin (0.1 UlmL, 0).
Washedrabbit
platelets were preincubated with DMSO 10.5%. control) or various
concentrations of YC-1 at 37°C for 3 minutes. The inducerwas then
added to trigger the aggregation. Percentages ofinhibition are presented as means f SEM (n = 5).
DMSO
luciferin, Dowex-l (100 to 200 mesh: X8, chloride)resin,fura-2acetoxymethyl ester, trichloroacetic acid(TCA), U46619, imidazole,
ethyleneglycol-bis-(~-aminoethylether) N,N,N’,N’-tetraacetic acid
(EGTA), prostaglandin E, (PGE,), 3-isobutyl-l-methylxanthine
(IBMX), creatine phosphate, creatine phosphokinase, Tris(hydroxymethyl) aminomethane, adenosine S’-trisphosphate
(ATP), adenosine
3’,S’-cyclic monophosphate (CAMP), guanosine5‘-trisphosphate
(GTP), guanosine 3’,S‘-cyclic monophosphate (cGMP), Ophiophagus hannah snake venom, sodiumnitroprusside,
forskolin, and
myo-inositol were
purchased
from
Sigma. My0-[2-~H]inositol,
[a-”P]ATP, [W~~PIGTP,
[’HICAMP,[3H]cGMP,andIP,radioimmunoassay kit were purchased from Amersham (Amersham,
UK).
Thromboxane B2, prostaglandin D*, CAMP,and cGMP enzyme immunoassay kits were from Cayman Chemical CO (AM Arbor, MI).
Protein assay kit and neutral alumina were obtained from
BioRad.
RESULTS
Effects of YC-l on aggregation and ATP release of washed
rabbit platelets. In washed rabbit platelets, U46619 (1
pmoVL), collagen (10 pg/mL), AA (100 pmoVL), PAF (3.6
nmoVL), and thrombin (0.1 U/mL) all caused approximately
90% aggregation.YC-1inhibitedU46619-,collagen-,
AA-,
PAF-, and thrombin-induced platelet aggregation in a concentration-dependent manner with IC,, values of 10.1 ? 1.7,
14.6 ? 1.4, 20.5 ? 2.8, 41.1 i. 2.9, and 57.3 IT 10.2 pmol/
L, respectively (Fig 2). PGE, (1 pmoVL) also inhibited completely platelet aggregation caused by these aggregation inducers. In contrast, sodium nitroprusside (10 pmol/L) inhibited U46619-, AA-, and collagen-induced platelet aggregation by 100.0% f O.O%, 45.1 t- 4.0%, and 46.9% ? 5.4%
(n = 6), but did not significantly affect PAF- and thrombininduced maximal platelet aggregation even when the concentration 1 mmol/L was used (6.7% 2 1.4% and 7.4% ? 1.1%
inhibition, respectively; n = 6). However, sodium nitroprusside (1 mmoVL) slowed the aggregation induced by PAF
and thrombin. It prolonged the latent periods of PAF- and
I
2
min
t
PAF
Fig 3. Effects of Y C l , PGE,, and sodium nitroprussideon platelet
disaggregation. Platelet ageregation was induced by PAF (3.6 nmoll
L). Platelets were preincubated at 37°C for 3 minutes before the addition of PAF. Y C 1 ( 6 0 pmol/L), PGE, (1 pmol/L), or sodium nitroprusside (1 mmol/L) was added 5 minutes later (at 8 minutes).
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VC-1, AN ACTIVATOR 4229
OF GUANYLATE CYCLASE
Table l . Effects of YC-1, PGE,, Sodium Nitroprusside, Indomethacin, and imidazole on the Thromboxane B2 Formation of Washed Rabbfi
Platelets Caused by AA, Collagen, PAF, Thrombin, and U46619
( n o X 10’ platelets)
Thromboxane B,
Treatment
Control
YC-1 (pmol/L)
15
793.9
30
408.8
60
150
PGE, (1 pmol/L)
Sodium
nitroprusside
(10 pmol/L)
Indomethacin (1 pmol/L)
Imidazole (1 mmol/L)
M
(100 prnolR)
886.3
569.9
2 161.5
101.8 2 247.2
2 158.6*
Collagen
PAF
( 10 pglrnLl
(3.6nmol/Ll
21.3t 72.8
21.1 2 7.6
-
292.5 2 61.0*
2 32.3t
21.9 2 9.5t
861.0 67.6
t 105.6
871.0 2 110.2
264.9
5.6 2 0.9t
156.2 2 45.4*
1.8 2 1 3
-
-
2 24.2t
0.71.0
2 0.1$
2 1.2$
-t 2.9
66.8*
U46619
Thrombin
(0.1 U h L l
( 1 pmollLl
1.6 -+ 0.1
2 8.0
5.0 2 1.5
3.1 2
2.6 2
0.3 2
1.82
2.8 2
0.4*
1.0$
O.lt
0.1*
0.8*
1.1 2 O.lt
2 0.1
1.6 2 0.1
DMSO (0.5%. control), PGE,, sodium nitroprusside, indomethacin, imidazole, or various concentrations of YC-1 was preincubated with platelets
at 37°C for 3 minutes and then the inducer was added. Aggregation and thromboxane formation were terminated by EDTA (2 mmol/L) and
indomethacin (50 pnol/L) 6 minutes after the addition of the inducer. The thromboxane B2 level of resting platelets was 0.6 2 0.2 ng/3 x 10’
platelets. Values are presented as means 2 SEM (n = 4 to 7).
P i .01 as compared with the control.
t P < .001 as compared with the control.
P < .05 as compared with the control.
*
caused by these five inducers even when the aggregation
and ATP release were blocked completely (data not shown).
Effects of YC-1 on platelet thromboxane B2 and prostaglandin D2formation. The thromboxane B2 level of resting
platelets was 0.6 ? 0.2 ng/3 X lo8 platelets. AA, collagen,
PAF, and thrombin markedly increased, whereas U46619
insignificantly affected, the thromboxane B2 level after incubation with platelets for 6 minutes. Although YC-1 inhibited
collagen-, PAF-, and thrombin-induced thromboxane B2 formation in a concentration-dependent manner, it did notaffect
AA-induced thromboxane B2 formation at a concentration
(30 pmoVL) that inhibited more than 70% of the aggregation
induced by AA. However, YC- 1at a concentration (60 pmoV
L) that completely blocked AA-induced platelet aggregation
partially suppressed AA-induced thromboxane B2 formation
(Table 1). Similarly, PGE, and sodium nitroprusside markedly suppressed collagen-, PAF-, and thrombin-induced
thromboxane B2 formation without affecting that formation
caused by AA (Table 1). In contrast, indomethacin (1 pmoV
L) and imidazole (1 mmol/L) markedly inhibited AA-induced thromboxane B2 formation (Table 1).
The prostaglandin D2 level of resting platelets (0.07 t
0.01 ng/3 X lo8 platelets) was increased in the presence of
AA (100 pmol/L) to 1.32 t 0.20 ng/3 X lo8 platelets. This
prostaglandin D2 formation was inhibited by indomethacin,
but was enhanced markedly by imidazole. YC-l (60 pmoVL)
did not inhibit this AA-induced prostaglandin D2 formation
(Table 2).
Effects of YC-1 on IP3formation. Resting platelets containing 0.9 t 0.1 pmol of IPJ109 cells. AA (100 pmol/L),
U46619 (1 pmoVL), PAF (3.6 nmol/L), and thrombin (0.1
U/mL) induced a rapid and transient formation of IP3, reaching its peak within 5 seconds. Collagen (10 pg/mL) also
caused IP3 formation of platelets with a slower rate, reaching
its peak within 30 seconds. The maximum production of
IP3 at 5 seconds after AA, U46619, PAF, and thrombin
stimulation was 3.4 2 0.3, 3.4 t 0.4, 3.8 2 0.3, and 4.1 5
0.7 pmoVlO’ platelets, respectively, whereas the maximum
production of IP3 at 30 seconds after collagen stimulation
was 3.9 -+ 0.5 pmoVlO’ platelets. YC-1 (60 or 300 pmol/L)
and PGE, (1 pmol/L) at concentrations that markedly inhibited platelet aggregation also suppressed the IP3 formation
caused by these five inducers (Fig 4).
Effects of YC-I on the intracellular calcium of platelets.
In fura-2-loaded platelets, AA, U46619, collagen, PAF, and
thrombin caused an increase of intracellular free calcium.
As shown in Fig 5 , this increase caused by these five inducers
was almost completely inhibited by YC-1 (60 pmol/L).
Effects of YC-I on the platelet CAMP and cGMP levels.
The CAMPand cGMP levels of resting platelets were 2.1 2
0.3 and 1.2 2 0.1 pmoVlO’ platelets, respectively. YC-l (15
to 300 pmoVL) increased both levels in a concentrationand time-dependent manner (Fig 6). After incubation with
platelets for 45 seconds, YC-1 (300 pmoVL) increased the
Table 2. Effect of YC-1, Indomethacin, and Imidazole
on the Prostaglandin D2 Formation of Washed Rabbk
Platelets Caused by AA
Treatment
Resting
Control ( A A 100 pmol/L)
YC-1 (60 pmol/L)
Indomethacin (1 pmol/L)
Imidazole (1 mmol/L)
Prostaglandin D,
(ngD x 10’ platelets)
0.07 5
1.32 5
1.20 2
0.10 5
49.60 5
0.01
0.20
0.40
0.101
6.90*
DMSO (0.5%. resting and control), YC-1, indomethacin, or imidazole
was preincubated with platelets at 37°C for 3 minutes and then AA
was added. Aggregation and prostaglandin D2formation were terminated by EDTA (2 mmol/L) and indomethacin (50 pmol/Lj 6 minutes
after the addition of AA. Values are presented as means 2 SEM (n =
5 to 7).
x P < .001 as compared with the control.
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KO ET AL
AA
platelet cAMP levels. Such an increase byYC-1wasnot
potentiated by IBMX. However, IBMX and YC-1 markedly
potentiated formation of cAMP caused by PGE,. Sodium
nitroprusside at a concentration 10 pmol/L also potentiated
formation of cAMP by PGE, with smaller potency (Table
3).
Effects of YC-1 on platelet phosphodiesterase. As shown
in Table 4, IBMX inhibited platelet cAMP and cGMP phosphodiesterases in a concentration-dependent manner. Inhibition of cAMP phosphodiesterase was more pronounced than
inhibition of cGMP phosphodiesterase. YC-l (30 to 300
pmol/L,) did not inhibit activities of cAMP and cGMP phosphodiesterase in platelets.
Effects of YC-I on platelet adenylate cyclase and guanylate cyclase activities. Adenylate cyclase activity of resting
platelets was 5.1 t 0.9 pmol cAMP formedmg proteidmin.
PGE, ( 1 pmoVL) and forskolin (10 pmoVL) increased this
basal activity to 47.1 t 4.0 and 72.3 t 8.1pmol cAMP
formed/mg proteidmin, respectively. However, YC-l (60 to
300 pmoVL) did not affect the platelet adenylate cyclase
activity (Table 5 ) .
Guanylate cyclase activities of platelet lysate and the soluble fraction were28.2 2 6.3 and15.1 t 3.3 pmol cGMP
formedhg proteidmin. YC-1 (6 to 150 pmoUL)and sodium
nitroprusside (10 and 100 pmol/L.) increased the activities
of guanylate cyclase of platelet lysate and soluble fraction
in a concentration-dependent manner (Table 5). However,
YC-1 (150 pmol/L) and sodium nitroprusside (10 and 100
pmol/L) did not increase the platelet particulate guanylate
cyclase activity (data not shown).
Effects of methemoglobin and superoxide dismutase on
the antiplutelet activity of YC-I. YC-l (60 pmol/L) and
sodium nitroprusside (10 pmolk) inhibited collagen (10 pgl
PAF Thrombin
U46619 Collagen
Fig 4. Inhibition of YC-1 and PGEl on the formation of IP3 in
washed rabbit platelets caused bysome aggregation inducers. Platelets werepreincubated with DMSO (0.5%, control [Dl), YC-1 (60
pmolfL for AA, U46619, and collagen, and 300 pmollL for PAF and
or PGEl (1 pmollL [HI) at 37°C for 3 minutes. AA 1100
thrombin [HI),
pmol/L), U46619 (1 pmol/L), PAF (3.6nmollLI, or thrombin (0.1 U/
mL) was then added for another 5 seconds, whereas collagen (10
pg/mL) was added for another 30 seconds. Increases in IP3 are presented asmeans f SEM (n = 4 or 5). **P < .01; ***P < ,001 as
compared with the respective control.
cAMP and cGMP levels to 40.2 t 10.5 and 117.2 t 44.8
pmol/109 platelets, respectively. The cGMP levelwas increased rapidly and reached a maximum at 15 seconds that
was maintained for 2 minutes after incubation of YC-l with
platelets. In contrast, YC-1 increased the cAMP level much
more slowly than it increased the cGMP level and attained
no maximal level within 2 minutes (Fig 6).
IBMX, sodium nitroprusside, and PGE, increased the
Control
1
i
1
YC- 1
W
h
"l<
4
Thrombin
TI"----
"Ir"---
A
A
PAF
Collagen
"l
Fig 5. Effectsof YC-1 on the
increase of intracellular calcium concentration of platelets
cauaed by some aggregation inducers.Fura-2-loaded platelets
were preincubated with DMSO
(0.5%, control) or YC-l (60pmoll
L) at 37°C for 3 minutes. Thrombin (0.1 UlmL), PAF (3.6nmoll
L), collagen (10 pglmLI, AA (100
pmol/L), or U46619 (l pmol/L)
was then added (arrows). Indomethacin (10 pmol/LI was pres"
l
7
ent in each medium except
A
thosechallenged with AA and
U46619
collagen.
"
l
r
A
AA
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423 1
YC-1, AN ACTIVATOR OF GUANYLATECYCLASE
A
Table 3. Interactions of YC-1, IBMX, Sodium Nitroprusside, and
Prostaglandin E, on the cAMP Level of Washed Rebbii Platelets
1401
Treatment
Resting
IBMX (300 pmol/L)
Sodium nitroprusside (10 pmol/L)
YC-1
300 pmol/L
300 pmol/L IBMX 300 prnol/L
PGE,
+
1 pmol/L
1 pmol/L
1 pmol/L
1 pmol/L
+ IBMX 300 pmol/L
+ sodium nitroprusside 10 pmol/L
+ YC-1 300 pmol/L
CAMP
Ipmol/W platelets)
2.1 2 0.3 (6)
18.9 i 4.4 (6)
11.9 t 4.6 (4)
37.0 2 4.5 (6)
52.7 i 13.7 (6)
127.3
3,063.1
331.9
2,031.0
i 26.7 (6)
2 841.8 (4)
i 39.6 (4)
t 310.3 (4)
Washed rabbit platelets (1 x lo9 platelets) were incubated for 45
seconds at 37°C with the additions indicated. Incubation was terminated by addition of EDTA (10 mmol/L), followed by immediate boiling for 5 minutes. cAMP content of platelets was determined by enzyme immunoassay and is expressed as means t SEM h ) .
300 1
164.0 t 49.0, 214.2 2 25.7, and 251.0 2 49.0 seconds,
respectively (n = 8 to 10).
l004
I
DISCUSSION
Incubation Period (min)
Fig 6. Concentration- and time-dependent increase of platelet
cAMP (01 and cGMP (01 levels caused by YC-1. Washed rabbRplatelets were preincubated at 37°C for 1 minute; various concentrations
of YC-l were then added for 45 seconds (A) or YC-l (300 pmollLI
was added for various periods (B). The reactions were terminated by
adding EDTA (10 mmoliL) followed by immediate boiling for 5 minutes. The cAMP and cGMP contents were determined by enzyme
immunoassay. Values are presented as means ? SEM (n = 4 to 6).
The present work shows that YC-1 inhibits platelet aggregation, ATP release, phosphoinositide breakdown, and increase of intracellular free calcium caused by various inducers. Its mechanisms of action include direct activation of
platelet soluble guanylate cyclase and indirect elevation of
platelet cAMP level.
Thromboxane A2 is an important mediator of release reaction and aggregation in latel let.'^ Formation of thromboxane
B*, a stable metabolite of thromboxane Az, induced by collagen, PM, and thrombin was inhibited by YC-l in a complete
Table 4. Effects of YC-1 and IBMX on Platelet cAMP and cGMP
mL)-induced platelet aggregation 87.4% 2 1.8%and 46.9%
Phosphodiusterase Activities
2 5.4% (n = 5), respectively. Hemoglobin (5 pmol/L) did
cGMP
cAMP
not affect this aggregation by itself. However, it reversed
Phosphodiesterase
Phosphodiesterase
Treatment
Activity (cpm)
Activity (cpm)
the antiplatelet activity of sodium nitroprusside (5.9% 2
1.0% inhibition, n = 5) without affecting that of YC-1
None (basal)
10,178 i 396
10,058 -C 291
YC-1 (pmol/L)
(87.4% 2 2.1% inhibition, n = 5).
10,196 t 120
30
10,692 t 701
YC-1 (6 pmol/L) and sodium nitroprusside (1 pmoVL)
10,385 i 111
60
10,227 2 507
inhibited collagen (10 pg/mL)-induced platelet aggregation
i 686
9,952 i 118
13.7% 2 2.4% and 17.8% 2 2.8% (n = 3 , respectively.10,452 150
10,123 t 130
300
10,463 i 568
Superoxide dismutase (20 U/mL) did not affect this aggregaIBMX (pmol/L)
tion, but it potentiated the antiplatelet activity of sodium
50
7,835 i 514*
nitroprusside (66.4% 2 1.3% inhibition, n = 5) without 5,941 100
i 415t
9,952 i 118
affecting that of YC-l (18.9% % 3.9% inhibition, n = 5 ) .
300
2,829 i 89t
9,412 C 420
Effects of YC-I on tail bleeding time of mice. Tail bleed500
1,692 i 132t
8,867 i 294$
ing time of untreated mice was 66.3 % 3.5 seconds (n = 8).
7,181 i 281t
1,000
DMSO (2.5 p u g ) alone had no effect on (78.1 ? 5.5 secPhosphodiesterase activity was measured as described in Materials
onds, n = lo), whereas indomethacin (3 mgkg) markedly
and Methods. Values are presented as means I
SEM (n = 4).
prolonged, the tail bleeding time of mice (499.0 2 53.4
* P < .01 as compared with the basal activity.
seconds, n = 10). YC-1 (3, 10, and 30 mg/kg) also markedly
t P < ,001 as compared with the basal activity.
and significantly prolonged the tail bleeding time of mice to
P < .05 as compared with thebasal activity.
*
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KO ET AL
4232
sodilators, such as sodium nitroprusside and nitroglycerin,
are believed to exert their effects via the release of nitric
oxide, which stimulates the formation of cGMP by soluble
guanylate ~yc1ase.z~
In biologic systems, superoxide dismuGuanylate Cyclase Activity
Adenylate
Cyclase Activity
(pmol
cGMPlmg
proteinlmin)
tase extends the half-life of nitric oxide; therefore, superox(pmol cAMPIrng
ide dismutase may promote the effectiveness of nitric oxide
Fraction
Soluble
Lysate
proteinlmin)
Treatment
____~
by bringing it to its active biologic form.” In contrast. meth28.2 2 6.3
15.1 t 3.3
5.1 t 0.9
None (basal)
of soluble guanylate cyclase
ylene blue inhibits activation
47.1 2 4.0*
PGE, (1pmol/L)
by nitric oxide and hemoglobin chelates nitric oxide.x The
Forskolin (10 pmol/L) 72.3 2 8.1*
antiplateletactivity of sodium nitroprussidewasinhibited
Sodium
by hemoglobinand potentiated by superoxidedismutase,
nitroprusside
respectively. However, the antiplatelet activity of YC- 1 was
(gmol/L)
38.2 5 4.2*
10
affected by neitherhemoglobinnor
superoxide dismutase.
97.8 t 26.0t
100
Thus, YC-l may directly activate platelet soluble guanylate
YC-l (pmol/L)
cyclase without causing release of nitric oxide.
75.0 I 7 . 2 t
35.6 2 9.6
6
YC-I andsodiumnitroprussidealsoelevatedplatelet
104.0 2 9.3*
51.3 t 9.3t
15
cAMP level. However, YC-I neither activated platelet ade133.9
f
11.4’
56.2 ? 8.0*
30
nylate cyclase nor inhibited platelet CAMP-phosphodiester190.0 t 21.0* 77.8 t 11.1*
60
5.5 t 2.1
ase activities. It has been reported by Maurice and Haslam’y
271.0 2 20.9* 85.3 -t 8.7*
5.7 t 0.4
150
that cGMPexertsimportant
functional effects througha
4.5 t 1.2
300
cGMP-inhibited low Km cAMP phosphodiesterase, an enAdenylate cyclase and guanylate cyclase activitieswere determined
zyme known to bepresent in many cells, including platelets.
as outlined in Materials andMethods. Values are presented a s means
The
nitrovasodilator sodium nitroprusside causes small int SEM ( n = 4).
creases in cAMP in rabbit platelets, in addition to the ex* P < ,001 as compared with the basal activity.
pectedlargeincreases
in &MP. In our results,platelet
t P < .01 as compared with the basal activity.
cAMP levelsincreased by YC-1 and sodiumnitroprusside
were not potentiated by IBMX, a nonselective phosphodiesterase inhibitor. In addition, YC- I , sodium nitroprusside, and
and concentration-dependent manner. However, YC-1 at a
IBMX potentiated cAMP formation causedby PGE, . Thus,
concentration 30 pmoVL inhibited more than 70% platelet
elevation of platelet cAMP level by YC-l may be mediated
aggregation induced by AA but did not affect AA-induced
by inhibition of cGMP-inhibited low Km cAMP phosphodithromboxane BZformation. Additionally, YC-1 also did not
esterase.
inhibit AA-induced prostaglandin D, formation.
Thus,
In conclusion, YC-l is a direct activator of platelet soluble
YC- 1 inhibits collagen-, PAF-, and
thrombin-induced thromguanylate
cyclase. It may also possess antithrombotic potenboxane B2 formation viaa step beforecyclooxygenase. Phostial in vivo because the tail bleeding times
of conscious mice
phoinositide breakdown is an important pathway in signal
transduction of agonist-induced platelet a c t i ~ a t i o n .This
~ ~ . ~ ~ were prolonged. However, furtherinvestigation of its in vivo
antithrombogenic activity is warranted.
processgeneratestwoactive
products,diacylglycerol and
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~
~
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From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
1994 84: 4226-4233
YC-1, a novel activator of platelet guanylate cyclase
FN Ko, CC Wu, SC Kuo, FY Lee and CM Teng
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