The novel compound MP407 inhibits platelet aggregation through cyclic AMP-dependent processes

Article abstract of DOI:10.1016/j.ejphar.2017.09.024

Platelet hyperactivity plays a critical role for initiating several vascular diseases such as atherothrombosis. Therefore, development of effective antiplatelet agents is necessary for ameliorating platelet-related diseases. In this study, we investigated the effects of the new synthesized compound, MP407 on platelet aggregation and further elucidated the underlying mechanisms. Our results demonstrated that MP407 dose-dependently inhibited collagen-induced platelet aggregation, thromboxane B₂ (TXB₂) production, intracellular Ca²⁺ mobilization, platelet membrane GPIIb/IIIa expression, and the phosphorylation of Akt, GSK3β, p38MAPK, and phospho (Ser) PKC substrate (p47). Moreover, MP407 is able to increase the cyclic AMP formation both in resting and activated platelets. However, blocking cyclic AMP formation with 2′5′-ddAdo, an inhibitor of adenylate cyclase, greatly reversed the antiplatelet activity of MP407 and related platelet-activating pathways. MP407 also enhanced VASP phosphorylation at Ser157 in collagen-stimulated platelets, which was attenuated by addition of 2′5′-ddAdo. Therefore, the antiplatelet activity of MP407 may be modulated by cyclic AMP-dependent regulation of Akt, GSK3β, p38MAPK and VASP phosphorylation. Notably, treatment with MP407 markedly reduced the pulmonary thrombosis and the numbers of paralysis and death in mice induced by ADP injection, but did not affect the bleeding time. Taken together, MP407 may be a potential candidate or lead compound for developing novel antiplatelet or antithrombotic agents for platelet hyperactivity-triggered disease therapy.

Full text of DOI:10.1016/j.ejphar.2017.09.024

Author’s Accepted Manuscript
The novel compound MP407 inhibits platelet
aggregation through cyclic AMP-dependent
processes
Chung-Shuen Lin, Tso-Hsiao Chen, I-Hsin Lin,
An-Rong Lee, Tz-Chong Chou
www.elsevier.com/locate/ejphar
PII:
S0014-2999(17)30612-X
http://dx.doi.org/10.1016/j.ejphar.2017.09.024
EJP71410
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 17 August 2017
Revised date: 15 September 2017
Accepted date: 15 September 2017
Cite this article as: Chung-Shuen Lin, Tso-Hsiao Chen, I-Hsin Lin, An-Rong Lee
and Tz-Chong Chou, The novel compound MP407 inhibits platelet aggregation
through cyclic AMP-dependent processes, European Journal of Pharmacology,
http://dx.doi.org/10.1016/j.ejphar.2017.09.024
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The novel compound MP407 inhibits platelet aggregation through cyclic
AMP-dependent processes
Chung-Shuen Lina,1, Tso-Hsiao Chenb,1, I-Hsin Lina, An-Rong Leec, Tz-Chong Choud,*
aSchool of Post-Baccalaureate Chinese Medicine, Tzu Chi University, Hualien, Taiwan
bDivision of Nephrology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical
University, Taipei, Taiwan; Department of Internal Medicine, School of Medicine, College of
Medicine, Taipei Medical University, Taipei, Taiwan
cSchool of Pharmacy, National Defense Medical Center, Taipei, Taiwan
dInstitute of Medical Sciences, Tzu Chi University, Hualien, Taiwan; Department of
Biotechnology, Asia University, Taichung, Taiwan; China Medical University Hospital, China
Medical University, Taichung, Taiwan
*Correspondence to: Ph.D, Institute of Medical Sciences, Tzu Chi University, 6F, Xie-Li
Building, No. 707, Sec. 3, Zhongyang Rd., Hualien, 97002, Taiwan. Tel.: 886-3-8561825, ext.
5620; fax: 886-3-8573710, chou195966@gmail.com
Abstract:
Platelet hyperactivity plays a critical role for initiating several vascular diseases such as
atherothrombosis. Therefore, development of effective antiplatelet agents is necessary for
ameliorating platelet-related diseases. In this study, we investigated the effects of the new
synthesized compound, MP407 on platelet aggregation and further elucidated the underlying
mechanisms. Our results demonstrated that MP407 dose-dependently inhibited
collagen-induced platelet aggregation, thromboxane B2 (TXB2) production, intracellular Ca2+
mobilization, platelet membrane GPIIb/IIIa expression, and the phosphorylation of Akt,
1
These authors contributed equally to this work.
1

GSK3 p38MAPK, and phospho (Ser) PKC substrate (p47). Moreover, MP407 is able to
increase the cyclic AMP formation both in resting and activated platelets. However, blocking
cyclic AMP formation with 2’5’-ddAdo, an inhibitor of adenylate cyclase, greatly reversed the
antiplatelet activity of MP407 and related platelet-activating pathways. MP407 also enhanced
VASP phosphorylation at Ser157 in collagen-stimulated platelets, which was attenuated by
addition of 2’5’-ddAdo. Therefore, the antiplatelet activity of MP407 may be modulated by
cyclic AMP-dependent regulation of Akt, GSK3p38MAPK and VASP phosphorylation.
Notably, treatment with MP407 markedly reduced the pulmonary thrombosis and the numbers
of paralysis and death in mice induced by ADP injection, but did not affect the bleeding time.
Taken together, MP407 may be a potential candidate or lead compound for developing novel
antiplatelet or antithrombotic agents for platelet hyperactivity-triggered disease therapy.
Key words:
platelet aggregation; MP407; cyclic AMP; GSK3VASP; thromboxane B2
1. Introduction
When blood vessels are damaged, platelets quickly adhere to the subcellular matrix at the
sites of vascular injury, leading to initiation and formation of haemostatic plug. Platelet
hyperactivity also plays a critical role in the development of circulatory disorders, including
atherothrombosis, myocardial infarction and cerebrovascular diseases (Ruggeri, 2002). Several
studies have reported that treatment with antiplatelet drugs can prevent and reduce the
incidence of stroke in patients with vascular diseases (Willoughby et al., 2002). Therefore,
inhibiting platelet hyperactivity has been regarded as a promising strategy for ameliorating
2

thrombosis-related cardiovascular diseases.
Platelet activation is a complex process modulated by a variety of molecules and signaling
pathways (Lazarus et al., 2003). Under normal condition, collagen, the most potent
physiological activator of platelets, is sequestered under the endothelium of blood vessel walls.
When the vessels are injury, the subendothelial collagen is exposed and binds to platelet
receptor GP-VI, resulting in Ca2+-dependent phospholipase A2 (PLA2) activation and
arachidonic acid (AA) release from membrane phospholipids. Then, AA is further converted to
thromboxane A2 (TXA2), a strong stimulator of platelet aggregation, via the actions of
cyclooxygenase (COX) and thromboxane synthase (Vezza et al., 2002). In response to agonists,
phospholipase C (PLC) is activated, leading to inositol 1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG) production, which in turn elevates intracellular Ca2+ concentration [Ca2+]i
and protein kinase C (PKC) activity that are essential for platelet aggregation and granule
secretion (Jardin et al., 2008; Harper et al., 2007). Activation of phosphatidylinositol 3-kinase
(PI3K)/Akt cascade is known to stimulate platelet aggregation through phosphorylation and
inactivation of glycogen synthase kinase 3 (GSK3), a negative regulator of platelet activation
(O'Brien et al., 2011; Li, et al., 2008). Notably, there are endogenous aggregation-inhibiting
molecules existing in platelets, such as cyclic adenosine monophosphate (cyclic AMP) that is
produced by adenylate cyclase. Cyclic AMP is able to inhibit the adhesion, aggregation, release
of granule contents, TXA2 formation, and intracellular Ca2+ mobilization of platelets via a
series of cellular signaling pathways (Jang et al., 2002). Several cyclic AMP-elevating agents
have been confirmed to inhibit platelet aggregation (Hayashi et al., 2009; Liu et al., 2009). The
major target of cyclic AMP is cyclic AMP-dependent protein kinase (PKA) that phosphorylates
vasodilator-stimulated phosphoprotein (VASP), thereby attenuating the affinity of VASP for
contractile protein filamentous actin and fibrinogen binding to platelet membrane glycoprotein
IIb/a (GPIIb/IIIa) (Laurent et al., 1999; Sudo, et al., 2003). Accordingly, the compounds with
3

an ability to increase cyclic AMP formation and inhibit Akt activation may have a potential to
inhibit platelet aggregation. Although many clinical antiplatelet drugs, such as aspirin, clopidogrel,
and tirofiban, were used in treating thromboembolic diseases, they still have some defects and
limitations (Tantry et al., 2005). After screening the effects of these new synthesized compounds on
platelet aggregation, we found that the compound, MP407, exerts a potent antiplatelet activity
mainly through elevation of cyclic AMP production, therefore regulating Akt/GSK3 cascade and
VASP phosphorylation. Similarly, inhibition of ADP-induced pulmonary thrombosis in mice by
MP407 was also observed.
2. Materials and methods
2.1.Materials
Collagen
(type
1,
equine
tendon),
AA,
imidazole,
indomethacin,
3-isobutyl-1-methylxanthine (IBMX), and Fura-2/AM were purchased from Sigma Chemical
Company (St. Louis, MO, USA). The 2’5’-dideoxy-adeonsine (2’5’ddAdo) was purchased
from Biomol company (Plymouth Meeting, PA, USA). Thromboxane B2 (TXB2), prostaglandin
E2 (PGE2), and cyclic AMP enzyme-linked immunosorbent assay (ELISA) kits were purchased
from Cayman Chemical Company, (Ann Arbor, MI, USA). The antibodies against p38MAPK,
phospho-p38 MAPK(Thr180/Tyr182), phospho (Ser) PKC substrate (p47), AKT,
phospho-AKT(Ser473), GSK3β, posopho-GSK3β, VASP, phospho-VASP(Ser157) were
purchased from Cell Signaling (Danvers, MA, USA). The compound of MP407,
5-(2-hydroxy-3-(4-methoxybenzyl) aminopropoxy-3,3-dinethyl-1,3-dihydroindol-2-one, was
provided by Prof, An-Rong Lee and it was synthesized as described previously (Lee et al.
1995). Briefly, p-Anisidine (1) was protected with benzyl bromide under basic conditions and
then reacted with bromine in acetic acid at 10 – 15℃ to obtain 2 (90%) that was treated with
equivalent amount of sodium hydride at 0-5℃ until the reaction was complete to give 3 (78%).
The compound 3 was cyclized to get 4 in dry toluene by treatment with tributyltin hydride
4

(TBTH), catalyzed with azobisisobutyronitrile (AIBN) under a reflux condition (87%).
Compound 4 was hydrolyzed by heating with 47% HBr to get 5 (66%). Treatment of 5 with
excessive epichlorohydrin afforded 6 (42%) that was then reacted with 4-methoxybenzylamine
under a reflux condition to obtain target MP407 (48%) (Fig.1). The crude MP407 was further
purified by column chromatography (silica gel, CH2Cl2/THF/MeOH: 12:1:1). Recrystallization
yielded pure MP407 that was characterized by mp (174) and specific spectra data (ir, nmr, and
ms). The requirements for elemental analysis (purity) for C, H, and N were within  0.4% of
the theoretical values and for HPLC chromatogram was essentially one peak. The MP407 was
dissolved in dimethylsulfoxide (DMSO) for subsequent tests. All other chemicals and reagents
were purchased from Sigma Chemical Company.
2.2.Platelet aggregation
The present study and all procedures of mice were approved by the Ethical Committee of
Animal Experiments (102069-A), Tzu Chi University. Blood was withdrawn from rabbit
marginal vein mixed with anticoagulant, EDTA (100 mM ,14:1 v/v) and centrifuged at 160 g,
25°C for 10 min to obtain platelet rich plasma (PRP). Then, platelet suspension was prepared
from the PRP according to the washing procedures described previously (Lai et al., 2010). The
platelet pellets were finally suspended in Tyrode's solution containing CaCl2 (1 mM), NaCl
(136.8 mM), KCl (2.7 mM), NaHCO3 (11.9 mM), MgCl2 (2.1 mM), NaH2PO4 (0.4 mM),
glucose (10 mM), and bovine serum albumin (0.35%). Platelet concentration was counted by
Coulter counter (Model ZM) and adjusted to 3.0x108 platelets/ml. The platelet aggregation was
measured turbidimetrically at 37°C with constant stirring at 1000 rpm by using an
aggregometer (Model 560, Chrono-Log Corporation, Havertown, PA, USA). The platelet
suspension was preincubated with drugs or solvent control (DMSO) for 3 min followed by
addition of collagen (10 g/ml) or AA (100M) for 6 min. The platelet aggregation was
evaluated by measuring the peak height of the aggregation curves and expressed as the
5

percentage of maximal aggregation. To avoid the effect of the solvent on the platelet
aggregation, the final concentration of DMSO was fixed at 0.5% (v/v).
2.3.Lactate dehydrogenase assay
Level of lactate dehydrogenase (LDH), acting as an index of platelet damage, was
measured. Briefly, platelets were preincubated with MP407 (10, 25 M) for 10 min and
centrifugalized at 10,000g for 5 min. Then, the supernatant was incubated with phosphate
buffer, containing 0.2 mg -NADPH for 20 min at room temperature followed by addition of
100 l pyruvate solution and the absorbance wavelength was read at 340 nm. LDH released
was compared with the total LDH activity of platelets dissolved in 0.1% Triton X-100.
2.4.Measurement of cyclic AMP and TXB2 levels
Platelet suspensions (3×108/ml) were incubated with drugs or solvent control at 37°C for 3
min, and the reaction was stopped by adding 10 mM EDTA and immediately boiling for 3 min.
After centrifugation at 10,000g for 5 min, the levels of cyclic AMP in the supernatants were
determined by ELISA kits, respectively. For determination of TXB2 formation, platelets were
preincubated with drugs or solvent control at 37°C for 3 min followed by addition of agonists
for 6 min. After boiling for 6 min, the amounts of TXB2 in the supernatants were measured by
using ELISA kit.
2.5.Measurement of COX activity
Various concentrations of MP407 were preincubated with platelets at 37℃ for 3 min in the
presence of imidazole (1 mM), an inhibitor of thromboxane synthase, then AA (100 M) was
added for another 6 min and boiled for 3 min. The amount of PGE2 in the samples was
measured using an ELISA kit to reflect the COX activity.
2.6.Western blotting
The isolated platelet proteins (20 g) of different groups were separated in 8 %
SDS-polyacrylamide gels and electrotransferred using semi dry transfer (Bio-Rad Laboratories
6

Inc., Hercules, California, USA). Then, various primary antibodies were incubated with the
transferred membranes for 1.5 h. A peroxidase-conjugated secondary antibody was then added
in a PBS-Tween 20 and incubation for 1 h. The protein bands were detected by the enhanced
chemiluminescence chemiluminescence (ECL) reagent (Amersham International Plc.,
Buckinghamshire, UK).
2.7.Measurement of Ca2+ mobilization in platelets
The PRP (3.0x108 platelets/ml) was incubated with Fluo-2 AM (5 M) for 30 min at 37°C
in the dark followed by centrifugation at 500g for 10 min. Then, the pellets were suspended in
2 ml Tyrode solution. The fluorescence intensity of 20,000 platelets per sample was analyzed
using a flow cytometer (FACScan, Becton Dickinson, Heidelberg, Germany).
2.8.Measurement of platelet surface glycoprotein IIb/IIIa expression
After platelet suspension was incubated with CD41/CD61-FITC that was raised against a
platelet glycoprotein IIb/IIIa (GPIIb/IIIa) complex, various drugs and collagen (10 μg/ml) was
added for 10 min. The reaction was stopped by adding 500 μl of 1% paraformaldehyde, and the
fluorescence intensity of 20,000 platelets of samples was analysed using a flow cytometer.
2.9.ADP-induced pulmonary thrombosis in mice
The thrombotic mice model was performed as described previously (Chen et al., 2015). The
ICR mice (20-25g) were randomly divided into 3 groups, including (1) the DMSO (solvent
control), (2) MP407 (2 mg/kg, i.p.)-treated mice, (3) MP407 (4 mg/kg, i.p.)-treated mice, and
each group contained 10 mice. After administration of solvent or MP407 for 30 min, the
thrombi occluded in the pulmonary vessels was induced by intravenous injection of ADP (300
mg/kg). The numbers of paralysis and death of each group were recorded at 24 h after ADP
injection.
2.10.
The bleeding time assay
After the mice were treated with solvent or MP407 (2 or 4 mg/kg, i.p.) for 15 min, the tail
7

was transected 3 mm from the tip and immersed into saline at 37℃. The accumulated bleed
time was recorded over a 20-min period.
2.11.
Statistical analysis
The data was expressed as means and their standard errors. One-way ANOVA with post hoc
Bonferroni test was used for statistical analysis. Results were considered significant difference
at a value of P<0.05.
3. Results
3.1.MP407 increased cyclic AMP formation but inhibited platelet aggregation
Treatment with MP407 significantly increased cyclic AMP formation both in resting and
collagen-activated platelets, but did not affect the cyclic GMP level (data not shown). The
elevation of cyclic AMP formation caused by MP407 in activated platelets was markedly
enhanced by forskolin, an adenylate cyclase activator. However, simultaneous addition of
3-Isobutyl-1-methylxanthine (IBMX), a cyclic AMP phosphodiesterase inhibitor, did not
further enhance the cyclic AMP formation (Table 1). Moreover, MP407 dose-dependently
inhibited the platelet aggregation induced by collagen or AA with IC50 values of 7.2±0.6 and
2.5±0.3 M, respectively. However, blocking cyclic AMP synthesis with 2’5’ddAdo (200 M),
an adenylate cyclase inhibitor, greatly diminished the antiplatelet activity of MP407 (Fig. 2),
suggesting the involvement of cyclic AMP in its antiplatelet activity. The effects of MP407
were not due to the cytotoxicity evidenced by no significant release of LDH (data not shown).
3.2.Effects of MP407 on TXB2 production and COX activity
To investigate the antiplatelet mechanisms of MP407, the TXB2 production was measured.
Our results revealed that MP407 markedly inhibited collagen-induced TXB2, a stable
metabolite of TXA2, production, which was reduced by 2’5’-ddAdo. However, MP407 did not
affect AA-induced TXB2 production (Table 2). In the presence of imidazole, MP407 had no
significant effect on the PGE2 production in AA-stimulaed platelets. On the contrary, addition
8

of indomethacin, a COX inhibitor, strongly decreased the PGE2 production in AA-stimulated
platelets (Table 3). Thus, the inhibition of TXB2 production by MP407 may be not due to
directly suppressing COX activity.
3.3.Effects of MP407 on Akt, GSK3 and VASP phosphorylation
To explore the molecular mechanisms of MP407, the important mediators for regulating
platelet aggregation, such as Akt, GSK3, VASP and p38MAPK were examined. As shown in
Fig. 3A, MP407 inhibited Akt and GSK3 phosphorylation and increased the VASP
phosphorylation(Ser157) in collagen-stimulated platelets, and the effects of MP407 were
reversed by addition of 2’5’ddAdo. Similarly, the inhibitory effects of MP407 on
collagen-induced p38MAPK and p47 phosphorylation were attenuated by 2’5’ddAdo (Fig. 3B).
These findings suggest that cyclic AMP involves MP407-regulaed platelet signaling.
3.4.MP407 reduced Ca2+ mobilization and platelet surface GPIIb/IIIa expression
The increase of intracellular Ca2+ mobilization and the GPIIb/IIIa expression is essential for
platelet aggregation. In this study, we confirmed that collagen-evoked Ca2+ mobilization in
Fluo-2-loaded platelets was inhibited by MP407 (Fig. 4A). Moreover, MP407-mediated
reduction of Ca2+ mobilization was similar in the presence of external Ca2+ (1 mM) or in the
Ca2+-free solution (data not shown). Compared to collagen-stimulated alone platelets, MP407
dose-dependently reduced platelet surface GPIIb/IIIa expression, and it was attenuated by
2’5’-ddAdo (Fig. 4B). This highlights the importance of cyclic AMP on the inhibition of
platelet activation by MP407.
3.5.Effects of MP407 on ADP-induced pulmonary thrombosis and bleeding time
Compared to vehicle-treated group, pre-treatment with MP407 greatly reduced the numbers
of paralysis and death caused by ADP injection (Table 4), supporting that MP407 exhibits an
antithrombotic effect in vivo. No significant difference was observed between MP407 (2 or 4
mg/kg, i.p.)-treated mice and the normal mice (11.9±1.8 min, 10.8±1.7 min versus 10.5 ±1.4
9

min), suggesting that MP407 may not have an obvious bleeding risk.
4. Discussion
pro-coagulant
cardiovascular disorders. It is well known
that cyclic AMP-elevating agents have an antiplatelet activity (Hayashi et al., 2009), implying
that development of this type of compounds is a promising strategy to discover novel
antiplatelet agents. In this study, we confirmed that MP407 exhibits a potent antiplatelet
activity due to elevation of cyclic AMP formation in collagen-stimulated platelets. The amount
of cyclic AMP is controlled by its synthesis and degradation mediated by adenylate cyclase and
cyclic AMP phosphodiesterase, respectively. Based on the fact that the increase of cyclic AMP
formation caused by MP407 was markedly enhanced by simultaneous addition of forskolin but
not by IBMX, we propose that inhibiting cyclic AMP phosphodiesterase activity may be a
major mechanism accounting for the increased cyclic AMP formation.
Blocking cyclic AMP formation with 2’5’-ddAdo greatly diminished the antiplatelet
activity of MP407, indicating that elevation of cyclic AMP involves its actions on platelet
aggregation. Next, the role of cyclic AMP and its-regulated signaling on the antiplatelet activity
of MP407 were investigated. The PI3K/Akt cascade modulates several cellular functions,
including metabolism, proliferation, survival, angiogenesis, and platelet aggregation (Sussman
et al., 2011; Guidetti et al., 2015; Chen et al., 2004). Upon PI3K activation, pyruvate
dehydrogenase kinase isozyme-1 (PDK1) and Akt, a family of serine/threonine kinases, are
recruited to the membrane, therefore stimulating Akt phosphorylation (activation) by PDK1,
which in turn induces platelet aggregation (Kim et al., 2001). Importantly, cyclic AMP can
10

suppress Akt phosphorylation by inhibiting PDK1 activity via preventing PDK1 translocation
to the plasma membrane (Kim et al., 2001). The collagen-induced phosphorylation of
Akt(Ser473) was inhibited by MP407, which is consistent with other cyclic AMP-elevating
agents (Hayashi et al., 2009). Similarly, the above effects of MP407 were markedly reversed by
2’5’-ddAdo. These findings indicate that cyclic AMP-dependent responses are closely linked to
the inhibition of MP407 on Akt activity and platelet aggregation. Moreover, attenuating
Akt-induced phosphodiesterase 3A (PDE3A) activation may be another possible mechanism for
enhancing platelet cyclic AMP level by MP407 (Zhang et al., 2007) by preventing cyclic AMP
degradation.
The GSK3, a serine-threonine kinase, has two isoforms (and , and the GSK3an
active form, is expressed predominantly in platelets. The platelets come from GSK3+/− mice
display increased platelet aggregation, dense granule secretion, fibrinogen binding, and thrombi
formed (Li et al., 2008), suggesting that GSK3is capable of inhibiting platelet aggregation.
Importantly, Akt also inhibits GSK3 activity by phosphorylating GSK3at Ser9 (van Weeren
et al., 1998). Thus, inhibition of GSK3Serphosphorylation by MP407 may be associated
with suppression of Akt activity, while the event was abolished by 2’5’-ddAdo. The functions
of VASP, an actin- and profilin-binding protein, are largely controlled by the phosphorylation
at the specific sites. The serine 157 and serine 239 of VASP are preferentially phosphorylated
by cyclic AMP and cyclic GMP-dependent kinases (PKA and PKG), respectively (Aszodi et al.,
1999), which ultimately suppresses the affinity of VASP for contractile protein filamentous
actin and fibrinogen binding to GPIIb/IIIa, thereby inhibiting platelet aggregation (Horstrup et
al., 1994). Our data showed that MP407 increased the VASP (Ser157) phosphorylation without
affecting the phosphorylation of VASP (Ser239) (data not shown). Collectively,
MP407-regulated the activity of GSK3and VASP in platelets is through a cyclic AMP
dependent manner.
11

Activation of p38 mitogen-activated protein kinase (p38MAPK) is thought a crucial
stimulator for collagen-induced platelet aggregation by activating PLA2/AA/TXA2 pathway
(Coulon et al., 2003). Of note, the PLA2/AA/TXA2 cascade is also suppressed by cyclic AMP
(Xing et al., 1999). A marked increase of p38MAPK phosphorylation in collagen-stimulated
platelets was inhibited by MP407, while the effect was attenuated by 2’5’ddAdo. Interestingly,
we found that only the TXB2 production induced by collagen not by AA was inhibited by
MP407 without affecting the COX activity evidenced by no significant change on AA-induced
PGE2 formation in imidazole-treated platelets. These results imply that the inhibition of TXB2
production by MP407 may mainly attribute to reduction of AA release and not due to directly
suppressing COX activity. The PLC is a key enzyme responsible for the generation of IP3 and
DAG by hydrolyzing the phosphatidylinositol 4,5-bisphosphate (PIP2). Then, DAG activates
PKC, causing substrate p47 phosphorylation and platelet activation. Importantly, cyclic AMP is
able to inhibit the functions of PLC by reducing PIP2 resynthesis (Ryningen, et al., 1998). In
this study, the increased p47 phosphorylation in collagen-stimulated platelets was inhibited by
MP407, and it was attenuated by 2’5’ddAdo. Therefore, the antiplatelet activity of MP407 may
be, at least partly, mediated by cyclic AMP-dependent suppression of p38MAPK and PKC
activity.
A rise of [Ca2+]i caused by Ca2+ release from intracellular stores and/or Ca2+ entry through
plasma membrane channels is required for agonist-induced platelet granule secretion and
aggregation (Rink et al., 1990). Activation of cyclic AMP/PKA is known to reduce
agonist-evoked platelet [Ca2+]i by phosphorylating ATP-dependent calcium pump, leading to
Ca2+ uptake into the dense tubular system, and inhibition of calcium release from the dense
tubular system via attenuating PLC-induced IP3 generation (Kim et al., 2014; Lazarowski et al.,
1989). Accordingly, the inhibition of the rise of Ca2+ mobilization in activated platelets by
MP407 may be related to elevation of cyclic AMP level. Moreover, MP407-mediated reduction
12

of Ca2+ mobilization was similar in the presence or absence of external Ca2+ (1 mM), implying
that the effect is mainly due to suppressing Ca2+ release from intracellular stores.
An exposure of platelet surface GPIIb/IIIa is considered as a common final step of platelet
aggregation and clot retraction through binding to soluble fibrinogen and bridging platelets
(Shattil, et al., 1998; Coller, 2001). Importantly, cyclic AMP also plays a crucial role on the
inhibition of platelet surface GPIIb/IIIa expression and activation by suppressing Akt activity
and GSK3 phosphorylationSer, but elevating VASP phosphorylation (Ser157) (Chen et al.,
2004; Horstrup et al., 1994). As expected, MP407 dose-dependent inhibited platelet surface
GPIIb/IIIa expression via a cyclic AMP dependent manner. In summary, cyclic AMP-dependent
inhibition of Akt, GSK3 p38, and p47 phosphorylation, and enhancement of VASP
phosphorylation(Ser157), thereby attenuating TXA2 production, Ca2+ mobilization, and
GPIIb/IIIa expression may all account for the antiplatelet activity of MP407 (Fig. 5).
Previous studies have confirmed that dihydropyridazinone derivatives such as indolidan
exhibit positive inotropic and vasodilatory activities, and ameliorate cardiovascular diseases, in
particular congestive heart failure (Corder et al., 1992; Amsallem et al., 2005). However, some
adverse effects, such as tachycardia were found after treatment with indolidan and its
analogues. In this study, we designed and synthesized various non-steroidal and
non-catecholamine cardiotonics that have a core structure deviated from the classical
dihydropyridazinone-containing compounds. Among these synthetic compounds, we have
demonstrated that MP407 has an inotropic activity in isolated ventricular tissues of Mongrel
dogs (<10% change in contractile force at 10 μM) and an antiplatelet activity mainly through
elevation of cyclic AMP formation. Notably, administration of MP407 markedly attenuated the
numbers of paralysis and death due to severe pulmonary thrombosis induced by ADP injection.
In addition, treatment with MP407 did not prolong the bleeding time, suggesting that MP407
has a low hemorrhagic risk. Taken together, MP407 may be a potential candidate or lead
13

compound for developing new inotropic, antiplatelet or antithrombotic agents for platelet
hyperactivity-related disease therapy.
Conflict of interest
The authors declare that there is no conflict of interests.
Acknowledgement
This work was supported by the grants from Tzu Chi University (TCMMPSP104-09-02).
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cascade
in
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Figure 1. Synthesis of MP407.
Figure 2. Effects of MP407 on collagen or AA-induced platelet aggregation. Washed platelets
were preincubated with Tyrode solution, MP407 (1-25 M) or MP407 combination with
℃
2’5’ddAdo (200 M) at 37
for 3 min, then collagen (10 g/ml) or AA (100M) was added
to induce the platelet aggregation. The inhibition of platelet aggregation was expressed as the
**
percentage of maximal aggregation. Data were expressed as mean ± S.E.M. (n=5). P<0.01,
***P<0.001 compared to respective MP407+collagen-treated platelets.
Figure 3. Effects of cyclic AMP on MP407-mediated platelet signaling. Washed platelets were
preincubated with Tyrode solution, MP407 or MP407 (25 M) combination with 2’5’ddAdo at
℃
37 for 3 min, followed by addition of collagen for 6 min. The protein expression of target
genes in various groups were examined (A, B). The blots were representative examples of five
experiments.
Figure 4. Effects of MP407 on intracellular Ca2+ mobilization and platelet membrance
GPIIa/IIIb expression. Fluo2-AM-loaded platelets or washed platelets were pretreated with
MP407 in the presence or absence of 2’5’ddAdo, followed by addition of collagen. The
intracellular Ca2+ mobilization and the expression of GPIIa/IIIb in platelets were analyzed by a
**
flow cytometer. Data were expressed as mean ± S.E.M. (n=5). P<0.01, ***P<0.001 compared
#
to the collagen-stimulated alone platelets. P<0.01 compared to respective MP407 (25
M)+collagen-treated platelets.
18

Figure 5. A schematic diagram represents the antiplatelet activity of MP407. MP407 increases
cyclic AMP formation through inhibition of cyclic AMP phosphodiesterase (PDE) activity,
resulting in suppressing Akt, GSK3 p38, and p47 phosphorylation, and increasing VASP
phosphorylation(Ser157), which in turn attenuates TXA2 production, Ca2+ mobilization, and
platelet surface GPIIb/IIIa expression. Taken together, the antiplatelet activity of MP407 may
be mediated by cyclic AMP-dependent responses.
H3CO
Br
O
H3CO
Br
H3CO
i) BnBr/NaHCO3/H2O
NaH/C2H5CCOCl/THF
N
ii) Br2/HOAc
90%
NHBn
78%
NH2
Bn
O
2
3
1
87%
TBTH/AIBN/toluene
O
HO
H3CO
O
47% HBr
epichlorohydrin/K2CO3/MeOH
42%
O
O
O
N
66%
N
N
Bn
Bn
Bn
4
5
6
4-methoxybenzylamine/MeOH
48%
H3CO
OH
O
H
N
O
N
Bn
MP407
Figure 1
19

Figure 2
A
B
20

Figure 3
A
B
Resting
50
40
30
20
10
0
Collagen
Collagen
+ MP407 (10 μM)
,
#
**
***
***
Collagen
+ MP407 (25 μM)
R
0
10
25
25
MP407 (M)
2'5'ddAdo
(200 M)
+
Collagen
collagen
+ MP407 (25 μM)
+ 2’5’ddAdo
Figure 4
21

Figure 5
Table 1. Effects of MP407 on cyclic AMP formation
Treatment
Cyclic AMP (pmol/108 platelets)
Resting
MP407 (25 μM)
11.5 ± 1.2
15.1 ± 1.2a
9.8 ± 0.4
collagen (10 μg/ml)
+ MP407 (10 μM)
+ MP407 (25 μM)
+ forskolin (10 μM)
+ forskolin + MP407 (25 μM)
+ IBMX (50 μM)
+ IBMX + MP407 (25 μM)
13.8 ± 0.7b
18.2 ± 0.6c
42.1 ± 0.4d
98.7 ± 5.6d,
16.9 ± 0.5b
20.1 ± 1.9c
e
Tyrode solution, MP407, forskolin, IBMX or the combination of forskolin + MP407 or IBMX
22

+ MP407 were preincubated with platelets at 37℃ for 3 min. Then, collagen was added for 6
min followed by boiling for 3 min. The amounts of cyclic AMP in supernatant were measured.
Values are presented as the mean ± S.E.M. (n=5). aP<0.05 vs resting platelets, bP<0.05, cP<0.01,
e
dP<0.001 vs collagen-stimulated alone platelets, P<0.001 vs collagen and MP407 (25
M)-treated platelets.
Table 2. Effects of MP407 on TXB2 formation induced by collagen or AA
TXB2 (ng/ml) formation induced by
Treatment
collagen (10 μg/ml)
1.2 ± 0.0
AA (100 μM)
1.4 ± 0.0
Resting
Control
168.0 ± 9.8
671.7 ± 32.7
651.3 ± 45.5
613.7 ± 28.5
MP407 (1 μM)
MP407 (5 μM)
MP407 (10 μM)
MP407 (25 μM)
MP407 (25 μM)
+ 2’5’-ddAdo (200 M)
29.0 ± 4.0a
6.7 ± 1.7b
38.7 ± 4.5a,c
Washed platelets were preincubated with Tyrode solution or MP407 at 37℃ for 3 min. Then,
collagen or AA was added for another 6 min to trigger TXB2 formation. Data were expressed as
the mean ± S.E.M. (n=5). aP<0.01, bP<0.001 vs collagen-stimulated alone platelets. cP<0.01 vs
collagen + MP407 (25 μM)-treated platelets.
23

Table 3. Effects of MP407 on cyclooxygenase activity
Treatment
PGE2 (ng/ml)
Resting
0.02 ± 0.00
AA (100 μM)
+ imidazole (1 mM)
+ imidazole + MP407 (1 μM)
+ imidazole + MP407 (5 μM)
+ imidazole + MP407 (10 μM)
+ indomethacin (2 μM)
139.1 ± 10.4
119.1 ± 24.6
123.6 ± 8.3
124.1 ± 15.0
11.6 ± 4.8
Tyrode solution or MP407 was preincubated with platelets at 37℃ for 3 min in the presence of
imidazole (1 mM). Then, AA was added for another 6 min followed by boiling for 3 min. The
amounts of PGE2 in the samples were measured using ELISA kit. Data were expressed as the
mean ± S.E.M. (n=5).
Table 4. MP407 inhibited ADP-induced pulmonary thrombosis in mice.
groups
Numbers of paralysis and death
Vehicle
MP407 (2 mg/kg, i.p.)
MP407 (4 mg/kg, i.p.)
9
4
2
The mice were treated with solvent or MP407 for 30 min followed by injection of ADP (300
mg/kg) to induce pulmonary thrombosis. Then, recorded the numbers of paralysis and death at
24 h after ADP injection. Each group contained 10 mice.
24