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\begin{document}

\title{Pharmacological actions of Dieckol on the modulation of platelet
functions and thrombus formation via integrin \selectlanguage{greek}α\selectlanguage{english}IIb\selectlanguage{greek}β\selectlanguage{english}3 and cAMP signaling\selectlanguage{english}}



\author[1]{Muhammad Irfan}%
\author[2]{Tae-Hyung  Kwon}%
\author[3]{Hyuk-Woo Kwon}%
\author[1]{Man Hee Rhee}%
\affil[1]{Kyungpook National University}%
\affil[2]{Chuncheon Bio Industry Foundation }%
\affil[3]{Far East University}%


\vspace{-1em}



  
  \date{May 07, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and purpose: Dieckol is a phlorotannin that can be found in
seaweeds, particularly in Eisenia bicyclis (brown algae) and is known to
have anti-oxidant, anti-inflammatory, and anti-microbial properties. It
also possesses anti-thrombotic and pro-fibrinolytic activities; however,
the mechanistic aspects of anti-platelet and anti-thrombotic activity
are yet to be explored. Experimental approach: We investigated the
pharmacological effects of dieckol on the modulation of platelet
functions using human, rat, and mice models. Inhibitory effects of
dieckol on platelet aggregation were assessed using platelet-rich plasma
and washed platelets, followed by measurement of dense granule
secretions, fibrinogen binding to integrin \selectlanguage{greek}α\selectlanguage{english}IIb\selectlanguage{greek}β\selectlanguage{english}3, fibronectin adhesion
assay, platelet spreading on immobilized fibrinogen, and clot
retraction. Cyclic nucleotide signaling events were evaluated, such as
cyclic-AMP production followed by vasodilator-stimulated phosphoprotein
(VASP) stimulation. The in vivo anti-thrombotic potential was evaluated
in mice using an acute pulmonary thromboembolism model and tail bleeding
assay. Key Results: Dieckol markedly inhibited platelet aggregation and
granule secretion; furthermore, it down-regulated integrin
\selectlanguage{greek}α\selectlanguage{english}IIb\selectlanguage{greek}β\selectlanguage{english}3--mediated inside-out and outside-in signaling events, including
platelet adhesion, spreading, and clot retraction, whereas it
upregulated the cAMP--PKA--VASP pathway. Dieckol-treated mice
significantly survived the thrombosis than vehicle-treated mice, without
affecting hemostasis. Histological examinations of lungs revealed
minimum occluded vasculature in dieckol-treated mice. Conclusion and
implications: Taken together, dieckol possesses strong anti-platelet and
anti-thrombotic properties and is a potential therapeutic drug candidate
to treat and prevent platelet-related cardiovascular disorders.%
\end{abstract}%



\sloppy


\textbf{Abbreviations}

ACD, acid citrate dextrose; ADP, adenosine diphosphate; ATP, adenosine
triphosphate; cAMP, 3',5'-cyclic adenosine monophosphate; DMSO, dimethyl
sulfoxide; PRP, platelet rich plasma; VASP, vasodilator-stimulated
phosphoprotein; WP, washed platelets.

\textbf{What is already known}

\begin{itemize}
\tightlist
\item
  Impaired hemostasis is a common complication of anti-platelet drugs
  while dealing with thrombotic events.
\item
  Surface integrins especially
  \selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}play critical role in modulation
  of platelet functions and thormbus formation.
\end{itemize}

\textbf{What does this study adds}

Dieckol inhibts platelet aggregation and thrombus formation without
affecting hemostasis by regulating integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3} and cAMP signaling.

\textbf{What is the clinical significance}

Dieckol can be a potential candiate to treat and prevent
platelet-related cardiovascular disorders.

\textbf{Introduction}

Cardiovascular disease (CVD) is considered among the leading causes of
death globally. In 2013, coronary heart disease caused almost one in
seven deaths, and heart failure caused one in nine deaths in the United
States (Mozaffarian et al., 2016; Shafiq, Tatinati, Ang, \& Veluvolu,
2016). In Europe, CVD causes 42\% of deaths in men and 52\% of deaths in
women (Pagidipati \& Gaziano, 2013). CVD includes a number of diseases
and conditions like atherosclerosis, coronary heart disease, heart
attack, hypertension, and stroke. Several risk factors are involved in
the pathophysiology of CVD, but hyperactive platelets are considered
main culprits as they cause hemostatic plug and stenosis of vasculature,
which may also lead to ischemic stroke. Under normal physiological
conditions, platelets play a vital role in maintaining hemostasis;
however, aberrant or hyper-activation of platelets may cause thrombotic
complications that contribute to the development of thrombosis or other
platelet-related CVD (Andrews \& Berndt, 2004; J.-H. Shin, Kwon, Rhee,
\& Park, 2019). Pharmacological suppression of platelets has been proven
effective in the treatment and prevention of CVD; however, available
treatment options are often hampered by side effects and complications.
For example, aspirin is a great antiplatelet agent but may cause severe
gastric ulcers or prolonged bleeding, while clopidogrel sometimes causes
aplastic anemia and thrombocytopenic purpura (Barrett et al., 2008),
while a significant part of population is resistant to these most
commonly used anti-platelet (Ferguson, Dokainish, \& Lakkis, 2008; Wang,
Bhatt, \& Topol, 2006), thus, this situation necessitates the
development of safer and efficacious therapeutic regimes with no or
minimum side effects. In this regard, ethno-medicinal application could
be one of the best approaches to hamper CVD and CVD-related
complications (Badimon, Vilahur, \& Padro, 2010). Natural herbal
compounds and the Mediterranean diet reportedly possess
anti-atherosclerotic and anti-thrombotic properties due to the presence
of bioactive compounds affecting platelet function, thus reducing the
future risk of thrombosis (Irfan, Kim, \& Rhee, 2020; J.-H. Kim, 2018;
Vilahur \& Badimon, 2013).

Adenosine diphosphate (ADP) is an important \emph{in vivo} agonist,
affecting two types of receptors in the platelet membrane, namely
P\textsubscript{2}Y\textsubscript{1} and
P\textsubscript{2}Y\textsubscript{12}. ADP causes calcium mobilization,
platelet shape change, and rapid reversible platelet aggregation through
P\textsubscript{2}Y\textsubscript{1}-mediated signaling, whereas it
triggers granule secretion and stable platelet aggregation via
P\textsubscript{2}Y\textsubscript{12}-mediated signaling pathway
(Michelson, 2008), Mitogen-activated protein kinases (MAPK) and the
PI3K/Akt signaling pathway have been proven to be involved in
agonist-stimulated platelet aggregation and thrombus formation (Kansra
\& Shukla, 2000). Cyclic nucleotide signaling triggers the elevation of
cyclic-AMP\emph{via} the phosphorylation of vasodilator-stimulated
phosphoprotein (VASP), which is followed by the modulation of integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}-mediated inside-out and
outside-in signaling, thus subsequently resulting in platelet activation
and stable thrombus formation (Li, Ajdic, Eigenthaler, \& Du, 2003).
Compounds that inhibit these signaling cascades would be very useful in
treating and preventing platelet-related cardiovascular disorders.

We have previously investigated the anti-platelet and anti-thrombotic
effects of \emph{Eisenia bicyclis} extract and found that it contained
several phlorotannins, including dieckol (Irfan, Kwon, Yun, Park, \&
Rhee, 2018). Dieckol~is a phlorotannin that can be found in sea weeds,
particularly in \emph{Eisenia bicyclis} (brown algae), \emph{Ecklonia
cava} , and\emph{Ecklonia stolonifera} (Kwon, Kim, Kim, \& Park, 2013).
It is known to possess antimicrobial (Eom, Kim, \& Kim, 2012),
anti-oxidant (T. Shin, Ahn, Hyun, Kim, \& Moon, 2014) and
anti-inflammatory (Yayeh et al., 2014) properties. A few years ago, Kim
et al. (T. H. Kim, Ku, \& Bae, 2012) reported that this compound
possesses anti-thrombotic and pro-fibrinolytic activities; however,
mechanistic aspects of its antiplatelet effects have not been explored
yet. Herein, we investigated its potential anti-platelet and
anti-thrombotic effects and elucidated its underlying mechanistic
aspects using human, rat, and mouse models.

\textbf{Materials and methods}

Extraction and isolation of dieckol from Eisenia bicyclis

Dieckol and other phlorotannins were procured from the ethyl acetate
fraction of \emph{E. bicyclis} (brown algae) as our previously described
detailed method (Irfan, Kwon, et al., 2018; Yayeh et al., 2014). After
purification using the HPLC system, several phlorotannins were procured
including dieckol, and their structure was determined by NMR. Chemical
structure of dieckol is shown in \textbf{Figure 1E} and other
phlorotannins \textbf{(Figure S1)} .

Chemicals and reagents

Collagen and ADP were obtained from Chrono-Log (Havertown, PA, USA).
Thrombin, epinephrine, fura-2-acetoxymethyl ester (fura-2/AM), and
dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). An ATP Assay Kit was obtained from Biomedical Research Service
Center (Buffalo, NY, USA). Cyclic-AMP and cyclic-GMP EIA kits were
obtained from Cayman Chemicals (Ann Arbor, MI, USA), and the serotonin
EIA kit was purchased from Labor Diagnostika Nord (Nordhorn, Germany).
Fibrinogen Alexa Fluor 488 conjugate was obtained from Molecular Probes
(Eugene, OR, USA). Fibronectin Adhesion Assay Kit was procured from Cell
Biolabs, Inc. (San Diego, CA, USA). Antibodies against
vasodilator-stimulated phosphoprotein (VASP, RRID:AB\_2213542),
phospho-VASP\textsuperscript{ser157} (RRID: AB\_331569), \selectlanguage{greek}β-\selectlanguage{english}actin (RRID:
AB\_330288), phospho-ERK (phospho-p44/42, RRID: AB\_331646), ERK
(p44/42, RRID: AB\_330744), stress-activated protein kinase (SAPK)/c-Jun
N-terminal protein kinase (JNK, RRID: AB\_330894), phospho-SAPK/JNK
(RRID: AB\_330894), Phspho-P38\textsuperscript{MAPK} (RRID: AB\_331641),
P38\textsuperscript{MAPK} (RRID: AB\_330713), phospho-PI3K (phospho-p85,
RRID: AB\_659940), PI3K (p85, RRID: AB\_329869), phospho-Akt (RRID:
AB\_329825), and Akt (RRID: AB\_329827) were acquired from Cell
Signaling Technology (Beverly, MA, USA), and PKA\selectlanguage{greek}αβγ \selectlanguage{english}(RRID: AB\_2691446)
was purchased from ThermoFisher Scientific (Massachusetts, USA). Dieckol
was dissolved in DMSO for further use in experiments. All chemicals were
of reagent grade.

Animals

Seven-week-old male Sprague--Dawley rats (RRID: RGD\_734476), weighing
250--260 g and 7 weeks old C57BL/6 mice (RRID: MGI:6199956) weighing
20--22 g were purchased from Orient Co. (Seoul, Korea). Rats and mice
were acclimatized for 1 week in an air-conditioned room with a 12 h/12 h
light/dark cycle at a temperature and humidity of 22\selectlanguage{ngerman}°C ± 2°C and 55\% ±
10\%, respectively. Animal experiments were conducted following IACUC
guidelines, and the protocols were approved by the Ethics Committee of
College of Veterinary Medicine, Kyungpook National University, Daegu,
Republic of Korea. Animal studies are reported in compliance with the
ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, \& Altman, 2010;
McGrath \& Lilley, 2015) and with the recommendations made by the
\emph{British Journal of Pharmacology} with all effort was taken to
minimize the number of animals used and their suffering.

Human Platelets Preparation

The human platelet rich plasma (PRP) from healthy human volunteers who
had provided informed consent was obtained from the Korean Red Cross
Blood Center (KRBC, Daegu, Republic of Korea), and its experimental use
was approved by the KRBC and the Korea National Institute for Bioethics
Policy Public Institutional Review Board (PIRB) at Kyungpook National
University, Daegu, Republic of Korea (PIRB17-1019-03). Anticoagulated
PRP was centrifuged for 10 min at 120\emph{g} to remove red blood cells
and centrifuged for 15 min at 360\emph{g} to obtain platelet pellets.
The platelets were either resuspended in platelet-poor plasma (PPP) or
washed twice with washing buffer (138-mM NaCl, 2.7-mM KCl, 12-mM
NaHCO\textsubscript{3}, 0.36-mM NaH\textsubscript{2}PO\textsubscript{4},
5.5-mM glucose, and 1-mM Na\textsubscript{2}EDTA, pH 6.5). The platelets
were then resuspended in suspension buffer (138-mM NaCl, 2.7-mM KCl,
12-mM NaHCO\textsubscript{3}, 0.36-mM
NaH\textsubscript{2}PO\textsubscript{4}, 0.49-mM MgCl\textsubscript{2},
5.5-mM glucose, 0.25 \% gelatin, pH 6.9) to a final concentration of 5 ×
10\textsuperscript{8}/mL. All of the above procedures were carried out
at room temperature (23°C ± 1°C).

Preparation of washed rat platelets

Whole blood was collected from rats \emph{via} heart puncture and
transferred to a tube containing acid citrate dextrose (ACD)
anticoagulant solution. Blood was centrifuged at 170\emph{g} for 7 min
to obtain PRP. Then, PRP was further centrifuged at 350\emph{g} for 7
min to isolate washed platelets (WP). The platelet concentration was
adjusted at 3 × 10\textsuperscript{8} cells/mL using Tyrode's buffer
(137-mM NaCl, 12-mM NaHCO\textsubscript{3}, 5.5-mM glucose, 2-mM KCl,
1-mM MgCl\textsubscript{2}, and 0.3-mM NaHPO\textsubscript{4}, pH 7.4)
for use in platelet aggregation assays. All preparation procedures were
performed at room temperature.

Platelet aggregation assay and scanning electron microscopy analysis

Anti-platelet activity of dieckol was assessed using standard \emph{in
vitro} techniques. Light transmission aggregometry was performed to
assess the effects of dieckol on agonist-stimulated platelet aggregation
using a Chrono-Log aggregometer (Havertown, PA, USA) as previously
described (Irfan, Jeong, Kwon, et al., 2018). Briefly, PRP or washed
platelets were pre-incubated with various concentrations of either
dieckol or vehicle for 1 min at 37°C in the presence of calcium chloride
(1-mM CaCl\textsubscript{2} for washed platelets and 10 mM for PRP).
Then, the mixture was stimulated with an agonist (ADP, collagen or
thrombin) and incubated for 5 min with continuous stirring. Vehicle
(DMSO) concentration was maintained at less than 0.1\%.

A field emission scanning electron microscope (SU8220; Hitachi, Japan)
was used to assess platelet aggregation by obtaining ultrastructure
images at the Center for Scientific Instruments, Kyungpook National
University, Daegu, Korea. For the platelet aggregation assay, the
platelet mixture was fixed with 0.5\% paraformaldehyde followed by
osmium tetroxide treatment; then, it was dehydrated with ascending
concentrations of ethanol, freeze dried, and then scanned as previously
described (Irfan, Jeong, Kwon, et al., 2018).

{[}Ca\textsuperscript{2+}{]}\textsubscript{i}mobilization

The intracellular calcium ion concentration
({[}Ca\textsuperscript{2+}{]}\emph{\textsubscript{i}} ) was evaluated
with fura-2/AM as previously described (Irfan, Jeong, Saba, et al.,
2018). Briefly, rat PRP was incubated with 5-\selectlanguage{greek}µ\selectlanguage{english}M fura-2/AM for 60 min at
. Then fura-2-loaded washed platelets were prepared and pre-incubated
with dieckol for 1 min at in the presence of 1-mM CaCl\textsubscript{2}
and were subsequently stimulated with for 3 min. Fura-2 fluorescence in
the cytosol was measured using a spectrofluorometer (F-2500, Hitachi,
Japan) as previously described by Schaeffer and Blaustein (Schaeffer \&
Blaustein, 1989) using the following formula:
{[}Ca\textsuperscript{2+}{]}\emph{\textsubscript{i}}= 224 nM \selectlanguage{ngerman}× (\emph{F}
\selectlanguage{english}-\emph{F\textsubscript{min}} )/(\emph{F\textsubscript{max}} \selectlanguage{english}- \emph{F}
), wherein 224 nM is the dissociation constant of the
fura-2-Ca\textsuperscript{2+}complex, and \emph{F\textsubscript{min}}and
\emph{F\textsubscript{max}} represent fluorescence intensity levels at
very low and very high Ca\textsuperscript{2+} concentrations,
respectively.

ATP release assay

Human PRP or washed rat platelets were incubated with either vehicle or
various concentrations of dieckol and CaCl\textsubscript{2} (1-mM
CaCl\textsubscript{2} for washed platelets and 10-mM for PRP) for 1 min
at and then stimulated with . Supernatants were obtained by centrifuging
the platelet mixture after the aggregation reaction terminated, and ATP
secretion was measured in a luminometer (GloMax 20/20, Promega, Madison,
WI, USA) using an ATP assay kit (Biomedical Research Service Center).

Measurement of Serotonin Concentration

Human PRP was pre-incubated with either vehicle or various
concentrations of dieckol in the presence of 10-mM
CaCl\textsubscript{2}for 1 min at and then stimulated with ADP for 5 min
under continuous stirring. After the aggregation reaction was
terminated, the supernatant was obtained by centrifuging the platelet
mixture, and serotonin secretion was measured with the Serotonin-EIA
kit, according to manufacturer's instructions.

Measurement of cyclic-AMP and cyclic-GMP

Washed rat platelets were pre-incubated with various concentrations of
dieckol, forskolin, , or vehicle in the presence of 1-mM
CaCl\textsubscript{2} for 1 min at and were then stimulated with for 5
min in a platelet aggregometer. The reaction was terminated, and the
solution was centrifuged at 2000\emph{g} for 5 min at , and the cAMP and
cGMP levels in the supernatant were determined with EIA Kits (Cayman
Chemical, Ann Arbor, MI, USA) according to the manufacturer's protocol.

Flow cytometry

Fibrinogen binding was measured using flow cytometry as previously
described (Irfan, Jeong, Kwon, et al., 2018). Fibrinogen binding to
integrin \selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3} was assessed using
fluorescein isothiocyanate-labeled anti-fibrinogen antibodies in the
presence or absence of various concentrations of dieckol. Washed rat
platelets were pre-treated with dieckol and stimulated with an agonist
for 5 min. Platelets were then fixed with 0.5\% paraformaldehyde for 30
min at 4\selectlanguage{ngerman}°C, washed three times with phosphate-buffered saline (PBS), and
then suspended in PBS. Flow cytometric analysis was performed using the
FACS Aria\selectlanguage{english} III flow cytometer(r) (Becton Dickinson Immunocytometry
Systems, San Jose, CA, USA), and data were analyzed using CellQuest
software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).

Fibronectin adhesion assay

To determine the effect of dieckol on platelet adhesion, fibronectin
adhesion assay was performed using Fibronectin Adhesion Assay Kit (Cell
Biolabs, Inc., San Diego, CA, USA), according to the manufacturer's
protocol as previously described (Irfan, Jeong, Kwon, et al., 2018).
Briefly, pretreated, washed rat platelets (1 x 10\textsuperscript{6}
cells/mL) were incubated in fibronectin-coated wells for 90 min at 37degC
and then washed with DDW. Next, the adhered cells were stained with
crystal violet for 10 min and then washed with DDW. Finally, the stained
adhered cells were solubilized with extraction solution. This lysate
solution was transferred to a 96-well plate, and absorbance was measured
at 540 nm with a spectrophotometer.

Platelet spreading on immobilized fibrinogen and confocal microscopy

To examine the inhibitory effect of dieckol on platelet adhesion and
spreading, platelets were allowed to adhere on immobilized fibrinogen as
previously described (Sakthivel Vaiyapuri et al., 2015). Coverslips were
coated with 100-\selectlanguage{greek}µ\selectlanguage{english}g/mL fibrinogen in PBS overnight at 4\selectlanguage{ngerman}°C, followed by
blocking with 1\% bovine serum albumin for 1 h at room temperature.
Washed rat platelets (1 × 10\textsuperscript{6}cells/mL) were pretreated
with vehicle, dieckol, or GR144053 {[}a glycoprotein IIb/IIIa (integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}) receptor antagonist{]} in the
presence of 1-mM CaCl\textsubscript{2} and were allowed to spread for 60
min at 37\selectlanguage{ngerman}°C on fibrinogen-coated cover glass; then, they were washed and
stained with SYTO-9 before analysis using a Nikon A1-R-confocal
microscope.

Clot retraction in human and rat platelet-rich plasma and kinetics of
clot retraction

Human PRP (400 \selectlanguage{greek}µ\selectlanguage{english}L) was pre-incubated with vehicle, dieckol, or Y-27632
(Rock inhibitor), in the presence of 10-mM CaCl\textsubscript{2} and
then stimulated with thrombin (1 U/mL). Clot retraction was observed for
90 min at room temperature and photographed. Rat PRP (250 \selectlanguage{greek}µ\selectlanguage{english}L) was mixed
with red blood cells (5 \selectlanguage{greek}µ\selectlanguage{english}L) and vehicle, dieckol, or Y-27632. Then,
Tyrode's buffer was added to bring the volume to 1 mL as previously
described (S. Vaiyapuri et al., 2013). Thrombin (1 U/mL) was added to
initiate fibrin clot formation, and then, clot retraction was observed
for 2 h at room temperature. Clot weight was measured as a marker of
clot retraction. Kinetics of clot retraction were assessed as previously
described (Misztal, Przes\selectlanguage{polish}ł\selectlanguage{english}aw, Rusak, \& Tomasiak, 2013). Pictures were
processed with Image-J software and clot surface areas were plotted as a
percentage of clot retraction.

Immunoblotting

Immunoblot analysis was performed as previously described. Washed
platelets were pre-incubated with various concentrations of dieckol in
the presence of 1-mM CaCl\textsubscript{2} for 1 min at and were then
stimulated with Platelet aggregation was terminated by adding lysis
buffer (PRO-PREP; iNtRON Biotechnology, Seoul, Korea), and then, protein
concentration was estimated using the BCS assay (PRO-MEASURE; iNtRON
Biotechnology). Total platelet proteins were separated in a 10\%
SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with
5\% skim milk, probed with respective primary and secondary antibodies,
and visualized using enhanced chemiluminescence.

Survival rate study

A collagen- and epinephrine-induced acute pulmonary thromboembolism
experiment was carried out in mice as previously described (Canobbio et
al., 2013). Briefly, mice were randomly divided (10 mice in each group)
and administered saline, dieckol (30 mg/kg i.p.), or ASA (100 mg/kg
i.p.) once a day for three days. Then, 1 h after the final injection,
the mice were challenged with 0.1 mL of a mixture containing collagen
(500 \selectlanguage{greek}μ\selectlanguage{english}g/mL) and epinephrine (60 \selectlanguage{greek}μ\selectlanguage{english}g/mL) by smooth injection into one of
the tail veins. The mortality of mice in each group was monitored for 10
min, and the data are presented as the percentage of surviving animals
in each treatment group. At the end of each experiment, surviving
animals were euthanized with an overdose of anesthesia.

Lung histology

Two minutes after the thrombotic challenge, mice were quickly euthanized
with an overdose of anesthesia, and the lungs were perfused with a
fixing solution (10\% formalin buffered with calcium carbonate). The
trachea was ligated and harvested together with the lungs, and the
organs were soaked in cold saline and fixed with 10\% formalin for 24 h.
The lungs were then embedded in paraffin, and 5--6-\selectlanguage{greek}µ\selectlanguage{english}m-thick sections
were cut and stained with hematoxylin and eosin for thrombus
identification. The samples were observed under a light microscope (Axio
Lab A1; Carl Zeiss MicroImaging, Jena, Germany) by a histopathologist
blinded to the treatment, and a minimum of 10 fields were observed in
each specimen.

Mice tail bleeding assay

Male mice were randomly divided (five mice in each group) and
administered with saline, dieckol (30 mg/kg i.p.), or ASA (50 mg/kg
i.p.) once a day for three days. After one hour of last administration,
mice were anesthetized, and tail bleeding assay was performed as
previously described (Endale et al., 2012). Briefly, the tail was
pre-warmed for 2--3 min in a normal saline solution at 37\selectlanguage{ngerman}°C. The tail
was precisely transected 3--5 mm from the tip to induce bleeding. The
distal portion of the tail was vertically immersed in normal saline
solution at 37°C. The bleeding time was recorded as the time between the
start of transection to cessation of bleeding.

Statistical analysis

To assess statistical significance among observed differences, the
obtained data were analyzed by one--way analysis of variance (ANOVA)
followed by \emph{post-hoc} Dunnett's test (SAS Institute Inc., Cary,
NC, USA). The given data are presented as the mean ± standard deviation
(SD). P-values of 0.05 or less were considered statistically
significant.

\textbf{Results}

Phlorotannin from Eisenia bicyclis inhibits platelet aggregation

Five phlorotannin compounds (6,6'-bieckol, 6,8'-bieckol, 8,8'-bieckol,
dieckol, and phlorofucofuroeckol-A) were obtained from the ethyl acetate
fraction of the \emph{Eisenia bicyclic} extract as previously described
(Irfan, Kwon, et al., 2018). We investigated whether these individual
compounds inhibit agonist-induced platelet aggregation. Consequently, we
found that all of them inhibited platelet aggregation, with dieckol
being the most potent inhibitor of platelet aggregation \textbf{(Fig.
S1)} . Thus, we selected dieckol for further study and elucidated its
underlying mechanism of modulating platelet functions.

Dieckol inhibited agonist-stimulated platelet aggregation

Anti-platelet activity of dieckol was assessed using standard \emph{in
vitro} techniques. Light transmission aggregometry was performed to
assess the effects of dieckol on agonist-stimulated platelet
aggregation. We evaluated our compound against different ligands
(collagen, ADP and thrombin) and found that in each case, dieckol
inhibited agonist-induced rat platelet aggregation in a dose-dependent
manner \textbf{(Fig. 1A)} (\emph{p} \textless{} 0.001). Similarly,
dieckol showed significant inhibitory tendency against collagen, ADP,
and thrombin-stimulated human platelet aggregation in a dose-dependent
manner \textbf{(Fig. 1B)} (\emph{p} \textless{} 0.001 \&
\emph{p}\textless{} 0.05). Dieckol also proved to strongly and
significantly inhibit platelet aggregation in human PRP in a
dose-dependent manner\textbf{(Fig. 1C) (} \emph{p} \textless{} 0.001).
We confirmed the anti-platelet effects of dieckol on platelet
aggregation using electron microscopy. \textbf{Figure 1D} shows platelet
shape changes in vehicle or dieckol-treated platelets. Figure 1D(a)
displays resting platelets, whereas Figure 1D(b) shows fully activated
platelets and fibrin meshwork in vehicle-treated platelets stimulated
with ADP while increasing concentrations of dieckol inhibited shape
change and platelet aggregation in a dose-dependent manner {[}Figure
1D(c-e){]}.

Dieckol inhibited {[}Ca\textsuperscript{2+}{]}\textsubscript{i}
elevation, and dense granule secretion

To assess whether our compound inhibited
{[}Ca\textsuperscript{2+}{]}\emph{\textsubscript{i}} mobilization, an
intracellular calcium mobilization assay was performed. As shown
in\textbf{Figure 2A} , dieckol markedly blocked
{[}Ca\textsuperscript{2+}{]}\textsubscript{\emph{i}} mobilization in
ADP-stimulated rat platelets in a dose-dependent manner
(\emph{p}\textless{} 0.001). Dieckol has also shown inhibitory effects
on ATP secretion (a marker for dense granule secretions). Dieckol
significantly hampered ATP release (\emph{p} \textless{} 0.01 and
\emph{p} \textless{} 0.001) in rats' washed platelets (\textbf{Fig. 2B}
) and human PRP (\textbf{Fig. 2D} ) in a dose-dependent manner. Similar
inhibitory effects were observed on serotonin secretion from platelets
in agonist-stimulated human PRP pre-treated with various concentrations
of dieckol than vehicle treated platelets (\textbf{Fig. 2C} ).

Dieckol attenuates fibrinogen binding and fibronectin adhesion

Our results show that various concentrations of dieckol significantly
inhibited fibrinogen binding to integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3} in a dose-dependent manner
(\textbf{Fig. 3A\&B} ). Also, it was observed that dieckol significantly
inhibited platelet adhesion to fibronectin in a dose-dependent
manner\textbf{(Fig. 3C)} and also attenuated platelet spreading on
fibrinogen\textbf{(Fig. 3D)} . These results suggest that dieckol blocks
integrin-mediated inside-out signaling and the early phase of outside-in
signaling.

Dieckol inhibits clot retraction

A chain of signaling events started after fibrinogen bound to integrin
proteins; this transduced signals into the cell and initiated platelet
spreading in the early phase, whereas in later phases of thrombus
formation, this triggers clot retraction, which may be termed as
late-stage outside-in signaling (Tucker, Sage, \& Gibbins, 2012). Our
results show that dieckol treatment significantly limited
thrombin-induced clot retraction in rat PRP\textbf{(Fig. 3E-G)} and in
human PRP \textbf{(Fig. 3H)} .

Dieckol attenuated MAPK and PI3K/Akt phosphorylation

To explain the underlying mechanism of the effect of dieckol on
ADP-stimulated platelet activation, we investigated the modulation of
the MAPK pathway. \textbf{Figure 4} shows that dieckol significantly
attenuated the activation of ERK and JNK but not of
p38\textsuperscript{MAPK}; furthermore, it also inhibited PI3K/Akt
phosphorylation in agonist-induced platelets in a dose-dependent manner.

Dieckol elevates intracellular cAMP level

Platelet activation suppresses cyclic-AMP generation and cyclic
nucleotide-dependent protein kinase activity, whereas the elevation of
intracellular cAMP levels has been proven to inhibit platelet
aggregation. Therefore, we measured the inhibitory effects of dieckol on
cAMP and cGMP generation and platelet activation. Our initial screening
results showed that dieckol only elevates cAMP levels and had no effect
on cGMP levels \textbf{(Fig. 5A)} . Further, cAMP levels were found to
be dose-dependently increased in the presence of increasing
concentrations of dieckol (\textbf{Fig. 5B} ).

Dieckol enhanced VASP and PKA\selectlanguage{greek}αβγ \selectlanguage{english}phosphorylation

VASP is a regulator of actin dynamics in platelets and is a substrate of
cyclic nucleotide (cAMP/cGMP)-dependent protein kinases (\emph{i.e.} ,
PKA/PKG), and the activation of VASP can hinder platelet aggregation due
to the enhanced activity of these kinases. Therefore, we investigated
the effect of our compound on VASP phosphorylation. The results showed
that dieckol and forskolin (\emph{i.e.} , a positive regulator of
adenylyl cyclase) significantly enhanced the phosphorylation of total
VASP as well as VASP\textsuperscript{ser157} (a site preferred by PKA).
To further explore whether these effects are mediated by the
cAMP--PKA--VASP\textsuperscript{ser157} pathway, we evaluated PKA\selectlanguage{greek}αβγ
\selectlanguage{english}phosphorylation, which was significantly augmented by various ascending
concentrations of dieckol (\textbf{Fig. 5C} ).

Dieckol protects mice from thrombosis without affecting hemostasis

Our results clearly show that dieckol considerably protected mice from
thrombosis and offered a significantly better survival rate (8/10)
compared with the saline-treated control group (3/10) mice. Histological
analysis revealed the occluded pulmonary vessels formed by platelet
aggregation in the control group, whereas dieckol treatment greatly
reduced thrombosis events. The number of occluded vessels was also
observed to be far lesser in intervention group mice than in control
group mice. Bleeding time was also increased in aspirin-treated mice
group, whereas saline and dieckol treatment did not prolonged tail
bleeding \textbf{(Fig. 6)} . These results suggest that dieckol
protected mice from thrombosis without affecting hemostasis.

\textbf{Discussion}

Platelets play an important role in sealing injured blood vessels by
forming a hemostatic plug and maintain hemostasis; however,
pathophysiological activation may lead to serious thrombotic
complications, including development of atherothrombosis and
thromboembolism. Cardiovascular disorders are among leading causes of
morbidity and mortality in modern societies, particularly in Western
countries, due to high rates of heart attack and stroke. Although
several pharmacological drugs are available to treat platelet-induced
CVD, they are associated with adverse effects and often lack efficacy in
some patients (S. Vaiyapuri et al., 2013). It has been reported that
natural therapeutic regimes and nutraceuticals are effective in reducing
the CVD risk, and these approaches have gained clinical interest in
treating CVD because of their proven pharmacological effects (Badimon et
al., 2010; Vilahur \& Badimon, 2013). Dieckol is a natural phlorotannin
found in marine algae, particularly in\emph{Eisenia bicyclis} , which is
a popular sea food in Korea, Japan, and China and reportedly possesses
several pharmacological properties. In the present study, we evaluated
its anti-platelet and anti-thrombotic potential. First, we analyzed
various phlorotannins contained in the\emph{Eisenia bicyclis} extract
and found dieckol to be most potent among them.

Collagen, thrombin and ADP induce platelet aggregation by triggering
granule secretion, platelet activation, and aggregation. These agonists
or adhesive proteins cause platelet activation by triggering various
intracellular signaling pathways which can be divided into the
following: (i) early receptor signaling, (ii) merging common signal
transduction and amplification, (iii) inside-out signaling (mainly
involves conformational change in integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3} structure and causes fibrinogen
binding and platelet adhesion), and (iv) outside-in signaling, which
enhances platelet aggregation and triggers clot retraction (Bye et al.,
2015; Estevez \& Du, 2017). Our results showed that dieckol
significantly inhibited platelet aggregation in human PRP and washed
platelets and also inhibited rat platelet aggregation induced by various
agonists.

Platelet contains dense (\selectlanguage{greek}δ\selectlanguage{english}) and alpha (\selectlanguage{greek}α\selectlanguage{english}) granules. \selectlanguage{greek}δ \selectlanguage{english}granules include
ADP, ATP, Ca2+, and serotonin, whereas \selectlanguage{greek}α \selectlanguage{english}granules contain fibronectin,
fibrinogen, vWF, and P-selectin. Several signaling events contribute to
granule secretion, such as: (i) Src family kinase signaling (considered
as early signaling in platelet activation), (ii) intracellular calcium
ion mobilization, (iii) cyclic nucleotide signaling (\emph{e.g.} ,
cAMP-VASP-PKA pathway); inside-out and outside-in signaling, (iv) PKC
activation, (v) MAPK and PI3K/Akt activation, and (vi) RhoA pathway
activation. All of the above mentioned signaling events contribute to
granule secretion, which causes enhanced platelet activation and shape
changes (Estevez \& Du, 2017).
{[}Ca\textsuperscript{2+}{]}\emph{\textsubscript{i}} mobilization plays
a key role in the modulation of platelet function that triggers other
signaling cascades involved in coagulation, primes platelet aggregation,
and thrombus formation (Smith, Negrelli, Manek, Hawes, \& Viera, 2015).
In our results, dieckol markedly reduced
{[}Ca\textsuperscript{2+}{]}\emph{\textsubscript{i}} mobilization and
ATP and serotonin secretion, which suggest its potential anti-platelet
mechanism.

Platelets express various integrin proteins on their surface, including
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}; conformational changes in this
protein during platelet activation causes fibrinogen to bind to it,
which is considered a marker for inside-out signaling (Calderwood,
2004). Fibronectin, which is another adhesive ligand, binds to integrins
and enhances platelet aggregation by forming cohesive aggregates that
lead to stable thrombus formation in vasculature by recruiting signaling
proteins in the vicinity of integrin cytoplasmic tails (Varga-Szabo,
Pleines, \& Nieswandt, 2008). Dieckol attenuated fibrinogen binding,
fibronectin adhesion, and platelet spreading. Further, small GTP
proteins belong to the Rho family and control the cell shape, motility,
and proliferation; further, Rho kinases (ROCKs) were found to activate
Rho-A followed by cytoskeletal changes via myosin light chain reaction
(MLC) phosphorylation. In addition, a role of Rho kinase in mediating
clot retraction has been previously described using a Rho kinase
inhibitor (Y-27632) (Liao, Seto, \& Noma, 2007). Dieckol inhibited clot
retraction in both rat and human PRP. These results suggest that dieckol
potentially inhibits platelet aggregation by inhibiting granule
secretion and modulating integrin \selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}
signaling. \textbf{Figure 7}summarizes the potential anti-platelet
effects of dieckol on platelet intracellular signaling.

Adam et al. (Adam, Kauskot, Rosa, \& Bryckaert, 2008) have established
that MAPK molecules (ERK, JNK and p38\textsuperscript{MAPK}) activated
in platelets by various agonists contribute to platelet aggregation.
PI3K/Akt has been reported to lie downstream of SFK and is known to
recruit and activate PLC\selectlanguage{greek}γ\selectlanguage{english}2, which consequently liberates IP3 and DAG,
thus leading to calcium mobilization, granule secretion, and platelet
aggregation (Senis, Mazharian, \& Mori, 2014). Our results have shown
that dieckol inhibited platelet activation by inhibiting the
phosphorylation of ERK, JNK, and PI3K/Akt \textbf{(Fig. 7)} .

Elevated levels of cAMP have been known to inhibit platelet activation
via increased phosphorylation of VASP\textsuperscript{ser157}; moreover,
VASP is a major substrate for cAMP-dependent PKA. VASP stimulation is
also known to inhibit integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}signaling (Coles et al., 2002).
Our results clearly state that dieckol upregulated the cAMP--PKA--VASP
pathway in a dose-dependent manner, thus indicating its potential
inhibitory mechanism on platelet activation \textbf{(Fig. 7)} .

Strong coagulation factor (\emph{e.g.} , thrombin, ADP, or collagen
combined with epinephrine)-induced thrombosis models via systematic
administration have been widely used to evaluate anti-thrombotic drugs
(Miao, Liu, \& Wang, 2010). Studies have shown that the intravenous
administration of collagen and epinephrine into mice veins induced
platelet aggregation and very lethal pulmonary thromboembolism in a
short period of time (Huang, Wang, Luo, Xie, \& Shi, 2007).
Konstantinides et al\emph{.}(Konstantinides, Scha\selectlanguage{english}fer, Neels, Dellas, \&
Loskutoff, 2004) reported that the histological analysis of
collagen--epinephrine-induced pulmonary thromboembolism in the lungs
showed occluded vasculature rich in platelet thrombi. Anti-thrombotic
effect of dieckol was evaluated by challenging mice with
collagen--epinephrine intravenous injection, an established model to
study defects of platelet functions and thrombosis (Miao et al., 2010).
Pulmonary thromboembolism was observed in the majority of controlled
mice, whereas dieckol- and ASA-treated mice significantly survived the
thrombosis. Moreover, dieckol did not increase the bleeding time,
indicating that it modulated platelet function without affecting
hemostasis.

\textbf{Conclusion}

Dieckol significantly inhibited agonist-induced human and rat platelet
aggregation, granule secretion, and integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}-mediated inside-out and
outside-in signaling; furthermore, it down-regulated MAPK and PI3K/Akt
pathways but upregulated the cAMP--PKA--VASP pathway. Moreover, it
protected mice from thrombosis without affecting hemostasis. Taken
together, these results indicate the clinical significance and potential
of dieckol as an anti-platelet and anti-thrombotic agent for treatment
and prevention of platelet-related cardiovascular disorders.

\textbf{Author Contributions}

MI and MHR designed and conceptualized the study. MI performed
experiments, analyzed data and wrote manuscript. H-WK and T-HK performed
partial experiments and analyzed data. MHR and T-HK supervised the
research work. All authors read and approved the final manuscript.

\textbf{Declaration of Transparency and Scientific Rigour}

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the BJP guidelines for Design \& Analysis and Animal
Experimentation, and as recommended by funding agencies, publishers and
other organizations engaged with supporting research.

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\textbf{Figure legends}

\textbf{Fig. 1.} \textbf{Dieckol inhibits agonist-induced platelet
aggregation} . Washed platelets (\textbf{A\&D} from rat;
\textbf{B\&C}from human) were pretreated with either dieckol or vehicle
for 1 min in the presence of 1-mM CaCl\textsubscript{2} and then
stimulated with collagen, ADP, or thrombin for 5 min. \textbf{(C)} Human
PRP was incubated with vehicle or various concentrations of dieckol in
the presence of 10-mM CaCl\textsubscript{2} for 1 min and then
stimulated with ADP (25~\selectlanguage{greek}µ\selectlanguage{english}M) for 5 min. \textbf{(D)} Scanning electron
microscopy was performed to assess the extent of platelet aggregation
and the ultrastructure. Representative scanning electron microscopy
images (5000\selectlanguage{ngerman}×) of platelets treated with dieckol or vehicle {[}(a)
resting, (b) vehicle, (c) dieckol (2.5~\selectlanguage{greek}µ\selectlanguage{english}M), (d) dieckol (5 \selectlanguage{greek}µ\selectlanguage{english}M), and (e)
dieckol (10 \selectlanguage{greek}µ\selectlanguage{english}M){]} and then stimulated with ADP (b-e). \textbf{(E)}
Chemical structure of dieckol. The graphs show the mean \selectlanguage{ngerman}± SD of at least
five independent experiments in duplicate. **\emph{p} \textless{} 0.01
and ***\emph{p} \textless{} 0.001 vs. control. WP, washed platelets.

\textbf{Fig. 2.} \textbf{Dieckol inhibits ADP-stimulated
{[}Ca\textsuperscript{2+}{]}\emph{\textsubscript{i}} elevation, ATP
release, and serotonin secretion.} \textbf{(A)} Washed platelets were
loaded with a calcium fluorophore (5~\selectlanguage{greek}µ\selectlanguage{english}M, fura-2/AM) for 1 h. Fura
2/AM-loaded platelets were pre-treated with dieckol for 2 min at 37\selectlanguage{ngerman}°C
and stimulated with ADP (10~\selectlanguage{greek}µ\selectlanguage{english}M). \textbf{(B)} Washed platelets were
pre-treated with various concentrations of dieckol or vehicle for 1 min
in the presence of 1-mM CaCl\textsubscript{2} and stimulated with ADP
(10 \selectlanguage{greek}µ\selectlanguage{english}M) for 5 min. \textbf{(C-D)} Human PRP was pre-treated with various
concentrations of dieckol or vehicle for 1 min in the presence of 10-mM
CaCl\textsubscript{2} and then stimulated with ADP (25 \selectlanguage{greek}µ\selectlanguage{english}M) for 5 min.
The concentration of ATP was assessed in a luminometer using an ATP
assay kit and serotonin secretion was assessed using an EIA kit. Results
represent the mean \selectlanguage{ngerman}± SD of experiments performed on four independent
days. * \emph{p} \textless{} 0.05, **\emph{p} \textless{} 0.01, and
***\emph{p} \textless{} 0.001 vs. control.

\selectlanguage{greek}\textbf{Fig. 3. Dieckol blocks integrin
α\textsubscript{IIb}β\textsubscript{3}-mediated inside-out and
outside-in signaling in human and rat platelets}\selectlanguage{english} . \textbf{(A)} Flow
cytometry was used to measure fibrinogen binding to rat platelets after
treatment with (a) resting, (b) vehicle, (c) dieckol (2.5~\selectlanguage{greek}µ\selectlanguage{english}M), (d)
dieckol (5 \selectlanguage{greek}µ\selectlanguage{english}M), (e) dieckol (10 \selectlanguage{greek}µ\selectlanguage{english}M), and (f) EGTA (10 \selectlanguage{greek}µ\selectlanguage{english}M), and then
platelet suspension was stimulated with ADP (b-f). \textbf{(B)} Bar
graph summarizing the inhibitory effect of dieckol on fibrinogen binding
to integrin \selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}. \textbf{(C)} Washed
rat platelets were pre-incubated with different concentrations of
dieckol, vehicle, or GR155053 (an
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}inhibitor) and then loaded onto a
fibronectin-coated plate and incubated for 90 min at 37\selectlanguage{ngerman}°C. Then, the
assay was performed as described in the methods section. Bar graphs
summarizing the inhibitory effect of dieckol on fibronectin adhesion.
\textbf{(D)} Washed rat platelets were pre-incubated with vehicle (a),
dieckol (b), or GR155053 (c) and then loaded onto a fibrinogen-coated
cover glass and incubated for 60 min at 37°C, washed and stained with
SYTO-9, and scanned under confocal microscope. \textbf{(E-G)} Effect of
dieckol on clot retraction in rat PRP was analyzed \emph{in vitro} .
Thrombin (1 U/mL) was added to initiate fibrin clot formation, and clot
retraction was observed for 2 h at room temperature. \textbf{(E)} A
representative image of clot retraction at 90 min in the presence or
absence of dieckol or Y-27632 (Rho kinase inhibitor) and, \textbf{(G)}
Kinetics of clot retraction were measured by Image-J software and clot
surface areas were plotted as a percentage of retraction. \textbf{(H)}
Human PRP was pre-incubated with vehicle (a), dieckol (b) or Y-27632
(c). Thrombin (1 U/mL) was added to initiate fibrin clot formation, and
clot retraction was observed for 90 min at room temperature. (B, C, F,
G) Results represent the mean ± SD of experiments performed on four
independent days. *\emph{p} \textless{} 0.05, **\emph{p} \textless{}
0.01, and ***\emph{p}\textless{} 0.001 vs. control.

\textbf{Fig. 4. Dieckol inhibited ADP-stimulated MAPK activation and
attenuated phosphorylation of PI3K/Akt in the
P\textsubscript{2}Y\textsubscript{12} signaling pathway.} Washed
platelets were pre-treated with different concentrations of dieckol and
stimulated with ADP. Following termination of platelet aggregation,
proteins were extracted from the platelets and analyzed for
phosphorylation of respective molecules by immunoblotting. Immunoblot
images are representative of the mean ± SD of experiments performed on
four independent days. * \emph{p} \textless{} 0.05,
**\emph{p}\textless{} 0.01, and ***\emph{p} \textless{} 0.001 vs.
control.

\textbf{Fig. 5.} \selectlanguage{greek}\textbf{Dieckol elevates cAMP production and enhances
VASP and PKAαβγ phosphorylation.}\selectlanguage{english} Washed rat platelets were pretreated
with various concentrations of dieckol, vehicle, forskolin or SNP for 1
min in the presence of 1-mM CaCl\textsubscript{2} and were then
stimulated with ADP for 5 min. \textbf{(A-B)} After terminating the
aggregation, the cAMP and cGMP immunoassays were performed using the
cAMP and cGMP EIA kits, according to the manufacturer's
protocol.\textbf{(C)} Proteins were extracted from the platelets and
analyzed for phosphorylation of total VASP,
p-VASP\textsuperscript{ser157}, and PKA\selectlanguage{greek}αβγ \selectlanguage{english}by immunoblotting. Shown are
representative immunoblot images and graphs of the means \selectlanguage{ngerman}± SD and the
data of at least four independent experiments. *\emph{p} \textless{}
0.05 and ***\emph{p} \textless{} 0.001 vs. control. R, resting; C,
control; FSK, forskolin; SNP, sodium nitroprusside.

\textbf{Fig. 6. Dieckol inhibits pulmonary thromboembolism in mice
without affecting hemostasis. (A-C)} Mice were pre-treated with saline,
dieckol (30 mg/kg i.p.), or ASA (100 mg/kg i.p.) once a day for 3 days
(n = 10 in each group). One hour after the final administration, mice
were challenged with a collagen + epinephrine mixture and checked for
survival at 10 min \textbf{(B)} . ***\emph{p} \textless{} 0.001 compared
to the agonist-treated group. Arrows show the microvasculature occluded
due to collagen-epinephrine induced thrombosis. \textbf{(C)} Graph shows
occluded lung microvasculature. At least ten fields were examined, and
the number of occluded vessels was counted in each group.
\textbf{(D)}Mice were administered dieckol (30 mg/kg), aspirin (a
positive control, 50 mg/kg), or saline (n = 5 in each group). Tail
bleeding assay was performed as described in method section. The graph
shows mean ± SD of at least five independent experiments performed.
***\emph{p} \textless{} 0.01 vs. control.

\textbf{Fig. 7. Summary of inhibitory effects of dieckol on platelet
intracellular signaling pathways.}

\textbf{Hosted file}

\verb`Figures.pptx`  available at \url{https://authorea.com/users/334567/articles/460534-pharmacological-actions-of-dieckol-on-the-modulation-of-platelet-functions-and-thrombus-formation-via-integrin-%CE%B1iib%CE%B23-and-camp-signaling}

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