Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets.

0.71 MB·English

by  Chang, Yi

Changetal.JournalofBiomedicalScience2013,20:4
http://www.jbiomedsci.com/content/20/1/4
RESEARCH Open Access
Caesalpinia sappan
Brazilin isolated from L. acts
as a novel collagen receptor agonist in human
platelets
Yi Chang1,2†, Steven Kuan-Hua Huang3†, Wan-Jung Lu4,5, Chi-Li Chung6,7, Wei-Lin Chen4,5, Shun-Hua Lu4,5,
Kuan-Hung Lin4,8* and Joen-Rong Sheu4,5*
Abstract
Background: Brazilin, isolated from theheartwood ofCaesalpinia sappan L., has been shown to possess multiple
pharmacologicalproperties.
Methods: In this study, platelet aggregation,flow cytometry, immunoblotting analysis, and electron spin resonance
(ESR) spectrometry were used to investigate theeffects of brazilin on platelet activation ex vivo. Moreover,
fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used inin vivo study.
Results: We demonstrated that relatively low concentrations ofbrazilin(1 to 10 μM) potentiated platelet
aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations ofbrazilin (20 to 50
μM)directlytriggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an
antagonist ofADP).It was not inhibited by yohimbine (anantagonist of epinephrine),by SCH79797 (an antagonist
ofthrombinprotease-activated receptor [PAR] 1), or by tcY-NH2 (anantagonist of PAR 4).Brazilindid not
significantly affect FITC-triflavin binding to theintegrin α β in platelet suspensions. Pretreatment of the platelets
IIb 3
withcaffeic acid phenethyl ester (anantagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies
raised against collagen receptor glycoproteinVI and integrin α β , respectively, abolished platelet aggregation
2 1
stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the
phosphorylation ofphospholipase C (PLC)γ2and Lyn, which were significantlyattenuated in thepresence ofJAQ1
and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. Anin vivo
mouse study showed that brazilin treatment (2and 4mg/kg)significantly shortened the occlusion time for platelet
plug formation inmesenteric venules.
Conclusion: To the best ofour knowledge, this study provides the first evidence that brazilinacts a novelcollagen
receptor agonist. Brazilin is a plant-based natural product, may offertherapeutic potentialas intended
anti-thrombotic agents for targeting of collagen receptors or to be used a useful toolfor the study of detailed
mechanisms incollagen receptors-mediated platelet activation.
Keywords: Brazilin, Collagen receptors, Lyn phosphorylation, Occlusion time, Platelet activation
*Correspondence:[email protected];[email protected]
†Equalcontributors
4DepartmentofPharmacology,CollegeofMedicine,TaipeiMedical
University,250Wu-HsingSt,Taipei11031,Taiwan
5GraduateInstituteMedicalSciences,CollegeofMedicine,TaipeiMedical
University,250Wu-HsingSt,Taipei11031,Taiwan
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Changetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Changetal.JournalofBiomedicalScience2013,20:4 Page2of11
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Background activation plays a crucial role in numerous cardiovascular
Brazilin (7,11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 andcerebrovasculardisorders.
(6H)-tetrol)isthemajorcomponentisolatedfromtheheart- Untilthisstudy,nodatahadbeenpublishedontheeffect
woodofCaesalpiniasappanL.(Leguminosae)(Figure1).C. of brazilin in platelet activation. One study reported that
sappan has long been widely used as an oriental traditional brazilin significantly inhibited thrombin-, collagen-, and
or folk medicine. It is considered an analgesic and anti- ADP-induced platelet aggregation in washed rat platelets
inflammatoryagentandhasbeenusedtotreatemmeniopa- [13]. By contrast, our preliminary study showed that
thy, sprains, and convulsions [1]; it has also been used to brazilin potentiated or stimulated platelet aggregation in
treat diabetic complications [2] and to improve blood circu- washed human platelets. This discrepancy might result
lation [3]. Extracts of C. sappan have been shown to exert from specie-specific characteristics of platelets. We thus
various pharmacological effects, including anti-hypercholes- systematicallyexaminedtheinfluenceofbrazilininhuman
terolemia, sedation, and depression of the central nervous platelets ex vivo and in platelet plug formation in vivo.
system [4]. In addition, it is an anti-hepatitis B surface anti- Thefindingswereusedtocharacterizethemechanismsof
gen (HBsAg) [5] and lowers the motility of human sperm brazilin-mediatedactivationinhumanplatelets.
[6]. Brazilin is also used as a natural red pigment for
histological staining [7]. Several studies have shown that the Methods
anti-hyperglycemic [8], anti-hepatotoxic [9], and anti- Materials
inflammatory effects of brazilin are caused by the inhibition ATP, caffeic acid phenethyl ester (CAPE), collagen (type I,
ofinduciblenitricoxidesynthase(NOS)inmacrophagecells bovine achilles tendon), heparin, fluorescein sodium,
[10],andvasorelaxationinducedbytheactivationofNOSin yohimbine, prostaglandin E (PGE ), arachidonic acid
1 1
endothelialcells[4]. (AA), ADP, thrombin, U73122, 5,5-dimethyl-1 pyrroline
Intravascular thrombosis is associated with several car- N-oxide (DMPO), and bovine serum albumin (BSA)
diovascular diseases. The initiation of an intraluminal were purchased from Sigma Chem. (St Louis, MO).
thrombosis is thought to involve platelet adherence and SCH79797 and trans-cinnamoyl-YPGKF-NH2 (tcY-NH )
2
aggregation. During normal circulation, platelets do not were obtained from TOCRIS Bioscience (Ellisville, MIS).
aggregate. However, when a blood vessel is damaged, pla- Anti-glycoprotein (GP) VI (JAQ1) and anti-integrin
teletsadheretothedisruptedsurfaceandreleasebiologic- α β (Sam.G4) monoclonal antibodies (mAbs) were
2 1
ally active constituents that induce aggregation [11]. obtained from Emfret Analytics (Würzburg, Germany);
Resting (circulating) plateletsare anuclear cells, discoidin anti-phospholipaseCγ2(PLCγ2)andanti-phospho(Tyr759)
shape, which originate from megakaryocytes in the bone PLCγ2 polyclonal antibodies (pAbs) from Cell Signaling
marrow. Platelets may be activated by various physio- (Beverly, MA); anti-Lyn and anti-phospho Lyn pAbs
logicalorpharmacologicalagents.Physiologicalagentsin- fromSantaCruz(SantaCruz,CA).TheHybond-Ppoly-
clude thrombin, collagen, ADP, platelet-activating factor vinylidene difluoride (PVDF) membrane, an enhanced
(PAF), and epinephrine, whereas pharmacological agents chemiluminescence (ECL) Western blotting detection
include calcium ionophores and cyclic endoperoxide ana- reagent and analysis system, horseradish peroxidase
logues. Upon activation, the platelets lose their discoid (HRP)-conjugated donkey anti-rabbit IgG, and sheep
shape and become more spherical, extending long, spiky anti-mouse IgG were from Amersham (Buckinghamshire,
pseudopods and bulky surface protrusions [12]. The vari- UK). The brazilin was purchased from MP Biomedical
ous agonists are thought to exert their effects by interact- (Solon, OH). We dissolved the brazilin in 0.5% dimethyl
ingwithspecificreceptorsonplateletmembranes.Platelet sulfoxide(DMSO)andstoreditat4°Cuntiluse.
Preparationofhumanplateletsuspensions
Human platelet suspensions were prepared as previously
described [14]. This study conformed to the principles
outlined in the Helsinki Declaration, and human volun-
teers gave informed consent. In brief, blood was col-
lected from healthy human volunteers who had taken no
medicine during the preceding 2 wk, and was mixed
with acid-citrate-dextrose (ACD) (9:1, v/v). After centri-
fugation, the supernatant (platelet-rich plasma; PRP) was
supplemented with PGE (0.5 μM) and heparin (6.4 IU/
1
ml) and incubated for 10 min. The mixture was then
centrifuged at 500 g; thereafter, the platelets were
Figure1Chemicalstructureofbrazilin. washed and suspended in aTyrode’s solution containing
Changetal.JournalofBiomedicalScience2013,20:4 Page3of11
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BSA (3.5 mg/ml). The final concentration of Ca+2 in the (25 and 50 μM), collagen (1 μg/ml) or a solvent control
Tyrode’ssolution was1mM. (0.5% DMSO) for 3 min. The reaction was allowed to
proceed for 5 min, followed by the addition of DMPO
Plateletaggregation (100 μM) fortheESR study.
A turbidimetric method was applied to measure platelet
aggregation [14], using a Lumi-Aggregometer (Payton, Fluoresceinsodium-inducedplateletthrombiin
Canada). Platelet suspensions (3.6 × 108 cells/ml) were mesentericmicrovesselsofmice
pretreated with or without reagents for 3 min, followed As described previously [14], mice were anesthetized,
by the addition of brazilin or various agonists to trigger and an external jugular vein was cannulated with PE-10
platelet activation. The reaction was allowed to proceed so that dye and medication could be administered by an
for at least 6 min, and the extent of aggregation was intravenous (i.v.) bolus. A segment of the small intestine
expressedinlight-transmissionunits. was placed onto a transparent culture dish for micro-
scopic observation. Venules (30 to 40 μm) were selected
Flowcytometricanalysis for irradiation to produce a microthrombus. Using the
Fluorescence-conjugated triflavin, an α β disintegrin, epi-illumination system, light from a 100-W mercury
IIb 3
was prepared as previously described [11]. Platelet sus- lamp was passed through a B-2A filter (Nikon, Tokyo,
pensions (3.6 × 108 cells/ml) were preincubated with Japan) with a DM 510 dichromic mirror (Nikon). Wave-
brazilin (25 and 50 μM) or a solvent control (0.5% lengths below 520 nm had been eliminated from the fil-
DMSO) for 3 min, followed by the addition of 2 μl of a tered light, which was used to irradiate a microvessel;
solution of FITC-triflavin (2 μg/ml). The suspensions the area of irradiation was approximately 100 μm in
were then assayed for fluorescein-labeled platelets, using diameter on the focal plane. Various dosages of brazilin
a flow cytometer (Beckman Coulter, Miami, FL). Data (2 and 4 mg/kg) or an isovolumetric solvent control
were collected from 50,000 platelets per experimental (0.5% DMSO) was administered 1 min after fluorescein
group, and the platelets were identified by their charac- sodium (15 μg/kg) administration. Five minutes after ad-
teristic forward and orthogonal light-scattering profiles. ministration of the fluorescein sodium, irradiation by fil-
All experiments were repeated at least 4 times to ensure tered light and the video timer were simultaneously
reliability. begun,andplateletaggregationwasobservedonatelevi-
sion monitor. The time lapse for inducing thrombus
Immunoblotting formation leading to the cessation of blood flow was
Washed platelets (1 × 109 cells/ml) were preincubated measured.
with reagents for 3 min, followed by the addition of ago-
nists to trigger platelet activation. The reaction was Statisticalanalysis
stopped, and platelets were immediately re-suspended in The experimental results are expressed as the means ±
200 μl of lysis buffer. Samples containing 80 μg of pro- S.E.M. and are accompanied by the number of observa-
tein were separated using a 12% sodium dodecylsulfate tions. Paired Student’s t-test was used to determine sig-
polyacrylamidegelelectrophoresis(SDS-PAGE);proteins nificant differences in the in vivo studies of platelet plug
were electrotransferred by semidry transfer (Bio-Rad, formation. The other experiments were assessed by the
Hercules, CA). Blots were blocked with TBST (10 mM method of analysis of variance (ANOVA). If this analysis
Tris-base, 100 mM NaCl, and 0.01% Tween 20) con- indicatedsignificantdifferencesamongthegroupmeans,
taining 5% BSA for 1 h and then probed with various then each group was compared using the Newman-
primary antibodies. Membranes were incubated with Keuls method. A p value of less than 0.05 was consid-
HRP-linked anti-mouse IgG or anti-rabbit IgG (diluted eredstatistically significant.
1:3000 in TBST) for 1 h. Immunoreactive bands were
detected using an ECL system. Bar graphs depicting Results
quantitative ratios were produced by scanning the react- Theinfluenceofbrazilinonplateletaggregationin
ive bands and quantifying their optical density using washedhumanplatelets
videodensitometry (Bio-profil; Biolight Windows Appli- Low concentrations of brazilin (1, 5, and 10 μM) poten-
cation V2000.01;Vilber Lourmat,France). tiated platelet aggregation in a concentration-dependent
manner induced by a sub-threshold concentration of col-
Measurementofhydroxylradicalsbyelectronspin lagen (0.1 μg/ml) in washed human platelets (Figure 2A).
resonance(ESR)spectrometry At higher concentrations (20, 30, and 50 μM), brazilin
The ESR method used a Bruker EMX ESR spectrometer directly triggered platelet aggregation in a concentration-
as described previously [15]. In brief, platelet suspen- dependent manner (Figure 2B). A high dose of brazilin
sions (3.6 × 108 cells/ml) were incubated with brazilin (50 μM) was equally potent in inducing platelet
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A B
brazilin
1 5 10
%)
0
1
collagen 20 30 50 ΔT(
brazilin min
C D
ATP
yohimbine
ATP yohimbine
%)
ADP brazilin epinephrine brazilin T(10
Δ
min
E
%)
0
1
T(
Δ
min
collagen 100
50
brazilin
Figure2Effectofbrazilininpotentiatingorstimulatinghumanplateletaggregationinwashedplateletsorplatelet-richplasma.
Washedplatelets(3.6×108/ml)wereincubatedwithbrazilin(1to50μM),eitherwith(A)orwithout(B)theadditionofcollagen(0.1μg/ml)to
triggerplateletaggregation.Inadditionalexperiments,washedplatelets(3.6×108/ml)werepreincubatedwith(C)ATP(50μM)or(D)yohimbine
(5μM);thiswasfollowedbytheadditionofADP(20μM),brazilin(50μM),orepinephrine(10μM)totriggerplateletaggregation.(E)
Furthermore,platelet-richplasmawasincubatedwithcollagen(1μg/ml)orbrazilin(50to10μM)totriggerplateletaggregation.Aggregation
profiles(AtoE)arerepresentativeexamplesof4similarexperiments.
Changetal.JournalofBiomedicalScience2013,20:4 Page5of11
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aggregation compared with collagen (1 μg/ml) (data not stimulated by epinephrine (10 μM), but not by brazilin
shown). As shown in Figure 2C, ATP (50 μM), an antag- (50 μM) (Figure 2D). However, brazilin (50 μM) did not
onist to ADP on platelets, inhibited platelet aggregation significantly induce platelet aggregation in PRP even at a
stimulated by ADP (20 μM) more effectively than that higher concentration of 100 μM (Figure 2E), indicating
stimulatedbybrazilin(50μM).Yohimbine(5μM),anα - that brazilin might have high binding ability with protein
2
adrenoceptor antagonist, inhibited platelet aggregation in plasma. Furthermore, we also found that there are no
A
SCH79797
tcY-NH2
thrombin
B
SCH79797 tcY-NH2
collagen
C
%)
SCH79797 tcY-NH2 10
T(
Δ
min
brazilin
Figure3Theinfluenceofantagonistsofthrombinreceptorsinbrazilin-mediatedplateletaggregation.Washedplatelets(3.6×108/ml)
werepreincubatedwithSCH79797(1μM)ortcY-NH2(100μM);thiswasfollowedbytheadditionof(A)thrombin(0.5U/ml),(B)collagen(1μg/
ml),or(C)brazilin(50μM)totriggerplateletaggregation.Aggregationprofiles(AtoC)arerepresentativeexamplesof4similarexperiments.
Changetal.JournalofBiomedicalScience2013,20:4 Page6of11
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significant differences in the platelet number between the significantly affect FITC-triflavin binding to the integrin
control(0.5%DMSO)andbrazilin-treatedvalues(control, α β in platelet suspensions (25 μM, 55.1 ± 5.2; 50 μM,
IIb 3
1376.8 ± 284.1 × 106/ml vs. 2 mg/kg brazilin, 1159.2 ± 54.3±4.5)(Figure4G,candd).Theseresultsshowedthat
383.4 × 106/ml, n=5, p > 0.05; control, 1252.0 ± 310.6 × the stimulatory effect of brazilin on platelet aggregation
106/mlvs.4mg/kgbrazilin,1020.4±328.7×106/ml,n=5, didnotaffectintegrinα β .Overall,ourfindingsprovide
IIb 3
p > 0.05) during the administration of various doses of evidencesthatbrazilinactsasacollagenreceptoragonist.
brazilin (2 and 4 mg/kg) into the tail vein of mice (ICR
strain) for 10 min. In addition, SCH79797 (1 μM), a BrazilinstimulatedPLCγ2andLynactivationthrough
thrombin protease-activated receptor (PAR) 1 antagonist, collagenreceptors
and tcY-NH2 (100 μM), a PAR 4 antagonist, both mark- PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate
edlydiminishedplateletaggregationstimulatedbythrom- (PIP ) to generate 2 secondary messengers: inositol
2
bin (0.5 U/ml) (Figure 3A), but not by collagen (1 μg/ml) 1,4,5-trisphosphate (IP ) and diacylglycerol (DAG).
3
(Figure3B).NeitherSCH79797(1μM)nortcY-NH2(100 These messengers trigger platelet activation [18]. The
μM) significantly affected platelet aggregation stimulated immunoblotting analysis showed that brazilin (50 μM)
by brazilin (50 μM) (Figure 3C). These results indicated treatment resulted in marked phosphorylation of PLCγ2
thatbrazilin-inducedplateletaggregationwasnotmediated compared with resting platelets (Figure 5A). Treatment
even partially by ADP receptors, α -adrenoceptors, or with JAQ1 (4 μg/ml) or Sam.G4 (4 μg/ml) significantly
2
thrombinPARreceptorsontheplateletmembranes. attenuated this phosphorylation stimulated by brazilin
(Figure 5A), but notby thrombin (0.5 U/ml) (Figure 5B). In
Collagenreceptorsinvolvedinbrazilin-inducedplatelet addition, Lyn was specifically phosphorylated by brazilin (50
aggregation μM)andcollagen(1μg/ml),butnotbythrombin(0.5U/ml)
GP VI and integrin α β are the main collagen receptors or ADP (20 μM) (Figure 5C). Brazilin (50 μM) stimulated
2 1
involved in platelet adhesion and aggregation [16]. Pre- Lyn phosphorylation was diminished in the presence of
treatment with JAQ1 (4 μg/ml) and Sam.G4 (4 μg/ml), JAQ1(4μg/ml)orSam.G4(4μg/ml)(Figure5D).
which are mAbs against GP VI and integrin α β re-
2 1
spectively, abolished platelet aggregation stimulated by Influenceofbrazilininhydroxylradicalformationinvitro
collagen (1 μg/ml) (Figure 4A) but not by thrombin andplateletplugformationinmicrovesselsofmice
(0.5U/ml)(Figure4B).U73122(10μM),aPLCinhibitor, A typical ESR signal of hydroxyl radical (OH●) formation
and indomethacin (25 μM), a cyclooxygenase inhibitor, was triggered in collagen-activated platelets compared to
both markedly diminished platelet aggregation stimu- restingplateletsor0.5%DMSO-treatedplatelets(Figures6A,
lated by collagen (1 μg/ml) and brazilin (50 μM) a, b, and c). However, treatment with brazilin (25 and 50
(Figures 4C and 4D), indicating that brazilin triggers μM) did not significantly trigger hydroxyl radical formation
platelet aggregation perhaps mediate by thromboxane (Figures6A,dande).
A -dependent mechanisms as collagen did. Furthermore, Ourobservationofthrombusformationinthemicroves-
2
caffeic acid phenethyl ester (CAPE; 25 and 50 μM) [17], sels of mice pretreated with fluorescein sodium (15 μg/kg)
an active component of propolis obtained from honey- showedthattherequiredocclusiontimewasapproximately
bee hives, acts as a collagen receptor antagonist; this 90s.Whenbrazilin(2and4mg/kg)wasadministeredafter
compound abolished platelet aggregation stimulated by pretreatment with fluorescein sodium, the occlusion time
either collagen (1μg/ml)orbrazilin (50μM) (Figure 4E). was significantly shorter compared with the solvent con-
Pretreatment with either JAQ1 (4 μg/ml) or Sam.G4 trols (occlusion time for 2 mg/kg brazilin was 74.4 ± 2.4 s
(4 μg/ml) abolished platelet aggregation stimulated by comparedwith91.9±2.3sfor0.5%DMSO;n=5,p<0.05;
brazilin (50 μM) (Figure 4F). These results showed that for 4 mg/kg brazilin, occlusion time was 72.7 ± 3.4 s com-
brazilin might activate platelet aggregation through col- pared with 91.2 ± 3.8 s for 0.5% DMSO; n=5, p < 0.05)
lagen receptors ontheplatelet membranes. (Figure6B).Theseresultsindicatedthatbrazilinstimulated
Triflavin is an α β disintegrin, which inhibits platelet plateletplugformationinvivo.
IIb 3
aggregationbydirectlyinterferingwithfibrinogenbinding
to the integrin α β [11]. We evaluated whether brazilin Discussion
IIb 3
would bind directly to the platelet integrin α β , leading Inthisstudy,uptoourknowledge,thisisanovelfinding
IIb 3
tointerruptionofplateletaggregation.Ourresultsshowed that brazilin, a plant-based natural product acts as a col-
that the relative intensity of the fluorescence of 2 μg/ml lagen receptor agonist induce platelet activation, other
FITC-triflavin bound directly to platelets was 55.2 ± 4.5 than that some collagen receptor agonists purified from
(Figure 4G, a). The fluorescent intensity was markedly thesnake venoms[19,20].
reducedinthepresenceof5mMEDTA(negativecontrol, Platelets are activated by a variety of physiological
5.2 ± 0.6)(Figure4G, b). Brazilin (25 and 50 μM) did not stimuli (e.g., thrombin, collagen, ADP, epinephrine, and
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A B
JAQ1 Sam.G4 JAQ1 Sam.G4
%)
0
1
T(
Δ
collagen thrombin min
C D
U73122 U73122 indomethacin indomethacin
%)
0
1
T(
collagen brazilin collagen brazilin Δmin
E F
CAPE CAPE
25 25 50 JAQ1 Sam.G4
%)
0
1
collagen brazilin brazilin ΔT(
min
G a b
s
l
l
e
c

f
o
100 101 102 100 101 102
r c d
e
b
m
u
N
100 101 102 100 101 102
Fluorescence intensity
Figure4Theinfluenceofantagonistsofcollagenreceptorsandintegrinα β inbrazilin-mediatedplateletaggregation.Washed
IIb 3
platelets(3.6×108/ml)werepreincubatedwithJAQ1(4μg/ml),Sam.G4(4μg/ml),U73122(10μM),indomethacin(25μM),orcaffeicacid
phenethylester(CAPE;25and50μM).Thiswasfollowedbytheadditionof(AandC-E)collagen(1μg/ml),(B)thrombin(0.5U/ml),or(C-F)
brazilin(50μM)totriggerplateletaggregation.(G)ThesolidlinerepresentsthefluorescenceprofilesofFITC-triflavin(a)intheabsenceofbrazilin
asapositivecontrol;(b)inthepresenceof5mMEDTAasanegativecontrol;(c)inthepresenceof25μMbrazilin;or(d)inthepresenceof50
μMbrazilin.Thereafter,2μg/mlFITC-triflavinwasaddedineachcase.Profiles(AtoG)arerepresentativeexamplesof4similarexperiments.
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A B
p-PLC 2 p-PLC 2
PLC 2 PLC 2
2.0 2.5
n n
o o
ti ti 2.0
a 1.5 a
yl ) yl )
r al r al
o s o s 1.5
h a h a
p b 1.0 p b
os s/ os s/
h d h d 1.0
p ol p ol
2 (f 0.5 2 (f
C C 0.5
L L
P P
0.0 0.0
brazilin thrombin
JAQ1 JAQ1
Sam.G4 Sam.G4
C D
p-Lyn p-Lyn
Lyn Lyn
p < 0.01
2.5 3.0
p < 0.01
n n 2.5
o 2.0 o
ti ti
yla al) yla al) 2.0
r s 1.5 r s
o a o a
h b h b 1.5
hosp olds/ 1.0 hosp olds/ 1.0
p f p f
n ( 0.5 n (
y y 0.5
L L
0.0 0.0
brazilin brazilin
collagen JAQ1
thrombin Sam.G4
ADP
Figure5Regulatoryeffectsofantagonistsofcollagenreceptorsinbrazilin-mediatedphospholipaseC(PLC)γ2andLyn
phosphorylationofwashedplatelets.Washedplatelets(1.2×109/ml)werepreincubatedwithJAQ1(4μg/ml)orSam.G4(4μg/ml),followed
bytheadditionof(A,C-D)brazilin(50μΜ)or(B-C)thrombin(0.5U/ml),collagen(1μg/ml),ADP(20μΜtotriggerplateletactivation.The
reactionswerestoppedbytheadditionofEDTA(10mM).Cellswerethencollectedandsubcellularextractswereanalyzedfor(A-B)PLCγ2and
(C-D)Lynphosphorylationbyimmunoblotting,asdescribedinthe“Materialsandmethods”section.Profiles(AtoD)arerepresentativeexamples
of4similarexperiments.Dataarepresentedasthemeans±S.E.M.(n=4);*p<0.05and**p<0.01,comparedtothesolvent(0.5%DMSO)-or
thrombin-treatedgroup;#p<0.05and##p<0.01,comparedtothebrazilin-treatedgroup.
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complexandthePARs[12].Ofthe4knownPARisoforms,
A
PAR1, PAR3, and PAR4 constitute the active thrombin
a
receptorsonhumanplatelets[22].PAR1andPAR4arees-
sential for thrombin-induced human platelet activation
b
[23].Furthermore,epinephrinecouldinduceplateletaggre-
* * gationinthepresenceofsub-physiologicalcalciumconcen-
c *
trations, as occurs in citrated plasma [24]. Aggregation as
*
monitored in the light transmission aggregometer occurs
d withoutprecedingshapechange(disctospheretransform-
ation)(Figure2D).Plateletspossessstimulatoryα -adreno-
2
e ceptorsandinhibitoryβ-adrenoceptors;inmostindividuals
theα -adrenoceptorspredominate.
2
Platelets adhere to the connective tissue protein colla-
gen, with a resulting change in shape and the release of
B
100 granules. Adhesion is partly dependent on the release of
(s) 80 * * ADPandTxA2,whereasaggregationisentirelydependent
e onthereleasethereof[21].Thematrixproteincollagenis
m
ti 60 present in the vascular subendothelium and vessel wall,
on and acts as a substrate for platelet adhesion; it is also an
si 40 endogenous platelet activator. Among the platelet recep-
u
ccl 20 torsknowntointeractdirectlywithcollagen,integrinα2β1
O
(GP Ia/IIa) and GP VI [19] appear to play a key role and
0
haverecentlygainedtheattentionofresearchers.GPVIis
ctl 2 ctl 4
widely recognized as a requisite factor for the formation
Brazilin(mg/kg)
of platelet aggregates on a collagen surface under blood
Figure6Regulatoryeffectsofbrazilininhydroxylradical
flow[25].Integrinα β isanothermajorcollagenreceptor
formationandtheocclusiontimerequiredforthrombus 2 1
formationinthemesentericvenulesofmice.(A)Fortheelectron onendothelialcellsandplatelets.Incellsexpressinginteg-
spinresonance(ESR)study,washedplateletswereincubatedwith rinα β ,manysignals(includingtyrosinephosphorylation
2 1
(a)Tyrode’ssolutiononly(restinggroup),orplateletswere andmatrixremodeling)areactivatedaftercelladhesionto
incubatedwith(b)thesolventcontrol(0.5%DMSO),(c)collagen(1 collagen [26]. Recent findings suggest that integrin α β
μg/ml),(d)brazilin(25μM),or(e)brazilin(50μM)totriggerhydroxyl 2 1
and GP VI might contribute to the overall processes of
radicalformation.Asterisk(*)indicatestheformationofhydroxyl
radical.(B)Micewereadministeredeitherbrazilin(2and4mg/kg) plateletadhesionandactivation[19,27,28].
orisovolumetric0.5%DMSO(ctl;solventcontrol).Thereafter, GP VIisaplateletmembraneproteinwithamolecular
mesentericvenuleswereselectedforirradiationtoinduce weight of 62 kDa. It has been identified as a physio-
microthrombusformation.Thebargraphs(A)presentthemeans±
logical collagen receptor and belongs to a membrane of
S.E.M.oftheocclusiontime(s)forinducingplateletplugformation
(n=5).*p<0.05comparedwiththeindividualsolventcontrol. the immunoglobulin superfamily, which forms a com-
plex with the Fc receptor γ-chain (FcRγ) containing
immunoreceptor tyrosine-based activation motifs (ITAM)
PAF). These agonists are thought to exert their effects and is phosphorylated by Src-family kinases such as Fyn
by interacting with specific receptors on the platelet and Lyn [16,25]. Tyrosine kinases (Fyn and Lyn) are
membranes. The primary effects of agonists may be involved in GP VI-dependent activation and might phos-
enhanced bysecondary effectscaused by the synthesisof phorylatetheFcRγ[29].FynandLynwereshowntobind
thromboxane A (TxA ) from the arachidonic acid (AA) to the Pro-rich domain of the GP VI cytoplasmic tail in
2 2
or by the secretion of ADP from the dense granules in platelets[30],suggestingthattheGPVI-dependentactiva-
platelets. ADP binds to 2 major purinergic receptors tion mechanism might be similar to that of the cytokine
(P2Y and P2Y ), which play an important role in po- receptors.Inthisprocess,receptor-boundtyrosinekinases
1 12
tentiating platelet activation induced by other aggregat- (suchasSrc)phosphorylatethecytoplasmictailsofrecep-
ing agonists [21]. Therefore, ATP, an antagonist to ADP, tors when the receptors become associated with each
might affect platelet aggregation stimulated by other other through ligand binding. This phosphorylation will
agonists,includingbrazilin (Figure 2C). initiate the signal transduction pathway. In platelets,
Thrombinisoneofthemostpotentactivatorsofplatelets cross-linking of the GP VI/FcRγ complex would enable
and its role in promoting thrombus formation has been the GP VI-bound Fyn or Lyn to move to a position close
clearly established. Thrombin activates platelets through enoughtoFcRγthatitwouldcatalyzethephosphorylation
multiple cell-surface receptors, including the GP Ib/V/IX of FcRγ ITAM. In turn, this triggers the phosphorylation
Changetal.JournalofBiomedicalScience2013,20:4 Page10of11
http://www.jbiomedsci.com/content/20/1/4
of downstream signals, including the linker for activation endothelial cells, exposure of sub-endothelial collagen
ofT-cells(LAT),leadingtothe activation ofa kinase cas- provides the major trigger to initiate platelet adhesion
cade(i.e.,PLCγ ). and aggregation at the site of injury. This is followed by
2
Our previous study [17] showed that the antiplatelet arterial thrombus formation [11]. When platelets aggre-
activity of CAPE might involve direct interference with gate, they release a number of substances including
the binding of collagen to its specific receptors on the TxA and ADP, both of which strengthen the platelet ac-
2
platelet membrane. The current study showed that tivation processes. He et al. [27] showed that integrin
CAPE markedly inhibited brazilin-induced platelet ag- α β -deficient mice exhibited delayed thrombus forma-
2 1
gregation. Furthermore, brazilin markedly stimulated tion following carotid artery injury. This result was con-
platelet aggregation and PLCγ and Lyn phosphoryl- sistent with the previously reported correlation between
2
ation. All these reactions were significantly diminished high levels of integrin α β expression and increased risk
2 1
by JAQ1 (anti-GP VI mAb) and Sam.G4 (anti-integrin for thrombosis involving the coronary and cerebral ves-
α β mAb). Interestingly, we also found that the relative sels [32,33]. Nieswandt et al. [34] reported that mice
2 1
fluorescence intensity of the FITC-collagen (1 μg/ml) depletedofGP VIwere completelyprotected from lethal
bound directly to platelets was 11.7 ± 1.9 (n=4) and collagen-induced pulmonary thromboemboli. Similarly,
hence the fluorescent intensity was markedly reduced in our study showed that brazilin potentiated platelet plug
the presence of 1 μg/ml collagen (1.6 ± 1.4, n=4); how- formation in the mesenteric venules of rats. Activated
ever pretreatment with brazilin (25, 50, and 100 μM) plateletsalsocontributetoenhancetheassemblyandac-
showed a significant increase in the relative fluorescence tivity of two major coagulation factor complexes which
intensity of FITC-collagen (25 μM, 33.8 ± 13.9; 50 μM, facilitatescoagulationandthrombus stabilization.There-
38.4 ± 10.6; 100 μM, 61.8 ± 9.8; n=4) (data not shown). fore, the coagulation factors may be involved in brazilin
These results suggest that brazilin may act at the allo- shortenedtheocclusiontime invivo.
steric site to display allosteric agonism on collagen
receptors, and subsequently enhances both the affinity Conclusions
andefficacy ofcollagen towards itsbinding sites. A simi- In conclusion, the key finding of this study was that bra-
lar model has been proposed in G-protein-coupled zilin acts as a collagen receptor agonist. However, the
receptors and predicts that allosteric ligands bind to a detailed mechanisms of brazilin-mediated signaling
topographically distinct site on a receptor to modulate events in platelet activation require further investigation.
orthosteric ligand affinity and/or efficacy [31]. A study Brazilin isanovel plant-basednatural product,mayoffer
also reported that some allosteric ligands can enhances therapeutic potential as intended anti-thrombotic agents
both affinity and efficacy, and it displays allosteric agon- for targeting of collagen receptors or to be used a useful
ism [31]. Therefore, we speculate that brazilin may serve tool for the study of detailed mechanisms in collagen
as an allosteric ligand for collagen receptors in platelets. receptors-mediatedplateletactivation.
Overall,theseresultsprovidedevidencethatthestimula-
Abbreviations
tion of platelet activation by brazilin might be the result
AA:Arachidonicacid;ATP:Adenosinetriphosphate;BSA:Bovineserum
ofdirect stimulationofcollagen receptorsontheplatelet albumin;CAPE:Caffeicacidphenethylester;DAG:Diacylglycerol;
membrane. However, our experiments did not rule out GP:Glycoprotein;iNOS:Induciblenitricoxidesynthase;PAF:Platelet-
activatingfactor;PAR:Proteinase-activatedreceptor;PLC:PhospholipaseC;
the possibility that other as-yet-unidentified mechanisms
PRP:Platelet-richplasma;PGE:ProstaglandinE;TxA:ThromboxaneA;
1 1 2 2
mightbeinvolvedinbrazilin-mediatedplateletactivation. TNF:Tumornecrosisfactor.
Reactive oxygen species (i.e., hydrogen peroxide and
Competinginterests
hydroxyl radicals) derived from platelet activation might
Theauthorsdeclarethattheyhavenocompetinginterests.
amplify platelet reactivity during in vivo thrombus for-
mation. Free radical species act as secondary messengers Authors’contributions
that increase cytosolic Ca2+ during the initial phase of YCandSKHHperformedresearchandwrotethemanuscript;WJL,CLC,WLC,
andSHLperformedthepartialexperimentsandanalyzeddata;KHLandJRS
platelet activation processes [15]. It is also evident that conceivedofthestudyanddesignedresearch.Allauthorsreadand
some of the hydrogen peroxide produced by platelets is approvedthefinalmanuscript.
converted into hydroxyl radicals, as platelet aggregation
Acknowledgements
can be inhibited by hydroxyl radical scavengers [15]. In ThisworkwassupportedbygrantsfromtheNationalScienceCouncilof
the present study, we found that brazilin did not signifi- Taiwan(NSC97-2320-B-038-016-MY3andNSC100-2320-B-038-021-MY3);Shin
KongWuHo-SuMemorialHospital(SKH-8302-99-DR-35andSKH-8302-102-
cantly induce hydroxyl radical formation as compared
NDR-04),andChi-MeiMedicalCenter-TaipeiMedicalUniversity(99CM-TMU-09).
with the collagen-stimulated platelets, indicating that
brazilin may have a differential characterization on free Authordetails
1DepartmentofAnesthesiology,ShinKongWuHo-SuMemorialHospital,95
radical formation apart from acting as the collagen re-
Wen-ChangRd,Taipei11101,Taiwan.2SchoolofMedicine,Fu-JenCatholic
ceptor agonist in platelets. Following an injury to the University,510Zhong-ZhengRd,Taipei24205,Taiwan.3DivisionofUrology,