Research Article Correlation between Platelet Gelsolin and...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 985746 7 pageshttpdxdoiorg1011552013985746

Research ArticleCorrelation between Platelet Gelsolin and PlateletActivation Level in Acute Myocardial Infarction Rats andIntervention Effect of Effective Components of ChuanxiongRhizome and Red Peony Root

Yue Liu Huijun Yin Yuerong Jiang Mei Xue Chunyu Guo Dazhuo Shi and Keji Chen

Cardiovascular Disease Centre Xiyuan Hospital China Academy of Chinese Medical Sciences Beijing 100091 China

Correspondence should be addressed to Huijun Yin huijunyinyahoocomcn

Received 21 August 2012 Revised 2 December 2012 Accepted 7 February 2013

Academic Editor Peng Nam Yeoh

Copyright copy 2013 Yue Liu et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The biological role of platelet gelsolin in platelet activation of acute myocardial infarction is not defined In order to providea potential new antiplatelet target for Chinese medicine and to elucidate the contribution of Xiongshao capsule the effectivecomponents of Chuanxiong rhizome and red peony root in this study we randomly allocated Sprague Dawley rats to left anteriordescending coronary artery ligation or sham surgery and different drug prophylaxis as control We found that gelsolin is highlyexpressed in platelet rich plasma and lowly expressed in platelet poor plasma accompanied by the high platelet activation level inmodel rats plasma actin filaments and mean fluorescence intensity (MFI) of platelet calcium ion increased and plasma vitaminD binding protein decreased in model rats Xiongshao capsule could inhibit the gelsolin expression in platelet rich plasma andischemic heart tissue simultaneously and reduce the level of plasma F-actin and MFI of platelet calcium ion Our study concludesthat platelet gelsolin is an important contributor to platelet activation and platelet gelsolin inhibition may form a novel target forantiplatelet therapy Xiongshao capsulemay be a promisingChinesemedicine drug for antiplatelet and aspirin-like cardioprotectioneffect

1 Introduction

Despite recent medical advances cardiovascular diseasesremain the predominant cause of morbidity and mortalityall over the world [1 2] Rupture of atherosclerotic plaqueand the ensuing thrombotic changes are the triggers for acutecoronary event Platelet activation and aggregation play cru-cial roles in this process of atherothrombosisThe emergenceof antiplatelet drug is themilestone of prevention and therapyof cardiovascular disease and provides the primary therapeu-tic strategy to combat cardiovascular diseases The properapplication of antiplatelet drug in reducing the mortalityand morbidity of acute myocardial infarction successfullyhas been verified by a large number of large-scale clinicaltrials [3] Antiplatelet drug such as aspirin now is rec-ommended for the secondary prevention of cardiovasculardisease (CVD) in patients with CVD because it decreases therisk of CVD events and mortality in clinical trials of men

and women with CVD [4] But many clinical problems arosealong with the wide range of application of antiplatelet drugs(such as aspirin and clopidogrel etc) during the past 10 years[5 6] Despite their proven benefits recurrent cardiovascularevents still occur in those taking antiplatelet drugs This hasled to the concept of ldquoantiplatelet drug resistancerdquo most com-monly aspirin or clopidogrel resistance The latest researchshows that aspirin prophylaxis in people without prior CVDdoes not lead to reductions in cardiovascular death for thebenefits are further offset by clinically important bleedingevents [7] which limit the clinical practice of antiplateletdrugs widely These phenomena suggest that other pathwayscapable of stimulating platelet activation may exist andprovide an impetus for developing new antiplatelet drugswhich possess higher efficacy and fewer adverse effects

Proteomics technology has been successfully appliedto platelet research during the past 5 years contributingto the emerging field of platelet proteomics which led to

2 Evidence-Based Complementary and Alternative Medicine

the identification of a considerable amount of novel plateletproteins many of which have been further studied at theirfunctional level [8] Our previous work [9] indicated thatplatelet gelsolin is the main platelet differential functionalprotein between patients of coronary heart disease andhealthy people by platelet proteomics Studies have alsoshown that platelet gelsolin is highly expressed in patientswith acute coronary syndrome (ACS) and the blood-stasissyndrome (BSS) of traditional Chinese medicine (TCM) [1011] Gelsolin is known to have one of the key roles in extra-cellular actin-scavenger system (EASS) [12] but the biolog-ical role of platelet gelsolin in platelet activation of acutemyocardial infarction (AMI) is unclear On the prevention ofatherosclerosis or vulnerable plaque Chinese medicine andWestern medicine agree on stabling plaque and promotingblood circulation Based on the agreed thoughts of theEastern andWestern worlds the application of Chinese herbsfor activating blood circulation (ABC herbs) has valuablesignificance in the exploration of reducing the risk of cardio-vascular event [13 14] Chuanxiong rhizome and Red peonyroot are the two classical ABC herbs in China and have beenused for thousands of years in the prevention and treatment ofCVD Xiongshao capsule (XSC) is a patent drug in China andis composed of effective components of Chuanxiong rhizomeand Red peony root Our previous studies have showed thatpaeoniflorin ferulic acid and total phenolic acid are themajoractive principles of the water extract from Xiongshao capsule[15 16] Clinical studies indicated that XSC can effectivelyprevent restenosis after percutaneous coronary intervention(PCI) [17] but the antiplatelet target of XSC is not defined

In the present study we used AMI as a disease modelto investigate the correlation between platelet gelsolin andplatelet activation level in rat model of AMI and the prophy-laxis mechanism of XSC in vivo

2 Materials and Methods

21 Drug and Reagents Xiongshao Capsule (XSC) whichcontained paeoniflorin (more than or equal to 28mg eachcapsule) ferulate (more than or equal to 35mg each capsule)and total phenolic acid (more than or equal to 34mg eachcapsule) 025 g per capsule were provided by Beijing Inter-national Institute of Biological Products (batch no 200091Beijing China) aspirin 01 g per capsule was purchased fromBayerHealthCareManufacturing (batch no BJ01653 BeijingChina) verapamil 004 g per tablet was purchased from theCentral Pharmaceutical Co Ltd (batch no 100402 TianjingChina) All the drugs were dissolved in pure water before use

Fluo-3AM was purchased from Sigma (St Louis MOUSA) rabbit anti-gelsolin polyclonal antibodywas purchasedfromAbcam (San Francisco USA) mouse anti-120573-actinmon-oclonal antibody was purchased from Sigma (St Louis MOUSA) FITC-Phalloidin was purchased from Sigma (St LouisMO USA) enzyme-linked immunosorbent assay (ELISA)kit of P-selectin gelsolin F-actin vitamin D binding protein(VDBP) CK-MB cTnI TXB

2 and COX-1 were purchased

from Huamei Biological Technology Company (WuhanHubei province China)

22 Animal Grouping and Treatment Sprague Dawley (SD)rats (male weight 220ndash250 g 119899 = 90) were obtained fromBeijing University Laboratory Animal Center (the animalcertificate No SCXK (Jing) 2006ndash0009) The rats werehoused in humidity-controlled (55 plusmn 5) rooms at (22 plusmn2)∘C with a 12 h on12 h off light cycle The animals weremaintained with free access to standard diet and tap water

After one week of adaptive feeding we randomly allo-cated the SD rats into six groups of 15 rats each as fol-lows Model group Sham group Aspirin group Xscd group(the high dose group) Xscx group (the low doses group)and Verapamil group Aspirin 40mgkgday verapamil4mgsdotkgminus1 dminus1 and XSC 390mgkgday 195mgkgday pergavage for 3 consecutive weeks were administrated to theaspirin verapamil Xscd and Xscx groups respectively Ratsin the Sham and Model groups received the same volumeof distilled water per gavage for 3 weeks After 3 weeksmyocardial infarction (MI) model was created in rats byligating the left anterior descending coronary artery (LAD) asdescribed before [18] The Animal Care and Use Committeeof Xiyuan hospital approved the experimental protocol

23 Sample Preparation After 3 hours of ligation all the ratswere killed after anesthesia by intraperitoneal injection of20 urethane (05mL100 g) Fresh blood (10mL) was drawnfrom the abdominal aorta and collected into vacutainer tubescontaining acid citrate dextrose (ACD) 9 vv (trisodiumcitrate 220 gL citric acid 80 gL dextrose 245 gL) as anti-coagulant The initial 2mL of blood was discarded to avoidspontaneous platelet activation The blood was centrifugedfor 10min at 150timesg at room temperature to obtain platelet-rich plasma (PRP) and the remaining blood centrifuged for20min at 800timesg to obtain platelet poor plasma (PPP)

Ischemic heart tissue was taken after blood collection andpreserved at minus80∘C for detection of gelsolin expression bywestern blotting

24 Enzyme-Linked Immunosorbent AssayAnalysis Thecon-centration of PRP and PPP of gelsolin plasma F-actin VDBPCK-MB cTnI TXB

2 COX-1 were determined by enzyme-

linked immunoadsorbent assay (ELISA) as per the manufac-turerrsquos instructions The absorbance was measured at 450 nmin an ELISA reader

25 Western Blotting Analysis The level of gelsolin in is-chemic heart tissues was determined byWestern blot analysisaccording to the standard procedure as described previously[19] 120573-actin was used as a loading control

26 Detection of MFI of Platelet Calcium Ion Platelet-richplasma was prepared and incubated with 4120583molL Fluo-3-AM (Sigma Saint Louis MO USA) at 37∘C for 40min Thecalcium concentration of platelets was determined using flowcytometry tomeasure themean fluorescence intensity (MFI)as previously described [20]

27 Statistical Analysis Data are presented asmeanplusmn SDTheSPSS Statistics 110 package was utilized to analyze the data

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The outcome among the different groups after ligation ofLAD

Group 119873 Dead rats (119899) Surviving rats (119899)Sham 15 6 9Model 15 6 9Aspirin 15 5 10Xscd 15 6 9Xscx 15 7 8Verapamil 15 7 8

Differences among groups were analyzed using the one-wayanalysis of variance (ANOVA) followed by multiple compar-isons by Least-Significant Difference (LSD) test Spearmanrsquoscorrelation coefficients were calculated to study the relationsbetween gelsolin concentration in PRP and plasma P-selectinlevel Differences between groups were at 119875 lt 005

3 Results

31 General Condition All the rats in the different groupssurvived and exhibited normal physical appearance andbehavior during the gavage period of different drugs Theoutcome among the different groups after ligation of LAD ispresented in Table 1

32 XSC Reduces the Concentration of Myocardial InjuryMarkers We chose CK-MB and cTnI as the myocardialinjury markers in rats with acute myocardial infarction(AMI) Compared with the Sham group the concentrationof CK-MB and cTnI of Model group increased significantlyafter ligation of LAD for 3 hours (119875 lt 001) The high doseof XSC (390mgkgday) can reduce the concentration of CK-MB and cTnImarkedly (119875 lt 005) this has similar effect withaspirin in vivo (see Table 2)

33 XSC Inhibits the Platelet Activation Level We choosethe plasma P-selectin as the marker of platelet activationlevel Compared with Sham group the plasma P-selectinconcentration of theModel group increased significantly afterligation of LAD for 3 hours (119875 lt 001) The high dose of XSCcan inhibit P-selection level markedly (119875 lt 005) this hassimilar effect with the Aspirin group (see Figure 1)

34 XSC Reduces the Platelet Gelsolin Level and Enhancesthe Activity of Extracellular Actin-Scavenger System (EASS)Plasma gelsolin and VDBP are the main components ofthe EASS which undertake the responsibility as scavengerof the abnormal increased extracellular filament actin (F-actin) Compared with the Sham group the plasma gelsolinand VDBP of the Model group was reduced significantly(119875 lt 005) and F-actin increased markedly (119875 lt 001)while platelet gelsolin it increased markedly (119875 lt 001) Highdose of XSC can reduce platelet gelsolin and F-actin level(119875 lt 005) while it increased plasma gelsolin and VDBPsignificantly (119875 lt 005) (see Figures 2 3 and 4)

0

5

10

15

20

25

Sham Model Aspirin Xscd Xscx Verapamil

P-se

lect

in co

ncen

trat

ion

(ng

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771 998771998771998771

lowastlowast

Figure 1 Effect of Xiongshao Capsule (XSC) on P-selectin con-centration of AMI rats lowastlowast119875 lt 001 compared to Sham group and998771119875 lt 005 or 998771998771119875 lt 001 compared to Model group

0

10

20

30

40

50

60

70

80

GSN in PRP GSN in PPP

Gels

olin

conc

entr

atio

n (n

gm

L)

ShamModelAspirin

XscdXscxVerapamil

daggerdaggerdagger

998771 998771 998771998771998771998771lowastlowast

lowastlowast

Figure 2 Effect of Xiongshao Capsule (XSC) on Gelsolin concen-tration among PRP and PPP of AMI rats lowastlowast119875 lt 001 compared toSham group 998771119875 lt 005 or 998771998771119875 lt 001 compared to Model groupand dagger119875 lt 005 or daggerdagger119875 lt 001 compared to Aspirin group

35 XSC Inhibits the Activation of TXB2and COX-1 Com-

paredwith Shamgroup the concentration of TXB2andCOX-

1 of Model group increased significantly after ligation of LADfor 3 hours (119875 lt 001) High dose of XSC can reduce COX-1and TXB

2level significantly (119875 lt 005) this has similar effect

with the Aspirin group (see Figure 5)

36 XSC Inhibits the MFI of Calcium Compared with Shamgroup the MFI of calcium of the Model group increasedmarkedly (119875 lt 001) High dose of XSC can inhibit plateletcalcium increase (119875 lt 005) This has similar effect to theVerapamil group (119875 lt 005) (see Figure 6)

37 XSC Attenuates the Expression of Gelsolin in InfarctedMyocardium Compared with Sham group the gelsolin ex-pression of infarcted myocardium of Model group increasedmarkedly and XSC can inhibit gelsolin expression ofinfarcted myocardium but verapamil has no such effect (seeFigure 7)


Table 2 Effect of Xiongshao Capsule (XSC) on the concentration of myocardial injury markers of AMI rats

Group 119873 CK-MB (ngmL) cTnI (pgmL)Sham 9 0279 plusmn 0074 981 plusmn 262Model 9 0386 plusmn 0043lowastlowast 1518 plusmn 43lowastlowast

Aspirin 10 0340 plusmn 0024dagger 1204 plusmn 119dagger

Xscd 9 0336 plusmn 0027dagger 1223 plusmn 141dagger

Xscx 8 0351 plusmn 0013 1385 plusmn 302Verapamil 8 0358 plusmn 0017 1443 plusmn 298lowastlowast119875 lt 001 compared to Sham group and dagger119875 lt 005 compared to Model group

0

1000

2000

3000

4000

5000

6000

7000


Plas

ma F

-act

in co

ncen

trat

ion

(pg

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

lowastlowast

Figure 3 Effect of Xiongshao Capsule (XSC) on Plasma F-actinconcentration of AMI rats lowastlowast119875 lt 001 compared to Sham groupand 998771119875 lt 005 compared to Model group

0

20

40

60

80

100

120

140

160


998771

lowast

998771998771

Plas

ma V

DBP

(ng

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

Figure 4 Effect of Xiongshao Capsule (XSC) on Plasma VDBPconcentration of AMI rats lowast119875 lt 005 compared to Sham group and998771119875 lt 005 or 998771998771119875 lt 001 compared to Model group

38 Analyses of Correlation between Platelet Gelsolin Con-centration and Plasma P-Selectin Level Next we investigatedany potential correlation between the platelet gelsolin con-centration and plasma P-selectin levels that may exist in theModel group and Xscd group Correlation analysis showedthat platelet gelsolin concentrations were high positivelycorrelated with plasma P-selectin levels in the Model group(see Figure 8(a)) and Xscd group (see Figure 8(b))

0

50

100

150

200

250

300

350

TXB2 Cox-1

ShamModelAspirin

XscdXscxVerapamil

998771 998771

998771998771

lowastlowast

lowastlowast

TXB2

and

COX-

1 co

ncen

trat

ion

(pg

mL)

Figure 5 Effect of Xiongshao Capsule (XSC) on Plasma TXB2and

COX-1 concentration of AMI rats lowastlowast119875 lt 001 compared to Shamgroup and 998771119875 lt 005 compared to Model group

4 Discussion

Gelsolin is a calcium-regulated actin filament (F-actin) sev-ering and capping protein which is expressed as both cyto-plasmic and plasma isoforms The functions of extracellulargelsolin are less well defined Gelsolin is also an importantcytoskeletal protein which is a key actin binding protein(ABPs) as well Increasingly evidence has shown that gelsolinhas close relationship with many diseases and pathologicalprocesses such as cancer apoptosis infection and inflam-mation pulmonary diseases and aging [21] During the past5 years many scholars began to focus on gelsolinrsquos possiblerole in the development of cardiovascular diseases [22]Activated platelets play a pivotal role in the formation ofarterial thrombi and antiplatelet drugs become the core inthe prevention and treatment of CVD Platelet activation notonly causes the changes ofmembrane protein but also a seriesof morphological changes from inviscid discotic circulatingplatelets to a paste-like protruding platelet jelly that affectsthe regulation of platelet cytoskeletal proteins

Using differential proteomics of platelet our previousstudy [9] indicated that platelet gelsolin was the mainplatelet differential functional proteins between patients with


0

2

4

6

8

10

12


MFI

of p

late

let c

alci

um io

n

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

998771998771

lowastlowast

Figure 6 Effect of Xiongshao Capsule (XSC) on MFI of plateletcalcium ion concentration of AMI rats lowastlowast119875 lt 001 compared toSham group and 998771119875 lt 005 or 998771998771119875 lt 001 compared to Modelgroup

Gelsolin

Model Sham Aspirin Xscd Xscx Verapamil

0

02

04

06

08

1

12

14


120573-actin

daggerdagger998771998771 daggerdagger998771998771

lowastlowast

Gels

olin

120573-a

ctin

Figure 7 Effect of Xiongshao Capsule (XSC) on protein level ofgelsolin in ischemic heart tissue ofAMI rats lowastlowast119875 lt 001 compared toSham group and 998771998771119875 lt 001 compared to Model group daggerdagger119875 lt 001compared to Aspirin group

coronary heart disease and healthy people In addition datafrom our previous clinical studies demonstrated that [10 11]platelet gelsolin was highly expressed in patients with acutecoronary syndrome (ACS) and the blood stasis syndrome(BSS) of traditional Chinese medicine Meanwhile basedon the Chinese medicine principle of ldquoprescription to syn-dromerdquo Platelet gelsolin may be viewed as a new target forABC herbs In this study we evaluated the biological role ofplatelet gelsolin in the development of platelet activation ina rat model of AMI and the potential contribution of XSCprophylaxis in this progress in vivo

As we know P-selectin is a 140 kD glycoprotein that ispresented in the granules of platelets and translocates rapidlyto the cell surface after platelet activation it is generallyconsidered as the gold marker of platelet activation [23] Inthis study after ligation of LAD for 3 hours the concentrationof CK-MB and cTnI and the P-selectin level of the Modelrats increased significantly compared with Sham rats whichindicated that the model rats had myocardial injury andplatelet activation

The actin cytoskeleton plays a central role in manyfundamental cellular processes involving the generation offorce and facilitation of movement which are enabled by theassembly of actin monomers into filaments and cooperationwith a wide variety of ABPs [21] including gelsolin Actinmonomers (G-actin) spontaneously associates to form F-actin under physiological conditions and vice versa Thisdynamic progress keeps in balance all the time In the pres-ence of tissue injury or cell death G-actin is released intothe circulation where it can interact with components of thehaemostatic and fibrinolytic systems or polymerize and formF-actin excessively Studies [24] have suggested that F-actincan lead to platelet aggregation directly in vitro and thepresence of excessive F-actin in blood vessels which canplug smaller vessels and decrease blood flow to promote theformation of blood clots can be fatal Infusion of high dosesof G-actin in rabbits caused the rapid and fatal formationof massive F-actin-containing thrombi in arterioles andcapillaries of pulmonary veins [25] An extracellular actin-scavenger system (EASS) [12] was therefore likely to existPlasma gelsolin together with vitamin D binding protein(VDBP) another extracellular ABPs were regarded as poten-tially important components of this systemThey are capableof removing F-actin from the circulation and inhibiting F-actin elongation to alleviate the vascular toxicity of excessiveF-actin In this study the concentration of gelsolin in PRPof AMI rats increased accompanied by the high plateletactivation and increased level of F-actin while gelsolin inPPP decreased which indicates the EASS of AMI rats wassuppressed Correlation analysis showed that platelet gelsolinconcentrations were high positively correlated with plasmaP-selectin levels in the Model group

Xiongshao capsule (XSC) is a patent drug developed fromXue Fu Zhu Yu Decoction It is the classic formula used foractivating blood circulation (ABC) in China for hundreds ofyears Clinical studies have shown that XSC can effectivelyprevent restenosis after percutaneous coronary intervention(PCI) [17] XSC was shown to enhance the protective effect ofischemic postconditioning on rat with myocardial ischemicreperfusion injury [26] It was also shown to stabilize ath-erosclerotic plaque by suppressing inflammation and theexpression of Fc120574RIIIA [27] But the potential antiplateletmechanism of XSC prophylaxis is unclear In this study wefound that high dose of XSC prophylaxis could decrease theconcentration of myocardial injury markers CK-MB andcTnI and reduce the plasma P-selectin level of AMI ratsas well The antiplatelet mechanism of aspirin involves theinhibition of COX-1 and TXA

2 our study shows that high

dosage of XSC can inhibit the activation of TXB2and COX-1

in vivo which has similar cardioprotection effect with aspirinin vivo Meanwhile high dosage of XSC prophylaxis inhibitedthe expression of platelet gelsolin in AMI rats by inhibitingthe platelet calcium influx but increased the concentrationof plasma gelsolin and plasma VDBP simultaneously so theEASS was activated and the concentration of F-actin in AMIrats decreased which indicated that the F-actin was beingremoved from the circulation Calcium ions not only promotegelsolin secretion but also play a vital role in the developmentof platelet activation Studies have shown increased platelet


0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30P-selectin concentration (ngmL)

Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L) 119903 = 0724

119875 = 0027 lt 005

(a)

0

10

20

30

40

50

60

70

80

0 5 10 15 20

Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L)

P-selectin concentration (ngmL)

119903 = 0747

119875 = 0021 lt 005

(b)

Figure 8 Correlation between gelsolin concentration in PRP and plasma P-selectin level of Model group and Xscd group (a) Model groupand (b) Xscd group

[Ca2+]119894in patients with CVD [28] and that calcium channel

blocker (CCB) could reduce platelet [Ca2+]119894and inhibit

platelet aggregation [29] Verapamil is a classic CCB agentand a previous study in vivo [30] shows that verapamilexhibits a dose-dependent inhibitory effect on platelet aggre-gation and thrombus formation in rats In this study ourresults show that high dosage of XSC can mimic the calciumchannel antagonist effect

We have also investigated the expression of the gelsolin ininfarcted myocardium of AMI rats The results indicate thatthe gelsolin expression of infarctedmyocardiumof theModelgroup increased markedly while XSC can inhibit gelsolinexpression of infarcted myocardium significantly verapamilor aspirin has no such effect holding that other pathwayexisted in the regulation of gelsolin as well Heart failure(HF) is the end stage of CVD (including after AMI) It isof great importance to know the effects and mechanism ofXSC on cardioprotection at earlier stages of CVDVentricularremodeling after AMI is the main pathological change of HFA previous study [31] has showed that gelsolin is an importantcontributor to heart failure progression through novel mech-anisms ofHIF-1120572 andDNase I activation and downregulationof antiapoptotic survival factors Based on these results andour study we propose that gelsolin inhibition is a promisingtarget for CVD therapy besides antiplatelet agent

5 Conclusion

We have provided experimental evidence supporting ourconclusion that high correlation between platelet gelsolin andplatelet activation level in AMI rats the aspirin-like cardio-protection and antiplatelet effects of XSC are related to itsinhibition on platelet gelsolin platelet calcium influx andactivated the EASS Taken together our results suggest thatplatelet gelsolin is a potential antiplatelet target and XSC is apromising lead compound for antiplatelet and cardiovasculartherapy

Conflicts of Interests

The authors declare that there is no conflict of interests

Author Contributions

H Yin conceived and designed the animal experiments andhelped to draft the paper Y Liu Y Jiang M Xue and C Guoperformed the experiments Y Liu participated in its designanalyzed the data and prepared the paper K Chen and DShi participated in its design and coordination and gave finalapproval for this paper to be published

Acknowledgments

This study was supported by the National Natural Sci-ence Foundation of China (nos 81073086 81030063 and81102845)

References

[1] J Choi and J C Kermode ldquoNew therapeutic approaches tocombat arterial thrombosisrdquoMolecular Interventions vol 11 no2 pp 111ndash123 2011

[2] S Tseeng and R Arora ldquoReviews aspirin resistance biologicaland clinical implicationsrdquo Journal of Cardiovascular Pharmacol-ogy andTherapeutics vol 13 no 1 pp 5ndash12 2008

[3] A D Michelson ldquoAntiplatelet therapies for the treatment ofcardiovascular diseaserdquo Nature Reviews Drug Discovery vol 9no 2 pp 154ndash169 2010

[4] C Baigent L Blackwell R Collins et al ldquoAspirin in the primaryand secondary prevention of vascular disease collaborativemeta-analysis of individual participant data from randomisedtrialsrdquoThe Lancet vol 373 no 9678 pp 1849ndash1860 2009

[5] D N Juurlink T Gomes D T Ko et al ldquoA population-basedstudy of the drug interaction between proton pump inhibitorsand clopidogrelrdquo Canadian Medical Association Journal vol180 no 7 pp 713ndash718 2009


[6] J L Mega S L Close S D Wiviott et al ldquoCytochrome P-450polymorphisms and response to clopidogrelrdquo New EnglandJournal of Medicine vol 360 no 4 pp 354ndash362 2009

[7] S R Seshasai S Wijesuriya R Sivakumaran et al ldquoEffect ofaspirin on vascular and nonvascular outcomes meta-analysisof randomized controlled trialsrdquo Archives of Internal Medicinevol 172 no 3 pp 209ndash216 2012

[8] A Garcıa ldquoClinical proteomics in platelet research challengesaheadrdquo Journal of Thrombosis and Haemostasis vol 8 no 8 pp1784ndash1785 2010

[9] X F Li Y R Jiang C FWu K J Chen and H J Yin ldquoStudy onthe correlation between platelet function proteins and symptomcomplex in coronary heart diseaserdquo Zhongguo Fen Zi Xin ZangBing Xue Za Zhi vol 9 no 6 pp 326ndash331 2009

[10] Y Liu H J Yin and K J Chen ldquoResearch on the correlationbetween platelet gelsolin and blood-stasis syndrome of coro-nary heart diseaserdquo Chinese Journal of Integrative Medicine vol17 no 8 pp 587ndash592 2011

[11] Y Liu H J Yin Y R JiangM Xue andK J Chen ldquoCorrelationbetween platelet gelsolin level and different types of coronaryheart diseaserdquo Chinese Science Bulletin vol 57 no 6 pp 631ndash638 2012

[12] WM Lee and RM Galbraith ldquoThe extracellular actin-scaven-ger system and actin toxicityrdquoNew England Journal of Medicinevol 326 no 20 pp 1335ndash1341 1992

[13] K J Chen ldquoExplore the possibilities of Chinese herb andformulas for promoting blood circulation and removing bloodstasis on reducing the cardiovascular riskrdquo Zhongguo Zhong XiYi Jie He Za Zhi vol 28 no 5 p 389 2008

[14] Y Liu H J Yin D Z Shi and K-J Chen ldquoChinese herb andformulas for promoting blood circulation and removing bloodstasis and antiplatelet therapiesrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2012 Article ID 184503 8pages 2012

[15] Z Zhang L M Qing and K J Chen ldquoStudy on the pharma-cokinetics of paeoniflorin contained in Xiongshao capsule incaninerdquo Zhongguo Shi Yan Fang Ji Xue Za Zhi vol 6 no 6 pp21ndash24 2000

[16] Z Zhang Y F Yan and K J Chen ldquoStudy on the pharmacok-inetics of ferulic acid in canine serum after giving an intragas-trical single dose of Xiongshao capsules to a dogrdquo Beijing ZhongYi Yao Da Xue Xue Bao vol 24 no 1 pp 25ndash28 2001

[17] K J Chen D Z Shi H Xu et al ldquoXS0601 reduces the incidenceof restenosis a prospective study of 335 patients undergoingpercutaneous coronary intervention in ChinardquoChinese MedicalJournal vol 119 no 1 pp 6ndash13 2006

[18] M Sun F DawoodW HWen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[19] GH Li Y Shi Y Chen et al ldquoGelsolin regulates cardiac remod-eling after myocardial infarction through DNase I-mediatedapoptosisrdquo Circulation Research vol 104 no 7 pp 896ndash9042009

[20] M M Zhuang Y X Wen S L Liu et al ldquoDetermination of thelevel of cytopplasmic free calcium in human platelets with flowcytometryrdquo Xi An Jiao Tong Da Xue Xue Bao vol 26 no 5 pp508ndash510 2005

[21] GH Li PDArora Y ChenCAMcCulloch andP Liu ldquoMul-tifunctional roles of gelsolin in health and diseasesrdquo MedicinalResearch Reviews vol 32 no 5 pp 999ndash1025 2012

[22] Y Liu Y R Jiang H J Yin et al ldquoGelsolin and cardiovasculardiseasesrdquo Zhongguo Fen Zi Xin Zang Bing Xue Za Zhi vol 11 no1 pp 50ndash53 2011

[23] A D Michelson and M I Furman ldquoLaboratory markers ofplatelet activation and their clinical significancerdquoCurrent Opin-ion in Hematology vol 6 no 5 pp 342ndash348 1999

[24] C A Vasconcellos and S E Lind ldquoCoordinated inhibition ofactin-induced platelet aggregation by plasma gelsolin and vita-min D-binding proteinrdquo Blood vol 82 no 12 pp 3648ndash36571993

[25] J G Haddad K D Harper M Guoth et al ldquoAngiopathicconsequences of saturating the plasma scavenger system foractinrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 87 no 4 pp 1381ndash1385 1990

[26] D W Zhang L Zhang J G Liu et al ldquoEffects of Xiongshaocapsule combined with ischemic postconditioning on mono-cyte chemoattractant protein-1 and tumor necrosis factor-120572 inrat myocardium with ischemic reperfusion injuryrdquo ZhongguoZhong Xi Yi Jie He Za Zhi vol 30 no 12 pp 1279ndash1283 2010

[27] Y Huang H J Yin X J Ma et al ldquoCorrelation betweenFc120574RIIIA and aortic atherosclerotic plaque destabilization inApoE knockout mice and intervention effects of effectivecomponents of Chuanxiong Rhizome and Red Peony RootrdquoChinese Journal of Integrative Medicine vol 17 no 5 pp 355ndash360 2011

[28] M Yoshimura T Oshima H Hiraga et al ldquoIncreased cytosolicfree Mg2+ and Ca2+ in platelets of patients with vasospasticanginardquo American Journal of Physiology vol 274 no 2 part 2pp R548ndashR554 1998

[29] A Fujinishi K Takahara C Ohba Y Nakashima and AKuroiwa ldquoEffects of nisoldipine on cytosolic calcium plateletaggregation and coagulationfibrinolysis in patients with coro-nary artery diseaserdquo Angiology vol 48 no 6 pp 515ndash521 1997

[30] W Li Y Liu Y Huang and Y Ji ldquoEffect of verapamil on thethrombogenes is and nitric oxide level in the serum of ratsrdquoNanjing Yi Ke Da Xue Xue Bao vol 27 no 10 pp 1080ndash10832007


Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the identification of a considerable amount of novel plateletproteins many of which have been further studied at theirfunctional level [8] Our previous work [9] indicated thatplatelet gelsolin is the main platelet differential functionalprotein between patients of coronary heart disease andhealthy people by platelet proteomics Studies have alsoshown that platelet gelsolin is highly expressed in patientswith acute coronary syndrome (ACS) and the blood-stasissyndrome (BSS) of traditional Chinese medicine (TCM) [1011] Gelsolin is known to have one of the key roles in extra-cellular actin-scavenger system (EASS) [12] but the biolog-ical role of platelet gelsolin in platelet activation of acutemyocardial infarction (AMI) is unclear On the prevention ofatherosclerosis or vulnerable plaque Chinese medicine andWestern medicine agree on stabling plaque and promotingblood circulation Based on the agreed thoughts of theEastern andWestern worlds the application of Chinese herbsfor activating blood circulation (ABC herbs) has valuablesignificance in the exploration of reducing the risk of cardio-vascular event [13 14] Chuanxiong rhizome and Red peonyroot are the two classical ABC herbs in China and have beenused for thousands of years in the prevention and treatment ofCVD Xiongshao capsule (XSC) is a patent drug in China andis composed of effective components of Chuanxiong rhizomeand Red peony root Our previous studies have showed thatpaeoniflorin ferulic acid and total phenolic acid are themajoractive principles of the water extract from Xiongshao capsule[15 16] Clinical studies indicated that XSC can effectivelyprevent restenosis after percutaneous coronary intervention(PCI) [17] but the antiplatelet target of XSC is not defined

In the present study we used AMI as a disease modelto investigate the correlation between platelet gelsolin andplatelet activation level in rat model of AMI and the prophy-laxis mechanism of XSC in vivo

2 Materials and Methods

21 Drug and Reagents Xiongshao Capsule (XSC) whichcontained paeoniflorin (more than or equal to 28mg eachcapsule) ferulate (more than or equal to 35mg each capsule)and total phenolic acid (more than or equal to 34mg eachcapsule) 025 g per capsule were provided by Beijing Inter-national Institute of Biological Products (batch no 200091Beijing China) aspirin 01 g per capsule was purchased fromBayerHealthCareManufacturing (batch no BJ01653 BeijingChina) verapamil 004 g per tablet was purchased from theCentral Pharmaceutical Co Ltd (batch no 100402 TianjingChina) All the drugs were dissolved in pure water before use

Fluo-3AM was purchased from Sigma (St Louis MOUSA) rabbit anti-gelsolin polyclonal antibodywas purchasedfromAbcam (San Francisco USA) mouse anti-120573-actinmon-oclonal antibody was purchased from Sigma (St Louis MOUSA) FITC-Phalloidin was purchased from Sigma (St LouisMO USA) enzyme-linked immunosorbent assay (ELISA)kit of P-selectin gelsolin F-actin vitamin D binding protein(VDBP) CK-MB cTnI TXB

2 and COX-1 were purchased

from Huamei Biological Technology Company (WuhanHubei province China)

22 Animal Grouping and Treatment Sprague Dawley (SD)rats (male weight 220ndash250 g 119899 = 90) were obtained fromBeijing University Laboratory Animal Center (the animalcertificate No SCXK (Jing) 2006ndash0009) The rats werehoused in humidity-controlled (55 plusmn 5) rooms at (22 plusmn2)∘C with a 12 h on12 h off light cycle The animals weremaintained with free access to standard diet and tap water

After one week of adaptive feeding we randomly allo-cated the SD rats into six groups of 15 rats each as fol-lows Model group Sham group Aspirin group Xscd group(the high dose group) Xscx group (the low doses group)and Verapamil group Aspirin 40mgkgday verapamil4mgsdotkgminus1 dminus1 and XSC 390mgkgday 195mgkgday pergavage for 3 consecutive weeks were administrated to theaspirin verapamil Xscd and Xscx groups respectively Ratsin the Sham and Model groups received the same volumeof distilled water per gavage for 3 weeks After 3 weeksmyocardial infarction (MI) model was created in rats byligating the left anterior descending coronary artery (LAD) asdescribed before [18] The Animal Care and Use Committeeof Xiyuan hospital approved the experimental protocol

23 Sample Preparation After 3 hours of ligation all the ratswere killed after anesthesia by intraperitoneal injection of20 urethane (05mL100 g) Fresh blood (10mL) was drawnfrom the abdominal aorta and collected into vacutainer tubescontaining acid citrate dextrose (ACD) 9 vv (trisodiumcitrate 220 gL citric acid 80 gL dextrose 245 gL) as anti-coagulant The initial 2mL of blood was discarded to avoidspontaneous platelet activation The blood was centrifugedfor 10min at 150timesg at room temperature to obtain platelet-rich plasma (PRP) and the remaining blood centrifuged for20min at 800timesg to obtain platelet poor plasma (PPP)

Ischemic heart tissue was taken after blood collection andpreserved at minus80∘C for detection of gelsolin expression bywestern blotting

24 Enzyme-Linked Immunosorbent AssayAnalysis Thecon-centration of PRP and PPP of gelsolin plasma F-actin VDBPCK-MB cTnI TXB

2 COX-1 were determined by enzyme-

linked immunoadsorbent assay (ELISA) as per the manufac-turerrsquos instructions The absorbance was measured at 450 nmin an ELISA reader

25 Western Blotting Analysis The level of gelsolin in is-chemic heart tissues was determined byWestern blot analysisaccording to the standard procedure as described previously[19] 120573-actin was used as a loading control

26 Detection of MFI of Platelet Calcium Ion Platelet-richplasma was prepared and incubated with 4120583molL Fluo-3-AM (Sigma Saint Louis MO USA) at 37∘C for 40min Thecalcium concentration of platelets was determined using flowcytometry tomeasure themean fluorescence intensity (MFI)as previously described [20]

27 Statistical Analysis Data are presented asmeanplusmn SDTheSPSS Statistics 110 package was utilized to analyze the data





3 Results





0

5

10

15

20

25


P-se

lect

in co

ncen

trat

ion

(ng

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771 998771998771998771

lowastlowast


0

10

20

30

40

50

60

70

80


Gels

olin

conc

entr

atio

n (n

gm

L)

ShamModelAspirin

XscdXscxVerapamil

daggerdaggerdagger

998771 998771 998771998771998771998771lowastlowast

lowastlowast















0

1000

2000

3000

4000

5000

6000

7000


Plas

ma F

-act

in co

ncen

trat

ion

(pg

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

lowastlowast


0

20

40

60

80

100

120

140

160


998771

lowast

998771998771

Plas

ma V

DBP

(ng

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8



0

50

100

150

200

250

300

350

TXB2 Cox-1

ShamModelAspirin

XscdXscxVerapamil

998771 998771

998771998771

lowastlowast

lowastlowast

TXB2

and

COX-

1 co

ncen

trat

ion

(pg

mL)



4 Discussion




0

2

4

6

8

10

12


MFI

of p

late

let c

alci

um io

n

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

998771998771

lowastlowast


Gelsolin


0

02

04

06

08

1

12

14


120573-actin


lowastlowast

Gels

olin

120573-a

ctin










0

10

20

30

40

50

60

70

80


Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L) 119903 = 0724

119875 = 0027 lt 005

(a)

0

10

20

30

40

50

60

70

80

0 5 10 15 20

Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L)


119903 = 0747

119875 = 0021 lt 005

(b)






5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results





0

5

10

15

20

25


P-se

lect

in co

ncen

trat

ion

(ng

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771 998771998771998771

lowastlowast


0

10

20

30

40

50

60

70

80


Gels

olin

conc

entr

atio

n (n

gm

L)

ShamModelAspirin

XscdXscxVerapamil

daggerdaggerdagger

998771 998771 998771998771998771998771lowastlowast

lowastlowast















0

1000

2000

3000

4000

5000

6000

7000


Plas

ma F

-act

in co

ncen

trat

ion

(pg

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

lowastlowast


0

20

40

60

80

100

120

140

160


998771

lowast

998771998771

Plas

ma V

DBP

(ng

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8



0

50

100

150

200

250

300

350

TXB2 Cox-1

ShamModelAspirin

XscdXscxVerapamil

998771 998771

998771998771

lowastlowast

lowastlowast

TXB2

and

COX-

1 co

ncen

trat

ion

(pg

mL)



4 Discussion




0

2

4

6

8

10

12


MFI

of p

late

let c

alci

um io

n

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

998771998771

lowastlowast


Gelsolin


0

02

04

06

08

1

12

14


120573-actin


lowastlowast

Gels

olin

120573-a

ctin










0

10

20

30

40

50

60

70

80


Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L) 119903 = 0724

119875 = 0027 lt 005

(a)

0

10

20

30

40

50

60

70

80

0 5 10 15 20

Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L)


119903 = 0747

119875 = 0021 lt 005

(b)






5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















0

1000

2000

3000

4000

5000

6000

7000


Plas

ma F

-act

in co

ncen

trat

ion

(pg

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

lowastlowast


0

20

40

60

80

100

120

140

160


998771

lowast

998771998771

Plas

ma V

DBP

(ng

mL)

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8



0

50

100

150

200

250

300

350

TXB2 Cox-1

ShamModelAspirin

XscdXscxVerapamil

998771 998771

998771998771

lowastlowast

lowastlowast

TXB2

and

COX-

1 co

ncen

trat

ion

(pg

mL)



4 Discussion




0

2

4

6

8

10

12


MFI

of p

late

let c

alci

um io

n

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

998771998771

lowastlowast


Gelsolin


0

02

04

06

08

1

12

14


120573-actin


lowastlowast

Gels

olin

120573-a

ctin










0

10

20

30

40

50

60

70

80


Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L) 119903 = 0724

119875 = 0027 lt 005

(a)

0

10

20

30

40

50

60

70

80

0 5 10 15 20

Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L)


119903 = 0747

119875 = 0021 lt 005

(b)






5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

12


MFI

of p

late

let c

alci

um io

n

119899 = 9 119899 = 9 119899 = 9119899 = 10 119899 = 8 119899 = 8

998771

998771998771

lowastlowast


Gelsolin


0

02

04

06

08

1

12

14


120573-actin


lowastlowast

Gels

olin

120573-a

ctin










0

10

20

30

40

50

60

70

80


Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L) 119903 = 0724

119875 = 0027 lt 005

(a)

0

10

20

30

40

50

60

70

80

0 5 10 15 20

Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L)


119903 = 0747

119875 = 0021 lt 005

(b)






5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

70

80


Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L) 119903 = 0724

119875 = 0027 lt 005

(a)

0

10

20

30

40

50

60

70

80

0 5 10 15 20

Plat

elet

gels

olin

conc

entr

atio

n (n

gm

L)


119903 = 0747

119875 = 0021 lt 005

(b)






5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Correlation between Platelet Gelsolin and...

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