)joebxj1vcmjtijoh$psqpsbujpo …downloads.hindawi.com/journals/bmri/2016/5675084.pdf · research...

Research ArticleIdentification of ITGA2B and ITGB3 Single-NucleotidePolymorphisms and Their Influences on the Platelet Function

Qian Xiang1 Shun-Dong Ji23 Zhuo Zhang1 Xia Zhao1 and Yi-Min Cui1

1Department of Pharmacy Base for Clinical Trial Peking University First Hospital Beijing 100034 China2Jiangsu Institute of Hematology Key Laboratory of Thrombosis and Hemostasis of Ministry of HealthThe First Affiliated Hospital of Soochow University Suzhou 215006 China3Collaborative Innovation Center of Hematology Soochow University Suzhou 215006 China

Correspondence should be addressed to Yi-Min Cui bdyyyljd126com

Received 3 February 2016 Accepted 18 April 2016

Academic Editor Robert Baiocchi

Copyright copy 2016 Qian Xiang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of the study was to investigate ITGA2B and ITGB3 genetic polymorphisms and to evaluate the variability in the plateletfunction in healthy Chinese subjects The genetic sequence of the entire coding region of the ITGA2B and ITGB3 genes wasinvestigated Adenosine diphosphate-induced platelet aggregation glycoprotein IIbIIIa content bleeding time and coagulationindexes were detected Thirteen variants in the ITGA2B locus and 29 variants in the ITGB3 locus were identified in the ChinesepopulationThe rs1009312 and rs2015049were associatedwith themean platelet volumeThe rs70940817was significantly correlatedwith the prothrombin timeThe rs70940817 and rs112188890were related with the activated partial thromboplastin time and ITGB3rs4642 was correlated with the thrombin time and fibrinogen The minor alleles of rs56197296 and rs5919 were associated withdecreased ADP-induced platelet aggregation and rs55827077 was related with decreased GPIIbIIIa per platelet The rs1009312rs2015049 rs3760364 rs567581451 rs7208170 and rs117052258 were related with bleeding time Further studies are needed toexplore the clinical importance of ITGA2B and ITGB3 SNPs in the platelet function

1 Introduction

Platelet aggregation plays a central role in the pathogenesisof acute thrombosis in coronary heart disease stroke andperipheral arterial disease The cellular events leading toplatelet aggregation aremediated by the binding of fibrinogento the glycoprotein (GP)IIbIIIa receptor of platelets as a finalcommon pathway GPIIbIIIa is a platelet-specific surfacemembrane receptor and also called alpha IIb beta 3 (aIIb1205733) in the integrin nomenclature and thus plays a primaryrole in both platelet adhesion and thrombus formation atthe vascular injury site [1] A large interindividual numbervariability for GPIIbIIIa receptors expressed on the plateletsurface is commonly observed [2 3] Moreover a defect inthe GPIIbIIIa complex or a qualitative abnormality of thiscomplex is seen inGlanzmannrsquos thrombasthenia patientswithimpaired platelet aggregation and increased bleeding [4]

The ITGA2B gene encodes the aIIb subunit (GPIIb)whereas the ITGB3 gene encodes 1205733 (GPIIIa) The ITGA2B

spanning 17 kb has 30 exons whereas the ITGB3 spanning46 kb has 15 exons they are closely located on chromosome17q2132 without evidence for coordinated expression [5]A few studies have linked single-nucleotide polymorphisms(SNPs) in ITGA2B and ITGB3 with increased or decreasedplatelet responses to various agonists and the risk of acutecoronary syndrome and atherosclerosis [6ndash8]

A common polymorphism in the ITGB3 known as thehuman platelet antigen 1 (HPA-1b PlA2 or rs5918) arisesfrom a single-nucleotide change at position 1565 in Exon2 ofITGB3 resulting in a leucine (PlA1) to proline (PlA2) substi-tution at residue 33 [9]The SNP has been extensively studiedas an inherited risk factor for acute coronary syndromeand for its effect on the platelet function [6ndash8] The HPA-3 (rs5910) polymorphism results from a thymine to guaninebase change that leads to the replacement of isoleucine (HPA-3a) by serine (HPA-3b) at codon 843 of GPIIb (ITGA2B)Thispolymorphism may potentially influence the activity of theGPIIbIIIa complex and associate with thrombosis [10]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 5675084 11 pageshttpdxdoiorg10115520165675084

2 BioMed Research International

According to the aforementioned studies GPIIbIIIa wasbelieved to be a platelet-platelet contact receptor playing animportant role in platelet aggregation and its SNPswere asso-ciated with platelet hyperreactivity and have an effect on thepharmacodynamics of antiplatelet drugs However limitedinformationwas available on other genetic polymorphisms ofITGA2B and ITGB3 in Asian populations or their associationwith the platelet function [11 12] Moreover the prevalenceof the PlA2 allele (rs5918) is dependent on ethnicity with afrequency of approximately 15100 in Caucasian populations[13] falling to less than 1100 in Oriental populations [12 14]

In this study regions of the exons of ITGA2B and ITGB3genes were sequenced in 86 unrelated healthy Chinese Inaddition the association between genetic variants of ITGA2Band ITGB3 exons and the adenosine diphosphate- (ADP-)induced platelet aggregation GPIIbIIIa content bleedingtime and coagulation indexes was investigated in 55 of the86 subjects

2 Materials and Methods

21 Study Design The research was conducted in compliancewith the Declaration of Helsinki The study protocol wasapproved by the Ethical Review Board of the Peking Univer-sity First Hospital All subjects gave their written informedconsent prior to participation in the study Healthy nativeChinese subjects (119899 = 86) between 18 and 45 years of age witha bodymass index (BMI) of 19ndash24 kgm2 were included in thestudy and their genotypes were unknown

All the subjects were considered healthy on the basis oftheirmedical history physical examination vital signs (bloodpressure pulse rate and temperature) safety laboratory tests(blood chemistry hematological tests and urinalysis) and 12-lead electrocardiography

TheADP-induced platelet aggregation (transmission119879max) GPIIbIIIa content (GPIIbIIIa per platelet) bleedingtime and coagulation indexes including the mean plateletvolume (MPV) platelet count (PLT) prothrombin time (PT)activated partial thromboplastin time (aPTT) fibrinogen(FIB) and thrombin time (TT) were detected in 55 of the 86subjects None of the donors had taken any medication for 2weeks before blood collection

22 ADP-Induced Platelet Aggregation Blood was collectedin 38 sodium citrate tubes and platelet-rich plasma (PRP)was obtained by centrifuging blood at 1000 revolutions perminute (rpm) for 10 minutes at room temperature The PRPwas collected in a fresh tube and platelet was counted byPlatelet Counter PL100 (Sysmex Kobe Japan) Platelet-poorplasma (PPP) was obtained by centrifuging the remainingblood at 3000 rpm for 10 minutes at room temperature andplatelet numbering PRP was adjusted to 250 times 109L by PPPDetection was completed within 1 hour after sampling

Platelet aggregation was determined by the turbidimetricmethod using 20 umolL ADP as the agonist After zerosetting with the PPP assays were performed in platelet-richplasma in a Chrono-Log aggregometer Platelet aggregationwas quantified as the maximum change in light transmissionoccurring within 5 minutes of addition of agonist

23 GPIIbIIIa Content GPIIbIIIa on the platelet surfacewas evaluated in the PRP as the maximal binding of a 125I-labeled GPIIbIIIa integrin antiplatelet antibody F(ab)

2[15]

The PRP was fixed with equal volume of anticoagulant-fixative solution (10mmolL EDTA-Na

2 02 glutaralde-

hyde and 002 sodium azide dissolved in PBS pH 74)After stored at 4∘C overnight platelet was washed byTyrodersquos solution twice and resuspended in Tyrode solu-tion containing 035 bovine serum albumin (concentrationadjusted to 1 times 1011 plateletsL) and sodium azide wasadded at final concentration of 002 (VV) to preserve theplatelet at 4∘C for a week-long before assay For detectionthe platelet suspension (100 120583L) was added to a 05mLEppendorf tube and then incubated with 100 120583L of antibody(containing 20000 cpm of labeled monoclonal antibody(McAb) and 08 120583g of unlabeled McAb) at 37∘C for 1 hourafter washed with 035 bovine serum albuminTyrodersquossolution for three times The radioactivity count of theprecipitate in the tube was measured Assuming that onemonoclonal antibody would only bind one GP moleculeon the platelet membrane the number of GP moleculeson the platelet membrane was calculated as number of GPmoleculesplatelet = (binding ratetimes amount of antibody (g)timesAvogadrorsquos number)(molecular weight of antibodytimes plateletcount)

24 Bleeding Time The skin bleeding time was carried outusing the Simplate-II device (General Diagnostics NJ USA)A sphygmomanometer cuff was inflated on the patientrsquos armto 40mmHg The arm was supported at the level of theheart and a muscular area on the volar aspect distal to theantecubital area was identified and swabbed with alcoholThe Simplate-II device was used to perform the incisionperpendicular to the antecubital fossa Bloodwas blottedwithsterile filter paper every 30 seconds until blood no longerstained the paper The time from incision to stopping ofbleeding was recorded

25 Genetic Analysis Genomic DNA was extracted fromperipheral whole blood samples of each subject using a DNAPurification Kit (Wizard Promega WI USA) After thepolymerase chain reaction (PCR) products were obtained allsamples were directly sequenced to determine the SNPs inITGA2B and ITGB3 (Life Technologies Biotechnology CoLtd China)

Primers required for PCR amplification and sequencingwere designed according to the wild-type ITGA2B andITGB3 sequences reported in theGenBank (NG 0083311 andNG 0083321 resp) Tables 1 and 2 describe the primer pairsused to amplify the promoters the 51015840- and 31015840-untranslatedregions (UTRs) the entire coding regions and the intron-exon junctions of the ITGA2B and ITGB3 genes

26 Data Analysis TheVariant Reporter v11 (Life Technolo-gies Biotechnology Co Ltd) software suite was used forthe initial analysis of the sequence including base callingfragment assembly SNP and sequence insertionsdeletionsdetection Polymorphisms of ITGA2B and ITGB3 geneswere named according to the genomic reference sequences

BioMed Research International 3

Table1Summaryof

GPIIb

(ITGA

2B)v

ariatio

nsdetected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0004

193

Nucleotidec

hange

NP00

04102

aminoacid

change

MA(F)

H-W119875

ITGA2B

-01

rs3760364lowast

51015840up

stream

end

44390436

-963

ctcccaaggg

AT

ctcatttaca

T(0052)

060

9ITGA2B

-02

New

51015840up

stream

end

44390152

-679

tagaccaagg

TCccattcacca

C(000

6)0957

ITGA2B

-03

New

51015840up

stream

end

44390081

-608

caagacggag

GA

aggagtgagg

A(0006)

0957

ITGA2B

-04

New

Exon

344

385910

354

tgatgagacc

CTgaaatgtagg

Arg108Stop

T(000

6)0957

ITGA2B

-05

New

Exon

1344

380960

1344

ctcccaggtcCAtggacagccc

Leu4

38Met

A(0006)

0957

ITGA2B

-06

rs201355504

Intro

n13

44380821

1393+5

8cttggcacttCTcagcgaatgt

A(0006)

0957

ITGA2B

-07

New

Exon

1444

380638

1401

tagacctgatCGgtgggagctt

Ile46

7Met

G(0006)

0957

ITGA2B

-08

rs850730

Intro

n21

44377095

2188-7Cgt

GccctcctcatCGtcccagatag

G(0477)

0289

ITGA2B

-09

New

Exon

2344

376336

2320

cgtgccggtcCTgggcagaggc

Arg774T

rpT(000

6)0957

ITGA2B

-10

rs117

870452

Exon

2344

376322

2334

gcagctccac

CTtgggcctctg

Gln778=

G(0012)

0913

ITGA2B

-11

rs5911

Exon

2644

375697

2621Tgt

Gggggctgggg

AC

tgggcagccc

Ile874Ser

G(0477)

0289

ITGA2B

-12

rs5910

Exon

3044

372421

3063Cgt

Tacccccaggt

CTggcttcttc

aVa

l1021=

T(0465)

0289

ITGA2B

-13

New

31015840-U

TR44

372213

lowast151CgtA

gctacccccc

CAtcctgctgcc

A(0017)

0869

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib

rium119875valueMA(F)minor

allele(fr

equency)G

Pglycop

rotein

lowastSN

Pswhich

related

with

bleeding

timeinthisstu

dy


Table 2 Primer sequences used in this study

DNAsequencenumber

Amplified orsequencedregion

Forward primer (51015840 to 31015840) Reverse primer (51015840 to 31015840) Amplified regionNC 00001711

Length(bp)

ITGA2B-1 TCCTCCTCTTCCGCTTACCG TACTACCACCGTGCTAGTCC 44389728ndash44390630 848ITGA2B-2 Exon1 CCAATATGGCTGGTTGAG AACTTCCCTTACGGCTCA 44389267ndash44390059 792ITGA2B-3 Exon1 CCAGTGCAGCTCACCTTCTA GATGAGGGAAATGGAACAGA 44388253ndash44389368 1115ITGA2B-4 Intron1 TATGAACCACTCCACCCT TTGGCACTCTTGATTCTG 44388169ndash44389006 837ITGA2B-5 Exon2 Exon3 ACCGCTGGTTCTTGTTGC CCTACGGGCGTCTTCTCA 44385649-44386479 830ITGA2B-6 Exon4ndashExon6 TACAGGGCACAGGGAACAATC AGAGGCTCTGGGAGGACACG 44384990ndash44385810 820ITGA2B-7 Exon7 Exon8 TCCTGGCGGCTATTATTTCT GCACCGACGACATATTCTGG 44384339ndash44385186 847ITGA2B-8 Exon9ndashExon11 ATTTGCGCCCTTGTCCTC AGCCGAATCGCCCATAGA 44383538ndash44384605 1067ITGA2B-9 Exon12 CCCTCTGTCTCCCTTTCC CATCCAGTCTCCCACCAA 44383189ndash44383838 649ITGA2B-10 Exon13 Exon14 CCTAGTCTCCTGGGATGTTC TCACGGGTGTCTTGGTCT 44380390ndash44381163 773ITGA2B-11 Exon14ndashExon17 TAATCGCCAATTCTGACCC CACATCCCACCTTCTCCTG 44379854ndash44380682 828ITGA2B-12 Exon18 ACCCACTGGACTTGTTCATC TGTGACTTGGCACTAACCC 44379610ndash44379957 347ITGA2B-13 Exon19 Exon20 TGGACGACAGAGCGAGAC GGCCATACCTCGACATTG 44378354ndash44378989 635ITGA2B-14 Exon21 CATGTGACAGTCCCTTGA AAAGTCACTCACCCAAGGA 44377551ndash44377933 382ITGA2B-15 Exon22 CTTGGAGGGTGAAGACTGG CAACTCCTGACCTCCAGTGA 44376833ndash44377179 346ITGA2B-16 Exon23ndashExon25 CCAGGTCTAACTTCAGTGTG GCTCTGGCAGGAAGATCTGT 44375629ndash44376523 894ITGA2B-17 Exon26 TCCGACCTGCTCTACATCC CGGGCTTGCTCACATAGTC 44375277ndash44375913 636ITGA2B-18 Exon27 Exon28 ATGACCCTCCCTGCATCTC CACCTTGACACCTGCCTTT 44374500ndash44375317 817ITGA2B-19 Exon29 GCACGCATGGTTCAACGT CCTCCCGAGTAGCTGAGATT 44374025ndash44374731 706ITGA2B-20 Exon30 AAAGGCATCCATTTGTGA TGTTGGTAAGGCTGGTCTC 44371896ndash44372566 670ITGB3-1 CAGGAGGTGGAGGATTGT GCTGGATTCTTGGGACAC 47252637ndash47253474 837ITGB3-2 Exon1 CGGTTCAGAGAAGGCATTCAG GCTCCAAGTCCGCAACTTGA 47253373ndash47254015 642ITGB3-3 Intron1 TTGGCGTAGGAGGTGAGTGA CCGCAGGAAGCCAAGTTGAA 47253929ndash47254518 589ITGB3-4 Intron1 TTGGCGTAGGAGGTGAGTGAG GAAGTTGCAGTGAGCCGAGAA 47253929ndash47255063 1134ITGB3-5 Exon2 ATTGGGAAAGTTGGGAAGG GAAAGGGCAGCAGTGGTT 47274335ndash47274773 438ITGB3-6 Exon3 AGGCTGGTCTTGAACTCTTG CTCCACCTTGTGCTCTAT 47283116ndash47283612 496ITGB3-7 Exon4 GGGCTTTCTGGTTTGCTT CATTTCCCTCCCATTCTC 47284329ndash47284978 649ITGB3-8 Exon5 TGTCTGGGTAACTGTGGT CATCTGCCTACTTTGCTG 47286124ndash47286725 601ITGB3-9 Exon6 TCCAAGGACTGGGACTGA ATGATGCTGCTGCTATGC 47286919ndash47287483 564ITGB3-10 Exon7 Exon8 AGCCCAAGCAAGATAAGT GGAGAAGGCAGTAAGACC 47289597ndash47290393 796ITGB3-11 Exon9 AAACTGGGCTCCAATAAC TGAGGGACTGAAGGTAAAG 47290561ndash47291406 845ITGB3-12 Exon10 CAGGGCAGGGAACAACTT GGATTGGTCCTTATACTCAAAA 47292050ndash47292720 670ITGB3-13 Exon11 GAGCAAGTCCTGCCATAC TCACAGAGTGTCCTCCATAA 47299101ndash47299890 789ITGB3-14 Exon12 CAGAAATGGCATAGGGTT TCTTGCTGAGTCTGTGGG 47300168ndash47300824 656ITGB3-15 Exon13 CTTGAATCTAGGCATCGT GTATTGAACTCCTGACCC 47302581ndash47303050 469ITGB3-16 Exon14 CCTCAAGTAGGTCCCAGTG AACATGACCACCCAAAGC 47307158ndash47307721 563ITGB3-17 Exon15 CTCATCTCCTCCTGTTATTT TGACATTCTCCCAACCTAC 47309941ndash47310337 396

NG 0083311 andNG 0083321 respectively Novel SNPswerenamed as ITGA2B-number or ITGB3-number in the presentstudy

27 Statistical Analysis The association between two param-eters was assessed by Pearsonrsquos two-tailed test Statisticalanalyses were performed using the Statistical Package forSocial Sciences software program forWindows (SPSS version160) A 119875 value of 005 was considered significant

3 Results

31 ITGA2B and ITGB3Variations Within the Chinese sam-ple 13 and 29 variants in ITGA2B and ITGB3were identifiedrespectively Two novel ITGB3 SNPs with MA(F) gt 002were found which were not reported in the National Cen-ter for Biotechnology Information (NCBI) dbSNP database(httpswwwncbinlmnihgovsnp) The allele frequenciesandHardyndashWeinberg equilibrium test results of the identifiedpolymorphic sites are shown in Tables 3 and 4


Table3Summaryof

ITGB

3varia

tions

detected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0002122

Nucleotidec

hange

NP00

04102am

ino

acid

change

MA(F)

H-W119875

ITGB3

-01

rs147363351

51015840-U

TR47253042

-820

agcttccaga

GA

gttttaagtc

A(0012)

0913

ITGB3

-02

rs3809862

51015840-U

TR47253062

-800

ctggggaaga

CTccagggactc

T(0424)

0822

ITGB3

-03

rs7208170lowast

51015840-U

TR47253393

-469

aaggcattc

aGA

cagatgtttg

A(0419)

0680

ITGB3

-04

New

51015840-U

TR47253360

-502

tagtgaataa

TAaaaggactga

A(0023)

0913

ITGB3

-05

rs7208055

51015840-U

TR47253461

-401

gtgaatgtgt

CAccaagaatcc

A(0221)

0273

ITGB3

-06

rs55827077lowast

51015840-U

TR47253717

-145

tagagaagcc

GC

gaggggagga

C(044

8)0494

ITGB3

-07

rs567581451lowast

51015840-U

TR47253771

-91

acccaccgcg

-TC

CCCtcccctcccc

insTCC

CC(0058)

0567

ITGB3

-08

rs117

052258lowast

51015840-U

TR47253855

-7cgcgggaggc

GC

gacgagatgc

C(0227)

0771

ITGB3

-09

New

Exon

147253882

21ggccgcggcc

CGcggccgctct

Pro7=

G(0407)

0567

ITGB3

-10

rs11871251

Intro

n1472540

6179+121

ctgggaatgc

GA

cgtgtcctgg

A(0453)

0771

ITGB3

-11

New

Intro

n1472540

8279+152

tggcgcggtC

Gggagccggga

G(0012)

0913

ITGB3

-12

rs112188890lowast

Intro

n147254101

79+161

gagctgggga

CTcttcctggcc

T(0116

)0864

ITGB3

-13

rs117

414137lowast

Intro

n147254192

79+2

52aggctgagcg

CGcttcccggcc

G(0116)

0864

ITGB3

-14

rs118

7144

7Intro

n147254252

79+3

12ccgcgctcac

CGcggggctgcg

G(0448)

0918

ITGB3

-15

New

Intro

n147254331

79+3

91tggggcttc

cGA

ggggttg

ttcA(0006)

0957

ITGB3

-16

rs1009312lowast

Intro

n147254774

79+8

34ggcacagccc

GA

gggttgctgc

G(0471)

000

0ITGB3

-17

rs2015049

Intro

n147254865

79+9

25ggccgcctctGA

cctcagagga

A(052

9)000

0ITGB3

-18

rs56197296lowast

Intro

n547287025

47287029

778-45

778-41

catggctgaa

TTTG

T-tttg

tctcct

delTTT

GT(0169)

0049

ITGB3

-19

rs5919lowast

Exon

647287174

882

ttgtccagccT

Caatgacgggc

Pro294=

C(0262)

0022

ITGB3

-20

rs41504748

Intro

n747290145

1036-40

accaccagctTCcctttggtaa

C(0070)

0334

ITGB3

-21

rs15908

Exon

947290971

1143

cttccagctcAG

Tactttagaac

Val381=

C(0599)

000

6ITGB3

-22

New

Exon

1047292177

1299

gaggctgtcc

CTcaggagaagg

Pro4

33=

T(000

6)0957

ITGB3

-23

rs46

42lowast

Exon

1047292411

1533AgtG

cagcaggacgaA

Gtgcagccccc

Glu511=

G(033

1)0214

ITGB3

-24

rs133064

87Ex

on10

47292422

1544

tgcagccccc

ACG

ggagggtcag

Arg515G

lnA(0012)

0913

ITGB3

-25

rs4634lowast

Exon

1047292423

1545

gcagcccccg

GA

gagggtcagc

Arg515=

A(033

1)0214

ITGB3

-26

New

Exon

1047292528

1659

gcgagtgtga

CTg

acttc

tcct

Asp550=

T(000

6)0957

ITGB3

-27

rs149823724

Exon

1147299519

1902

cagatgcctg

CTaccttta

aga

Cys634=

T(000

6)0957

ITGB3

-28

rs118

70252

Intro

n11

47300459

1914-19

cctta

atcacT

Cgtgtcctctc

C(0035)

0869

ITGB3

-29

rs70940817lowast

Exon

1247300524

1960

cctacatgac

GA

aaaatacctg

Glu654Lys

A(0076)

0432

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib


allele(fr

equency)

lowastSN

Pswhich

related

with

ADPindu

cedplateletsa

ggregatio

nGPIIbII

Iacontentbleeding

timeor

coagulationindexesinthisstu

dy


Table 4 Association between individual ITGA2B and ITGB3 SNPs and the ADP induced platelets aggregation GPIIbIIIa content bleedingtime PLT and MPV (mean plusmn SD)

dbSNP Genotypes 119873Aggregation max119879

GPIIb-IIIa perplatelet Bleeding time (s) PLT (109L) MPV (fL)

rs3760364 AA 48 366 plusmn 153 459371 plusmn 152140 2813 plusmn 974 2770 plusmn 7087 948 plusmn 084

AT 7 274 plusmn 161 410609 plusmn 155607 3643 plusmn 873 2577 plusmn 837 951 plusmn 066

119875 0148 0433 0038 0512 0911

rs7208170GG 18 385 plusmn 162 488350 plusmn 137460 3267 plusmn 1008 2551 plusmn 621 914 plusmn 074

GA 24 351 plusmn 143 457149 plusmn 157831 2888 plusmn 866 2906 plusmn 828 963 plusmn 080

AA 13 317 plusmn 171 397090 plusmn 155451 2492 plusmn 1091 2721 plusmn 604 968 plusmn 084

119875 0229 0105 0030 0428 0051


GC 22 351 plusmn 130 471507 plusmn 137069 2795 plusmn 1024 2919 plusmn 855 955 plusmn 085

CC 14 306 plusmn 181 365181 plusmn 166619 2871 plusmn 1133 2801 plusmn 552 966 plusmn 093

119875 0091 0021 0401 0251 0141

rs567581451 -- 49 350 plusmn 144 453176 plusmn 148926 2822 plusmn 991 2773 plusmn 737 956 plusmn 080

-insTCCCC 6 385 plusmn 244 453068 plusmn 191360 3700 plusmn 648 2523 plusmn 568 888 plusmn 062

119875 0609 0999 0040 0428 0054




119875 0165 0539 0028 0764 0288

rs1009312aGG 19 393 plusmn 159 464707 plusmn 137986 3268 plusmn 842 2764 plusmn 950 913 plusmn 061



119875 0189 0160 0027 0944 0029

rs56197296TTTGTTTTGT 42 377 plusmn 159 453981 plusmn 145647 2850 plusmn 947 2805 plusmn 763 941 plusmn 081

TTTGTdel 11 309 plusmn 102 452629 plusmn 187224 3300 plusmn 1162 2496 plusmn 571 978 plusmn 079

deldel 2 115 plusmn 78 438955 plusmn 164310 2250 plusmn 636 2880 plusmn 212 930 plusmn 113

119875 0014 0916 0695 0433 0416

rs5919TT 31 388 plusmn 161 453425 plusmn 140291 2990 plusmn 966 2837 plusmn 788 934 plusmn 084

TC 16 349 plusmn 125 476965 plusmn 175449 3150 plusmn 1056 2477 plusmn 586 964 plusmn 073


119875 0015 0634 0130 0728 0148Note Pearsonrsquos two-tailed test was used to analyze correlation between genotype and parameter ars1009312 was completely linked with rs2015049

32 Linkage Disequilibrium Analysis Using the detectedpolymorphisms greater than 001 in frequency [MA(F) gt002] linkage disequilibrium (LD) was analyzed for |1198631015840|and 1199032 values (Figure 1) For ITGA2B rs5911 was completelylinked with rs850730 (1199032 = 100) and rs5910 was stronglylinked with rs850730 and rs5911 (1199032 = 095) For ITGB3 com-plete LD was observed between rs4642 and rs4634 (1199032 =100) Other LD results are shown in Figure 1 A new SNPITGB3-09 was found for which MA(F) in this study was0402

The HardyndashWeinberg equilibrium 119875 values of rs1009312and rs2015049 were lt0001 The possible reason is that thesample in this study may have a Chinese population of otherethnic groups besides the Han population Those two SNPswere not included in the LD analysis

33 Correlation of ADP-Induced Platelet Aggregation GPIIbIIIa Content Bleeding Time and Coagulation Indexes TheGPIIbIIIa contents present as average numbers of GPIIbIIIa receptor in each platelet were associated with ADP-induced platelet aggregation (119903 = 0280 119875 = 0038) PLT(119903 = minus0522 119875 lt 0001) PT (119903 = 0453 119875 = 0001) andTT (119903 = 0545 119875 lt 0001) (Figures 1 and 2) The relationshipbetween GPIIbIIIa content and APTT was not significant(119903 = 0242 119875 = 0075) while the association between ADP-induced platelet aggregation and APTT was moderatelysignificant (119903 = 0267 119875 = 0049) (Figure S1) The bleedingtime had no association with other parameters It had anegative association trend with MPV but was not significant(119903 = minus0244 119875 = 0073) The PLT was associated with PT(119903 = minus0415 119875 = 0002) and TT (119903 = minus0363 119875 = 0007)


rs59

10

rs59

11

rs11

7870

452

rs85

0730

rs37

6036

4

Block 1 (4kb)1 2 3 4 5

(a)

rs14

7363

351

rs38

0986

2

rs72

0817

0

rs72

0805

5

rs55

8270

77

ITG

B3-0

9

rs11

7052

258

rs56

7581

451

rs11

8712

51

rs12

1888

90

rs11

7414

137

rs11

8714

47

rs56

1972

96

rs59

19

rs41

5047

48

rs15

908

rs46

42

rs46

34

rs70

9408

17

6666

9191

43 9494

643

9394

6284

8797

9794

95

6866

7777

6668

9494

9495

2325

2635

8248

47

6728

2822

1125

2566

6558

4073

7340

116

2418

1434

345

6264

69

2222

3939

6695

9090

9090

4040

2996

8766

9443

28

6565

4844

7049

2826

9455

5577

7712

12891 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 22

Block 1 (0kb) Block 2 (0kb) Block 3 (0kb) Block 4 (0kb) Block 5 (5kb)

(b)

Figure 1 Linkage disequilibrium (LD)map of ITGB2A and ITGB3 SNPs along with their locations in the ITGB2A and ITGB3 genes Variantspresent at gt1 frequency were included in the LD analysis using the statistics |1198631015840| and 1199032 Red depicts a significant linkage between an SNPpair Numbers inside the square indicate 1199032 times 100 (a) ITGB2A (b) ITGB3

The FIB was negatively correlated with TT (119903 = minus0409119875 = 0002)

34 Impact of ITGA2B and ITGB3 SNPs on ADP-InducedPlatelet Aggregation GPIIbIIIa Content Bleeding Time andCoagulation Indexes For ITGA2B only one SNP rs3760364was related with the bleeding time (119875 = 0038) (Table 4)No significant correlation or trend was observed betweenITGA2B SNPs and other parameters

For ITGB3 the mean values of ADP-induced plateletaggregation of homozygous mutant genotypes in rs56197296

and rs5919 were lower than those of heterozygous mutationsand wild type Moreover the GPIIbIIIa per platelet wasassociated with rs55827077 A trend of GPIIbIIIa per plateletwas observed among rs70940817 GG (45) GA (9) andAA (1)carriers (438077 plusmn 141575 510868 plusmn 193680 61277 resp119875 = 0093) although it did not reach a significant levelThe bleeding time was significantly related with rs3760364rs7208170 rs567581451 and rs117052258 (Table 4)

In the present study SNPs related with PLT were notfound ITGB3 rs1009312 which is completely linked withrs2015049 was significantly associated with the MPV


0

10

20

30

40

50

60

70

80

0 10000 20000 30000 40000 50000 60000 70000 80000GPIIb-IIIa per platelet

r = 0280 P = 0038

Agg

rega

tion

max

T

Figure 2 Correlations of the maximal level of ADP-inducedplatelet aggregation and GPIIbIIIa content in healthy volunteers119903 coefficient of correlation 119875 significance of correlation (Pearsonrsquostest here and elsewhere)

(Table 4) ITGB3 rs70940817 was correlated with the PT andAPTT (119875 = 0002 and 119875 = 0003 resp) The coagulationindexes and their 119875 values are summarized in Table 5

4 Discussion

In this study 13 variants in the ITGA2B locus and 29 variantsin the ITGB3 locus in the Chinese population were observedTwo of the 29 variants located in ITGB3 were novel SNPswith MA(F) gt 002 Variants in ITGA2B and ITGB3 genesdisplayed significant interethnic differences in the globalpopulations (Table 6) The C allele frequency of SNP rs5918(TC) located in the ITGB3 gene was only inhomogeneouslydistributed at 07 in the Chinese population while it iscommon in whites and Africans (allele frequency 137versus 128) The following alleles of SNPs rs7208170Ars55827077C rs11871251A rs1009312G and rs2015049G werefound to be more common in the Han Chinese and Africans(37sim50) than in the European ancestry (10sim20) Inthe ITGA2B gene the rs5911 was completely linked withrs850730 and strongly linked with rs5910 in the Chinesepopulation in this study and all the three SNPswere commonin whites and Asians However the rs5911 was not found inAfricans According to the ethnicity difference described inthe preceding text resequencing of the ITGA2B and ITGB3genes in the Chinese population was believed to be veryimportant to reveal the function of SNPs

Variation in theMPV or PLT can have a profound impacton differences in the platelet function between individuals[16 17] and these traits have a strong genetic component [18ndash21] However limited information is available for Asians Inthe present study the A alleles of rs1009312 and rs2015049located in ITGB3 were positively associated with the MPV(119875 = 0029) The MPV had a trend in rs7208170 alleles butdid not reach a significant level (119875 = 0051) The differ-ence between PLT and SNPs in ITGA2B or ITGB3 was notsignificant

The APTT and PT are clinical tests commonly used toindicate coagulation factor deficiencies [22 23] activatedcoagulation and risk of venous thromboembolism [24 25]

To date two genome-wide association studies (GWAS) ofAPTT and PT conducted in the European ancestry have beenreported One study identified genome-wide significant asso-ciations of APTT and variants at F12 (MIM 610619) KNG1(MIM 612358) and HRG (MIM 142640) [26] In the otherresearch the GWAS for APTT and PT was conducted andreplicated genome-wide significant associations at KNG1HRG F11 F12 and ABO for APTT and identified significantassociations at the F7 and PROCREDEM2 regions for PTEight genetic loci accounted for sim29 of the variance inAPTT and two loci accounted for sim14 of the variance in PT[27] In this study the association of APTT PT FIB and TTwith ITGA2B and ITGB3SNPs was investigated The A alleleof ITGB3 rs70940817 was found to significantly correlate withthe elevated APTT and PT (119875 = 0003 and 119875 = 0002 resp)The T allele of ITGB3 rs112188890 was related with APTT(119875 = 0029) and the G allele of ITGB3 rs4642 was associatedwith TT and FIB (119875 = 0015 and 119875 = 0029 resp)

TheADP-induced platelet aggregation test was often usedto identify the platelet function and the efficacy of antiplateletdrugs Jones et al detected 1327 SNPs and investigated theircorrelation with ADP or collagen-related peptide-inducedplatelet aggregation in 500 healthy northern European sub-jects This identified 17 novel associations with the plateletfunction (119875 lt 0005) accounting for approximately 46 ofthe variation in response [28] In this study the minor alleleof rs56197296 and rs5919 was found to be associated with thedecreased ADP-induced platelet aggregation (119875 = 0014 and119875 = 0015 resp)

GPIIbIIIa is the central receptor of platelet aggrega-tion The variations of GPIIbIIIa amount expressed on aplatelet surface might affect the platelet-aggregating activityMany studies reported that the rs5918 correlated with theGPIIbIIIa receptor expression [29] however many conflictstudies have also been published [3 30] OrsquoHalloran et alinvestigated whether three polymorphisms of the GPIIIapromoter (minus468GA minus425AC and minus400CA) influencedthe RNA expression and receptor density in the plateletsof patients with cardiovascular disease [3] They found athreefold variation between the subjects in the number ofGPIIbIIIa receptors expressed per platelet although noassociation between the receptor density and the PlA2 orthe three promoter polymorphisms was demonstrated In thepresent study rs55827077 which was located at the promoterregion of the GPIIIs gene at position minus145 was related withGPIIbIIIa per platelet

The bleeding time is a test that evaluates the plateletfunction in vivo A prolonged bleeding time can result fromplatelet abnormality Von Willebrand factor deficiency orvascular disorders such as EhlersndashDanlos syndrome A dis-ruption of platelet aggregation results in a prolongation of thebleeding time [31] In this study the bleeding time was relatedwith rs3760364 of ITGA2B and rs7208170 rs567581451 andrs117052258 of ITGB3 (119875 lt 005) No previous study report-ing on the effect of SNPs on the bleeding time was foundThemechanism of the relationship needs to be further examinedHowever a negative trend between the bleeding time andMPV (119903 = minus0244 119875 = 0073) was observed Moreoverrs1009312 and rs2015049 were found to significantly associate


Table 5 Association between individual ITGA2B and ITGB3 SNPs and the coagulation indexes (mean plusmn SD)

Genotypes 119873 PT (s) APTT (s) TT (s) FIB (gL)

rs112188890aCC 42 109 plusmn 108 312 plusmn 213 157 plusmn 222 252 plusmn 045

CT 12 112 plusmn 135 322 plusmn 273 147 plusmn 159 272 plusmn 044

TT 1 125 367 165 272119875 0126 0029 0299 0173

rs4642bAA 27 109 plusmn 120 309 plusmn 225 161 plusmn 236 243 plusmn 042

AG 21 113 plusmn 108 320 plusmn 234 152 plusmn 171 268 plusmn 044

GG 7 104 plusmn 101 323 plusmn 279 142 plusmn 139 276 plusmn 045

119875 0770 0088 0015 0029

rs70940817GG 45 107 plusmn 108 311 plusmn 219 152 plusmn 201 252 plusmn 042

GA 9 118 plusmn 102 328 plusmn 226 168 plusmn 234 275 plusmn 054

AA 1 127 372 1575 310119875 0002 0003 0078 0069MPV mean platelet volume PLT platelet count PT prothrombin time APTT activated partial thromboplastin time FIB fibrinogen TT thrombin timeNote Pearsonrsquos two-tailed test was used to analyze correlation between genotype and parameteraStrong LD was observed between rs112188890 and rs117414137 (1199032 = 099) bcompletely LD was observed between rs4642 and rs4634

Table 6 Comparison of ITGA2B and ITGB3 allele frequencies in different ethnic groups

dbSNP MA(F)a

Number This study Chinese Asianb European-ancestryc Africand

ITGA2B-01 rs3760364 T(0052) T(0012) T(0) T(0)ITGA2B-08 rs850730 G(0477) G(0467) G(0297) G(0437)ITGA2B-11 rs5911 G(0477) G(0467) G(0305) G(0)ITGA2B-12 rs5910 T(0465) T(0442) T(0376) T(0432)ITGB3-03 rs7208170 A(0419) A(0496) A(0194) A(0431)ITGB3-05 rs7208055 A(0221) A(0225) A(0117) A(0356)ITGB3-06 rs55827077 C(0448) C(0417) C(0158) C(039)ITGB3-08 rs117052258 C(0227) C(0208) NA NAITGB3-10 rs11871251 A(0453) A(0482) A(0199) A(0373)ITGB3-12 rs12188890 T(0116) T(0) T(0023) T(0)ITGB3-16 rs1009312 G(0417) A(0434) A(0125) A(0432)ITGB3-17 rs2015049 G(0471) A(0478) A(0138) A(0413)NF rs5918 C(0) C(0007) C(0137) C(0128)ITGB3-18 rs56197296 delTTTGT(0169) NA NA NAITGB3-19 rs5919 C(0262) C(0232) C(0049) C(0187)ITGB3-23 rs4642 G(0331) G(031) G(0283) G(0306)ITGB3-25 rs4634 A(0331) A(0350) A(0283) A(0331)ITGB3-29 rs70940817 A(0076) NA NA NAdbSNP single nucleotide polymorphism database MA(F) minor allele (frequency) SNP single nucleotide polymorphisms NF not found NA not availableaResource of MP(F)s was from GenBank and HapMap httpswwwncbinlmnihgovsnp bHan Chinese in Beijing China (CHB) or CHB + Japanese inTokyo Japan (JPT) if there is no CHB data in the SNP cUtah residents with Northern and Western European ancestry from the CEPH collection (CEU)dYoruba in Ibadan Nigeria (YRI)

with both bleeding time and MPV Further the minor allelesof rs7208170 and rs567581451 whichwere significantly relatedwith the bleeding time had a trend with the MPV amonggenotypes although they did not reach a significant level (119875 =0051 and 119875 = 0054 resp) However no relationship wasobserved among SNPs bleeding time andMPV in rs3760364and rs117052258

In this study a positive correlation of the level of ADP-induced aggregation and GPIIbIIIa content was detectedin healthy volunteers This correlation is consistent with a

previous report Yakushkin et al investigated the relationshipbetween the number of GPIIbIIIa and the level of ADP-induced aggregation in a group of 35 healthy volunteers andfound positive and significant correlations between the levelof platelet aggregation induced by different ADP doses (from125 to 20mmolL) and the number of GPIIbIIIa [32]

A strong positive correlation was observed betweenGPIIbIIIa per platelet and PLT PT and TT (119875 lt 001)Although the TT was strongly correlated with GPIIbIIIa perplatelet (119903 = 0545 119875 lt 0001) the relationship of SNPsTT


and SNPsGP (IIbIIIa) content is not linked For exampleITGB3 rs4642 was significantly related with the TT (119875 =0015) while the value of GPIIbIIIa per platelet had no trendamong rs4642 genotypes (119875 = 0789) The other coagulationindexes were the same Therefore the relationship betweenSNPs and GPIIbIIIa content cannot be deduced from thecorrelation of coagulation indexes and SNPs

Some of the significant correlations between SNPs andplatelet function parameters in the present study were notreported previously which may be due to the reason thatSNPs with low MA(F) in the European ancestry were notselected as candidate SNPs in previous studies For examplethe GWAS-genotyping platforms lack the coverage of lowfrequencyrare variants [33] Moreover LDmight be anotherreason if different genetic patterns (haplotypes) are presentin different populations [3]

A possible limitation of the current study is the limitednumber of subjects After stratification the number of sub-jects in each genotypic group was small thereby limitingfurther haplotype association analysis and accession of smalleffects of an SNP with a small minor allele frequency

5 Conclusion

In summary as ethnicity difference might limit the interpre-tation of the function of SNPs resequencing the ITGA2B andITGB3 genes and investigating their functions in the Chinesepopulation are very important In the present study nineSNPs were found to associate with indexes of platelet andcoagulation haemostasis Newer studies are needed particu-larly to further assess the clinical importance of the above-discussed SNPs in disease susceptibility and antiplateletdrugs pharmacodynamics Further studies should pay moreattention to the roles of ITGA2B and ITGB3 SNPs in ethnicvariations

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This studywas supported by grants from theNational NaturalScience Foundation of China (no 81273592 no 81202592and no 81373487)

References

[1] J J Calvete ldquoClues for understanding the structure and functionof a prototypic human integrin the platelet glycoprotein IIbIIIacomplexrdquo Thrombosis and Haemostasis vol 72 no 1 pp 1ndash151994

[2] C LWagnerM AMascelli D S Neblock H FWeisman B SColler and R E Jordan ldquoAnalysis of GPIIbIIIa receptor num-ber by quantification of 7E3 binding to human plateletsrdquo Bloodvol 88 no 3 pp 907ndash914 1996

[3] A M OrsquoHalloran R Curtin F OrsquoConnor et al ldquoThe impactof genetic variation in the region of the GPIIIa gene on Plexpression bias and GPIIbIIIa receptor density in plateletsrdquo

British Journal of Haematology vol 132 no 4 pp 494ndash5022006

[4] S Bellucci and J Caen ldquoMolecular basis ofGlanzmannrsquosThrom-basthenia and current strategies in treatmentrdquo Blood Reviewsvol 16 no 3 pp 193ndash202 2002

[5] M Fiore A T Nurden P Nurden and U Seligsohn ldquoClinicalutility gene card for Glanzmann thrombastheniardquo EuropeanJournal of Human Genetics vol 20 no 10 p 1101 2012

[6] C N Floyd B H Ellis and A Ferro ldquoThe PlA1A2 polymor-phismof glycoprotein IIIa as a risk factor for stroke a systematicreview and meta-analysisrdquo PLoS ONE vol 9 no 7 Article IDe100239 2014

[7] CN Floyd andA Ferro ldquoThePlA1A2 polymorphismof glyco-protein IIIa in relation to efficacy of antiplatelet drugs a sys-tematic review and meta-analysisrdquo British Journal of ClinicalPharmacology vol 77 no 3 pp 446ndash457 2014

[8] C N Floyd A Mustafa and A Ferro ldquoThe PlA1A2 polymor-phism of glycoprotein IIIa as a risk factor for myocardial infarc-tion a meta-analysisrdquo PLoS ONE vol 9 no 7 article e1015182014

[9] P J Newman R S Derbes and R H Aster ldquoThe humanplatelet alloantigens PlA1 and PlA2 are associated with aleucine33proline33 amino acid polymorphism in membraneglycoprotein IIIa and are distinguishable by DNA typingrdquoJournal of Clinical Investigation vol 83 no 5 pp 1778ndash17811989

[10] J-H Wu D-W Zhang X-L Cheng H Shi and Y-P FanldquoPlatelet glycoprotein IIb HPA-3 ab polymorphism is associ-ated with native arteriovenous fistula thrombosis in chronichemodialysis patientsrdquoRenal Failure vol 34 no 8 pp 960ndash9632012

[11] M-P Li Y Xiong A Xu et al ldquoAssociation of platelet ITGA2Band ITGB3 polymorphisms with ex vivo antiplatelet effectof ticagrelor in healthy Chinese male subjectsrdquo InternationalJournal of Hematology vol 99 no 3 pp 263ndash271 2014

[12] Y Zhang Y Han L Dong et al ldquoGenetic variation of ITGB3is associated with asthma in chinese han childrenrdquo PLoS ONEvol 8 no 2 article e56914 2013

[13] S Simsek N M Faber P M Bleeker et al ldquoDeterminationof human platelet antigen frequencies in the Dutch populationby immunophenotyping and DNA (allele-specific restrictionenzyme) analysisrdquo Blood vol 81 no 3 pp 835ndash840 1993

[14] J Lim S Lal K CNg K-SNgN Saha andC-KHeng ldquoVari-ation of the platelet glycoprotein IIIa PIA1A2 allele frequenciesin the three ethnic groups of Singaporerdquo International Journalof Cardiology vol 90 no 2-3 pp 269ndash273 2003

[15] Z Yiming J Shundong D Ningzheng H Yang and RChanggeng ldquoMeasurement of affinity constant of humanizedmonoclonal antibody 813-F(ab)2 binding to platelets of humanmacaque and dosrdquo SuzhouUniversity Journal ofMedical Sciencevol 27 no 1 pp 8ndash10 2007

[16] PMW Bath andR J Butterworth ldquoPlatelet sizemeasurementphysiology and vascular diseaserdquo Blood Coagulation and Fibri-nolysis vol 7 no 2 pp 157ndash161 1996

[17] T J Kunicki S AWilliams D J Nugent andM Yeager ldquoMeanplatelet volume and integrin alleles correlate with levels of inte-grins 120572 iIb120573 3 and 120572 2120573 1 in acute coronary syndrome patientsand normal subjectsrdquoArteriosclerosisThrombosis and VascularBiology vol 32 no 1 pp 147ndash152 2012

[18] K Shameer J C Denny K Ding et al ldquoA genome- andphenome-wide association study to identify genetic variants


influencing platelet count and volume and their pleiotropiceffectsrdquo Human Genetics vol 133 no 1 pp 95ndash109 2014

[19] R Qayyum B M Snively E Ziv et al ldquoA meta-analysis andgenome-wide association study of platelet count and meanplatelet volume inAfricanAmericansrdquo PLoSGenetics vol 8 no3 Article ID e1002491 2012

[20] N Soranzo A Rendon C Gieger et al ldquoAnovel variant onchromosome 7q223 associated with mean platelet volumecounts and functionrdquoBlood vol 113 no 16 pp 3831ndash3837 2009

[21] C Meisinger H Prokisch C Gieger et al ldquoA genome-wideassociation study identifies three loci associated with meanplatelet volumerdquo American Journal of Human Genetics vol 84no 1 pp 66ndash71 2009

[22] G C White II ldquoThe partial thromboplastin time defining anera in coagulationrdquo Journal ofThrombosis and Haemostasis vol1 no 11 pp 2267ndash2270 2003

[23] S Kitchen A McCraw andM EchenaguciaDiagnosis of Hem-ophilia and Other Bleeding Disorders A Laboratory ManualWorld Federation of Hemophilia (WFH) Montreal Canada2nd edition 2010

[24] G Hron S Eichinger A Weltermann P Quehenberger W MHalbmayer and P A Kyrle ldquoPrediction of recurrent venousthromboembolism by the activated partial thromboplastintimerdquo Journal of Thrombosis and Haemostasis vol 4 no 4 pp752ndash756 2006

[25] N A Zakai T Ohira RWhite A R Folsom andM CushmanldquoActivated partial thromboplastin time and risk of future venousthromboembolismrdquo American Journal of Medicine vol 121 no3 pp 231ndash238 2008

[26] L M Houlihan G Davies A Tenesa et al ldquoCommon variantsof large effect in F12 KNG1 and HRG are associated withactivated partial thromboplastin timerdquoTheAmerican Journal ofHuman Genetics vol 86 no 4 pp 626ndash631 2010

[27] W Tang C Schwienbacher L M Lopez et al ldquoGenetic asso-ciations for activated partial thromboplastin time and pro-thrombin time their gene expression profiles and risk of coro-nary artery diseaserdquo American Journal of Human Genetics vol91 no 1 pp 152ndash162 2012

[28] C I Jones S Bray S F Garner et al ldquoA functional genomicsapproach reveals novel quantitative trait loci associated withplatelet signaling pathwaysrdquo Blood vol 114 no 7 pp 1405ndash14162009

[29] J S Bennett F Catella-Lawson A R Rut et al ldquoEffect of theP1A2 alloantigen on the function of 1205733-integrins in plateletsrdquoBlood vol 97 no 10 pp 3093ndash3099 2001

[30] O V Sirotkina S G Khaspekova A M Zabotina Y VShimanova and A V Mazurov ldquoEffects of platelet glycoproteinIIb-IIIa number and glycoprotein IIIa Leu33Pro polymorphismon platelet aggregation and sensitivity to glycoprotein IIb-IIIaantagonistsrdquo Platelets vol 18 no 7 pp 506ndash514 2007

[31] A Fuentes A Rojas K B Porter G Saviello andW F OrsquoBrienldquoThe effect of magnesium sulfate on bleeding time in preg-nancyrdquo American Journal of Obstetrics and Gynecology vol 173no 4 pp 1246ndash1249 1995

[32] V V Yakushkin I T Zyuryaev S G Khaspekova O V Sirotk-ina M Y Ruda and A V Mazurov ldquoGlycoprotein IIb-IIIa con-tent and platelet aggregation in healthy volunteers and patientswith acute coronary syndromerdquo Platelets vol 22 no 4 pp 243ndash251 2011

[33] B J Grady and M D Ritchie ldquostatistical optimization of phar-macogenomics association studies key considerations from

study design to analysisrdquo Current Pharmacogenomics and Per-sonalized Medicine vol 9 no 1 pp 41ndash66 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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EndocrinologyInternational Journal of



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BioMed Research International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


According to the aforementioned studies GPIIbIIIa wasbelieved to be a platelet-platelet contact receptor playing animportant role in platelet aggregation and its SNPswere asso-ciated with platelet hyperreactivity and have an effect on thepharmacodynamics of antiplatelet drugs However limitedinformationwas available on other genetic polymorphisms ofITGA2B and ITGB3 in Asian populations or their associationwith the platelet function [11 12] Moreover the prevalenceof the PlA2 allele (rs5918) is dependent on ethnicity with afrequency of approximately 15100 in Caucasian populations[13] falling to less than 1100 in Oriental populations [12 14]

In this study regions of the exons of ITGA2B and ITGB3genes were sequenced in 86 unrelated healthy Chinese Inaddition the association between genetic variants of ITGA2Band ITGB3 exons and the adenosine diphosphate- (ADP-)induced platelet aggregation GPIIbIIIa content bleedingtime and coagulation indexes was investigated in 55 of the86 subjects

2 Materials and Methods

21 Study Design The research was conducted in compliancewith the Declaration of Helsinki The study protocol wasapproved by the Ethical Review Board of the Peking Univer-sity First Hospital All subjects gave their written informedconsent prior to participation in the study Healthy nativeChinese subjects (119899 = 86) between 18 and 45 years of age witha bodymass index (BMI) of 19ndash24 kgm2 were included in thestudy and their genotypes were unknown

All the subjects were considered healthy on the basis oftheirmedical history physical examination vital signs (bloodpressure pulse rate and temperature) safety laboratory tests(blood chemistry hematological tests and urinalysis) and 12-lead electrocardiography

TheADP-induced platelet aggregation (transmission119879max) GPIIbIIIa content (GPIIbIIIa per platelet) bleedingtime and coagulation indexes including the mean plateletvolume (MPV) platelet count (PLT) prothrombin time (PT)activated partial thromboplastin time (aPTT) fibrinogen(FIB) and thrombin time (TT) were detected in 55 of the 86subjects None of the donors had taken any medication for 2weeks before blood collection

22 ADP-Induced Platelet Aggregation Blood was collectedin 38 sodium citrate tubes and platelet-rich plasma (PRP)was obtained by centrifuging blood at 1000 revolutions perminute (rpm) for 10 minutes at room temperature The PRPwas collected in a fresh tube and platelet was counted byPlatelet Counter PL100 (Sysmex Kobe Japan) Platelet-poorplasma (PPP) was obtained by centrifuging the remainingblood at 3000 rpm for 10 minutes at room temperature andplatelet numbering PRP was adjusted to 250 times 109L by PPPDetection was completed within 1 hour after sampling

Platelet aggregation was determined by the turbidimetricmethod using 20 umolL ADP as the agonist After zerosetting with the PPP assays were performed in platelet-richplasma in a Chrono-Log aggregometer Platelet aggregationwas quantified as the maximum change in light transmissionoccurring within 5 minutes of addition of agonist

23 GPIIbIIIa Content GPIIbIIIa on the platelet surfacewas evaluated in the PRP as the maximal binding of a 125I-labeled GPIIbIIIa integrin antiplatelet antibody F(ab)

2[15]

The PRP was fixed with equal volume of anticoagulant-fixative solution (10mmolL EDTA-Na

2 02 glutaralde-

hyde and 002 sodium azide dissolved in PBS pH 74)After stored at 4∘C overnight platelet was washed byTyrodersquos solution twice and resuspended in Tyrode solu-tion containing 035 bovine serum albumin (concentrationadjusted to 1 times 1011 plateletsL) and sodium azide wasadded at final concentration of 002 (VV) to preserve theplatelet at 4∘C for a week-long before assay For detectionthe platelet suspension (100 120583L) was added to a 05mLEppendorf tube and then incubated with 100 120583L of antibody(containing 20000 cpm of labeled monoclonal antibody(McAb) and 08 120583g of unlabeled McAb) at 37∘C for 1 hourafter washed with 035 bovine serum albuminTyrodersquossolution for three times The radioactivity count of theprecipitate in the tube was measured Assuming that onemonoclonal antibody would only bind one GP moleculeon the platelet membrane the number of GP moleculeson the platelet membrane was calculated as number of GPmoleculesplatelet = (binding ratetimes amount of antibody (g)timesAvogadrorsquos number)(molecular weight of antibodytimes plateletcount)

24 Bleeding Time The skin bleeding time was carried outusing the Simplate-II device (General Diagnostics NJ USA)A sphygmomanometer cuff was inflated on the patientrsquos armto 40mmHg The arm was supported at the level of theheart and a muscular area on the volar aspect distal to theantecubital area was identified and swabbed with alcoholThe Simplate-II device was used to perform the incisionperpendicular to the antecubital fossa Bloodwas blottedwithsterile filter paper every 30 seconds until blood no longerstained the paper The time from incision to stopping ofbleeding was recorded

25 Genetic Analysis Genomic DNA was extracted fromperipheral whole blood samples of each subject using a DNAPurification Kit (Wizard Promega WI USA) After thepolymerase chain reaction (PCR) products were obtained allsamples were directly sequenced to determine the SNPs inITGA2B and ITGB3 (Life Technologies Biotechnology CoLtd China)

Primers required for PCR amplification and sequencingwere designed according to the wild-type ITGA2B andITGB3 sequences reported in theGenBank (NG 0083311 andNG 0083321 resp) Tables 1 and 2 describe the primer pairsused to amplify the promoters the 51015840- and 31015840-untranslatedregions (UTRs) the entire coding regions and the intron-exon junctions of the ITGA2B and ITGB3 genes

26 Data Analysis TheVariant Reporter v11 (Life Technolo-gies Biotechnology Co Ltd) software suite was used forthe initial analysis of the sequence including base callingfragment assembly SNP and sequence insertionsdeletionsdetection Polymorphisms of ITGA2B and ITGB3 geneswere named according to the genomic reference sequences


Table1Summaryof

GPIIb

(ITGA

2B)v

ariatio

nsdetected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0004

193

Nucleotidec

hange

NP00

04102

aminoacid

change

MA(F)

H-W119875

ITGA2B

-01

rs3760364lowast

51015840up

stream

end

44390436

-963

ctcccaaggg

AT

ctcatttaca

T(0052)

060

9ITGA2B

-02

New

51015840up

stream

end

44390152

-679

tagaccaagg

TCccattcacca

C(000

6)0957

ITGA2B

-03

New

51015840up

stream

end

44390081

-608

caagacggag

GA

aggagtgagg

A(0006)

0957

ITGA2B

-04

New

Exon

344

385910

354

tgatgagacc

CTgaaatgtagg

Arg108Stop

T(000

6)0957

ITGA2B

-05

New

Exon

1344

380960

1344


Leu4

38Met

A(0006)

0957

ITGA2B

-06

rs201355504

Intro

n13

44380821

1393+5


A(0006)

0957

ITGA2B

-07

New

Exon

1444

380638

1401


Ile46

7Met

G(0006)

0957

ITGA2B

-08

rs850730

Intro

n21

44377095

2188-7Cgt


G(0477)

0289

ITGA2B

-09

New

Exon

2344

376336

2320


Arg774T

rpT(000

6)0957

ITGA2B

-10

rs117

870452

Exon

2344

376322

2334

gcagctccac

CTtgggcctctg

Gln778=

G(0012)

0913

ITGA2B

-11

rs5911

Exon

2644

375697

2621Tgt

Gggggctgggg

AC

tgggcagccc

Ile874Ser

G(0477)

0289

ITGA2B

-12

rs5910

Exon

3044

372421

3063Cgt

Tacccccaggt

CTggcttcttc

aVa

l1021=

T(0465)

0289

ITGA2B

-13

New

31015840-U

TR44

372213

lowast151CgtA

gctacccccc

CAtcctgctgcc

A(0017)

0869

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib


allele(fr

equency)G

Pglycop

rotein

lowastSN

Pswhich

related

with

bleeding

timeinthisstu

dy



DNAsequencenumber



Length(bp)




3 Results



Table3Summaryof

ITGB

3varia

tions

detected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0002122

Nucleotidec

hange

NP00

04102am

ino

acid

change

MA(F)

H-W119875

ITGB3

-01

rs147363351

51015840-U

TR47253042

-820

agcttccaga

GA

gttttaagtc

A(0012)

0913

ITGB3

-02

rs3809862

51015840-U

TR47253062

-800

ctggggaaga

CTccagggactc

T(0424)

0822

ITGB3

-03

rs7208170lowast

51015840-U

TR47253393

-469

aaggcattc

aGA

cagatgtttg

A(0419)

0680

ITGB3

-04

New

51015840-U

TR47253360

-502

tagtgaataa

TAaaaggactga

A(0023)

0913

ITGB3

-05

rs7208055

51015840-U

TR47253461

-401

gtgaatgtgt

CAccaagaatcc

A(0221)

0273

ITGB3

-06

rs55827077lowast

51015840-U

TR47253717

-145

tagagaagcc

GC

gaggggagga

C(044

8)0494

ITGB3

-07

rs567581451lowast

51015840-U

TR47253771

-91

acccaccgcg

-TC

CCCtcccctcccc

insTCC

CC(0058)

0567

ITGB3

-08

rs117

052258lowast

51015840-U

TR47253855

-7cgcgggaggc

GC

gacgagatgc

C(0227)

0771

ITGB3

-09

New

Exon

147253882

21ggccgcggcc

CGcggccgctct

Pro7=

G(0407)

0567

ITGB3

-10

rs11871251

Intro

n1472540

6179+121

ctgggaatgc

GA

cgtgtcctgg

A(0453)

0771

ITGB3

-11

New

Intro

n1472540

8279+152

tggcgcggtC

Gggagccggga

G(0012)

0913

ITGB3

-12

rs112188890lowast

Intro

n147254101

79+161

gagctgggga

CTcttcctggcc

T(0116

)0864

ITGB3

-13

rs117

414137lowast

Intro

n147254192

79+2

52aggctgagcg

CGcttcccggcc

G(0116)

0864

ITGB3

-14

rs118

7144

7Intro

n147254252

79+3

12ccgcgctcac

CGcggggctgcg

G(0448)

0918

ITGB3

-15

New

Intro

n147254331

79+3

91tggggcttc

cGA

ggggttg

ttcA(0006)

0957

ITGB3

-16

rs1009312lowast

Intro

n147254774

79+8

34ggcacagccc

GA

gggttgctgc

G(0471)

000

0ITGB3

-17

rs2015049

Intro

n147254865

79+9

25ggccgcctctGA

cctcagagga

A(052

9)000

0ITGB3

-18

rs56197296lowast

Intro

n547287025

47287029

778-45

778-41

catggctgaa

TTTG

T-tttg

tctcct

delTTT

GT(0169)

0049

ITGB3

-19

rs5919lowast

Exon

647287174

882

ttgtccagccT

Caatgacgggc

Pro294=

C(0262)

0022

ITGB3

-20

rs41504748

Intro

n747290145

1036-40


C(0070)

0334

ITGB3

-21

rs15908

Exon

947290971

1143

cttccagctcAG

Tactttagaac

Val381=

C(0599)

000

6ITGB3

-22

New

Exon

1047292177

1299

gaggctgtcc

CTcaggagaagg

Pro4

33=

T(000

6)0957

ITGB3

-23

rs46

42lowast

Exon

1047292411

1533AgtG

cagcaggacgaA

Gtgcagccccc

Glu511=

G(033

1)0214

ITGB3

-24

rs133064

87Ex

on10

47292422

1544

tgcagccccc

ACG

ggagggtcag

Arg515G

lnA(0012)

0913

ITGB3

-25

rs4634lowast

Exon

1047292423

1545

gcagcccccg

GA

gagggtcagc

Arg515=

A(033

1)0214

ITGB3

-26

New

Exon

1047292528

1659

gcgagtgtga

CTg

acttc

tcct

Asp550=

T(000

6)0957

ITGB3

-27

rs149823724

Exon

1147299519

1902

cagatgcctg

CTaccttta

aga

Cys634=

T(000

6)0957

ITGB3

-28

rs118

70252

Intro

n11

47300459

1914-19

cctta

atcacT

Cgtgtcctctc

C(0035)

0869

ITGB3

-29

rs70940817lowast

Exon

1247300524

1960

cctacatgac

GA

aaaatacctg

Glu654Lys

A(0076)

0432

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib


allele(fr

equency)

lowastSN

Pswhich

related

with

ADPindu

cedplateletsa

ggregatio

nGPIIbII

Iacontentbleeding

timeor


dy







119875 0148 0433 0038 0512 0911




119875 0229 0105 0030 0428 0051




119875 0091 0021 0401 0251 0141



119875 0609 0999 0040 0428 0054




119875 0165 0539 0028 0764 0288




119875 0189 0160 0027 0944 0029




119875 0014 0916 0695 0433 0416









rs59

10

rs59

11

rs11

7870

452

rs85

0730

rs37

6036

4

Block 1 (4kb)1 2 3 4 5

(a)

rs14

7363

351

rs38

0986

2

rs72

0817

0

rs72

0805

5

rs55

8270

77

ITG

B3-0

9

rs11

7052

258

rs56

7581

451

rs11

8712

51

rs12

1888

90

rs11

7414

137

rs11

8714

47

rs56

1972

96

rs59

19

rs41

5047

48

rs15

908

rs46

42

rs46

34

rs70

9408

17

6666

9191

43 9494

643

9394

6284

8797

9794

95

6866

7777

6668

9494

9495

2325

2635

8248

47

6728

2822

1125

2566

6558

4073

7340

116

2418

1434

345

6264

69

2222

3939

6695

9090

9090

4040

2996

8766

9443

28

6565

4844

7049

2826

9455

5577

7712

12891 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 22


(b)








0

10

20

30

40

50

60

70

80


r = 0280 P = 0038

Agg

rega

tion

max

T



4 Discussion













TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Summaryof

GPIIb

(ITGA

2B)v

ariatio

nsdetected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0004

193

Nucleotidec

hange

NP00

04102

aminoacid

change

MA(F)

H-W119875

ITGA2B

-01

rs3760364lowast

51015840up

stream

end

44390436

-963

ctcccaaggg

AT

ctcatttaca

T(0052)

060

9ITGA2B

-02

New

51015840up

stream

end

44390152

-679

tagaccaagg

TCccattcacca

C(000

6)0957

ITGA2B

-03

New

51015840up

stream

end

44390081

-608

caagacggag

GA

aggagtgagg

A(0006)

0957

ITGA2B

-04

New

Exon

344

385910

354

tgatgagacc

CTgaaatgtagg

Arg108Stop

T(000

6)0957

ITGA2B

-05

New

Exon

1344

380960

1344


Leu4

38Met

A(0006)

0957

ITGA2B

-06

rs201355504

Intro

n13

44380821

1393+5


A(0006)

0957

ITGA2B

-07

New

Exon

1444

380638

1401


Ile46

7Met

G(0006)

0957

ITGA2B

-08

rs850730

Intro

n21

44377095

2188-7Cgt


G(0477)

0289

ITGA2B

-09

New

Exon

2344

376336

2320


Arg774T

rpT(000

6)0957

ITGA2B

-10

rs117

870452

Exon

2344

376322

2334

gcagctccac

CTtgggcctctg

Gln778=

G(0012)

0913

ITGA2B

-11

rs5911

Exon

2644

375697

2621Tgt

Gggggctgggg

AC

tgggcagccc

Ile874Ser

G(0477)

0289

ITGA2B

-12

rs5910

Exon

3044

372421

3063Cgt

Tacccccaggt

CTggcttcttc

aVa

l1021=

T(0465)

0289

ITGA2B

-13

New

31015840-U

TR44

372213

lowast151CgtA

gctacccccc

CAtcctgctgcc

A(0017)

0869

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib


allele(fr

equency)G

Pglycop

rotein

lowastSN

Pswhich

related

with

bleeding

timeinthisstu

dy



DNAsequencenumber



Length(bp)




3 Results



Table3Summaryof

ITGB

3varia

tions

detected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0002122

Nucleotidec

hange

NP00

04102am

ino

acid

change

MA(F)

H-W119875

ITGB3

-01

rs147363351

51015840-U

TR47253042

-820

agcttccaga

GA

gttttaagtc

A(0012)

0913

ITGB3

-02

rs3809862

51015840-U

TR47253062

-800

ctggggaaga

CTccagggactc

T(0424)

0822

ITGB3

-03

rs7208170lowast

51015840-U

TR47253393

-469

aaggcattc

aGA

cagatgtttg

A(0419)

0680

ITGB3

-04

New

51015840-U

TR47253360

-502

tagtgaataa

TAaaaggactga

A(0023)

0913

ITGB3

-05

rs7208055

51015840-U

TR47253461

-401

gtgaatgtgt

CAccaagaatcc

A(0221)

0273

ITGB3

-06

rs55827077lowast

51015840-U

TR47253717

-145

tagagaagcc

GC

gaggggagga

C(044

8)0494

ITGB3

-07

rs567581451lowast

51015840-U

TR47253771

-91

acccaccgcg

-TC

CCCtcccctcccc

insTCC

CC(0058)

0567

ITGB3

-08

rs117

052258lowast

51015840-U

TR47253855

-7cgcgggaggc

GC

gacgagatgc

C(0227)

0771

ITGB3

-09

New

Exon

147253882

21ggccgcggcc

CGcggccgctct

Pro7=

G(0407)

0567

ITGB3

-10

rs11871251

Intro

n1472540

6179+121

ctgggaatgc

GA

cgtgtcctgg

A(0453)

0771

ITGB3

-11

New

Intro

n1472540

8279+152

tggcgcggtC

Gggagccggga

G(0012)

0913

ITGB3

-12

rs112188890lowast

Intro

n147254101

79+161

gagctgggga

CTcttcctggcc

T(0116

)0864

ITGB3

-13

rs117

414137lowast

Intro

n147254192

79+2

52aggctgagcg

CGcttcccggcc

G(0116)

0864

ITGB3

-14

rs118

7144

7Intro

n147254252

79+3

12ccgcgctcac

CGcggggctgcg

G(0448)

0918

ITGB3

-15

New

Intro

n147254331

79+3

91tggggcttc

cGA

ggggttg

ttcA(0006)

0957

ITGB3

-16

rs1009312lowast

Intro

n147254774

79+8

34ggcacagccc

GA

gggttgctgc

G(0471)

000

0ITGB3

-17

rs2015049

Intro

n147254865

79+9

25ggccgcctctGA

cctcagagga

A(052

9)000

0ITGB3

-18

rs56197296lowast

Intro

n547287025

47287029

778-45

778-41

catggctgaa

TTTG

T-tttg

tctcct

delTTT

GT(0169)

0049

ITGB3

-19

rs5919lowast

Exon

647287174

882

ttgtccagccT

Caatgacgggc

Pro294=

C(0262)

0022

ITGB3

-20

rs41504748

Intro

n747290145

1036-40


C(0070)

0334

ITGB3

-21

rs15908

Exon

947290971

1143

cttccagctcAG

Tactttagaac

Val381=

C(0599)

000

6ITGB3

-22

New

Exon

1047292177

1299

gaggctgtcc

CTcaggagaagg

Pro4

33=

T(000

6)0957

ITGB3

-23

rs46

42lowast

Exon

1047292411

1533AgtG

cagcaggacgaA

Gtgcagccccc

Glu511=

G(033

1)0214

ITGB3

-24

rs133064

87Ex

on10

47292422

1544

tgcagccccc

ACG

ggagggtcag

Arg515G

lnA(0012)

0913

ITGB3

-25

rs4634lowast

Exon

1047292423

1545

gcagcccccg

GA

gagggtcagc

Arg515=

A(033

1)0214

ITGB3

-26

New

Exon

1047292528

1659

gcgagtgtga

CTg

acttc

tcct

Asp550=

T(000

6)0957

ITGB3

-27

rs149823724

Exon

1147299519

1902

cagatgcctg

CTaccttta

aga

Cys634=

T(000

6)0957

ITGB3

-28

rs118

70252

Intro

n11

47300459

1914-19

cctta

atcacT

Cgtgtcctctc

C(0035)

0869

ITGB3

-29

rs70940817lowast

Exon

1247300524

1960

cctacatgac

GA

aaaatacctg

Glu654Lys

A(0076)

0432

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib


allele(fr

equency)

lowastSN

Pswhich

related

with

ADPindu

cedplateletsa

ggregatio

nGPIIbII

Iacontentbleeding

timeor


dy







119875 0148 0433 0038 0512 0911




119875 0229 0105 0030 0428 0051




119875 0091 0021 0401 0251 0141



119875 0609 0999 0040 0428 0054




119875 0165 0539 0028 0764 0288




119875 0189 0160 0027 0944 0029




119875 0014 0916 0695 0433 0416









rs59

10

rs59

11

rs11

7870

452

rs85

0730

rs37

6036

4

Block 1 (4kb)1 2 3 4 5

(a)

rs14

7363

351

rs38

0986

2

rs72

0817

0

rs72

0805

5

rs55

8270

77

ITG

B3-0

9

rs11

7052

258

rs56

7581

451

rs11

8712

51

rs12

1888

90

rs11

7414

137

rs11

8714

47

rs56

1972

96

rs59

19

rs41

5047

48

rs15

908

rs46

42

rs46

34

rs70

9408

17

6666

9191

43 9494

643

9394

6284

8797

9794

95

6866

7777

6668

9494

9495

2325

2635

8248

47

6728

2822

1125

2566

6558

4073

7340

116

2418

1434

345

6264

69

2222

3939

6695

9090

9090

4040

2996

8766

9443

28

6565

4844

7049

2826

9455

5577

7712

12891 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 22


(b)








0

10

20

30

40

50

60

70

80


r = 0280 P = 0038

Agg

rega

tion

max

T



4 Discussion













TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















DNAsequencenumber



Length(bp)




3 Results



Table3Summaryof

ITGB

3varia

tions

detected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0002122

Nucleotidec

hange

NP00

04102am

ino

acid

change

MA(F)

H-W119875

ITGB3

-01

rs147363351

51015840-U

TR47253042

-820

agcttccaga

GA

gttttaagtc

A(0012)

0913

ITGB3

-02

rs3809862

51015840-U

TR47253062

-800

ctggggaaga

CTccagggactc

T(0424)

0822

ITGB3

-03

rs7208170lowast

51015840-U

TR47253393

-469

aaggcattc

aGA

cagatgtttg

A(0419)

0680

ITGB3

-04

New

51015840-U

TR47253360

-502

tagtgaataa

TAaaaggactga

A(0023)

0913

ITGB3

-05

rs7208055

51015840-U

TR47253461

-401

gtgaatgtgt

CAccaagaatcc

A(0221)

0273

ITGB3

-06

rs55827077lowast

51015840-U

TR47253717

-145

tagagaagcc

GC

gaggggagga

C(044

8)0494

ITGB3

-07

rs567581451lowast

51015840-U

TR47253771

-91

acccaccgcg

-TC

CCCtcccctcccc

insTCC

CC(0058)

0567

ITGB3

-08

rs117

052258lowast

51015840-U

TR47253855

-7cgcgggaggc

GC

gacgagatgc

C(0227)

0771

ITGB3

-09

New

Exon

147253882

21ggccgcggcc

CGcggccgctct

Pro7=

G(0407)

0567

ITGB3

-10

rs11871251

Intro

n1472540

6179+121

ctgggaatgc

GA

cgtgtcctgg

A(0453)

0771

ITGB3

-11

New

Intro

n1472540

8279+152

tggcgcggtC

Gggagccggga

G(0012)

0913

ITGB3

-12

rs112188890lowast

Intro

n147254101

79+161

gagctgggga

CTcttcctggcc

T(0116

)0864

ITGB3

-13

rs117

414137lowast

Intro

n147254192

79+2

52aggctgagcg

CGcttcccggcc

G(0116)

0864

ITGB3

-14

rs118

7144

7Intro

n147254252

79+3

12ccgcgctcac

CGcggggctgcg

G(0448)

0918

ITGB3

-15

New

Intro

n147254331

79+3

91tggggcttc

cGA

ggggttg

ttcA(0006)

0957

ITGB3

-16

rs1009312lowast

Intro

n147254774

79+8

34ggcacagccc

GA

gggttgctgc

G(0471)

000

0ITGB3

-17

rs2015049

Intro

n147254865

79+9

25ggccgcctctGA

cctcagagga

A(052

9)000

0ITGB3

-18

rs56197296lowast

Intro

n547287025

47287029

778-45

778-41

catggctgaa

TTTG

T-tttg

tctcct

delTTT

GT(0169)

0049

ITGB3

-19

rs5919lowast

Exon

647287174

882

ttgtccagccT

Caatgacgggc

Pro294=

C(0262)

0022

ITGB3

-20

rs41504748

Intro

n747290145

1036-40


C(0070)

0334

ITGB3

-21

rs15908

Exon

947290971

1143

cttccagctcAG

Tactttagaac

Val381=

C(0599)

000

6ITGB3

-22

New

Exon

1047292177

1299

gaggctgtcc

CTcaggagaagg

Pro4

33=

T(000

6)0957

ITGB3

-23

rs46

42lowast

Exon

1047292411

1533AgtG

cagcaggacgaA

Gtgcagccccc

Glu511=

G(033

1)0214

ITGB3

-24

rs133064

87Ex

on10

47292422

1544

tgcagccccc

ACG

ggagggtcag

Arg515G

lnA(0012)

0913

ITGB3

-25

rs4634lowast

Exon

1047292423

1545

gcagcccccg

GA

gagggtcagc

Arg515=

A(033

1)0214

ITGB3

-26

New

Exon

1047292528

1659

gcgagtgtga

CTg

acttc

tcct

Asp550=

T(000

6)0957

ITGB3

-27

rs149823724

Exon

1147299519

1902

cagatgcctg

CTaccttta

aga

Cys634=

T(000

6)0957

ITGB3

-28

rs118

70252

Intro

n11

47300459

1914-19

cctta

atcacT

Cgtgtcctctc

C(0035)

0869

ITGB3

-29

rs70940817lowast

Exon

1247300524

1960

cctacatgac

GA

aaaatacctg

Glu654Lys

A(0076)

0432

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib


allele(fr

equency)

lowastSN

Pswhich

related

with

ADPindu

cedplateletsa

ggregatio

nGPIIbII

Iacontentbleeding

timeor


dy







119875 0148 0433 0038 0512 0911




119875 0229 0105 0030 0428 0051




119875 0091 0021 0401 0251 0141



119875 0609 0999 0040 0428 0054




119875 0165 0539 0028 0764 0288




119875 0189 0160 0027 0944 0029




119875 0014 0916 0695 0433 0416









rs59

10

rs59

11

rs11

7870

452

rs85

0730

rs37

6036

4

Block 1 (4kb)1 2 3 4 5

(a)

rs14

7363

351

rs38

0986

2

rs72

0817

0

rs72

0805

5

rs55

8270

77

ITG

B3-0

9

rs11

7052

258

rs56

7581

451

rs11

8712

51

rs12

1888

90

rs11

7414

137

rs11

8714

47

rs56

1972

96

rs59

19

rs41

5047

48

rs15

908

rs46

42

rs46

34

rs70

9408

17

6666

9191

43 9494

643

9394

6284

8797

9794

95

6866

7777

6668

9494

9495

2325

2635

8248

47

6728

2822

1125

2566

6558

4073

7340

116

2418

1434

345

6264

69

2222

3939

6695

9090

9090

4040

2996

8766

9443

28

6565

4844

7049

2826

9455

5577

7712

12891 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 22


(b)








0

10

20

30

40

50

60

70

80


r = 0280 P = 0038

Agg

rega

tion

max

T



4 Discussion













TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table3Summaryof

ITGB

3varia

tions

detected

inthisstu

dy

Sequ

ence

number

obtained

inthisstu

dydb

SNP

Locatio

nNC

00001711

NM

0002122

Nucleotidec

hange

NP00

04102am

ino

acid

change

MA(F)

H-W119875

ITGB3

-01

rs147363351

51015840-U

TR47253042

-820

agcttccaga

GA

gttttaagtc

A(0012)

0913

ITGB3

-02

rs3809862

51015840-U

TR47253062

-800

ctggggaaga

CTccagggactc

T(0424)

0822

ITGB3

-03

rs7208170lowast

51015840-U

TR47253393

-469

aaggcattc

aGA

cagatgtttg

A(0419)

0680

ITGB3

-04

New

51015840-U

TR47253360

-502

tagtgaataa

TAaaaggactga

A(0023)

0913

ITGB3

-05

rs7208055

51015840-U

TR47253461

-401

gtgaatgtgt

CAccaagaatcc

A(0221)

0273

ITGB3

-06

rs55827077lowast

51015840-U

TR47253717

-145

tagagaagcc

GC

gaggggagga

C(044

8)0494

ITGB3

-07

rs567581451lowast

51015840-U

TR47253771

-91

acccaccgcg

-TC

CCCtcccctcccc

insTCC

CC(0058)

0567

ITGB3

-08

rs117

052258lowast

51015840-U

TR47253855

-7cgcgggaggc

GC

gacgagatgc

C(0227)

0771

ITGB3

-09

New

Exon

147253882

21ggccgcggcc

CGcggccgctct

Pro7=

G(0407)

0567

ITGB3

-10

rs11871251

Intro

n1472540

6179+121

ctgggaatgc

GA

cgtgtcctgg

A(0453)

0771

ITGB3

-11

New

Intro

n1472540

8279+152

tggcgcggtC

Gggagccggga

G(0012)

0913

ITGB3

-12

rs112188890lowast

Intro

n147254101

79+161

gagctgggga

CTcttcctggcc

T(0116

)0864

ITGB3

-13

rs117

414137lowast

Intro

n147254192

79+2

52aggctgagcg

CGcttcccggcc

G(0116)

0864

ITGB3

-14

rs118

7144

7Intro

n147254252

79+3

12ccgcgctcac

CGcggggctgcg

G(0448)

0918

ITGB3

-15

New

Intro

n147254331

79+3

91tggggcttc

cGA

ggggttg

ttcA(0006)

0957

ITGB3

-16

rs1009312lowast

Intro

n147254774

79+8

34ggcacagccc

GA

gggttgctgc

G(0471)

000

0ITGB3

-17

rs2015049

Intro

n147254865

79+9

25ggccgcctctGA

cctcagagga

A(052

9)000

0ITGB3

-18

rs56197296lowast

Intro

n547287025

47287029

778-45

778-41

catggctgaa

TTTG

T-tttg

tctcct

delTTT

GT(0169)

0049

ITGB3

-19

rs5919lowast

Exon

647287174

882

ttgtccagccT

Caatgacgggc

Pro294=

C(0262)

0022

ITGB3

-20

rs41504748

Intro

n747290145

1036-40


C(0070)

0334

ITGB3

-21

rs15908

Exon

947290971

1143

cttccagctcAG

Tactttagaac

Val381=

C(0599)

000

6ITGB3

-22

New

Exon

1047292177

1299

gaggctgtcc

CTcaggagaagg

Pro4

33=

T(000

6)0957

ITGB3

-23

rs46

42lowast

Exon

1047292411

1533AgtG

cagcaggacgaA

Gtgcagccccc

Glu511=

G(033

1)0214

ITGB3

-24

rs133064

87Ex

on10

47292422

1544

tgcagccccc

ACG

ggagggtcag

Arg515G

lnA(0012)

0913

ITGB3

-25

rs4634lowast

Exon

1047292423

1545

gcagcccccg

GA

gagggtcagc

Arg515=

A(033

1)0214

ITGB3

-26

New

Exon

1047292528

1659

gcgagtgtga

CTg

acttc

tcct

Asp550=

T(000

6)0957

ITGB3

-27

rs149823724

Exon

1147299519

1902

cagatgcctg

CTaccttta

aga

Cys634=

T(000

6)0957

ITGB3

-28

rs118

70252

Intro

n11

47300459

1914-19

cctta

atcacT

Cgtgtcctctc

C(0035)

0869

ITGB3

-29

rs70940817lowast

Exon

1247300524

1960

cctacatgac

GA

aaaatacctg

Glu654Lys

A(0076)

0432

dbSN

Psin

glen

ucleotidep

olym

orph

ismdatabaseH

-W119875H

ardyndashW

einb

ergequilib


allele(fr

equency)

lowastSN

Pswhich

related

with

ADPindu

cedplateletsa

ggregatio

nGPIIbII

Iacontentbleeding

timeor


dy







119875 0148 0433 0038 0512 0911




119875 0229 0105 0030 0428 0051




119875 0091 0021 0401 0251 0141



119875 0609 0999 0040 0428 0054




119875 0165 0539 0028 0764 0288




119875 0189 0160 0027 0944 0029




119875 0014 0916 0695 0433 0416









rs59

10

rs59

11

rs11

7870

452

rs85

0730

rs37

6036

4

Block 1 (4kb)1 2 3 4 5

(a)

rs14

7363

351

rs38

0986

2

rs72

0817

0

rs72

0805

5

rs55

8270

77

ITG

B3-0

9

rs11

7052

258

rs56

7581

451

rs11

8712

51

rs12

1888

90

rs11

7414

137

rs11

8714

47

rs56

1972

96

rs59

19

rs41

5047

48

rs15

908

rs46

42

rs46

34

rs70

9408

17

6666

9191

43 9494

643

9394

6284

8797

9794

95

6866

7777

6668

9494

9495

2325

2635

8248

47

6728

2822

1125

2566

6558

4073

7340

116

2418

1434

345

6264

69

2222

3939

6695

9090

9090

4040

2996

8766

9443

28

6565

4844

7049

2826

9455

5577

7712

12891 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 22


(b)








0

10

20

30

40

50

60

70

80


r = 0280 P = 0038

Agg

rega

tion

max

T



4 Discussion













TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















119875 0148 0433 0038 0512 0911




119875 0229 0105 0030 0428 0051




119875 0091 0021 0401 0251 0141



119875 0609 0999 0040 0428 0054




119875 0165 0539 0028 0764 0288




119875 0189 0160 0027 0944 0029




119875 0014 0916 0695 0433 0416









rs59

10

rs59

11

rs11

7870

452

rs85

0730

rs37

6036

4

Block 1 (4kb)1 2 3 4 5

(a)

rs14

7363

351

rs38

0986

2

rs72

0817

0

rs72

0805

5

rs55

8270

77

ITG

B3-0

9

rs11

7052

258

rs56

7581

451

rs11

8712

51

rs12

1888

90

rs11

7414

137

rs11

8714

47

rs56

1972

96

rs59

19

rs41

5047

48

rs15

908

rs46

42

rs46

34

rs70

9408

17

6666

9191

43 9494

643

9394

6284

8797

9794

95

6866

7777

6668

9494

9495

2325

2635

8248

47

6728

2822

1125

2566

6558

4073

7340

116

2418

1434

345

6264

69

2222

3939

6695

9090

9090

4040

2996

8766

9443

28

6565

4844

7049

2826

9455

5577

7712

12891 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 22


(b)








0

10

20

30

40

50

60

70

80


r = 0280 P = 0038

Agg

rega

tion

max

T



4 Discussion













TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















rs59

10

rs59

11

rs11

7870

452

rs85

0730

rs37

6036

4

Block 1 (4kb)1 2 3 4 5

(a)

rs14

7363

351

rs38

0986

2

rs72

0817

0

rs72

0805

5

rs55

8270

77

ITG

B3-0

9

rs11

7052

258

rs56

7581

451

rs11

8712

51

rs12

1888

90

rs11

7414

137

rs11

8714

47

rs56

1972

96

rs59

19

rs41

5047

48

rs15

908

rs46

42

rs46

34

rs70

9408

17

6666

9191

43 9494

643

9394

6284

8797

9794

95

6866

7777

6668

9494

9495

2325

2635

8248

47

6728

2822

1125

2566

6558

4073

7340

116

2418

1434

345

6264

69

2222

3939

6695

9090

9090

4040

2996

8766

9443

28

6565

4844

7049

2826

9455

5577

7712

12891 2 3 4 5 6 7 8 9 10 11 12 15 16 17 18 19 20 22


(b)








0

10

20

30

40

50

60

70

80


r = 0280 P = 0038

Agg

rega

tion

max

T



4 Discussion













TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

70

80


r = 0280 P = 0038

Agg

rega

tion

max

T



4 Discussion













TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















TT 1 125 367 165 272119875 0126 0029 0299 0173




119875 0770 0088 0015 0029





dbSNP MA(F)a











5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion


Competing Interests


Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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