# Identification ofITGA2B andITGB3 Single-Nucleotide Polymorphisms and Their Influences on the Platelet Function

Authors: Qian Xiang; Shun-Dong Ji; Zhuo Zhang; Xia Zhao; Yi-Min Cui
Journal: BioMed Research International
 (2016)

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## Abstract

The aim of the study was to investigateITGA2B andITGB3 genetic polymorphisms and to evaluate the variability in the platelet function in healthy Chinese subjects. The genetic sequence of the entire coding region of theITGA2B andITGB3 genes was investigated. Adenosine diphosphate-induced platelet aggregation, glycoprotein IIb/IIIa content, bleeding time, and coagulation indexes were detected. Thirteen variants in theITGA2B locus and 29 variants in theITGB3 locus were identified in the Chinese population. The rs1009312 and rs2015049 were associated with the mean platelet volume. The rs70940817 was significantly correlated with the prothrombin time. The rs70940817 and rs112188890 were related with the activated partial thromboplastin time, andITGB3 rs4642 was correlated with the thrombin time and fibrinogen. The minor alleles of rs56197296 and rs5919 were associated with decreased ADP-induced platelet aggregation, and rs55827077 was related with decreased GPIIb/IIIa per platelet. The rs1009312, rs2015049, rs3760364, rs567581451, rs7208170, and rs117052258 were related with bleeding time. Further studies are needed to explore the clinical importance ofITGA2B andITGB3 SNPs in the platelet function.


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## Body

## 1. Introduction

Platelet aggregation plays a central role in the pathogenesis of acute thrombosis in coronary heart disease, stroke, and peripheral arterial disease. The cellular events leading to platelet aggregation are mediated by the binding of fibrinogen to the glycoprotein (GP)IIb/IIIa receptor of platelets as a final common pathway. GPIIb/IIIa is a platelet-specific, surface membrane receptor and also called alpha IIb beta 3 (aIIbβ3) in the integrin nomenclature and thus plays a primary role in both platelet adhesion and thrombus formation at the vascular injury site [1]. A large interindividual number variability for GPIIb/IIIa receptors expressed on the platelet surface is commonly observed [2, 3]. Moreover, a defect in the GPIIb/IIIa complex or a qualitative abnormality of this complex is seen in Glanzmann’s thrombasthenia patients with impaired platelet aggregation and increased bleeding [4].TheITGA2B gene encodes the aIIb subunit (GPIIb), whereas theITGB3 gene encodes β3 (GPIIIa). TheITGA2B spanning 17 kb has 30 exons, whereas theITGB3 spanning 46 kb has 15 exons; they are closely located on chromosome 17q21.32 without evidence for coordinated expression [5]. A few studies have linked single-nucleotide polymorphisms (SNPs) inITGA2B andITGB3 with increased or decreased platelet responses to various agonists and the risk of acute coronary syndrome and atherosclerosis [6–8].A common polymorphism in theITGB3, known as the human platelet antigen 1 (HPA-1b, PlA2, or rs5918), arises from a single-nucleotide change at position 1565 in Exon2 ofITGB3, resulting in a leucine (PlA1) to proline (PlA2) substitution at residue 33 [9]. The SNP has been extensively studied as an inherited risk factor for acute coronary syndrome and for its effect on the platelet function [6–8]. The HPA-3 (rs5910) polymorphism results from a thymine to guanine base change that leads to the replacement of isoleucine (HPA-3a) by serine (HPA-3b) at codon 843 of GPIIb (ITGA2B). This polymorphism may potentially influence the activity of the GPIIb/IIIa complex and associate with thrombosis [10].According to the aforementioned studies, GPIIb/IIIa was believed to be a platelet-platelet contact receptor playing an important role in platelet aggregation, and its SNPs were associated with platelet hyperreactivity and have an effect on the pharmacodynamics of antiplatelet drugs. However, limited information was available on other genetic polymorphisms ofITGA2B andITGB3 in Asian populations or their association with the platelet function [11, 12]. Moreover, the prevalence of the PlA2 allele (rs5918) is dependent on ethnicity, with a frequency of approximately 15/100 in Caucasian populations [13] falling to less than 1/100 in Oriental populations [12, 14].In this study, regions of the exons ofITGA2B andITGB3 genes were sequenced in 86 unrelated healthy Chinese. In addition, the association between genetic variants ofITGA2B andITGB3 exons and the adenosine diphosphate- (ADP-) induced platelet aggregation, GPIIb/IIIa content, bleeding time, and coagulation indexes was investigated in 55 of the 86 subjects.


## 2. Materials and Methods

### 2.1. Study Design

The research was conducted in compliance with the Declaration of Helsinki. The study protocol was approved by the Ethical Review Board of the Peking University First Hospital. All subjects gave their written informed consent prior to participation in the study. Healthy native Chinese subjects (n=86) between 18 and 45 years of age with a body mass index (BMI) of 19–24 kg/m2 were included in the study, and their genotypes were unknown.All the subjects were considered healthy on the basis of their medical history, physical examination, vital signs (blood pressure, pulse rate, and temperature), safety laboratory tests (blood chemistry, hematological tests, and urinalysis), and 12-lead electrocardiography.The ADP-induced platelet aggregation (transmission,T% max), GPIIb/IIIa content (GPIIb/IIIa per platelet), bleeding time, and coagulation indexes, including the mean platelet volume (MPV), platelet count (PLT), prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FIB), and thrombin time (TT) were detected in 55 of the 86 subjects. None of the donors had taken any medication for 2 weeks before blood collection.


### 2.2. ADP-Induced Platelet Aggregation

Blood was collected in 3.8% sodium citrate tubes, and platelet-rich plasma (PRP) was obtained by centrifuging blood at 1000 revolutions per minute (rpm) for 10 minutes at room temperature. The PRP was collected in a fresh tube and platelet was counted by Platelet Counter PL100 (Sysmex, Kobe, Japan). Platelet-poor plasma (PPP) was obtained by centrifuging the remaining blood at 3000 rpm for 10 minutes at room temperature, and platelet numbering PRP was adjusted to 250 × 109/L by PPP. Detection was completed within 1 hour after sampling.Platelet aggregation was determined by the turbidimetric method using 20 umol/L ADP as the agonist. After zero setting with the PPP, assays were performed in platelet-rich plasma in a Chrono-Log aggregometer. Platelet aggregation was quantified as the maximum change in light transmission occurring within 5 minutes of addition of agonist.


### 2.3. GPIIb/IIIa Content

GPIIb/IIIa on the platelet surface was evaluated in the PRP as the maximal binding of a125I-labeled GPIIb/IIIa integrin antiplatelet antibody F(ab)2 [15]. The PRP was fixed with equal volume of anticoagulant-fixative solution (10 mmol/L EDTA-Na2, 0.2% glutaraldehyde, and 0.02% sodium azide dissolved in PBS, pH 7.4). After stored at 4°C, overnight, platelet was washed by Tyrode’s solution twice and resuspended in Tyrode solution, containing 0.35% bovine serum albumin (concentration adjusted to 1 × 1011 platelets/L), and sodium azide was added at final concentration of 0.02% (V/V) to preserve the platelet at 4°C for a week-long before assay. For detection, the platelet suspension (100 μL) was added to a 0.5 mL Eppendorf tube and then incubated with 100 μL of antibody (containing 20,000 cpm of labeled monoclonal antibody (McAb) and 0.8 μg of unlabeled McAb) at 37°C for 1 hour, after washed with 0.35% bovine serum albumin/Tyrode’s solution for three times. The radioactivity count of the precipitate in the tube was measured. Assuming that one monoclonal antibody would only bind one GP molecule on the platelet membrane, the number of GP molecules on the platelet membrane was calculated as number of GP molecules/platelet = (binding rate × amount of antibody (g) × Avogadro’s number)/(molecular weight of antibody × platelet count).


### 2.4. Bleeding Time

The skin bleeding time was carried out using the Simplate-II device (General Diagnostics, NJ, USA). A sphygmomanometer cuff was inflated on the patient’s arm to 40 mmHg. The arm was supported at the level of the heart, and a muscular area on the volar aspect distal to the antecubital area was identified and swabbed with alcohol. The Simplate-II device was used to perform the incision perpendicular to the antecubital fossa. Blood was blotted with sterile filter paper every 30 seconds until blood no longer stained the paper. The time from incision to stopping of bleeding was recorded.


### 2.5. Genetic Analysis

Genomic DNA was extracted from peripheral whole blood samples of each subject using a DNA Purification Kit (Wizard; Promega, WI, USA). After the polymerase chain reaction (PCR) products were obtained, all samples were directly sequenced to determine the SNPs inITGA2B andITGB3 (Life Technologies Biotechnology Co., Ltd., China).Primers required for PCR amplification and sequencing were designed according to the wild-typeITGA2B andITGB3 sequences reported in the GenBank (NG_008331.1 and NG_008332.1, resp.). Tables 1 and 2 describe the primer pairs used to amplify the promoters, the 5′- and 3′-untranslated regions (UTRs), the entire coding regions and the intron-exon junctions of theITGA2B andITGB3 genes.Table 1
Summary of GPIIb (ITGA2B) variations detected in this study.
Sequence number obtained in this study dbSNP Location NC_000017.11 NM_000419.3 Nucleotide change NP_000410.2 amino acid change MA(F) H-WP ITGA2B-01 rs3760364∗ 5′ upstream end 44390436 -963 ctcccaaggg A/T ctcatttaca  T(0.052) 0.609 ITGA2B-02 New 5′ upstream end 44390152 -679 tagaccaagg T/C ccattcacca  C(0.006) 0.957 ITGA2B-03 New 5′ upstream end 44390081 -608 caagacggag G/A aggagtgagg  A(0.006) 0.957 ITGA2B-04 New Exon3 44385910 354 tgatgagacc C/T gaaatgtagg Arg108Stop T(0.006) 0.957 ITGA2B-05 New Exon13 44380960 1344 ctcccaggtc C/A tggacagccc Leu438Met A(0.006) 0.957 ITGA2B-06 rs201355504 Intron13 44380821 1393+58 cttggcactt C/T cagcgaatgt  A(0.006) 0.957 ITGA2B-07 New Exon14 44380638 1401 tagacctgat C/G gtgggagctt Ile467Met G(0.006) 0.957 ITGA2B-08 rs850730 Intron21 44377095 2188-7C>G ccctcctcat C/G tcccagatag  G(0.477) 0.289 ITGA2B-09 New Exon23 44376336 2320 cgtgccggtc C/T gggcagaggc Arg774Trp T(0.006) 0.957 ITGA2B-10 rs117870452 Exon23 44376322 2334 gcagctccac C/T tgggcctctg Gln778= G(0.012) 0.913 ITGA2B-11 rs5911 Exon26 44375697 2621T>G ggggctgggg A/C tgggcagccc Ile874Ser G(0.477) 0.289 ITGA2B-12 rs5910 Exon30 44372421 3063C>T acccccaggt C/T ggcttcttca Val1021= T(0.465) 0.289 ITGA2B-13 New 3′-UTR 44372213 ∗151C>A gctacccccc C/A tcctgctgcc  A(0.017) 0.869 dbSNP, single nucleotide polymorphism database; H-WP, Hardy–Weinberg equilibrium P value; MA(F), minor allele (frequency); GP, glycoprotein. ∗SNPs which related with bleeding time in this study.Table 2
Primer sequences used in this study.
DNA sequence number Amplified or sequenced region Forward primer (5′ to 3′) Reverse primer (5′ to 3′) Amplified region NC_000017.11 Length (bp) ITGA2B-1  TCCTCCTCTTCCGCTTACCG TACTACCACCGTGCTAGTCC 44389728–44390630 848 ITGA2B-2 Exon1 CCAATATGGCTGGTTGAG AACTTCCCTTACGGCTCA 44389267–44390059 792 ITGA2B-3 Exon1 CCAGTGCAGCTCACCTTCTA GATGAGGGAAATGGAACAGA 44388253–44389368 1115 ITGA2B-4 Intron1 TATGAACCACTCCACCCT TTGGCACTCTTGATTCTG 44388169–44389006 837 ITGA2B-5 Exon2, Exon3 ACCGCTGGTTCTTGTTGC CCTACGGGCGTCTTCTCA 44385649-44386479 830 ITGA2B-6 Exon4–Exon6 TACAGGGCACAGGGAACAATC AGAGGCTCTGGGAGGACACG 44384990–44385810 820 ITGA2B-7 Exon7, Exon8 TCCTGGCGGCTATTATTTCT GCACCGACGACATATTCTGG 44384339–44385186 847 ITGA2B-8 Exon9–Exon11 ATTTGCGCCCTTGTCCTC AGCCGAATCGCCCATAGA 44383538–44384605 1067 ITGA2B-9 Exon12 CCCTCTGTCTCCCTTTCC CATCCAGTCTCCCACCAA 44383189–44383838 649 ITGA2B-10 Exon13, Exon14 CCTAGTCTCCTGGGATGTTC TCACGGGTGTCTTGGTCT 44380390–44381163 773 ITGA2B-11 Exon14–Exon17 TAATCGCCAATTCTGACCC CACATCCCACCTTCTCCTG 44379854–44380682 828 ITGA2B-12 Exon18 ACCCACTGGACTTGTTCATC TGTGACTTGGCACTAACCC 44379610–44379957 347 ITGA2B-13 Exon19, Exon20 TGGACGACAGAGCGAGAC GGCCATACCTCGACATTG 44378354–44378989 635 ITGA2B-14 Exon21 CATGTGACAGTCCCTTGA AAAGTCACTCACCCAAGGA 44377551–44377933 382 ITGA2B-15 Exon22 CTTGGAGGGTGAAGACTGG CAACTCCTGACCTCCAGTGA 44376833–44377179 346 ITGA2B-16 Exon23–Exon25 CCAGGTCTAACTTCAGTGTG GCTCTGGCAGGAAGATCTGT 44375629–44376523 894 ITGA2B-17 Exon26 TCCGACCTGCTCTACATCC CGGGCTTGCTCACATAGTC 44375277–44375913 636 ITGA2B-18 Exon27, Exon28 ATGACCCTCCCTGCATCTC CACCTTGACACCTGCCTTT 44374500–44375317 817 ITGA2B-19 Exon29 GCACGCATGGTTCAACGT CCTCCCGAGTAGCTGAGATT 44374025–44374731 706 ITGA2B-20 Exon30 AAAGGCATCCATTTGTGA TGTTGGTAAGGCTGGTCTC 44371896–44372566 670 ITGB3-1  CAGGAGGTGGAGGATTGT GCTGGATTCTTGGGACAC 47252637–47253474 837 ITGB3-2 Exon1 CGGTTCAGAGAAGGCATTCAG GCTCCAAGTCCGCAACTTGA 47253373–47254015 642 ITGB3-3 Intron1 TTGGCGTAGGAGGTGAGTGA CCGCAGGAAGCCAAGTTGAA 47253929–47254518 589 ITGB3-4 Intron1 TTGGCGTAGGAGGTGAGTGAG GAAGTTGCAGTGAGCCGAGAA 47253929–47255063 1134 ITGB3-5 Exon2 ATTGGGAAAGTTGGGAAGG GAAAGGGCAGCAGTGGTT 47274335–47274773 438 ITGB3-6 Exon3 AGGCTGGTCTTGAACTCTTG CTCCACCTTGTGCTCTAT 47283116–47283612 496 ITGB3-7 Exon4 GGGCTTTCTGGTTTGCTT CATTTCCCTCCCATTCTC 47284329–47284978 649 ITGB3-8 Exon5 TGTCTGGGTAACTGTGGT CATCTGCCTACTTTGCTG 47286124–47286725 601 ITGB3-9 Exon6 TCCAAGGACTGGGACTGA ATGATGCTGCTGCTATGC 47286919–47287483 564 ITGB3-10 Exon7, Exon8 AGCCCAAGCAAGATAAGT GGAGAAGGCAGTAAGACC 47289597–47290393 796 ITGB3-11 Exon9 AAACTGGGCTCCAATAAC TGAGGGACTGAAGGTAAAG 47290561–47291406 845 ITGB3-12 Exon10 CAGGGCAGGGAACAACTT GGATTGGTCCTTATACTCAAAA 47292050–47292720 670 ITGB3-13 Exon11 GAGCAAGTCCTGCCATAC TCACAGAGTGTCCTCCATAA 47299101–47299890 789 ITGB3-14 Exon12 CAGAAATGGCATAGGGTT TCTTGCTGAGTCTGTGGG 47300168–47300824 656 ITGB3-15 Exon13 CTTGAATCTAGGCATCGT GTATTGAACTCCTGACCC 47302581–47303050 469 ITGB3-16 Exon14 CCTCAAGTAGGTCCCAGTG AACATGACCACCCAAAGC 47307158–47307721 563 ITGB3-17 Exon15 CTCATCTCCTCCTGTTATTT TGACATTCTCCCAACCTAC 47309941–47310337 396


### 2.6. Data Analysis

The Variant Reporter v1.1 (Life Technologies Biotechnology Co., Ltd.) software suite was used for the initial analysis of the sequence, including base calling, fragment assembly, SNP, and sequence insertions/deletions detection. Polymorphisms ofITGA2B andITGB3 genes were named according to the genomic reference sequences NG_008331.1 and NG_008332.1, respectively. Novel SNPs were named asITGA2B-number orITGB3-number in the present study.


### 2.7. Statistical Analysis

The association between two parameters was assessed by Pearson’s two-tailed test. Statistical analyses were performed using the Statistical Package for Social Sciences software program for Windows (SPSS version 16.0). AP value of 0.05 was considered significant.


## 2.1. Study Design

The research was conducted in compliance with the Declaration of Helsinki. The study protocol was approved by the Ethical Review Board of the Peking University First Hospital. All subjects gave their written informed consent prior to participation in the study. Healthy native Chinese subjects (n=86) between 18 and 45 years of age with a body mass index (BMI) of 19–24 kg/m2 were included in the study, and their genotypes were unknown.All the subjects were considered healthy on the basis of their medical history, physical examination, vital signs (blood pressure, pulse rate, and temperature), safety laboratory tests (blood chemistry, hematological tests, and urinalysis), and 12-lead electrocardiography.The ADP-induced platelet aggregation (transmission,T% max), GPIIb/IIIa content (GPIIb/IIIa per platelet), bleeding time, and coagulation indexes, including the mean platelet volume (MPV), platelet count (PLT), prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (FIB), and thrombin time (TT) were detected in 55 of the 86 subjects. None of the donors had taken any medication for 2 weeks before blood collection.


## 2.2. ADP-Induced Platelet Aggregation

Blood was collected in 3.8% sodium citrate tubes, and platelet-rich plasma (PRP) was obtained by centrifuging blood at 1000 revolutions per minute (rpm) for 10 minutes at room temperature. The PRP was collected in a fresh tube and platelet was counted by Platelet Counter PL100 (Sysmex, Kobe, Japan). Platelet-poor plasma (PPP) was obtained by centrifuging the remaining blood at 3000 rpm for 10 minutes at room temperature, and platelet numbering PRP was adjusted to 250 × 109/L by PPP. Detection was completed within 1 hour after sampling.Platelet aggregation was determined by the turbidimetric method using 20 umol/L ADP as the agonist. After zero setting with the PPP, assays were performed in platelet-rich plasma in a Chrono-Log aggregometer. Platelet aggregation was quantified as the maximum change in light transmission occurring within 5 minutes of addition of agonist.


## 2.3. GPIIb/IIIa Content

GPIIb/IIIa on the platelet surface was evaluated in the PRP as the maximal binding of a125I-labeled GPIIb/IIIa integrin antiplatelet antibody F(ab)2 [15]. The PRP was fixed with equal volume of anticoagulant-fixative solution (10 mmol/L EDTA-Na2, 0.2% glutaraldehyde, and 0.02% sodium azide dissolved in PBS, pH 7.4). After stored at 4°C, overnight, platelet was washed by Tyrode’s solution twice and resuspended in Tyrode solution, containing 0.35% bovine serum albumin (concentration adjusted to 1 × 1011 platelets/L), and sodium azide was added at final concentration of 0.02% (V/V) to preserve the platelet at 4°C for a week-long before assay. For detection, the platelet suspension (100 μL) was added to a 0.5 mL Eppendorf tube and then incubated with 100 μL of antibody (containing 20,000 cpm of labeled monoclonal antibody (McAb) and 0.8 μg of unlabeled McAb) at 37°C for 1 hour, after washed with 0.35% bovine serum albumin/Tyrode’s solution for three times. The radioactivity count of the precipitate in the tube was measured. Assuming that one monoclonal antibody would only bind one GP molecule on the platelet membrane, the number of GP molecules on the platelet membrane was calculated as number of GP molecules/platelet = (binding rate × amount of antibody (g) × Avogadro’s number)/(molecular weight of antibody × platelet count).


## 2.4. Bleeding Time

The skin bleeding time was carried out using the Simplate-II device (General Diagnostics, NJ, USA). A sphygmomanometer cuff was inflated on the patient’s arm to 40 mmHg. The arm was supported at the level of the heart, and a muscular area on the volar aspect distal to the antecubital area was identified and swabbed with alcohol. The Simplate-II device was used to perform the incision perpendicular to the antecubital fossa. Blood was blotted with sterile filter paper every 30 seconds until blood no longer stained the paper. The time from incision to stopping of bleeding was recorded.


## 2.5. Genetic Analysis

Genomic DNA was extracted from peripheral whole blood samples of each subject using a DNA Purification Kit (Wizard; Promega, WI, USA). After the polymerase chain reaction (PCR) products were obtained, all samples were directly sequenced to determine the SNPs inITGA2B andITGB3 (Life Technologies Biotechnology Co., Ltd., China).Primers required for PCR amplification and sequencing were designed according to the wild-typeITGA2B andITGB3 sequences reported in the GenBank (NG_008331.1 and NG_008332.1, resp.). Tables 1 and 2 describe the primer pairs used to amplify the promoters, the 5′- and 3′-untranslated regions (UTRs), the entire coding regions and the intron-exon junctions of theITGA2B andITGB3 genes.Table 1
Summary of GPIIb (ITGA2B) variations detected in this study.
Sequence number obtained in this study dbSNP Location NC_000017.11 NM_000419.3 Nucleotide change NP_000410.2 amino acid change MA(F) H-WP ITGA2B-01 rs3760364∗ 5′ upstream end 44390436 -963 ctcccaaggg A/T ctcatttaca  T(0.052) 0.609 ITGA2B-02 New 5′ upstream end 44390152 -679 tagaccaagg T/C ccattcacca  C(0.006) 0.957 ITGA2B-03 New 5′ upstream end 44390081 -608 caagacggag G/A aggagtgagg  A(0.006) 0.957 ITGA2B-04 New Exon3 44385910 354 tgatgagacc C/T gaaatgtagg Arg108Stop T(0.006) 0.957 ITGA2B-05 New Exon13 44380960 1344 ctcccaggtc C/A tggacagccc Leu438Met A(0.006) 0.957 ITGA2B-06 rs201355504 Intron13 44380821 1393+58 cttggcactt C/T cagcgaatgt  A(0.006) 0.957 ITGA2B-07 New Exon14 44380638 1401 tagacctgat C/G gtgggagctt Ile467Met G(0.006) 0.957 ITGA2B-08 rs850730 Intron21 44377095 2188-7C>G ccctcctcat C/G tcccagatag  G(0.477) 0.289 ITGA2B-09 New Exon23 44376336 2320 cgtgccggtc C/T gggcagaggc Arg774Trp T(0.006) 0.957 ITGA2B-10 rs117870452 Exon23 44376322 2334 gcagctccac C/T tgggcctctg Gln778= G(0.012) 0.913 ITGA2B-11 rs5911 Exon26 44375697 2621T>G ggggctgggg A/C tgggcagccc Ile874Ser G(0.477) 0.289 ITGA2B-12 rs5910 Exon30 44372421 3063C>T acccccaggt C/T ggcttcttca Val1021= T(0.465) 0.289 ITGA2B-13 New 3′-UTR 44372213 ∗151C>A gctacccccc C/A tcctgctgcc  A(0.017) 0.869 dbSNP, single nucleotide polymorphism database; H-WP, Hardy–Weinberg equilibrium P value; MA(F), minor allele (frequency); GP, glycoprotein. ∗SNPs which related with bleeding time in this study.Table 2
Primer sequences used in this study.
DNA sequence number Amplified or sequenced region Forward primer (5′ to 3′) Reverse primer (5′ to 3′) Amplified region NC_000017.11 Length (bp) ITGA2B-1  TCCTCCTCTTCCGCTTACCG TACTACCACCGTGCTAGTCC 44389728–44390630 848 ITGA2B-2 Exon1 CCAATATGGCTGGTTGAG AACTTCCCTTACGGCTCA 44389267–44390059 792 ITGA2B-3 Exon1 CCAGTGCAGCTCACCTTCTA GATGAGGGAAATGGAACAGA 44388253–44389368 1115 ITGA2B-4 Intron1 TATGAACCACTCCACCCT TTGGCACTCTTGATTCTG 44388169–44389006 837 ITGA2B-5 Exon2, Exon3 ACCGCTGGTTCTTGTTGC CCTACGGGCGTCTTCTCA 44385649-44386479 830 ITGA2B-6 Exon4–Exon6 TACAGGGCACAGGGAACAATC AGAGGCTCTGGGAGGACACG 44384990–44385810 820 ITGA2B-7 Exon7, Exon8 TCCTGGCGGCTATTATTTCT GCACCGACGACATATTCTGG 44384339–44385186 847 ITGA2B-8 Exon9–Exon11 ATTTGCGCCCTTGTCCTC AGCCGAATCGCCCATAGA 44383538–44384605 1067 ITGA2B-9 Exon12 CCCTCTGTCTCCCTTTCC CATCCAGTCTCCCACCAA 44383189–44383838 649 ITGA2B-10 Exon13, Exon14 CCTAGTCTCCTGGGATGTTC TCACGGGTGTCTTGGTCT 44380390–44381163 773 ITGA2B-11 Exon14–Exon17 TAATCGCCAATTCTGACCC CACATCCCACCTTCTCCTG 44379854–44380682 828 ITGA2B-12 Exon18 ACCCACTGGACTTGTTCATC TGTGACTTGGCACTAACCC 44379610–44379957 347 ITGA2B-13 Exon19, Exon20 TGGACGACAGAGCGAGAC GGCCATACCTCGACATTG 44378354–44378989 635 ITGA2B-14 Exon21 CATGTGACAGTCCCTTGA AAAGTCACTCACCCAAGGA 44377551–44377933 382 ITGA2B-15 Exon22 CTTGGAGGGTGAAGACTGG CAACTCCTGACCTCCAGTGA 44376833–44377179 346 ITGA2B-16 Exon23–Exon25 CCAGGTCTAACTTCAGTGTG GCTCTGGCAGGAAGATCTGT 44375629–44376523 894 ITGA2B-17 Exon26 TCCGACCTGCTCTACATCC CGGGCTTGCTCACATAGTC 44375277–44375913 636 ITGA2B-18 Exon27, Exon28 ATGACCCTCCCTGCATCTC CACCTTGACACCTGCCTTT 44374500–44375317 817 ITGA2B-19 Exon29 GCACGCATGGTTCAACGT CCTCCCGAGTAGCTGAGATT 44374025–44374731 706 ITGA2B-20 Exon30 AAAGGCATCCATTTGTGA TGTTGGTAAGGCTGGTCTC 44371896–44372566 670 ITGB3-1  CAGGAGGTGGAGGATTGT GCTGGATTCTTGGGACAC 47252637–47253474 837 ITGB3-2 Exon1 CGGTTCAGAGAAGGCATTCAG GCTCCAAGTCCGCAACTTGA 47253373–47254015 642 ITGB3-3 Intron1 TTGGCGTAGGAGGTGAGTGA CCGCAGGAAGCCAAGTTGAA 47253929–47254518 589 ITGB3-4 Intron1 TTGGCGTAGGAGGTGAGTGAG GAAGTTGCAGTGAGCCGAGAA 47253929–47255063 1134 ITGB3-5 Exon2 ATTGGGAAAGTTGGGAAGG GAAAGGGCAGCAGTGGTT 47274335–47274773 438 ITGB3-6 Exon3 AGGCTGGTCTTGAACTCTTG CTCCACCTTGTGCTCTAT 47283116–47283612 496 ITGB3-7 Exon4 GGGCTTTCTGGTTTGCTT CATTTCCCTCCCATTCTC 47284329–47284978 649 ITGB3-8 Exon5 TGTCTGGGTAACTGTGGT CATCTGCCTACTTTGCTG 47286124–47286725 601 ITGB3-9 Exon6 TCCAAGGACTGGGACTGA ATGATGCTGCTGCTATGC 47286919–47287483 564 ITGB3-10 Exon7, Exon8 AGCCCAAGCAAGATAAGT GGAGAAGGCAGTAAGACC 47289597–47290393 796 ITGB3-11 Exon9 AAACTGGGCTCCAATAAC TGAGGGACTGAAGGTAAAG 47290561–47291406 845 ITGB3-12 Exon10 CAGGGCAGGGAACAACTT GGATTGGTCCTTATACTCAAAA 47292050–47292720 670 ITGB3-13 Exon11 GAGCAAGTCCTGCCATAC TCACAGAGTGTCCTCCATAA 47299101–47299890 789 ITGB3-14 Exon12 CAGAAATGGCATAGGGTT TCTTGCTGAGTCTGTGGG 47300168–47300824 656 ITGB3-15 Exon13 CTTGAATCTAGGCATCGT GTATTGAACTCCTGACCC 47302581–47303050 469 ITGB3-16 Exon14 CCTCAAGTAGGTCCCAGTG AACATGACCACCCAAAGC 47307158–47307721 563 ITGB3-17 Exon15 CTCATCTCCTCCTGTTATTT TGACATTCTCCCAACCTAC 47309941–47310337 396


## 2.6. Data Analysis

The Variant Reporter v1.1 (Life Technologies Biotechnology Co., Ltd.) software suite was used for the initial analysis of the sequence, including base calling, fragment assembly, SNP, and sequence insertions/deletions detection. Polymorphisms ofITGA2B andITGB3 genes were named according to the genomic reference sequences NG_008331.1 and NG_008332.1, respectively. Novel SNPs were named asITGA2B-number orITGB3-number in the present study.


## 2.7. Statistical Analysis

The association between two parameters was assessed by Pearson’s two-tailed test. Statistical analyses were performed using the Statistical Package for Social Sciences software program for Windows (SPSS version 16.0). AP value of 0.05 was considered significant.


## 3. Results

### 3.1.ITGA2B andITGB3 Variations

Within the Chinese sample, 13 and 29 variants inITGA2B andITGB3 were identified, respectively. Two novelITGB3 SNPs with MA(F) > 0.02 were found, which were not reported in the National Center for Biotechnology Information (NCBI) dbSNP database (https://www.ncbi.nlm.nih.gov/snp/). The allele frequencies and Hardy–Weinberg equilibrium test results of the identified polymorphic sites are shown in Tables 3 and 4.Table 3
Summary ofITGB3variations detected in this study.
Sequence number obtained in this study dbSNP Location NC_000017.11 NM_000212.2 Nucleotide change NP_000410.2 amino acid change MA(F) H-WP ITGB3-01 rs147363351 5′-UTR 47253042 -820 agcttccaga G/A gttttaagtc  A(0.012) 0.913 ITGB3-02 rs3809862 5′-UTR 47253062 -800 ctggggaaga C/T ccagggactc  T(0.424) 0.822 ITGB3-03 rs7208170∗ 5′-UTR 47253393 -469 aaggcattca G/A cagatgtttg  A(0.419) 0.680 ITGB3-04 New 5′-UTR 47253360 -502 tagtgaataa T/A aaaggactga  A(0.023) 0.913 ITGB3-05 rs7208055 5′-UTR 47253461 -401 gtgaatgtgt C/A ccaagaatcc  A(0.221) 0.273 ITGB3-06 rs55827077∗ 5′-UTR 47253717 -145 tagagaagcc G/C gaggggagga  C(0.448) 0.494 ITGB3-07 rs567581451∗ 5′-UTR 47253771 -91 acccaccgcg -/TCCCC tcccctcccc  insTCCCC(0.058) 0.567 ITGB3-08 rs117052258∗ 5′-UTR 47253855 -7 cgcgggaggc G/C gacgagatgc  C(0.227) 0.771 ITGB3-09 New Exon1 47253882 21 ggccgcggcc C/G cggccgctct Pro7= G(0.407) 0.567 ITGB3-10 rs11871251 Intron1 47254061 79+121 ctgggaatgc G/A cgtgtcctgg  A(0.453) 0.771 ITGB3-11 New Intron1 47254082 79+152 tggcgcggt C/G ggagccggga  G(0.012) 0.913 ITGB3-12 rs112188890∗ Intron1 47254101 79+161 gagctgggga C/T cttcctggcc  T(0.116) 0.864 ITGB3-13 rs117414137∗ Intron1 47254192 79+252 aggctgagcg C/G cttcccggcc  G(0.116) 0.864 ITGB3-14 rs11871447 Intron1 47254252 79+312 ccgcgctcac C/G cggggctgcg  G(0.448) 0.918 ITGB3-15 New Intron1 47254331 79+391 tggggcttcc G/A ggggttgttc  A(0.006) 0.957 ITGB3-16 rs1009312∗ Intron1 47254774 79+834 ggcacagccc G/A gggttgctgc  G(0.471) 0.000 ITGB3-17 rs2015049 Intron1 47254865 79+925 ggccgcctct G/A cctcagagga  A(0.529) 0.000 ITGB3-18 rs56197296∗ Intron5 47287025_47287029 778-45_778-41 catggctgaa TTTGT/- tttgtctcct  delTTTGT(0.169) 0.049 ITGB3-19 rs5919∗ Exon6 47287174 882 ttgtccagcc T/C aatgacgggc Pro294= C(0.262) 0.022 ITGB3-20 rs41504748 Intron7 47290145 1036-40 accaccagct T/C cctttggtaa  C(0.070) 0.334 ITGB3-21 rs15908 Exon9 47290971 1143 cttccagctc A/G/T actttagaac Val381= C(0.599) 0.006 ITGB3-22 New Exon10 47292177 1299 gaggctgtcc C/T caggagaagg Pro433= T(0.006) 0.957 ITGB3-23 rs4642∗ Exon10 47292411 1533A>G cagcaggacga A/G tgcagccccc Glu511= G(0.331) 0.214 ITGB3-24 rs13306487 Exon10 47292422 1544 tgcagccccc A/C/G ggagggtcag Arg515Gln A(0.012) 0.913 ITGB3-25 rs4634∗ Exon10 47292423 1545 gcagcccccg G/A gagggtcagc Arg515= A(0.331) 0.214 ITGB3-26 New Exon10 47292528 1659 gcgagtgtga C/Tgacttctcct Asp550= T(0.006) 0.957 ITGB3-27 rs149823724 Exon11 47299519 1902 cagatgcctg C/T acctttaaga Cys634= T(0.006) 0.957 ITGB3-28 rs11870252 Intron11 47300459 1914-19 ccttaatcac T/C gtgtcctctc  C(0.035) 0.869 ITGB3-29 rs70940817∗ Exon12 47300524 1960 cctacatgac G/A aaaatacctg Glu654Lys A(0.076) 0.432 dbSNP, single nucleotide polymorphism database; H-WP, Hardy–Weinberg equilibrium P value; MA(F), minor allele (frequency). ∗SNPs which related with ADP induced platelets aggregation, GPIIb/IIIa content, bleeding time, or coagulation indexes in this study.Table 4
Association between individual ITGA2B and ITGB3 SNPs and the ADP induced platelets aggregation, GPIIb/IIIa content, bleeding time, PLT, and MPV (mean ± SD).
dbSNP Genotypes N Aggregation maxT% GPIIb-IIIa per platelet Bleeding time (s) PLT (109/L) MPV (fL) rs3760364 AA 48 36.6 ± 15.3 45937.1 ± 15214.0 281.3 ± 97.4 277.0 ± 70.87 9.48 ± 0.84 AT 7 27.4 ± 16.1 41060.9 ± 15560.7 364.3 ± 87.3 257.7 ± 83.7 9.51 ± 0.66 P   0.148 0.433 0.038 0.512 0.911 rs7208170 GG 18 38.5 ± 16.2 48835.0 ± 13746.0 326.7 ± 100.8 255.1 ± 62.1 9.14 ± 0.74 GA 24 35.1 ± 14.3 45714.9 ± 15783.1 288.8 ± 86.6 290.6 ± 82.8 9.63 ± 0.80 AA 13 31.7 ± 17.1 39709.0 ± 15545.1 249.2 ± 109.1 272.1 ± 60.4 9.68 ± 0.84 P   0.229 0.105 0.030 0.428 0.051 rs55827077 GG 18 39 . 9 ± 16 . 2 49297.7 ± 14047.7 315.0 ± 86.2 253 . 2 ± 62.6 9.24 ± 0.65 GC 22 35 . 1 ± 13 . 0 47150.7 ± 13706.9 279.5 ± 102.4 291.9 ± 85 . 5 9.5 5 ± 0.85 CC 14 30 . 6 ± 18 . 1 36518.1 ± 16661.9 287.1 ± 113.3 280.1 ± 55.2 9.66 ± 0.93 P   0.091 0.021 0.401 0.251 0.141 rs567581451 -/- 49 35.0 ± 14.4 45317.6 ± 14892.6 282.2 ± 99.1 277.3 ± 73.7 9.56 ± 0.80 -/insTCCCC 6 38.5 ± 24.4 45306.8 ± 19136.0 370.0 ± 64.8 252.3 ± 56.8 8.88 ± 0.62 P   0.609 0.999 0.040 0.428 0.054 rs117052258 GG 29 36.7 ± 16.3 44811.1 ± 14492.1 319.7 ± 93.6 277.1 ± 79.2 9.37 ± 0.82 GC 21 37.3 ± 13.3 48026.0 ± 15188.4 268.6 ± 105.4 267.9 ± 59 . 9 9.58 ± 0.80 CC 4 18.3 ± 14.8 31964.5 ± 18104.4 232.5 ± 61.8 309.7 ± 84.4 9.70 ± 1.01 P   0.165 0.539 0.028 0.764 0.288 rs1009312a GG 19 39.3 ± 15.9 46470.7 ± 13798.6 326.8 ± 84.2 276.4 ± 95.0 9.13 ± 0.61 GA 22 34.0 ± 14.6 48883.3 ± 14927.5 287.7 ± 101.3 272.7 ± 58.6 9.64 ± 0.88 AA 14 32.3 ± 16.6 38145.0 ± 16040.8 250.7 ± 105.0 275.0 ± 59.1 9.71 ± 0.82 P   0.189 0.160 0.027 0.944 0.029 rs56197296 TTTGT/TTTGT 42 37.7 ± 15.9 45398.1 ± 14564.7 285.0 ± 94.7 280 . 5 ± 76 . 3 9.41 ± 0.81 TTTGT/del 11 30.9 ± 10.2 45262.9 ± 18722.4 330.0 ± 116.2 249.6 ± 57.1 9.78 ± 0.79 del/del 2 11.5 ± 7.8 43895.5 ± 16431.0 225.0 ± 63.6 288.0 ± 21.2 9.30 ± 1.13 P   0.014 0.916 0.695 0.433 0.416 rs5919 TT 31 38.8 ± 16.1 45342.5 ± 14029.1 299.0 ± 96.6 283.7 ± 78.8 9.34 ± 0.84 TC 16 34.9 ± 12.5 47696.5 ± 17544.9 315.0 ± 105.6 247 . 7 ± 58.6 9.64 ± 0.73 CC 8 23.3 ± 13.8 40455.6 ± 15470.6 217.5 ± 67.6 292 . 9 ± 60.5 9.73 ± 0.85 P   0.015 0.634 0.130 0.728 0.148 Note: Pearson’s two-tailed test was used to analyze correlation between genotype and parameter.ars1009312 was completely linked with rs2015049.


### 3.2. Linkage Disequilibrium Analysis

Using the detected polymorphisms greater than 0.01 in frequency [MA(F) > 0.02], linkage disequilibrium (LD) was analyzed forD′ and r2 values (Figure 1). ForITGA2B, rs5911 was completely linked with rs850730 (r2=1.00), and rs5910 was strongly linked with rs850730 and rs5911 (r2=0.95). ForITGB3, complete LD was observed between rs4642 and rs4634 (r2=1.00). Other LD results are shown in Figure 1. A new SNPITGB3-09 was found for which MA(F) in this study was 0.402.Figure 1
Linkage disequilibrium (LD) map ofITGB2A andITGB3 SNPs along with their locations in theITGB2A andITGB3 genes. Variants present at >1% frequency were included in the LD analysis using the statistics D′ and r2. Red depicts a significant linkage between an SNP pair. Numbers inside the square indicate r2×100. (a)ITGB2A. (b)ITGB3.
(a) (b)The Hardy–Weinberg equilibriumP values of rs1009312 and rs2015049 were <0.001. The possible reason is that the sample in this study may have a Chinese population of other ethnic groups besides the Han population. Those two SNPs were not included in the LD analysis.


### 3.3. Correlation of ADP-Induced Platelet Aggregation, GPIIb/IIIa Content, Bleeding Time, and Coagulation Indexes

The GPIIb/IIIa contents, present as average numbers of GPIIb/IIIa receptor in each platelet, were associated with ADP-induced platelet aggregation (r=0.280, P=0.038), PLT (r=-0.522, P<0.001), PT (r=0.453, P=0.001), and TT (r=0.545, P<0.001) (Figures 1 and 2). The relationship between GPIIb/IIIa content and APTT was not significant (r=0.242, P=0.075), while the association between ADP-induced platelet aggregation and APTT was moderately significant (r=0.267, P=0.049) (Figure S1). The bleeding time had no association with other parameters. It had a negative association trend with MPV but was not significant (r=-0.244, P=0.073). The PLT was associated with PT (r=-0.415, P=0.002) and TT (r=-0.363, P=0.007). The FIB was negatively correlated with TT (r=-0.409, P=0.002).Figure 2
Correlations of the maximal level of ADP-induced platelet aggregation and GPIIb/IIIa content in healthy volunteers.r, coefficient of correlation; P, significance of correlation (Pearson’s test here and elsewhere).


### 3.4. Impact ofITGA2B andITGB3 SNPs on ADP-Induced Platelet Aggregation, GPIIb/IIIa Content, Bleeding Time, and Coagulation Indexes

ForITGA2B, only one SNP rs3760364 was related with the bleeding time (P=0.038) (Table 4). No significant correlation or trend was observed betweenITGA2B SNPs and other parameters.ForITGB3, the mean values of ADP-induced platelet aggregation of homozygous mutant genotypes in rs56197296 and rs5919 were lower than those of heterozygous mutations and wild type. Moreover, the GPIIb/IIIa per platelet was associated with rs55827077. A trend of GPIIb/IIIa per platelet was observed among rs70940817 GG (45), GA (9), and AA (1) carriers (43807.7±14157.5, 51086.8±19368.0, 61277, resp., P=0.093), although it did not reach a significant level. The bleeding time was significantly related with rs3760364, rs7208170, rs567581451, and rs117052258 (Table 4).In the present study, SNPs related with PLT were not found.ITGB3 rs1009312, which is completely linked with rs2015049, was significantly associated with the MPV (Table 4).ITGB3 rs70940817 was correlated with the PT and APTT (P=0.002 and P=0.003, resp.). The coagulation indexes and their P values are summarized in Table 5.Table 5
Association between individualITGA2B and ITGB3 SNPs and the coagulation indexes (mean ± SD).
Genotypes N PT (s) APTT (s) TT (s) FIB (g/L) rs112188890a CC 42 10.9 ± 1.08 31.2 ± 2.13 15.7 ± 2.22 2.52 ± 0.45 CT 12 11.2 ± 1.35 32.2 ± 2.73 14.7 ± 1.59 2.72 ± 0.44 TT 1 12.5 36.7 16.5 2.72 P   0.126 0.029 0.299 0.173 rs4642b AA 27 10.9 ± 1.20 30.9 ± 2.25 16.1 ± 2.36 2.43 ± 0.42 AG 21 11.3 ± 1.08 32.0 ± 2.34 15.2 ± 1.71 2.68 ± 0.44 GG 7 10.4 ± 1.01 32.3 ± 2.79 14.2 ± 1.39 2.76 ± 0.45 P   0.770 0.088 0.015 0.029 rs70940817 GG 45 10.7 ± 1.08 31.1 ± 2.19 15.2 ± 2.01 2.52 ± 0.42 GA 9 11.8 ± 1.02 32.8 ± 2.26 16.8 ± 2.34 2.75 ± 0.54 AA 1 12.7 37.2 15.75 3.10 P   0.002 0.003 0.078 0.069 MPV, mean platelet volume; PLT, platelet count; PT, prothrombin time; APTT, activated partial thromboplastin time; FIB, fibrinogen; TT, thrombin time. Note: Pearson’s two-tailed test was used to analyze correlation between genotype and parameter. aStrong LD was observed between rs112188890 and rs117414137 (r2=0.99); bcompletely LD was observed between rs4642 and rs4634.


## 3.1.ITGA2B andITGB3 Variations

Within the Chinese sample, 13 and 29 variants inITGA2B andITGB3 were identified, respectively. Two novelITGB3 SNPs with MA(F) > 0.02 were found, which were not reported in the National Center for Biotechnology Information (NCBI) dbSNP database (https://www.ncbi.nlm.nih.gov/snp/). The allele frequencies and Hardy–Weinberg equilibrium test results of the identified polymorphic sites are shown in Tables 3 and 4.Table 3
Summary ofITGB3variations detected in this study.
Sequence number obtained in this study dbSNP Location NC_000017.11 NM_000212.2 Nucleotide change NP_000410.2 amino acid change MA(F) H-WP ITGB3-01 rs147363351 5′-UTR 47253042 -820 agcttccaga G/A gttttaagtc  A(0.012) 0.913 ITGB3-02 rs3809862 5′-UTR 47253062 -800 ctggggaaga C/T ccagggactc  T(0.424) 0.822 ITGB3-03 rs7208170∗ 5′-UTR 47253393 -469 aaggcattca G/A cagatgtttg  A(0.419) 0.680 ITGB3-04 New 5′-UTR 47253360 -502 tagtgaataa T/A aaaggactga  A(0.023) 0.913 ITGB3-05 rs7208055 5′-UTR 47253461 -401 gtgaatgtgt C/A ccaagaatcc  A(0.221) 0.273 ITGB3-06 rs55827077∗ 5′-UTR 47253717 -145 tagagaagcc G/C gaggggagga  C(0.448) 0.494 ITGB3-07 rs567581451∗ 5′-UTR 47253771 -91 acccaccgcg -/TCCCC tcccctcccc  insTCCCC(0.058) 0.567 ITGB3-08 rs117052258∗ 5′-UTR 47253855 -7 cgcgggaggc G/C gacgagatgc  C(0.227) 0.771 ITGB3-09 New Exon1 47253882 21 ggccgcggcc C/G cggccgctct Pro7= G(0.407) 0.567 ITGB3-10 rs11871251 Intron1 47254061 79+121 ctgggaatgc G/A cgtgtcctgg  A(0.453) 0.771 ITGB3-11 New Intron1 47254082 79+152 tggcgcggt C/G ggagccggga  G(0.012) 0.913 ITGB3-12 rs112188890∗ Intron1 47254101 79+161 gagctgggga C/T cttcctggcc  T(0.116) 0.864 ITGB3-13 rs117414137∗ Intron1 47254192 79+252 aggctgagcg C/G cttcccggcc  G(0.116) 0.864 ITGB3-14 rs11871447 Intron1 47254252 79+312 ccgcgctcac C/G cggggctgcg  G(0.448) 0.918 ITGB3-15 New Intron1 47254331 79+391 tggggcttcc G/A ggggttgttc  A(0.006) 0.957 ITGB3-16 rs1009312∗ Intron1 47254774 79+834 ggcacagccc G/A gggttgctgc  G(0.471) 0.000 ITGB3-17 rs2015049 Intron1 47254865 79+925 ggccgcctct G/A cctcagagga  A(0.529) 0.000 ITGB3-18 rs56197296∗ Intron5 47287025_47287029 778-45_778-41 catggctgaa TTTGT/- tttgtctcct  delTTTGT(0.169) 0.049 ITGB3-19 rs5919∗ Exon6 47287174 882 ttgtccagcc T/C aatgacgggc Pro294= C(0.262) 0.022 ITGB3-20 rs41504748 Intron7 47290145 1036-40 accaccagct T/C cctttggtaa  C(0.070) 0.334 ITGB3-21 rs15908 Exon9 47290971 1143 cttccagctc A/G/T actttagaac Val381= C(0.599) 0.006 ITGB3-22 New Exon10 47292177 1299 gaggctgtcc C/T caggagaagg Pro433= T(0.006) 0.957 ITGB3-23 rs4642∗ Exon10 47292411 1533A>G cagcaggacga A/G tgcagccccc Glu511= G(0.331) 0.214 ITGB3-24 rs13306487 Exon10 47292422 1544 tgcagccccc A/C/G ggagggtcag Arg515Gln A(0.012) 0.913 ITGB3-25 rs4634∗ Exon10 47292423 1545 gcagcccccg G/A gagggtcagc Arg515= A(0.331) 0.214 ITGB3-26 New Exon10 47292528 1659 gcgagtgtga C/Tgacttctcct Asp550= T(0.006) 0.957 ITGB3-27 rs149823724 Exon11 47299519 1902 cagatgcctg C/T acctttaaga Cys634= T(0.006) 0.957 ITGB3-28 rs11870252 Intron11 47300459 1914-19 ccttaatcac T/C gtgtcctctc  C(0.035) 0.869 ITGB3-29 rs70940817∗ Exon12 47300524 1960 cctacatgac G/A aaaatacctg Glu654Lys A(0.076) 0.432 dbSNP, single nucleotide polymorphism database; H-WP, Hardy–Weinberg equilibrium P value; MA(F), minor allele (frequency). ∗SNPs which related with ADP induced platelets aggregation, GPIIb/IIIa content, bleeding time, or coagulation indexes in this study.Table 4
Association between individual ITGA2B and ITGB3 SNPs and the ADP induced platelets aggregation, GPIIb/IIIa content, bleeding time, PLT, and MPV (mean ± SD).
dbSNP Genotypes N Aggregation maxT% GPIIb-IIIa per platelet Bleeding time (s) PLT (109/L) MPV (fL) rs3760364 AA 48 36.6 ± 15.3 45937.1 ± 15214.0 281.3 ± 97.4 277.0 ± 70.87 9.48 ± 0.84 AT 7 27.4 ± 16.1 41060.9 ± 15560.7 364.3 ± 87.3 257.7 ± 83.7 9.51 ± 0.66 P   0.148 0.433 0.038 0.512 0.911 rs7208170 GG 18 38.5 ± 16.2 48835.0 ± 13746.0 326.7 ± 100.8 255.1 ± 62.1 9.14 ± 0.74 GA 24 35.1 ± 14.3 45714.9 ± 15783.1 288.8 ± 86.6 290.6 ± 82.8 9.63 ± 0.80 AA 13 31.7 ± 17.1 39709.0 ± 15545.1 249.2 ± 109.1 272.1 ± 60.4 9.68 ± 0.84 P   0.229 0.105 0.030 0.428 0.051 rs55827077 GG 18 39 . 9 ± 16 . 2 49297.7 ± 14047.7 315.0 ± 86.2 253 . 2 ± 62.6 9.24 ± 0.65 GC 22 35 . 1 ± 13 . 0 47150.7 ± 13706.9 279.5 ± 102.4 291.9 ± 85 . 5 9.5 5 ± 0.85 CC 14 30 . 6 ± 18 . 1 36518.1 ± 16661.9 287.1 ± 113.3 280.1 ± 55.2 9.66 ± 0.93 P   0.091 0.021 0.401 0.251 0.141 rs567581451 -/- 49 35.0 ± 14.4 45317.6 ± 14892.6 282.2 ± 99.1 277.3 ± 73.7 9.56 ± 0.80 -/insTCCCC 6 38.5 ± 24.4 45306.8 ± 19136.0 370.0 ± 64.8 252.3 ± 56.8 8.88 ± 0.62 P   0.609 0.999 0.040 0.428 0.054 rs117052258 GG 29 36.7 ± 16.3 44811.1 ± 14492.1 319.7 ± 93.6 277.1 ± 79.2 9.37 ± 0.82 GC 21 37.3 ± 13.3 48026.0 ± 15188.4 268.6 ± 105.4 267.9 ± 59 . 9 9.58 ± 0.80 CC 4 18.3 ± 14.8 31964.5 ± 18104.4 232.5 ± 61.8 309.7 ± 84.4 9.70 ± 1.01 P   0.165 0.539 0.028 0.764 0.288 rs1009312a GG 19 39.3 ± 15.9 46470.7 ± 13798.6 326.8 ± 84.2 276.4 ± 95.0 9.13 ± 0.61 GA 22 34.0 ± 14.6 48883.3 ± 14927.5 287.7 ± 101.3 272.7 ± 58.6 9.64 ± 0.88 AA 14 32.3 ± 16.6 38145.0 ± 16040.8 250.7 ± 105.0 275.0 ± 59.1 9.71 ± 0.82 P   0.189 0.160 0.027 0.944 0.029 rs56197296 TTTGT/TTTGT 42 37.7 ± 15.9 45398.1 ± 14564.7 285.0 ± 94.7 280 . 5 ± 76 . 3 9.41 ± 0.81 TTTGT/del 11 30.9 ± 10.2 45262.9 ± 18722.4 330.0 ± 116.2 249.6 ± 57.1 9.78 ± 0.79 del/del 2 11.5 ± 7.8 43895.5 ± 16431.0 225.0 ± 63.6 288.0 ± 21.2 9.30 ± 1.13 P   0.014 0.916 0.695 0.433 0.416 rs5919 TT 31 38.8 ± 16.1 45342.5 ± 14029.1 299.0 ± 96.6 283.7 ± 78.8 9.34 ± 0.84 TC 16 34.9 ± 12.5 47696.5 ± 17544.9 315.0 ± 105.6 247 . 7 ± 58.6 9.64 ± 0.73 CC 8 23.3 ± 13.8 40455.6 ± 15470.6 217.5 ± 67.6 292 . 9 ± 60.5 9.73 ± 0.85 P   0.015 0.634 0.130 0.728 0.148 Note: Pearson’s two-tailed test was used to analyze correlation between genotype and parameter.ars1009312 was completely linked with rs2015049.


## 3.2. Linkage Disequilibrium Analysis

Using the detected polymorphisms greater than 0.01 in frequency [MA(F) > 0.02], linkage disequilibrium (LD) was analyzed forD′ and r2 values (Figure 1). ForITGA2B, rs5911 was completely linked with rs850730 (r2=1.00), and rs5910 was strongly linked with rs850730 and rs5911 (r2=0.95). ForITGB3, complete LD was observed between rs4642 and rs4634 (r2=1.00). Other LD results are shown in Figure 1. A new SNPITGB3-09 was found for which MA(F) in this study was 0.402.Figure 1
Linkage disequilibrium (LD) map ofITGB2A andITGB3 SNPs along with their locations in theITGB2A andITGB3 genes. Variants present at >1% frequency were included in the LD analysis using the statistics D′ and r2. Red depicts a significant linkage between an SNP pair. Numbers inside the square indicate r2×100. (a)ITGB2A. (b)ITGB3.
(a) (b)The Hardy–Weinberg equilibriumP values of rs1009312 and rs2015049 were <0.001. The possible reason is that the sample in this study may have a Chinese population of other ethnic groups besides the Han population. Those two SNPs were not included in the LD analysis.


## 3.3. Correlation of ADP-Induced Platelet Aggregation, GPIIb/IIIa Content, Bleeding Time, and Coagulation Indexes

The GPIIb/IIIa contents, present as average numbers of GPIIb/IIIa receptor in each platelet, were associated with ADP-induced platelet aggregation (r=0.280, P=0.038), PLT (r=-0.522, P<0.001), PT (r=0.453, P=0.001), and TT (r=0.545, P<0.001) (Figures 1 and 2). The relationship between GPIIb/IIIa content and APTT was not significant (r=0.242, P=0.075), while the association between ADP-induced platelet aggregation and APTT was moderately significant (r=0.267, P=0.049) (Figure S1). The bleeding time had no association with other parameters. It had a negative association trend with MPV but was not significant (r=-0.244, P=0.073). The PLT was associated with PT (r=-0.415, P=0.002) and TT (r=-0.363, P=0.007). The FIB was negatively correlated with TT (r=-0.409, P=0.002).Figure 2
Correlations of the maximal level of ADP-induced platelet aggregation and GPIIb/IIIa content in healthy volunteers.r, coefficient of correlation; P, significance of correlation (Pearson’s test here and elsewhere).


## 3.4. Impact ofITGA2B andITGB3 SNPs on ADP-Induced Platelet Aggregation, GPIIb/IIIa Content, Bleeding Time, and Coagulation Indexes

ForITGA2B, only one SNP rs3760364 was related with the bleeding time (P=0.038) (Table 4). No significant correlation or trend was observed betweenITGA2B SNPs and other parameters.ForITGB3, the mean values of ADP-induced platelet aggregation of homozygous mutant genotypes in rs56197296 and rs5919 were lower than those of heterozygous mutations and wild type. Moreover, the GPIIb/IIIa per platelet was associated with rs55827077. A trend of GPIIb/IIIa per platelet was observed among rs70940817 GG (45), GA (9), and AA (1) carriers (43807.7±14157.5, 51086.8±19368.0, 61277, resp., P=0.093), although it did not reach a significant level. The bleeding time was significantly related with rs3760364, rs7208170, rs567581451, and rs117052258 (Table 4).In the present study, SNPs related with PLT were not found.ITGB3 rs1009312, which is completely linked with rs2015049, was significantly associated with the MPV (Table 4).ITGB3 rs70940817 was correlated with the PT and APTT (P=0.002 and P=0.003, resp.). The coagulation indexes and their P values are summarized in Table 5.Table 5
Association between individualITGA2B and ITGB3 SNPs and the coagulation indexes (mean ± SD).
Genotypes N PT (s) APTT (s) TT (s) FIB (g/L) rs112188890a CC 42 10.9 ± 1.08 31.2 ± 2.13 15.7 ± 2.22 2.52 ± 0.45 CT 12 11.2 ± 1.35 32.2 ± 2.73 14.7 ± 1.59 2.72 ± 0.44 TT 1 12.5 36.7 16.5 2.72 P   0.126 0.029 0.299 0.173 rs4642b AA 27 10.9 ± 1.20 30.9 ± 2.25 16.1 ± 2.36 2.43 ± 0.42 AG 21 11.3 ± 1.08 32.0 ± 2.34 15.2 ± 1.71 2.68 ± 0.44 GG 7 10.4 ± 1.01 32.3 ± 2.79 14.2 ± 1.39 2.76 ± 0.45 P   0.770 0.088 0.015 0.029 rs70940817 GG 45 10.7 ± 1.08 31.1 ± 2.19 15.2 ± 2.01 2.52 ± 0.42 GA 9 11.8 ± 1.02 32.8 ± 2.26 16.8 ± 2.34 2.75 ± 0.54 AA 1 12.7 37.2 15.75 3.10 P   0.002 0.003 0.078 0.069 MPV, mean platelet volume; PLT, platelet count; PT, prothrombin time; APTT, activated partial thromboplastin time; FIB, fibrinogen; TT, thrombin time. Note: Pearson’s two-tailed test was used to analyze correlation between genotype and parameter. aStrong LD was observed between rs112188890 and rs117414137 (r2=0.99); bcompletely LD was observed between rs4642 and rs4634.


## 4. Discussion

In this study, 13 variants in theITGA2B locus and 29 variants in theITGB3 locus in the Chinese population were observed. Two of the 29 variants located inITGB3 were novel SNPs with MA(F) > 0.02. Variants inITGA2B andITGB3 genes displayed significant interethnic differences in the global populations (Table 6). The C allele frequency of SNP rs5918 (T/C), located in theITGB3 gene, was only inhomogeneously distributed at 0.7% in the Chinese population, while it is common in whites and Africans (allele frequency 13.7% versus 12.8%). The following alleles of SNPs, rs7208170A, rs55827077C, rs11871251A, rs1009312G, and rs2015049G, were found to be more common in the Han Chinese and Africans (37%~50%) than in the European ancestry (10%~20%). In theITGA2B gene, the rs5911 was completely linked with rs850730 and strongly linked with rs5910 in the Chinese population in this study, and all the three SNPs were common in whites and Asians. However, the rs5911 was not found in Africans. According to the ethnicity difference described in the preceding text, resequencing of theITGA2B andITGB3 genes in the Chinese population was believed to be very important to reveal the function of SNPs.Table 6
Comparison ofITGA2B and ITGB3 allele frequencies in different ethnic groups.
dbSNP MA F a Number This study Chinese Asia n b European-ancestr y c Africa n d ITGA2B-01 rs3760364 T(0.052) T(0.012) T(0) T(0) ITGA2B-08 rs850730 G(0.477) G(0.467) G(0.297) G(0.437) ITGA2B-11 rs5911 G(0.477) G(0.467) G(0.305) G(0) ITGA2B-12 rs5910 T(0.465) T(0.442) T(0.376) T(0.432) ITGB3-03 rs7208170 A(0.419) A(0.496) A(0.194) A(0.431) ITGB3-05 rs7208055 A(0.221) A(0.225) A(0.117) A(0.356) ITGB3-06 rs55827077 C(0.448) C(0.417) C(0.158) C(0.39) ITGB3-08 rs117052258 C(0.227) C(0.208) NA NA ITGB3-10 rs11871251 A(0.453) A(0.482) A(0.199) A(0.373) ITGB3-12 rs12188890 T(0.116) T(0) T(0.023) T(0) ITGB3-16 rs1009312 G(0.417) A(0.434) A(0.125) A(0.432) ITGB3-17 rs2015049 G(0.471) A(0.478) A(0.138) A(0.413) NF. rs5918 C(0) C(0.007) C(0.137) C(0.128) ITGB3-18 rs56197296 delTTTGT(0.169) NA NA NA ITGB3-19 rs5919 C(0.262) C(0.232) C(0.049) C(0.187) ITGB3-23 rs4642 G(0.331) G(0.31) G(0.283) G(0.306) ITGB3-25 rs4634 A(0.331) A(0.350) A(0.283) A(0.331) ITGB3-29 rs70940817 A(0.076) NA NA NA dbSNP, single nucleotide polymorphism database; MA(F), minor allele (frequency); SNP, single nucleotide polymorphisms; NF, not found; NA, not available. aResource of MP(F)s was from GenBank and HapMap, https://www.ncbi.nlm.nih.gov/snp/; bHan Chinese in Beijing, China (CHB), or CHB + Japanese in Tokyo, 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).Variation in the MPV or PLT can have a profound impact on differences in the platelet function between individuals [16, 17], and these traits have a strong genetic component [18–21]. However, limited information is available for Asians. In the present study, the A alleles of rs1009312 and rs2015049, located inITGB3, were positively associated with the MPV (P=0.029). The MPV had a trend in rs7208170 alleles but did not reach a significant level (P=0.051). The difference between PLT and SNPs inITGA2B orITGB3 was not significant.The APTT and PT are clinical tests commonly used to indicate coagulation factor deficiencies [22, 23], activated coagulation, and risk of venous thromboembolism [24, 25]. To date, two genome-wide association studies (GWAS) of APTT and PT conducted in the European ancestry have been reported. One study identified genome-wide significant associations of APTT and variants at F12 (MIM 610619), KNG1 (MIM 612358), and HRG (MIM 142640) [26]. In the other research, the GWAS for APTT and PT was conducted and replicated genome-wide significant associations at KNG1, HRG, F11, F12, and ABO for APTT and identified significant associations at the F7 and PROCR/EDEM2 regions for PT. Eight genetic loci accounted for ~29% of the variance in APTT, and two loci accounted for ~14% of the variance in PT [27]. In this study, the association of APTT, PT, FIB, and TT withITGA2B andITGB3SNPs was investigated. The A allele ofITGB3 rs70940817 was found to significantly correlate with the elevated APTT and PT (P=0.003 and P=0.002, resp.). The T allele ofITGB3 rs112188890 was related with APTT (P=0.029), and the G allele ofITGB3 rs4642 was associated with TT and FIB (P=0.015 and P=0.029, resp.).The ADP-induced platelet aggregation test was often used to identify the platelet function and the efficacy of antiplatelet drugs. Jones et al. detected 1327 SNPs and investigated their correlation with ADP or collagen-related peptide-induced platelet aggregation in 500 healthy northern European subjects. This identified 17 novel associations with the platelet function (P<0.005) accounting for approximately 46% of the variation in response [28]. In this study, the minor allele of rs56197296 and rs5919 was found to be associated with the decreased ADP-induced platelet aggregation (P=0.014 and P=0.015, resp.).GPIIb/IIIa is the central receptor of platelet aggregation. The variations of GPIIb/IIIa amount expressed on a platelet surface might affect the platelet-aggregating activity. Many studies reported that the rs5918 correlated with the GPIIb/IIIa receptor expression [29]; however, many conflict studies have also been published [3, 30]. O’Halloran et al. investigated whether three polymorphisms of the GPIIIa promoter (−468G/A, −425A/C, and −400C/A) influenced the RNA expression and receptor density in the platelets of patients with cardiovascular disease [3]. They found a threefold variation between the subjects in the number of GPIIb/IIIa receptors expressed per platelet, although no association between the receptor density and the PlA2 or the three promoter polymorphisms was demonstrated. In the present study, rs55827077, which was located at the promoter region of the GPIIIs gene at position −145, was related with GPIIb/IIIa per platelet.The bleeding time is a test that evaluates the platelet functionin vivo. A prolonged bleeding time can result from platelet abnormality, Von Willebrand factor deficiency, or vascular disorders such as Ehlers–Danlos syndrome. A disruption of platelet aggregation results in a prolongation of the bleeding time [31]. In this study, the bleeding time was related with rs3760364 ofITGA2B and rs7208170, rs567581451, and rs117052258 ofITGB3 (P<0.05). No previous study reporting on the effect of SNPs on the bleeding time was found. The mechanism of the relationship needs to be further examined. However, a negative trend between the bleeding time and MPV (r=-0.244, P=0.073) was observed. Moreover, rs1009312 and rs2015049 were found to significantly associate with both bleeding time and MPV. Further, the minor alleles of rs7208170 and rs567581451, which were significantly related with the bleeding time, had a trend with the MPV among genotypes, although they did not reach a significant level (P=0.051 and P=0.054, resp.). However, no relationship was observed among SNPs, bleeding time, and MPV in rs3760364 and rs117052258.In this study, a positive correlation of the level of ADP-induced aggregation and GPIIb/IIIa content was detected in healthy volunteers. This correlation is consistent with a previous report. Yakushkin et al. investigated the relationship between the number of GPIIb/IIIa and the level of ADP-induced aggregation in a group of 35 healthy volunteers and found positive and significant correlations between the level of platelet aggregation induced by different ADP doses (from 1.25 to 20 mmol/L) and the number of GPIIb/IIIa [32].A strong positive correlation was observed between GPIIb/IIIa per platelet and PLT, PT, and TT (P<0.01). Although the TT was strongly correlated with GPIIb/IIIa per platelet (r=0.545, P<0.001), the relationship of SNPs/TT and SNPs/GP (IIb/IIIa) content is not linked. For example,ITGB3 rs4642 was significantly related with the TT (P=0.015), while the value of GPIIb/IIIa per platelet had no trend among rs4642 genotypes (P=0.789). The other coagulation indexes were the same. Therefore, the relationship between SNPs and GPIIb/IIIa content cannot be deduced from the correlation of coagulation indexes and SNPs.Some of the significant correlations between SNPs and platelet function parameters in the present study were not reported previously, which may be due to the reason that SNPs with low MA(F) in the European ancestry were not selected as candidate SNPs in previous studies. For example, the GWAS-genotyping platforms lack the coverage of low frequency/rare variants [33]. Moreover, LD might be another reason, if different genetic patterns (haplotypes) are present in different populations [3].A possible limitation of the current study is the limited number of subjects. After stratification, the number of subjects in each genotypic group was small, thereby limiting further haplotype association analysis and accession of small effects of an SNP with a small minor allele frequency.


## 5. Conclusion

In summary, as ethnicity difference might limit the interpretation of the function of SNPs, resequencing theITGA2B andITGB3 genes and investigating their functions in the Chinese population are very important. In the present study, nine SNPs were found to associate with indexes of platelet and coagulation haemostasis. Newer studies are needed, particularly, to further assess the clinical importance of the above-discussed SNPs in disease susceptibility and antiplatelet drugs pharmacodynamics. Further studies should pay more attention to the roles ofITGA2B andITGB3 SNPs in ethnic variations.



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*Source: 5675084-2016-11-14.xml*