Platelet Glycoprotein IIb HPA-3 a/b Polymorphism Is Associated with Native Arteriovenous Fistula Thrombosis in Chronic Hemodialysis Patients

Abstract

Objective: The aim of this study was to investigate the association of Glycoprotein IIb (GPIIb) human platelet antigen-3 (HPA-3) a/b polymorphism with end-stage renal disease (ESRD) on hemodialysis (HD) and native Arteriovenous fistula (AVF) thrombosis. Methods: The polymorphism in the GPIIb subunit of the receptor HPA-3 (a and b alleles) was identified by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 145 HD patients and 120 healthy controls from a Chinese Han population. The HD patients were classified into two groups: G1 and G2. G1 included 65 HD patients presented at least one AVF thrombosis episode and G2 included 80 HD patients without any episode of AVF thrombosis. Results: There were no significant differences in either HPA-3 a/b genotypes (aa, ab, and bb) frequency distribution (p = 0.396) or allele (a and b) frequency distribution (p = 0.146) between HD patients and control groups. However, there were significant differences in both HPA-3 a/b genotypes (aa, ab, and bb) distribution (χ² = 6.127, p = 0.047) and allele (a and b) frequency distribution (χ² = 5.954, p = 0.015) between G1 and G2. The relative risk of native AVF dysfunction in ab + bb patients compared with that of aa patients was 2.31 (95% confidence interval: 1.18–4.52). Conclusions: These findings suggest an association between AVF thrombosis and the HPA-3b allele, and it is likely that HPA-3 a/b polymorphisms could be useful markers for potential risk of native AVF thrombosis in HD patients.

• human platelet antigen
• genetic polymorphism
• arteriovenous fistula
• thrombosis
• hemodialysis

INTRODUCTION

The surgically placed dialysis arteriovenous fistula (AVF) is considered to be the ideal choice for hemodialysis (HD) access by the Kidney Disease Outcomes Quality Initiative (KDOQI) and the Fistula First Breakthrough Initiative. HD vascular access failure is one of the most important complications of kidney failure.¹ AVF dysfunction increases morbidity and hospitalizations² in HD patients, of which more than 80% of cases are due to thrombosis.³

Platelet adhesion, activation, and aggregation are the principal functions of platelets implied in the pathogenesis of thrombotic diseases.⁴ Human platelets carry membrane glycoproteins (GPs) that control platelet aggregation and activation. As a member of the integrin family, the membrane complex GPIIb/IIIa recognizes fibrinogen, von Willebrand factor, fibronectin, and vitronectin as ligands, and it is involved in platelet aggregation and thrombus formation. Platelet membrane GPs are highly polymorphic and can be recognized as alloantigens or autoantigens, and most of their polymorphisms are caused by single base-pair substitution, resulting in an amino acid replacement.⁵ The GPIIb gene polymorphism consists of a substitution of serine for isoleucine at position 843 [human platelet antigen-3 (HPA-3) a/b bp T2622G, amino acid Ile843Ser].⁶ A large population study suggested that heritable factors play a major role in determining platelet aggregation⁷; of the overall variance in platelet aggregation, 21–30% was due to heritable factors. The development of thrombotic disorders in humans is one of the most common causes of morbidity and mortality in HD patients. Perturbation of hemostasis is central to the pathogenesis of this disease and may be in part genetically determined. Moreover, there are no data on the significance of HPA-3 alleles in the genesis of end-stage renal disease (ESRD) on HD or native AVF thrombosis.

Therefore, the aim of this study was to investigate the frequency of HPA-3 a/b polymorphisms of the platelet receptor GPIIb/IIIa in HD patients with or without native AVF thrombosis in a Chinese Han population.

METHODS

Patients

A total of 145 patients undergoing chronic maintenance HD were eligible for this study. The subjects were enrolled at the Affiliated Hospital of Nantong University (Nantong, PR China), maintained on dialysis for 46 ± 33 months. A radiocephalic fistula with side-to-end anastomosis under local infiltration anesthesia was constructed in all of the enrolled patients. All patients required regular HD sessions for 4–5 h, 3 times a week. Blood flow was usually 200–400 mL/min with a dialysate flow at a constant rate of 500 mL/min. All patients received regular doses of standard heparin (100–150 UI/kg) before HD session. The patients were classified into two groups: G1 and G2. G1 included 65 HD patients presented at least one native AVF thrombosis episode, which was defined as the absence of blood flow and the impossibility for hemodialysis. G2 included 80 HD patients without any episode of native AVF thrombosis. Loss of primary functional patency of the AVF (loss of primary functional patency within 6 months of first use) occurred in 23 (35.38%) of total 65 cases in G1. A total of 120 age- and gender-matched healthy individuals were used as controls in the study. Patients receiving oral anticoagulation or antiplatelet therapy; with prior history of arterial or venous thrombosis, acute or chronic hepatic disease, malignant diseases, acute infections, and history of renal transplantation; and pregnant women were automatically excluded from the study. Subjects whose first-degree relatives had ESRD were excluded from the control group. As ethnic differences may influence the results in single-nucleotide polymorphism (SNP) studies, only Chinese Hans were enrolled in this project. A detailed history and clinical variables such as age, gender, body mass index (BMI), presence or absence of diabetes, and using erythropoietin or not were recorded. Total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides were measured using standard techniques. The study was approved by the Medical Ethics Committee of Nantong University, and written informed consent was obtained from all participants.

Genotyping Protocols

DNA was extracted from peripheral blood using a small amount of genomic DNA extraction kit (Songon, Shanghai, China) with appropriate revision in experimental methods. Sequence-specific primers (SSPs) were used for the genotyping as described previously.⁸ Allele-specific primer sequences used for HPA-3 polymorphisms were: HPA-3a (5′-GGG GGA GGG GCT GGG GA-3′), HPA-3b (5′-GGG GGA GGG GCT GGG GC-3′), and HPA-3 common (5′-GAC CTG CTC TAC ATC CTG GA-3′). Specific primers amplifying a fragment of human growth hormone (HGH) were used as positive controls: HGH-A (5′-TGC CTT CCC AAC CAT TCC CTT A-3′) and HGH-B (5′-CCA CTC ACG GAT TTC TGT TGT GTT TC-3′). The size of the polymerase chain reaction (PCR) products was 230 base pairs for HPA-3 and 434 base pairs for HGH.

A total of 50 μL of the reaction volume containing final reaction component concentrations of 0.2 mmol/L for each of the four dNTPs, 2.0 mmol/L MgCl₂, 0.4 μL of each allele-specific primer pairs, 0.1 mg/L for genomic DNA, and 1.5 U Taq polymerase (Sangon) was added to each well of a 96-well plate. PCR cycles were carried out as follows: after denaturation for 10 min at 94°C, the amplification cycle (denaturation step at 94°C for 50 s, annealing step at 60°C for 50 s, and extension step at 72°C for 50 s) was repeated 35 times and followed by final extension for 10 min at 72°C.

PCR products were determined by electrophoresis in 2.5% agarose gel with ethidium bromide, and run at 150 V for 25 min, visualized under UV illumination and documented by photography. Fifty amplification products were sent to direct DNA sequencing.

Statistical Analyses

All continuous variables are expressed as mean and standard deviation. Student’s t-test was used to compare continuous variables from the two groups. Genotype and allele frequencies were obtained by direct count. Differences in the distribution of alleles and genotypes between the groups and deviation from Hardy–Weinberg equilibrium were assessed by χ² test. All significant tests were two-tailed and were considered statistically significant at p < 0.05. Statistical software package SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

RESULTS

The clinical characteristics of all participants enrolled in the study are depicted in Table 1. There were no significant differences in gender, age, or BMI between the HD and control groups. The characteristics of the HD patients are given in Table 2. No significant differences were found regarding clinical variables, cholesterol, and triglycerides between G1 and G2.

Table 1.  Clinical characteristics of HD and control subjects.

	HD patient	Control	p
Case number	145	120
Age (years)	53.81 ± 11.78	51.26 ± 11.24	0.075
Gender (% female)	57 (39.31)	52 (43.33)	0.508
Body mass index (kg/m^2)	23.01 ± 2.78	23.63 ± 2.66	0.067

Note: HD, hemodialysis.

Table 2.  Patient profiles in G1 and G2.

	G1	G2	p
Case number	65	80
Age (years)	53.12 ± 11.99	54.36 ± 11.64	0.530
Gender (% female)	24 (36.92)	33 (41.25)	0.596
Body mass index (kg/m^2)	22.71 ± 2.83	23.26 ± 2.72	0.238
Using erythropoietin (%)	57 (87.69)	68 (85.00)	0.640
Total cholesterol (mmol/L)	4.42 ± 1.14	4.21 ± 1.05	0.239
High-density lipoprotein cholesterol (mmol/L)	1.21 ± 0.33	1.24 ± 0.29	0.522
Low-density lipoprotein cholesterol (mmol/L)	2.59 ± 0.76	2.43 ± 0.78	0.208
Triglycerides (mmol/L)	1.30 ± 0.50	1.23 ± 0.61	0.431
Diabetes (% number)	6 (9.23)	6 (7.50)	0.768

Table 3 summarizes the distributions of HPA-3 a/b genotypes and allele frequencies for all participants. The genotype distribution among the subjects was in Hardy–Weinberg equilibrium in both the HD group (p > 0.05) and the control group (p > 0.05). G1 and G2 also were in Hardy–Weinberg proportions (p > 0.05). The distribution of the HPA-3 a/b genotypes (aa, ab, and bb) was 48.28%, 35.86%, and 15.85% for HD subjects and 55.00%, 34.17%, and 10.83% for the controls, respectively. The derived allele frequencies for the “a” and “b” alleles were 66.21% and 33.79% in the HD subjects and 72.08% and 27.92% in the control subjects, respectively. There were no significant differences in either genotype frequency distribution (χ² = 1.855, p = 0.396) or allele frequency distribution (χ² = 2.115, p = 0.146) between these two groups, which suggested that the HPA-3 a/b polymorphism of the platelet receptor GPIIb/IIIa was not significantly associated with ESRD.

Table 3.  Distributions of HPA-3 a/b genotypes and alleles in control and HD groups and their subgroups.

	Genotype frequency [n (%)]			Allele frequency [n (%)]
Group	aa	Ab	Bb	A	B
Control (n = 120)	66 (55.00)	41 (34.17)	13 (10.83)	173 (72.08)	67 (27.92)
HD (n = 145)	70 (48.28)	52 (35.86)	23 (15.86)	192 (66.21)	98 (33.79)
G1	24 (36.92)	28 (43.08)	13 (20.00)	76 (58.46)	54 (41.54)
G2	46 (57.50)	24 (30.00)	10 (12.50)	116 (72.50)	44 (27.50)

Note: HD, hemodialysis.

On the other hand, the distribution of the HPA-3 a/b genotypes (aa, ab, and bb) was 36.92%, 43.08%, and 20.00% for G1 and 57.50%, 30.00%, and 12.50% for G2, respectively. The derived allele frequencies for the “a” and “b” alleles were 58.46% and 41.54% in G1 and 72.50% and 27.50% in G2, respectively. There were significant differences in both genotype distribution (χ² = 6.127, p = 0.047) and allele frequency distribution (χ² = 5.954, p = 0.015) between G1 and G2. The relative risk of native AVF dysfunction in ab + bb patients compared with that of aa patients was 2.31 [95% confidence interval (CI) = 1.18–4.52].

DISCUSSION

The forearm radiocephalic fistula is considered as the optimal first choice for a native fistula. However, this fistula has the disadvantage that it often fails to mature in older patients, women, and those with significant underlying vascular disease, particularly diabetics.^2,9,10 Once mature and functional, AVFs are the preferred form of HD vascular access because of their relative lack of infection.¹¹ The major complications are later venous stenosis followed by thrombosis. ESRD and HD may actually increase the risk of AVF dysfunction. Among other factors, the increasing thrombotic trend in HD patients is due to the activation of platelets and clotting factors.^12,13 Although AVF thrombosis is primarily related to mechanical or surgical events, the genetic background of the patient may also significantly contribute to the occurrence of those episodes.

GPIIb/IIIa molecules were abundantly expressed on the platelet surface, had a central role in platelet aggregation,¹⁴ and carry HPA-3 alloantigens. The studies to determine whether the HPA-3 a/b polymorphism might be associated with increased thrombotic risk are less, and some studies found that HPA-3b was associated with a higher thrombotic risk,^15,16 whereas others did not.^17,18 The association between ESRD as well as native AVF thrombosis and the HPA-3 a/b polymorphism has not been studied in the Chinese population. In this study, we examined the association of the HPA-3 a/b polymorphism in Chinese Han patients suffering from ESRD.

Despite the limited number of participants enrolled in this study, no significant difference was observed in either genotype frequency distribution (p = 0.396) or allele frequency distribution (p = 0.146) between the ESRD and control groups, suggesting that the HPA-3 a/b polymorphism is not significantly associated with ESRD. In this study, significant differences of HPA-3 a/b genotypes and allele frequencies were found between patients with native AVF thrombosis and without this complication. We further demonstrated that patients with the ab + bb genotype had greater native AVF thrombosis risk than those with the aa genotype. The HPA-3b allele was associated with native AVF thrombosis. This result was not in agreement with the results of the only reported study carried out in Tunisian patients,¹⁹ which does not reveal an association between the HPA-3 a/b polymorphisms and the occurrence of thrombosis in AVF. The discrepancy may be explained, at least in part, by ethnic differences of the populations, the inadequate control of other risk factors for thrombosis, as well as the limited sample size assayed in the different studies.

In conclusion, our data support that platelet GbIIb HPA-3 a/b polymorphism is associated with native AVF thrombosis, and the b allele is a potential risk for native AVF thrombosis in Chinese Han HD patients. Given the inherent limitations of case–control studies and the complex nature of genetic susceptibility for chronic degenerative diseases, further studies are still needed to be conducted in individual ethnic groups to verify the relevance of this polymorphism to the development of disease.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.