The Relationship between P-Selectin Polymorphisms and Thrombosis in Antiphospholipid Syndrome: A Pilot Case-Control Study

Objective: The selectins are cell adhesion molecules that mediate the interactions among leukocytes, activated platelets, and endothelial cells. We aimed to investigate whether P-selectin polymorphisms are associated with thrombosis in patients with antiphospholipid syndrome (APS). Materials and Methods: The diagnosis and classification of APS were based on the report of an international workshop. Genomic DNA was extracted from citrated blood samples of all subjects. Three single nucleotide polymorphisms associated with the P-selectin coding region (S290N, c.1087G>A; N562D, c.1902G>A; T715P, c.2363A>C) were assessed. Results: There were 26 APS (65%) patients with thrombosis. The number of patients without thrombosis was 14 (35%). The frequency of the N562D-DN genotype was significantly higher in patients with APS than in healthy controls (p=0.003). The frequency of this genotype was significantly higher in patients with APS with thrombosis compared with patients with no thrombosis (p=0.03). The N562D-NN genotype was found at a higher frequency in patients with APS than in healthy controls (p=0.004). Conclusion: Our results suggest that the N562D polymorphism of the DN genotype of P-selectin is associated with an increased risk of thrombosis in patients with APS.


Introduction
Antiphospholipid syndrome (APS) is an autoimmune disease characterized by pregnancy morbidity and arterialvenous thrombosis in the presence of antiphospholipid antibodies (aPLAs) [1]. Although the relationship between aPLAs and thrombosis is known, the mechanisms of thrombosis in APS has not been fully elucidated. Selectins, which are cell adhesion molecules, mediate the interactions among leukocytes, activated platelets, and endothelial cells. P-selectin, which can be identified as a soluble form in plasma, intercedes in the attachment and rolling of leukocytes on activated endothelial cells and is involved in the recruitment of leukocytes to thrombi [2,3]. Novel data suggest that the levels of soluble P-selectin (sP-selectin) are increased in APS patients with thrombosis [4]. In this study, we aim to investigate whether P-selectin polymorphisms are associated with thrombosis in patients with APS.

Patients and Controls
Forty adult patients with APS and 40 healthy subjects with no history of thrombosis or autoimmune disease were included in the study. The history of disease, physical examination, and screening of lupus anticoagulant (LA) and serum anticardiolipin IgG and IgM levels were assessed for all patients. The diagnosis and classification of APS were based on an international consensus statement [1]. The subjects participating in the study had no LA and/or serum anticardiolipin-related systemic diseases or risk factors such as hypertension or hyperlipidemia for thrombosis.
LA was diagnosed using activated partial thromboplastin time, kaolin clotting time, and Russell's viper venom test according to published criteria [5]. IgG and IgM anticardiolipin antibodies were determined by enzyme-linked immunosorbent assay [6], and levels equal to or greater than 4 standard deviations were regarded as positive. The anticardiolipin antibody and LA tests were repeated 3 months after the first determination. APS patients with thrombosis had arterial and/or venous thrombosis as well as aPLA positivity. aPLA-positive patients with no thrombosis suffered from either first trimester fetal losses or thrombocytopenia and had persistently positive aPLA test results but no thrombotic complications over at least 3 years of follow-up.
The study protocol was approved by the local ethics committee, and written and signed informed consent was obtained from all participants.

Genotyping
Genomic DNA was extracted from citrated blood samples.

Statistical Analysis
Data are expressed as mean ± SD, number (%), or median (range). Test statistics were computed using the Mann-Whitney U test and the Kruskal-Wallis test. The chi-square test and odds ratio were used to calculate the 95% confidence intervals. Correlation coefficients and significance were calculated by Spearman's test to assess the differences between groups. For all tests, a 2-tailed p-value of <0.05 was considered statistically significant. Statistical analyses were performed using the software package SPSS 15 running on Windows NT.

Results
There were 26 APS (65%) patients with thrombosis, 12 (46%) of which cases involved veins, 10 (38%) arteries, and 4 (15%) veins and arteries together. The number of APS patients without thrombosis was 14 (35%). The mean age of patients (80% female) was 39.4±9.5 years. The characteristics of patients are seen in Table 1. The frequency of the N562D-DN genotype was significantly higher in patients with APS than healthy controls (p=0.003). The frequency of this genotype was significantly higher in patients with APS with thrombosis compared to patients without thrombosis (p=0.03). The N562D-NN genotype was found at a higher frequency in patients with APS than in healthy controls (p=0.004). The frequency of the N562D-NN genotype was not different between patients without thrombosis and control subjects (p=0.21). On the other hand, S290N and T715P polymorphisms were not different between patient and control groups (Tables 2 and 3). There was no relationship between aPLA, thrombocytopenia, or pregnancy loss and any polymorphism.

Discussion
P-selectin is expressed on activated platelets and endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) is found in neutrophils and monocytes, and these are from microparticles. Connecting P-selectin/PSGL-1 activated leukocytes for endothelium rolling, and providing the release of tissue factor initiates thrombosis [7]. Previous studies suggest that sP-selectin levels increased in patients with thrombosis after a finding of the association of P-selectin and thrombosis in an animal model [8,9,10]. Additionally, P-selectin polymorphisms were detected in patients with thrombosis, but not always together with high sP-selectin levels. High sP-selectin levels were detected in patients with systemic lupus erythematosus and APS [11,12]. According to other studies, P-selectin polymorphisms play a role in the pathogenesis of systemic lupus erythematosus [13,14].
On the other hand, the relationship between polymorphism of PSGL-1 and ischemic cerebrovascular disease was shown previously [15]. Roldan et al. demonstrated that short alleles of PSGL-1 protect against cardiovascular disease [16]. v Additionally, the relationship PSGL-1 VNRT polymorphisms and risk of thrombosis in APS patients was shown by Diz-Kucukkaya et al. [17]. The relationship between P-selectin polymorphism and thrombosis in APS patients was shown for the first time in our study. The c.1087G>A, c.1902G>A, and c.2363A>C polymorphisms lead to S290N, N562D, and T715P P-selectin gene variations, respectively. These variations are in the genetic region encoding the repeated part of the P-selectin gene and may be effective in binding P-selectin to PSGL-1. Therefore, our data are valuable in order to determine risk factors other than traditional ones for thrombosis in APS despite the fact that we did not estimate sP-selectin levels.
In conclusion, our results suggest that the N562D polymorphism DN genotype of P-selectin is associated with an increased risk of thrombosis in patients with APS. The NN genotype of the same polymorphism might be protective against thrombosis in those patients. The effect of N562D polymorphism on sP-selectin levels will be studied in future work.

Conflict of Interest Statement
The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.