Introduction
Venous thromboembolism (VTE), including deep-vein thrombosis (DVT) and pulmonary embolism (PE), is the third most common acute cardiovascular syndrome and its disease burden is growing as the population and life expectancy expand [
1]. DVT is defined as venous thrombus forming in a large vein, such as the leg or pelvis, while PE is developed when the thrombus dislodges and spreads through the heart to the pulmonary arteries [
2]. VTE has become a global public health concern, with an estimated 300,000–600,000 individuals affected each year in the United States, offering high morbidity and mortality rates [
3]. Notably, patients with coronavirus disease 2019 (COVID-19) were proven to be at significantly higher risk of developing VTE due to concomitant prothrombotic status [
4].
The etiology of VTE is complicated and varied, and its onset may be attributed to the disruption of the coagulation homeostasis. Several factors may be involved in the development of VTE, including intrinsic (e.g., thrombophilia), acquired (e.g., obesity, cancer, prothrombotic medication) and external (e.g., reduced mobility due to surgery, trips lasting more than four hours) [
5]. For patients with suspected VTE, it is recommended to combine clinical pretest probability assessment, D-dimer test, and imaging for diagnosis [
6]. Patients with VTE would benefit from prompt anticoagulation treatment based on bleeding risk assessment. Different pharmaceutical regimens such as heparin, low-molecular-weight heparin (LMWH), fondaparinux, or the direct oral anticoagulants (rivaroxaban or apixaban) should be applied on the basis of the patient's individual condition and duration of treatment [
7]. However, given the aggressive nature of VTE episodes, early risk assessment and refined management for patients would be imperative. In this scenario, it is meaningful to explore individualized biomarkers for the meticulous management of VTE patients.
Given the crucial role of genetic factors in disease pathogenesis, single nucleotide polymorphisms (SNPs) have become a hot topic of research at present. In previous study, we reported the potential value of combining tumor necrosis factor-α (
TNF-α) -308G/A gene polymorphism with neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in predicting the efficacy and safety of anti-TNF-α therapy [
8]. Some inherited polymorphisms have been identified as high-risk factors for VTE, such as 4G or 5G polymorphism of plasminogen activator inhibitor 1 (PAI-1), G1691A mutation in the
F5 gene (Factor V Leiden (FVL)), G20210A of the
F2 (prothrombin) gene, and C677T of the methylenetetrahydrofolate reductase (MTHFR) gene.
PAI-1 gene (
SERPINE1) is located on chromosome 7q which encodes the secreted protein containing 402 amino acids [
9]. As the primary inhibitor of tissue-type and urokinase-type plasminogen activator, PAI-1 is an integral member of the fibrinolysis system [
10]. Abnormally increased PAI-1 can impair plasminogen activation resulting in excessive fibrin accumulation in the blood vessels, which further leads to thrombosis. A single nucleotide insertion or deletion (4G or 5G) polymorphism at 675 bp upstream of the PAI-1 gene (
SERPINE1) transcription start site has been reported to be associated with the expression of PAI-1, with the 4G allele being favored for elevated expression levels [
11,
12]. The 4G/5G polymorphism of the PAI-1 has been reported to be associated with the risk of venous thrombosis [
13], ischemic stroke [
14], femoral necrosis [
15], diabetic nephropathy [
16], cancers [
17], and systemic lupus erythematosus [
18]. Unfortunately, the association of the PAI-1 4G/5G polymorphism with susceptibility, treatment efficacy and recurrence status of VTE has not been well investigated, especially in Chinese population.
Factor V is a crucial component involved in the coagulation process and it can be cleaved at amino acid 506 by activated protein C (APC). However, when the nucleotide at position 1691 of the
F5 gene is converted from guanine to adenine, the arginine at position 506 is substituted by glutamine, which is known as the FVL mutation. In this case, APC fails to cleave factor V and thus blocks the anticoagulant effect. It has been reported that FVL heterozygote and homozygote carriers have a sevenfold and 80-fold increased risk of VTE, respectively, relative to individuals without FVL mutation [
19]. Notably, FVL appears to be relatively rare in Asian populations, particularly in the Chinese population [
20]. However, given the tremendous risk of thrombosis associated with FVL mutation, the assessment of FVL genotypes was also included in this study. In addition, there was a report claiming that FVL may interact with the PAI-1 4G/5G polymorphism to determine the risk of recurrence of VTE in a Swedish population [
21]. On this basis, the roles of the PAI-1 4G/5G and FVL polymorphisms in Chinese VTE patients were desired to be explored in this study.
Thus, we evaluated the accuracy of TaqMan-minor groove binder (MGB) reverse transcription-polymerase chain reaction (RT-PCR) method for detecting PAI-1 4G/5G and FVL polymorphism using sequencing method as the gold standard. Besides, we also provided a new perspective on the potential utility of the PAI-1 4G/5G polymorphism in relation to VTE risk, treatment efficacy and recurrence status in Chinese population.
Discussion
With the development of genomics technology, the role of gene single nucleotide polymorphisms (SNPs) in the pathogenesis of disease is gradually being appreciated. Growing studies have highlighted the association of SNPs with disease susceptibility and prognosis, which is of great significance for achieving individualized medicine. VTE is closely involved with hypercoagulable status of the blood, in which genetic factors cannot be ignored. In this context, the present study focused on the association of the PAI-1 4G/5G polymorphism with VTE susceptibility, treatment efficacy and recurrence status in Chinese populations.
PAI-1 4G/5G polymorphisms have been reported in numerous studies, but there are a variety of methods for determining genotypes, including allele-specific PCR [
25], TaqMan PCR [
26], PCR-restriction fragment length polymorphism [
27]. However, most studies have not evaluated the methodology. To ascertain the accuracy of genotype identification for FVL and PAI-1 4G/5G polymorphisms, all subjects were genotyped using sequencing (gold standard) and TaqMan-MGB RT-PCR in this study. The results showed that TaqMan-MGB RT-PCR method was highly accurate in identifying FVL and PAI-1 genotypes, consistent with the results of the sequencing method, and was not disturbed by jaundice and lipids in the samples. The TaqMan-MGB RT-PCR technique has been available for decades and is growing in development [
28]. It is worth noting that the major advantages of the TaqMan-MGB probe are that it forms a stable binding to the target gene and suppresses the production of non-specific amplification products [
29,
30]. Recently, TaqMan-MGB RT-PCR has been used to detect drug resistance in
Helicobacter pylori [
31], subtype analysis of swine influenza virus [
32], mutation analysis of hereditary optic neuropathy [
33] and SNPs detection in diabetes susceptibility genes [
34]. Given that there are currently no standardized detection procedures for FVL and PAI-1 genotypes in China, our study may lay the foundation for the practical application of the TaqMan-MGB RT-PCR method in the future.
Factor V Leiden is an inherited condition that causes resistance to the anticoagulant effect of APC, which in turn increases the risk of venous thrombosis [
35]. Previous research has demonstrated that FVL SNP variant (rs6025) is highly associated with the risk of VTE by using Genome Wide Association Studies (GWAS) [
36]. Nevertheless, no FVL mutation was identified in all subjects in this study. Our results were consistent with the report by Wang et al. [
24], implying that FVL mutation was relatively rare in Chinese population. Ethnicity appears to be an essential influence on the frequency of FVL mutation. According to a previous multiracial survey in the United States, the frequency of FVL mutation was highest in Hispanic-Americans at 1.65% and slightly lower in African-Americans at 0.87%, while no mutations were observed in Asian-Americans or Native-Americans [
37].
Impairment of the fibrinolytic system plays an influential contribution in the progression of thrombotic disease. PAI-1, as the inhibitor of plasminogen, has been extensively studied. On the other hand, the role of its genetic polymorphisms in thrombotic disease has been of increasing interest. Unfortunately, the association of the PAI-1 4G/5G polymorphism with VTE susceptibility remains controversial. Many studies have proved that the PAI-1 4G/5G polymorphism was not significantly associated with VTE, while significant associations were discovered in several other studies [
38]. A recent meta-analysis has shown that the PAI-1 4G/5G polymorphism was associated with an increased risk of VTE, especially in Asian populations [
13]. Paradoxically, no obvious correlation of the PAI-1 4G/5G polymorphism with VTE was observed in our study by using five genetic models (allele, genotype, dominant, recessive and additive). The patient's underlying disease and risk factors may be vital points in explaining this discrepancy. Tsantes et al. reported that the association of the PAI-1 4G/5G polymorphism with VTE was evident in patients with genetic risk factors (such as family history of hereditary diseases), whereas this association was no longer elucidated in patients (such as antiphospholipid antibody syndrome, Bechet disease) which had no genetic risk factors [
39]. In our study, there were no explicit genetic risk factors in the majority of cases, and the venous thrombosis developed secondary to tumors or autoimmune diseases. In addition to this, the limited sample size and the differences in ethnicity in various regions of China were not negligible elements. Therefore, future multicenter studies could contribute to a more profound understanding of the effect of the PAI-1 4G/5G polymorphism in the susceptibility to VTE in Chinese patients.
To further explore the laboratory characteristics of VTE patients with different genotypes of PAI-1, we analyzed the heterogeneity of hematological markers. Most laboratory parameters do not differ among patients with various genotypes. Patients carrying the 4G/4G genotype had lower RBC than those with the 4G/5G genotype. Considering the potential effect of gender on RBC counts, we re-evaluated this difference by performing multiple regression analysis adjusted for age and gender. It is interesting to note that this difference is no longer significant through the multiple regression analysis. Considering the limited sample size of this study, it would be desirable to conduct regional collaborative studies to clarify the effect of PAI-1 genotypes on RBC counts.
Additionally, we also observed that individuals with the 4G/5G genotype had lower neutrophil counts and NLR than the 5G/5G genotype while lymphocyte counts and PLR did not change significantly. The interaction of inflammation and coagulation suggested the subtle role of neutrophils in thrombosis. Kushnir et al. reported that persistent neutrophilia was a marker for non-malignancy, non-infected VTE patients [
40]. Mechanistically, the neutrophil-derived enzymes may inhibit anticoagulant factors such as APC, antithrombin and tissue factor pathway inhibitor [
41]. Furthermore, the neutrophil serine proteases and extracellular nucleosomes can enhance tissue factor- and factor XII-dependent coagulation pathways [
42]. The 5G/5G genotype carriers have higher levels of neutrophils perhaps related to the fact that most of them are tumor patients, whereas tumors are thought to be closely associated with the activation of the coagulation system and the formation of thrombus. The role of neutrophils in the PAI-1 4G/5G polymorphism deserves further investigation to elucidate the complex involvement of blood cells in the coagulation, fibrinolytic and inflammatory pathways.
Currently, the management of VTE is mainly dependent on anticoagulation therapy. Nevertheless, few studies have reported the association between the PAI-1 4G/5G polymorphism and treatment efficacy. In this study, we found that the patients with the 5G/5G genotype were more likely to achieve complete recanalization compared to the 4G/4G genotype, hinting that individual with the 5G/5G genotype may be able to benefit more from treatment. Similarly, Fernandez-Cadenas et al. have reported that patients with the 4G/4G genotype had higher rates of re-occlusion compared to patients with other genotypes, heralding poor prognosis after thrombolytic therapy in patients with ischemic stroke [
43]. Several studies have illustrated that the PAI-1 4G/5G polymorphism can affect the expression of PAI-1, with the 4G/4G genotype being the most highly abundant and the 5G/5G genotype being the least abundant [
44,
45]. As the inhibitor of plasmin formation, high concentrations of PAI-1 may contribute to the deposition of fibrin in the vessel rather than being lysed, thus preventing complete recanalization. Another interesting finding was that individuals with the 4G/5G genotype had a reduction in D-dimer levels after treatment. D-dimer has been extensively studied as the degradation product of fibrin formation resulting from the dissolution of thrombi by the fibrinolytic system [
46]. Therefore, the decrease in D-dimer may be indicative of effective treatment and thrombus lysis. Intriguingly, the D-dimer graph seems to show a decreasing trend after treatment as a whole in all genotypes. It is just that some cases are significantly increased in patients with the 4G/4G genotype and some increased in patients with the 5G/5G genotype. In this context, we have reviewed the medical records of individuals whose D-dimer did not decline after treatment. D-dimer is known to be a non-specific indicator that may be elevated in a variety of physiological and pathological conditions. We found the decline in D-dimer may be influenced by the patient's underlying diseases, the invasive nature of the treatment and the duration of bed rest.
There is a broad consensus on the critical nature of genetic factors in reoccurring thrombosis. As for the recurrent status of VTE, the PAI-1 4G/5G polymorphism also appears to be of potential utility. Our results indicated that individuals carrying the 5G/5G genotype were more likely to develop a recurrence-free status as compared to individuals with the 4G/4G or 4G/5G genotypes. The presence of the PAI-1 4G allele has been reported to increase the risk of thrombosis in patients with other thrombotic defects, such as protein C (PC) and protein S (PS) defects [
47]. Similarly, another study has shown that the PAI-1 4G allele was a risk factor for the development of PE in patients with PS deficiency [
48]. Thus, these studies may imply the association of the PAI-1 4G allele with the PC/PS complex deficiency. It is well known that PC, PS and phospholipids can form a complex that inactivates FVa which is considered as a pro-coagulation factor; therefore, defects in PC/PS are often closely linked to recurrent VTE [
49]. We hypothesized that the relapse vulnerability of individuals carrying the 4G allele might be associated with reduced PC/PS activity. Unfortunately, studies on the correlation between PAI-1 polymorphisms and PC or PS activity levels are still scarce. Taken together, it would be meaningful to explore the association of 4G alleles with PC/PS activity levels in patients with recurrent VTE in future studies. Interestingly, the complete recanalization rate was higher in the order 5G/5G, 4G/5G, 4G/4G, which seems to be related to PAI-1 4G/5G polymorphism and the amount of PAI-1 in the blood, whilst 4G/5G was more common than 4G/4G when looking at recurrence rate. we considered that the patient's general condition, underlying disease, lifestyle habits, genetic factors and economic situation may influence the patient's prognosis and explain the above discrepancy.
When comparing the differences in PAI-1 expression levels, we found that the VTE group had relatively higher concentrations of PAI-1 than HC group. Frischmuth et al. reported that higher plasma PAI-1 levels were associated with an increased risk of future incident VTE [
50]. Yang et al. suggested that plasma PAI-1 had a higher predictive value for VTE than D-dimer [
51]. Thus, our results together with the above reports, may imply that plasma PAI-1 may be a potential biomarker in the diagnosis of VTE. However, when we made group comparisons by PAI-1 genotypes, no differences in plasma PAI-1 antigen levels were observed among the different subgroups in either the VTE or HC groups. Theoretically, the 4G allele could bind to the transcriptional enhancer while the 5G allele binds to the transcriptional repressor thereby resulting in higher levels of PAI-1 expression in individuals carrying the 4G/4G genotype and lower levels of PAI-1 expression in individuals with the 5G/5G genotype [
13]. Nevertheless, the situation became quite diverse in terms of various diseases and populations. Chi et al. reported that individuals with the 4G/4G genotype had higher plasma PAI-1 levels relative to individuals with the 5G5G genotype in patients with severe burn sepsis [
52]. In another study, the 5G/5G genotype group showed lower levels of PAI-1 compared to the 4G/4G genotype group in obese women [
53]. In contrast, Sabino et al. found that young patients with ischemic stroke in Brazil had higher levels of PAI-1 compared with controls, but PAI-1 expression was not affected by the PAI-1 4G/5G polymorphism [
54]. Remarkably, a study including 113 patients with rheumatoid arthritis showed that individuals with the 4G/4G genotype had higher PAI-1 mRNA levels compared to the 4G/5G or 5G/5G genotype carriers, but plasma PAI-1 levels were not significantly different [
55]. Taken together, we hypothesize that PAI-1 may play an important role in the development of VTE, but its expression is not solely determined by the PAI-1 4G/5G polymorphism; factors such as patient condition, co-morbidity and ethnicity may also combine to regulate PAI-1 expression levels. Interestingly, in our study, the 4G/4G carriers appeared to have higher median PAI-1 expression levels whilst the 5G/5G carriers had lower median expression levels in the VTE group. Hence, larger sample sizes and regional comprehensive researches would be required in the future to clarify the effect of the PAI-1 4G/5G polymorphism on plasma PAI-1 expression levels in Chinese patients with VTE.
Nevertheless, there are some limitations to our study. The limited sample size necessitated caution in the interpretation of our results. However, compared to other studies, our study systematically compared the differences in the individual distribution, laboratory parameters, treatment efficacy and prognosis of the various genotypes, which could be beneficial for future applications. Future multi-center, large-scale, long dimensional studies are imperative to further delineate the role of PAI-1 4G/5G polymorphisms in VTE. Additionally, in exploring the association of the PAI-1 4G/5G polymorphism with PAI-1, we only examined the antigen expression levels of PAI-1 but not the activity of PAI-1. In the future, we would collect more samples to assess the level of PAI-1 antigen/activity in VTE patients with different PAI-1 genotypes and their changes between pre- and post-treatment. Moreover, only the roles of PAI-1 4G/5G in VTE susceptibility, treatment and prognosis were investigated in this study; other genetic mutations such as F2 G20210A and MTHFR C677T will be explored in the next step. Finally, incomplete information on some patients excluded from the study may lead to biased results.
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