MPO is a marker of the presence of NETs and cellular remnants of platelet-activating neutrophils, which are implicated in thrombotic processes.18 The formation of a thrombus and the inflammatory cascade involves biochemical pathways that intersect at different points and are indicated by many of their enzymes; few studies have implicated MPO as an inhibitor of pro-coagulant activity and with a neutral role.19,20 The activity levels differ between antioxidant enzymes; an increase in superoxide dismutase activity and decrease in glutathione peroxidase activity has been reported in patients with venous thrombosis.21
In a comparative analysis of the transcriptome of leukocytes from patients with thrombosis with and without post-thrombotic syndrome, a difference in gene expression was found and sequenced in 12 different genes, messenger RNA and non-coding RNAs.22
Different biomarkers, based on endothelial dysfunction, increased inflammatory activity, dysfunction in the coagulation cascade were tested and found differences in serum dosages of CRP, ICAM-1, and E-selectin that were increased in patients with post-thrombotic syndrome while MMP9 and MCP-1 were decreased.23
We hypothesized that an increase in MPO gene expression may also contribute to PTS sequelae owing to the presence of thrombus remnants, thereby causing a possible capture of NETs consistently over time; however, we cannot verify this hypothesis with our results. Although MPO level is elevated in pro-coagulant states, such as in cancer and pregnancy,24,25 its gene expression may not necessarily be increased, since this enzyme may be obtained from the degranulation of leukocytes and is present in NETs.
FLT4 gene expression was decreased in participants of both groups two and three. Studies have reported indications of lymphatic proliferation associated with arterial obstructions,24 as seen in a study comparing peri-coronary adipose tissue from infarcted hearts with non-coronary perivascular adipose tissue (internal mammary artery used for revascularization). A considerable increase in FLT4 mRNA expression has been previously observed (0.13 vs 0.07, p = 0.02).11
In an experimental study on different phases of angiogenesis, the VEGF C/Flt4/ERK axis has been shown to promote cell cycle arrest and the initiation of the sprouting phase. Accordingly, the decrease in FLT4 expression in the present study may be physiologically explained by the persistent formation of new vessels, especially in patients with PTS.25 This finding cannot be interpreted as a possible characteristic to compose a diagnostic panel as it would require a larger number of participants in the study to rule out type I statistical error.
The increased activity of F13A1 in patients with venous thrombosis has already been described; however, studies have not evaluated its role in PTS. Several factors must be considered, such as the presence of variability between samples due to polymorphisms, as well as possible protection against thromboembolism in patients with a Val34Leu polymorphism in the F13A1 gene.26 Genotyping was not performed for the samples in the present study.
The relationship between increased F13A1 activity and acute myocardial infarction in young patients was studied, and a possible risk factor for thrombosis was found.27 This corroborates the hypothesis suggested by our research in the sense that there is a possible higher activity of coagulation FXIII in more severe thrombosis as seen in enhanced F13A1 expression. An observational study on a cohort of 98 patients with coronavirus disease 2019 was recently published, in which a concentration considerably below the normal limit of Factor XIII protein was found. The authors attributed the decreased level to high consumption of coagulation factors; however, low synthesis or deficiency owing to immunological destruction was not completely ruled out.28 These observations suggest that the concentration of Factor XIII alone is not enough to clarify its action, and studies on the expression of the genes involved, such as F13A1, can provide complementary data.
The finding of new biomarkers will provide data for the construction of models for predicting and preventing post-thrombotic syndrome, as is already being built with technology using machine learning models aimed at post-thrombotic syndrome.29
However, our results cannot be interpreted as a biomarker diagnostic panel, as it would require a greater number of participants in the study to rule out type I statistical error. There are risks of bias inherent to cross-sectional studies and risks of selection bias besides inability to generalize the findings to different populations at this moment.
To the best of our knowledge, results related to increased expression of F13A1 in patients with PTS are obtained for the first time, despite the results were obtained using few samples. However, consistent and compatible statistical analyses were performed with small sample groups and normality test.
We aimed to find a relationship between increased expression of three different genes (MPO, FLT4, and F13A1); however, we found an increase in F13A1 gene expression and a decrease in FLT4 expression in patients with a Villalta’s score above 4 points (patients with PTS). However, despite including homogeneous groups, our sample had a reduced number of participants per group. Our research provides a proof of concept that contributes to the identification of biomarkers for PTS. Therefore, additional studies on the expression of MPO, FLT4, and F13A1 in patients with PTS are required to validate these results.
Patients with venous thrombosis may benefit from being warned about the possibility of developing post-thrombotic syndrome right at the beginning of the condition, preventing the worsening of conditions with venous hypertension accompanied by edema and ulcers in the lower limbs. Our research will move on to a more relevant next step, increasing the number of samples from each group. New studies focusing on the gene expression of F13A1 and Flt4 and the dosage of the proteins themselves are necessary to ascertain their real usefulness as a biomarker to be applied at the population level.