Exposure to poly- and perfluoroalkyl substances and its association with the coagulation system: a mini-review

Introduction

Poly- and perfluoroalkyl substances (PFASs) are a large group of chemicals (>4000) in which carbon-hydrogen bonds are replaced, fully or in part, by carbon-fluorine bonds.¹ They can be found in everyday consumer products, such as food and food packaging, water, carpets, surfactants, textile coating, clothing, paint, paper, dental floss, cosmetic products, cookware coating, and fire-extinguishing foam. Human exposure occurs through the digestive system (or through breast milk), respiratory system, or skin. In humans, PFASs have been detected in serum, tissues, and urine.^1-4 Most of these substances are highly resistant to degradation and metabolism, and as persistent organic pollutants, they can accumulate in living organisms over long periods of time.^1,2,4 In effect, humans are most often exposed to a mixture of PFASs, making it difficult to attribute the ultimate adverse effects to a single substance. Chronic exposure to these substances was reported to be associated with certain malignancies, lower birth weight, sex hormone disruption, brain degeneration, and liver damage.^1-4 At the same time, metabolic changes, dyslipidemia, insulin resistance, and endothelial dysfunction were shown to contribute to cardiovascular complications.⁵ Coagulation disorders, particularly thrombosis, can cause acute coronary syndromes, stroke, pulmonary embolism, and peripheral ischemia. However, the direct effect of PFAS exposure on the coagulation system has not been extensively studied. Therefore, the aim of this paper was to review the available literature investigating the relationship between PFASs and coagulation.

Methods

Prior to the literature search, a Population, Exposure, Comparator, and Outcome (PECO) statement and a search protocol were developed. The PECO statement for this study is outlined in Table 1. Only original human studies and experimental animal studies were included in this review.

Table 1. Population, Exposure, Comparator, and Outcome statement

Population	Humans or animals exposed to a known PFAS
Exposure	Exposure to a PFAS through the digestive system, respiratory system, skin, or during experimental research
Comparator	Humans / animals exposed to lower (or none) levels of PFASs compared with highly exposed humans / animals
Outcome	Any effect on coagulation disorders (platelet count and function, clot formation, impaired fibrinolysis) not directly related to exposure to other substances or a health condition
Abbreviations: PFAS, poly- and perfluoroalkyl substance

The PubMed, Scopus, Embase, Trip Database, Global Health, Cochrane Library, and ProQuest Environmental databases were searched to identify relevant studies published between January 2017 and May 2024. Combinations of 2 key words were used during the search, including the chemical group name (ie, PFAS), and the effect on the coagulation system (ie, thrombosis, platelet function, blood clotting disorders, fibrinolysis, plasminogen activator inhibitor-1 [PAI-1], thrombin generation, fibrin clot structure, and blood clot properties).

Results

The search yielded only 96 records. Of those, 7 studies were ultimately included in the current review. After few meticulous screening of the retrieved full-text articles, some studies were excluded from analysis due to insufficient information about the impact of PFASs on the coagulation system and platelet function. Others were found ineligible due to a risk of bias or issues related to data extraction. In addition, some records were excluded during data screening. Finally, 6 human studies and 1 animal study were identified as potentially eligible for inclusion.

Platelet count and function

We identified only 1 study that provided direct evidence for the accumulation of various PFASs in platelets and their impact on increased platelet aggregation.⁶ It was a cross-sectional study combining in vitro and ex vivo analysis. The authors studied the effect of exposure to perfluorooctanoic acid (PFOA) on platelet function. Platelet aggregation was stimulated using the following agonists: thrombin receptor activator for peptide 6 (TRAP-6 ), arachidonic acid (ASPI), and adenosine diphosphate (ADP).

To confirm whether platelet aggregation was stimulated by PFOA, the authors evaluated the aggregation profile of blood samples from 48 male participants (mean [SD] age, 18.7 [0.6] years) who were chronically exposed to PFOA (mean [SD] serum level, 128 [48.5] ng/ml), and compared them with samples obtained from 30 controls (mean [SD] age, 22.1 [1.3] years) with low exposure to PFOA (mean [SD] serum level, 4.7 [2.1] ng/ml). Aggregometry results showed no abnormalities in either group. In addition, there were no significant differences in the ADP test values between the highly exposed and control participants. However, the highly exposed participants scored higher than the controls on the ASPI and TRAP-6 tests, which may suggest that exposure to PFOA leads to an increase in platelet aggregation.⁶

Through liquid chromatography coupled with triple quadrupole mass spectrometry, the authors demonstrated that platelet cell membrane was the primary site of PFOA accumulation.⁶ In order to assess the significance of this finding, they performed an in vitro analysis of platelets pretreated with 400 ng/ml of PFOA (a concentration observed in chronically exposed humans). Stimulation of PFOA-incubated platelets with TRAP-6 resulted in a greater increase in cytosolic calcium levels, as compared with the platelets not activated with PFOA. Free cytosolic calcium release plays an important role in the promotion of platelet activation, degranulation, and aggregation in response to soluble agonists and ligands of platelet adhesion receptors. In order to demonstrate the biological effects of the altered calcium signal, platelet degranulation was examined in both resting and stimulated conditions. The platelets incubated with 400 ng/ml of PFOA expressed the same amount of P-selectin on their surface as the platelets that were not activated with PFOA. Aggregation and cell-cell communication can be facilitated by P-selectin interacting with ligands produced in monocytes and endothelial cells.⁶

In an experimental in vitro study by Minuz et al,⁷ human platelets from healthy participants were incubated with C6O4 (2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoro- methoxy)-1,3-dioxolan-4-yl)oxy)-, ammonium salt) at different concentrations (1–500 ng/ml). The effects of C6O4 on platelet microparticle activation, production, phenotype, and aggregation under flow were investigated. C6O4 is a new-generation PFAS, often found in food contact packaging. Significant concentrations of C6O4 have also been found in rivers.⁷ In this study, pretreatment of platelet-rich plasma with 100–200 ng/ml of C6O4 significantly increased platelet aggregation under flow, as compared with both control conditions and added presence of acetylsalicylic acid. On turbidimetric evaluation, arachidonic acid, ADP, and collagen induced higher maximal aggregation when platelet-rich plasma was pretreated with 100–500 ng/ml of C6O4. Aggregation induced by the tested agonists was nearly suppressed by acetylsalicylic acid. Finally, pretreatment with C6O4 increased the number of platelet microparticles in resting conditions and in the presence of ADP or TRAP. Acetylsalicylic acid tended to reduce platelet microparticle generation.

In another study, Lin et al⁸ investigated the link between plasma levels of perfluorooctanesulfonic acid (PFOS) and increased carotid intima-media thickness. They assessed circulating endothelial and platelet microparticles in 848 participants aged 12–30 years. Circulating microparticles excreted with urine were assessed as a biomarker of platelet activation, and urinary 8-hydroxydeoxyguanosine levels were assessed as a surrogate marker of oxidative stress–derived DNA damage. Platelet-derived microparticles express the prothrombin complex on the surface, while endothelium-derived microparticles express the tissue factor. In addition, platelet-derived microvesicles express receptors, such as fibrinogen receptor, P-selectin, and von Willebrand factor receptor glycoprotein Ib, which enable interactions with the blood cells and endothelium.^9-11 Multiple linear regression analyses showed that the number of platelet- and endothelium-derived microparticles increased significantly across the quartiles of PFOS, and that their elevation correlated with an increased odds ratio of greater carotid intima-media thickness with higher serum PFOS levels. However, it is important to note that due to the small size of microparticles and their susceptibility to interference from other factors, the results should be interpreted with caution, as they may be affected by the presence of artifacts, sample preparation techniques, material insulation procedure, and analytical methods employed.¹²

Thrombogram, a component of the hemogram provided by automated analyzers, is a simple method for estimating platelet count and size. It also provides an indirect assessment of platelet function. Several studies explored the association between PFAS levels and thrombograms. In their cross-sectional study, Lin et al¹³ investigated the relationship between PFAS concentrations and complete thrombograms in a large cohort of 1779 Taiwanese individuals aged 12–64 years. The study population was divided based on the presence of coronary artery disease. Serum levels of 12 different PFASs were measured in all participants. However, there were 8 PFASs with serum levels 70% below the limit of detection. Therefore, only PFOA, PFOS, perfluorononanoic acid, and perfluoroundecanoic acid were included in the analysis. The median (interquartile range [IQR] PFOA level was 4.2 (5.8–8.9) ng/ml, and the median (IQR) PFOS level was 4.4 (4.1–8) ng/ml. A negative correlation was oserved between platelet count and the levels of all 4 PFASs in both study groups, and positive correlations were found between PFOS levels and platelet distribution width, mean platelet volume, and platelet-large cell ratio. Although there was no association between PFAS levels and platelet function, the authors reported that PFOS had a stronger relationship with thrombography results than other PFASs.¹³ Of note, the study had numerous limitations, as described by the authors. First, due to the cross-sectional design, causality could not be inferred. In addition, the authors did not account for several important potential confounders, such as comorbidities (cardiovascular disease, cerebral stroke, respiratory disease, chronic renal failure, gastrointestinal disorders, rheumatoid disease, diabetes, inflammatory disorders, and cancer), medication use, and exposure to other environmental chemicals that could affect platelet count, size, or function. Moreover, although the correlations between PFAS levels and platelet parameters were statistically significant, they did not reach clinical relevance.

Importantly, other studies did not confirm the relationship between exposure to PFASs and various platelet parameters. Olsen et al¹⁴ studied the relationship between occupational exposure of 263 workers with mean serum PFOS and PFOA levels of 910 ng/ml (range, 60–10060 ng/ml) and 1130 ng/ml (range, 40–12700 ng/ml), respectively. There was no significant correlation between PFOA or PFOS levels and platelet count. Similarly, Emmet et al¹⁵ assessed serum PFOA levels (median [IQR], 354 [181-571] ng/ml) in 371 participants from a United States community and did not find a significant correlation between the PFOA concentration and platelet count. However, the population size in these 2 studies was smaller than in the work by Lin et al.^13-15

In conclusion, moderate-quality evidence supports the hypothesis that platelet-dependent thrombus formation might be a determinant of an increased prevalence of cardiovascular events in individuals exposed to PFASs.

Fibrinolytic dysfunction

PAI-1 is a known marker of fibrosis, thrombosis, and increased cardiovascular risk. It plays a crucial role in the coagulation system, as it inhibits fibrinolysis in the circulation. Elevated PAI-1 levels can lead to thrombosis and atherosclerosis.¹⁶ However, data on the association between PFAS exposure and fibrinolytic disorders are scarce, with only 1 paper reporting such a relationship.

In their experimental study, Deng et al¹⁷ showed that acute exposure to a mixture of polychlorinated biphenyl (PCB)-126 and PFOS, and to each substance alone, resulted in increased plasma PAI-1 levels in mice. Liver injury was observed only in the mice exposed to the mixture of PFOS and PCB-126. Additionally, the mixture had a synergistic effect on serum PAI-1 levels and liver damage. Of note, the doses of PCB-126 (0.5 mg/kg) and PFOS (250 mg/kg) were intentionally higher than human environmental exposure levels. It is unknown whether similar findings would be observed if human PFAS concentrations were used. On the other hand, the half-lives of PFOS range from days (in mice)¹⁸ to years (in humans),¹⁹ and human exposure to these pollutants may increase due to slower elimination. Thus, it is possible that the effect in humans would be similar to that seen in mice.

General conclusions

Studies have shown that PFASs can accumulate in the lipid membrane of platelets and accelerate their aggregation. However, it is important to note that the occurrence and intensity of the analyzed health outcomes (eg, fibrinolytic dysfunction or platelet-dependent thrombus formation) is a result of individual predisposition as well as several independent factors, including PFAS exposure. In certain occupational groups, the exposure to PFASs is greater due to the presence of these substances in clothing and other products used by people from these groups. Available literature lacks information concerning the presence of PFASs in elements of building infrastructure and effects of this type of exposure on the coagulation system. The studies included in the present review focused primarily on 2 PFASs: PFOA and C6O4, and only one of them demonstrated a direct effect on selected platelet functions. In order to draw definitive conclusions, the finding has to be reproduced in future experiments. Currently available data are insufficient to confirm the effect of PFASs on other coagulation parameters. In addition, humans are continuously exposed to several thousand PFASs mixed with other chemicals that may act synergistically on the coagulation system, so further studies are needed to assess the effects of these PFASs and their mixtures. Finally, more research is needed to assess the effects of these substances on other coagulation parameters that may promote thrombosis (Figure 1).