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The interpretations below are provided by Donna Castellone, MS, MT (ASCP) SH for Aniara Diagnostica, Inc.

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INTRODUCTION

Microplastics were defined in 2004 when the accumulation of plastic fragments and fibers were found in marine environments.1 Exposure to microplastics particles (MP) less than 5mm in size has become a significant problem present in society.² Plastic production increased from 1.5 million tons to 390 million tons from 1950-2021, with utilization rising 20 times higher in the past 50 years.³ At this rate the ratio of trash to fish will be 1:3 by 2050, meaning there will be more plastic than fish in the ocean.¹

MP can be classified as primary in that they occur as a byproduct of manufactured plastics or secondary and formed by mechanical or photochemical degradation of larger plastics in the environment.² Exposure occurs through inhalation, ingestion and skin contact. They are found in food, water and even things like teabags and food packaging.³

A study in 2023 in the UK found that the level of microplastics in humans is about 74,000 – 121,000 particles per person per year via inhalation and ingestion. When identified via blood tests polyethylene terephthalate and polystyrene were found to be the most common plastics.⁴

MPs have been investigated in various human biological samples, including colectomy specimens, saliva, sputum, lungs, liver, breast milk, and feces. The detection of MPs in systemic tissues suggests that MPs may also be detected in the bloodstream, which supplies the entire body. Recent studies have suggested that exposure to MP could be associated with coagulation function and cardiovascular events.¹

COAGULATION AND MICROPLASTICS

What is the impact of micro/nanoplastics entering the blood stream and the physiological impact on coagulation. The most common surface modifications of polystyrene explored include non-functionalized polystyrene (nPS), aminated polystyrene (aPS), and carboxylated polystyrene (cPS). Research is being conducted using purified coagulation factors, human plasma, computational models, human whole blood, and in-vivo animal models of thrombosis. However, a lack of consensus exists regarding the type of particles, surface modification and shape and their impact on clotting.²

THROMBOELASTOGRAPHY (TEG):

Thromboelastography (TEG) uses a whole blood system which enables the study of the complexity of in-vivo hemostasis/thrombosis cellular (ex. red blood cells, white blood cells, and platelets) and protein components using a shear environment. Using TEG analysis eliminates the possible complexities that endothelial cells may introduce. TEG was used to looking at clotting in the samples including parameters of R-time (clotting cascade activation), K-time (time to initial fibrin polymerization), Maximum Amplitude (MA; clot strength), and Angle (rate of fibrin polymerization). Additional TEG parameters collected included Time to Maximum Amplitude, Clotting Index, and G. ²

TEG enabled testing of polystyrene particle surface modifications, sizes, and particle concentrations. The study collected blood samples from 3 healthy volunteers. Three groups of microparticles (MP) were explored – aminated PS (aPS), carboxylated PS (cPS), and non-functionalized PS (nPS). Both nPS and cPS were acquired in 500, 100, and 50 nm sizes, while aPS was only manufactured in 500 and 100 nm sizes. Concentrations in WB included 25, 100, and 250 μg/ml. Samples were run for 60 minutes, run four times and performed over 3 days. Each day was dedicated to testing MP of a particular size across all concentrations and surface modifications.²

Results show that cPS consistently activated the clotting cascade, demonstrating increased fibrin polymerization rates, and enhanced clot strength in a size and concentration-dependent manner. The generation of clots were at or above baselines.² cPS operates via activation of the intrinsic pathway as an activator for FXII, kallikrein and HMWK to generate activated factor XII.⁵ It is believed that the ability of cPS to behave as an activator is dependent on the geometries of the particles, in particular the surface curvature with procoagulant effect seen with particles of at least 50 mm in size. Platelet activation has also been demonstrated as seen by the high MA readouts on TEG relating to clot strength.² Another possibility is that the cPS negatively charged surface could also enable the prothrombinase complex (factors Xa, Va, and prothrombin) could effectively assemble and activate thrombin since individual components have an affinity for negatively charged surfaces.⁶

nPS had minimal effects on clotting dynamics except for 50  nm particles at the lowest concentration. This size impacted the speed of the cascade activation, fibrin deposits and clot strength, but effects tapered off at larger sizes and higher concentrations of nPS. The exact mechanism for this is not known.² The clotting effects of aPS (100  nm particles) resembled those of cPS but were diminished in the 500 nm aPS group. Studies have shown that these aPS particles and those with similar surface chemistries are able to increase rates of thrombosis as well a promote increased platelet aggregation and activation.⁷

Limitations to the study include the limited sample size of healthy donors that do not capture the diversity of microplastics that humans are exposed to. Additionally, TEG displays overall clotting and platelet activity is limited in that is doesn't look at the coagulation factors, platelet behavior or clot microarchitecture as well as the physiological clotting that occurs in the presence of shear.¹

COAGULATION AND INFLAMMATORY FACTORS:

MP can impact coagulation by increasing inflammatory and coagulation markers. These can contribute to the buildup of fatty plaques and increase the risk of blood clots, MI and strokes. Caroxylated polystyrene particles (cPS) can activate the clotting cascade increasing fibrin polymerization rates and clot strength. MP have been found in blood clots from stroke, MI and DVT patients with the level of MP correlating with the severity of the stroke.¹

A study was conducted in 36 healthy Korean adults (20-60 years and 72% female). Questionnaires included plastic-related lifestyle factors such as physical activity, consumption of seafood, and usage of plastic food containers. MP were analyzers using Fourier-transform infrared (µ-FTIR) spectroscopy. Coagulation and inflammatory markers in blood samples were analyzed, including C-reactive protein, prothrombin time, activated partial prothrombin time (aPTT), antithrombin III, platelet count, erythrocyte sedimentation rate, and fibrinogen.¹

Assessing the amount of MP exposure is challenging. Analysis was performed by µ-FTIR which uses infrared (IR) images and data analyzed using software that could detect MPs. It compares the IR spectrum versus a reference and compares spectra from polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polyvinylchloride (PVC), polyurethane (PU), polyamide (PA), poly(methyl methacrylate) (PMMA), and polycarbonate (PC). They were then classified into groups. Samples with a major-to-minor axis ratio greater than 3 were classified as fibers, while the remaining samples were classified as fragments.¹

Using µ-FTIR MP were detected in 88.9% of participants (32/36) Using another method known as µ-Raman, MPs were found in two additional samples. Particles sizes ranged from 20-50 μm were found to be higher in subjects with higher education levels. This also correlated with altered coagulability. Higher MP (≥ 3/ml) had a longer aPTT and a positive associate with hsCRP and fibrinogen.¹ Animal studies showed that increasing PS MPs in plasma increased PT as well as clotting factors VII, IX, VIII, XI, protein S and C. MP could also impact the liver which is the organ mostly responsible for producing clotting factors, decreased liver function could also be associated with the presence of MP. MP and elevated platelet counts were significantly associated. The chemical composition of MP could affect the balance of the coagulation pathway. PS nanoparticles could induce thrombin generation, but amine-modified nanoparticles could decrease thrombin formation.⁸ While a study of 1482 pregnant women showed that urinary phthalate metabolites were associated with a prolonged aPTT.⁹

Atherosclerosis could be a result of chronic inflammation and promote lipid core formation. A study of 257 patients who had carotid endarterectomy found that 58.4% of patients who presented with MPs in plaques had a higher risk of CV events than those without. An additional study of 82 ACS patients found that those who had higher MP also had inflammation related cytokines (IL-6 and IL12p70).¹

Limitations of this study were samples were specifically recruited, there may have been samples that were contaminated and MP that were smaller than 5–20 μm could not be detected by µ-FTIR.¹ Higher MP levels are associated with longer aPTTs and lower AT levels which indicate impaired blood clotting. Higher CRP levels are present suggesting that these trigger inflammatory responses. Further research is needed to investigate the health effects of MP in cardiovascular and blood coagulation.¹⁰

CONCLUSIONS:

Studying microplastics is difficult due to the challenges with isolating them and testing them in particular for the cardiovascular system and thrombosis/hemostasis. Conclusions are from in-vitro and a few in-vivo studies. Little is known about the interplay between microplastics and human health from a coagulation point of view. However, a correlation between the presence of MP and inflammation has been determined, which can impact coagulation factors as well as platelets. It is important to be aware of this phenomenon that looks to be an ongoing issue and may further complication diagnosis of coagulopathies.

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REFERENCES:

1. Dong-Wook Lee, Jaehak Jung, Seul-ah Park, Yunjeong Lee, Juyang Kim, Changwoo Han, Hwan-Cheol Kim, Joon Hee Lee & Yun-Chul Hong , Microplastic particles in human blood and their association with coagulation markers Scientific Reports volume 14, (2024) December
2. Alexei Christodoulides 1, Abigail Hall 1, Nathan J Alves 1,2,*, Exploring microplastic impact on whole blood clotting dynamics utilizing thromboelastography, Front Public Health, 2023 Jul 13;11:1215817 https://pmc.ncbi.nlm.nih.gov/articles/PMC10372794/
3. Study uncovers the grave impact of microplastics on blood clotting TOI Lifestyle Desk / etimes.in / Dec 10, 2024, 23:36 IST https://timesofindia.indiatimes.com/life-style/health-fitness/health-news/study-uncovers-the-grave-impact-of-microplastics-on-blood-clotting/articleshow/116183945.cms
4. Leslie HA, van Velzen MJM, Brandsma SH, Vethaak AD, Garcia-Vallejo JJ, Lamoree MH. Discovery and quantification of plastic particle pollution in human blood. Environ Int. (2022) 163:107199.
5. Sanfins E, Augustsson C, Dahlbäck B, Linse S, Cedervall T. Size-dependent effects of nanoparticles on enzymes in the blood coagulation cascade. Nano Lett. (2014) 14:4736–44.
6. Qureshi SH, Yang L, Manithody C, Rezaie AR. Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex. Biochemistry. (2009) 48:5034–41.
7. 7Dobrovolskaia MA, Patri AK, Simak J, Hall JB, Semberova J, de Paoli Lacerda SH, et al. Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro. Mol Pharm. (2012) 9:382–93.
8. Ali, N. et al. The potential impacts of micro-and-nano plastics on various organ systems in humans. EBioMedicine. 99 (2024)
9. Jiang, M. et al. Urinary concentrations of phthalate metabolites associated with changes in clinical hemostatic and hematologic parameters in pregnant women. Environ. Int. 120, 34–42 (2018).
10. Lee, D.-W. et al. (2024). "Microplastic particles in human blood and their association with coagulation markers." Nature

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