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Interference of C604 on platelet aggregation pathways


Perfluoroalkyl substances (PFAS) are synthetic compounds with surfactant properties, which you can find in many products like fire-extinguisher foams, soil-extraction additives, household detergents, and films, waterproof clothing, and coatings for cookware.

These compounds accumulate in the environment and humans, causing potential health issues. People living in nearby production plants of long-chain legacy PFAS such as perfluoro-octane sulfonic acid (PFOS) and perfluoro-octanoic acid (PFOA) are more likely to develop reproductive, metabolic, and cardiovascular diseases. The risks of exposure to short-chain PFAS are unknown.

Due to these concerns, the manufacturers discontinued PFOS and PFOA production. The fluorinated compound acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro 5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy)-, ammonium salt (1:1), C6O4, could be a suitable substitute since it has lower environmental and tissue persistence due to its novel cycle di-ether structure.

However, reports have detected C604 in water and workers’ blood samples. So, researchers at the University of Padova, Italy, investigated the effects of C604 on platelet activation profile.


Platelet adhesion and aggregation under flow

The researchers performed microfluidic experiments using a Microfluidic Cellix Platform composed of a pump, an optic microscope equipped with a fluorescence microscopy camera, and Vena8 Fluoro+ biochips (Cellix).

First, they coated the biochip channels with collagen overnight at 4 ◦C, blocked them with BSA, and washed them with saline solution. Then, they treated platelet-rich plasma (PRP) with increasing doses of C604 and diluted it with physiological saline solution to obtain a final concentration of 1x108/mL platelets. After that, they perfused each specimen through the channels with a shear stress of 10 dyne/cm2 for 3 min.

They also performed experiments with PRP pre-incubated with ASA (100 umol/L) to block cyclooxygenase-1 activity and prevent Thromboxane A2 (TXA2) generation.

After the perfusion, they took 20 images for each experiment and analysed them with software. They calculated platelet adhesion, expressed as areas occupied by cell aggregates.


  • Platelets firmly adhering generate small aggregates upon the interaction and adherence to collagen fibers.

  • Pre-incubation with C604 100 ng/mL (+0.60 AU, 95% CI: +0.21 to +0.99 AU; P < 0.05 vs control) and 200 ng/mL (+0.66 AU, 95% CI: +0.15 to +1.17 AU; P < 0.05 vs control) significantly increased platelet aggregates (Fig 2.A).

  • Incubation of PRP with ASA 100 umol/L significantly reduced platelets adhesion and aggregation to immobilised collagen under flow when PRP was not pretreated with C6O4 (-0.40 AU, 95% CI: - 0.11 to - 0.71 AU; P < 0.05) and when it was treated with 100 ng/mL C6O4 (-0.60 AU, 95% CI: - 0.11 to 0- 1.08 AU; P < 0.05 or 200 ng/mL C6O4 (- 0.74 AU, 95% CI: - 0.06 to - 1.48 AU; P < 0.05). (Fig 2.B)

Fig. 2. Microfluidics analysis of platelet adhesion and aggregation (A). Representative images showing platelet adhesion to immobilized collagen under different experimental conditions and platelet thrombus formation. Flowing in collagen-coated microchannels adhering platelets recruit circulating platelets to generate large stable aggregates. Before the analysis, PRP was incubated for 30 min at 37 ◦C without or with 100 or 200 ng/mL C6O4, in the presence or absence of ASA 100 μmol/L. (B) Quantitative analysis of the area covered by adhering platelets under control conditions and in the presence of C6O4 with and without acetylsalicylic acid (ASA). The ratio of collagen-adhering platelet aggregates generated from treated PRP to aggregates from untreated PRP flowing at the same time in two parallel microchannels are calculated. Data are presented as individual values normalized versus control conditions representing the platelet-covered area. Mean and standard deviation (Mean ± SD) are shown. One-way ANOVA followed by Tukey’s test as post hoc analysis were applied. Significance: *=P < 0.05.

Other experiments in this study

Platelet aggregation and release of platelets-derived microvesicles

Using PRP from sodium citrate anticoagulated blood, the researchers measured platelet aggregation using a four-chamber aggregometer. As agonists, they used collagen, adenosine diphosphate (ADP), and arachidonic acid (AA).

The researchers also determined the in vitro release of pro-coagulant platelet-derived microvesicles (PMV) in PRP. The assay involved applying low intensity-shear stress to PRP, stimulated or not, with a platelet agonist.

Main findings of these experiments

  • Exposure to C604 accelerated platelet aggregation induced by AA.

  • C604 and ASA inhibited platelet aggregation induced by AA.

  • C604 also altered platelet aggregation induced by ADP and collagen.

  • All the tested agonists increased in vitro PMV generation, particularly AA and collagen.

  • Pre-treatment of PRP with C604 increased PMV from resting PRP without platelet agonists.

  • There was no difference in PMV release after stimulation with AA between platelets exposed to C6O4 compared to the naïve sample. On the other hand, there was a difference for platelets stimulated with ADP.

  • ASA reduced MPV generation in all tested conditions.

This study showed that C604 alters the cell's major biophysical properties, such as its fluidity and electrostatic charge distribution, with detrimental effects on platelet aggregation profile and platelet micro-vesicle release.

For more details about these experiments, access the complete study here.

How to get started?

Would you like to run similar experiments in your lab? This is the minimum experimental setup you’ll need:

  • Vena8 Fluoro+ biochips – to mimic human blood vessels and model blood clots.

  • Mirus Evo pump – to control flow rates in the biochip. You may set the shear rate to model thrombosis in microcapillaries or other vessels.

  • Microenvironmental chamber – a temperature-controlled frame that keeps the biochip at 370C. The microenvironmental chamber sits on the microscope stage.

  • Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.

  • Digital camera – to capture images and video recordings. We supply the Prime BSI Express with both the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.

  • Image Pro Cell Analysis software – for your image and video analysis.

If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. We have options that suit all budgets. You can check them out on our eShop.


Minuz, P., De Toni, L., Dall'Acqua, S., Di Nisio, A., Sabovic, I., Castelli, M., ... & Foresta, C. (2021). Interference of C6O4 on platelet aggregation pathways: Cues on the new-generation of perfluoro-alkyl substance. Environment International, 154, 106584.


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