Interference of C6O4 on platelet aggregation pathways: Cues on the new-generation of perfluoro-alkyl substance

Health concerns associated with the exposure to legacy perfluoro-alkyl substances (PFAS) led to the development of new-generation PFAS, such as C6O4. Here we investigated the possible effects of C6O4 on the platelet's activation profile, by incubating human platelets from healthy donors with C6O4 at different concentrations and evaluating the effects on activation, production and phenotype of platelets micro-particles (MPV) and aggregation under-flow. Based on the eventual platelet pro-aggregation profile detected, the preventive effect of acetylsalicylic acid (ASA) was also explored.

Adhesion-induced platelet aggregation of platelet rich plasma (PRP) under flow was evaluated on collagen-coated microchip at a shear stress of 10 Dyne. The turbidimetric method was used to investigate platelet aggregation. Finally, the in vitro generation of pro-coagulant MPV in PRP was evaluated by flow cytometry, as characterized by CD41 and annexin V positive events, under resting conditions and after stimulation with agonists at low shear stress.

The generation of platelet aggregates under flow was significantly increased by the pretreatment of PRP with 100-200 ng/mL C6O4, compared to both the control condition and the experiment performed in presence of ASA. Arachidonic acid (AA), ADP and collagen induced an higher maximal aggregation, at turbidimetric evaluation, when PRP was pretreated with 100-500 ng/mL C6O4. In addition, PRP stimulated with AA also showed a steeper slope of the aggregation curve. The aggregation induced by the tested agonists was almost abolished by ASA. Finally, pretreatment with C6O4 increased the number of MPV in resting conditions and in presence of ADP and TRAP. ASA tended to reduce MPV generation.

Exposure to C6O4 associates with an increased platelet response to agonists, translating into a possible increased risk of cardiovascular events. Pending a further clarification on the toxicokinetics of this compound, our results claim the possible prophylactic use of ASA.