OC 39.4 - Use of an in vitro platelet production approach to assess the effects of chronic cigarette and e-cigarette exposure on platelet generation and function.

Background: In recent years electronic cigarette use has become more prevalent, with people perceiving it as a risk-free smoking alternative. However, data suggest an association between E-cigarette usage and cardiovascular pathology, including platelet function. Epigenetic changes are possible responses to exposure, as we have shown for exposure to cigarette smoke leading to altered F2LR3 (PAR4) methylation. Current studies include human cohort studies or in vivo exposure systems in mouse models. In vitro however, longer-term exposure on platelets has not been possible to investigate. Our recently developed approaches to generate large numbers of platelets in vitro have enabled assessment of longer-term exposure.

Aims: To understand the effects of acute and longer-term exposure to extracts of E-cigarettes and cigarettes on platelet generation and function in vitro.

Methods: Haemopoietic stem cells were isolated from mice and cultured in the presence or absence of e-cigarette and cigarette extract. Platelets were generated and isolated by perfusion of megakaryocytes through a novel e x vivo heart-lung model system (https://www.biorxiv.org/content/10.1101/2021.11.01.466743v1). FACS-based analysis was used to monitor platelet generation and function (CD62P and activated IIb3 integrin).

Results: Acute exposure of ‘native’ mouse platelets leads to a pro-thrombotic phenotype, showing decreased EC50 values when compared to control P selectin mobilisation and integrin aIIbb3 activation upon stimulation with PAR4-activating peptide when compared to air infused. Importantly however, culture of megakaryocytes in the presence of e-cigarette extract had no effect upon platelet generation or function .

Conclusion(s): We conclude that acute exposure of platelets to cigarette and e-cigarette extract leads to a pro-thrombotic phenotype. However, culture of megakaryocytes in the presence of these extracts has no effect on platelet generation or functional responsiveness of generated platelets. The work highlights a novel technique that could be used for toxicological assessment of compounds or exposures on megakaryocytes their ability to generate functional platelets.