In Vitro and Ex Vivo Approaches to Evaluate Next-Generation Tobacco and Non-Tobacco Products on Human Blood Platelets

Abstract

Human blood platelets are major hemostatic regulators in the circulation and important in the mediation of chronic inflammation and immunomodulation. They are key elements that promote cardiovascular pathogenesis that leads to atherosclerosis, thrombosis, myocardial infarction, and stroke. New information on tobacco use and platelet dysregulation shows that these highly understudied vascular cells are dysregulated by tobacco smoke. Thus, platelet function studies should be an important consideration for the evaluation of existing and next-generation tobacco and non-tobacco products. Novel in vitro approaches are being sought to investigate these products and their influence on platelet function. Platelets are ideally suited for product assessment, as robust and novel in vitro translational methods are available to assess platelet function. Furthermore, the use of human biological systems has the advantage that risk predictions will better reflect the human condition.

Keywords: cardiovascular disease; in vitro testing; inflammation; nicotine; platelet; tobacco.

FIG. 1.

Hemostasis involves a balance of platelet activation. Too little platelet activation can lead to bleeding on disruption of vascular integrity. However, too much platelet activation can lead to thrombosis.

FIG. 2.

Platelet activation. Platelets are smooth discoid-shaped cells in their inactive state and undergo shape change on activation. Platelets can be activated by vessel wall damage and other traditional hemostatic stimuli, foreign toxicants, such as cigarette smoke, as well as by inflammatory signals and ensuing CVD. Platelet microvesicles are shed from the surface of activated platelets, and various mediators are additionally secreted in response to stimulation. CD40L, CD40 ligand; CD62P, P-selectin; CVD, cardiovascular disease; PDGF, platelet-derived growth factor; PF4, platelet factor 4; RANTES, regulated on activation, normal T cell express and secreted; TGF-β, transforming growth factor beta.

FIG. 3.

High-throughput in vitro platelet assays. Whole blood is collected from healthy human donors and centrifuged to obtain platelet-rich plasma. Either platelet-rich plasma or washed platelets are plated into a 96-well plate and treated with combinations of chemicals and activators. Cell-free supernatants can be analyzed for mediator release by immunoassay, and platelets can be evaluated for cell surface activation markers by flow cytometry.

FIG. 4.

Platelet dysregulation by tobacco products. Tobacco products can alter many aspects of platelet function, including hemostasis, inflammation, and immunity, and disease pathology.

FIG. 5.

Tobacco products alter human platelet spreading. Washed platelets freshly isolated from healthy human donors were treated with vehicle (media alone), 100 nM nicotine, or 6.5 U/mL cigarette smoke extract for 30 minutes, then were allowed to spread on fibrinogen-coated coverslips for 45 minutes. Coverslips were washed with PBS, then fixed with 4% paraformaldehyde, and visualized with DIC microscopy. Cigarette smoke extracts were generated in the University of Rochester Smoke Inhalation Facility using 3R4F reference cigarettes (University of Kentucky) and an automated cigarette smoking machine (Jaeger-Baumgartner). DIC, differential interference contrast.