Short-Term Exposure to Waterpipe/Hookah Smoke Triggers a Hyperactive Platelet Activation State and Increases the Risk of Thrombogenesis

Abstract

Objective: Cardiovascular disease is a major public health problem. Among cardiovascular disease's risk factors, tobacco smoking is considered the single most preventable cause of death, with thrombosis being the main mechanism of cardiovascular disease mortality in smokers. While tobacco smoking has been on the decline, the use of waterpipes/hookah has been rising, mainly due to the perception that they are less harmful than regular cigarettes. Strikingly, there are few studies on the negative effects of waterpipes on the cardiovascular system, and none regarding their direct contribution to thrombus formation. Approach and Results: We used a waterpipe whole-body exposure protocol that mimics real-life human exposure scenarios and investigated its effects, relative to clean air, on platelet function, hemostasis, and thrombogenesis. We found that waterpipe smoke (WPS)-exposed mice exhibited both shortened thrombus occlusion and bleeding times. Further, our results show that platelets from WPS-exposed mice are hyperactive, with enhanced agonist-induced aggregation, dense and α-granule secretion, αIIbβ3 integrin activation, phosphatidylserine expression, and platelet spreading, when compared with clean air-exposed platelets. Finally, at the molecular level, it was found that Akt (protein kinase B) and ERK (extracellular signal-regulated kinases) phosphorylation are enhanced in the WPS and in nicotine-treated platelets.

Conclusions: Our findings demonstrate that WPS exposure directly modulates hemostasis and increases the risk of thrombosis and that this is mediated, in part, via a state of platelet hyperactivity. The negative health impact of WPS/hookah, therefore, should not be underestimated. Moreover, this study should also help in raising public awareness of the toxic effects of waterpipe/hookah.

Keywords: hookah; platelets; smokers; thrombosis; tobacco smoking; waterpipe.

Figure 1:

WPS exposure results in systemic delivery of nicotine (cotinine) and shortens the bleeding time in the tail bleeding time assay, and the time to occlusion in the ferric chloride in vivo thrombosis model. (A) The concentrations of cotinine were measured in serum from WPS and clean air exposed mice. Each bar represents the mean ± SD (n = 10; ****P<0.0001). (B) This data illustrates the results of tail bleeding time assay (as described in the “Methods”) comparing WPS and clean air exposed mice. Each point represents the tail bleeding time of a single animal (clean air, n=7; and WTS, n=7; ***P<0.001). (C) This data illustrates the results of the ferric chloride–induced thrombosis model (as described in the “Methods”) comparing WPS and clean air exposed mice time to occlusion. Each point represents the occlusion time of a single animal (clean air, n=7; and WPS, n=7; ***P<0.001). WPS indicates Waterpipe Smoke.

Figure 2:

Platelet aggregation and dense granule secretion are enhanced in WPS exposed mice, using two different brands. Platelets from WPS (two separate brands) and clean air exposed mice were stimulated with 0.1 U/ml thrombin, or 5 μmol/L ADP, before their aggregation (A-D) and dense granule secretion (E-H) responses were measured in a lumi-aggregometer. Platelets were incubated with luciferase/luciferin (12.5 μL) for the dense granules measurements. The experiment was repeated 3 times, with blood pooled from at least 8 mice each time (*P <0.05; **P <0.01). WPS indicates Waterpipe Smoke.

Figure 3:

Platelets alpha granule secretion and integrin GPIIb-IIIa activation are increased in WPS exposed mice. Platelets from WPS and clean air exposed mice were prepared and washed. (A, B) Platelets were incubated with fluorescein isothiocyanate–conjugated CD62P antibody (for α granules), and the fluorescent intensities were measured by flow cytometry after stimulation with 0.1 U/ml thrombin or 5 μmol/L ADP. (C, D) Platelets were incubated with fluorescein isothiocyanate–conjugated JON/A antibody, and the fluorescent intensities were measured by flow cytometry after stimulation with 0.1 U/ml thrombin or 5 μmol/L ADP. Average mean fluorescence intensities shown (****P < 0.0001; NS. nonsignificant). Each experiment was repeated 3 times, with blood pooled from at least 8 mice each time. WPS indicates Waterpipe Smoke.

Figure 4:

Platelet phosphatidylserine (PS) exposure is enhanced in WPS exposed mice. Platelets from WPS and clean air exposed mice were prepared and washed. Platelets were incubated with fluorescein isothiocyanate-conjugated Annexin V antibody and the fluorescent intensities were measured by flow cytometry after stimulation with 0.1 U/ml thrombin (A) or 5 μmol/L ADP (B). Average mean fluorescence intensities shown (****P < 0.0001; NS. nonsignificant). Each experiment was repeated 3 times, with blood pooled from at least 8 mice each time. WPS indicates Waterpipe Smoke.

Figure 5:

Platelet spreading is enhanced in WPS exposed mice. Platelets from WPS and clean air exposed mice were allowed to adhere to fibrinogen-coated coverslips for 5 minutes after stimulation with thrombin (0.1 U/mL). (A) Tetramethylrhodamine-conjugated phalloidin was used to stain for F-actin and imaged using Leica DMi8 inverted widefield fluorescence microscope with integrated high precision focus drive. Images were processed using LAS X Wizard imaging software. (B) Quantification of the spreading data. Data are representative of 3 independent experiments. Each experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time. WPS indicates Waterpipe Smoke.

Figure 6:

Platelet Akt and ERK activation (phosphorylation) are enhanced in WPS exposed and nicotine treated mice. Platelets from WPS and clean air exposed mice as well as from nicotine and vehicle injected mice were prepared and washed. Platelets were stimulated with 0.1 U/ml thrombin (A) or 5 μmol/L ADP (C) for 3 minutes, and proteins were lysed using 1X sample buffer. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis before being subjected to immunoblotting with anti-Akt, anti-pAkt (Ser473), anti-ERK, and anti-pERK antibodies. (B. D) Data quantification of agonist-induced ERK and Akt phospgorylation (****P < 0.0001; ***P < 0.001; NS. nonsignificant). Each experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time. WPS indicates Waterpipe Smoke.

Figure 7:

WPS does not affect plasma fibrinogen or protein expression levels of GPIIb-IIIa, PAR4 or p85 (PI3K), whereas continine enhances aggregation and dense granule secretion. Plasma was collected from WPS and clean air controls before the levels of fibrinogen were measured (A); data is presented as mean ± SD (n = 5). Mice were injected with 1 μM cotinine or vehicle, once daily for 1 week before platelets were harvested, and stimulated with thrombin (0.1 U/ml), and aggregation (B) and ATP release (C) were monitored using Lumi-aggregometer. (D) Platelets from WPS and clean air exposed mice were prepared and washed. Proteins were lysed using 1X sample buffer, and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis before being subjected to immunoblotting with anti-GPIIb-IIIa (αIIbβ3), anti-PAR4 or anti-p85/PI3K antibodies; (E) data quantification of GPIIb-IIIa,PAR4 and p85/PI3K expression; NS: nonsignificant. Each experiment was repeated at least 3 times, with blood pooled from a group of 8 mice each time. WPS indicates Waterpipe Smoke.