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Abstract

Introduction

It is well documented that cardiovascular disease (CVD) is the leading cause of death in the US and worldwide, with smoking being the most preventable cause. Additionally, most smokers die from thrombotic-based diseases, in which platelets play a major role. To this end, because of the proven harm of smoking, several novel tobacco products such as electronic(e)-waterpipe have been gaining popularity among different sectors of the population, partly due to their “false” safety claims. While many investigators have focused on the negative health effects of traditional cigarettes and e-cigarettes on the cardiovascular system, virtually little or nothing is known about e-waterpipes, which we investigated herein.

Methods and Materials

To investigate their occlusive CVD effects, we employed a whole-body mouse exposure model of e-waterpipe vape/smoke and exposed C57BL/6J male mice (starting at 7 weeks of age) for 1 month, with the controls exposed to clean air. Exposures took place seven times a week, according to the well-known Beirut protocol, which has been employed in many studies, as it mimics real-life waterpipe exposure scenarios; specifically, 171 puffs of 530 ml volume of the e-liquid at 2.6 s puff duration and 17 s puff interval.

Results

The e-waterpipe exposed mice had shortened bleeding and occlusion times, when compared to the clean air controls, indicating a prothrombotic phenotype. As for the mechanism underlying this phenotype, we found that e-waterpipe exposed platelets exhibited enhanced agonist-triggered aggregation and dense granule secretion. Also, flow cytometry analysis of surface markers of platelet activation showed that both P-selectin and integrin GPIIb-IIIa activation were enhanced in the e-waterpipe exposed platelets, relative to the controls. Finally, platelet spreading and Akt phosphorylation were also more pronounced in the exposed mice.

Conclusion

We document that e-waterpipe exposure does exert untoward effects in the context of thrombosis-based CVD, in part, via promoting platelet hyperreactivity.

Keywords

electronic waterpipe platelets thrombosis occlusive cardiovascular disease

Introduction

Risk factors for developing cardiovascular diseases (CVDs)—which refer to a group of diseases affecting both the heart and blood vessels—include high blood pressure, high cholesterol, diabetes, obesity, smoking, among others. CVD has been documented for years as a major cause of death in the United States (U.S.) and the world at large,¹ with smoking being a major contributor. In general, CVDs are associated with significantly high mortality and morbidity rates, representing about 17.9 million deaths in 2019, which accounts for 32% of the world's deaths.² This has culminated in huge costs, for example, approximately $216 billion in healthcare services.²

Although there has been a significant reduction in the rate of tobacco use in many countries, a rapid increase in population has led to an exponential rise in cigarette smokers globally, from 721 million in 1980 to 967 million in 2012.³ Given the well-known negative health effects associated with cigarette smoking, its popularity has decreased considerably among U.S. adults within the last several decades. This—at least in part—led the tobacco companies to develop/invent alternatives in the form of emerging tobacco-related products (ETRPs), which include electronic/e-cigarettes, e-waterpipes (also known as e-hookah), heat-not-burn devices, among others. These ETRPs are/were claimed to be “safe(r)” relative to traditional cigarettes.⁴ Although e-cigarette use is common with young people, waterpipe usage/waterpipe smoking remains a significant research focus as its use and popularity continue to rise in the US and beyond.⁵ For instance, in the U.S., it is estimated that approximately 10 million adults use traditional combustible waterpipes, of which about 1.4 million use it daily or some days. To this end, the youth (aged 18-24 years) show the highest rate of waterpipe smoking at an estimated rate of 12.2%, whereas 10% of college students reported their use, making waterpipe usage a major public health concern.^6,7 In addition, European and Eastern Mediterranean countries have the highest rate of waterpipe use, especially among teenagers, who smoke it more than adults.⁸ Its popularity is in part due to the misperception of its “safety.”⁶ Other reasons for this trend are the social acceptability, peer pressure, and youth curiosity.^7,9

It has been documented that a single waterpipe session may involve about 50-100 times smoke inhalation in comparison to traditional cigarettes.^10–12 Consequently, e-waterpipes were invented in 2014—in part as an improvement to traditional waterpipes⁶—with the main advertised advantage of providing an “emission-free” option to users.¹³ Hence, it is widely marketed that the e-waterpipes have an even more reduced health risk than the traditional waterpipes.¹⁴ These devices combine a traditional waterpipe bowl and a rechargeable e-head containing the e-liquid, which delivers flavored aerosols to the user, with or without nicotine.⁶ Importantly however, when this e-liquid is heated at very high temperatures, it undergoes thermal decomposition, which contributes to the toxicant profile associated with using e-waterpipes.⁴ This new “innovation”/Electronic Nicotine Delivery System (ENDS) is gradually becoming more and more common among the youth, and a major reason for this trend is that the e-liquids come in different yet appealing flavors.¹⁵ To this end, there is ample literature that traditional waterpipe use could negatively impact human health, including the cardiovascular system; for instance, we have previously shown that they modulate platelet reactivity and increase the risk of thrombus formation.¹⁶ However, studies in the context of e-waterpipes are very limited.¹³ Hence, the objective of this study is to investigate the impact of e-waterpipe exposure (for 1 month) on platelet function and the risk of thrombotic diseases.

Materials and Methods

Reagents and Materials

Thrombin, chrono-lume mixture, stir bars, and other disposables were purchased from Chronolog Corporation (Havertown, PA), whereas adenosine diphosphate (ADP) was from Sigma Aldrich (St Louis, MO). Fluorescein isothiocyanate/FITC-conjugated anti-P-selectin, Akt (protein kinase B), and phospho-Akt (Ser473) antibodies were purchased from Cell Signaling Technology, Inc (Danvers, MA). The phycoerythrin-conjugated GPIIb-IIIa (αIIbβ3) antibody was obtained from Emfret Analytics (Würzburg, Germany). The enzyme-linked immunosorbent assay (ELISA) cotinine detection kit was purchased from Calbiotech (El Cajon, CA). The e-liquid, e-head, and other e-waterpipe accessories were purchased from Smoking hookah (Houston, TX).

Animals

C57BL/6J (7-week-old male) mice were procured from the Jackson Laboratory (Bar Harbor, ME) and housed a maximum of 5 mice/ cage with unlimited access to food and water, under 12/12 light/dark cycles, at 24 °C. All mice experiments were approved by the Institutional Animal Care and Use Committee of Texas A&M University, College Station.

E-waterpipe Exposure Protocol

The e-waterpipe exposure protocol involved whole-body exposures within a chamber of 40 × 30 × 25 cm, L × W × H dimensions, and which is connected to a programmable machine, an e-waterpipe apparatus, and a rechargeable e-waterpipe head, as we previously described.¹⁷ The machine was programed to deliver, daily, for 1 month, 171 puffs of 530 mL volume, 2.6 s puff duration, and 17 s interpuff interval as per the Beirut method.^18–20 This protocol has been employed in various studies worldwide and is also popular among investigators because it mimics real-life waterpipe/e-waterpipe environments.^21–23 The e-liquid used for this study was the popular human brand known as Ecto and contained 24 mg of nicotine and a Double Apple flavor, along with other ingredients such as a propylene glycol (PG): vegetable glycerin (VG; 50:50) vehicle, citric acid, and artificial flavors. The e-head was set at a voltage of 6.0 V.

Cotinine Assay

Cotinine, a metabolite of nicotine, was quantified in both e-waterpipe and clean air-exposed mice, by using an ELISA Cotinine kit as described by the manufacturers.

Tail Bleeding Time

This was carried out as previously described.^24,25 Briefly, the tail of mice that were anesthetized using isoflurane was clipped 5 mm from the tip and placed in saline solution with a temperature of 37 °C. The time needed for the bleeding to stop was recorded.

In Vivo FeCl₃ Model for Thrombosis

The details have been previously described.^24,25 Mice were anesthetized with avertin, after which the left carotid artery was isolated, and blood flow established. Next, a filter paper saturated with 7.5% ferric chloride was used to induce artery injury, and the occlusion time recorded using a flow probe.

Peripheral Blood Cell/ Platelet Counts

The blood count, including platelets, was performed using a HEMAVET 950FS apparatus (Erba Diagnostics, Miami Lakes, FL).

Platelet-rich Plasma Preparation

Citrated blood was pooled together from each of the exposed and clean air group separately and centrifuged at 180 g for 11 min and platelet-rich plasma (PRP) collected. Platelets were counted with the HEMAVET.

Washed Platelet Preparation

Equal amounts of blood were mixed with Tyrodes buffer of pH 7.4 in a 2 ml Eppendorf tube. This mixture was centrifuged at 180 g for 7 min. PRP was recovered and centrifuged a second time at 400 g. The recovered pellets were mixed in 1 ml of HEPES Tyrodes, centrifuged, and platelets were counted.

In Vitro Platelet Aggregation

PRP from exposed and clean air mice was activated with the agonists collagen (1.25 µg/ml) or thrombin (0.1 U/ml). Aggregation was measured using a model 700 aggregometer, and experiments were repeated three times. The percentage maximum aggregation—which is the highest peak of the aggregation curve—was compared for both groups.

Dense Granule Release

Dense granule release was measured by adding 12.5 µL of luciferase mixture to PRP, before stimulation with collagen (1.25 µg/ml) or thrombin (0.1 U/ml). Release of ATP was measured using a model 700 aggregometer, and experiments were repeated three times.

Flow Cytometric Analysis

Briefly, washed platelets were prepared as described above and 1 µl of 1 mm Ca²⁺ was added to Eppendorf tubes in triplicates for both groups. Afterwards, washed platelets at a concentration of 100,000 platelets/µl were added and incubated with FITC-conjugated CD62P (P-selectin) or phycoerythrin-conjugated rat anti-mouse JON/A antibodies. Finally, platelets were stimulated with either the collagen-related peptide (CRP; 2.5 µg/ml) or thrombin (0.04 U/ml) and intensities measured using a BD Accuri C6 flow cytometer. Each experiment was repeated three times from pooled blood samples of five mice for each group.

Platelet Spreading

Briefly, platelets were incubated with fibrinogen-coated coverslips, stimulated with thrombin (0.005 U/mL) for 10 min, and immediately fixed with 4% v/v paraformaldehyde. They were then permeabilized with 0.2% of saponin in phosphate buffer. The coverslips were mounted on the microscopic glass slides containing a drop of cell mounting media (Vectashield containing Phalloidin with TRITC fluorophore) and sealed using a clear nail polish. Images were taken with an inverted microscope Nikon T1, and a Xyla 5.2 Andor camera was used to capture images. ImageJ software (Open-source NIH software) was used to process images, and Shape Index Map method was employed to generate images of polarized and polymerized actin.

Immunoblotting

Washed platelets were stimulated with ADP (2 µM) for 3 min, lysed, and their proteins separated by gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked using 4% bovine serum albumin before being probed with the primary antibodies (pAkt/tAkt) and visualized with horseradish peroxidase (either anti-rabbit or anti-mouse immunoglobulin G).

Statistical Analysis

GraphPad Prism Version 7 and/or Image J software was employed for all experiments. A normality test was performed before each analysis and based on this result, the Mann-Whitney test was used in bleeding and occlusion times experiments, whereas the differences in aggregation and secretion were analyzed using student's t-test. Flow cytometry data was analyzed using one-way ANOVA with Tukey's test; platelet spreading and immunoblotting assay were analyzed using Image J software. Experiments were performed at least three times each. Statistical significance was fixed at P < 0.05 for all analysis.

Results

E-waterpipe Exposure Does Not Affect Total Blood Cell Counts

We did not observe (Table 1) any detectable difference in the platelet count from e-waterpipe and clean air-exposed mice, which were 804.6 ± 72 and 875 ± 49.22 thousand/µl, for the clean air and e-waterpipe mice, respectively (P-value = 0.44). Likewise, no differences were detected/observed in other blood cells (Table 1).

Table 1.

Total Blood Cell Counts from Clean Air and E-Waterpipe Exposed Mice.

Cell type	Clean air	E-waterpipe	P-value
Platelets	804.6 ± 72	875 ± 49.22	0.44
Mean platelet value (MPV)	5.40 ± 0.07	5.44 ± 0.06	0.68
Red blood cells (RBCs)	6.82 ± 0.51	7.34 ± 0.37	0.44
Lymphocytes	122.20 ± 10.12	110.40 ± 3.31	0.30
Monocytes	33.71 ± 2.20	31.80 ± 1.19	0.47
Hematocrit (HCT)	34.36 ± 2.16	36.86 ± 1.90	0.41

Blood was collected by cardiopuncture and measured with a Hemavet Hematology Analyzer. Counts were expressed as thousands per microliter, whereas RBCs counts were expressed as millions per microliter. Data is presented as mean ± SEM.

E-waterpipe Exposure Delivers Cotinine to Exposed Mice

To validate our exposure conditions, cotinine, a metabolite of nicotine, was measured using an ELISA kit. A significant presence of cotinine was observed in the exposed mice (p = 0.0065), whereas—as expected—it was not detected in the clean air control animals (Figure 1a).

Figure 1.

Exposure to e-waterpipe results in the delivery of nicotine (cotinine) and shortens the bleeding and occlusion times. (a) Cotinine levels were measured in the serum of e-waterpipe and clean air-exposed mice (**P < 0.01). (b) The tail bleeding assay and the (c) ferric chloride-induced thrombosis model (as described in the Methods) comparing e-waterpipe and clean air-exposed mice. Each point shows the bleeding time (*P < 0.05) and the occlusion time (*P < 0.05) of a single animal.

E-waterpipe Exposure Modulates Hemostasis and Thrombosis in Mice

While it was previously shown that ETRPs, namely e-cigarettes have the capacity to increase the risk of thrombotic CVDs,²⁴ whether e-waterpipes produce similar effects has yet to be investigated. Our results showed that the tail bleeding time was shortened in the e-waterpipe exposed mice, compared to the clean air; specifically, 83.75 ± 23.71 versus 244.6 ± 44.2 s, respectively (Figure 1b), which indicates a prothrombotic phenotype. Next, we examined if these e-waterpipe exposed mice would be susceptible to thrombosis. Again, the e-waterpipe exposed mice exhibited a shortened occlusion time, compared to the control mice; specifically, 205.8 ± 28.22 versus 373.4 ± 51.02 s, respectively (Figure 1c). These data document—for the first time—that exposure to e-waterpipes indeed modifies hemostasis and thrombosis in mice.

E-waterpipe Exposure Enhances Agonist-induced Platelet Aggregation

Since our in vivo assays revealed that e-waterpipe exposure could modulate hemostasis and thrombosis, we next sought to understand the mechanism of the observed prothrombotic phenotype. Hence, we first assessed the aggregation response in both groups. Our results showed that platelets from mice exposed to e-waterpipes did exhibit enhanced platelet aggregation, relative to clean air, in response to collagen and thrombin stimulation (Figure 2a and b).

Figure 2.

Platelet aggregation and dense granule secretion are enhanced in the e-waterpipe exposed mice. Platelets were stimulated with either collagen or thrombin before their aggregation (a, b) and dense granule release (c, d) responses were measured with a lumi-aggregometer. Platelets were incubated with 12.5 µL of luciferase/luciferin for dense granules measurements. Experiments were repeated three times with blood pooled from five mice each time (*P < 0.05 and **P < 0.01). The % maximum aggregation is the % of aggregation indicated by the aggregometry system once the aggregation response reaches the highest level/maximum.

E-waterpipe Exposure Enhances Agonist-induced Platelet Secretion

Platelet activation is highly dependent on granule release through a process of exocytosis.^26,27 Given our previous demonstration that exposure to traditional waterpipes, and e-cigarettes enhances platelet dense and alpha granule secretion, and the close association between these smoking/vaping products and e-waterpipes, we investigated the latter's impact on these two platelet functional responses. Our results show that both ATP secretion (Figure 2c and d) and P-selectin expression (Figure 3a and b) were both enhanced in response to agonist stimulation as a result of e-waterpipe exposures, which further suggests a hyperactive platelet state.

Figure 3.

Platelet α-granule secretion is enhanced in the e-waterpipe exposed mice. Platelets were incubated with fluorescein isothiocyanate-conjugated CD62P antibody before stimulation with either the collagen-related peptide (CRP) (a) or thrombin (b). Fluorescence intensities were measured by flow cytometry analysis. Experiments were repeated three times with blood pooled from five mice each time (****P < 0.0001).

E-waterpipe Exposure Enhances Integrin GPIIb-IIIa/αIIbβ3 Activation

Platelet aggregation requires an increase in integrin αIIbβ3 expression,²⁶ hence, and in light of the enhanced platelet aggregation response, we sought to investigate if our whole-body e-waterpipe exposure enhances activation of this integrin. Our result showed that integrin activation was enhanced in the exposed mice in response to CRP or thrombin (Figure 4a and b).

Figure 4.

Platelet integrin αIIbβ3 activation is enhanced in the e-waterpipe exposed mice compared to control. Platelets were incubated with phycoerythrin-conjugated JON/A antibody before stimulation with CRP (a) or thrombin (b), and fluorescence intensities were measured. Experiments were repeated three times with blood pooled from five mice each time (****P < 0.0001).

E-waterpipe Exposure Enhances Platelet Spreading

Platelet spreading is a process where platelets recruited at the site of vascular injury flatten and increase in area by deforming the plasma membrane.²⁸ This involves increases in intracellular Ca²⁺, resulting in elevation of actin and myosin levels, and culminating in platelet adhesion.²⁹ Afterwards, there is the formation of filopodia and lamellipodia, which strengthen contact with other platelets.²⁹ The effect of e-waterpipe exposure on this response was investigated herein, and it was found that filopodia and lamellipodia formation are higher in the e-waterpipe exposed platelets, in response to thrombin (Figure 5a; data quantificaton is shown in b).

Figure 5.

Platelets spreading is enhanced in the e-waterpipe exposed mice compared. Platelets were placed on fibrinogen-coated coverslips before stimulation with thrombin. Platelets were fixed, and fluorescently labeled with phalloidin-FITC labeled and imaged using inverted microscope (Nikon Ti). Images were processed in ImageJ software and Shape Index Tool of ImageJ was used to observe polarized actin (a). (b) Quantification of activated platelets. Each scatter plot represents mean values ± STDEV of activated platelets counted per image (n = 30- images; ****P value < 0.0001).

E-waterpipe Exposure Enhances Akt Phosphorylation

The phosphorylation of Akt is known to be an important pathway for thrombus formation. Hence, we investigated if exposure to e-waterpipe would enhance Akt phosphorylation. Our results show that phosphorylation of Akt is more evident in the e-waterpipe exposed platelets when stimulated with ADP, providing biochemical evidence, and further validating their hyperactive platelets phenotype (Figure 6a and b).

Figure 6.

Platelet Akt phosphorylation is enhanced in the e-waterpipe exposed mice compared to control. (a, b) Proteins were separated by SDS gel electrophoresis and transferred to a nitrocellulose membrane before being probed with anti-pAkt, tAkt, and anti-actin anti-rabbit antibodies (*P < 0.05).

Discussion

Platelets are key players in physiological hemostasis and are the blood cells responsible for arresting excessive blood loss from an injured blood vessel.²⁹ However, any shift in this function can lead to pathological thrombosis, which occurs when a formed clot occludes blood flow to vital organs. Indeed, tobacco smoke exposure can increase the risk of coronary plaque rupture, and thrombus formation, which may ultimately cause acute coronary death (ACS) and lead to sudden cardiac death.^30,31 Regarding “waterpipes,” in a previous report, we demonstrated that exposure to traditional waterpipe does elevate the risk of thrombosis, in mice.¹⁶ However, virtually nothing is known regarding e-waterpipe effects on platelet biology and cardiovascular function. Also, whether there are “health” advantages in the context of occlusive CVD for transiting from using flavored tobacco to electrically heated e-liquid,¹³ as in the case of switching from traditional waterpipe to e-waterpipe, remains unknown. Given the close association between traditional waterpipes and e-waterpipes, and the latter's overlap with e-cigarettes, we hypothesized that e-waterpipe exposure will also modulate platelet function and increase the risk for thrombosis.

With regard to the toxicant profile of ETRPs, this was described in a previous report⁴; it is important to note that many of them require an e-liquid for use. To this end, the e-liquid used for the e-waterpipe is comparable to that of e-cigarettes, in terms of concentrations of PG:VG, nicotine, and other flavonoids that are included. Although there has been no direct study on the toxicological profile of e-waterpipes, most of these ETRPs emit hazardous substances that can lead to severe health problems and have toxic chemicals that are overlapping.³² Moreover, it has been documented that traditional waterpipes have a toxicant profile that is comparable to cigarette.³³ Given the close association between e-cigarettes and e-waterpipes on one hand as well as traditional waterpipes and e-waterpipes on the other hand, it is possible that there is overlap in their negative health effects.

Based on the above gap in literature, we defined the effects of e-waterpipe exposure-for 1 month—on platelet reactivity by using a novel whole-body exposure protocol that mimics real-life settings, based on the Beirut protocol.^18,19,34 Of note, the puffing topography, puff interval, and number of puffs applied should simulate human exposure. The advantage of this protocol is that it allows the mice to roam and “vape” freely within the exposure chamber, without much human handling resulting in less stress on the animals. Consequently, our model provides both basic and translational results applicable to human e-waterpipe use/vaping patterns.³⁵ We used a mouse model—in part—since the literature provides ample evidence regarding the clinical and translational relevance of using such animal models as an effective comparison to human tobacco exposure.^35,36

First, we investigated the levels of cotinine, a metabolite of nicotine, in the serum to assess its delivery to the exposed mice and effectiveness of our exposure protocol. Our results showed elevated levels of cotinine in the serum of the exposed mice (642 ng/ml), whereas—expectedly—it was not detectable in the clean air control mice. This result supports the notion that our exposure protocol is realistic—as it yields levels that are consistent with that of human users of ENDS. A similar report which summarized plasma cotinine levels in three different studies observed high levels of cotinine in ENDS users comparable with the levels obtained in our study.^37,38 This also disputes the notion that e-waterpipes are emission-free vaping devices, and that they do not deliver substantial amounts of nicotine to the user.⁶ Given the known health effects of nicotine, our results suggest that e-waterpipes may not be a safe(r) alternative to tobacco smoking.

After validation of our exposure model, we next investigated the impact of e-waterpipes on hemostasis, which is one of the primary functions of platelets. Our data reveals for the very first time a significantly reduced tail bleeding time in the e-waterpipe exposed mice, relative to the clean air controls, indicating a prothrombotic phenotype. The latter was investigated by carrying out the ferric chloride-induced thrombosis model. Again, our data showed a shortened occlusion time in the exposed mice, indicating an increased risk for thrombosis. These findings clearly support the notion that e-waterpipes may not be as safe(r) as advertised even with a relatively short exposure period. They are also consistent with our previous studies with traditional waterpipes and e-cigarettes,^16,24 albeit these studies were of a much shorter duration (1 week as opposed to 1 month). It is important to note that the effect of nicotine on mice tail bleeding and thrombosis (in humans) varies depending on the length of exposure, dose, and subject susceptibility. Irrespective of these conditions, many investigators have documented that novel tobacco products, including waterpipes, can modulate the tail bleeding by stimulating nicotine release from nicotinic acetylcholine receptors in the vascular smooth muscles, leading to norepinephrine release.³⁹ In addition, a separate study by Lyytinen et al (2021) found that exposure to nicotine from e-cigarettes increased platelet thrombus formation, within 15 and 60 minutes after exposure, in human participants.⁴⁰ Importantly, and regarding the total blood count, we did not see any differences between the exposed and the clean air mice. This finding indicates that exposure to e-waterpipes, at least at the present exposure conditions, does not affect the platelet life cycle. We have previously shown that there is no blood count difference when mice were exposed to traditional waterpipes,¹⁶ which was also documented by another study.⁴¹

Given the increased risk of thrombosis and altered hemostasis in the exposed mice, we next investigated the mechanism that may be responsible for the observed phenotype. Our results reveal that agonist-induced platelet aggregation, ATP secretion, p-selectin expression/α-granule release, and integrin GPIIb-IIIa activation were all enhanced because of e-waterpipe exposure, further indicating that the observed prothrombotic phenotype was—at least in part—because of platelet hyperactivity. This was also observed in platelets from mice exposed to traditional waterpipes and e-cigarettes.^16,24,42

Our data shows that exposure to ETRPs, including e-waterpipes can trigger platelet activation and secretion of P-selectin, a cell adhesion molecule, which facilitates adhesion to the endothelium and contributes to thrombus formation. When platelets get activated after a vascular injury, they undergo spreading to cover a wider surface area of the site of vessel injury, which promotes the formation of a stable blood clot. Our results showed that platelets exposed to e-waterpipe exhibited an enhanced spreading response, which further supports a state of hyperactivity.

We next investigated whether exposure to e-waterpipe would upregulate Akt activation.^43,44 Our results revealed an increase in agonist-induced phosphorylation of Akt in the exposed platelets. Of note, we have documented in a previous report that exposure to e-cigarettes did increase Akt phosphorylation.²⁴

In conclusion, we document—for the first time—that e-waterpipes modulate hemostasis and increase the risk of thrombosis-based CVD. Indeed, this is the “first of its kind” investigation looking at the negative health effects of e-waterpipe in this context. The mechanism seems to involve a hyperactive platelet phenotype, in terms of aggregation, secretion, integrin activation, spreading and Akt phosphorylation. These results also cast doubt about the safety claims of these products, which we believe should not be marketed as a safe(r) alternative to cigarettes. Finally, these findings should serve as the basis for cessation efforts and policies to regulate exposure to ETRPs/e-waterpipes.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Institute of Environmental Health Sciences, the National Heart, Lung, And Blood Institute and the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Awards Number R03ES030486, R01HL145053, R21ES029345, R56HL158730, R21HD105187, R21ES034512 and. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This research was also supported by startup funds provided by the School of Pharmacy, Texas A&M University (to Khasawneh and Alshbool).

ORCID iD

Fadi T. Khasawneh

Data Availability Statement

All relevant data is included in this manuscript. We are happy to share any other data related to e-waterpipe characterization upon a reasonable request to the corresponding authors.

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Exposure to Electronic Waterpipes Increases the Risk of Occlusive Cardiovascular Disease in C57BL/6J Mice

Abstract

Introduction

Methods and Materials

Results

Conclusion

Keywords

Introduction

Materials and Methods

Reagents and Materials

Animals

E-waterpipe Exposure Protocol

Cotinine Assay

Tail Bleeding Time

In Vivo FeCl3 Model for Thrombosis

Peripheral Blood Cell/ Platelet Counts

Platelet-rich Plasma Preparation

Washed Platelet Preparation

In Vitro Platelet Aggregation

Dense Granule Release

Flow Cytometric Analysis

Platelet Spreading

Immunoblotting

Statistical Analysis

Results

E-waterpipe Exposure Does Not Affect Total Blood Cell Counts

E-waterpipe Exposure Delivers Cotinine to Exposed Mice

E-waterpipe Exposure Modulates Hemostasis and Thrombosis in Mice

E-waterpipe Exposure Enhances Agonist-induced Platelet Aggregation

E-waterpipe Exposure Enhances Agonist-induced Platelet Secretion

E-waterpipe Exposure Enhances Integrin GPIIb-IIIa/αIIbβ3 Activation

E-waterpipe Exposure Enhances Platelet Spreading

E-waterpipe Exposure Enhances Akt Phosphorylation

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

Data Availability Statement

References

In Vivo FeCl₃ Model for Thrombosis