Nicotine (high-performance liquid chromatography grade with ≥98%; Figure 1) was purchased from Aladdin (Shanghai, China). It was dissolved in normal saline (NS) to prepare a working solution with a final concentration of 5 mM throughout the experiments. Thrombin, prostaglandin E1 (PGE1), bovine serum albumin (BSA), Fluorescein isothiocyanate (FITC)-labeled phalloidin, EDTA and tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin were purchased from Sigma Chemicals (St. Louis, MO). The antibodies against phospho-AKT (Ser473), phospho-PI3K (Tyr458), phospho-ERK1/2 (Tyr202/204), total-AKT, total-PI3K, and total- ERK1/2 were purchased from Cell Signaling (Beverly, MA). Biolegend (San Diego, CA, United States) supplied the FITC-conjugated anti-P-selectin (CD62P) and FITC-conjugated anti-PAC-1 antibodies. Fluo-3 AM was obtained from MCE (Shanghai, China). For thromboelastometry assays, recombinant human thromboplastin (Dade-Innovin) was purchased from DRNX-III, Dingrun Medical Equipment Corp (Chongqing, China). All other chemicals used in the current study were of analytical reagent grade.

Obtaining washed human platelets for our research was done by collecting 20 mL of whole blood from volunteer college students between the ages of 18 and 25, both genders. Participants were required to be free of drugs and abstain from any substances that could influence their platelet function (e.g., alcohol, caffeine, aspirin and so on) for a minimum of 2 weeks prior to the collection, and to have no habits of excessive alcohol consumption, smoking, or other addictive behaviors. Prior to the collection process, we ensured that informed consent was secured from each participant, and all procedures involving human subjects and blood samples were conducted in accordance with ethical standards. The research adhered to the Helsinki Declaration’s guidelines and garnered the green light from the Wuhan University of Science and Technology’s Ethics Board (No. 2022158, September 2022).

Utilizing a minuscule amount of tissue factor, whole blood thromboelastometry (minTF TEM) was initiated with a diluted form of human recombinant thromboplastin, reaching a final dilution ratio of 1–7,200. During each trial, a blend of 330 μL of whole blood was combined with 20 μL of 0.2 mol/L calcium chloride (CaCl₂) and 10 μL of the aforementioned diluted thromboplastin, along with saline as vehicle control and nicotine (10, 20, 50 μM) as the drug intervention group, which were all added at the beginning of the experiment. After a 5-min incubation, detection was initiated within the ROTEM analysis cups using a ROTEM^® delta thromboelastograph system. The assessment process commenced 2 s post-mixing, and a suite of thromboelastometric metrics was ascertained, including Clotting Time (CT), Clot Formation Time (CFT), α-angle, and Maximum Clot Firmness (MCF). CT refers to the interval from the launch of the sample analysis to the emergence of the first discernible clot, marked by an amplitude of 2 mm. CFT spans from the CT to the moment when the clot’s firmness hits a benchmark of 20 mm. The α-angle quantifies the dynamics of clot formation, while MCF denotes the peak vertical deflection observed in the thromboelastometry graph, indicative of the clot’s ultimate strength and stability. The data collection process was permitted to extend up to a maximum of 2 hours.

Blood samples were drawn from a pool of healthy participants. These samples were treated with a 3.8% sodium citrate mixture at a 9:1 volume-to-volume ratio to prevent clotting and then spun at 950 revolutions per minute for 14 min to isolate the platelets. Post-separation, the platelets were rinsed twice and reconstituted in Tyrode’s buffer—a solution containing 13.8 mM NaHCO₃, 13.7 mM NaCl, 0.36 mM NaH₂PO₄, 2.5 mM KCl, 5.5 mM glucose, and 20 mM HEPES, all balanced to a pH of 7.4. The resulting platelet concentration was standardized to 3 × 10⁸ platelets/mL (Zhu et al., 2022). The entire preparation procedure was carried out at ambient temperature. Prior to application, the cleaned platelets were left to sit at room temperature for 30 min.

Wash platelets and adjust to a concentration of 3 × 10⁸ platelets/mL. The washed platelet suspension was dispensed into EP tubes and CaCl₂ solution was added to give a final concentration of 1 mM. Different groups were set up and labelled, including background blank wells (Tyrode’s buffer, no cells), sample control wells (untreated control cells without drug treatment), toxicity maximal wells (untreated control wells used for detection of maximal toxicity), and drug-treated wells (including a drug concentration gradient). Two parallel duplicate wells were set up for each group, and different concentrations of nicotine (10 μM, 20 μM, 40 μM, 80 μM, 160 μM) were added to the drug-treated wells and incubated at 37C to detect LDH release. One hour before the scheduled assay time, LDH release reagent was added to the most toxic wells in an amount of 10% (25 μL) of the total system and incubated at 37C. After incubation, platelet counts were performed, followed by centrifugation at 480 g, and the supernatant from each well was added to a 96-well plate with Lactate dehydrogenase (LDH) assay workup. Incubate for 30 min at room temperature away from light and shake slowly using a horizontal shaker, followed by absorbance measurement at 490 nm and dual-wavelength measurement using 600 nm as the reference wavelength. Finally, mortality was calculated based on the actual absorbance of each well minus the absorbance of the background blank wells, and the cytotoxicity curve was plotted, where the vertical coordinate was the actual absorbance and the horizontal coordinate was the drug concentration.

Platelet aggregation and ATP release tests were conducted using an aggregometer (Chrono-Log, Havertown, PA), following established protocols. The washed platelets (3 × 10⁸ platelets/mL) (Zhu et al., 2022) were then preincubated with 1 mM of CaCl₂ and normal saline (as vehicle control), specific inhibitors or varying concentrations of nicotine (10 μM, 20 μM, 50 μM) for 5 minutes while being stirred at 1000 RPM at 37°C. Subsequently, the platelets were activated with different agonists. ATP release from the platelets was quantified using a luciferin-luciferase reagent (Final concentration of 1 μM, Chrono-lume, Chrono-Log). The aggregation and ATP release data were meticulously recorded and analyzed with the help of aggregolink software (Chrono-Log).

Washed platelets were brought to a concentration of 2 × 10⁷ platelets/mL (Song et al., 2019) and subsequently positioned on 24-well tissue culture slides that had been pre-coated with fibrinogen (25 μg/mL) or with bovine serum albumin (as negative control). Subsequently, 2.5 μL of CaCl₂ (final concentration 1 mM Ca²⁺) and 2.5 μL of MnCl₂ (final concentration 1 mM Mn²⁺) were added. According to the predetermined grouping, an equal volume of solvent control or nicotine (10 μM, 20 μM, 50 μM) was added to each group. These slides were then placed in a humidified incubator, maintained at 37°C Celsius and a CO₂ level of 5% for a spell of 45 min. Following a couple of rinses with phosphate-buffered saline, the attached platelets were secured using 2% paraformaldehyde solution. They underwent further processing with a 0.1% Triton X-100 treatment before being dyed with TRITC-conjugated phalloidin (Final concentration of 1 μg/mL). Fluorescent imagery of the specimens was obtained with an Olympus fluorescence microscope at a magnification of ×100, hailing from Japan, and the images were later scrutinized using ImageJ software to gauge the mean coverage area of the spread platelets and the size of individual platelets that had attached.

A suspension of washed platelets, at a concentration of 500 μL and containing 3 × 10⁸ platelets/mL (Zhu et al., 2022), was compounded. This mixture included 400 μg of 400 μg/mL fibrinogen and 1 mM CaCl₂. The mixture was pre-treated with different nicotine concentrations (10 μM, 20 μM, 50 μM) or a vehicle control (saline) for 5 min. Platelets without agonist were used as a negative control. The clot contraction process was initiated by introducing 0.2 U/mL of thrombin, after which the mixture was placed in an incubator set at 37°C Celsius and took photos of the samples at 0, 15, 30, and 60 min.

Washed platelets (5 × 10⁷ platelets/mL) (Song et al., 2019) were initially pretreated with varying concentrations of nicotine (10 μM, 20 μM, 50 μM), specific inhibitors or vehicle control (saline) for 5 min prior to being activated with thrombin (0.08 U/mL) for another 5 min at 37°C. Following this activation, the platelets were incubated at room temperature, in the dark, for 15 min with either FITC-conjugated anti-P-selectin (CD62P, Final concentration of 1 μM) or FITC-conjugated anti-PAC-1 (CD41/CD61, Final concentration of 1 μM) antibody. The analysis involved counting 10,000 events within the specified population using flow cytometry, and the data were processed using a BD Bioscience flow cytometer.

Washed human platelets (5×10⁷ cells/mL) (Song et al., 2019) labeled with Fluo-3 AM (Final concentration of 1 μM) were measured using flow cytometry, and platelets were incubated with vehicle control (saline) or nicotine for 5 min 50 μL of platelets after incubation with the drug were taken in a flow tube, and the reaction was terminated by adding 450 μL Buffer. The samples were mixed well and detected by BD flow cytometer, and after the baseline was smooth for about 20 s, 5 μL 10 mM CaCl₂ (final concentration 1 mM Ca²⁺) and agonist were added and mixed well and the detection was continued for 4 min. The average fluorescence intensity of calcium ions at each 20s time point was analyzed by using the FlowJo software, and the curves were fitted by using the Sigmaplot software.

The platelets were treated with 2 × lysis buffer to extract the desired material. The supernatant obtained after centrifugation was assessed for protein concentration using a BCA protein assay kit. The proteins were then separated via 10% SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and subjected to primary and secondary antibody incubation. The bands were ultimately detected using an ECL kit and quantified using ImageJ software.

The statistical analysis was conducted using GraphPad Prism 10, a software developed by GraphPad in the United States. The mean values were presented alongside the standard error of the mean (SEM) for every set of data. The student's t-test was employed to compare two groups, while the one-way ANOVA was used to compare multiple groups. Any p-value that was less than 0.05 was deemed statistically significant.

Thromboelastometry is a valuable tool for monitoring changes in coagulation (except for vascular endothelial factors). Our study aimed to explore the effects of varying concentrations of nicotine on clot strength and timing, specifically looking at the rate of fibrin formation, clot strength, and clot stability. By examining representative Thromboelastometry at different nicotine concentrations, we were able to observe the procoagulant effect of nicotine (Figure 2A). Interestingly, the addition of nicotine shortened clotting time (CT), with the most notable effect seen at a concentration of 20 μM (p < 0.0001, Figure 2B). While there was a tendency for 10 μM and 20 μM nicotine to shorten coagulation formation time (CFT) without significant difference. However, a concentration of up to 50 μM did lead to a prolonging of coagulation time (Figure 2C). The selected concentration of nicotine had a significant impact on maximum coagulation firmness (MCF), with 20 μM nicotine having the most positive effect (p < 0.0001, Figure 2D). Notably, there was no significant effect on α Angle (Figure 2E). The most significant differences were observed in the MCF values, which reflect platelet function. As such, our study focused on the effects of nicotine on platelet function.

Aimed at the poison of various concentrations of nicotine to platelets, exploring the LDH exposure of platelets. LDH leakage is a recognized method for evaluating cytotoxicity, with the level of LDH indicative of the degree of cellular disruption. 10–160 μM concentration, almost had no impact on the cell mortality and number of platelets, and the cell mortality of 160 μM Nicotinethe was 13.84% ± 0.4% (VS control p < 0.0001, Figures 3A, B).

Platelet aggregation is the experiment that best reflects the function of platelets. The aggregometer reflects the degree of platelet aggregation by detecting the change in turbidity after platelet aggregation. We observed that nicotine alone did not induce platelet aggregation (Supplementary Figure S1). To investigate further, we tested washed platelet aggregation was induced with different agonists (Thrombin, Arachidonic Acid, Collagen), and it was found that nicotine did not significantly promote Arachidonic Acid and Collagen-induced aggregation (Supplementary Figure S2). Whereas, aggregation of washed platelets incubated with a concentration gradient (10 μM, 20 μM, 50 μM) of nicotine, was induced by thrombin at a final concentration of 0.08 U (Figure 3C). Statistical analysis showed that nicotine promoted platelet aggregation, and although there was no difference at a nicotine concentration of 10 μM, both 20 μM and 50 μM nicotine significantly promoted platelet aggregation, and the effect was most pronounced at a concentration of 20 μM (VS control p < 0.0001, Figure 3D). Similarly, to examine the effects of different physiological environments on platelet aggregation, we induced platelet-rich plasma aggregation using various agonists, including thrombin, ADP, arachidonic acid, and collagen. The results demonstrated that nicotine enhanced thrombin-induced platelet-rich plasma aggregation, while it had no significant effect on aggregation induced by the other agonists (Supplementary Figure S3).

By analyzing ATP release (the marker of dense granule) (Figure 3E), we discovered that nicotine enhanced ATP release to the maximum extent at a concentration of 20 μM (VS control p < 0.05; 69.03% ± 1.65% after 20 μM nicotine incubation, p < 0.0001; 66.37% ± 1.32% after 50 μM nicotine incubation, p < 0.001. (Figure 3H). The median fluorescence intensity (MFI) of P-Selectin expression increased significantly, from 1,057 ± 38.34 in the vehicle group to 2,354 ± 32.17 in the 20 μM nicotine group, p < 0.0001 (Supplementary Figure S4).

Platelets exhibit an “inside-out” signaling mechanism that agonist stimulation triggers a series of internal signaling events in platelets, such as rearrangement of the platelet backbone, changes in morphology, protein synthesis, and release of granules. Ultimately, this leads to a change in the affinity of the platelet integrin receptor αIIbβ3 for fibrinogen, transitioning from a low to high-affinity state. In humans, the high-affinity state of αIIbβ3 can be detected through the use of PAC-1 antibody, and the activation of integrin αIIbβ3 can be detected by measuring the binding strength of PAC-1 via flow cytometry (Figure 4A). Results from the study showed that final concentrations of 20 μM and 50 μM nicotine led to an increase in PAC-1 expression, while 10 μM nicotine tended to promote PAC-1 expression, but no significant difference was observed (Figure 4B). The MFI of PAC-1 expression demonstrated a significant increase, rising from 354.3 ± 9.597 in the vehicle group to 1741 ± 29.14 in the 20 μM nicotine group, p < 0.0001 (Supplementary Figure S4). Platelet activation is primarily driven by the integrin αIIbβ3, which facilitates platelet signaling from the “outside-in” signaling mechanism. To examine the impact of nicotine on platelet adhesion and expansion, platelets were exposed to varying concentrations of nicotine, 1% BSA (as a blank control), fibrinogen (as a Negative control) and vehicle (as a solvent control) (Figure 4C). The staining of the platelet skeleton using ghost pen cyclic peptides showed a significant rise in platelet adhesion and spreading on fibrinogen among the nicotine-treated group across all tested concentrations, and this promotion was not attributable to the solvent. The most striking variation emerged at 20 μM, with the number of adhering platelets increasing from 49.11 ± 2.30 platelets/0.01 mm² to 86.12 ± 2.14 platelets/0.01 mm². Additionally, there was a noticeable growth in the average expansion area of individual platelet, which increased from 49.87 ± 1.59 μm² to 54.07 ± 1.08 μm² (Figures 4D, E). These results suggest that nicotine promotes platelet adhesion and expansion on fibrinogen. Thromboconstriction occurs following primary and secondary hemostasis. The findings from the plaque retraction assay demonstrated that all three concentrations of nicotine facilitated the contraction of platelet plugs after 60 min. Notably, the 20 μM concentration of nicotine exerted the most pronounced promotional effect (Figures 4F, G).

The signaling of platelet intracellular calcium ions plays a critical role in the activation of platelets. Upon activation, agonists cause an increase in the concentration of intracellular calcium ions within platelets. This heightened concentration is a result of the release of intracellular calcium pools, as well as the inward flow of extracellular calcium ions. By utilizing the Flou-3-AM dye to bind to free intracellular calcium ions and measuring the resulting fluorescence intensity through flow cytometry, changes in platelet intracellular calcium ion concentration due to nicotine were detected (Figure 5A). All concentration gradients of nicotine were found to promote an increase in intracellular calcium ion concentration in platelets, with the highest concentration observed at 20 μM nicotine (Figure 5B).

To determine which receptors on platelet membranes were bound by nicotine, utilizing molecular docking simulation software. This method involves creating a simulation of the forces that occur between receptors and ligands, and binding them together in a way that maximizes their complementary shapes and properties. The results showed that nicotine had a stronger binding score with PAR1, PAR4 receptors. Molecular docking was used to map the interaction of nicotine with two receptors, PAR1 and PAR4, visualizing through a schematic of the 3D interaction (Figures 6A, B). Additionally, the flow cytometry assay demonstrated that nicotine alone could activate platelet P-Selectin expression (Figure 6C), indicating the release of α-granules. Importantly, the facilitation of this process could not be reversed by SCH 530348 (PAR1 receptor inhibitor), but could be reversed by BMS-986120 (PAR4 receptor inhibitor) (Figure 6D). The MFI of P-Selectin was measured at 1775 ± 21.94 following the administration of SCH 530348, which did not show a significant reversal compared to the 20 μM nicotine group (1802 ± 19.13). In contrast, the MFI of P-Selectin significantly decreased to 120.3 ± 7.68 with the application of BMS-986120 (Supplementary Figure S4). Furthermore, BMS-986120 effectively countered the pro-aggregation effect of nicotine in the thrombin-induced platelet-rich plasma aggregation assay, while SCH 530348 was unable to produce a similar reversal (Supplementary Figure S5).

Upon platelet activation, a series of intracellular signal transduction events converge on the cytoplasmic tail of αIIbβ3, causing the extracellular structural domain of αIIbβ3 to transition to a high-affinity state for its extracellular ligand. The activation status of αIIbβ3 was assessed via flow cytometry (Figure 7A), and it was found that nicotine alone induced the activation of the platelet integrin receptor αIIbβ3, a state that could only be reversed by BMS-986120 (Figure 7B). The MFI of PAC-1 was 876.7 ± 3.71 following treatment with SCH 530348, showing no significant reversal compared to the 20 μM nicotine group (890 ± 5.56). In contrast, the MFI of P-Selectin decreased markedly to 13.67 ± 1.45 after treatment with BMS-986120 (Supplementary Figure S4). To delve deeper into the impact of nicotine on these targets, Western blotting was utilized to gauge the phosphorylation levels of specific proteins (Figure 7C). The findings revealed a significant increase in the phosphorylation levels of PI3K (Figure 7D), AKT (Figure 7E), and ERK1/2 (Figure 7F) induced by nicotine.