1,8-Cineole inhibits platelet-leukocyte aggregate formation by reducing P-selectin expression

Abstract

Introduction: Platelets, traditionally recognized for their role in hemostasis, have increasingly been implicated in cancer progression, including head and neck squamous cell carcinoma (HNSCC). Beyond releasing growth factors and chemokines, platelets modulate leukocyte-mediated proinflammatory responses and effector functions through direct or indirect contact. These processes promote tumor cell proliferation, survival, epithelial to mesenchymal transition (EMT) and extravasation. Consequently, targeting platelet-leukocyte aggregate (PLA) formation represents a promising pharmacological strategy to interfere with platelet-mediated pro-tumorigenic effects. 1,8-cineole, a plant-derived metabolite found in several botanical sources, has shown potent anti-platelet effects through modulation of the adenosine A_2A receptor signaling. However, its influence on PLA formation has not been investigated.

Methods: In this study, we analyzed platelet activation and PLA formation in HNSCC patients compared to healthy donors. A co-culture system combined with blocking antibodies was employed to elucidate the mechanisms of PLA formation. Moreover, the pharmacological effects of 1,8-cineole were compared with those of conventional anti-platelet drugs.

Results: The results revealed elevated P-selectin expression and enhanced PLA formation in HNSCC patients. PLA formation was predominantly mediated through P-selectin-PSGL-1 interactions. Ex vivo studies demonstrated that 1,8-cineole significantly reduced PLA formation by inhibiting P-selectin expression on platelets. Notably, traditional anti-platelet agents did not significantly inhibit PLA formation, despite effectively reducing platelet aggregation.

Discussion: These findings identify a pharmacological effect of 1,8-cineole in disrupting platelet-leukocyte interactions via suppression of the P-selectin-PSGL-1 axis. This suggests that 1,8-cineole offers potential pharmacological benefits in mitigating platelet-mediated inflammation and tumor progression.

Keywords: 1,8-cineole; anti-platelet drugs; head and neck squamous cell carcinoma; platelet-leukocyte aggregates; platelets.

FIGURE 1

Platelets from HNSCC patients exhibit a prothrombotic phenotype. (A-D) Whole blood was collected from healthy donors (n=13) and HNSCC patients (n=12). The samples were immediately stained to determine the prevalence of aggregates between platelets (CD41⁺) and (A) monocytes, (B) eosinophils, (C) neutrophils and (D) T-cells. (E) Age was compared between HD and HNSCC patients. (F) Correlation between CD41⁺ monocytes and age were identified. Data were fitted with a simple linear regression, with the corresponding R² and p-value indicated. (G) Platelets isolated from healthy donors and HNSCC patients were stained for P-selectin expression on the cell surface. (H-J) HD and HNSCC platelets were stimulated ex vivo with ADP (H), TRAP (I) or IV.3+Fab (J) for 10 min and maximal aggregation was measured. Data is presented as box plot with mean and minimum to maximum values. *P < 0.05, ****P < 0.0001. Mann-Whitney test was used for comparison between healthy donors and HNSCC patients.

FIGURE 2

Platelet activation enhances PLA formation. Platelets were either left unstimulated or stimulated with 5 µM TRAP for 5 min before being co-cultured with leukocytes at a ratio of 1:50 for 15 min. The presence of CD41⁺ leukocyte subpopulations was subsequently identified and analyzed via flow cytometry as outlined in Supplementary Figure S3. Data is presented as mean ± SEM. Statistical significance is denoted as *P < 0.05. Unpaired Students’ t-tests were used for comparison between untreated and treated groups.

FIGURE 3

P-selectin expression mediates PLA formation in an in vitro co-culture assay. (A) Platelets were pretreated with 5 μM TRAP for 2 min, followed by the addition of the indicated blocking antibodies (10 μg/mL) for additional 5 min. Subsequently, platelets were co-cultured with leukocytes at a ratio of 1:50 for 15 min. For PSGL-1 blockade, leukocytes were pretreated with anti-PSGL-1 (10 μg/mL) for 10 min. PLAs were analyzed via flow cytometry (n=3-7). (B, C) The expression levels of PSGL-1 (B) and CD11b (C) were measured on the surface of freshly isolated leukocytes using flow cytometry (n=3). (D, E) The percentages of CD61+ leukocytes were plotted against the expression levels of PSGL-1 (D) or CD11b (E). Data were fitted with a simple linear regression, with the corresponding R2 and p-value (P) indicated (n=3). Data are presented as mean ± SEM. Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001. One-way ANOVA followed by Dunnett’s post-hoc test was used for analysis in (A) and Tukey’s post-hoc test for (B) and (C).

FIGURE 4

PLA formation is affected by 1,8-cineole. Platelets were pretreated with or without 1,8-cineole or the indicated antithrombotic drugs and subsequently activated with TRAP for 10 min. (A) PLA formation was measured with 1,8-cineole-treated platelets after 15 min co-incubation with leukocytes by flow cytometry (n=4). (B) PLA formation was determined with antithrombotic-treated platelets after 15 min of co-culture by flow cytometry (n=3). (C) Aggregation was determined with treated or untreated platelets for 10 min (n=3). Representative Graph (left panel) and area under the curve [AUC] (right panel) are shown (n=5). (D) P-selectin expression on the cell surface was measured. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001. One-way ANOVA followed by Dunnett’s post-hoc test was used.

FIGURE 5

1,8-cineole abrogated TRAP-induced platelet-leukocyte aggregate formation via P-selectin regulation in comparison to classical antithrombotics. PLA formation is induced by the activation of platelets via subsequent P-selectin expression on the cell surface, binding to PSGL-1 expressed on leukocytes. 1,8-cineole inhibits P-selectin expression, thereby reducing PLA formation. Tirofiban blocks the binding of fibrinogen to the GPIIb/IIIa receptor, fully inhibiting aggregation, whereas P-selectin expression and PLA formation remain fully active. Cangrelor antagonizes the ADP receptor, thus inhibiting the platelet activation enhancement, but not the first step of activation. ASA blocks COX-1 activity and subsequent TXA₂ formation, leading to the inhibition of the platelet activation enhancement, while it does not interfere with the initial step of platelet activation. Created with https://BioRender.com.