Chrysoeriol Exerts Antiplatelet Effects by Regulating cAMP/cGMP and PI3K/MAPK Pathway

Abstract

Chrysoeriol, a flavonoid naturally found in several plants, including Danggui Susan, a traditional herbal medicine, exhibits promising anti-inflammatory and antioxidant properties. Its potential to prevent cardiovascular diseases, primarily through inhibiting platelet activation and aggregation, has attracted significant interest. This study aimed to investigate the molecular mechanisms underlying the antiplatelet effects of chrysoeriol. The compound effectively suppressed collagen-induced platelet aggregation without inducing cytotoxicity. Chrysoeriol elevated intracellular levels of cyclic AMP (cAMP) and cyclic GMP (cGMP), enhanced inositol 1,4,5-trisphosphate receptor (IP₃R) phosphorylation, and reduced cytosolic calcium (Ca²⁺) mobilization, all of which contributed to its antiplatelet action. Furthermore, chrysoeriol inhibited the phosphorylation of PI3K, Akt, JNK, and p38 MAPK, pathways involved in the activation of cytosolic phospholipase A2 (cPLA₂) and thromboxane A2 (TXA₂) production. These effects were accompanied by reduced TXA₂ production and secretion of dense granules (ATP and serotonin). Chrysoeriol also impaired thrombin-induced clot retraction, further suggesting its capacity to regulate platelet responses and cytoskeletal rearrangements. These findings highlight chrysoeriol’s multi-target mechanisms, including modulation of cyclic nucleotides, kinase pathways, and platelet function, offering potential as a therapeutic agent to prevent thrombotic cardiovascular events.

INTRODUCTION

Cardiovascular disease (CVD), including heart attacks and strokes, remains leading causes of death worldwide. According to the 2025 Heart Disease and Stroke Statistics Update, the incidence of ischemic heart disease, stroke, hypertensive heart disease, and cardiomyopathy/myocarditis increased by 68%, 44%, 87%, and 46%, respectively, between 1990 and 2021 (Martin et al., 2025). CVD currently accounts for approximately one in three deaths in the United States (Martin et al., 2025). A key pathological feature of many CVD is thrombosis, where excessive platelet activation and coagulation lead to intravascular clot formation. Platelets rapidly adhere, aggregate, and initiate hemostasis after vascular injury, followed by fibrin stabilization. However, when thrombi obstruct vessels supplying the heart or brain, they can cause myocardial infarction or stroke. Even small thrombi can be fatal in narrowed arteries. Thus, regulating platelet activity is crucial in CVD prevention and treatment.

Endothelial-derived prostacyclin (PGI₂) and nitric oxide (NO) raise platelet cAMP and cGMP levels, activating protein kinases A (PKA) and G (PKG) to suppress platelet reactivity (Beck et al., 2014). These kinases inhibit intracellular calcium signaling by phosphorylating inositol 1,4,5-trisphosphate receptor (IP₃R), thereby attenuating activation (Lopes-Pires et al., 2015). They also phosphorylate vasodilator-stimulated phosphoprotein (VASP), inhibiting integrin αIIb/β₃ activation and fibrinogen binding, preventing aggregation (Unsworth et al., 2017; Worth et al., 2010). Platelet aggregation involves multiple intracellular signaling pathways, including mitogen-activated protein kinases (MAPKs: ERK, JNK, p38) and PI3K/Akt signaling (Irfan et al., 2018; Patel and Naik, 2020). These pathways regulate key processes such as integrin activation, dense granule release (ATP, serotonin), and thromboxane A₂ (TXA₂) production through the phosphorylation of cytosolic phospholipase A₂ (cPLA₂) (Adam et al., 2008). Additionally, PI3K/Akt signaling is known to elevate intracellular calcium levels, further promoting platelet aggregation (Guidetti et al., 2015). Therefore, platelet function is tightly regulated by the balance between stimulatory and inhibitory signals through these signaling pathways.

Chrysoeriol, a flavonoid present in various plants and the herbal formula Danggui Susaun, has known anti-inflammatory, antioxidant, and anticancer effects (Kim et al., 2019). Recent studies suggest chrysoeriol may suppress platelet function, possibly through modulation of the TXA₂ pathway (Lee et al., 2023). However, its effects on key signaling mechanisms—such as cAMP/cGMP pathways, IP₃R or VASP phosphorylation—remain poorly understood. Prior research has emphasized its antioxidant role, but its potential as a platelet inhibitor is largely unexplored.

This study aims to elucidate how chrysoeriol modulates platelet activation and aggregation. Specifically, we investigate its effects on cAMP–PKA and cGMP–PKG signaling cascades, modulation of PI3K/Akt and MAPK pathways, and inhibition of cPLA₂ activity and thromboxane A₂ (TXA₂) production. These findings may support the development of chrysoeriol as a novel antithrombotic agent for cardiovascular applications.

MATERIALS AND METHODS

Preparation of human platelets

Platelet-rich plasma (PRP) was isolated via centrifugation at 1,650×g for 5 min. The plasma was subsequently washed using a buffer containing 138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO₃, 0.36 mM NaH₂PO₄, 0.49 mM MgCl₂, and 5.5 mM glucose (pH 7.4). The resulting washed platelets were suspended in the same buffer at 25°C and adjusted to a density of 10⁸ cells/mL (Schwarz et al., 2001). PRP samples were obtained from the Korean Red Cross Blood Center (Suwon, Korea), and all human-derived procedures were conducted under the approval of Namseoul University’s IRB (1041479-HR-201803-003).

Platelet aggregation test

Chrysoeriol (4, 8, 16, 24, 32 μM) was added to the platelet suspensions (10⁸ cells/mL) along with 2 mM CaCl₂. The mixture was incubated for 3 min at 37°C. Platelet aggregation was initiated with 2.5 μg/mL collagen and monitored for 5 min at 1,000 rpm using a Chrono-Log aggregometer. Aggregation was quantified by maximum amplitude (%). DMSO (0.1%) served as the vehicle control in all samples (Irfan et al., 2018). For comparative experiments, the clinically used PDE inhibitors dipyridamole and cilostazol were included as positive controls and tested at the same concentrations as chrysoeriol (32 and 64 µM) under identical assay conditions.

Cytotoxicity assessment

Platelets treated with chrysoeriol at 8-32 μM were incubated for 1 h. Samples were centrifuged (10,000×g, 2 min), and supernatants were used to assess LDH release using a commercial LDH assay (Cayman Chemical, USA). Absorbance was read at 490 nm on a TECAN microplate reader (Kim et al., 2019). The LDH Lysis Solution (10% Triton X-100) was used to achieve complete cell lysis and define the maximum enzyme release (100% cytotoxicity). Cell viability (%) was calculated as 100% − cytotoxicity (%). DMSO-treated platelets served as the negative control, while Triton X-100 (0.1%) was used as the positive control for comparison. To confirm the assay linearity and reliability, partial lysis (0.005% Triton X-100) was also tested.

Measurement of cAMP and cGMP

To evaluate cyclic nucleotide levels, platelets were exposed to chrysoeriol (8-32 μM) in the presence of 2 mM CaCl₂ for 3 min at 37°C. Collagen (2.5 μg/mL) was then added for 5 min. Reactions were stopped by adding 80% ethanol, followed by centrifugation (10,000×g, 10 min). The supernatants were concentrated to dryness in a SpeedVac concentrator (55°C, IR heat, vacuum, lids open). The dried residues were reconstituted in ELISA buffer and used for the quantification of cAMP and cGMP using commercial ELISA kits (Cayman Chemical, USA) according to the manufacturer’s instructions. Absorbance was measured at 410 nm with a TECAN microplate reader (Gambaryan, 2022). The measured absorbance values were converted into B/B₀ (%) ratio, where B represents the sample absorbance and B₀ the absorbance of the zero standard. These ratios were used to calculate the amount of cAMP or cGMP bound to the antiserum, and the final concentrations were determined from standard curves generated with known standards provided in the kit.

Western blot analysis

After platelet stimulation, 1× Cell Lysis Buffer was used to terminate reactions. Protein levels were measured via BCA assay (Pierce Biotechnology). Equal amounts (15 µg) were separated on 8% SDS-PAGE and transferred to PVDF membranes. PVDF membranes were pre-wetted in methanol to activate the membrane surface and reduce hydrophobicity. After blocking with 3% BSA in TBST for 1 h at room temperature, the membranes were incubated with primary antibodies (1:1,000) overnight at 4°C (approximately 18 h). After washing three times with TBST, HRP-conjugated secondary antibodies (1:2,000) were applied for 1 h at room temperature. Detection was performed using the WesternBright ECL system (Advansta), and band density was quantified via Quantity One (Bio-Rad) (Nieswandt and Watson, 2003).

Intracellular calcium monitoring

The intracellular calcium ion concentration ([Ca²⁺]_i) was measured with Fura-2/AM as previously described (Grynkiewicz et al., 1985). PRP was incubated with 5 μM of Fura-2/AM for 60 min at 37°C and washed. The Fura-2-loaded platelets were then pre-incubated with chrysoeriol (8-32 μM) for 3 min at 37°C in the presence of 2 mM CaCl₂, and subsequently stimulated with collagen (2.5 μg/mL) for 5 min. Fluorescent signals were recorded using a Hitachi F-7000 fluorescence spectrofluorometer (F-7000, Hitachi, Japan). Light emission was measured at 510 nm, with simultaneous excitation at 340 and 380 nm that changed every 0.5 s. Fura-2 fluorescence in the cytosol measured with the spectrofluorometer was calculated as previously described by Grynkiewicz with the following formula: [Ca²⁺]_i 224 nM×(F−Fmin)/(Fmax−F), in which 224 nM is the dissociation constant of the Fura-2-Ca²⁺ complex, and Fmin and F max represent the fluorescence intensity levels at very low and very high Ca²⁺ concentrations, respectively. In our experiment, Fmax was the intensity of the Fura-2-Ca²⁺ complex fluorescence at 510 nm after the platelet suspension containing 2 mM of CaCl₂ had been solubilized with Triton X-100 (0.1%). Fmin was the fluorescence intensity of the Fura-2-Ca²⁺ complex at 510 nm, after the platelet suspension containing 20 mM Tris/3 mM of EGTA had been solubilized with Triton X-100 (0.1%). F represented the intensity of Fura-2 complex fluorescence at 510 nm after the platelet suspension which was stimulated with collagen with or without chrysoeriol (8-32 μM) in the presence of 2 mM CaCl₂.

Flow cytometry for fibrinogen binding

Platelets were treated with chrysoeriol (8-32 μM) and 2 mM CaCl₂ at 37°C for 3 min, followed by stimulation with collagen and Alexa Fluor 488-conjugated fibrinogen (30 µg/mL) for 5 min. After incubation, an equal volume of 0.5% paraformaldehyde was added to fix the cells. The entire aggregation reaction, including stimulation and fixation, was performed under dark conditions to prevent fluorophore bleaching. All samples were diluted with 1× PBS to maintain an optimal event rate prior to flow-cytometric acquisition, and approximately 10,000 events were collected per sample. Binding of fibrinogen to αIIb/β₃ was analyzed via flow cytometry (BD Biosciences) using CellQuest software (Wentworth et al., 2006).

TXA₂ quantification

Because thromboxane A₂ (TXA₂) is highly unstable and rapidly hydrolyzed to its inactive metabolite, thromboxane B₂ (TXB₂), the latter was quantified as a reliable indicator of TXA₂ synthesis. Human platelet suspensions (1×10⁸ cells/mL) were preincubated with various concentrations of chrysoeriol in the presence of CaCl₂ at 37°C for 3 min to induce activation. The reactions were then terminated by the immediate addition of ice-cold ethanol to prevent further enzymatic activity. Subsequently, the amount of TXB₂ generated was determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical, Ann Arbor, MI, USA), and the absorbance was measured at 410 nm according to the manufacturer’s instructions (Hsia et al., 2024).

ATP and serotonin secretion

To terminate the release reactions of ATP and serotonin from activated platelets, EDTA (2 mM) was added to chelate calcium ions and halt further aggregation processes. The samples were then subjected to centrifugation at 10,000×g for 10 min to remove cellular debris, and the resulting supernatants were carefully collected for biochemical analysis. ATP concentrations were quantified using a luminescence-based ATP Detection Kit (Cayman Chemical, USA), according to the manufacturer’s instructions. The emitted light intensity, which correlates directly with ATP content, was measured using a luminometer. In parallel, serotonin levels were determined through a colorimetric ELISA assay (Abcam, UK) at an absorbance of 410 nm, providing a sensitive and specific assessment of granule secretion (Irfan et al., 2019).

Clot retraction analysis

To evaluate the effect of chrysoeriol on fibrin clot retraction, 400 μL of platelet-rich plasma (PRP) was carefully transferred into polypropylene tubes containing varying concentrations of chrysoeriol (16, 32, and 64 μM) along with 2 mM CaCl₂ to initiate platelet activation. The reaction was triggered by adding thrombin (0.05 U/mL), which converts fibrinogen to fibrin and promotes platelet-mediated clot retraction. The samples were then incubated at 37°C for 15 min under static conditions to allow complete clot formation and retraction. Following incubation, the extent of clot retraction was visually documented using a high-resolution digital camera under standardized lighting and distance conditions. The residual clot area, representing the degree of retraction, was subsequently analyzed and quantified using ImageJ software (NIH, version 1.46) according to the method (Li et al., 2010).

Network pharmacological and drug-likeliness analysis

Network pharmacological and drug-likeness analysis was performed as reported previously (Akram et al., 2025). Briefly, the canonical SMILES notation for chrysoeriol was retrieved from PubChem (PubChem CID: 5280666; Accessed on 25^th July 2025). The potential chrysoeriol targets were identified using SwissADME target prediction and mapped via STRING using UniProt IDs. “Platelet activation” genes were retrieved from GeneCards and compared with chrysoeriol targets using Venny 2.1 to find overlaps. These common genes were analyzed in STRING to generate a protein–protein interaction (PPI) network and identify related KEGG pathways. The chrysoeriol gene pathway network was then constructed and visualized in Cytoscape to explore the mechanistic link between chrysoeriol and platelet activation. SwissADME was used to evaluate the pharmacokinetics and drug-likeness of chrysoeriol, and the BOILED-Egg model predicted intestinal absorption and blood–brain barrier permeability.

Statistical analysis

All experimental data were presented as mean ± standard deviation (SD). Intergroup comparisons were made using one-way ANOVA, followed by Tukey–Kramer post hoc analysis. Statistical processing was conducted in SPSS (v21.0; SPSS Inc., Chicago, IL, USA), and p-values <0.05 were regarded as statistically significant.

RESULTS

Inhibitory effect of chrysoeriol on platelet aggregation and cytotoxicity

When human platelets were stimulated with collagen (2.5 μg/mL), aggregation levels increased markedly to 83.0% (Fig. 1A). Chrysoeriol treatment led to a dose-dependent decrease in platelet aggregation, with inhibition observed at 4, 8, 16, 32, and 64 µM concentrations, reaching a maximum inhibition of 94.0% at the highest dose (Fig. 1A). When platelets were treated with chrysoeriol alone (8-64 µM) without collagen stimulation, the aggregation response was comparable to that of the DMSO-treated negative control group (Fig. 1A). As expected, resting platelets exhibit minimal basal signaling without external agonists; therefore, chrysoeriol alone did not induce or alter aggregation. The calculated IC₅₀ was 23.74 µM (Fig. 1B), demonstrating strong anti-aggregatory potency. Additionally, cell viability across all concentrations exceeded 98%, indicating that chrysoeriol did not induce cytotoxicity (Fig. 1C). To further clarify the mechanism, we directly compared chrysoeriol with clinically used PDE inhibitors, dipyridamole and cilostazol, under identical assay conditions (Fig. 1D). At matched concentrations (32 and 64 µM), chrysoeriol showed stronger inhibition than dipyridamole at both doses and exhibited a comparable effect to cilostazol at 64 µM, although cilostazol remained slightly more potent overall. Furthermore, to evaluate the broader inhibitory potential of chrysoeriol, we examined its effects on platelet aggregation induced by other representative agonists (U46619 and thrombin) in addition to collagen (Fig. 1E). Chrysoeriol significantly suppressed aggregation induced by both agonists, supporting its efficacy across multiple activation pathways.

Fig. 1.

Chrysoeriol’s effect on platelet aggregation and cytotoxicity. (A) Inhibitory effect of chrysoeriol on collagen-induced platelet aggregation and aggregation response in unstimulated platelets. (B) IC₅₀ value of chrysoeriol against collagen-induced platelet aggregation. (C) Cytotoxicity of chrysoeriol in suspended platelets. (D) Comparison of chrysoeriol with the PDE inhibitors dipyridamole and cilostazol at matched concentrations (32 and 64 μM) under collagen-induced platelet aggregation. (E) Real-time inhibition of platelet aggregation induced by collagen, U46619, and thrombin by chrysoeriol. Data are presented as mean ± SD (n=4). Statistical significance is indicated by *p<0.05 and **p<0.01 compared with collagen-stimulated platelets.

Effects of chrysoeriol on cAMP/cGMP levels, IP₃R phosphorylation, and calcium mobilization

As cyclic nucleotides play a key role in inhibiting platelet activation, we assessed their levels after chrysoeriol treatment. Both cAMP and cGMP levels rose significantly in a dose-dependent manner, with cAMP showing a more prominent increase (Fig. 2A, 2B). IP₃R phosphorylation was also enhanced following treatment (Fig. 2C), which correlated with a reduction in intracellular calcium levels (Fig. 2D). These findings suggest that chrysoeriol strengthens the inhibitory signaling in platelets through cyclic nucleotide elevation and calcium suppression.

Fig. 2.

Chrysoeriol’s effect on cyclic nucleotide formation, IP₃R phosphorylation, and intracellular Ca²⁺ mobilization. (A) Chrysoeriol’s enhancement of cAMP production. (B) Chrysoeriol’s enhancement of cGMP production. (C) Chrysoeriol’s promotion of IP₃R phosphorylation. (D) Chrysoeriol’s inhibition of intracellular calcium mobilization. Data are presented as mean ± SD (n=4). Statistical significance is indicated by ^ap<0.05 compared with non-stimulated platelets, and *p<0.05 and **p<0.01 compared with collagen-stimulated platelets.

Stimulation of VASP phosphorylation and inhibition of fibrinogen binding by chrysoeriol

Following the increase in cAMP and cGMP levels, we investigated the effect of chrysoeriol on VASP phosphorylation. Phosphorylation at Ser 157 (regulated by cAMP) and Ser 239 (regulated by cGMP) increased in a concentration-dependent manner (Fig. 3A). Notably, phosphorylation at Ser 157 was more pronounced than at Ser 239, consistent with the stronger elevation of cAMP over cGMP. Furthermore, based on this increase in VASP phosphorylation, we assessed whether chrysoeriol affected αIIb/β₃-fibrinogen binding. Fibrinogen binding in collagen-stimulated platelets increased to 80.0%, while chrysoeriol treatment reduced fibrinogen proportionally to concentration, with final binding percent of 34.1%, 18.5%, 12.3%, and 5.9% at concentrations of 8, 16, 32, and 64 µM, respectively (Fig. 3B, 3C). These findings suggest that chrysoeriol promotes cAMP/cGMP production and VASP phosphorylation, thereby reducing fibrinogen binding to αIIb/β₃ integrin.

Fig. 3.

Chrysoeriol’s effect on VASP phosphorylation and fibrinogen binding. (A) Chrysoeriol’s promotion of VASP phosphorylation. (B) Histogram from flow cytometry analysis of fibrinogen binding. a: Base (intact platelets), b: collagen (2.5 µg/mL), c: collagen (2.5 µg/mL)+Chrysoeriol (8 µM), d: collagen (2.5 µg/mL)+Chrysoeriol (16 µM), e: collagen (2.5 µg/mL)+Chrysoeriol (32 µM), f: collagen (2.5 µg/mL)+Chrysoeriol (64 µM). (C) Chrysoeriol’s inhibition of fibrinogen binding (%) induced by collagen. Data are presented as mean ± SD (n=4). Statistical significance is indicated by ^ap<0.05 compared with non-stimulated platelets, and *p<0.05 and **p<0.01 compared with collagen-stimulated platelets.

Effects of chrysoeriol on PI3K/Akt and MAPK signaling pathways

We evaluated the effect of chrysoeriol on the PI3K/Akt and MAPK pathways, which are critical in platelet aggregation. Phosphorylation of PI3K and Akt was significantly reduced following chrysoeriol treatment compared to collagen-stimulated controls (Fig. 4A), suggesting that chrysoeriol inhibits platelet aggregation by suppressing the PI3K/Akt pathway. Additionally, chrysoeriol significantly attenuated phosphorylation of JNK and p38 MAPKs (Fig. 4B). However, ERK phosphorylation was not notably induced by collagen stimulation and was unaffected by chrysoeriol treatment, indicating that ERK may have a relatively minor role in this context. These results suggest that chrysoeriol suppresses platelet aggregation primarily through inhibition of the PI3K/Akt and MAPK (JNK and p38) signaling pathways.

Fig. 4.

Chrysoeriol’s inhibitory effect on PI3K/Akt and MAPK phosphorylation. (A) Chrysoeriol’s suppression of PI3K/Akt phosphorylation. (B) Chrysoeriol’s suppression of MAPK phosphorylation. Data are presented as mean ± SD (n=4). Statistical significance is indicated by ^ap<0.05 compared with non-stimulated platelets, and *p<0.05 and **p<0.01 compared with collagen-stimulated platelets.

Effects of chrysoeriol on cPLA2 phosphorylation and TXA2 release

Chrysoeriol decreased cPLA2 phosphorylation in a concentration-dependent manner (Fig. 5A), which is associated with its suppression of the PI3K/Akt and MAPK pathways. Since cPLA2 plays a key role in releasing arachidonic acid from platelet membranes and subsequently producing TXA2, we also measured TXA2 levels. Chrysoeriol treatment led to a concentration-dependent reduction in TXA2 release (Fig. 5B). These results indicate that chrysoeriol suppresses cPLA2 phosphorylation, thereby inhibiting TXA2 generation and release, ultimately contributing to the inhibition of platelet aggregation.

Fig. 5.

Chrysoeriol’s inhibitory effect on cPLA₂ phosphorylation and TXA₂ production. (A) Chrysoeriol’s suppression of cPLA₂ phosphorylation. (B) Chrysoeriol’s suppression of TXA₂ production. Data are presented as mean ± SD (n=4). Statistical significance is indicated by ^ap<0.05 compared with non-stimulated platelets, and *p<0.05 and **p<0.01 compared with collagen-stimulated platelets.

Reduction of platelet granule secretion by chrysoeriol

ATP and serotonin release, both associated with dense granule secretion and further platelet activation, were significantly elevated upon collagen stimulation. However, chrysoeriol treatment led to a concentration-dependent decline in both ATP and serotonin levels (Fig. 6). This suggests that chrysoeriol suppresses not only early platelet signaling but also the amplification mechanisms driven by granule secretion.

Fig. 6.

Chrysoeriol’s inhibitory effect on granule release in platelets. (A) Chrysoeriol’s inhibition of ATP secretion. (B) Chrysoeriol’s inhibition of serotonin secretion. Data are presented as mean ± SD (n=4). Statistical significance is indicated by ^ap<0.05 compared with non-stimulated platelets, and *p<0.05 and **p<0.01 compared with collagen-stimulated platelets.

Inhibitory effects of chrysoeriol on platelet-mediated fibrin clot retraction

Upon activation, platelets aggregate and form stable thrombi in cooperation with coagulation factors, ultimately leading to fibrin clot retraction. Therefore, we assessed the effect of Chrysoeriol on thrombin-induced fibrin clot retraction. Thrombin stimulation markedly enhanced clot retraction, while Chrysoeriol significantly and concentration-dependently suppressed fibrin clot retraction (Fig. 7). These findings indicate that Chrysoeriol inhibits thrombin-induced, platelet-mediated fibrin clot retraction, contributing to its overall antithrombotic effect.

Fig. 7.

Chrysoeriol’s inhibitory effect on platelet-mediated clot retraction. (A) Chrysoeriol’s suppression of fibrin clot retraction induced by thrombin. (B) Chrysoeriol’s reduction of clot retraction area. Data are presented as mean ± SD (n=4). Tube diameter: 5 mm. Statistical significance is indicated by ^ap<0.05 compared with non-stimulated platelets, and *p<0.05 and **p<0.01 compared with thrombin-stimulated platelets.

Network pharmacology and in silico drug-likeness prediction of chrysoeriol

To elucidate the potential molecular mechanisms underlying the antiplatelet effects of chrysoeriol, we performed a network pharmacological analysis. Venn analysis revealed 17 overlapping targets between chrysoeriol-predicted proteins and platelet activation-related genes, suggesting a direct mechanistic link (Fig. 8A). Protein–protein interaction (PPI) network analysis identified key hub proteins, including AKT1, PIK3R1, EGFR, SRC, and GSK3B, all of which are central regulators of platelet signaling, adhesion, integrin activation, and cytoskeletal remodeling (Fig. 8B). Particularly, SRC plays a critical role in outside-in signaling via integrin αIIb/β₃ and downstream phosphorylation of FAK and PI3K, essential for thrombus formation (Durrant et al., 2017; Senis et al., 2014). Pathway enrichment using Cytoscape indicated strong involvement of these targets in multiple relevant pathways such as platelet activation, PI3K/Akt signaling, MAPK signaling, and arachidonic acid metabolism, aligning well with our experimental findings (Fig. 8C). The SwissADME analysis further confirmed the drug-likeness of chrysoeriol, showing no violations of major drug-likeness rules, high gastrointestinal absorption, and acceptable water solubility. The BOILED-Egg model also predicted favorable intestinal permeability without blood–brain barrier penetration (Fig. 8D). The compound is moderately soluble and easily synthesizable, with no violation of Lipinski’s rule of five and no structural alerts for pan-assay interference compounds (PAINS) (Fig. 8E). These findings collectively support chrysoeriol as a multi-target modulator of key thrombo-inflammatory pathways in platelets, offering a scientific rationale for its potential as a natural antiplatelet therapeutic agent.

Fig. 8.

Network pharmacology and in silico prediction of chrysoeriol targets associated with platelet activation. (A) Venn diagram showing the overlap between predicted chrysoeriol targets (22 proteins) and platelet activation-related genes (5218 genes from GeneCards), revealing 17 common targets. (B) Protein–protein interaction (PPI) network of the 17 overlapping genes constructed using the STRING database. Key hub proteins such as AKT1, PIK3R1, EGFR, SRC, and PTK2 are highlighted due to their central roles in platelet signaling. (C) Compound–target–pathway network generated using Cytoscape, illustrating the connections between chrysoeriol overlapping protein targets, and enriched KEGG pathways. Major pathways include platelet activation, PI3K-Akt signaling, MAPK signaling, arachidonic acid metabolism, and focal adhesion. (D) BOILED-Egg model from SwissADME, predicting chrysoeriol’s passive gastrointestinal absorption and lack of blood–brain barrier permeability based on WLOGP and topological polar surface area (tPSA). (E) Bioavailability radar plot showing chrysoeriol’s physicochemical properties within the optimal range for drug-likeness across six key descriptors (lipophilicity, size, polarity, solubility, saturation, and flexibility).

DISCUSSION

Chrysoeriol, a naturally derived flavonoid, has been noted for its anti-inflammatory, antioxidant, and anticancer activities (Kim et al., 2019). Prior studies have indicated that Chrysoeriol suppresses platelet aggregation by inhibiting the TXA₂ pathway (Lee et al., 2023) but the detailed molecular mechanisms behind this effect remain largely unexplored. In the present study, we sought to elucidate the intracellular signaling events modulated by chrysoeriol that contribute to its antiplatelet activity.

Under physiological conditions, platelet quiescence is primarily maintained by endothelial-derived prostacyclin (PGI₂) and nitric oxide (NO), which activate intracellular cAMP–PKA and cGMP–PKG signaling pathways, respectively (Gambaryan, 2022). These pathways converge on the phosphorylation of IP₃R and IRAG, suppressing intracellular Ca²⁺ release and downstream platelet activation (Shevchuk et al., 2021). In contrast, upon vascular injury, collagen becomes exposed and binds to the glycoprotein VI (GPVI) receptor on platelets, initiating a cascade involving the FcRγ chain and PLCγ2 that leads to Ca²⁺ mobilization. This signal is amplified by PI3K/Akt and MAPK pathways, resulting in integrin αIIb/β₃ activation, granule secretion, and thrombus formation (Nieswandt and Watson, 2003). As our study employed collagen as the primary agonist to stimulate platelet activation, these signaling pathways were considered the key targets to evaluate the inhibitory effects of chrysoeriol.

Chrysoeriol significantly elevated intracellular cAMP and cGMP levels, leading to downstream activation of PKA and PKG, respectively. This activation was accompanied by enhanced phosphorylation of IP₃R, which suppresses intracellular Ca²⁺ mobilization and platelet activation. The rapid onset of cyclic nucleotide accumulation—within 3 min—suggests that chrysoeriol may act through direct enzymatic modulation rather than gene expression. While the exact mechanism remains to be clarified, chrysoeriol may stimulate adenylyl cyclase (AC) or soluble guanylyl cyclase (sGC) activity and/or inhibit phosphodiesterases (PDEs), enzymes responsible for cAMP and cGMP degradation. This possibility is supported by the fact that Chrysoeriol is the 3′-O-methylated derivative of luteolin, sharing both structural and functional similarities with its parent compound (Aboulaghras et al., 2022). Given that luteolin and other flavonoids are known to inhibit phosphodiesterase (PDE) activity, chrysoeriol may similarly act as a PDE inhibitor, thereby sustaining intracellular cyclic nucleotide levels (Ko et al., 2004). Clinically used PDE inhibitors such as dipyridamole and cilostazol also support the pharmacological relevance of targeting this pathway (Li et al., 2010; Maurice et al., 2014). Building on this possibility, we directly compared chrysoeriol with the PDE inhibitors dipyridamole and cilostazol in collagen-induced platelet aggregation. At matched concentrations (32 and 64 μM), chrysoeriol exerted stronger inhibition than dipyridamole and showed a comparable level of inhibition to cilostazol at 64 μM. Although this comparison does not directly establish PDE inhibition by chrysoeriol, the similar inhibitory effects observed under collagen-induced aggregation are consistent with the rapid elevation of cAMP/cGMP found in our study. Collectively, these results support the possibility that PDE-related mechanisms may contribute to the antiplatelet action of chrysoeriol.

VASP phosphorylation at Ser¹⁵⁷ (PKA target) and Ser²³⁹ (PKG target) was significantly increased by chrysoeriol, supporting functional activation of cyclic nucleotide signaling. This modification is known to interfere with fibrinogen binding to integrin αIIb/β₃, thereby attenuating platelet aggregation (Napeñas et al., 2013; Wentworth et al., 2006). Consistently, flow cytometric analysis revealed that chrysoeriol suppressed fibrinogen binding in a dose-dependent manner, indicating inhibition of inside-out signaling and integrin activation.

Chrysoeriol inhibited the phosphorylation of key kinases upstream of TXA₂ synthesis, including PI3K, Akt, JNK, and p38 MAPK, which regulate cytosolic phospholipase A₂ (cPLA₂) activation. Correspondingly, cPLA₂ phosphorylation was reduced, leading to decreased TXA₂ production. Although ERK phosphorylation was minimally affected, this is consistent with prior findings that ERK plays a limited role in collagen-induced platelet activation (Irfan et al., 2019). Moreover, ERK is known to undergo transient activation within 1-3 min post-stimulation (Li et al., 2006). Our 5-minute sampling time may have missed this transient peak, but previous studies using identical timing have successfully detected ERK inhibition, supporting the methodological validity of our approach (Choi et al., 2024). Nevertheless, to gain a clearer understanding of ERK’s time-dependent changes in collagen-stimulated platelets, future studies could assess their phosphorylation at shorter intervals (e.g., 1, 3, and 5 min) to capture any potential transient activation peak.

Additionally, our data showed that chrysoeriol significantly decreased the secretion of ATP and serotonin, two granule-stored agonists that further potentiate platelet aggregation. This suggests that chrysoeriol not only inhibits early activation signaling but also limits downstream amplifying responses that sustain thrombus growth. Recent studies have elucidated the complexity of these pathways, highlighting the dynamic regulation of platelet inhibition through cAMP/PKA and cGMP/PKG signaling cascades (Hsia et al., 2024).

To identify potential molecular targets through which chrysoeriol exerts its antiplatelet effects, a network pharmacology analysis was conducted. In our case, the analysis revealed 17 overlapping targets between predicted chrysoeriol-binding proteins and genes associated with platelet activation. These targets were not only highly interconnected in the PPI network but also mapped to key signaling pathways such as PI3K-Akt, MAPK, cAMP, and integrin-mediated outside-in signaling. These are well-established regulators of platelet function, and their involvement strengthens the mechanistic plausibility of chrysoeriol’s antiplatelet effects. Furthermore, pharmacokinetic predictions confirmed that chrysoeriol exhibits drug-like properties, including high gastrointestinal absorption and lack of toxicity alerts, enhancing its therapeutic potential. Although these computational approaches cannot replace experimental validation, they provide a comprehensive mechanistic framework that supports and deepens our in vitro observations. Overall, the network pharmacology analysis complements our experimental data by confirming that chrysoeriol exerts its antiplatelet activity through multi-target modulation of key signaling pathways. This integrative approach reinforces the robustness of our mechanistic understanding and highlights chrysoeriol as a promising candidate for further development as an antiplatelet agent.

Pharmacokinetic studies in rats have shown that the plasma concentration of chrysoeriol reaches approximately 0.3 µM after oral administration (Chen et al., 2012). Although these data provide useful reference information for understanding in vivo concentrations, the concentrations used in our in vitro experiments (8-64 µM) were not intended to reflect achievable systemic levels. Rather, they were selected to investigate the intrinsic antiplatelet activity of chrysoeriol under conditions free from metabolic and protein-binding influences. In this context, our findings provide an experimental basis demonstrating that chrysoeriol itself possesses potential antiplatelet properties, serving as a starting point for further pharmacological development. Determining the concentrations that can produce similar effects in vivo remains an important task for future research. Therefore, additional pharmacokinetic and pharmacodynamic studies, such as monitoring plasma levels and platelet reactivity in animal models, will be necessary to bridge the gap between the in vitro findings and their in vivo relevance.

In conclusion, chrysoeriol enhanced intracellular cAMP and cGMP concentrations in human platelets, leading to the phosphorylation of IP3R and VASP. This process effectively attenuated cytosolic Ca²⁺ mobilization and suppressed the activation of integrin αIIb/β3. Furthermore, chrysoeriol demonstrated antiplatelet potential by suppressing the phosphorylation of key signaling proteins, including those in the PI3K/Akt and MAPK cascades, thereby reducing granule secretion. It also markedly reduced thrombin-driven fibrin clot formation (Fig. 9). Collectively, these findings indicate that chrysoeriol could serve as a promising candidate for preventing platelet aggregation via regulation of these signaling pathways.

Fig. 9.

Schematic diagram of the antithrombotic mechanisms of chrysoeriol. Chrysoeriol increases cAMP and cGMP levels, activating PKA and PKG, which reduces intracellular Ca²⁺ and inhibits MLC phosphorylation, disrupting platelet aggregation and clot retraction. It also promotes VASP phosphorylation, which reduces the affinity between integrin αIIbβ3 and fibrinogen. Furthermore, chrysoeriol suppresses PI3K/Akt and MAPK pathways, reducing granule secretion (ATP, serotonin) and TXA₂ synthesis, which also contributes to the inhibition of platelet aggregation and clot retraction. Created with BioRender.com