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Applied Biological Chemistry
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Anti-thrombotic effects of arteanoflavone by regulating cyclic nucleotides and aggregation on human platelets

Applied Biological Chemistry volume 67, Article number: 61 (2024) Cite this article

Abstract

Excessive clotting or abnormal platelet accumulation can lead to serious cardiovascular disorders such as atherosclerosis, stroke, and thrombosis. Therefore, it is imperative to identify compounds capable of controlling or impeding platelet aggregation to prevent the onset of cardiovascular diseases. Arteanoflavone, a compound extracted from Artemisia iwayomogi, has not garnered scientific recognition for its potential health benefits, recent studies have substantiated its anti-inflammatory, antioxidant, and anti-allergic properties. However, the precise mechanisms by which arteanoflavone influences platelet aggregation and blood clot formation have not been conclusively established. This research investigates arteanoflavone’s role in these processes, particularly in platelets induced by collagen. The study reveals a significant increase in the production of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) correlating with the administered dosage of arteanoflavone. Concurrently, a noticeable escalation is observed in substrates of cAMP-dependent kinase and cGMP-dependent kinase, specifically VASP and inositol 1,4,5-trisphosphate receptor (IP3R).

Arteanoflavone demonstrates its ability to limit Ca2+ movement in the dense tubular system through IP3R phosphorylation. Moreover, phosphorylated VASP inhibits the binding of fibrinogen to αIIb/β3, thus suppressing platelet activity. Arteanoflavone also stimulates the phosphorylation of PI3K/Akt, a protein linked to platelet granule release, and MAPK (ERK, JNK, and p38) protein, associated with both platelet granule release and TXA2 production.

Lastly, arteanoflavone impedes collagen-induced platelet aggregation and blood clot formation by inhibiting fibrin production in thrombin-induced platelets. Hence, it is suggested that arteanoflavone could be valuable as an agent that effectively deters platelet inhibition and blood clot formation through antiplatelet mechanisms.

Introduction

Cardiovascular disease stands as the primary reason for death worldwide, with conditions such as coronary heart disease being responsible for one-seventh of all deaths, while heart failure contributes to one-ninth of all fatalities, focusing solely on the United States [1]. During vascular damage, platelets are crucial for maintaining hemostasis by aiding in the creation of clots to halt bleeding. Yet, if platelet activation and accumulation are not properly controlled, it can result in the undesired generation of blood clots. These blood clots bear a significant association with the development of cardiovascular issues such as atherosclerosis, disease of the coronary artery, heart failure, and cerebrovascular accidents, also known as strokes [2]. As such, managing the activation of platelets is crucial in limiting their aggregation, which becomes a significant factor in preventing and treating cardiovascular diseases.

Pharmacological compounds used in the treatment of thrombotic complications primarily act by inhibiting platelets, and while they are effective in current cardiovascular disease (CVD) prevention and treatment, their usage is associated with significant complications and side effects [3]. Recognizing these challenges, there is growing interest in exploring alternatives, an one promising approach involves the use of natural bioactive compounds [4]. Compounds derived from the traditional Mediterranean diet and plants with a history of medicinal use have demonstrated antiplatelet and cardioprotective properties, presenting a potential avenue for more effective and safer CVD prevention [5,6,7].

Inositol 1,4,5-trisphosphate (IP3), a compound originating from phospholipids found in platelet membranes, influences the Ca2+ reservoir (dense tubular system) within platelets to heighten the release of Ca2+ into the cytosol. Increased Ca2+ levels trigger the functioning of the Ca2+-dependent protein kinase PKC and the Ca2+/calmodulin-dependent enzyme known as myosin light chain (MLC) kinase (MLCK). This ultimately leads to the phosphorylation process involving MLC. The phosphorylation of MLC triggers morphological transformations in platelet cells, thus instigating platelet aggregation. This process, however, can be hindered by cyclic nucleotides cAMP and cGMP, which function as secondary messengers. cAMP instigates A-kinase, whereas cGMP spurs G-kinase, and both enzymes orchestrate the phosphorylation of the IP3 channel receptor, subsequently constraining the release of calcium into the cytosol [8]. Furthermore, by diminishing the attraction between fibrinogen and Glycoprotein IIb/IIIa (αIIb/β3), a receptor known for promoting platelet aggregation via its binding to fibrinogen, A-kinase/G-kinase efficiently impede platelet aggregation [9]. The activation of αIIb/β3 is also hindered by the phosphorylation of VASP at Ser157, influenced by cAMP, and at Ser239, guided by cGMP. Thus, in the evaluation of substances with antithrombotic characteristics, it’s vital to assess their impact on cAMP/cGMP-dependent signaling target molecules.

During platelet activation, a variety of substances discharged from platelet granules serve to amplify platelet aggregation. Fibrinogen and serotonin are prime examples of such substances. The binding of collagen to platelet membrane receptors stimulates the PI3K/Akt pathway, which is instrumental in the release of granule substances and the ensuing platelet aggregation [10]. Also, MAPKs (ERK, JNK, and p38) function as intracellular signal transducers when platelets are activated. The phosphorylation of MAPKs triggers granule secretion and bolsters platelet aggregation [11]. Evaluating the influence of a substance on PI3K/Akt and MAPK phosphorylation can contribute to determining its antithrombotic potential.

Arteanoflavone, derived from Artemisia iwayomogi, is a member of the flavonoid family [12]. A. iwayomogi has exhibited a wide array of biological traits, including anti-inflammatory, antioxidant, anti-allergic, anti-obesity, and hepatoprotective properties against CCl4-induced hepatic fibrosis [13,14,15,16,17]. However, no studies to date have confirmed the antithrombotic and antiplatelet effects of either A. iwayomogi or arteanoflavone, nor their underlying processes. Flavonoids, recognized for their positive impacts on cardiovascular health, have drawn interest for potential therapeutic applications [18]. Some flavonoids have shown to impede platelet aggregation, and their antithrombotic activity is believed to be linked to their binding to cellular receptors [19, 20]. Thus, the goal of our research was to investigate the potential antiplatelet and antithrombotic characteristics of arteanoflavone, a flavonoid isolated from A. iwayomogi, and decipher the mechanism by which arteanoflavone regulates platelet aggregation in human platelets.

Methods

Platelet suspension production and preparation

The Korean Red Cross Blood Center, located in Suwon, South Korea, supplied the human platelet-rich plasma (PRP) used in this study. Following the methods of previous studies, The PRP was subjected to centrifugation at a speed of 1,300 x g for a total of 10 min to aid in platelet collection [21]. Post-centrifugation, the harvested platelets were rinsed twice with a buffer solution. The washing buffer used in this study had the following composition: 2.7 mM of potassium chloride (KCl), sodium chloride (NaCl) at 138 mM, 12 mM of sodium bicarbonate (NaHCO3), 0.36 mM of sodium dihydrogen phosphate (NaH2PO4), and 1 mM of ethylenediaminetetraacetic acid (Na2EDTA), and 5.5 mM of glucose, all balanced to achieve a pH of 6.9. Once the washing phase was completed, the platelets were subsequently suspended. The suspension buffer was a concoction of 2.7 mM KCl, 138 mM NaCl, 12 mM NaHCO3, 0.49 mM of magnesium chloride (MgCl2), 0.36 mM NaH2PO4, and 5.5 mM glucose, 0.25% gelatin, all adjusted to maintain a pH of 7.4. All platelets were then suspended in the previously mentioned buffer solution, achieving a final platelet concentration of 108 cells/mL. A constant temperature of 25 °C was maintained throughout the procedure to avert unneeded platelet aggregation. The utilization of all human biological materials, inclusive of the PRP, was approved by the Committee of Journalists (1,041,479-HR-202110-002) at Namseoul University, Korea.

Analysis of aggregation in platelet samples

The platelet suspension, with a concentration of 108 cells/mL, was incubated with varying dosages of arteanoflavone. This suspension was held at a temperature of 37 °C for a span of 3 min. Upon completion of the incubation period, 2 mM CaCl2 was added to the platelet suspension. To trigger the aggregation response, collagen was introduced into the suspension and the reaction was permitted to continue for 5 min. The measurement of platelet aggregation was conducted using an aggregometer from Chrono-Log Co., based in Havertown, Pennsylvania, USA. The aggregometer’s stirring speed was maintained at 1000 rpm. The aggregation degree was ascertained by the observed increase in light transmittance during the reaction process. Since light transmittance is inversely proportional to platelet aggregation, a surge in light transmittance signifies a reduction in platelet aggregation. A suspension buffer with 0% transmittance served as the reference point for the control value. Dimethyl sulfoxide (DMSO) was used at a concentration of 0.1% to dissolve arteanoflavone. The same DMSO concentration was used across all experiments.

Measurement of cytotoxicity

The cytoplasmic existence of lactate dehydrogenase (LDH) serves as a trustworthy cytotoxicity marker. A concoction of platelets was formulated at a concentration of 108 cells/mL and subjected to a room temperature incubation period lasting two hours. Various doses of arteanoflavone were introduced to the suspension during this incubation period. Once the incubation was completed, the platelet solution was centrifuged at a speed of 12,000 x g for a duration of 2 min. The supernatant obtained post-centrifugation was then collected for further analysis. An LDH Enzyme Immunoassay (EIA) kit was employed to assess the LDH levels within the supernatant. The task of measuring the LDH concentration was conducted with the help of a multi-reader instrument known as Synergy HT, an innovation from BioTek Instruments, based in the town of Winooski in Vermont, USA.

Measurement of cyclic nucleotide production (cAMP and cGMP)

Platelets were prepared in a suspension, with a density of 108 cells/mL, and were treated with differing quantities of arteanoflavone. The suspension was then held at a constant temperature of 37 degrees Celsius for an interval of 3 min. After this period of incubation, the suspension was exposed to 2 mM CaCl2, succeeded by the introduction of collagen to prompt platelet stimulation. The procedure was sustained for five minutes before being abruptly halted by the infusion of 1 M HCl. After this interruption, a unique Enzyme Immunoassay (EIA) kit, which specializes in the analysis of cyclic nucleotides (cAMP and cGMP), was utilized to determine their concentrations. The quantification of these cyclic nucleotides was performed utilizing the Synergy HT multi-reader, an instrument developed by BioTek, based in Winooski, Vermont, USA.

Assessment of intracellular calcium movement

In this study, Fura 2-AM, a common fluorescent marker for cytosolic calcium, was utilized. The procedure involved creating a concoction containing 5 µM Fura 2-AM and platelet-rich plasma (PRP), which was subsequently allowed to incubate for an hour at a temperature of 37 °C. This process allowed for the Fura 2-AM to be effectively loaded into the platelets. As per earlier description, a suspension of platelets was arranged, cleansed, and then maintained at 37 °C for a span of 3 min with the supplement of 2 µM CaCl2. Following this, collagen was incorporated into the suspension for the purpose of inciting the platelets, and this provocation was permitted to last for a duration of 5 min. The luminosity of Fura 2 was quantified using an SFM 25 spectrophotometer, produced by the BioTek company based in the United States, specifically in Winooski, Vermont. The device was configured to initiate the wavelength scan from 340 nm, proceeding onwards to 380 nm in increments of 0.5 s, with the detection of fluorescence emission set at 510 nm. The calculation of the released calcium concentration was determined from the fluorescence readings of Fura 2, utilizing the Grynkiewicz formula, which connects the ratio of fluorescence to the concentration of calcium. This equation allows the conversion of fluorescence ratios into meaningful units of calcium concentration ([Ca2+]i) in the cytosol, providing a quantitative measurement of intracellular calcium mobilization.

Fibrinogen binding measurement

The interaction between fibrinogen and platelets is a pivotal process in the accumulation of platelets, a process that can be observed using flow cytometry and fibrinogen marked with fluorescence. For this specific procedure, a suspension of human platelets was prepared with a cell concentration of 108/mL, and was subsequently subjected to a 2 mM CaCl2 treatment. This was followed by the introduction of Alexa Fluor 488-human fibrinogen, a variant of fibrinogen labeled with a fluorescent marker, into the suspension of platelets. The platelets were then stimulated with collagen, a process that induces platelet activation and fibrinogen binding, and allowed to react for 5 min. The reaction was stopped using phosphoric acid-buffered saline (PBS) at pH 7.4, and the platelets were subsequently fixed using 0.5% paraformaldehyde to preserve the current state of the cells for analysis. Flow cytometry was utilized to monitor the interaction between fibrinogen and the platelets. The level of detected fluorescence is directly linked to the quantity of Alexa Fluor 488-human fibrinogen that has bound to the platelets, offering a method to quantify the degree of fibrinogen’s interaction with platelets. The flow cytometry was conducted using equipment provided by BD Biosciences in San Jose, California, USA. The data generated from the flow cytometry were analyzed using the Cell-Quest software, a program specifically designed for processing flow cytometry data.

TXB2 production measurement

During this study, a suspension consisting of 108 cells/mL of human platelets was formulated. It was then subjected to varying concentrations of arteanoflavone and incubated for 3 min at a temperature of 37 °C. To trigger activation, the platelets were subsequently exposed to collagen. This activation, in turn, prompts the platelets to produce TXA2 (and consequently TXB2). After allowing the reaction to proceed for 5 min, an enzyme immunoassay (EIA) specific for TXB2 was used to quantify the level of TXB2 production, which reflects the amount of TXA2 produced by the platelets.

This assay is highly specific and sensitive, and allows for the detection of very small amounts of TXB2. Measurements were carried out using the Synergy HT Multi-Reader, a device made by BioTek in Winooski, Vermont, USA, that can perform multiple types of readings, including absorbance and fluorescence.

Evaluating the release of ATP and serotonin

In this investigation, the discharge of ATP and serotonin from platelets was quantified. A human platelet suspension was initially created at a density of 108 cells/mL. These platelets underwent treatment with assorted quantities of arteanoflavone and were subsequently maintained at 37 °C for a span of 3 min. Post incubation, a solution of 2 mM CaCl2 was introduced to stimulate calcium-dependent signaling routes, while collagen was utilized to prompt platelet activation. Post a 5-minute reaction interval, the process was terminated by incorporating 2 mM EDTA in chilled water. EDTA serves the function of a chelating agent that associates with Ca2+ ions, thereby disrupting Ca2+-reliant signaling and effectively putting a halt to the reaction. The mixture was then centrifuged to separate the platelets from the supernatant, which contains the released serotonin and ATP. Enzyme immunoassay (EIA) kits specific for ATP and serotonin were used to quantify the amount of these molecules present in the supernatant, thus indicating the degree of platelet activation and granule release. The measurements were conducted using a Synergy HT Multi-Reader, a device made by BioTek, based in Winooski, Vermont, USA, capable of absorbance and fluorescence readings.

Western immunoblotting measurement

This procedure describes the steps involved in Western blot analysis, a common technique used to detect specific proteins in a sample. After the reaction was stopped with a 1X dissolution buffer, the platelets were lysed to release the proteins inside them. Protein concentrations in the lysate were quantified using a bicinchoninic acid (BCA) protein assay kit provided by Pierce Biotechnology. Subsequently, a total of 20 µg of protein was fractionated utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a method dedicated to segregating proteins by their molecular mass. Proteins, once separated, were relocated onto polyvinylidene difluoride (PVDF) membranes, offering a consistent platform for additional evaluations. Following this, the membranes were subjected to an incubation process involving a primary antibody, designed to latch onto the target protein under investigation. A secondary antibody, which can bind to the primary antibody, was then added. In the final stage, an enhanced chemiluminescence (ECL) reagent, courtesy of Thermo Fisher Scientific, was employed to reveal the bands of protein on the membranes.

Determination of platelet-mediated fibrin thrombus formation

Thrombin, a potent enzyme in the blood clotting process, was added to the PRP along with CaCl2 to induce the formation of a fibrin thrombus. The use of polyethylene tubes is crucial here because it reduces the chances of unwanted clotting due to the tube surface, which could interfere with the experimental results. The mixture was allowed to react at 37 °C, approximately human body temperature, for a quarter of an hour to facilitate the formation of thrombi. Afterward, the resulting fibrin clots were imaged and documented using a digital photography device. The area of the clot, which can provide a measure of the clot’s size and therefore the extent of clotting, was calculated using ImageJ software. ImageJ is a freely available, open-source image analysis software developed by the National Institutes of Health (NIH).

Analysis of statistical data

The outcomes of all experiments are communicated as the average ± standard deviation. The t-test by Student or ANOVA was employed to ascertain statistical significance in the results, with a P value under 0.05 denoting significance. If ANOVA revealed notable differences in the means across groups, we carried out additional analysis using Scheffe’s post hoc test.

Results

Influence of arteanoflavone on aggregation and cytotoxicity in human platelets

Aggregation reactions were investigated in human platelets stimulated by collagen (2.5 µg/mL). Collagen-induced platelet aggregation was recorded at 77.5%, which demonstrated a significant aggregation reaction. The addition of arteanoflavone dose-dependently inhibited these platelet aggregation reactions. Arteanoflavone was purchased from Avention Co., Ltd. (CAS Number: 68710-17-8) and was extracted from Artemisia iwayomogi, and HPLC & H-NMR analysis confirmed 97% purity. The levels of aggregation inhibition directly correlated with increasing doses of arteanoflavone, with inhibition percentages as follows: 5.8% at 60 µM, 23.5% at 120 µM, 59.7% at 180 µM, and 89.7% at 240 µM. As a result, the IC50 of arteanoflavone was confirmed to be about 142.6 μm, and this result was confirmed to be similar to or lower than that of flavonoids derived from plants of the Artemisia genus whose platelet aggregation effect has been confirmed [22,23,24,25]. Furthermore, arteanoflavone did not exhibit any cytotoxic effects on human platelets. Consequently, arteanoflavone effectively inhibits platelet aggregation reactions induced by collagen (2.5 µg/mL) without triggering undesired cytotoxicity (Fig. 1D).

Fig. 1
figure 1

Arteanoflavone’s effect on agonists-induced human platelet aggregation. (A) The structure of arteanoflavone (B) Arteanoflavone’s effect on collagen-induced human platelet aggregation. (C) The IC50 value of arteanoflavone on platelet aggregation. (D) Cytotoxicity of arteanoflavone. The results are presented as mean ± SD (n = 4). *p < 0.05, **p < 0.001 compared to the collagen-induced platelet suspension

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Arteanoflavone’s influence on the formation of cyclic nucleotides, intracellular Ca2+ shifts, and phosphorylation of IP3R

The pivotal function of cyclic nucleotides (cGMP and cAMP) in impeding platelet activation is well-established. This study delved into the capacity of arteanoflavone to boost the synthesis of these specific cyclic nucleotides (cGMP and cAMP). In the presence of arteanoflavone, collagen-triggered platelets showed a proportionate upsurge in the generation of cGMP and cAMP. This observation suggests arteanoflavone’s potential to thwart platelet activation by escalating the levels of cGMP and cAMP in platelets activated by collagen (Fig. 2A). Additionally, when collagen-stimulated platelets were pretreated with arteanoflavone, there was a significant phosphorylation of the IP3 receptor (Fig. 2C). This corresponded with a substantial reduction in the mobilization of Ca2+ that was previously enhanced by collagen (Fig. 2B). Thus, arteanoflavone appears to inactivate the Ca2+ channel via the phosphorylation of the IP3 receptor, leading to the suppression of Ca2+ mobilization.

Fig. 2
figure 2

Arteanoflavone’s effect on cAMP/cGMP production, intracellular Ca2+ mobilization, and related phophoprotein. (A) Arteanoflavone’s effect on cAMP/cGMP production. (B) Effects of Arteanoflavone on intracellular Ca2+ mobilization. (C) Effects of Arteanoflavone on IP3R phosphorylation. (D) Arteanoflavone’s effect on VASP phosphorylation. The results are presented as mean ± SD (n = 4). ap < 0.05 compared to non-stimulated platelet suspension, *p < 0.05, **p < 0.001 compared to the collagen-induced platelet suspension

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Exploring the effect of arteanoflavone on the phosphorylation of VASP and fibrinogen adherence

Given the finding that the introduction of arteanoflavone to collagen-stimulated platelets increases cGMP and cAMP, an investigation was conducted to understand its impact on cGMP and cAMP-dependent VASP. With escalating doses of arteanoflavone, the phosphorylation of VASP, influenced by cAMP, at the Ser157 position, showed a surge in a concentration-dependent manner (Fig. 2D). Correspondingly, noteworthy shifts were witnessed in the phosphorylation at the Ser239 location in VASP reliant on cGMP. The changes were notably significant with doses of 60 µM or more. This suggests that the rise in cAMP/cGMP instigated by arteanoflavone contributes to a marked amplification in the phosphorylation at the Ser157 site in cAMP-responsive VASP and at the Ser239 site in cGMP-responsive VASP. Considering arteanoflavone’s function in increasing the production of cAMP/cGMP and phosphorylation at the Ser157 site of cAMP-responsive VASP as well as the Ser239 site of cGMP-responsive VASP, further studies were performed to scrutinize its influence on fibrinogen’s interaction with αIIb/β3. With collagen being present, the bond between fibrinogen and αIIb/β3 spiked to an average of 92.1 ± 1.4%. However, the inclusion of arteanoflavone led to a suppression of fibrinogen binding to αIIb/β3, displaying a dose-dependent relationship where higher doses of arteanoflavone corresponded to greater levels of inhibition (Fig. 3). Among the tested doses, a significant inhibition rate of 93.3% was achieved with 120 µM of arteanoflavone.

Fig. 3
figure 3

Arteanoflavone’s effects on the binding of fibrinogen. (A) The histograms of flow cytometry on the binding of fibrinogen. a: Intact platelets(base), b: collagen, c: collagen + Arteanoflavone (60 µM), d: collagen + Arteanoflavone (120 µM), e: collagen + Arteanoflavone (180 µM), f: collagen + Arteanoflavone (240 µM). (B) Arteanoflavone’s effect on the binding (%) of fibrinogen induced by collagen. The results are presented as mean ± SD (n = 4). ap < 0.05 compared to non-stimulated platelet suspension, *p < 0.05, **p < 0.001 compared to the collagen-induced platelet suspension

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Alterations in MAPK and PI3K/Akt phosphorylation induced by arteanoflavone

The influence of arteanoflavone on the phosphorylation of the protein PI3K/Akt, which plays a role in platelet granule release, was examined. In platelets stimulated with collagen, the phosphorylation levels of both PI3K and Akt notably elevated. Nonetheless, arteanoflavone pretreatment caused a substantial decrease in PI3K and Akt phosphorylation (Fig. 4A). Additionally, the impact of arteanoflavone on MAPK proteins - p38, ERK, and JNK, which are recognized to be involved in the release of platelet granules and production of TXA2, was also explored. Their phosphorylation levels displayed a significant increase in collagen-stimulated platelets. Nonetheless, the phosphorylation of these MAPK proteins (p38, ERK, and JNK) was noticeably suppressed in the presence of arteanoflavone (Fig. 4B). This observation affirms that arteanoflavone impacts the phosphorylation of PI3K/Akt and MAPK proteins (ERK, JNK, p38) in collagen-stimulated platelets.

Fig. 4
figure 4

Arteanoflavone’s effect on the phosphorylation of PI3K/Akt and MAPK. (A) Arteanoflavone’s effect on PI3K/Akt phosphorylation. (B) Arteanoflavone’s effect on MAPK phosphorylation. The results are shown as mean ± SD (n = 4). The results are presented as mean ± SD (n = 4). ap < 0.05 compared to non-stimulated platelet suspension, *p < 0.05, **p < 0.001 compared to the collagen-induced platelet suspension

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Effect of arteanoflavone on granule release from human platelets

The discharge of granules from platelets, particularly ATP and serotonin, is intricately linked to the aggregation process of platelets. Thus, an investigation was conducted to determine how arteanoflavone impacts granule release. In platelets stimulated by collagen (2.5 µg/mL), there was a notable increase in the release of ATP from 0.2 µM to 4.5 µM, and serotonin release rose from 20 ng/108 to approximately 150 ng/108. However, it was observed that the release of both ATP and serotonin was inhibited with escalating concentrations of arteanoflavone (Fig. 5). Therefore, the finding suggests that arteanoflavone’s inhibitory action on the release of granules containing ATP and serotonin is linked to its ability to mitigate platelet aggregation and thrombosis.

Fig. 5
figure 5

Arteanoflavone’s effect on the secretion of granules, TXA2 production and cPLA2 phosphorylation. (A) Arteanoflavone’s effect on ATP release. (B) Arteanoflavone’s effect on the release of serotonin. (C) Arteanoflavone’s effect on TXA2 production. (D) Arteanoflavone’s effect on cPLA2 phosphorylation. The results are presented as mean ± SD (n = 4). ap < 0.05 compared to non-stimulated platelet suspension, *p < 0.05, **p < 0.001 compared to the collagen-induced platelet suspension

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Effect of arteanoflavone on platelet-mediated fibrin coagulation

Fibrin clot formation initiates in platelets via activation and aggregation, prompting signals from external pathways. In another study that confirmed the efficacy of platelets, it was confirmed that the effect on clot retraction was thrombin induction [26]. Because the use of thrombin rather than collagen is more effective in inducing coagulation, we investigated the formation of fibrin coagulation induced by thrombin. Consequently, an experiment was carried out to investigate the impact of arteanoflavone on platelet aggregation induced by thrombin. When platelets were exposed to thrombin, fibrin clots readily formed. However, the introduction of varying doses of arteanoflavone exhibited a concentration-dependent reduction in fibrin clot formation, as verified through experimental procedures (Fig. 6). This observation suggests that arteanoflavone is an effective agent in impeding the formation of thrombi.

Fig. 6
figure 6

Arteanoflavone’s effect on platelet-mediated fibrin clot formation. (A) Arteanoflavone’s effect on thrombin-retracted fibrin clot photographs. (B) Arteanoflavone’s effect on thrombin-retracted fibrin clot area. The results are presented as mean ± SD (n = 4). ap < 0.05 compared to non-stimulated platelet suspension, *p < 0.05, **p < 0.001 compared to the thrombin-induced platelet suspension

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Discussion

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the platelet cell membrane is subjected to the action of phospholipase C-γ2 (PLC-γ2). As a result, inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DG) are independently produced [27]. IP3 initiates the discharge of calcium (Ca2+) from the dense tubular system into the cytoplasm. This available calcium contributes to the phosphorylation of myosin light chains and focal adhesion proteins, crucial steps in platelet aggregation [28].

Protein kinases like protein kinase A (PKA) and protein kinase G (PKG) that are dependent on cyclic nucleotides are activated by cAMP and cGMP. These activated kinases play key regulatory roles in platelet aggregation. Interestingly, cAMP and cGMP, apart from their kinase activation role, also contribute to reducing intracellular calcium levels [29]. Our study corroborates that arteanoflavone significantly augments the production of cyclic nucleotides cAMP and cGMP and inhibits the accumulation of intracellular calcium [Ca2+]i. The elevation of cAMP and cGMP contributes to PKA activation, leading to the phosphorylation of a series of substrates, including inositol 1,4,5-triphosphate receptors (IP3R) [30]. With the addition of arteanoflavone, the phosphorylation of IP3R increases dose-dependently, as demonstrated in Fig. 3B. The cAMP/PKA/IP3R pathway is stimulated by arteanoflavone, resulting in reduced levels of intracellular calcium. Also, this compound enhances the phosphorylation of vasodilator-stimulated phosphoproteins (VASPs) via increased production of cAMP and cGMP, facilitated by PKA activation. VASP is crucial for PKA and PKG functioning, and it adjusts platelet activation through the regulation of secretion and adhesion of platelets. Consequently, the inactivation of integrin αIIb/β3 occurs through the phosphorylation of VASP, which inhibits platelet aggregation [31, 32].

Upon considering these results, the conclusion was drawn that αIIb/β3’s ability to bind with fibrinogen is obstructed when arteanoflavone is introduced. This inhibition can be associated with arteanoflavone’s capacity to trigger the cAMP/PKA/VASP pathways, leading to the eventual phosphorylation of VASP at Ser157/Ser239. Subsequently, this sequence of events inhibits the ability of fibrinogen to bind with αIIb/β3.

Further research will be required to elucidate how the introduction of arteanoflavone leads to an increase in cAMP. The activation of cyclases (adenyl/guanyl) and phosphodiesterase (PDE), influencing the levels of cyclic nucleotides (cAMP and cGMP), could be a plausible mechanism [9]. When platelet aggregation occurs, if phosphodiesterase (PDE) is obstructed, there is a noticeable increase in cyclic nucleotide levels. It is noteworthy that PDE inhibitors have shown efficacy in thrombosis treatment [33]. In clinical settings, the amplification of cyclic nucleotides is often facilitated by the utilization of several widely used phosphodiesterase (PDE) inhibitors such as treprostinil, dipyridamole, and cilostazol [34]. Consequently, it’s reasonable to postulate that arteanoflavone might exert similar therapeutic effects.

Significant roles in key platelet functions are played by the pathway of PI3K/Akt and the kinase of mitogen-activated protein (MAPK) [35, 36]. Research by Mei-Chi and colleagues emphasized the critical nature of MAPKs phosphorylation, particularly p38, in TXA2 production. This process incites the secretion of arachidonic acid, a forerunner to TXA2, and boosts platelet aggregation by inducing the phosphorylation of cytosolic PLA2 [37]. The production of TXA2 functions as a powerful autocrine agent that triggers further platelet activation and aggregation. It serves as a significant indicator when evaluating compounds or elements that curtail platelet function [37].

In this study, arteanoflavone showed a significant inhibitory effect on collagen-induced platelet aggregation in a concentration-dependent manner. Furthermore, arteanoflavone decreased the phosphorylation of cytosolic PLA2 in a concentration-dependent manner, which in turn markedly inhibited the collagen-induced TXA2 production (Fig. 5). arteanoflavone significantly diminished the release of serotonin and ATP, both of which are indicators of intracellular granule secretion. Additionally, arteanoflavone significantly attenuated the phosphorylation of PI3K/Akt and MAPK. By inhibiting the intracellular granule release of serotonin and ATP, reducing TXA2 production, and impeding the phosphorylation of phosphoproteins like PI3K/Akt and MAPK, arteanoflavone is able to effectively curb platelet aggregation.

Moreover, the release of integrin αIIb/β3 and granules triggers augmented signaling that transforms the cytoskeleton of platelets, ultimately impacting platelet aggregation and thrombus formation, a critical component in repairing damaged blood vessels. When platelets become activated, they cluster together, and together with fibrin, they form a blood clot. The process of fibrin clot contraction takes about 30–60 min, culminating in the formation of a clot plug. The formation of fibrin clots primarily depends on the interaction of fibrinogen-1 with αIIb/β3. Blocking the activity of αIIb/β3 can thus interfere with or stop blood clot creation [38]. Thrombin plays a role in blood clotting and platelet αIIb/β3 activation, which enhances the ability of fibrinogen to bind with αIIb/β3, causing the formation of a thrombus. This study revealed that arteanoflavone’s antiplatelet influence results in a dose-dependent suppression of fibrin clotting triggered by thrombin. These findings propose that arteanoflavone might potentially act as an effective substance exhibiting antiplatelet activity, with the capacity to hinder or even stop the development of blood clots.

To summarize, the study provides evidence that arteanoflavone can elevate the levels of cAMP/cGMP in human platelets, stimulate phosphorylation of IP3R and VASP, and markedly reduce the build-up of intracellular Ca2+ while suppressing the activation of integrin αIIb/β3 in the cytoplasm. Moreover, arteanoflavone exerts its antiplatelet activity by modulating the phosphorylation of phosphoproteins implicated in signal transduction, such as PI3K/Akt and MAPK, which leads to a reduction in TXA2 production and granule release. Ultimately, arteanoflavone has been shown to markedly inhibit thrombin-induced fibrin thrombus formation, suggesting its potential as an influential compound with antiplatelet properties that could potentially decelerate or prevent the formation of blood clots. Further studies are warranted to delve deeper into arteanoflavone’s precise mechanism of action and its potential therapeutic utility in conditions associated with thrombosis and platelet hyperactivity. If we determine whether the increase in cAMP and cGMP, which is an important point in the antiplatelet action of arteanoflavone, is due to activation of adenylate cyclase and guanylate cyclase or inhibition of phosphodiesterase, the point of action of arteanoflavone will be able to be confirmed. It is also very important to confirm how effective it will be when actually ingested, and for this purpose, we plan to conduct animal dietary experiments in the future.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

References

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Acknowledgements

This study was supported by the National Research Foundation of Korea Grant funded by the Korean government (Grant no. NRF-2020R1F1A1048567).

Funding

This study was supported by the National Research Foundation of Korea Grant funded by the Korean government (Grant no. NRF-2020R1F1A1048567).

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Author notes

  1. Ho Keun Choi and Ga Yeon Kim contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Laboratory Science, College of Health and Biomedical Services, Sangji University, Wonju, Korea

    Ho Keun Choi

  2. Department of Public Health, Dankook University Graduate School, Cheonan, Chungnam, Republic of Korea

    Ga Yeon Kim

  3. Department of Biomedical Laboratory Science, Namseoul University, Cheonan, Chungnam, Republic of Korea

    Ga Hee Lee, Hee su Jang, Da Hyeon Kang, Jin Pyo Lee & Dong-Ha Lee

  4. Molecular Diagnostics Research Institute, Namseoul University, Cheonan, Chungnam, Republic of Korea

    Dong-Ha Lee

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Contributions

HKC & GYK: Conceptualization, Methodology, Writing-Original draft preparation. GHL & HSJ: Data curation, Formal analysis, Writing-Original draft preparation. DHK: Visualization, Investigation. JPL: Validation, Writing-Reviewing and Editing. DHL: Supervision, Writing- Reviewing and Editing, Funding acquisition.

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Correspondence to Dong-Ha Lee.

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Choi, H.K., Kim, G.Y., Lee, G.H. et al. Anti-thrombotic effects of arteanoflavone by regulating cyclic nucleotides and aggregation on human platelets. Appl Biol Chem 67, 61 (2024). https://doi.org/10.1186/s13765-024-00914-6

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