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The Inhibitory Effects of Protaetia brevitarsis seulensis Larvae Extract on Human Platelet Aggregation and Glycoprotein IIb/IIIa Expression
1Department of Biomedical Laboratory Science and 2Microbiological Resource Research Institute, Far East University, Chungbuk 27601, Korea
3Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
4Cardiovascular Research Institute, School of Medicine, Kyungpook National University, Daegu 41944, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Prev Nutr Food Sci 2023; 28(3): 328-334
Published September 30, 2023 https://doi.org/10.3746/pnf.2023.28.3.328
Copyright © The Korean Society of Food Science and Nutrition.
Abstract
Keywords
INTRODUCTION
Platelets form blood clots to maintain cellular hemostasis. Therefore, correct platelet regulation is required to suppress harmful events during cardiovascular disease, thus antithrombosis therapies are required to specifically target platelet inhibition pathways (Jackson, 2011). However, contrary to expectations, many antiplatelet agents do not ameliorate cardiovascular disease mortality rates (Lee et al., 2021), which often occur due to sudden symptom onset. Therefore, researchers must focus on prevention rather than cure, and identify natural materials which prevent these diseases. Generally, natural materials elicit few side effects, and those products with antiplatelet effects can prevent thrombosis and cardiovascular disease via regular administration (Irfan et al., 2020; Sharifi-Rad et al., 2020). Therefore, in an attempt to identify new antithrombosis drugs, we focused on the antiplatelet activity of
Damaged blood vessels contain exposed collagen fibers which bind to integrins on platelets (Moroi and Jung, 2004), initiates platelet activation, and elevates calcium (Ca2+) concentrations in the cytosol. The first mechanism involved in calcium regulation is Ca2+ mobilization. Platelet activation thus releases calcium from the endoplasmic reticulum into the cytosol (Varga-Szabo et al., 2009). Next, a deficiency of stored Ca2+ in the endoplasmic reticulum can facilitate extracellular Ca2+ influx. Elevated intracellular Ca2+ concentrations ([Ca2+]i) activate Ca2+-dependent kinases which trigger granule release (Farndale, 2006). This agonist-induced signaling cascade or “inside-out signaling” activates glycoprotein IIb/IIIa (integrin αIIb/β3), which then binds to other platelets via adhesive proteins (fibrinogen and fibronectin), and αIIb/β3-mediated signaling triggers platelet aggregation (Phillips et al., 2001). Therefore, in this study, we examined the inhibitory effects of PBE on platelet aggregation.
MATERIALS AND METHODS
Materials
Platelet aggregation analysis
Platelets were separated and washed in washing buffer (pH 6.5) and adjusted in suspension buffer (pH 6.9) to 108/mL. PBE was poorly soluble in water, therefore it was dissolved in dimethyl sulfoxide (0.1%). Platelets (108/mL) were preincubated with different PBE concentrations (75, 100, 150, and 200 μM) at 37°C while stirring, and collagen was added for full platelet aggregation using an aggregometer (Chrono-Log Corp.).
Cytotoxicity analysis
We investigated if PBE concentrations affected lactate dehydrogenase (LDH) levels in platelets. Platelets (108/mL) were preincubated with different PBE concentrations for 15 min at 37°C while stirring. After centrifugation at 12,000
cAMP and cGMP analysis
Platelets (108/mL) were preincubated with different PBE concentrations (75, 100, 150, and 200 μM) for 5 min at 37°C. After platelet aggregation was stopped by ethanol (80%), platelets were centrifuged at 500
Ca2+ mobilization and influx analysis
To measure [Ca2+]i, the Grynkiewicz method (Grynkiewicz et al., 1985) was used. Platelets were incubated with Fura-2 AM for 20 min, washed, and platelet concentrations adjusted to 108/mL using suspension buffer. Platelets (108/mL) were incubated with different PBE concentrations (75, 100, 150, and 200 μM) at 37°C for 5 min and then stimulated with collagen (2.5 μg/mL). To detect Ca2+ influx, platelets were stimulated with 1 μM thapsigargin in the presence of 100 μM EGTA, and 2 mM calcium was added at 3 min. Ca2+ concentrations were analyzed using a fluorescence spectrophotometer (F-2700, Hitachi).
Western blotting
To investigate phosphorylation events, platelet aggregation was performed and platelet lysates quantified. Proteins were separated by electrophoresis and then transferred to polyvinylidene fluoride membranes. Primary antibodies were incubated with membranes overnight at 4°C, and after washing (Tris-buffered saline plus 0.1% Tween 20), a secondary antibody was added and incubated with membranes at room temperature for 2 h. Then, protein signals were developed in a darkroom. Western blotting results were calculated using the Quantity One program (Bio-Rad Laboratories).
Analyzing αIIb/β3 binding to fibrinogen
To examine fibrinogen binding, fibrinogen dye (Alexa Fluor 488) was used in platelet aggregation experiments. During platelet aggregation, artificial fibrinogen binds to activated platelet integrin αIIb/β3 and induces strong aggregation. Platelet binding to fibrinogen dye increases fluorescence and if αIIb/β3 activity is inhibited by PBE, fluorescence is reduced. We tested platelet aggregation using different PBE concentrations (75, 100, 150, and 200 μM) for 5 min. Then, platelet and fibrinogen dye binding was fixed in paraformaldehyde, transferred to flow cytometry tubes, and binding forces analyzed using a BD Biosciences flow cytometer.
Analyzing αIIb/β3 adhesion to fibronectin
Fibronectin is a plasma protein and functions as an adhesive protein to bind platelet integrin αIIb/β3. Therefore, we analyzed αIIb/β3 activity in fibronectin-coated wells. Platelets and different PBE concentrations (75, 100, 150, and 200 μM) were added to fibronectin-coated wells and stimulated by collagen. In normal reactions, platelets adhere to fibronectin-coated wells to form thin films. After reactions, wells were washed twice in buffer, and platelet layers stained using cell staining solution. After this, extract solution was added to extract stained platelet layers and absorbances analyzed using an ELISA plate reader to determine platelet adhesion.
Analyzing thromboxane A2 (TXA2)
Activated platelets synthesize TXA2 via an “inside-out signaling cascade.” TXA2 acts as a strong agonist and is quickly converted to thromboxane B2 (TXB2), which was measured. After collagen-induced platelet aggregation with PBE, indomethacin was added to stop reactions and mixtures centrifuged briefly to generate TXB2-containing supernatants, which were analyzed using an ELISA plate reader.
Data analysis
Results were expressed as the mean±standard deviation; the number of observations varied between different groups. To determine significant differences between groups, we used one way analysis of variance, and the Tukey–Kramer method was used for
RESULTS
Platelet aggregation and cytotoxicity
We used collagen (2.5 μg/mL) to promote full platelet aggregation; platelets (108/mL) were stirred for 2 min with different PBE concentrations, reacted for 5 min, and then collagen added. As shown Fig. 1A, platelets stimulated with collagen were strongly aggregated, but were dose-dependently inhibited by PBE; the half maximal inhibitory concentration was 119.5 μg/mL (Fig. 1B). To confirm PBE cytotoxic effects toward platelets, LDH release after platelet incubation with PBE was analyzed. As shown Fig. 1C, PBE did not affect cytotoxicity.
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Figure 1.
Protaetia brevitarsis seulensis extract (PBE) effects on platelet aggregation. (A) PBE effects on collagen-induced human platelet aggregation. (B) Half maximal inhibitory concentration (IC50) value of PBE on collagen-induced human platelet aggregation. (C) PBE effects on cytotoxicity. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. collagen-stimulated human platelets. NS, not significant.
Variations in cAMP and cGMP levels
The most well-known inhibitory molecules secreted form platelets are cyclic nucleotides (cAMP and cGMP), which are synthesized from nitric oxide and prostacyclin in endothelial cells (Haslam et al., 1978). In platelets, inositol 1,4,5-triphosphate receptor (IP3R), Rap1b, glycoprotein Ibβ, phosphodiesterase 3, and VASP are major cAMP and cGMP substrates. These signaling molecules can affect [Ca2+]i mobilization and αIIb/β3 activity (Schwarz et al., 2001). As shown Fig. 2, PBE increased cAMP and cGMP concentrations.
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Figure 2.
Protaetia brevitarsis seulensis extract (PBE) effects on cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) production. (A) PBE effects on collagen-induced cAMP production. (B) PBE effects on collagen-induced cGMP production. Data are presented as the mean±SD (n=4). *P <0.05 vs. collagen-stimulated human platelets.
IP3R-, ERK-, and p38-phosphorylation
We examined calcium concentrations and the phosphorylation of Ca2+-related signaling molecules. We first focused on Ca2+ mobilization. As shown Fig. 3A, collagen addition increased Ca2+ mobilization but was dose-dependently suppressed by PBE. Ca2+ regulation also occurs via Ca2+ influx, therefore, we investigated if PBE affected this. As shown Fig. 3B, thapsigargin-elevated Ca2+ influx was dose-dependently decreased by PBE.
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Figure 3.
Protaetia brevitarsis seulensis extract (PBE) effects on [Ca2+]i mobilization and IP3R/ERK/p38 phosphorylation. (A) PBE effects on collagen-induced [Ca2+]i mobilization. (B) PBE effects on thapsigargin-induced Ca2+ influx. (C) PBE effects on collagen-induced IP3R and ERK phosphorylation. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. agonist (collagen or thapsigargin-stimulated human platelets).
Next, we investigated if PBE could control IP3R phosphorylation and ERK dephosphorylation. IP3R is located on the surface of the endoplasmic reticulum and cAMP and cGMP are negative regulators of Ca2+ mobilization. IP3R phosphorylation by cAMP/cGMP-dependent kinases can block Ca2+ mobilization. In addition, the depletion of stored Ca2+ can initiate Ca2+ influx, and ERK is an important factor controlling this (Rosado and Sage, 2001). We observed that PBE increased IP3R phosphorylation and decreased ERK phosphorylation when induced by collagen (Fig. 3C).
TXA2, cPLA2-, and p38-phosphorylation
TXA2 acts as an agonist which stimulates platelet activation (Needleman et al., 1976). As shown Fig. 4A, TXA2 was dose-dependently inhibited by PBE. It is accepted that two signaling molecules affect TXA2 synthesis; cPLA2 and mitogen-activated protein kinase p38 (p38) are TXA2 regulators (Kramer et al., 1996). As shown Fig. 4B, collagen-elevated cPLA2 and p38 phosphorylation was inhibited by PBE.
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Figure 4.
Protaetia brevitarsis seulensis extract (PBE) effects on thromboxane A2 generation and cPLA2/p38 phosphorylation. (A) PBE effects on collagen-induced thromboxane A2 generation. (B) PBE effects on collagen-induced cPLA2/p38 phosphorylation. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. collagen-stimulated human platelets.
Fibrinogen binding and fibronectin adhesion
Next, we examined αIIb/β3 function, which can affect platelet aggregation and adhesion. As shown Fig. 5A and 5B, PBE suppressed collagen-elevated binding forces. To confirm PBE effects on αIIb/β3 activity, we analyzed its activity using fibronectin. As shown Fig. 5C, PBE strongly suppressed platelet adhesion and reduced αIIb/β3 activity. Thus, PBE inhibited αIIb/β3 structural changes.
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Figure 5.
Protaetia brevitarsis seulensis extract (PBE) effects on fibrinogen binding to αIIβ/β3 and fibronectin adhesion. (A) Flow cytometry histograms show fibrinogen binding. (B) PBE effects on collagen-induced fibrinogen binding (%). (C) PBE effects on collagen-induced fibronectin adhesion. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. collagen-stimulated human platelets.
PI3K-, Akt-, GSK-3-, SYK-phosphorylation, and VASP-dephosphorylation
We next analyzed the phosphorylation of αIIb/β3-related signaling molecules (PI3K/Akt/GSK-3/SYK/VASP), which are essential regulators of the αIIb/β3-mediated signaling cascade (Sudo et al., 2003; Guidetti et al., 2015). We confirmed that PBE significantly reduced PI3K/Akt/GSK-3/SYK-phosphorylation and elevated VASP (Ser157, Ser239) phosphorylation (Fig. 5).
DISCUSSION
In normal circulation, vascular endothelial cells release prostaglandin I2 and nitric oxide to promote cAMP and cGMP nucleotide production, which are the most important second messengers involved in the negative feedback of platelet actions (Smolenski, 2012) and are regulated by adenylate, guanylate cyclase, and phosphodiesterases (Haslam et al., 1999; Gresele et al., 2011). The cAMP/cGMP-dependent protein kinase phosphorylates various substrates such as actin binding protein, heat shock protein 27, G-protein α13 subunit, glycoprotein Ibβ subunit, inositol 1,4,5-trisphosphate receptor, Rap1b, caldesmon, VASP, and phosphodiesterase 3 (Schwarz et al., 2001). Therefore, we investigated if PBE affected cAMP and cGMP production and showed that it increased these levels (Fig. 5B).
Next, we examined if PBE regulated [Ca2+]i mobilization and Ca2+ influx levels via phosphorylation and dephosphorylation mechanisms, as shown Fig. 3A, PBE inhibited [Ca2+]i mobilization. Another Ca2+ regulation pathway occurs via influx, therefore, we evaluated thapsigargin-induced Ca2+ influx effects by PBE. As shown Fig. 3B, thapsigargin-induced Ca2+ influx was suppressed by PBE. Additionally, we confirmed that PBE regulated Ca2+ signaling by regulating IP3R phosphorylation and ERK dephosphorylation (Fig. 3C).
TXA2 is a platelet activator, therefore we examined if PBE could regulate TXA2 concentrations and associated signaling molecules (cPLA2 and p38). p38 activates cPLA2 via phosphorylation (Kramer et al., 1996). Activated cPLA2 hydrolyzes polyunsaturated fatty acids in membrane phospholipids, with arachidonic acid release. Next, cyclooxygenase-1 and TXA2 synthase generate TXA2, which is released from the platelet cytoplasm and activates other platelets (FitzGerald, 1991). We confirmed that PBE suppressed TXA2 levels via the dephosphorylation of signaling molecules (Fig. 4).
The αIIb/β3-mediated signaling cascade is important in hemostasis. Therefore, we investigated αIIb/β3 activity and related signaling molecules. As shown Fig. 5, PBE suppressed fibrinogen binding and fibronectin adhesion. We next evaluated if PBE could inhibit αIIb/β3-related signaling molecules. PI3K/Akt/GSK-3 is a signaling molecule that facilitates αIIb/β3 activation (Chen et al., 2004; Moroi and Watson, 2015; Valet et al., 2016; Moore et al., 2021). SYK is a 72 kDa tyrosine kinase that is stimulated and phosphorylated by platelet agonists adenosine diphosphate, thrombin, and collagen, and also functions after αIIb/β3 activation and promotes platelet aggregation (Clark et al., 1994; Keely and Parise, 1996). VASP helps regulate actin filament dynamics and platelet shape, but its phosphorylation can inhibit platelets. We investigated PBE effects on PI3K/Akt/GSK-3 phosphorylation and showed (Fig. 6) that PBE inhibited collagen-elevated PI3K, Akt (Ser473 and Thr308), and GSK-3α/β phosphorylation. Additionally, PBE decreased collagen-induced SYK and elevated VASP (Ser157, Ser239) phosphorylation. Therefore, PBE inhibited αIIb/β3 activation via αIIb/β3-related signaling molecules.
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Figure 6.
Protaetia brevitarsis seulensis extract (PBE) effects on PI3K/Akt/GSK-3α/β, SYK, and VASP phosphorylation. Data are presented as the mean±SD (n=4). *P <0.05 vs. collagen-stimulated human platelets.
To characterize PBE effects on platelet aggregation, component analysis research was conducted and alkaloid components identified (Lee et al., 2017). Among alkaloids, 5-hydroxyindolin-2-one (5-HI) and (1R,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid showed antiplatelet and anticoagulation activity and also bleeding time elongation (Lee et al., 2017; Choi et al., 2019). In our previous study, we used 5-HI to evaluate antiplatelet mechanisms in platelets and identified several inhibitory signaling molecules (Kwon et al., 2022). Therefore, antiplatelet PBE effects may be due to indole alkaloid effects, thus these putative PBE components should be characterized and confirmed.
Finally, we showed that PBE decreased platelet aggregation, intracellular Ca concentrations, and αIIb/β3 activation via cyclic nucleotides and phosphoprotein regulation. Therefore,
FUNDING
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF 2020R1I1A1A01067709).
AUTHOR DISCLOSURE STATEMENT
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Concept and design: JHS. Analysis and interpretation: HWK. Data collection: HWK. Writing the article: MHR, JHS. Critical revision of the article: HWK. Final approval of the article: all authors. Statistical analysis: HWK. Obtained funding: JHS. Overall responsibility: JHS.
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Article
Original
Prev Nutr Food Sci 2023; 28(3): 328-334
Published online September 30, 2023 https://doi.org/10.3746/pnf.2023.28.3.328
Copyright © The Korean Society of Food Science and Nutrition.
The Inhibitory Effects of Protaetia brevitarsis seulensis Larvae Extract on Human Platelet Aggregation and Glycoprotein IIb/IIIa Expression
Hyuk-Woo Kwon1,2 , Man Hee Rhee3,4 , Jung-Hae Shin3,4
1Department of Biomedical Laboratory Science and 2Microbiological Resource Research Institute, Far East University, Chungbuk 27601, Korea
3Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
4Cardiovascular Research Institute, School of Medicine, Kyungpook National University, Daegu 41944, Korea
Correspondence to:Jung-Hae Shin, E-mail: mlsjshin@naver.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The white-spotted flower chafer, Protaetia brevitarsis seulensis, is used as a traditional remedy against liver cirrhosis, hepatitis, and hepatic cancer. In this study, we investigated if P. brevitarsis extract (PBE) inhibited platelet aggregation via integrin αIIb/β3 regulation. We observed that PBE inhibited αIIb/β3 activation by regulating the cyclic nucleotides, cyclic adenosine monophosphate and cyclic guanosine monophosphate. Additionally, PBE affected phosphatidylinositol-3 kinase, Akt, SYK, glycogen synthase kinase-3α/β, cytosolic phospholipase A2, and p38 expression, which are signal transduction molecules expressed by platelets, and consequently suppressed αIIbβ3 activity and thromboxane A2 generation. Taken together, PBE showed strong antiplatelet effects and may be used to block thrombosis- and platelet-mediated cardiovascular diseases.
Keywords: cyclic nucleotides, glycoprotein IIb/IIIa, platelet aggregation, thrombosis
INTRODUCTION
Platelets form blood clots to maintain cellular hemostasis. Therefore, correct platelet regulation is required to suppress harmful events during cardiovascular disease, thus antithrombosis therapies are required to specifically target platelet inhibition pathways (Jackson, 2011). However, contrary to expectations, many antiplatelet agents do not ameliorate cardiovascular disease mortality rates (Lee et al., 2021), which often occur due to sudden symptom onset. Therefore, researchers must focus on prevention rather than cure, and identify natural materials which prevent these diseases. Generally, natural materials elicit few side effects, and those products with antiplatelet effects can prevent thrombosis and cardiovascular disease via regular administration (Irfan et al., 2020; Sharifi-Rad et al., 2020). Therefore, in an attempt to identify new antithrombosis drugs, we focused on the antiplatelet activity of
Damaged blood vessels contain exposed collagen fibers which bind to integrins on platelets (Moroi and Jung, 2004), initiates platelet activation, and elevates calcium (Ca2+) concentrations in the cytosol. The first mechanism involved in calcium regulation is Ca2+ mobilization. Platelet activation thus releases calcium from the endoplasmic reticulum into the cytosol (Varga-Szabo et al., 2009). Next, a deficiency of stored Ca2+ in the endoplasmic reticulum can facilitate extracellular Ca2+ influx. Elevated intracellular Ca2+ concentrations ([Ca2+]i) activate Ca2+-dependent kinases which trigger granule release (Farndale, 2006). This agonist-induced signaling cascade or “inside-out signaling” activates glycoprotein IIb/IIIa (integrin αIIb/β3), which then binds to other platelets via adhesive proteins (fibrinogen and fibronectin), and αIIb/β3-mediated signaling triggers platelet aggregation (Phillips et al., 2001). Therefore, in this study, we examined the inhibitory effects of PBE on platelet aggregation.
MATERIALS AND METHODS
Materials
Platelet aggregation analysis
Platelets were separated and washed in washing buffer (pH 6.5) and adjusted in suspension buffer (pH 6.9) to 108/mL. PBE was poorly soluble in water, therefore it was dissolved in dimethyl sulfoxide (0.1%). Platelets (108/mL) were preincubated with different PBE concentrations (75, 100, 150, and 200 μM) at 37°C while stirring, and collagen was added for full platelet aggregation using an aggregometer (Chrono-Log Corp.).
Cytotoxicity analysis
We investigated if PBE concentrations affected lactate dehydrogenase (LDH) levels in platelets. Platelets (108/mL) were preincubated with different PBE concentrations for 15 min at 37°C while stirring. After centrifugation at 12,000
cAMP and cGMP analysis
Platelets (108/mL) were preincubated with different PBE concentrations (75, 100, 150, and 200 μM) for 5 min at 37°C. After platelet aggregation was stopped by ethanol (80%), platelets were centrifuged at 500
Ca2+ mobilization and influx analysis
To measure [Ca2+]i, the Grynkiewicz method (Grynkiewicz et al., 1985) was used. Platelets were incubated with Fura-2 AM for 20 min, washed, and platelet concentrations adjusted to 108/mL using suspension buffer. Platelets (108/mL) were incubated with different PBE concentrations (75, 100, 150, and 200 μM) at 37°C for 5 min and then stimulated with collagen (2.5 μg/mL). To detect Ca2+ influx, platelets were stimulated with 1 μM thapsigargin in the presence of 100 μM EGTA, and 2 mM calcium was added at 3 min. Ca2+ concentrations were analyzed using a fluorescence spectrophotometer (F-2700, Hitachi).
Western blotting
To investigate phosphorylation events, platelet aggregation was performed and platelet lysates quantified. Proteins were separated by electrophoresis and then transferred to polyvinylidene fluoride membranes. Primary antibodies were incubated with membranes overnight at 4°C, and after washing (Tris-buffered saline plus 0.1% Tween 20), a secondary antibody was added and incubated with membranes at room temperature for 2 h. Then, protein signals were developed in a darkroom. Western blotting results were calculated using the Quantity One program (Bio-Rad Laboratories).
Analyzing αIIb/β3 binding to fibrinogen
To examine fibrinogen binding, fibrinogen dye (Alexa Fluor 488) was used in platelet aggregation experiments. During platelet aggregation, artificial fibrinogen binds to activated platelet integrin αIIb/β3 and induces strong aggregation. Platelet binding to fibrinogen dye increases fluorescence and if αIIb/β3 activity is inhibited by PBE, fluorescence is reduced. We tested platelet aggregation using different PBE concentrations (75, 100, 150, and 200 μM) for 5 min. Then, platelet and fibrinogen dye binding was fixed in paraformaldehyde, transferred to flow cytometry tubes, and binding forces analyzed using a BD Biosciences flow cytometer.
Analyzing αIIb/β3 adhesion to fibronectin
Fibronectin is a plasma protein and functions as an adhesive protein to bind platelet integrin αIIb/β3. Therefore, we analyzed αIIb/β3 activity in fibronectin-coated wells. Platelets and different PBE concentrations (75, 100, 150, and 200 μM) were added to fibronectin-coated wells and stimulated by collagen. In normal reactions, platelets adhere to fibronectin-coated wells to form thin films. After reactions, wells were washed twice in buffer, and platelet layers stained using cell staining solution. After this, extract solution was added to extract stained platelet layers and absorbances analyzed using an ELISA plate reader to determine platelet adhesion.
Analyzing thromboxane A2 (TXA2)
Activated platelets synthesize TXA2 via an “inside-out signaling cascade.” TXA2 acts as a strong agonist and is quickly converted to thromboxane B2 (TXB2), which was measured. After collagen-induced platelet aggregation with PBE, indomethacin was added to stop reactions and mixtures centrifuged briefly to generate TXB2-containing supernatants, which were analyzed using an ELISA plate reader.
Data analysis
Results were expressed as the mean±standard deviation; the number of observations varied between different groups. To determine significant differences between groups, we used one way analysis of variance, and the Tukey–Kramer method was used for
RESULTS
Platelet aggregation and cytotoxicity
We used collagen (2.5 μg/mL) to promote full platelet aggregation; platelets (108/mL) were stirred for 2 min with different PBE concentrations, reacted for 5 min, and then collagen added. As shown Fig. 1A, platelets stimulated with collagen were strongly aggregated, but were dose-dependently inhibited by PBE; the half maximal inhibitory concentration was 119.5 μg/mL (Fig. 1B). To confirm PBE cytotoxic effects toward platelets, LDH release after platelet incubation with PBE was analyzed. As shown Fig. 1C, PBE did not affect cytotoxicity.
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Figure 1.
Protaetia brevitarsis seulensis extract (PBE) effects on platelet aggregation. (A) PBE effects on collagen-induced human platelet aggregation. (B) Half maximal inhibitory concentration (IC50) value of PBE on collagen-induced human platelet aggregation. (C) PBE effects on cytotoxicity. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. collagen-stimulated human platelets. NS, not significant.
Variations in cAMP and cGMP levels
The most well-known inhibitory molecules secreted form platelets are cyclic nucleotides (cAMP and cGMP), which are synthesized from nitric oxide and prostacyclin in endothelial cells (Haslam et al., 1978). In platelets, inositol 1,4,5-triphosphate receptor (IP3R), Rap1b, glycoprotein Ibβ, phosphodiesterase 3, and VASP are major cAMP and cGMP substrates. These signaling molecules can affect [Ca2+]i mobilization and αIIb/β3 activity (Schwarz et al., 2001). As shown Fig. 2, PBE increased cAMP and cGMP concentrations.
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Figure 2.
Protaetia brevitarsis seulensis extract (PBE) effects on cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) production. (A) PBE effects on collagen-induced cAMP production. (B) PBE effects on collagen-induced cGMP production. Data are presented as the mean±SD (n=4). *P <0.05 vs. collagen-stimulated human platelets.
IP3R-, ERK-, and p38-phosphorylation
We examined calcium concentrations and the phosphorylation of Ca2+-related signaling molecules. We first focused on Ca2+ mobilization. As shown Fig. 3A, collagen addition increased Ca2+ mobilization but was dose-dependently suppressed by PBE. Ca2+ regulation also occurs via Ca2+ influx, therefore, we investigated if PBE affected this. As shown Fig. 3B, thapsigargin-elevated Ca2+ influx was dose-dependently decreased by PBE.
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Figure 3.
Protaetia brevitarsis seulensis extract (PBE) effects on [Ca2+]i mobilization and IP3R/ERK/p38 phosphorylation. (A) PBE effects on collagen-induced [Ca2+]i mobilization. (B) PBE effects on thapsigargin-induced Ca2+ influx. (C) PBE effects on collagen-induced IP3R and ERK phosphorylation. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. agonist (collagen or thapsigargin-stimulated human platelets).
Next, we investigated if PBE could control IP3R phosphorylation and ERK dephosphorylation. IP3R is located on the surface of the endoplasmic reticulum and cAMP and cGMP are negative regulators of Ca2+ mobilization. IP3R phosphorylation by cAMP/cGMP-dependent kinases can block Ca2+ mobilization. In addition, the depletion of stored Ca2+ can initiate Ca2+ influx, and ERK is an important factor controlling this (Rosado and Sage, 2001). We observed that PBE increased IP3R phosphorylation and decreased ERK phosphorylation when induced by collagen (Fig. 3C).
TXA2, cPLA2-, and p38-phosphorylation
TXA2 acts as an agonist which stimulates platelet activation (Needleman et al., 1976). As shown Fig. 4A, TXA2 was dose-dependently inhibited by PBE. It is accepted that two signaling molecules affect TXA2 synthesis; cPLA2 and mitogen-activated protein kinase p38 (p38) are TXA2 regulators (Kramer et al., 1996). As shown Fig. 4B, collagen-elevated cPLA2 and p38 phosphorylation was inhibited by PBE.
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Figure 4.
Protaetia brevitarsis seulensis extract (PBE) effects on thromboxane A2 generation and cPLA2/p38 phosphorylation. (A) PBE effects on collagen-induced thromboxane A2 generation. (B) PBE effects on collagen-induced cPLA2/p38 phosphorylation. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. collagen-stimulated human platelets.
Fibrinogen binding and fibronectin adhesion
Next, we examined αIIb/β3 function, which can affect platelet aggregation and adhesion. As shown Fig. 5A and 5B, PBE suppressed collagen-elevated binding forces. To confirm PBE effects on αIIb/β3 activity, we analyzed its activity using fibronectin. As shown Fig. 5C, PBE strongly suppressed platelet adhesion and reduced αIIb/β3 activity. Thus, PBE inhibited αIIb/β3 structural changes.
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Figure 5.
Protaetia brevitarsis seulensis extract (PBE) effects on fibrinogen binding to αIIβ/β3 and fibronectin adhesion. (A) Flow cytometry histograms show fibrinogen binding. (B) PBE effects on collagen-induced fibrinogen binding (%). (C) PBE effects on collagen-induced fibronectin adhesion. Data are presented as the mean±SD (n=4). *P <0.05, **P <0.01 vs. collagen-stimulated human platelets.
PI3K-, Akt-, GSK-3-, SYK-phosphorylation, and VASP-dephosphorylation
We next analyzed the phosphorylation of αIIb/β3-related signaling molecules (PI3K/Akt/GSK-3/SYK/VASP), which are essential regulators of the αIIb/β3-mediated signaling cascade (Sudo et al., 2003; Guidetti et al., 2015). We confirmed that PBE significantly reduced PI3K/Akt/GSK-3/SYK-phosphorylation and elevated VASP (Ser157, Ser239) phosphorylation (Fig. 5).
DISCUSSION
In normal circulation, vascular endothelial cells release prostaglandin I2 and nitric oxide to promote cAMP and cGMP nucleotide production, which are the most important second messengers involved in the negative feedback of platelet actions (Smolenski, 2012) and are regulated by adenylate, guanylate cyclase, and phosphodiesterases (Haslam et al., 1999; Gresele et al., 2011). The cAMP/cGMP-dependent protein kinase phosphorylates various substrates such as actin binding protein, heat shock protein 27, G-protein α13 subunit, glycoprotein Ibβ subunit, inositol 1,4,5-trisphosphate receptor, Rap1b, caldesmon, VASP, and phosphodiesterase 3 (Schwarz et al., 2001). Therefore, we investigated if PBE affected cAMP and cGMP production and showed that it increased these levels (Fig. 5B).
Next, we examined if PBE regulated [Ca2+]i mobilization and Ca2+ influx levels via phosphorylation and dephosphorylation mechanisms, as shown Fig. 3A, PBE inhibited [Ca2+]i mobilization. Another Ca2+ regulation pathway occurs via influx, therefore, we evaluated thapsigargin-induced Ca2+ influx effects by PBE. As shown Fig. 3B, thapsigargin-induced Ca2+ influx was suppressed by PBE. Additionally, we confirmed that PBE regulated Ca2+ signaling by regulating IP3R phosphorylation and ERK dephosphorylation (Fig. 3C).
TXA2 is a platelet activator, therefore we examined if PBE could regulate TXA2 concentrations and associated signaling molecules (cPLA2 and p38). p38 activates cPLA2 via phosphorylation (Kramer et al., 1996). Activated cPLA2 hydrolyzes polyunsaturated fatty acids in membrane phospholipids, with arachidonic acid release. Next, cyclooxygenase-1 and TXA2 synthase generate TXA2, which is released from the platelet cytoplasm and activates other platelets (FitzGerald, 1991). We confirmed that PBE suppressed TXA2 levels via the dephosphorylation of signaling molecules (Fig. 4).
The αIIb/β3-mediated signaling cascade is important in hemostasis. Therefore, we investigated αIIb/β3 activity and related signaling molecules. As shown Fig. 5, PBE suppressed fibrinogen binding and fibronectin adhesion. We next evaluated if PBE could inhibit αIIb/β3-related signaling molecules. PI3K/Akt/GSK-3 is a signaling molecule that facilitates αIIb/β3 activation (Chen et al., 2004; Moroi and Watson, 2015; Valet et al., 2016; Moore et al., 2021). SYK is a 72 kDa tyrosine kinase that is stimulated and phosphorylated by platelet agonists adenosine diphosphate, thrombin, and collagen, and also functions after αIIb/β3 activation and promotes platelet aggregation (Clark et al., 1994; Keely and Parise, 1996). VASP helps regulate actin filament dynamics and platelet shape, but its phosphorylation can inhibit platelets. We investigated PBE effects on PI3K/Akt/GSK-3 phosphorylation and showed (Fig. 6) that PBE inhibited collagen-elevated PI3K, Akt (Ser473 and Thr308), and GSK-3α/β phosphorylation. Additionally, PBE decreased collagen-induced SYK and elevated VASP (Ser157, Ser239) phosphorylation. Therefore, PBE inhibited αIIb/β3 activation via αIIb/β3-related signaling molecules.
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Figure 6.
Protaetia brevitarsis seulensis extract (PBE) effects on PI3K/Akt/GSK-3α/β, SYK, and VASP phosphorylation. Data are presented as the mean±SD (n=4). *P <0.05 vs. collagen-stimulated human platelets.
To characterize PBE effects on platelet aggregation, component analysis research was conducted and alkaloid components identified (Lee et al., 2017). Among alkaloids, 5-hydroxyindolin-2-one (5-HI) and (1R,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid showed antiplatelet and anticoagulation activity and also bleeding time elongation (Lee et al., 2017; Choi et al., 2019). In our previous study, we used 5-HI to evaluate antiplatelet mechanisms in platelets and identified several inhibitory signaling molecules (Kwon et al., 2022). Therefore, antiplatelet PBE effects may be due to indole alkaloid effects, thus these putative PBE components should be characterized and confirmed.
Finally, we showed that PBE decreased platelet aggregation, intracellular Ca concentrations, and αIIb/β3 activation via cyclic nucleotides and phosphoprotein regulation. Therefore,
FUNDING
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF 2020R1I1A1A01067709).
AUTHOR DISCLOSURE STATEMENT
The authors declare no conflict of interest.
AUTHOR CONTRIBUTIONS
Concept and design: JHS. Analysis and interpretation: HWK. Data collection: HWK. Writing the article: MHR, JHS. Critical revision of the article: HWK. Final approval of the article: all authors. Statistical analysis: HWK. Obtained funding: JHS. Overall responsibility: JHS.
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