Modulation of platelet functions by European toad (Bufo Bufo) skin secretions components

Iryna Udovychenko; Tetiana Halenova; Oleksandr Artemenko; Tetiana Vovk; Nataliia Raksha; Savchuk Olexii; Liudmyla Ostapchenko

doi:10.4103/bbrj.bbrj_362_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 7 | Issue : 1 | Page : 60-66

Modulation of platelet functions by European toad (Bufo Bufo) skin secretions components

Iryna Udovychenko¹, Tetiana Halenova¹, Oleksandr Artemenko², Tetiana Vovk¹, Nataliia Raksha¹, Savchuk Olexii¹, Liudmyla Ostapchenko¹
¹ Department of Biochemistry, Educational and Scientific Center, Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine
² Department of Biophysics and Medical Informatics, Educational and Scientific Center, Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

Date of Submission	12-Nov-2022
Date of Decision	23-Dec-2022
Date of Acceptance	11-Jan-2023
Date of Web Publication	14-Mar-2023

Correspondence Address:
Tetiana Halenova
64/13, Volodymyrska Street, Kyiv 01033
Ukraine

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_362_22

Abstract

Background: A growing number of reports indicate that amphibian skin secretions may have a remarkable medical importance; however, the effects of the components of some dermal secretions on blood platelets and hemostasis are inadequately recognized. Since our previous studies demonstrated that the general Bufo bufo skin secretions induced platelet aggregation in platelet-rich plasma, this work was designed to study the effects of the components of some fractions on platelet functions to comprehend its possible mechanism of action as platelet modulators. Methods: Chromatographic separation of B. bufo general skin secretions was carried out using size exclusion chromatography. Rabbit platelets were purified by column chromatography on Sepharose 4B. Various aspects of platelet function such as activation, aggregation, and adhesion were evaluated. Results: One fraction, out of 7, dose-dependently induced aggregation of isolated platelets and was used in further experiments. The studied fraction was shown to induce platelet adhesion onto fibrinogen-coated surface. Furthermore, the results demonstrated the effects of the fraction on some processes that involved in platelets activation: The fraction components facilitated (Ca²⁺)_i mobilization and attenuated platelets Akt phosphorylation, but had no effect on platelet serotonin secretion. Membrane integrity was determined using lactate dehydrogenase (LDH) assay. No increased LDH release was recorded that means no platelet damage, which could lead to misinterpretation of the data, occurred. Conclusion: The results suggest that components of the B. bufo skin secretions may be a promising source of natural compounds which can modulate platelet functions.

Keywords: Amphibian skin secretions, platelet activation, platelet adhesion, platelet aggregation, platelets

How to cite this article:
Udovychenko I, Halenova T, Artemenko O, Vovk T, Raksha N, Olexii S, Ostapchenko L. Modulation of platelet functions by European toad (Bufo Bufo) skin secretions components. Biomed Biotechnol Res J 2023;7:60-6

How to cite this URL:
Udovychenko I, Halenova T, Artemenko O, Vovk T, Raksha N, Olexii S, Ostapchenko L. Modulation of platelet functions by European toad (Bufo Bufo) skin secretions components. Biomed Biotechnol Res J [serial online] 2023 [cited 2023 Jun 2];7:60-6. Available from: https://www.bmbtrj.org/text.asp?2023/7/1/60/371698

Introduction

Recently, the naturally derived compounds that are able to activate platelets are widely being used as diagnostic tools to study normal platelets functions and/or to examine the course of various pathological conditions.^{[1],[2],[3],[4]} Regarding this, the search for new pharmacological approaches to modulate platelet functions is underway. For instance, the compounds derived from snake venoms are known worldwide and are used in medicine to detect different blood diseases.^[5],[6] Lately, there have been lots of researches that indicate that amphibian glandular secretions are an excellent source of diverse kind of pharmacologically and therapeutically significant compounds.^[7],[8] Thus, a few studies report that the skin secretions of certain amphibians are a great source of various active compounds, which are nowadays used for the production of painkillers, antimicrobial and anti-viral drugs, as well as the anti-cancerous medicines.^[9],[10] Furthermore, several research projects illustrate the application of compounds derived from the amphibian glandular secretions in the treatment of cardiovascular ailments and neurological problems.^[9],[11] The results of our previous studies have proved that the components of general skin secretions of Bombina bombina, Bauhinia variegata, Bufo bufo and Bonellia viridis affected the plasma clotting function in vitro.^[12],[13] Our findings also showed that the components of B. variegata, B. bufo and B. viridis skin secretions in a dose-dependent manner induced platelet aggregation.^[12] In another research of ours, we have proved that certain flow-through fractions of amphibian skin secretions of the genus Bombina are a potential source of bioactive constituents that can affect different stages of the hemostasis system.^[14]

Although a considerable amount of research has been elaborated to study the composition of the skin secretions of the bufonid toads, and many biological effects of the secretion's constituents have been revealed, it remains unclear whether the components of their glandular secretions affect the platelet functions. In view of these facts, the aim of the present study was to fractionate the B. bufo general skin secretions and to study the potential effects of the obtained fractions on the platelet functions to comprehend its possible mechanism of action as platelet modulators.

Materials

Collection of amphibian skin secretions

Adult specimens of the European toad (B. bufo, n = 50) were collected during spring spawning (males and females) on the lake Didorivka in Holosiivskyi district of Kyiv, Ukraine. Amphibians were authenticated by the Department of Zoology and Ecology of Taras Shevchenko National University of Kyiv, Ukraine. In view of the specificity of the localization of the glands on the toad's body (the greatest concentration of the glands is on the parotids), as well as an extremely long period of restoration of the parotoid gland secretions, amphibians were removed from nature for a short time and after the extraction of the secretions, all animals were released. To collects the skin secretions toads were put into a petri dish and after mechanical stimulation with fingers for 1–2 min the released secretions were collected by washing the dorsal region of each toad with a small amount of ultra-pure water (5 mL). Water suspensions of skin secretions were centrifuged at 2500 g for 15 min to remove debris. The supernatants were lyophilized (Telstar LyoQuest) and kept at 4°C till use.

Ethical statement

The experimental work with animals followed the international recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986) and was confirmed by the Ethical Committee of ESC “Institute of Biology and Medicine” of Taras Shevchenko National University of Kyiv (protocol № 2 approved August 19, 2021).

Chromatographic separation of general skin secretions

Lyophilized skin secretions of B. bufo (30 mg) were dissolved in 1 mL 0.05 M Tris-HCl buffer (pH 7.4), containing 0.13 M NaCl (tris-buffered saline [TBS]) and centrifuged at 10,000 g for 5 min. Protein concentration in the supernatant was assayed by the Bradford method, using a commercial reagent (BioRad) and bovine serum albumin as a standard. Chromatographic separation was performed using a system for liquid chromatography (BioRad, USA). The sample was applied to a Superdex 75 pg (GE Healthcare, HiLOad^™ 16/60) gel filtration column (flow rate 0.75 mL/min) equilibrated with TBS (pH 7.4). The elution was performed with the same buffer, and the fractions that correspond the appearance of peaks at 280 nm were collected in the plastic tubes.

Blood collection and platelet-rich plasma preparation

Healthy adult rabbits were supplied by the vivarium of Taras Shevchenko National University of Kyiv, Ukraine. Blood from healthy rabbits was drawn from the ear artery into a polyethylene tube with 3.8% sodium citrate (1:10 citrate, v/v). After centrifugation for 10 min at 300 g at room temperature, platelet-rich plasma (PRP) was transferred to a separate tube, and the platelet count was assessed with photo-optical aggregometer AT-02 (Medtech, Russian Federation). PRP was further processed for platelet isolation.

Platelet isolation with the Sepharose 4B column

Platelet were isolated according to the previously described procedures.^[15] A 40-mL Sepharose 4B column (Pharmacia Fine Chemicals, Uppsala, Sweden) was equilibrated with 50 mL of Tyrode/HEPES solution (137 mM NaCl, 5 mM HEPES, 5.5 mM glucose, 4.2 mM NaH₂PO₄, 27 mM KCl, 1 mM MgCl₂, and 119 mM NaHCO₃, pH 7.35), preheated at 37°C. After that, the 2 mL PRP was gently layered on the semi-dry top of the Sepharose 4B gel column. After the PRP had entered the gel, the column was rinsed with Tyrode/HEPES solution for the elution of platelets. Approximately 3 mL of platelet-rich eluate (3 × 10⁸ platelets per milliliter) were sampled and CaCl₂ (1 mM) was added. Then, the purified platelet suspension was transferred into a 37°C water bath for a 30-min resting period.

Platelet aggregation assay

The effects of the flow-through fractions on platelet aggregation were performed in vitro, using photo-optical aggregometer AT-02 (Medtech, Russia) to the described procedures.^[16] Briefly, 380 μL purified platelet suspension (230–250 cells/mL) and 20 μL samples of the 2 mL fractions were incubated at 37°C in the aggregometer cuvette and the aggregation process was monitored for 10 min. The maximum degree of aggregation was recorded and compared with the degree of aggregation in response to one of the platelet physiological inducers – adenosine 5'-diphosphate (ADP) (Sigma, USA) in the final concentration of 5 × 10⁻⁶ M. The aggregation response induced by ADP was considered 100%. The fraction that induced platelet aggregation was used in further experiments to study the effects on platelet functions.

Platelet adhesion assay

The adhesion of platelets to collagen, fibrinogen, and albumin was determined according to Tuszynski and Murphy.^[17] Using a 96-well plate, 0.5 mg/mL of collagen in 0.1 M CH₃COOH, 2 mg/mL of fibrinogen in 0.9% NaCl or 2 mg/mL of bovine albumin in phosphate-buffered saline (PBS) (10 mM Na₂HPO₄, 2 mM KH₂PO₄, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) were incubated at 4°C overnight. The application volume of the substrates was 100 μL per well. After incubation, to remove the unbound proteins, the microplatelet was washed three times with 200 μL of PBS. Then, 200 μL of 1% bovine albumin was added to the coated wells. The microplatelet was incubated for 2 h at 37°C. The excess of bovine albumin was poured off and the microplate was washed three times with 200 μL of PBS. To each well, 70 μL of platelet suspension (3 × 10⁸ platelets/mL) was added. In order to activate the purified platelets, 10 μL of thrombin (final concentration – 0.6 U/mL), ADP (5 × 10⁻⁶ mol/L), or the fraction of amphibian skin secretion were added to the platelet suspension. The microplatelet was incubated for 1 h at 37°C and washed at least three times with 200 μL PBS to remove unattached platelets. Subsequently, 140 μL of the substrate solution containing 1 mg/ml p-nitrophenyl phosphate in citrate buffer (0.2 M sodium citrate, 0.1 M citric acid, and 0.1% (w/v) Triton X-100, pH 5.4) was added to each well. The reaction was stopped after 1 h incubation at 25°C and the color was developed by the addition of 100 μL of NaOH (2 M). The absorbance of the reaction product, p-nitrophenol, was measured at 405 nm using a platelet reader (μQuant, BioTek Instruments Inc., USA). A calibration curve was used to relate the platelet numbers to their acid phosphatase activity. This was performed according to the described procedures.^[2]

Measurement of intracellular ionized calcium concentration

The (Ca²⁺)_i was determined with Fura 2-AM as described previously.^[18] Briefly, the purified platelets (3 × 10⁸ platelets/mL) were incubated with Fura 2-AM (5 μL/1 mL of platelets) for 50 min at 25°C. To remove the unbound Fura-2, platelets were centrifuged at 700 g for 10 min at 25°C. Then, the precipitate was washed with Tyrode/HEPES solution containing 0.1% bovine serum albumin, pH 6.5, centrifuged at 700 g for 10 min at 25°C, and washed for the second time with the same buffer but with different pH – 7.4. The Fura-2-loaded platelets were stimulated with thrombin (3 U/mL), collagen (4 mg/mL), or with the fraction of amphibian skin secretion for 3 min at 25°C in the presence of 1 mM CaCl₂. Fura-2 fluorescence was measured by spectrofluorophotometer (RF-1501, Shimadzu, Japan) with an excitation wavelength in the range of 340–380 nm, changing every 0.5 s, and with an emission wavelength of 510 nm. The (Ca²⁺)_i was calculated by the method of Schaeffer and Blaustein: [Ca²⁺]_i in cytosol = 224 nM × (F−F_min)/(F_max−F), where 224 nM is the dissociation constant of the Fura-2-Ca²⁺ complex, and F_min and F_max represent the fluorescence intensity levels at very low and very high Ca²⁺ concentrations, respectively. In our experiment, F_max is the fluorescence intensity of the Fura-2-Ca²⁺ complex at 510 nm after the platelet suspension containing 1 mM CaCl₂ had been treated with digitonin (0.1%). F_min is the fluorescence intensity of the Fura-2-Ca²⁺ complex at 510 nm after the platelet suspension containing 0.1 M of EGTA had been treated with digitonin (0.1%). F represents the fluorescence intensity of the Fura-2-Ca²⁺ complex at 510 nm after the platelet suspension was stimulated by thrombin, collagen or the studied fraction of amphibian skin secretion, in the presence of 1 mM CaCl₂.

Immunoblotting

Purified platelets were treated with the fraction of amphibian skin secretion, collagen (2 mg/mL), or ADP (5 × 10⁻⁶ mol/L), incubated for 10 min at 25°C, and centrifuged 5,000 g for 5 min. The precipitate was lysed with buffer containing 1% Triton X-100, incubated for 15 min at 4°C, and centrifuged 5000 g for 5 min. Aliquots of platelet lysates containing the same amount of protein were solubilized and dissolved in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) sample buffer (0.125 M Tris–HCl pH 6.8, 2% SDS, 2% β-mercaptoethanol, 20% glycerol, and 0.02% bromophenol blue).^[19] Later, the samples were separated in a 10% SDS-PAGE and transferred to nitrocellulose membrane in a transfer buffer (25 mM Tris, 192 mM glycine, pH 8.5, 96% ethanol and 0.1% SDS). Immunoblot was blocked with 20 mM Tris-HCl, pH 7.5, containing 5% nonfat milk and incubated overnight at 4°C with primary antibodies anti-Akt1/2/3 (Ser473) (Santa Cruz Biotechnology, USA). After, the membrane was washed, incubated with corresponding secondary antibodies (Sigma, USA), and proteins were visualized with a 3,3′-diaminobenzidine (DAB) reagent (Sigma, USA).

Serotonin secretion assay

Purified platelets were treated with the fraction of amphibian skin secretion, collagen (2 mg/mL), or ADP (5 × 10⁻⁶ mol/L), incubated for 10 min at 25°C, and centrifuged 5000 g for 5 min. Aliquots of supernatant were mixed with HClO₄ in a 1:5 ratio. After centrifugation for 5 min at 800 g at 0°C, aliquots of supernatant were diluted with 8 M HCl and cooled on ice. The secretion concentration was measured by spectrofluorophotometer (RF-1501, Shimadzu, Japan) with an excitation wavelength of 295 nm and with an emission wavelength of 550 nm.

Measurement of lactate dehydrogenase release

The integrity of platelets after incubation with a fraction of amphibian skin secretion was verified using a commercial kit for measurement of lactate dehydrogenase (LDH) activity (Felicit Diagnostics, Ukraine). The maximum level of LDH release was expressed as the value of lysis under the action of 0.1% Triton X-100.

Statistical analysis

All experiments were performed in parallel and repeated at least three times each. The data from three replicated experiments are expressed as mean ± standard deviation. Statistical analysis was carried out using one-way analysis of variance followed by a Bonferroni's test. Differences were considered to be statistically significant when P < 0.05.

Results

Supernatants of B. bufo skin secretions were fractionated by Superdex 75 pg column. Seven fractions were collected and all of them were studied on the ability to affect the process of purified platelet aggregation. The chromatogram of B. bufo general skin secretions fractionation is illustrated in [Figure 1]. Fraction #4 in a dose-dependent manner induced platelet aggregation. [Figure 2] shows the degree of platelet aggregation under the action of the components of this fraction in the final concentration of 250 μg of total protein in 1 mL of purified platelets corresponds to the degree of aggregation in the response to the action of 5 × 10⁻⁶ M ADP. Thus, fraction #4 was used in further experiments to study its effect on platelet functions and to investigate the possible mechanism of its action. The other flow-through fractions showed no inducing effect on the process of platelet aggregation.

Figure 1: Chromatogram of Bufo bufo general skin secretions fractionation. The chromatographic analysis was performed as described in the “Materials and Methods section”

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Figure 2: The effect of the fraction #4 on platelet aggregation. Purified platelets (10⁸/mL) were incubated with the fraction #4 in the presence of 1 mM CaCl₂. The effect of 5 × 10⁻⁶ M ADP was used as a control (100%). Bar graph shows mean ± SEM of at least 5 independent experiments performed. *P < 0.05 versus ADP-activated control. SEM: Structural equation modeling

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The extent of adhesion of platelets incubated with the B. bufo fraction #4 onto collagen, fibrinogen, or albumin surface was studied using acid phosphatase assay. The results are shown in [Figure 3]. The studied fraction #4 was shown to induce platelet adhesion onto fibrinogen-coated surface in a statistically significant manner, but not onto collagen- and albumin-coated surfaces. As demonstrated by the graph, the number of cells that adhere to the fibrinogen surface on the fraction #4 treatment was 109 × 10³ platelets/μL. In comparison with the positive controls, it was 50% and 63% lower than in the result of thrombin and ADP stimulation, respectively. The results of the same experiment on the collagen- and albumin-coated surfaces demonstrated that the number of cells that adhered on the fraction #4 incubation was insignificant (14 × 10³ platelets/μL and 5 × 10³ platelets/μL, respectively). In comparison with the controls the adhesion onto collagen was 91% and 93% lower than after thrombin and ADP stimulation, respectively, and a similar tendency was observed while studying the adhesion potency of the platelets to albumin: the values were 97% and 98% lower than on thrombin and ADP treatment, respectively.

Figure 3: The number of platelets that adhere onto fibrinogen-, collagen-, and albumin-coated surfaces upon thrombin (3 U/mL), ADP (5 × 10⁻⁶ M), or the fraction #4 treatment. *P < 0.05 versus adhesion value under thrombin action, ^#P < 0.05 versus adhesion value under ADP action

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Since it has been established that intracellular calcium concentration plays a crucial role in platelet aggregation and activation, we also studied the effects of the fraction #4 on intracellular calcium level. As shown in [Figure 4], fraction #4 facilitated the mobilization of intracellular ionized calcium. The released (Ca²⁺)_i level in platelets treated with the fraction #4 was the same as in the platelets stimulated by collagen and approximately 30% higher than in the untreated platelets sample. The released (Ca²⁺)_i level was threefold higher in platelets treated with thrombin compared with the platelets treated with the studied fraction.

Figure 4: Effects of the Bufo bufo fraction #4 on platelets intracellular Ca²⁺ concentration (Ca²⁺). Platelets were loaded with Fura 2-AM as described in “Materials and Methods section.” The platelets (10⁸/mL) were incubated with or without the fraction #4, thrombin (3 U/ml), or collagen (0.2 mg/mL) in the presence of 1 mM CaCl₂ for 5 min at 37°C (Ca²⁺)_i levels were determined as described in “Materials and Methods section.” Bar graphs show mean ± SEM of at least 3 independent experiments performed. *P < 0.05 versus intact platelets; ⁺P < 0.05 versus thrombin-activated control; ^#P < 0.05 versus collagen-activated control. SEM: Structural equation modeling

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Since it has been a well-established fact that the PI3K/Akt signaling pathway has been implicated in playing an important role in platelet activation during hemostasis and thrombosis involving platelet-matrix interaction and platelet aggregation, we determined whether studied fraction #4 induced platelet Akt phosphorylation. As demonstrated in [Figure 5], the B. bufo fraction #4 attenuated platelets Akt phosphorylation. The same result was observed while applying 2 μg/mL collagen.

Figure 5: Bufo bufo fraction #4 induced platelet PI3K/Akt signaling phosphorylation. Platelets (10⁸/mL) were incubated with the studied fraction #4, collagen (2 μg/mL) or ADP (5 × 10⁻⁶ M) for 10 min with constant stirring at 37°C. Platelets were lysed and immunoblotted using the corresponding antibodies recognizing phosphorylated Akt (Ser473 or Thr308). Data are representatives of at least 3 independent experiments

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Since serotonin is secreted from activated platelets during platelet aggregation and are generally recognized as a critical marker of this process, we examined whether preincubation with the fraction #4 affects the granule secretion. As demonstrated in [Figure 6], no statistically significant differences on platelet serotonin secretion levels were recorded while incubation of the fraction #4 with platelets. On the other hand the platelets incubation with collagen at a concentration of 2 μg/mL approximately threefold induced serotonin secretions level.

Figure 6: Effects of the Bufo bufo fraction #4 on platelets serotonin secretion. Platelets (10⁸/mL) were incubated with the studied fraction #4 or collagen (2 μg/mL) for 10 min with constant stirring at 37°C. Platelets were lysed, and the serotonin levels were determined as described in “Materials and Methods section.” Bar graph shows mean ± SEM of at least 3 independent experiments performed. *P < 0.05 versus intact platelets; ^#P < 0.05 versus collagen-activated control. SEM: Structural equation modeling

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Discussion

This study provides the first report on the ability of the components of B. bufo parotoid gland secretion to modulate platelets functions. Using several approaches, we have studied the effects of the fraction #4 of B. bufo skin secretions on the adhesion, activation, and aggregation processes of the isolated rabbit platelets in vitro, and characterized the possible mechanism that underly its action.

In our previous experiments, we have proved that the B. bufo general skin secretions cause platelet aggregation.^[13] After the chromatographic separation of these glandular secretions [Figure 1], the flow-through fractions were studied on the ability to induce platelet aggregation [Figure 2]. The fraction which dose-dependently induced isolated platelets aggregation was used in further experiments to study its effects on platelet functions.

On the first step to establish a possible mechanism of a platelet aggregation, we studied the effect of the fraction #4 on the platelet adhesion to collagen, fibrinogen, and albumin surfaces [Figure 3]. Platelet adhesion is realized in response to vascular damage. During this stage, platelets bind through specific membrane receptors to cellular and extracellular components of vascular walls. Apart from the crucial role in the hemostasis implementation, the adhesive properties of platelets are also involved in the manifestation of various pathophysiological processes.^[20] It is generally recognized that the interaction of platelet receptors with adhesive proteins results in the initiation of intracellular signaling reactions and platelet activation. Such processes are accompanied by the synthesis and release of platelet agonists such as TxA2, ADP, and other granular proteins, and also provide exposure of GP Ib and GP IIb/IIIa receptors on the platelet membrane.^[21] Our results suggest that the components of the studied fraction induce platelet adherence to immobilized fibrinogen-coated surface. It is generally known that the adhesion of platelets to fibrinogen is dependent on integrin α_IIbβ₃. The inside-out signaling of α_IIbβ₃ can be initiated by various soluble agonists, such as ADP, thrombin, or thromboxane A2 (TxA2), which bind to G protein-coupled seven-transmembrane domain receptors and triggers phospholipase C beta (PLC-β) activation.

Platelet activation is triggered by various agonists, including subendothelial collagens, TxA2 and ADP released from activated platelets, and thrombin generated by the coagulation cascade. Although these agonists act on different platelet G protein-coupled receptors and trigger various signaling pathways, all lead to an increase in the intracellular Ca²⁺ concentration.^[22] This agonist-induced calcium mobilization from intracellular stores and Ca²⁺ entry through the plasma membrane plays a crucial role in platelet aggregation and activation during hemostasis and thrombosis. This process involves PLC-mediated production of inositol-1, 4, 5-trisphosphate (IP3), which in turn releases Ca²⁺ from the intracellular stores through IP3 receptor channels.^[23] The increase in the intracellular Ca²⁺ concentration in platelets pretreated with B. bufo fraction #4 suggests that the effect on platelet functions might be mediated by cytoplasmic calcium increase [Figure 4].

Activated G protein-coupled receptors also stimulate the PI3K/Akt signaling pathway, which has been implicated in playing an important role in platelet activation.^[24] Extensive studies, using various experimental setups, have indicated the critical role of serine/threonine kinase Akt in the regulation of platelet degranulation and aggregation. Being the major effector of PI3K, Akt has three closely related isoforms (Akt1, Akt2, and Akt3) that are expressed in platelets. In our experiment, we determined whether studied fraction-induced platelet Akt phosphorylation is modulated as a signaling pathway in the platelet functions activity. Our result suggests that the studied B. bufo fraction attenuates platelets Akt phosphorylation and might involve PI3K/Akt signaling pathway while platelet activation [Figure 5].

Serotonin is secreted by the platelet-dense granules during platelet activation and plays a crucial role in platelet aggregation promotion and vasoconstriction of surrounding blood vessels, facilitating hemostasis. Our results demonstrate no statistically significant increase in serotonin secretion levels after incubation of the fraction #4 with platelets [Figure 6].

To make sure that no platelet damage, which could lead to misinterpretation of the data, occurred, membrane integrity was determined using LDH assay. No increased LDH release was recorded while testing the studied fraction #4 (data not shown).

To summarize, the present study provided a scientific basis to the role of the proteins and enzymes present in the B. bufo skin secretion and gave a background for further potential medical and pharmaceutical applications of the components of their glandular secretions.

Conclusion

The present study demonstrates that the fraction #4 of B. bufo skin secretions can modulate the platelet functions. This effect might be due to the binding of the fraction components with the G protein-coupled receptors on the platelet membrane with further PLC-β activation, calcium mobilization, and activation of protein kinase C which stimulate the PI3K/Akt pathway. The results suggest that the components of European toad parotoid gland secretions may be a promising source of potentially useful natural biologically active substances which can modulate platelet functions and might be used for the treatment of diseases that involve aberrant platelet function.

Limitation of the study

One of the limitations of the current study was the lack of information about the exact structure of bioactive compound within fraction #4 of B. bufo skin secretions responsible for the effects on platelet function.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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