Artocarpesin Prevents Collagen Induced Platelet Aggregation and Clot Retraction Through Cyclic Nucleotides and Dephosphorylation of MAPKs&nbsp;&nbsp;

doi:10.21203/rs.3.rs-258116/v1

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Artocarpesin Prevents Collagen Induced Platelet Aggregation and Clot Retraction Through Cyclic Nucleotides and Dephosphorylation of MAPKs

https://doi.org/10.21203/rs.3.rs-258116/v1

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Background

The cardiovascular diseases (CVDs) are becoming a critical threat to our lives in these years. It is now widely accepted that platelets play an important role in cardiovascular disease as they have a fundamental role in thrombosis. Therefore, many drugs or natural substances have been developed to treat CVDs. Cudrania tricuspidata (C. tricuspidata) is a regional plant containing various flavonoids and xanthones, and various physiological activities have been reported. Therefore, we evaluated antiplatelet effects using artocarpesin isolated from C. tricuspidata.

Methods

The in vitro effects of artocarpesin on platelets was assessed using measurement of calcium mobilization and serotonin release, glycoprotein IIb/IIIa activation, clot retraction and phosphorylation of signaling molecules.

Results

Artocarpesin inhibited human platelet aggregation, calcium mobilization, glycoprotein IIb/IIIa activation and thrombin-induced clot retraction through the regulation of associated signaling molecules such as vasodilator-stimulated phosphoprotein (VASP) and inositol 1, 4, 5-triphosphate receptor I (IP₃RI), and on the dephosphorylation of cytosolic phospholipase A₂ (cPLA₂), mitogen-activated protein kinases p38, JNK and phosphoinositide 3-kinase (PI3K)/Akt.

Conclusions

This study highlights that artocarpesin has inhibitory effects on platelet activities and thrombus formation and has potential value for preventing platelet-induced cardiovascular diseases.

Integrative & Complementary Medicine

Internal Medicine

Artocarpesin

Ca2+ mobilization

Serotonin secretion

αIIb/β3 affinity

Clot retraction

cAMP and cGMP.

In normal circulation of blood, collagen cannot bind with platelets. However, damaged vascular wall shows collagen fibers and interact with circulatory platelets to start hemostasis [1, 2]. After platelet activation, phosphatidylinositol 4,5-bisphosphate hydrolyzes into inositol 1,4,5-trisphosphate (IP₃) and IP₃ induced calcium mobilization affecting granule release [3–5]. These signaling cascades are called “inside-out signaling” and activated platelets occurs structural change of glycoprotein IIb/IIIa (αIIb/β3). The signaling mechanism induced by activated αIIb/β3 is called “outside-in signaling pathway”. During inside-out signaling, endogenous enzyme produces thromboxane A₂ affecting circulatory platelets [6–8]. Therefore, since platelets cause hemostasis and thrombosis, it is important to balance platelet activity [9] and there is a need to develop various substances to inhibit platelets to reduce CVDs [10].

In normal blood circulation, vascular endothelial cells release nitric oxide and prostaglandin I₂ which makes the platelets inactive. These molecules elevate nucleotides such as cyclic-adenosine monophosphate (cAMP) and cyclic-guanosine monophosphate (cGMP) within circulatory platelets and activate dependent kinases [11]. Vasodilator-stimulated phosphoprotein (VASP) and inositol 1, 4, 5-triphosphate receptor type I (IP₃RI) are major substrates of protein kinase A and protein kinase G and VASP contributes to αIIb/β₃ affinity and IP₃RI affects [Ca²⁺]_i mobilization. However, It has been reported that if cAMP/cGMP-dependent kinases phosphorylate VASP and IP₃RI, αIIb/β₃ activation and [Ca²⁺]_i mobilization are inhibited [12, 13].

Cudrania tricuspidate (C. tricuspidate) has been investigated various substances and biological activities. Therefore, we searched for a new substance from C. tricuspidate. We have confirmed the effects of isoderrone and steppogenin in previous studies [14, 15]. In addition, it has been reported that root extract of C. tricuspidate inhibited rat platelet aggregation [16]. Therefore, we investigated a more diverse material in C. tricuspidate and found artocarpesin.

Chemicals and reagents

Artocarpesin was purchased from ChemFaces (Wuhan, China). Collagen was purchased form Chrono-Log Co. (Havertown, PA, USA). Fura 2-AM (2-acetoxymethyl) and alexa fluor 488-conjugated fibrinogen were obtained from Invitrogen (Eugene, OR, USA). Serotonin ELISA kit was purchased from Labor Diagnostika Nord GmbH and CO. (Nordhorn, Germany). Bicinchoninic acid protein assay kit was purchased form Pierce Biotechnology (IL, USA). Cayman chemical (Ann Arbor, MI, USA) offered thromboxane B₂ assay kit, cAMP, cGMP enzyme immunoassay kit. Cell signaling (Beverly, MA, USA) supplied the lysis buffer and antibodies against phospho-p38, phosphor-JNK (1/2), phospho-VASP (Ser¹⁵⁷), phospho-VASP (Ser²³⁹), phospho-cPLA₂(Ser⁵⁰⁵), phosphor-PI3K (Tyr⁴⁵⁸), phospho-Akt (Ser⁴⁷³), phospho-inositol-3-phosphate receptor type I (Ser¹⁷⁵⁶), phosphor-PLC_γ₂ (Tyr⁷⁵⁹), β-actin, and anti-rabbit secondary antibody. Fibronectin-coated cell adhesion kit as procured from Cell Biolabs (San Diego, CA, USA).

Human platelets suspension

Korean Red Cross Blood Center (Suwon, Korea) supplied human platelet-rich plasma (PRP) for research, and study protocols were approved by the Public Institutional Review Board at the National Institute for Bioethics Policy (PIRB-P01-201812-31-007, Seoul, Republic of Korea). The suspension of platelets was adjusted to 5 × 10⁸/mL concentration according to the previous research [17, 18].

Platelet aggregation

For in vitro platelet aggregation, human platelets suspension (10⁸/mL) was pre-incubated for 3 min in presence or absence of artocarpesin along with 2 mM CaCl₂ at 37°C, then collagen (2.5 μg/mL) was added for stimulation. Dimethyl sulfoxide solution (0.1%) was used to dissolve the artocarpesin. Platelet aggregation was measured for 7 minutes under stirring condition. The change in light transmission is converted into the aggregation rate (%). Platelet aggregation was monitored using an aggregometer (Chrono-Log, Havertown, PA, USA).

Cytotoxicity measurement

Cytotoxicity of artocarpesin was conducted through lactate dehydrogenase leakage assay. Human platelets (10⁸/mL) was incubated with artocarpesin (40 to 100 μM) for 1 hour and centrifuged at 12,000g. The supernatant was used to detect the lactate dehydrogenase using ELISA reader (TECAN, Salzburg, Austria).

Calcium mobilization

The Fura 2-AM (5 μM) added PRP and incubated for 60 min. After incubation, human platelets suspension was washed with washing buffer. After washing step, platelets were suspended using suspending buffer and the suspension of platelets was adjusted to 5 × 10⁸/mL concentration. The Fura 2-AM loaded platelet suspension was pre-incubated with artocarpesin (40 to 100 μM) for 3 min at 37°C then added collagen (2.5 μg/mL). The calcium mobilization was measured using a spectro-fluorometer (Hitachi F-2700, Tokyo, Japan) and Grynkiewicz method was used for calculate the [Ca²⁺]_i values [19].

Measurement of Thromboxane B₂production

Thromboxane A₂ (TXA₂) is synthesized in platelets and quickly transforms into thromboxane B₂ (TXB₂), therefore, TXA₂ production was measured by detecting TXB₂ production. After platelet activation, the reaction was stopped by adding indomethacin (0.2 mM) in EDTA (5 mM). The TXB₂ was detected using ELISA reader (TECAN, Salzburg, Austria).

Serotonin release detection

Platelet aggregation was conducted for 7 min at 37°C with artocarpesin, then reaction cuvette place onto ice in order to terminate serotonin release for 3 min. After termination, the reaction mixture was centrifuged and the supernatant was used. The serotonin was detected using ELISA reader (TECAN, Salzburg, Austria).

Western blotting analysis

After platelet aggregation, platelets are dissolved using lysis buffer. The amount of dissolved protein was calculated and proteins (15 μg) were divided by 8% SDS-PAGE. After electrophoresis, proteins are transferred onto membranes and treated primary (1:1,000) and secondary antibodies (1:10,000). Western blotting was performed using the same sample separated after the platelet aggregation experiment. Western blotting analysis was conducted by using the Quantity One, Ver. 4.5 (BioRad, Hercules, CA, USA).

Fibrinogen binding to αIIb/β3

After platelet aggregation for 7 min, the reaction mixture was incubated with alexa flour 488-conjugated fibrinogen for 5 mins. After incubation, 0.5% paraformaldehyde was added to fix the binding between platelet integrin and fibrinogen marker. All procedures of fibrinogen binding assay were conducted in the dark condition. The binding assay was measured using flow cytometry (BD Biosciences, San Jose, CA, USA), and results were presented by the CellQuest software (BD Biosciences).

Fibronectin adhesion assay

Human platelets (10⁸/mL) was placed in fibronectin coated wells (bovine serum albumin coated well is used as a negative control) and incubated with artocarpesin in the presence of collagen (2.5 μg/mL) for 1h at 37°C. After incubation, wells were washed using PBS buffer and added cell stain solution for 10 min. After that, extraction solution was added and each extraction was measured by ELISA reader (TECAN, Salzburg, Austria).

Platelet-mediated fibrin clot retraction

Human platelet-rich plasma (300 μL) was incubated with artocarpesin for 30 min at 37°C, and clot retraction was triggered by adding thrombin (0.05 U/mL). After reacting for 15 min, pictures of fibrin clot were taken using a digital camera. Image J Software (v1.46) was used to calculate the clot area (National Institutes of Health, USA).

Statistical analyses

Experimental data have been presented as the mean ± standard deviation included with the various number of observations. To determine major differences among groups, Analysis of variance was performed followed by Tukey-Kramer method. SPSS 21.0.0.0 software (SPSS, Chicago, IL, USA) was employed for statistical analysis and p<0.05 values were considered as statistically significant.

Effects of artocarpesin on human platelets aggregation and cytotoxicity

Platelets suspension was incubated with various concentrations of artocarpesin (40 to 100 µM, Fig. 1A) without stimulation of collagen for 7 min, but the light transmission was not changed (Fig. 1B). However, collagen-induced platelet aggregation treated with artocarpesin (40 to 100 µM) was decreased dose-dependently. Its inhibitory degree was 22.1, 51.2, 85.8, and 96.7%, respectively and half maximal inhibitory concentration (IC₅₀) was 58.3 µM (Fig. 1C). To investigate the cytotoxicity of artocarpesin, we used various concentrations of artocarpesin. As shown in Fig. 1D, artocarpesin (40 to 100 µM) did not affect lactate dehydrogenase release.

Effects of artocarpesin on [Ca²⁺]_imobilization, IP₃RI phosphorylation, serotonin secretion, JNK phosphorylation

Intracellular ([Ca²⁺]_i) is a crucial essential factor for platelet aggregation and activation, thus we focused the effect of artocarpesin on [Ca²⁺]_i mobilization. As shown in Fig. 2A, [Ca²⁺]_i mobilization were elevated from 105.2 ± 0.6 nM to 770.6 ± 8.4 nM by collagen (2.5 µg/mL). However, artocarpesin dose-dependently reduced the increased [Ca²⁺]_i levels. To confirm the [Ca²⁺]_i mobilization regulation, we investigated Ca²⁺ control signaling molecule, inositol 1, 4, 5-triphosphate receptor type I (IP₃RI). As shown in Fig. 2B, artocarpesin (80 to 100 µM) increased IP₃RI phosphorylation in collagen-induced human platelets. This result means that the decrease of [Ca²⁺]_i level by artocarpesin is due to change of IP₃RI. Next, we examined whether artocarpesin affect serotonin release in δ-granules. As shown in Fig. 2C, artocarpesin dose-dependently inhibited collagen-stimulated serotonin secretion. The JNK1 is involved in platelet secretion [20], thus we investigated JNK (1/2) phosphorylation by artocarpesin. As shown in Fig. 2D, artocarpesin decreased JNK1 phosphorylation in collagen-induced human platelets.

Effects of artocarpesin on fibrinogen binding to integrin αIIb/β₃, fibronectin adhesion and VASP phosphorylation and PI3K/Akt dephosphorylation

Next, we investigated αIIb/β₃ activation, leading integrin-mediated outside-in signaling. Collagen elevated the αIIb/β₃ activation, with a binding rate of 82.5 ± 3.1% (Fig. 3A, 3B). However, artocarpesin decreased the binding force of fibrinogen dose-dependently (Fig. 3A, 3B). The αIIb/β3 can interact with fibronectin. Therefore, we examined whether artocarpesin affect fibronectin adhesion. As shown in Fig. 3C, fibronectin adhesion was suppressed by artocarpesin dose-dependently.

It is well known that phosphorylated VASP (Ser¹⁵⁷, Ser²³⁹) acts as a negative signaling in αIIb/β₃ and phosphorylated phosphoinositide 3-kinase (PI3K)/Akt has been known as a positive signaling in αIIb/β₃ [21, 22]. Thus, we examined whether artocarpesin affects its phosphorylation. Collagen-induced VASP phosphorylation was increased by artocarpesin dose-dependently (Fig. 3D, 3E) but, PI3K/Akt phosphorylation was suppressed by artocarpesin dose-dependently (Fig. 3F, 3G). These results mean that the decrease of αIIb/β₃ affinity by artocarpesin is due to VASP (Ser¹⁵⁷, Ser²³⁹) phosphorylation and PI3K (Tyr⁴⁵⁸)/Akt (Ser⁴⁷³) dephosphorylation.

Measurement thromboxane A₂production, dephosphorylation of cPLA₂, p38 and cyclic nucleotides

We investigated TXA₂ production associated signaling molecule. Collagen (2.5 µg/mL) stimulated human platelet produced TXA₂ (determined as TXB₂) from 1.2 ± 0.2 nM to 48.0 ± 0.2 ng/10⁸ platelets. However, artocarpesin inhibited TXA₂ production dose-dependently (Fig. 4A). Next, we investigated TXA₂ associated signaling molecules, cytosolic phospholipase A₂ (cPLA₂) and p38 mitogen-activated protein kinases (p38). As shown in Fig. 4B and 4C, the cPLA₂ and p38 are phosphorylated by collagen, but artocarpesin inhibited cPLA₂ and p38 phosphorylation dose-dependently. These results mean that the decrease of TXA₂ production by artocarpesin is due to cPLA₂ and p38 dephosphorylation.

Next, we investigated the production of cAMP and cGMP in platelets. As shown in Fig. 4D and 4E, the production of cAMP and cGMP was increased by artocarpesin dose-dependently. These results mean that artocarpesin can increase cAMP and cGMP level in human platelet and activates cAMP/cGMP dependent signaling pathways affecting [Ca²⁺]_i mobilization and αIIb/β3 activation.

Effects Of Artocarpesin On Clot Retraction And Plc Phosphorylation

[Ca²⁺]_i mobilization leads inside-out signaling pathway and activated integrin αIIb/β3 facilitates outside-in signaling pathway which trigger various actions in platelets such as spreading, granule secretion, adhesion and clot retraction. Therefore, we examined the inhibitory effects of artocarpesin on clot retraction. Figure 5A and 5B shows thrombin-induced fibrin clot formation and contraction. Thrombin induced platelet rich plasma was contracted with an inhibition rate of 90.3% compare with unstimulated platelet rich plasma. However, the retraction was suppressed by artocarpesin (40 to 100 µM) dose-dependently, with inhibitory degrees of 74.9, 67.1, 59.2 and 50.0%, respectively, compared with unstimulated platelet rich plasma (Fig. 5B). αIIbβ3 is an important medium for causing clot retraction. Activated αIIbβ3 triggers tyrosine phosphoryation of β3 integrin tail and activates phospholipase C_γ2 (PLC_γ2). The PLC_γ2 has been reported to be crucial for spreading action of platelets and mediating clot retraction [23]. Therefore, we examined whether artocarpesin affects the phosphorylation of PLC_γ2. As shown in Fig. 5C, collagen elevated PLC_γ2 phosphorylation was suppressed by artocarpesin dose-dependently.

C. tricuspidate is widespread throughout East Asia and used in ethnomedicine. In China, C. tricuspidate have been used as herbal teas for a long time. In Korea, C. tricuspidate have been widely used as traditional medicine against eczema, mumps and tuberculosis. Recently, about medical efficacy of C. tricuspidate, various studies are continuously being conducted and it has been reported that C. tricuspidate have various physiological activities including inflammation, diabetes, obesity, and tumor [24]. It has been reported that isoderrone, steppogenin and cudratricusxanthone A isolated from C. tricuspidate have anti-platelets effects [14, 15, 25]. Thus, we searched new substances from C. tricuspidate to find new anti-platelet drug and we investigated that whether substances have antiplatelet effect on collagen-induced human platelets. We investigated 8 single compounds such as alboctalol, cudraxanthone D, cudraflavanon B, isolupalbigenin, xanthone V1a, cudraflavone B, shuterin, and artocarpesin and we found artocarpesin was an anti-platelet substance. Artocarpesin potently inhibited collagen-induced platelet aggregation (Table 1). Therefore, we checked Ca²⁺ mobilization, serotonin release, αIIb/β₃ affinity, clot retraction and associated signaling molecules.

Table 1

Effects of compounds isolated from *Cudrania tricuspidata* on collagen-induced human platelet aggregation
Treatment	Aggregation (%)	Treatment	Aggregation (%)
Collagen (2.5 µg/mL)	91.8 ± 1.3
Cudraxanthone D		Xanthone V1a
40 uM	91.1 ± 1.1	40 uM	88.0 ± 1.5
60 uM	91.2 ± 1.2	60 uM	91.0 ± 1.1
80 uM	90.2 ± 1.1	80 uM	90.3 ± 1.5
100 uM	92.2 ± 0.5	100 uM	92.3 ± 1.4
Cudraflavanone B		Cudraflavone B
40 uM	91.0 ± 0.7	40 uM	91.7 ± 0.5
60 uM	91.5 ± 0.5	60 uM	90.0 ± 1.1
80 uM	89.6 ± 1.2	80 uM	91.7 ± 1.3
100 uM	89.4 ± 1.1	100 uM	92.0 ± 1.5
Alboctalol		Shuterin
40 uM	89.7 ± 1.3	40 uM	90.3 ± 1.5
60 uM	90.7 ± 0.8	60 uM	92.1 ± 0.8
80 uM	91.3 ± 0.9	80 uM	88.1 ± 1.1
100 uM	91.6 ± 1.4	100 uM	90.4 ± 1.2
Isolupalbigenin		Artocarpesin
40 uM	92.3 ± 1.5	40 uM	71.5 ± 1.3
60 uM	91.7 ± 1.2	60 uM	44.8 ± 1.0*
80 uM	89.7 ± 1.4	80 uM	13.0 ± 2.2*
100 uM	90.0 ± 0.8	100 uM	3.0 ± 0.8**
Results are expressed as % of aggregation induced by thrombin. The data are expressed as the mean ± standard deviation (n = 4). ^p < 0.05, ^*p < 0.01 versus the collagen-stimulated human platelets.

Artocarpesin suppressed [Ca²⁺]_i level and serotonin release through IP₃RI (Ser¹⁷⁵⁶) phosphorylation (Fig. 2B) and dephosphorylation of JNK1 (Fig. 2D). The activation of αIIb/β₃ leads to a rapid binding to fibrinogen and fibronectin and triggers outside-in signaling. Our results clarified that artocarpesin downregulated αIIb/β3 activity (Fig. 3A, 3C) through upregulation of phosphorylation of VASP (Fig. 3D, 3E) and downregulation of PI3K/Akt phosphorylation (Fig. 3F, 3G). Artocarpesin also suppressed TXA₂ production through dephosphorylation of cPLA₂ and p38 dose-dependently (Fig. 4B, 4C). Intracellular cAMP and cGMP are strong negative molecules and regulated by enzymes such as cyclic adenylate/guanylate cyclase, and phosphodiesterases. These cyclic nucleotides inhibit αIIb/β3 affinity and [Ca²⁺]_i mobilization. In our study, artocarpesin increased cAMP and cGMP concentration (Fig. 4D, 4E) and these cyclic nucleotides can elevate the phosphorylation of VASP (Ser¹⁵⁷, Ser²³⁹) and IP₃RI (Ser¹⁷⁵⁶).

The interaction between αIIb/β3 and fibrin affect the clot formation [5]. Therefore, we investigated that whether artocarpesin affect thrombin-induced fibrin clot retraction. As shown in Fig. 5A, artocarpesin strongly suppressed the retraction. This result is achieved through inhibition of Ca²⁺ mobilization, thromboxane A₂ production and αIIb/β3 inactivation. We confirmed these results through inhibition-related signaling molecules such as IP₃RI, JNK1, VASP, PI3K/Akt, cPLA₂ and p38. Therefore, we confirmed that inhibitory effects of artocarpesin on anti-platelet function and anti-thrombus functions are due to the elevated cyclic nucleotides and dephosphoryalation of MAPKs. Through the all experimental results, we believe that artocarpesin is valuable as a potential treatment for cardiovascular diseases. As evidence, PDE inhibitors (cilostazol, dipyridamole) have been reported to have therapeutic effects on thrombosis to increase cyclic nucleotides production [27, 28].

This study found that artocarpesin decreases calcium mobilization, fibrinogen-binding to αIIb/β₃, fibronectin adhesion and thrombin-facilitated clot retraction through the regulation of associated signaling molecules such as IP₃RI, JNK, cPLA₂, p38, VASP, PI3K/Akt and PLC_γ2. Therefore, we suggest that artocarpesin from the root and stems of C. tricuspidata would be a useful compound for prevention of thrombosis.

CVDs

cardiovascular diseases; VASP:vasodilator-stimulated phosphoprotein; IP₃RI:inositol 1, 4, 5-triphosphate receptor I; MAPKs:mitogen-activated protein kinases; cAMP:cyclic adenosine monophosphate; cGMP:cyclic guanosine monophosphate; TXA₂:Thromboxane A₂; PLCγ₂:phospholipase Cγ₂; PI3K:phosphoinositide 3-kinase

Ethics approval and consent to participate

Ethical approval for the study was acquired from the Public Institutional Review Board at the National Institute for Bioethics Policy (PIRB-P01-201812-31-007, Seoul, Republic of Korea).

Consent for publication

Not applicable.

Availability of data and materials

The data will be accessible by contacting the corresponding author of this study.

Competing interests

The authors declare no conflict of interest.

Funding

This study was supported by a grant (NRF-2018R1C1B5083580) from the Basic Science Research Program via the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology, Korea.

Authors’ contributions

Conception and design of the experiment: MHR, HWK. Performance of the experiment: JHS, HWK. Analysis and arrangement of the data: JHS, MI, YYL, HWK. Contribution of reagents, materials, and tool: JHS, HWK. Contribution of manuscript preparation: MHR, HWK. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to express their sincere gratitude to all the participants.

Author’s information

¹Department of Biomedical Laboratory Science, Catholic Kwandong University, Gangneung 25601, Korea. ²Laboratory of Physiology and Cell Signaling, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea. ³Department of Oral Biology, University of Illinois at Chicago, Chicago, Illinois. ⁴Department of Biomedical Laboratory Science, Far East University, Eumseong 27601, Korea.

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Artocarpesin Prevents Collagen Induced Platelet Aggregation and Clot Retraction Through Cyclic Nucleotides and Dephosphorylation of MAPKs

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