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

Cudrania tricuspidata (C. is a regional plant containing various avonoids and xanthones, and various physiological activities have reported. Therefore, we evaluated antiplatelet effects using artocarpesin isolated from C. tricuspidata.

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 I 2 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, 5triphosphate receptor type I (IP 3 RI) are major substrates of protein kinase A and protein kinase G and VASP contributes to αIIb/β 3 a nity and IP 3 RI affects [Ca 2+ ] i mobilization. However, It has been reported that if cAMP/cGMP-dependent kinases phosphorylate VASP and IP 3 RI, αIIb/β 3 activation and [Ca 2+ ] 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 con rmed 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.

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 × 10 8 /mL concentration according to the previous research [17,18].

Platelet aggregation
For in vitro platelet aggregation, human platelets suspension (10 8 /mL) was pre-incubated for 3 min in presence or absence of artocarpesin along with 2 mM CaCl 2 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 (10 8 /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 × 10 8 /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-uorometer (Hitachi F-2700, Tokyo, Japan) and Grynkiewicz method was used for calculate the [Ca 2+ ] i values [19].

Measurement of Thromboxane B 2 production
Thromboxane A 2 (TXA 2 ) is synthesized in platelets and quickly transforms into thromboxane B 2 (TXB 2 ), therefore, TXA 2 production was measured by detecting TXB 2 production. After platelet activation, the reaction was stopped by adding indomethacin (0.2 mM) in EDTA (5 mM). The TXB 2 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 our 488-conjugated brinogen for 5 mins. After incubation, 0.5% paraformaldehyde was added to x the binding between platelet integrin and brinogen marker. All procedures of brinogen binding assay were conducted in the dark condition. The binding assay was measured using ow cytometry (BD Biosciences, San Jose, CA, USA), and results were presented by the CellQuest software (BD Biosciences).

Fibronectin adhesion assay
Human platelets (10 8 /mL) was placed in bronectin 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 brin 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 brin 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 signi cant.
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 [Ca 2+ ] i mobilization and αIIb/β3 activation.

Effects Of Artocarpesin On Clot Retraction And Plc Phosphorylation
[Ca 2+ ] i mobilization leads inside-out signaling pathway and activated integrin αIIb/β3 facilitates outsidein 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 brin 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) dosedependently, 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 dosedependently.

Discussion
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 e cacy of C. tricuspidate, various studies are continuously being conducted and it has been reported that C. tricuspidate have various physiological activities including in ammation, 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 nd 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, cudra avanon B, isolupalbigenin, xanthone V1a, cudra avone 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 Ca 2+ mobilization, serotonin release, αIIb/β 3 a nity, clot retraction and associated signaling molecules.  2B) and dephosphorylation of JNK1 (Fig. 2D). The activation of αIIb/β 3 leads to a rapid binding to brinogen and bronectin and triggers outside-in signaling. Our results clari ed 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 TXA 2 production through dephosphorylation of cPLA 2 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 a nity and [Ca 2+ ] i mobilization. In our study, artocarpesin increased cAMP and cGMP concentration (Fig. 4D, 4E) and these cyclic nucleotides can elevate the phosphorylation of VASP (Ser 157 , Ser 239 ) and IP 3 RI (Ser 1756 ).
The interaction between αIIb/β3 and brin affect the clot formation [5]. Therefore, we investigated that whether artocarpesin affect thrombin-induced brin clot retraction. As shown in Fig. 5A, artocarpesin strongly suppressed the retraction. This result is achieved through inhibition of Ca 2+ mobilization, thromboxane A 2 production and αIIb/β3 inactivation. We con rmed these results through inhibitionrelated signaling molecules such as IP 3 RI, JNK1, VASP, PI3K/Akt, cPLA 2 and p38. Therefore, we con rmed 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].

Conclusion
This study found that artocarpesin decreases calcium mobilization, brinogen-binding to αIIb/β 3 , bronectin adhesion and thrombin-facilitated clot retraction through the regulation of associated signaling molecules such as IP 3 RI, JNK, cPLA 2 , 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.