Derrone Inhibits Platelet Aggregation, Granule Secretion, Thromboxane A2 Generation, and Clot Retraction: An In Vitro Study

Cudrania tricuspidata (C. tricuspidata) is widespread throughout East Asia and in China and Korea, and it is widely used as a traditional remedy against eczema, mumps, and tuberculosis. With regard to the aforementioned medical efficacy, various studies are continuously being conducted, and it has been reported that C. tricuspidata extract has various actions against inflammation, diabetes, obesity, and tumors. Therefore, we evaluated antiplatelet effects using derrone in C. tricuspidata. We examined the effect of derrone on the phosphorylation of 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 (p38MAPK), and Akt, which affects platelet function and thrombus formation. Various agonists-induced human platelets were inhibited by derrone without cytotoxicity, and it also decreased the intracellular calcium level through the signaling molecule phosphorylations. In addition, derrone inhibited glycoprotein IIb/IIIa (αIIb/β3) affinity. Thus, in the present study, derrone suppressed human platelet aggregation and thrombin-induced clot formation.


Introduction
In normal circulation of blood, platelets are necessary for hemostasis and thrombosis. [1]. e 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. erefore, many drugs or natural substances have been developed to treat CVDs. [2,3]. Inhibition of platelets has influence on the CVDs; however, the survival rate is still low.
us, discovery of various substances is required [4]. In addition, there is an ever-increasing interest toward different traditional medicines (mainly, herbal remedies) in different diseases, including CVDs [5][6][7]. Various ginsenosides have been used as a traditional herbal medicine against several diseases, especially CVDs [8]. Reviewing these options could enable scientists to examine and then introduce novel efficacious medicines.
Regarding the effects of Cudrania tricuspidata (C. tricuspidata) extracts for improving blood circulation, research has reported that the C. tricuspidata extract has antiplatelet effects on rat platelet aggregation [15]. erefore, in this study, we evaluate the potential efficacy of derrone from unripe fruits of C. tricuspidata.

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). e platelets in suspension were adjusted to 5 × 10 8 /mL concentration according to the previous research [16,17].

Platelet Aggregation Analysis.
For in vitro platelet aggregation, platelets suspension (10 8 /mL) was preincubated with derrone (15 to 60 μM) at 37°C for 2 min, and then agonists were added for stimulation. Derrone was dissolved in 0.1% dimethyl sulfoxide. Platelet aggregation was measured for 5 minutes. e change in light transmission is calculated into the aggregation rate (%). Platelet aggregation was conducted using an aggregometer under stirring condition (Chrono-Log, Havertown, PA, USA).

Cytotoxicity Analysis.
Cytotoxicity of derrone was conducted through lactate dehydrogenase leakage assay. Human platelets (10 8 /mL) was incubated with derrone (15 to 60 μM) for 1 hour and centrifuged at 12,000 g. e supernatant was used to detect the lactate dehydrogenase using ELISA reader (TECAN, Salzburg, Austria).

Intracellular Calcium Level.
e Fura 2-AM loaded platelet suspension was preincubated with derrone (15 to 60 μM) at 37°C for 2 min. After incubation, we added collagen (2.5 μg/mL) for calcium release from endoplasmic reticulum in platelets. e calcium mobilization was measured using a spectrofluorometer (Hitachi F-2700, Tokyo, Japan), and Grynkiewicz method was used for calculating the [Ca 2+ ] i concentration [18].

Detection of Serotonin.
Platelet aggregation was conducted for 7 min at 37°C with derrone (15 to 60 μM), and then reaction cuvette was placed onto ice in order to terminate serotonin release for 3 min. After termination, the reaction mixture was centrifuged, and the supernatant was used.

Immunoblotting.
After platelet aggregation, platelets are dissolved using lysis buffer. e 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 analysis was conducted by Quantity One program (BioRad, Hercules, CA, USA).

Fibrinogen
Binding to α IIb β 3 . After platelet aggregation for 7 min, the reaction mixture was incubated with fibrinogen (Alexa flour 488-conjugated) for 5 mins. After incubation, 0.5% paraformaldehyde in PBS was added to fix the binding between platelet integrin and fibrinogen marker. All procedures of fibrinogen binding assay were conducted in the dark condition. e flow cytometry measures the binding (BD Biosciences, San Jose, CA, USA).

Fibronectin Adhesion.
Human platelet suspension (10 8 / mL) was placed in fibronectin coated wells (bovine serum albumin coated well is used as a negative control) and preincubated with derrone (15 to 60 μM) and collagen (2.5 μg/mL) for 1 h 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).

Fibrin Clot Retraction.
Human platelet-rich plasma (300 μL) was incubated with derrone (15 to 60 μM) for 30 min at 37°C, and thrombin (0.05 U/mL) triggers the clot retraction. After reacting for 15 min, pictures of fibrin clot were taken using a digital camera. ImageJ (v1. 46) was used to convert to clot area (National Institutes of Health, USA).

Statistical Analyses.
All data are presented as the mean ± standard deviation with various numbers of observations. To determine major differences among groups, analysis of variance was performed, followed by the Tukey-Kramer method. SPSS 21.0.0.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis, and p < 0.05 was considered statistically significant.
us, we examined whether derrone-treated platelets can adhere to fibronectin-coated well. As shown in Figure 5(c), derrone suppressed fibronectin adhesion.

Discussion
C. tricuspidata is a perennial plant of the Moraceae family, and its roots, leaves, bark, stems, and fruits contain various physiological substances. Among the phytochemicals, xanthones and flavonoids are the major constituents of C. tricuspidata and have antiobesity, antidiabetic, and antitumor effects [22]. A study involving platelets, cudratricusxanthone A from C. tricuspidata extract has antiplatelet activity in thrombin-induced mouse platelets and anticoagulation activity [23]. Because the C. tricuspidata extract showed antiplatelet effects, we recently searched for a new candidate and confirmed that derrone has antiplatelet effects.
We examined whether derrone affects collagen-stimulated platelet activities and associated signaling molecules.
On the platelet surface, αIIb/β 3 is the most abundant receptor and is an important binding and adhesion molecule for fibrin-platelet mesh construction and platelet-monocyte interaction.
According to the results of studies on the antiplatelet effects of C. tricuspidata, administration of extract of C. tricuspidate (50 and 100 mg/kg) reduced hyperaggregated platelet aggregation without any hepatotoxicity in high-fat diet-(HFD-) fed rats. Moreover, a decrease in thromboxane A 2 production was observed in vivo [15]. In addition, we confirmed the antiplatelet effects of cudraxanthone L, euchrestaflavanone A, and cudraxanthone B [26][27][28]. eir inhibitory mechanism was similar to that of derrone, but cudraxanthone L and euchrestaflavanone A increased only the cAMP levels. Our study had some limitations, in that it was conducted in vitro and did not confirm the antiplatelet effect in vivo. Although the experiment was conducted using a low concentration (15-45 μM) of derrone in vitro, it is difficult to reach this concentration in blood by ingestion. Moreover, since our study is not an in vivo study using the human body, it is difficult to prove its effect in the human body; thus, we cannot prove that these effects would be the same in people with high and low platelet counts or cardiovascular disease. However, based on the in vitro effect, we suggest that derrone has the potential to inhibit thrombosis-mediated CVDs.
In conclusion, we confirmed that derrone suppressed collagen-stimulated platelet aggregation through   downregulation of intracellular calcium concentration, αIIb/ β 3 affinity, and clot retraction, which were achieved by the regulation of IP 3 RI (Ser 1756 ), VASP (Ser 157 and Ser 239 ), cPLA 2 (Ser 505 ), and p38 MAPK . In addition, derrone increases cAMP and cGMP levels in human platelets. ese two cyclic nucleotides are key mediators of the antiplatelet effects. us, we confirmed that derrone could be a potential phytochemical for the prevention of platelet-mediated illnesses.

Data Availability
e data used to support the findings of this study are included within the article.