Effects of Glycosaminoglycan from Urechis Unicinctus on the P₂Y₁₂ Receptor Signaling Pathway in Rat Platelets

Experimental animals
 
Specific pathogen-free SD rats (263 ± 19 g) were purchased from the Experimental Animal Center of the Military Medical Science Academy of the Chinese People’s Liberation Army, with SCXK-(Army) 2012-0004 animal certification.
 
Major drugs and reagents
 
Glycosaminoglycan from U. unicinctus was purified in the Laboratory of Oceanic Bioactive Material Utilization of Tianjin University of Science and Technology, China. The purity was 97.32% and the contents of sulfate, uronic acid and amino sugar in GAG were 30.26%, 25.25% and 7.58%, respectively (Yuan et al., 2014). Sodium ozagrel (SO) was obtained from Shijiazhuang Siyao Co., Ltd. and the approval number is H20052521. Rat platelet thromboxane B₂ (TXB₂), cyclic adenosine monophosphate (cAMP) and glycoprotein IIb/IIIa (GPIIb/IIIa) ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute. TRizol, RNaseOUT^™ Recombinant Ribonuclease Inhibitor, M-MLV RT and other molecular reagents were acquired from Invitrogen Company. The GAPDH, protein kinase A (PKA), p-PKA, vasodilator-stimulated phosphoprotein (VASP) and p-VASP antibodies were purchased from Cell Signaling Technology (USA). All primers were obtained from Huada Gene Technology Service Co., Ltd. The primer sequences were shown in Table 1.
 

 
Experimental methods
 
A total of 50 rats were randomly divided into five groups, control, positive control (SO) and GAG (high, medium and low concentrations), with 10 rats in each group. Normal saline, SO (4 mg/kg) and GAG (4, 8 and 16 mg/kg) were administered through the caudal vein with a total volume of 0.5 mL for each injection. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared and used to determine the concentrations of cAMP, TXB₂ and GPIIb/IIIa in the rat platelets (Miao, et al., 2018).
        
Furthermore, 50 rats were randomly divided into five groups, that is, resting, control, GAG, MRS2395 (P₂Y₁₂ inhibitor) and MRS2395+GAG, with 10 rats in each group. PRP was prepared and incubated for 10 min with normal saline (resting and control groups), GAG, MRS2395 and MRS2395+GAG, respectively. In addition to the resting group, the PRP of the other groups was stimulated for 5 min using ADP and the stimulation was stopped with an ice bath. Then, the PRP of all groups was used for the subsequent analysis of the phosphorylation levels of VASP and PKA. The PRP in control, GAG and MRS2395 groups was used for the research on P₂Y₁₂ gene expression.
 
Preparation of PRP and PPP
 
After 30 min administration, blood was collected from the femoral artery and anticoagulated with 3.8% sodium citrate (the blood to anticoagulant ratio was 9:1). The anticoagulant blood was centrifuged at 200×g for 5 min to obtain the PRP. Subsequently, the PRP was centrifuged at 1200×g for 10 min to obtain the PPP (Miao, et al., 2018).
 
Determination of cAMP in rat platelets
 
The platelet suspension (1×10⁹/mL) was placed in a plastic centrifuge tube and ADP was added as an inducer (the final concentration was 1 µmol/L). The stimulation was stopped with an ice bath. Then, 200 µL of 0.1 mol/L hydrochloric acid was added to the platelet suspension. Liquid nitrogen was frozen and thawed repeatedly at 37°C for five times. Centrifugation was conducted at room temperature (1200×g, 10 min). A 100 μL supernatant was used to determine the cAMP concentration of platelets in accordance with the kit instructions.
 
Determination of TXB₂ in rat platelets
 
The sample treatment was the same as that of the cAMP. The concentration of TXB₂ was determined in accordance with the kit instructions.
 
Determination of GPIIb/IIIa in rat platelets
 
The platelet suspension (1×10⁹/mL) was placed in a plastic centrifuge tube and ADP was added as an inducer (the final concentration was 5µmol/L). The stimulation was stopped with an ice bath. Centrifugation was conducted at room temperature (1200×g, 10 min). The precipitated platelets were lysated by freezing and thawing and centrifuged at 10000×g for 5 min. The supernatant was used to determine the GPII b/IIIa content.
 
Determination of the phosphorylation levels of VASP and PKA in rat platelets through Western blot
 
The platelet suspension (5×10⁹/mL) was added into ADP (the final concentration was 5 μmol/L) and the stimulation was stopped with an ice bath. Platelet precipitation was obtained via centrifugation at 1200×g for 10 min (4°C) and the precipitate was washed twice by using PBS. The platelet lysate was prepared in accordance with the RIPA lysate instructions and centrifuged for 5 min at 1200×g. The protein separated via SDS-polyacrylamide gel electrophoresis was transferred to a polyvinylidene fluoride (PVDF) film. The PVDF film was placed in 5% bovine serum albumin (BSA); shaken for 1 h at room temperature; incubated overnight at 4°C with prediluted rabbit anti-VASP, p-VASP, PKA and p-PKA and cleaned for 5 min. Then, the PVDF film was incubated at room temperature for 1 h with a Goat Anti-Rabbit IgG antibody labeled with horseradish peroxidase (HRP) and cleaned for 5 min. ECL chemiluminescent liquid (BeyoECL Star, Beyotime) was used for color reaction and chemiluminescence detection and image preservation were conducted using a chemiluminescent imager. ImageQuant TL density analysis software was adopted to analyze the gray values of protein bands in the image.
 
Analysis of P2Y12 gene expression in rat platelets using reverse-transcription PCR (RT-PCR)
 
The platelets precipitate were added into TRIzol, allowed to stand for 5 min at room temperature and centrifuged for 5 min at 10000×g. The supernatant was added into chloroform and allowed to stand for 15 min at room temperature after oscillating mixing. The supernatant was added into isopropanol, allowed to stand for 10 min at room temperature after mixing and centrifuged for 10 min (4°C, 10000×g). The precipitate was added into 75% ethanol, shaken gently and centrifuged for 5 min (4°C, 10000×g). Then, the precipitate was dissolved in water treated with diethyl pyrocarbonate and stored at -80°C after subpackaging.
        
The 5 µL PCR buffer, 1 µL DNTPs (10Mm), 1 µL Taq Ploymerase (5U/µL), 0.5 µL Primer-F,  0.5 µL Primer-R, 1 µL Template and 41 µL RNase-free water were added into 50 µL reaction system. The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, 94°C for 30 s; PCR amplification at 58°C for 30 s, 72°C for 40 s, 35 cycles; extension at 72°C for 5 min.
        
The PCR product was analyzed by agarose gel electrophoresis. The gray value of electrophoresis strips was analyzed using Image J software and the expression intensities of P₂Y₁₂ gene were expressed by the ratio of the area integral of P₂Y₁₂ and b-actin.
 
Analysis of P₂Y₁₂ gene expression in rat platelets via quantitative PCR (qPCR)

The 10 µL Master Mix (Pre-adding ROX), 0.5 µL Primer-F, 0.5 µL Primer-R, 1 µL Template and 8 µL ddH₂O were added into 20 µL reaction system.
        
The reaction conditions were as follows: pre-denaturation at 95°C for 120 s, 95°C for 10 s; PCR amplification at 60°C for 30 s, 72°C for 30 s, 40 cycles; Melt curve at 95°C for 60 s, 55°C for 60 s, 55-98°C  (10 s/cycle, 0.5°C/cycle).
 
Statistical analysis
 
One-way analysis of variance was used for data analysis followed by SPSS 17. All data were expressed in mean ± SD.

Effect of GAG on the cAMP concentration in rat platelets
 
As shown in Fig 1, the different concentrations of GAG and positive drug could significantly increase the cAMP concentration in rat platelets (p < 0.05, p < 0.01). With the increase in GAG concentration, the cAMP concentration in rat platelets showed an increasing trend, but it increased slowly when the GAG concentration was 16 mg/kg. A significant difference in cAMP concentration was observed between the same SO and GAG concentrations (p < 0.05).
 

 
Effect of GAG on the TXB₂ concentration in rat platelets
 
As shown in Fig 2, with the increase in GAG concentration, the TXB₂ concentration in rat platelets showed a decreasing trend, but it decreased slowly when the GAG concentration was 16 mg/kg. Middle and high concentrations of GAG and positive drugs could significantly reduce the TXB₂ concentration in rat platelets (p < 0.05, p < 0.01). A significant difference in TXB₂ concentration was observed between the same SO and GAG concentrations (p < 0.05), because the action mechanism of SO involves the inhibition of thromboxane A₂ production.
 

 
Effect of GAG on the GPIIb/IIIa concentration in rat platelets
 
The result was shown in Fig 3, different concentrations of GAG and positive drugs could significantly reduce the GPIIb/IIIa concentration in rat platelets (p < 0.05, p < 0.01). With the increase in GAG concentration, the GPIIb/IIIa concentration in rat platelets showed an increasing trend, but it increased slowly when the GAG concentration was 16 mg/kg. A significant difference in GPIIb/IIIa concentration was observed between the same SO and GAG concentrations (p < 0.01).
 

 
The premise of platelet aggregation is activation. The bases of platelet activation are platelet surface receptor and its corresponding intracellular signal transduction system and ultimately comes down to platelet membrane glycoprotein GPIIb/IIIa. GAG has an enhanced anti-platelet aggregation effect in rats. 
 
Effect of GAG on the phosphorylation level of PKA in rat platelets
 
The phosphorylation level of platelet PKA in the resting group was significantly higher than that in the control group (p < 0.01). GAG and MRS2395 significantly increased the phosphorylation level of PKA (p < 0.01). After GAG and MRS2395 were combined, the phosphorylation level of platelet PKA was higher than that of GAG or MRS2395 alone (Fig 4). These results indicated that GAG had a synergistic effect with MRS2395.
 

 
Effect of GAG on the phosphorylation level of VASP in rat platelets
 
In comparison with the control group, GAG significantly improved the phosphorylation level of platelet VASP (p < 0.01). MRS2395 alone or in combination with GAG significantly improved the phosphorylation level of VASP in rat platelets (p < 0.01), which is also higher than that of GAG alone (Fig 5). These results suggest that GAG and MRS2395 had a synergistic effect.
 

 
Effect of GAG on the expression level of P₂Y₁₂ receptor gene in rat platelets (RT-PCR)
 
The expression level of P₂Y₁₂ gene in GAG and MRS2395 groups was significantly lower (p < 0.01) than that in the control group. The expression level of P₂Y₁₂ gene in the MRS2395 group was lower than that in the GAG group, but no significant difference (p > 0.05) was observed (Fig 6).
 

 
Effect of GAG on the expression level of P₂Y₁₂ receptor gene in rat platelets (qPCR)
 
As shown in Fig 7, the expression levels of P₂Y₁₂ receptor gene on the platelet membrane in GAG and MRS2395 groups were significantly lower (p < 0.01) than that in the control group. The expression level of P₂Y₁₂ receptor gene on the platelet membrane in the MRS2395 group was significantly lower than that in the GAG group, but no significant difference (p > 0.05) was observed.
 

 
The cAMP is a cyclic nucleotide. The platelet aggregation function is regulated by the content of cAMP in platelets. When the content of cAMP increases, it can activate protein kinase A and excite calcium pump and inhibit the release of Ca²⁺ from storage, thus inhibiting platelet aggregation (Li, 2013).
        
The glycoprotein GPIIbIIIa is the ultimate common pathway for platelet activation and platelets are connected to one another and adhere to a group through the GPIIbIIIa receptor sites. PKA, also known as a cAMP-dependent protein kinase, exists in an inactive total enzyme state in the absence of cAMP in the surrounding environment. The regulatory subunit of PKA binds to cAMP after their encounter, transfers the phosphate groups from ATP to the serine or threonine residue of a particular protein for phosphorylation and alters the activities of these proteins. The role of almost all cAMPs in eukaryotic cells is achieved by activating PKA and phosphorylating its substrate protein (Jin 2006).VASP is an actin regulatory protein whose phosphorylation blocks the platelet GPIIb/IIIa receptor, thereby inhibiting platelet aggregation (Zhang et al., 2013). The P₂Y₁₂ receptor antagonist could attenuate the platelet microaggregation induced by low-dose ADP (Zhuang et al., 2010). Although ADP is a weak agonist, it plays an important role in platelet aggregation because it is largely stored in platelet dense granules and is released in large quantities after platelet activation, thereby enhancing the role of other agonists (Mangin et al., 2003). P₂Y₁₂ is the most important ADP receptor in the platelet membrane. The P₂Y₁₂ receptor is bound and activated by ADP and coupled with Gi protein. As a result, the activity of adenylate cyclase is inhibited and the cAMP concentration, PKA activity and phosphorylation of target proteins, such as VASP, are reduced. Moreover, the inhibitory effect of VASP on cell deformation and aggregation is relieved and platelet aggregation is enhanced (Zhai et al., 2017). TXA₂, which has strong platelet aggregation and vasoconstriction functions and is one of the strongest vasoconstrictive substances and platelet aggregation agents found thus far, is produced through the catalysis of thromboxane synthase in platelets. Considering that TXA₂ is unstable, TXB₂, which is a stable metabolite of TXA₂, is often used as an indicator of its concentration. The P₂Y₁₂ receptor is coupled with Gi protein after ADP binding, stimulates platelet granules to secrete TXA₂ and activates the fibrinogen receptor (GPIIb/IIIa receptor), thereby causing platelet aggregation (Guidetti et al., 2008; Shrestha et al., 2010).
        
In this study, we found that GAG significantly increased the cAMP concentration in rat platelets, significantly reduced platelet TXB₂ and GPIIb/IIIa concentrations, significantly increased the phosphorylation levels of platelet PKA and VASP and significantly inhibited the expression of platelet P₂Y₁₂ gene. It is presumed that glycosaminoglycan from U. unicinctus may inhibit platelet aggregation in rats through the signaling pathway of platelet P₂Y₁₂ receptor y.

This research was financially supported by Tianjin Application-based & Cutting-edge Technology Research Plan (Grant NO. 16JCZDJC33800) and Innovation Team for Tianjin Modern Agricultural Industry Technical System (Grant NO. ITTFRS2017006).