Influence of glycosaminoglycan from Mactra veneriformis on antiplatelet aggregation

The incidence rate of thrombosis has increased rapidly in the global scale in recent years and platelet activation is a key factor in causing atherosclerosis and arterial thrombosis (Zhu, 2011). A series of platelet reactions, such as adhesion, aggregation and release, may occur after being stimulated by adenosine diphosphate (ADP), acrylic acid (AA), collagen and other inducers, leading to the formation of thrombosis (Hua et al., 2012). Therefore, in the process of finding platelet aggregation inhibitory substances, the development of new antithrombotic drugs has become a popular topic in pharmaceutical research. Glycosaminoglycan (GAG) is the general term used to refer to the glycan part in proteoglycan, consisting of repeat units of disaccharide that are often amino sugar and uronic acid. The hydroxy in GAG is often sulfated. GAG possesses extensive biological functions of antioxidation, anticoagulation and antitumor, as well as lowers blood glucose (Li et al., 2012; Chang et al., 2009; Hu et al., 2009; Fan et al., 2008). Previous research indicated that GAG from M. veneriformis exhibited strong anti-clotting and antithrombotic effects (Cui et al., 2013, 2014; Yuan et al., 2015). Based on this result, we explored the inhibitory effects of GAG on ADP- induced platelet aggregation in rats, to provide theoretical basis for further utilization of GAG.

Experimental materials

Experimental animals
 
Wister rats weighting 234.6 ± 6.86 g, which were specific pathogen free (SPF) were purchased from the Experimental Animal Center of the Military Medical Science Academy of the Chinese People’s Liberation Army (certificate No. SCXK-(Army) 2012-0004.
 
Main drugs and reagents
 
GAG from M. veneriformis was separated and purified at the Applied Marine Biology Laboratory, Tianjin University of Science and Technology. The purity and relative molecular mass of GAG were 96.5% and 11.67 kD, respectively. The sulfate, uronic acid and amino sugar contents in GAG were 25.83, 47.61 and 2.53%, respectively. Rat cyclic adenosine monophosphate (cAMP) ELISA kit was obtained from Enzo Life Sciences under catalog No. ADI-900-066. Rat thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1a) ELISA kits were purchased from Shanghai Jiang Lai Biotechnology Co., Ltd. Ozagrel sodium for injection was 20 mg/branch, the national pharmaceutical production approval number of which was H20058330 (Nanjing Haijing Pharmaceutical Co., Ltd.). (Fura-2/AM) was procured from Dojindo Molecular Technologies, Inc. ADP and Triton X-100 were obtained from Sigma-Aldrich Co. EGTA was purchased from Amresco.
 
Main instruments
 
The Model-680 Microplate Reader was from Bio-Rad Laboratory, Inc.; the Allegra X-15R tabletop refrigerated centrifuge was from Beckman Coulter (USA) Co., Ltd.; the PAM-3 dual-channel platelet aggregation instrument was from Jiangsu Danyang Radio Factory; the RF-5301 fluorescence spectrophotometer was from Shimadzu Corporation; and the LBY-F5 Platelet adhesion instrument was from Beijing Pulishing Medical Instrument Co., Ltd.
 
Experimental methods

Platelet aggregation and adhesion assay in vitro and in vivo 

In vitro
 
Ten rats were randomly selected. Blood was collected from the femoral artery and then anticoagulated with 3.8% sodium citrate (the ratio of blood and anticoagulant was 9:1). The blood was centrifuged at 1000 rpm for 10 min to prepare platelet-rich plasma (PRP) and centrifuged at 3000 rpm for 10 min to prepare platelet-poor plasma (PPP). PRP was adjusted to 5×109/mL with PPP. Platelet preparations were incubated for 10 min at 37°C with normal saline, ozagrel sodium and GAG (high, middle and low concentrations) and then stimulated with ADP for 5 min (final concentration was 1 µmol/L). PAM-3 dual-channel platelet aggregation instrument was used to conduct the platelet aggregation experiment. The aggregation curve was drawn and the percentage of maximum platelet aggregation was calculated.

Separation and count of platelet were the same as the above. A total of 200 µL platelet preparations were incubated for 10 min at 37°C with 20 µL normal saline, ozagrel sodium and glycosaminoglycan (final concentrations: 1, 2 and 4 mg/mL), respectively. The reaction mixture was placed in glass beads of the platelet adhesion apparatus and then rotated for 15 min. The platelet number was determined again. The platelet adhesion rate (PAR) was calculated based on the following formula:
 

 
In vivo
 
Rats were randomly divided into control group, positive control group (ozagrel sodium) and GAG groups (high, middle and low concentrations), with 10 rats in each group. Normal saline, ozagrel sodium (4 mg/kg) and glycosaminoglycan (2, 4 and 8 mg/kg) were administered through caudal vein injection with 0.5 mL injection volume. After 30 min, blood was collected from the femoral artery, PRP and PPP plasma were prepared and the maximum platelet aggregation and platelet adhesion rates were determined in accordance to the aforementioned method.
 
Determination of platelet cAMP in vitro
 
Based on the literature (Hua et al., 2012), platelet preparations were incubated for 10 min at 37°C with normal saline, ozagrel sodium and GAG (high, middle and low concentrations) and then stimulated with ADP (final concentration was 1 µmol/L). The mixed solution was treated with 0.1 mol/L hydrochloric acid (200 µL) and then frozen with liquid nitrogen and thawed at 37°C repeatedly and centrifugated at 4000 rpm for 10 min. Platelet cAMP level was measured according to ELISA kit instructions.
 
Determination of [Ca²⁺]i in vitro
 
Based on a slight modification of the literature (Yang et al., 2010), platelet preparations were added into 5 µmol/L Fura-2/AM and incubated for 40 min. Centrifugation precipitations were washed two times with Hepes buffer and again suspended with Hepes buffer and then the platelet was adjusted to 5×10⁹/mL.

Load platelet suspensions were incubated for 10 min at 37°C with normal saline, ozagrel sodium and GAG (high, middle and low concentrations) in the presence of 1 mmol/L CaCl₂ and then stimulated with ADP (final concentration was 1 µmol/L). Fluorescence signals were recorded using the fluorescence spectrofluorometer.

Fluorescence emission was determined at 510 nm, with simultaneous excitation at 340 and 380 nm, changing every 0.5 s. Platelet [Ca²⁺]i was calculated based on the following formula:
 

In the formula, Kd represents the dissociation constant of the Fura-2-Ca²⁺ complex, which is 224 nmol/L. R represents the fluorescence intensity of the Fura-2-complex at 510 nm after the platelet suspension was stimulated by ADP, with and without GAG, in the presence of 1 mM CaCl₂.  Rmax is the fluorescence intensity of the Fura-2-Ca²⁺ complex at 510 nm after the platelet suspension containing 1 mmol/L of CaCl₂ had been solubilized by Triton X-100. Rmin is the fluorescence intensity of the Fura-2-Ca²⁺ complex at 510 nm, after the platelet suspension containing 20 mmol/L Tris/3 mM of EGTA had been solubilized by Triton X-100. SFB is the ratio of Rmin and Rmax at 380 nm.
 
Determination of TXB2 and 6-keto-PGF1a concentrations of rat plasma in vivo
 
After administration for 30 min, blood was collected from the femoral artery and anticoagulated with sodium citrate and centrifuged at 3000 rpm for 30 min. The plasma was then prepared. TXB2 and 6-keto-PGF1a concentrations were measured based on the kit instructions.
 
Statistical analysis
 
One-way analysis of variance was used for data analysis followed by SPSS 17. All data were expressed in mean ± SD. Significant differences between experiment groups were expressed at a significant level of 0.05 and 0.01.

Effects of GAG on platelet aggregation and adhesion rate of rats in vitro
 
The experimental results were shown in Table 1. Compared with the control group, the platelet aggregation percentage and adhesion rates of rats in the GAG groups were significantly reduced (P<0.01) indicating that GAG had observable inhibitory effect on ADP-induced platelet aggregation and platelet adhesion. The ozagrel sodium significantlydecreased platelet aggregation and adhesion rates (P<0.01). However, no significant difference was noted compared with the same concentration of GAG group (P >0.05).


 
Effects of GAG on platelet aggregation and adhesion rates of rats in vivo
 
The results were shown in Table 2. Different concentrations of GAG and ozagrel sodium could significantly reduce the platelet aggregation and platelet adhesion rates (P< 0.05, P <0.01). The extent of decrease was smaller than that in vitro. This result might be the reason for the larger contact opportunities between GAG and sodium ozagrel and platelet in vitro. Moreover, the same GAG concentration as the positive drug had a lower inhibitory effect on platelet aggregation and adhesion rates of rats, although no significant difference was noted (P >0.05).

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Effect of GAG on platelet cAMP concentration of rats in vitro
 
As seen in Fig 1, compared with the control group, the concentration of GAG and ozagrel sodium could significantly increase platelet cAMP level in rats  (P<0.05, P<0.01) and the influence degree of positive drug was greater than that of the same concentration of GAG, although the difference was not significant (P> 0.05). It was supposed that GAG might inhibit the role of platelet activation by increasing the platelet cAMP level, thereby inhibiting ADP-induced platelet aggregation.


 
GAG attenuated ADP-induced [Ca²⁺]i elevation
 
Platelet activation is marked by the release of platelet granular contents. Therefore, we determined whether GAG used at various concentrations attenuated the release of Ca²⁺ from dense granules in platelets and internal flow of Ca²⁺. GAG extensively diminished Ca²⁺ concentration in platelets (P<0.01) stimulated with ADP. The same inhibitory effect on ozagrel sodium was observed, although no significant difference between ozagrel sodium and the same GAG concentration was noted (Fig 2).

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Effects of GAG on TXB2 and 6-keto-PGF1a concentrations of rat plasma in vivo
 
The results of TXB2 and 6-keto-PGF1a concentration in rat plasma were shown in Table 3. Different concentrations of GAG and positive drug could significantly reduce the TXB2 concentration in the plasma (P<0.01) and remarkably increase the 6-keto-PGF1a concentration in the plasma (P<0.05, P<0.01). The influence degree of the same GAG concentration was lower than that of the positive drug, although the significant level was not reached (P >0.05).

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Platelets plays an important role in thrombus formation at the site of damaged blood vessels, especially arterial and microvascular thrombi, which are major causes of cardiovascular and cerebrovascular diseases (Fan et al., 2010). Platelet aggregation is a complex process. Platelet activation is mediated mainly through platelet adhesion at the site of injury, as well as the action of endogenous agonists, such as ADP, collagen, and thrombin, and the release of TXA2 acting as amplification factor. These agonists stimulate platelet aggregation by the specificity receptor on platelet membrane (Park et al., 2012).

The cAMP in the platelet is the second messenger of intracellular signal transmission. Platelet aggregation function is regulated by the cAMP content in the platelet.

The increase in cAMP content can activate protein kinases, induce phosphorylation, excite calcium pump, and inhibit the release of Ca²⁺, thus inhibiting platelet aggregation (Li et al., 2013). As transfer of activation information, calcium ions can activate the phospholipase C and phospholipase A2 which catalyze the hydrolysis of phospholipids to produce the important medium such as thromboxane A2 (TXA2) or platelet-activating factor (PAF). These medium can make platelets deformate, secret and release platelet α-granule membrane protein-140 (GMP-140), TXB2, platelet 4 factor (PF4) and b- thromboglobulin (b-TG), leading to the occurrence of the platelet aggregation reaction (Nesbitt et al., 2003). The current study demonstrated that GAG could reduce calcium ion concentration in rat platelets and significantly increase platelet cAMP concentration, thereby inhibiting platelet aggregation induced by ADP and significantly reducing the percentage of platelet aggregation in rats. Zhang et al., (1991) reported that Stichopus japonicus acidic mucopolysaccharide (SJAMP) could inhibit the increase of the content of platelet cAMP induced by PGE1 and the inhibition action was associated with platelet aggregation induced by SJAMP.

TXA2 is generated under the catalysis of thromboxane synthetase, which has very strong platelet aggregation and vasoconstriction action. Moreover, TXA2 is one of the strongest vasoconstrictor and platelet aggregation agents. Prostaglandin I2 (PGI2) is mainly generated from endothelial cells of the vessel wall and is a strong inhibitor of platelet aggregation. PGI2 play an important role in inhibiting adhesion, aggregation, release reaction of platelet, and inhibiting procoagulant activity of platelet. The dynamic equilibrium between TXA2 and PGI2 is the foundation to maintain body hemorrhage and coagulation function (Pang et al., 2004). TXA2 and PGI2 are not stable, thus,TXB2 and 6-keto-PGF1a, which are stable metabolites of TXA2 and PGI2, act as indexes to judge the concentration of TXA2 and PGI2 (Liu et al., 2012; Wang et al., 2014). Intravenous administration of low molecular weight fucoidan significantly could suppress TXB2 level and elevate 6-ketoPGF1a level in rats, accompanied with the decrease of T/K, and produce effective inhibition of rats platelet aggregation induced by thrombin (Zhao et al., 2012). The current study indicated that different concentrations of GAG significantly decreased the content of TXB2 and significantly increased the content of 6-keto-PGF1a in rat plasma (P<0.05, P<0.01). It was demonstrated that the anticoagulant effect of GAG might be achieved through the inhibition of platelet aggregation and release of platelet thromboxane and vascular endothelial prostacyclin.

This research was financially supported by Tianjin Application-based & Cutting-edge Technology Research Plan (Grant NO. 13JCZDJC29600).