Panax notoginseng saponin is superior to aspirin in inhibiting platelet adhesion to injured endothelial cells through COX pathway in vitro

doi:10.1016/j.thromres.2016.03.022

Thrombosis Research

Volume 141, May 2016, Pages 146-152

https://doi.org/10.1016/j.thromres.2016.03.022 Get rights and content

Abstract

Objective

This study was designed to investigate the effect of Panax notoginseng saponin (PNS) on platelet adhesion to injured endothelial cells (ECs) and platelet activation induced by injured ECs, and to explore its underlying mechanisms.

Methods

Human umbilical vein endothelial cells (HUVECs) pretreated with aspirin (ASA,15 μg/mL) or PNS (160 μg/mL), or neither, were exposed to oxidized low-density lipoprotein (ox-LDL,80 mg/L) for 16 h. Platelets were then added and co-cultured with HUVECs for 5 min. Platelet adhesion to ECs, platelet CD62p expression, and HUVEC apoptosis were assessed by fluorescence activated cell sorting (FACS)·Supernatant concentration of 6-keto-PGF1α and thromboxane 2 (TXB₂) were measured by radioimmunoassay. Cyclooxygenase-1 (COX-1) and COX-2 protein expression were measured by western blotting.

Results

The inhibitory effect of PNS on platelet activation was similar to ASA, but the inhibitory effect of PNS on platelet adhesion to ECs was superior to ASA. PNS modulated COX-2 expression, and increased 6-keto-PGF1α concentration in HUVECs, while down-regulated COX-1 expression and decreased supernatant TXB₂ concentration in platelets. Co-culturing of injured HUVECs with platelets increased HUVEC apoptosis induced by ox-LDL compared with HUVECs cultured without platelets; ASA increased HUVEC apoptosis induced by ox-LDL when cultured without platelets, while decreased the apoptosis when co-cultured with platelets.

Conclusions

EC protection by ASA is closely associated with its inhibitory effect on platelet activation. PNS is superior to ASA in protecting ECs and in inhibiting platelet adhesion to injured ECs, and the regulation of COX pathway in both ECs and platelets might be the underlying mechanisms of PNS.

Introduction

Interaction between platelets and injured endothelial cells (ECs) plays a vital role in thrombogenesis [1], [2]. Aspirin (ASA) has potent inhibitory effects on platelet activation by irreversibly acetylating platelet COX-1 and inhibiting thromboxane B₂, and also up-regulates the COX-2/prostacyclin (PGI₂) pathway in ECs, which contributes to EC survival [3]. However, its effect on platelet adhesion to injured ECs and platelet activation induced by injured ECs remain unclear. Although ASA is commonly used as an effective antiplatelet drug in clinical practice, it is frequently accompanied with “aspirin resistance” in approximately 5.5% to 60% patients [4], so dual antiplatelet regimen (ASA plus clopidogrel) and even triple antiplatelet regimen (ASA plus clopidogrel plus another more powerful antiplatelet agent) have been introduced [5]. Clopidogrel, another commonly used antiplatelet drug, has been also reported with 16.8% to 21% resistance occurrence [6]. Safe and powerful antiplatelet agents are still in need.

Panax notoginseng saponin (PNS) is active compound extracted from pseudo-ginseng including notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1 [7]. Many Chinese herbal patent medicines (containing a mix of PNS), such as Xueshuantong injection and Xueshuantong capsule, have been widely used in clinical practice in China for more than 20 years [8]. Previous studies have shown that PNS inhibits platelet activation induced by ADP [9] in vivo and reduces the adhesion of injured ECs induced by ox-LDL in vitro, which was supposed to be derived mainly from the active component notoginsenoside R1 [10], [11]. However, little is known about whether PNS reduces platelet adhesion to injured ECs.

The present experiment was designed to compare the effects of PNS and ASA on platelet adhesion to the injured ECs, and to explore the underlying mechanisms focusing on COX pathway.

Section snippets

Agents

Ox-LDL was purchased from Guangzhou Yiyuan Biotechnology Co, Ltd (Guangzhou, China); ASA and PNS were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China; see HPLC analysis of PNS in Fig. 1); Endothelial culture medium (ECM) was purchased from Cell Signaling Technology (Boston, MA, USA); 6-keto-PGF_1α and TXB₂ radioimmunoassay kits were purchased from Huaying Biotechnology Institute (Beijing, China); antibodies against CD61 and CD62p,

Platelet CD62p expression

Treatment with ox-LDL led to an increased CD62p expression when compared with the control group (P < 0.05, Fig. 2). Both ASA and PNS treatment decreased CD62p expression significantly compared with the model group (P < 0.05).

Platelet adhesion to HUVECs

Treatment with ox-LDL led to an increased MFI when compared with the control group (P < 0.05, Fig. 3), indicating that HUVEC injury triggered platelet adhesion to HUVECs. Both ASA and PNS treatment decreased the MFI (P < 0.05) compared with the model group. Moreover, PNS treatment

Discussion

The platelet adhesion to ECs is an essential phase of thrombogenesis. The present study showed that (1) PNS was superior to ASA in inhibiting platelet adhesion to injured ECs through modulation of the EC COX-2/PGI₂ pathway and down-regulation of platelet COX-1/TXA₂ pathway, for the first time to our knowledge; (2) Platelet activation induced by injured ECs exacerbated the EC apoptosis, and ASA protected ECs through its inhibition of platelet activation; (3) ASA inhibited platelet activation

Limitations

There are two limitations in our study should be noted. One is that the ADP signaling pathway in platelet has not been explored with PNS. ADP pathway is of much importance as it is targeted by clopidogrel. The effects of PNS on ADP pathway are worth to be investigated in future. The second limitation is that the combination effect of ASA and PNS on platelet adhesion was not investigated. ASA targets COX pathway in platelet, and the antiplatelet effect of PNS in the present study showed that it

Conclusions

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81273933 and 81102722), the Eleven Five-year Plan National Science and Technology Support Project (2006BAI04A01-2), the Jilin Province Major Science and Technology Achievement Transforming Project (11ZDZH005). We would like to thank Editage [http://online.editage.cn/] for English language editing.

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Effects of panax notoginseng combinating with dual antiplatelet drugs on the major adverse cardiovascular events in pafients undergoing percutaneous coronary intervention procedure

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^☆: Prof. Dazhuo Shi has made substantial contributions to the conception and design of this study, with revising and final approve of the article.

^☆☆: Assoc. Prof. Mei Xue has made substantial contributions to the design of this study, with data analysis of the study and revising of the article.

^☆☆☆: Mingming Wang has contributed to the conduction and data analysis of this study, and the article drafting.

^★: Yu Miao and Na Kou have contributed to the umbilical preparation and HUVEC culture.

^★★: Yonggang Xu has contributed to FACS detection and analysis.

^★★★: Lin Yang and Ying Zhang have contributed to the drug material supply and blood sampling and procession.

¹: Ming-ming Wang and Mei Xue are co-first authors.

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Full Length ArticlePanax notoginseng saponin is superior to aspirin in inhibiting platelet adhesion to injured endothelial cells through COX pathway in vitro☆,☆☆,☆☆☆,★,★★,★★★

Abstract

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Agents

Platelet CD62p expression

Platelet adhesion to HUVECs

Discussion

Limitations

Conclusions

Acknowledgments

J. Am. Coll. Cardiol.

Am. Heart J.

Prostaglandins Other Lipid Mediat.

J. Am. Coll. Cardiol.

J. Thromb. Haemost.

Microb. Pathog.

Platelets in inflammation and atherogenesis

Front. Immunol.

Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin

Proc. Natl. Acad. Sci. U. S. A.

Anti-platelet therapy: cyclooxygenase inhibition and the use of aspirin with particular regard to dual anti-platelet therapy

Br. J. Clin. Pharmacol.

Aspirin and clopidogrel resistance: methodological challenges and opportunities

Vasc. Health Risk Manag.

Effects of panax notoginseng combinating with dual antiplatelet drugs on the major adverse cardiovascular events in pafients undergoing percutaneous coronary intervention procedure

Chin. Med.

Full Length Article
Panax notoginseng saponin is superior to aspirin in inhibiting platelet adhesion to injured endothelial cells through COX pathway in vitro☆,☆☆,☆☆☆,★,★★,★★★