Platelet Protease Activated Receptor 1 Is Involved in the Hemostatic Effect of 20(S)-Protopanaxadiol by Regulating Calcium Signaling.

He Zhang,Daian Pan,Xiangyan Li,Daqing Zhao,Xueyuan Bai,Xiaolei Tang,Liwei Sun,Bailin Song,Wenjie Su,Xingquan Wu

doi:10.3389/fphar.2020.549150

Abstract

Panax notoginseng (Burk.) F.H. Chen has long been used to stop bleeding for hundreds of years in China. At present, only dencichine, notoginsenoside Ft1, and 20(S)-protopanaxadiol (PPD) showed hemostatic effect. However, the molecular mechanism of PPD on the platelet aggragetion needs to be further investigated. The study aims to evaluate the hemostatic effect of PPD and reveal its interacting targets using a series of experiments. In this study, the bleeding time was measured in mouse tail amputation and liver scratch models to evaluate hemostatic effect of PPD. The routine blood and plasma coagulation parameters in NS, HC, and PPD (2, 4, and 8 mg/kg) groups were measured using a blood analyzer. Platelet aggregation rate and ATP release were analyzed by a platelet aggregometer. Subsequently, the degranulation marker CD62P and PAC-1, and the concentrations of cytosolic Ca2+ ([Ca2+]i), cAMP, cGMP, and PAC-1 expressions were also assessed. We found that PPD shorted the bleeding time on the mouse tail amputation and liver scratch models and mainly increased blood platelet count in the rats after subcutaneous injection for 4 h. Meanwhile, PPD decreased APTT, increased FIB content, and directly induced platelet aggregation in vitro. In the absence of Ca2+, PPD induced the increase of [Ca2+]i and slightly increased the levels of CD62P and PAC-1. After the addition of 1 mM Ca2+, PPD treatment markedly promoted platelet activation by promoting ATP level, releasing CD62P and increasing PAC-1 binding in washed platelets. Excitingly, PPD-induced changes including platelet aggregation, decreased cAMP content, and the increases of CD62P and PAC-1 were significantly reversed by protease-activated receptor 1 (PAR-1) antagonist, vorapaxar, which showed similar function as thrombin. In addition, molecular docking analysis and ELISA assay demonstrated that PPD had a promising docking score with -6.6 kcal/mol and increased PAR-1 expression in human platelets, which indicated that PAR-1 is involved in PPD-induced platelet aggregation by regulating calcium signaling. Collectively, our study could provide the new insights of PPD as an essential hemostatic ingredient in Panax notoginseng for the treatment of hemorrhagic disease.

Highlights

Hemostasis is a pivotal process that prevents blood loss after blood vessel injury
The modern medical research found that dencichine and notoginsenoside Ft1 showed the hemostatic effect (Huang et al, 2014; Liu et al, 2019)
We evaluated the hemostatic effects of PPD in P. notoginseng on human/rat platelets and coagulation parameters

Summary

Introduction

Hemostasis is a pivotal process that prevents blood loss after blood vessel injury This process is tightly regulated and depends on an intricate series of events involving platelets, vascular components, and plasma coagulation factors (Ivanciu and Stalker, 2015). As a consequence of vessel wall damage, subendothelial matrixes (such as collagen, von Willebrand factor (vWF), fibronectin, etc.) are exposed to the flowing blood; circulating platelets adhere to the subendothelial surfaces (Corral et al, 2002). During this process, the platelets change its shape, release its granule contents, and gradually form platelet-platelet aggregation by adhering with each other (de Queiroz et al, 2017). Granules components of platelet release, including 5-hydroxytryptamine (5-HT), ADP, adenosine triphosphate (ATP), histamine, CD63, P-selectin, platelet factor 4 (PF4), and vWF, could bind to their relevant receptors to maintain and amplify the initial platelet response and stimulate more circulating platelets that are recruited to form the aggregation (Blair and Flaumenhaft, 2009; Smyth et al, 2009)

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