Abstract

BackgroundColumbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo.MethodsAggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study.ResultsCBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca2+]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin αIIbβ3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin αIIbβ3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin β3, Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation.ConclusionThis study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.

Highlights

  • Arterial thrombosis can lead to the development of cardiovascular diseases (CVDs) such as myocardial infarction, atherosclerosis, and even ischemic stroke

  • Platelet activation stimulated by various agonists induces a conformational change in integrin αIIbβ3, enabling it to bind to its ligands, resulting in the onset of platelet aggregation; this process is known as inside-out signal transduction [1]

  • Materials Collagen, luciferin-luciferase, arachidonic acid (AA), U46619, Adenosine diphosphate (ADP), fibrinogen, phorbol-12,13-dibutyrate (PDBu), heparin, prostaglandin E1 (PGE1), fluorescein isothiocyanate (FITC)-phalloidin, nitroglycerin (NTG), aspirin and thrombin were purchased from Sigma

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Summary

Introduction

Arterial thrombosis can lead to the development of cardiovascular diseases (CVDs) such as myocardial infarction, atherosclerosis, and even ischemic stroke. Platelet activation stimulated by various agonists induces a conformational change in integrin αIIbβ, enabling it to bind to its ligands, resulting in the onset of platelet aggregation; this process is known as inside-out signal transduction [1]. The binding of fibrinogen to activated integrin αIIbβ initiates a series of intracellular signaling events, such as tyrosine phosphorylation of numerous proteins and cytoskeleton reorganization; this process is referred to as outside-in signaling [1]. These outside-in reactions, originating in the integrin αIIbβ bound to fibrinogen, are required for maximal secretion, procoagulation, and clot retraction [1]. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo

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