The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbβ3-Mediated Inside-Out and Outside-In Signals in Human Platelets.

Wei-Chieh Huang,Chih-Hsuan Hsia,Joen-Rong Sheu,Chih-Wei Hsia,Kao-Chang Lin,Shaw-Min Hou,Ting-Yu Chen,Periyakali Saravana Bhavan

doi:10.3390/ijms22073643

Abstract

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2–8 μM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbβ3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbβ3-mediated outside-in signaling, such as integrin β3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbβ3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

Highlights

Arterial thrombosis can cause cardiovascular diseases (CVDs), including myocardial infarction, atherosclerosis, ischemic stroke, venous thromboembolism, and peripheral artery diseases, which are the leading causes of mortality worldwide
Data are presented as the mean ± standard deviation (n = 4). *** p < 0.001, compared with the bovine serum albumin (BSA); # p < 0.05, ## p < 0.01 and ### p < 0.001, compared with the 0.1%
PAC-1 reacts with the activation-induced conformational epitope of integrin αIIbβ3 [23], and PAC-1 binding was markedly reversed by PTE treatment stimulated by collagen

Summary

Introduction

Arterial thrombosis can cause cardiovascular diseases (CVDs), including myocardial infarction, atherosclerosis, ischemic stroke, venous thromboembolism, and peripheral artery diseases, which are the leading causes of mortality worldwide. PAC-1 reacts with the activation-induced conformational epitope of integrin αIIbβ3 [23], and PAC-1 binding was markedly reversed by PTE treatment stimulated by collagen. PTE noticeably abolished platelet adhesion and spreading and clot retraction as well as the phosphorylation of integrin β3, Src, and FAK on immobilized fibrinogen in the absence of platelet agonists. In a vascular thrombotic mouse model [28], mesenteric venules were continuously irradiated with fluorescein sodium throughout the experimental period, which severely damaged the endothelium, whereas treatment with 2 mg/kg PTE significantly extended the occlusion time These data are consistent with the fact that platelet aggregation is a crucial factor causing vascular thrombosis. PTE can be a potential natural compound for treating thromboembolic disorders

Materials

Platelet Preparation and Aggregation Study

Confocal Microscopy Analysis of Platelet Adhesion and Spreading

Measurement of Hydroxyl Radicals Through Electron Spin Resonance Spectrometry

Findings

Conclusions