Platycodin D inhibits platelet function and thrombus formation through inducing internalization of platelet glycoprotein receptors

Abstract

BackgroundPlatycodin D (PD) is one of the major bioactive components of the roots of Platycodon grandiflorum and possesses multiple biological and pharmacological properties, such as antiviral, anti-inflammatory, and anti-cancer activities. However, whether it affects platelet function remains unclear. This study aims to evaluate the role of PD in platelet function and thrombus formation.MethodsPlatelets were treated with PD followed by measuring platelet aggregation, activation, spreading, clot retraction, expression of glycoprotein receptors. Moreover, mice platelets were treated with PD and infused into wild-type mice for analysis of in vivo hemostasis and arterial thrombosis.ResultsPlatycodin D treatment significantly inhibited platelet aggregation in response to collagen, ADP, arachidonic acid and epinephrine, reduced platelet P-selectin expression, integrin αIIbβ3 activation, spreading on fibrinogen as well as clot retraction, accompanied with decreased phosphorylation of Syk and PLCγ2 in collagen-related peptide or thrombin-stimulated platelets. Moreover, PD-treated mice platelets presented significantly impaired in vivo hemostasis and arterial thrombus formation. Interestingly, PD induced internalization of glycoprotein receptors αIIbβ3, GPIbα and GPVI. However, GM6001, cytochalasin D, BAPTA-AM and wortmannin did not prevent PD-induced internalization of receptors.ConclusionsOur study demonstrates that PD inhibits platelet aggregation, activation and impairs hemostasis and arterial thrombosis, suggesting it might be a potent anti-thrombotic drug.

Salidroside inhibits platelet function and thrombus formation through AKT/GSK3β signaling pathway

“…Informed consent was acquired from all participants prior to the study. Platelets were isolated from human and mouse blood as described previously [57,58]. For human platelets, ACD-anti-coagulated venous blood was centrifuged for 20 min at 120 х g at room temperature to obtain platelet-rich plasma (PRP) which was centrifuged at 1,350 х g for 15 min, washed three times with CGS buffer and resuspended in Tyrode's buffer.…”

“…Clot retraction was performed by addition of thrombin (1 U/ml), 2 mM Ca 2+ and 0.5 mg/ml fibrinogen at 37°C as described previously [57,58]. Images were captured every 30 min.…”

“…Salidroside (20 mg/kg) was intraperitoneally injected into mice followed by analysis of tail bleeding time after 30 min as described previously [57,58].…”

“…Salidroside (20 μM) or vehicle-treated mouse platelets (1 x 10 8 ) were labelled with calcein and administrated into salidroside-treated mice or vehicle-treated mice respectively via tail vein injection. After 30 min, mesenteric arterioles were challenged with 10% w/v (final concentration) FeCl3 to induce thrombus formation which was monitored by a fluorescence microscopy (Olympus BX53) [57,58].…”

All-Trans Retinoic Acid Impairs Platelet Function and Thrombus Formation and Inhibits Protein Kinase CßI/δ Phosphorylation

“…After treatment with ATRA, FITC-conjugated anti-CD42b antibody (GPIb), FITC-conjugated mouse anti-human CD41a antibody ( IIb ) or anti-human GPVI antibody (detected by FITC-conjugated goat anti-mouse IgG) were added and incubated for 30 min at room temperature followed by flow cytometry analysis of the expression of platelet receptors as described previously (21).…”