Glaucocalyxin A Inhibits Platelet Activation and Thrombus Formation Preferentially via GPVI Signaling Pathway

Abstract

Platelets play a pivotal role in atherothrombosis and the antiplatelet agents have been proved to be useful in preventing onset of acute clinical events including myocardial infarction and stroke. Increasing number of natural compounds has been identified to be potential antiplatelet agents. Here we report the antiplatelet effect of glaucocalyxin A (GLA), an ent-diterpenoid that we isolated and purified from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, and investigate the molecular mechanisms by which GLA inhibits platelet activation and thrombus formation. The effect of GLA on platelet activation was measured using platelets freshly isolated from peripheral blood of healthy donors. Results showed that pretreatment of human platelets with lower concentrations of GLA (0.01μg/ml, 0.1μg/ml) significantly inhibited platelet aggregation induced by collagen (P<0.001) and CRP (P<0.01), a synthetic GPVI ligand, but not by ADP and U46619. Accordingly, GLA inhibited collagen-stimulated tyrosine phosphorylation of Syk, LAT, and phospholipase Cγ2, the signaling events in collagen receptor GPⅥ pathway. GLA also inhibited platelet p-selectin secretion and integrin activation by convulxin, a GPVI selective ligand. Additionally, GLA was found to inhibit low-dose thrombin-induced platelet activation. Using a flow chamber device, GLA was found to attenuate platelet adhesion on collagen surfaces in high shear condition. In vivo studies showed that GLA administration increased the time for complete occlusion upon vascular injury in mice, but did not extend tail-bleeding time when mice were administered with relatively lower doses of GLA. Therefore, the present results provide the molecular basis for the inhibition effect of GLA on platelet activation and its in vivo effect on thrombus formation, suggesting that GLA could potentially be developed as an antiplatelet and antithrombotic agent.

Natural Products for Antithrombosis

“…GLA inhibited platelet aggregation induced by collagen-related peptide (CRP), a GPVI specific agonist in a dose-dependent manner and reduced collagen-induced phosphorylation of three major molecules, tyrosine kinase Syk, LAT, and phospholipase C γ 2 in GPVI signaling pathway. Therefore, GLA can be developed and used as an collagen receptor antagonist for antiplatelet aggregation [ 108 ]. Salvianolic acid B (SB) is an active component isolated from Danshen ( Salvia miltiorrhiza ), a TCM widely used for the treatment of cardiovascular disorders.…”

Glaucocalyxin A Ameliorates Myocardial Ischemia-Reperfusion Injury in Mice by Suppression of Microvascular Thrombosis

“…Glaucocalyxin A (GLA) [7a,14b-dihydroxy-ent-kaur-16-en-3,15-dione], a natural ent-kaurane diterpenoid with a molecular formula of C20H28O4, was isolated from Rabdosia japonica, as previously described [ 13 ]. Briefly, stems and leaves of Rabdosia japonica (Burm.…”

“…Initially isolated from this perennial herb in the early 1990s, a diterpenoid compound named glaucocalyxin A (GLA) was shown to have therapeutic effect in ischemic disease, leukemia, neurodegenerative disorders, and breast cancer [ 14 ]. We recently reported that GLA inhibits platelet activation and aggregation, with minimal bleeding risk [ 13 ]. Moreover, GLA was also shown to protect cardiomyocytes against oxidative stress and to ameliorate neuroinflammation [ 15 , 16 ].…”

Pharmacological actions of miltirone in the modulation of platelet function

“…Platelet membrane antigens were determined by flow cytometric analysis previously reported [21]. Washed platelets (5 × 10 7 cells/ ml) were preincubated with different concentrations of miltirone or vehicle for 5 min prior to activation with collagen (2.0 µg/ml) for 5 min at 37°C.…”