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 (0.01 μg/ml, 0.1 μg/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.