Background: Nobiletin is a polymethoxylated flavone which possesses a wide range of beneficial effects and a low toxicity. Specifically, inhibition of platelet aggregation and adhesion by Nobiletin was demonstrated, hence, this compound may be used in antiplatelet therapy. However, the exact mechanisms of Nobiletin effects on platelets are not fully defined.

Aims: Here we investigated molecular mechanisms of Nobiletin effects on platelets.

Methods: Washed platelets from healthy donors were incubated with Nobiletin and analyzed by flow cytometry or Western blotting. Phosphatidylserine (PS) surface expression and αIIbβ3 integrins activation were assessed by flow cytometry. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation and caspase-3 activation were analyzed by Western blotting.

Results: Nobiletin dose-dependently (25-75 µM) inhibited αIIbβ3 integrins activation induced by Thrombin or CRP (collagen receptor peptide). Nobiletin did not cause PS exposure or caspase-3 cleavage indicating that inhibitory effect was not related to platelet apoptosis and stipulated by induction of intracellular inhibitory pathways in platelets. The major inhibition mechanisms in platelets are mediated by adenylate (AC) and guanylate cyclases (GC) activation and consequent synthesis of cyclic AMP and GMP which activate protein kinases A and G respectively. Activation of the inhibitory pathways can be monitored by the phosphorylation of the substrate protein VASP. We showed that Nobiletin induces VASP phosphorylation in less than 1 minute after incubation, and the phosphorylation is blocked by AC inhibitor SQ22536 but not GC inhibitor ODQ. We suggested that AC activation in this case is mediated by surface receptors activation. We examined four inhibitors (ZM241385, BWA868C, CAY10441, and L161982) for A₂A, DP1, IP and PGE₂ receptors which activate AC in platelets. Only DP1 receptor inhibition blocked Nobiletin-induced VASP phosphorylation.

Conclusions: In conclusion, we showed that inhibitory effect of Nobiletin on platelets is mediated by activation of DP1 receptor. The study was funded by RFBR (19-315-90102).

« Back to ISTH 2021 Congress

ISTH Congress Abstracts - https://abstracts.isth.org/abstract/antiplatelet-effect-of-nobiletin-is-mediated-by-activation-of-dp1-receptor/