Ferulenol, a Prenylated Coumarin, Suppresses Collagen-Induced Platelet Activation via PLCγ2-Mediated cPLA2 Signaling in Humans

Background:Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide and are strongly associated with atherothrombotic events. Thrombus formation results from the interplay between platelet activation and the coagulation cascade. While anticoagulant systems are essential for maintaining hemostasis, these systems can also increase the risk of bleeding complications. Coumarins led to the development of clinically important oral anticoagulants such as warfarin, which inhibits vitamin K epoxide reductase complex subunit 1 (VKORC1), a key enzyme in vitamin K recycling and normal blood clotting. Ferulenol, a prenylated coumarin derived from Ferula communis, has been reported to inhibit VKORC1, indicating a similar anticoagulant mechanism. Indeed, the close interplay between coagulation and platelet activation suggests that ferulenol may also modulate platelet function. However,the role of ferulenol in platelet activation has not yet been fully clarified.Methods:In this study, washed platelets obtained from healthy human donors were used to examine whether ferulenol suppresses platelet activation. To further clarify the underlying mechanisms, this study performed immunoblotting and confocal microscopy. In addition, this study evaluated the in vivo antithrombotic and hemostatic effects of ferulenol using a vascular thrombosis model and a tail bleeding time assay in mice.Results:Collagen (2 μg/mL)-induced platelet aggregation was reduced by ferulenol in a concentration-dependent manner from 10 to 40 μM, with near-complete inhibition at 40 μM. Ferulenol produced only moderate inhibition of arachidonic acid (AA, 60 μM)-induced aggregation at 80 μM and did not significantly affect by thrombin (0.02 U/mL)-induced platelet responses. Moreover, ferulenol markedly reduced collagen-induced platelet activation markers, including P-selectin expression, intracellular Ca2+ mobilization, and adenosine triphosphate (ATP) release. These inhibitory effects were accompanied by reduced phosphorylation of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), and mitogen-activated protein kinase (MAPK) family members, including extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Ferulenol also suppressed activation of the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β) signaling pathway. In mice, ferulenol extended the time to thrombotic platelet plug formation, without significantly increasing bleeding time.Conclusions:This study, provides the first evidence that ferulenol inhibits platelet activation. Mechanistically, ferulenol may inhibit collagen-induced platelet activation, at least in part, by suppressing PLCγ2 activation, thereby reducing phosphorylation of cPLA2, MAPKs, and PI3K/Akt/GSK3β signaling proteins. These inhibitory effects were reflected by reduced P-selectin exposure on the platelet surface, suggesting suppression of α-granule release. In addition, ferulenol reduced dense-granule ATP release and limited the rise in intracellular Ca2+ levels, thereby contributing to the inhibition of platelet aggregation. Therefore, ferulenol may be a promising candidate for preventing thromboembolic events associated with CVDs.