is implicated in regulation of platelet activation processes and can be a new promising target for antiplatelet therapy. However, investigations of 12-LOX were restricted by the lack of specific and potent 12-LOX inhibitors and by controversial data concerning the role of 12-LOX metabolites in platelet functions. A novel specific 12-LOX inhibitor ML355 was shown to inhibit platelet aggregation without adverse side effects on hemostasis; however, the molecular mechanisms of its action on platelets are poorly understood. Here, we showed that ML355 inhibited platelet activation induced by thrombin or thromboxane A 2 , but not by collagenrelated peptide. ML355 blocked protein kinase B, phosphoinositide 3-kinase, and extracellular signal-regulated kinase, but not p38 kinase, spleen tyrosine kinase (Syk), or phospholipase Cc2 phosphorylation in activated platelets. The main inhibitory effect of low doses of ML355 (1-20 lM) on thrombin activated platelets was mediated by the decrease in reactive oxygen species level, whereas high doses of ML355 (50 lM) caused cyclic adenosine monophosphate activation. ML355 did not affect the activity of nitric oxide-dependent soluble guanylyl cyclase, nor did it affect the relaxation of preconstricted aortic rings in mice. ML355 itself did not affect platelet viability, but at 50 lM dose blocked caspase-dependent apoptosis induced by B-cell lymphoma II inhibitor ABT-737. SIGNIFICANCE STATEMENTThe current paper provides novel and original data concerning molecular mechanisms of 12-LOX inhibitor ML355 action on platelets. These data reveal antiplatelet and protective effects of ML355 on platelets and may be of importance for both antiplatelet and anticancer therapy.

No abstract

One new compound isoembinin 1 along with ten known compounds 2-11 were isolated from the terrestrial parts of Iris lactea Pall. All of the compound structures were determined through extensive 1D and 2D NMR experiments along with HR-ESIMS analysis and comparison with literature data. Because many flavonoids exert antiplatelet and antioxidant activity we tested the effects of the isolated flavone C-glycosides 1-9 on platelet activation and reactive oxygen species (ROS) production. Platelet reactivity was assessed by activation of αIIbβ3 integrins activation and ROS production by DCF-DA fluorescence. For the analysis of whether protein kinase A or G are involved in the platelet inhibition, the activity of these kinases was analyzed by phosphorylation of their common substrate in platelets. In all experiments apigenin, which inhibit platelet activation was used as a positive control. All isolated flavone C-glycosides inhibited platelet αIIbβ3 integrins activation with IC50 in the µM range, however this inhibitory effect was found to not be mediated through the prevention of ROS formation or by the activation of cyclic nucleotide pathways. Structure-activity comparison between apigenin and compounds 1-9 shows that the presence of C-glycoside and O-glycoside residues on the aglycone apigenin diminish the degree of platelet inhibition.