Background: Overexpression of 12-lypoxygenase (12-LOX) is a common feature of tumor cells. Inhibition of 12-LOX induces apoptosis in several cancer cell lines and appears to be a promising strategy in anticancer therapy. However, 12-LOX inhibition can impair platelet activation during therapy and cause bleeding in patients.

Aims: The aim of our study was to investigate effects of a novel selective 12-LOX inhibitor ML355 on platelet functions and apoptosis.

Methods: Washed platelets from healthy donors were incubated with ML355 for indicated time and analyzed by flow cytometry or Western blotting. Phosphatidylserine (PS) surface expression (Annexin V-PE) and αIIbβ3 integrins activation (PAC-1-FITC) were assessed by flow cytometry. Vasodilator-stimulated phosphoprotein (VASP), PKB, p38 phosphorylation and caspase-3 activation were analyzed by Western blotting.

Results: We showed that ML355 (10 µM) impaired platelet αIIbβ3 integrins activation induced by thrombin, and inhibited PKB and p38 phosphorylation. 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. ML355 caused VASP phosphorylation starting from 20 µM meaning that inhibition of platelet activation in lower doses was mediated by 12-LOX inhibition and consequent dose elevation lead to PKA or PKG activation. VASP phosphorylation induced by ML355 was prevented by GC but not AC inhibition. NO-synthase (NOS) inhibition by LNAME did not affect ML355-mediated VASP phosphorylation. These data imply NO-independent mechanism of GC activation induced by ML355. Additionally, ML355 dose-dependently blocked PS exposure and caspase-3 activation, induced by a high affinity BCL-X_L inhibitor ABT-737.

Conclusions: In summary, we showed that ML355 inhibits platelet activation via NO-independent GC activation and blocks platelet apoptosis, induced by ABT-737.
The reported study was funded by RFBR, project number 19-315-90102.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/ml355-inhibits-platelets-via-gc-activation-and-blocks-caspase-dependent-apoptosis-in-platelets/