An anti-platelet agent, OPC-29030, inhibits translocation of 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid production in human platelets.

1. In human platelets, arachidonic acid is mainly metabolized by the two enzyme systems; cyclo-oxygenase and 12-lipoxygenase. Cyclo-oxygenase produces prostaglandin H(2) which is further converted to thromboxane B(2). 12-Lipoxygenase synthesizes 12(S)-hydroperoxyeicosatetraenoic acid which is reduced to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 2. An anti-platelet compound, OPC-29030, dose-dependently inhibited 12(S)-HETE production with an IC(50) of 0.06+/-0.01 microM, but not synthesis of thromboxane B(2) in human platelets. Although the compound suppressed 12(S)-HETE production in human platelets, cytosolic 12-lipoxygenase activity was not inhibited up to 10 microM. Essentially identical data were obtained with a 12-lipoxygenase of human erythroleukaemia cells which had megakaryocyte/platelet-like properties. 3. OPC-29030 also suppressed production of 5(S)-HETE, a 5-lipoxygenase product, in rat basophilic leukaemia cells without inhibiting enzyme activity. It has been shown that 5-lipoxygenase binds to membrane 5-lipoxygenase-activating protein (FLAP) to produce 5(S)-HETE, and thus FLAP inhibitor suppresses cellular 5(S)-HETE production. 4. A FLAP inhibitor, L-655,238, suppressed platelet 12(S)-HETE production, but had no effect on the 12-lipoxygenase activity. 5. Western blot analysis showed that platelet 12-lipoxygenase translocated from cytosol to membranes upon thrombin stimulation, and OPC-29030 suppressed this process in a dose-dependent manner. 6. These results suggest that the 12-lipoxygenase of human platelets binds to FLAP or a similar protein, and OPC-29030 suppresses 12(S)-HETE production by inhibiting a certain step of the 12-lipoxygenase translocation.