Non-Enzymatic Role of Platelet 12-LOX in Platelet Activation

Background 12-lipoxygenase (12-LOX) plays an essential role in regulation of vascular disease and human platelet function through oxylipin generation such as 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE) and 12-hydroxyeicosatetraenoic acid (12-HETE). Previous literature has demonstrated that 12-LOX enzymatic function such as oxylipin generation is important for collagen- or thrombin-mediated signaling events. However, the addition of 12(S)-HETE does not completely restore the inhibitory effect of pharmacologically or genetically inhibiting 12-LOX on platelet activation, indicating that 12-LOX may have a non-enzymatic function in platelets. Therefore, we tested the non-enzymatic role of 12-LOX in platelet activation.

Method 12 LOX was co-immunoprecipitated (co-IP) from washed human platelets following FcγRIIa or thrombin stimulation. The bound proteins were eluted by laemmli buffer and the elutes were analyzed on SDS-PAGE. Proteins co-IP with 12-LOX at various times were identified by mass spectrometry and confirmed by western blot analysis.

Result Silver-stained gel of 12-LOX co-IP indicated the presence of several protein bands in complex association with 12-LOX at different time points. These bands were analyzed by mass spectroscopy and several proteins observed in complex with 12-LOX were identified including integrin β3, Talin-1, Myh9, Nexilin, Gelsolin, Actin, cytoplasmic 2, and Actin related protein 2/3 (ARP 2/3) . To confirm the mass spectroscopy identified protein, these proteins were immunoblotted following co-IP of 12-LOX. to confirm their association with 12-LOX prior to and following platelet activation. These interactions were further confirmed with a reverse co-IP. Finally, to determine if the association pattern of these proteins with 12-LOX is disrupted in the presence of a 12-LOX inhibitor already shown to limit platelet activation and which is currently in clinical trials for treatment of HIT (ML355/VLX-1005), we were able to demonstrate that the inhibitor appears to lock the protein complex into place.

Conclusion Our study identified several proteins that are present in 12-LOX complex such as integrin β3, talin-1, Myh9 and ARP 2/3. 12-LOX interacts with Integrin β3, Talin-1 and cytoskeletal proteins. Furthermore, the dynamic movement of the proteins in this complex into and out of association with 12-LOX is essential for normal platelet activation suggesting 12-LOX plays an important non-enzymatic role in regulation of agonist-mediated platelet activation.