Abstract 57: 12-Lipoxygenase is a Critical Enzyme Regulating FcyRIIa-Mediated Platelet Activation

Abstract

Introduction: FcγRIIa plays a key role in platelet activation mediated by immune complexes containing IgG and platelet factor 4 (PF4) bound to heparin. One of the primary mechanisms by which FcγRIIa mediates it effects in the platelet is through its ITAM motif. 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of platelet function through GPVI, which also signals in part through an ITAM motif. We therefore hypothesize that 12-LOX can play an important role in regulation of FcγRIIa-mediated platelet activation resulting in aggregation and thrombosis. Identifying the importance of this regulatory interaction is essential for understanding the role of 12-LOX in regulation of hemostasis and thrombosis, and identifying a possible target for prevention of FcγRIIa -induced platelet activation. Objectives: Determine the requirement of 12-LOX activation in FcγRIIa -mediated platelet activation and identify the underlying mechanism by which 12-LOX activation regulates FcγRIIa. Methods: FcγRIIa-mediated Platelet aggregation was measured in washed human platelets and mouse platelets in the presence of a 12-LOX inhibitor or in FcγRIIa +/+ mice crossed with 12-LOX -/- mice. PLCγ and PKC activation were measured in the absence of 12-LOX activity. Additionally, Rap1, calcium, and αIIbβ3 activity was measured in the presence or absence of 12-LOX activity following stimulation of FcγRIIa with CD9 or goat anti-mouse IV.3 antibody (IV.3). Results: Both human and FcγRIIa +/+ mouse platelets were able to fully aggregate following stimulation with CD9 or IV.3. Pharmacological inhibition of 12-LOX or absence of 12-LOX significantly delayed and attenuated platelet aggregation. Additionally, PLCγ, calcium, Rap1 and activation of αIIbβ3 were inhibited. Conclusions: These observations support our hypothesis that 12-LOX is an essential enzyme in the regulation of FcγRIIa-mediated platelet activation. Further investigation of this regulatory pathway will help to confirm 12-LOX as a viable target for inhibition of FcγRIIa and other ITAM-mediated platelet activation.