Abstract/Summary

Introduction: Formyl peptide receptor 2/ALX (FPR2/ALX), a member of seven transmembrane G protein-coupled receptors superfamily, exhibits dual effects of pro- and anti-inflammatory responses based on the nature of stimulating ligand type. Recently, FPRs have been shown to be present in platelets and regulate their function. However, the role of FPR2/ALX and the effects of its anti-inflammatory ligands on the regulation of platelet function have not yet been addressed. Platelets play significant roles in the regulation of inflammation and host defence in addition to their significant roles in haemostasis and thrombosis. They act as sentinels through regulating inflammatory responses due to their large number in the circulation and their ability to rapidly release different kinds of immunomodulatory cytokines, and other inflammatory mediators. Given the anti-inflammatory effects of specific FPR2/ALX ligands, we sought to study the role of its anti-inflammatory ligand, lipoxin A4 analogue, BML-111 in the modulation of platelet function and thrombus formation. Results: The expression of FPR2/ALX in human and mouse platelets as well as megakaryocytes was confirmed using immunoblot analysis. The subcellular distribution of FPR2/ALX in platelets was demonstrated by immunocytochemistry. FPR2/ALX lipid raft association was also established. BML-111 inhibited a range of platelet activities such as aggregation, fibrinogen binding to integrin αIIbβ3, ! -granule secretion, dense granule secretion, Ca2+ mobilisation and integrin αIIbβ3-mediated outside-in signalling as reflected on clot retraction and platelet spreading. The selectivity of BML-111 for FPR2/ALX was confirmed using FPR2/ALX deficient mice and FPR2/ALX selective antagonist, WRW4. In vitro thrombus formation was also inhibited by various concentrations of BML-111. Moreover, the levels of vasodilator stimulated phosphorylation (VASP-S157) as an indicator of protein kinase A (PKA) activation were augmented substantially after BML-111 treatment in resting and activated platelets. However, this elevation was independent from the levels cyclic adenosine monophosphate (cAMP). Abstract III Conclusion: This study demonstrates the significance of FPR2/ALX and its anti-inflammatory ligand, BML-111 in the modulation of platelet function. This suggests that BML-111 may act as a potential modulator of platelet function using FPR2/ALX as a target. Overall, FPR2/ALX may act as novel target to control thrombo-inflammatory conditions.