iPLA2β Contributes to Activation of Human and Mouse Platelets Via ITAM Receptors Fcγ RIIA and GPVI

Platelet activation by ITAM receptors contributes to hemostasis, thrombosis, vascular integrity and host defense. In the course of our studies of FcγRIIA-mediated platelet activation, we became interested in those mechanisms that require neither full Syk activation nor changes in intracellular calcium. Calcium-independent phospholipase family member iPLA2β has been observed to modulate phospholipid remodeling and second messenger generation in human platelets (Beckett, Thromb Res 2007; Duvernay, Biochem 2015), while iPLA2γ has been studied in knockout mouse platelets (Yoda, JTH 2014) with modest effects noted on thrombosis and hemostasis. These enzymes do not require increased cytoplasmic calcium for their activity in cleaving the acyl group from the sn2 position of phospholipids to yield a free fatty acid and a lysophospholipid. However, the precise role of iPLA2β in human and mouse platelet activation has not been elucidated. Neither has the contribution of iPLA2β in the response to FcγRIIA-mediated activation been reported.

We identified the presence of iPLA2β protein in western blots of human and FcγRIIA transgenic mouse platelets. Of interest, multiple isoforms arising from proteolytic cleavage were detected. We treated washed human and FcγRIIA transgenic mouse platelets with agonists to FcγRIIA (IV.3 + GAM) and to GPVI (collagen or collagen-related peptide) in the absence and presence of pharmacologic inhibitors of iPLA2β. At a range of agonist doses up to 3X threshold, we observed significant inhibition of aggregation, dense granule secretion and alpha granule secretion (p<0.05 vs. vehicle only, n = 3 to 4 each). Inhibition occurred with either S-BEL (bromo-enolactone) or with FKGK18 (a fluoroketone), two chemically distinct iPLA2β inhibitor molecules with different modes of action. The IC50 for S-BEL was found to be 1.02 uM for human FcγRIIA, 2.04 uM for human GPVI, and 2.76 uM for transgenic mouse FcγRIIA activated platelets. FKGK18 was less potent, with IC50s at 7.88 uM for human FcγRIIA. In contrast, iPLA2γ inhibitor R-BEL was able to inhibit FcγRIIA -mediated activation, but at an IC50 of 2.62 uM. Notably, iPLA2β inhibition could eliminate ATP secretion from dense granules downstream of FcγRIIA and GPVI activation. When we added ADP to FcγRIIA stimulation in the presence of inhibitory doses of S-BEL, we overcame the inhibition.

We have identified for the first time that iPLA2β contributes to aggregation and secretion of both human and FcγRIIA transgenic mouse platelets. The platelets were slightly more sensitive to FcγRIIA than to GPVI inhibition. In other activatable secretory cells, iPLA2β plays both a homeostatic and signaling role. The mechanisms of iPLA2β action in platelets merit further study. Studies are in progress with genetic knockdown and knockout of the enzyme, to complement the findings with inhibitors.