With a newer, more selective and efficacious cytosolic phospholipase A<inf>2</inf>α (cPLA<inf>2</inf>α) inhibitor available, we revisited the role of cPLA<inf>2</inf>α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA<inf>2</inf>α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A<inf>2</inf> receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA<inf>2</inf>α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA<inf>2</inf>α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA<inf>2</inf>α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA<inf>2</inf>α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA<inf>2</inf>α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses.
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