Objective—The rate-limiting step in the biosynthesis of thromboxane A2(TxA2) and 12-hydroxyeicosatetraenoic acid (12-HETE) by platelets is activation of cytosolic phospholipase A2α(cPLA2α), which releases arachidonic acid, which is the substrate for cyclooxygenase-1 (COX-1) and 12-lipoxygenase. We evaluated signaling via the human platelet thrombin receptors, protease-activated receptor (PAR) 1 and PAR4, to the activation of cPLA2α, which provides a substrate for the biosynthesis of TxA2and 12-HETE.Methods and Results—Stimulating washed human platelets resulted in delayed biosynthesis of 12-HETE, which continues after maximal formation of TxA2is completed, suggesting that 12-HETE is not formed by the same pool of arachidonic acid that provides a substrate to COX-1. PAR1-induced formation of TxA2was inhibited by the phosphatidylinositol kinase inhibitor LY294002, whereas this inhibitor did not block 12-HETE biosynthesis. Both 1-butanol and propranolol also blocked TxA2biosynthesis but did not inhibit 12-HETE formation.Conclusion—The concerted evidence indicates that the platelet thrombin receptors signal activation of cPLA2αcoupled to COX-1 by a pathway different from that signaling activation of the cPLA2αcoupled to 12-lipoxygenase.