THROMBIN-INDUCED PHOSPHORYLATION AND ACTIVATION OF CA2-SENSITIVE CYTOSOLIC PHOSPHOLIPASE-A(2) IN HUMAN PLATELETS()

Receptor-mediated activation of human platelets by thrombin initiates a series of rapid biochemical events that include activation of phosph olipase A2 to liberate arachidonic acid for further conversion to thro mboxane A2. The identity of the phospholipase A2 involved has not been clear. Here we show by immunochemical analysis that human platelets c ontain significant amounts (60 ng/10(9) platelets) of the recently ide ntified Ca2+-sensitive cytosolic phospholipase A2 (cPLA2). Metabolic l abeling of human platelets with P-33i revealed that the extent of phos phorylation of cPLA2 was greatly increased after thrombin treatment. U pon stimulation of platelets with thrombin, cPLA2 exhibits enhanced ca talytic activity, as well as a change in its electrophoretic and chrom atographic properties compared with cPLA2 in resting platelets. These alterations of cPLA2 are reversed by treatment with phosphatase, demon strating that they are the consequence of thrombin-stimulated phosphor ylation. Thrombin-induced phosphorylation and activation of cPLA2 is r apid (half-maximal by 1 min at 1 unit/10(9) platelets) and dose-depend ent. Agonist-induced phosphorylation of cPLA2 is more sensitive to thr ombin than the generation of thromboxane A2, suggesting that it may be an early event in the sequence of steps leading to the mobilization a nd further metabolism of arachidonic acid. By comparing the functional properties of cPLA2 from control versus thrombin-stimulated platelets , we found that while activated cPLA2 exhibits the same Ca2+ requireme nt and apparent substrate affinity (K(m)), its catalytic activity (V(m ax)) is increased compared with control cPLA2. We conclude that 1) cPL A2 is likely to play an important role in agonist-induced mobilization of arachidonic acid and 2) thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity.