TRANSLOCATION AND PHOSPHORYLATION OF CYTOSOLIC PHOSPHOLIPASE A(2) IN ACTIVATED PLATELETS

The release of arachidonic acid, and its subsequent conversion to thro mboxane A(2), is an important component of platelet activation. The pr ecise mechanism of arachidonic acid release is unknown although cytoso lic phospholipase A(2) (cPLA(2)) has been implicated. In the present s tudy the effects of three agonists, the serine protease thrombin, the protein kinase C stimulant PMA and the calcium ionophore A23187 have b een examined on the translocation and phosphorylation of cPLA(2) and t hese have been correlated with arachidonic acid release. Thrombin, but neither PMA nor A23187, caused the release of [C-14]-arachidonic acid from unstirred, prelabeled platelets. Immunoblot analysis was carried out on cytosolic and membrane fractions from control and activated pl atelets using an anti-cPLA(2) antibody. In platelets stimulated by thr ombin or A23187, but not by PMA, there was a translocation of cPLA(2) to the membrane fraction. Immunoprecipitation of cPLA(2), from [P-32]- orthophosphate-prelabeled platelets, indicated enhanced phosphorylatio n on serine residues of cPLA(2) from thrombin- or PMA-stimulated plate lets. These results are consistent with two synergistic pathways media ting cPLA(2) activity. Increased cytosolic calcium causes the transloc ation of cPLA(2) to the membrane, and protein kinase either directly, or indirectly, phosphorylates the enzyme. Activation of both pathways, as occurs in response to thrombin, is required for arachidonic acid l iberation. (C) 1998 Elsevier Science Ltd.