Cytosolic phospholipase A2 is phosphorylated in collagen- and thrombin-stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase.
Human platelets pretreated with indomethacin release arachidonic acid predominantly through the activity of cytosolic phospholipase A2 (cPLA2), an 85-kDa protein. This enzyme is regulated by an increase in intracellular Ca2+, a necessary condition of for arachidonic acid liberation, and by phosphorylation. Phosphorylation of cPLA2 enhanced the Ca(2+)-induced arachidonic acid release in platelets stimulated by the ionophore A23187 and phorbol ester (phorbol 12,13-dibutyrate (PDBu)). In thrombin-stimulated platelets, however, phosphorylation appeared not to be necessary for arachidonic acid release since the latter response was not impaired in the presence of staurosporine, which inhibited phosphorylation. Collagen, thrombin, and PDBu induced phosphorylation of platelet cPLA2 as well as activation of mitogen-activated protein kinase (MAPK; p42mapk and p44mapk). cPLA2 activation was not dependent on protein kinase C (PKC) in thrombin- and collagen-stimulated platelets, as preincubation with the PKC inhibitor Ro 31-8220 neither interfered with cPLA2 phosphorylation nor reduced arachidonic acid release. However, collagen- and thrombin-induced activation of MAPK was inhibited by Ro 31-8220, indicating that PKC is necessary for MAPK stimulation in platelets. Although MAPK may underlie phosphorylation of cPLA2 in PDBu-activated human platelets, our results provide evidence for PKC- and MAPK-independent phosphorylation of cPLA2 in platelets stimulated by the physiological activators collagen and thrombin.
Börsch-Haubold
A G
AG
Department of Pharmacology, University of Oxford, United Kingdom.
Kramer
R M
RM
Watson
S P
SP
D016718:Arachidonic Acid / Q000096:biosynthesis*; D001792:Blood Platelets / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003094:Collagen / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D015539:Platelet Activation / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D007476:Ionophores; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D003094:Collagen; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; D002118:Calcium