Abstract
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.
Highlights
Human platelets pretreated with indomethacin release arachidonic acid predominantly through the activity of cytosolic phospholipase A2, an 85-kDa protein
The results demonstrate that cytosolic phospholipase A2 (cPLA2) undergoes phosphorylation in collagen- and thrombin-stimulated platelets independent of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) and that Ca2ϩ has a major role in its regulation independent of phosphorylation
Release of [3H]Arachidonic Acid from Platelets Is Dependent on Ca2ϩ—The release of [3H]arachidonic acid was measured as an index of cPLA2 activity in human platelets
Summary
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 for arachidonic acid liberation, and by phosphorylation. Phosphorylation of cPLA2 enhanced the Ca2؉-induced arachidonic acid release in platelets stimulated by the ionophore A23187 and phorbol ester (phorbol 12,13dibutyrate (PDBu)). 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. MAPK activity is regulated through MAPK kinase [13,14,15], which is itself activated downstream of PKC-dependent and -independent pathways [16, 17]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.