Suppression by wortmannin of platelet responses to stimuli due to inhibition of pleckstrin phosphorylation.
Studies were made of inhibition by wortmannin, a fungal metabolite, of human platelet responses to various stimuli. Wortmannin at concentrations as low as 1-100 nM inhibited several receptor-agonist-induced 5-hydroxytryptamine release from platelets, without affecting agonist-induced increases in the intracellular concentration of Ca2+. Phorbol 12-myristate 13-acetate (PMA), an active tumour promoter, caused 5-hydroxytryptamine release when combined with a low concentration of ionomycin, and platelet aggregation by itself; these effects of the phorbol ester were also inhibited by wortmannin as well as by staurosporine, a potent, although non-specific, protein kinase C (PKC) inhibitor, in a similar molar concentration range. The platelet responses to the receptor agonists or PMA were accompanied by increased incorporation of [32P]Pi into pleckstrin, a protein selectively expressed in platelets and other blood cells arising from haematopoietic stem cells, as a result of PKC activation in the intact cells. The pleckstrin phosphorylation was inhibited by wortmannin in ways mostly similar to those in which it inhibited the 5-hydroxytryptamine-release responses. Nevertheless, wortmannin failed to inhibit PKC activity measurable in a cell-free assay system which is highly susceptible to staurosporine. Nor did it inhibit the translocation of cytosolic PKC to membranes induced by addition of PMA to platelet cells. Thus wortmannin, which is not a direct inhibitor of PKC, could interfere with the kinase-dependent phosphorylation of pleckstrin, which may play an important role in the cellular responses to receptor stimulation.
Yatomi
Y
Y
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hazeki
O
O
Kume
S
S
Ui
M
M
D000470:Alkaloids / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D001798:Blood Proteins / Q000378:metabolism*; D002118:Calcium / Q000097:blood; D003094:Collagen / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D010710:Phosphates / Q000097:blood; D010750:Phosphoproteins; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood; D012701:Serotonin / Q000097:blood; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin
D000470:Alkaloids; D000730:Androstadienes; D001798:Blood Proteins; D010710:Phosphates; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; C039077:platelet protein P47; D012701:Serotonin; D015759:Ionomycin; D003094:Collagen; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D000077191:Wortmannin