Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors.
Stimulation of platelets with thrombin leads to rapid degradation of inositol phospholipids, generation of diacylglycerol (DAG) and subsequent activation of protein kinase C (PKC). Previous studies indicated that prior activation of PKC with phorbol myristate acetate (PMA) desensitizes platelets to thrombin stimulation, as indicated by a decreased production of inositol phosphates and decreased Ca2+ mobilization. This suggests that PKC activation generates negative-feedback signals, which limit the phosphoinositide response. To test this hypothesis further, we examined the effects of PKC activators and inhibitors on thrombin-stimulated DAG mass formation in platelets. Pretreatment with PMA abolishes thrombin-stimulated DAG formation (50% inhibition at 60 nM). Pretreatment of platelets with the PKC inhibitors K252a or staurosporine potentiates DAG production in response to thrombin (3-4-fold) when using concentrations required to inhibit platelet PKC (1-10 microM). K252a does not inhibit phosphorylation of endogenous DAG or phosphorylation of a cell-permeant DAG in unstimulated platelets, indicating that DAG over-production is not due to inhibition of DAG kinase. Sphingosine, a PKC inhibitor with a different mechanism of action, also potentiates DAG formation in response to thrombin. Several lines of evidence indicate that DAG formation under the conditions employed occurs predominantly by phosphoinositide (and not phosphatidylcholine) hydrolysis: (1) PMA alone does not elicit DAG formation, but inhibits agonist-stimulated DAG formation; (2) thrombin-stimulated DAG formation is inhibited by neomycin (1-10 mM) but not by the phosphatidate phosphohydrolase inhibitor propranolol; and (3) no metabolism of radiolabelled phosphatidylcholine was observed upon stimulation by thrombin or PMA. These data provide strong support for a role of PKC in limiting the extent of platelet phosphoinositide hydrolysis.
Bishop
W R
WR
Department of Microbiology, Schering-Plough Research, Bloomfield, NJ 07003.
August
J
J
Petrin
J M
JM
Pai
J K
JK
D000470:Alkaloids / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002227:Carbazoles / Q000494:pharmacology; D004075:Diglycerides / Q000097:blood*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D005246:Feedback; D005989:Glycerides / Q000097:blood*; D006801:Humans; D026121:Indole Alkaloids; D009355:Neomycin / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000097:blood; D010713:Phosphatidylcholines / Q000097:blood; D010766:Phosphorylation; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D015290:Second Messenger Systems; D013110:Sphingosine / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
C011439:1,2-diacylglycerol; D000470:Alkaloids; D002227:Carbazoles; D004075:Diglycerides; D005989:Glycerides; D026121:Indole Alkaloids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; C049985:staurosporine aglycone; D011433:Propranolol; D011493:Protein Kinase C; D013917:Thrombin; D019311:Staurosporine; D009355:Neomycin; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate