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PMID: 2386487 Published · ppublish English Journal Article

Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors.

The Biochemical journal ·Vol. 269 ·No. 2 ·1990-07-15 ·Pages 465-73

Bishop WR, August J, Petrin JM, Pai JK

Abstract

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.

MeSH Terms
Alkaloids/pharmacology Blood Platelets/drug effects,metabolism Carbazoles/pharmacology Diglycerides/blood Drug Synergism Enzyme Activation/drug effects Feedback Glycerides/blood Humans Indole Alkaloids Neomycin/pharmacology Phosphatidic Acids/blood Phosphatidylcholines/blood Phosphorylation Propranolol/pharmacology Protein Kinase C/antagonists & inhibitors Second Messenger Systems Sphingosine/pharmacology Staurosporine Tetradecanoylphorbol Acetate/pharmacology Thrombin/pharmacology
Chemicals
1,2-diacylglycerol Alkaloids Carbazoles Diglycerides Glycerides Indole Alkaloids Phosphatidic Acids Phosphatidylcholines staurosporine aglycone Propranolol Protein Kinase C Thrombin Staurosporine Neomycin Sphingosine Tetradecanoylphorbol Acetate
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Bishop W R
Department of Microbiology, Schering-Plough Research, Bloomfield, NJ 07003.
August J
Petrin J M
Pai J K
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2690465
Published
1990-07-15
Pages
465-73
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1131600
Subset
IM
External Links
PubMed source DOI: 10.1042/bj2690465 PMC Full Text View journal details
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