Correlation between platelet aggregation and dephosphorylation of a 68 kDa protein revealed through the use of putative PKC inhibitors.
The efficacy of two structurally and functionally unrelated protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, was assessed in intact human platelets by studying platelet aggregation in response to stimulation with phorbol 12-myristate 13-acetate (PMA) or the thromboxane-A2 mimetic, U46619. Surprisingly, both inhibitors increased aggregation in response to PMA, but decreased aggregation in response to U46619. To further explore this phenomenon, gel electrophoresis of 32P-labelled proteins from PMA- or U46619-stimulated platelets in the presence and absence of the two putative PKC inhibitors was performed. Although neither chelerythrine nor calphostin C proved to be effective PKC inhibitors in intact human platelets, a strong correlation between the dephosphorylation of a 68 kDa protein and the rate of platelet aggregation was observed. From these results, the indiscriminate use of PKC inhibitors in whole platelets is questioned and attention is drawn to the role of protein dephosphorylation in platelet activation. The 68 kDa protein was the major phosphorylated substrate in resting platelets. Okadaic acid increased phosphorylation of this band, indicating active phosphate group turnover under resting conditions.
Turini
M E
ME
Department of Nutritional Sciences, University of Guelph, Ontario, Canada.
Gaudette
D C
DC
Holub
B J
BJ
Kirkland
J B
JB
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000470:Alkaloids; D053119:Benzophenanthridines; D001798:Blood Proteins / Q000378:metabolism*; D006801:Humans; D008297:Male; D008970:Molecular Weight; D009281:Naphthalenes; D010617:Phenanthridines / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000494:pharmacology; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000031:analogs & derivatives* / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D001798:Blood Proteins; D009281:Naphthalenes; D010617:Phenanthridines; D010761:Phosphorus Radioisotopes; D010975:Platelet Aggregation Inhibitors; D011083:Polycyclic Compounds; D011450:Prostaglandin Endoperoxides, Synthetic; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; C016299:chelerythrine; D011493:Protein Kinase C; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate