Weak inhibition of protein kinase C coupled with various non-specific effects make sphingosine an unsuitable tool in platelet signal transduction studies.
The effects of sphingosine, the newly described inhibitor of the enzyme protein kinase C, on human platelet activation, were studied in order to gain further information on the role of protein kinase in platelet responses. Concentrations of the drug (5-20 microM) which had little effect on protein kinase C activation as measured by the phosphorylation of the 45 kDa and 20 kDa protein substrates induced by phorbol 12-myristate 13-acetate (PMA) and thrombin, strongly inhibited platelet aggregation induced by these agonists, as well as aggregation induced by ADP and ionomycin, which caused no detectable protein kinase C activation or 5-hydroxy[14C]tryptamine[( 14C]5HT) secretion. At approx. 10-fold higher concentrations (150-200 microM), sphingosine had significant inhibitory effects on PMA and thrombin-induced 45 kDa and 20 kDa protein phosphorylation. However, at these high concentrations, the drug caused extensive membrane damage/leakiness as suggested by the substantial release of [14C]5HT and [3H]adenine from pre-loaded platelets (50-70% release of both markers), and the total quenching of quin2 fluorescence by Mn2+ in the presence of the drug. Due to the increased membrane leakiness in the presence of the drug, an apparent potentiation of agonist-induced intracellular Ca2+ elevations in quin2-loaded platelets, as well as an increase in quin2 fluorescence with the drug alone (more than 50 microM) were also observed. Despite this, however, thrombin-induced [3H]arachidonate release was severely reduced in the presence of sphingosine, underlining the inhibitory effects at the membrane level. It is concluded that the weak, if any, inhibitory effects on protein kinase C at concentrations not affecting membrane integrity, as well as the inhibitory effects of sphingosine on platelet aggregation, make it an unsuitable compound as a tool for studies on platelet stimulus-response coupling.
Krishnamurthi
S
S
Thrombosis Research Unit, King's College School of Medicine and Dentistry, Rayne Institute, London, U.K.
Patel
Y
Y
Kakkar
V V
VV
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000097:blood; D004987:Ethers / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010750:Phosphoproteins / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood*; D012701:Serotonin / Q000097:blood; D015398:Signal Transduction / Q000187:drug effects*; D013110:Sphingosine / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001095:Arachidonic Acids; D001798:Blood Proteins; D004987:Ethers; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D016718:Arachidonic Acid; D012701:Serotonin; D015759:Ionomycin; D011493:Protein Kinase C; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium