Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.
Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.
Benistant
C
C
Department of Pathology and Cell Biology, Jefferson Medical College, Philadelphia, PA 19107.
Rubin
R
R
D000244:Adenosine Diphosphate / Q000494:pharmacology; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002118:Calcium / Q000097:blood; D004075:Diglycerides / Q000097:blood; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007295:Inositol Phosphates / Q000097:blood; D015759:Ionomycin / Q000494:pharmacology; D008854:Microscopy, Electron; D010712:Phosphatidic Acids / Q000097:blood; D015539:Platelet Activation / Q000187:drug effects; D011493:Protein Kinase C / Q000097:blood*; D012701:Serotonin / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000097:blood*
D001095:Arachidonic Acids; D004075:Diglycerides; D007295:Inositol Phosphates; D010712:Phosphatidic Acids; D016718:Arachidonic Acid; D012701:Serotonin; D000431:Ethanol; D015759:Ionomycin; D000244:Adenosine Diphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D007213:Indomethacin