Evidence that activation of phospholipase D can mediate secretion from permeabilized platelets.
Studies on electropermeabilized human platelets indicated that any two of three distinct factors must be present for marked secretion of dense or alpha-granule constituents to occur. These factors are Ca2+, activation of protein kinase C (PKC) and activation of an unidentified GTP-binding protein ('GE'). Thus, in the absence of Ca2+, phorbol ester and GTP[S] acted synergistically to promote secretion, whereas in the presence of Ca2+, either activation of PKC or addition of GTP[S] was sufficient. In all cases, secretion correlated with the activation of phospholipase D (PLD), as detected by the formation of [3H]phosphatidic acid (PA) in the absence of ethanol or of [3H]phosphatidylethanol (PEt) in the presence of ethanol. Secretion did not correlate with phospholipase C (PLC) activity or with the accumulation of 1,2-diacylglycerol (DAG), both of which required Ca2+ and were inhibited by phorbol ester. Ethanol partially inhibited secretion in the absence of Ca2+. BAPTA, a known inhibitor of Ca(2+)-independent secretion in permeabilized cells, caused parallel inhibitions of secretion and PLD activity. GTP[S] enhanced PKC activity, as indicated by pleckstrin phosphorylation, apparently by stimulating the formation of PA in the absence of Ca2+, as well as of DAG in the presence of Ca2+. PA and stable analogues, including PEt, stimulated the Ca(2+)-independent phosphorylation of pleckstrin and other proteins in platelet supernatant fraction. The results suggest that PA formed by activation of PLD may mediate secretion from permeabilized platelets by PKC-dependent and independent mechanisms. However, in intact platelets stimulated by thrombin, PLD accounted for only 10-20% of the total PA formed and can only play a major role in secretion if this PA fraction is distinct from that formed by the combined actions of PLC and DAG kinase.
Haslam
R J
RJ
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Coorssen
J R
JR
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D000431:Ethanol / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D010739:Phospholipase D / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001798:Blood Proteins; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000431:Ethanol; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium