No direct correlation between Ca2+ mobilization and dissociation of Gi during platelet phospholipase A2 activation.
Stimulation of human platelets with thrombin is accompanied by activation of both phospholipases C and A2. These have been considered to be sequential events, with phospholipase A2 activation resulting from the prior hydrolysis of inositol phospholipids and mobilization of intracellular Ca2+ stores. However, our and other laboratories have recently questioned this proposal, and we now present further evidence that these enzymes may be activated by separate mechanisms during thrombin stimulation. Alpha-thrombin induced the rapid hydrolysis of inositol phospholipids, and formation of inositol trisphosphate and phosphatidic acid. This was paralleled by mobilization of Ca2+ from internal stores. These responses were blocked by about 50% by prostacyclin. In contrast, the liberation of arachidonic acid induced by alpha-thrombin was totally inhibited by prostacyclin. The less-effective agonists, platelet activating factor (PAF) and gamma-thrombin also both stimulated phospholipase C, but whereas PAF evoked a rapid and transient response, that of gamma-thrombin was delayed and more sustained. The abilities of these agonists to induce the release of Ca2+ stores closely paralleled phospholipase C activation. However, the maximal intracellular Ca2+ concentrations achieved by these two agents were the same. Despite this, gamma-thrombin and not PAF, was able to release a small amount of arachidonic acid. When alpha-thrombin stimulation of platelets was preceded by epinephrine, there was a potentiation of phospholipase C activation, Ca2+ mobilization and aggregation. The same was true for gamma-thrombin and PAF. However, unlike alpha-thrombin, the gamma-thrombin-stimulated arachidonic acid release was not potentiated by epinephrine, but rather somewhat reduced. These results suggested that phospholipase C and phospholipase A2 were separable events in activated platelets. The mechanism by which alpha-thrombin stimulated phospholipase A2 did not appear to be through dissociation of the inhibitory GTP-binding protein, Gi, since gamma-thrombin decreased the pertussis toxin-induced ADP-ribosylation of the 41 kDa protein as much as did alpha-thrombin, but was a much less effective agent than alpha-thrombin at inducing arachidonic acid liberation.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D037342:Pertussis Toxin; D010712:Phosphatidic Acids / Q000097:blood; D010740:Phospholipases / Q000097:blood*; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000494:pharmacology; D010974:Platelet Aggregation; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007295:Inositol Phosphates; D010712:Phosphatidic Acids; D010972:Platelet Activating Factor; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; D002118:Calcium